Cyclooxygenase-2 Inhibition Enhances Proliferation of NKT Cells Derived from Patients with Laryngeal Cancer.

PubMed

Klatka, Janusz; Grywalska, Ewelina; Hymos, Anna; Guz, Małgorzata; Polberg, Krzysztof; Roliński, Jacek; Stepulak, Andrzej

2017-08-01

The aim of this study was to analyze whether inhibition of cyclooxygenase-2 by celecoxib and the subsequent enhancement in the proliferation of natural killer T (NKT) cells could play a role in dendritic cell (DC)-based laryngeal cancer (LC) immunotherapy. Peripheral blood mononuclear cells were obtained from 48 male patients diagnosed with LC and 30 control patients without cancer disease. Neoplastic cell lysate preparations were made from cancer tissues obtained after surgery and used for in vitro DCs generation. NKT cells proliferation assay was performed based on 3 H-thymidine incorporation assay. An increased proliferation of NKT cells was obtained from control patients compared to NKT cells obtained from LC patients regardless of the type of stimulation or treatment. In the patient group diagnosed with LC, COX-2 inhibition resulted in a significantly enhanced proliferation of NKT cells when stimulated with autologous DCs than NKT cells stimulated with DCs without COX-2 inhibition. These correlations were not present in the control group. Higher proliferation rate of NKT cells was also observed in non-metastatic and highly differentiated LC, which was independent of the type of stimulation or treatment. COX-2 inhibition could be regarded as immunotherapy-enhancing tool in patients with LC. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Current-Controlled Electrical Point-Source Stimulation of Embryonic Stem Cells

PubMed Central

Chen, Michael Q.; Xie, Xiaoyan; Wilson, Kitchener D.; Sun, Ning; Wu, Joseph C.; Giovangrandi, Laurent; Kovacs, Gregory T. A.

2010-01-01

Stem cell therapy is emerging as a promising clinical approach for myocardial repair. However, the interactions between the graft and host, resulting in inconsistent levels of integration, remain largely unknown. In particular, the influence of electrical activity of the surrounding host tissue on graft differentiation and integration is poorly understood. In order to study this influence under controlled conditions, an in vitro system was developed. Electrical pacing of differentiating murine embryonic stem (ES) cells was performed at physiologically relevant levels through direct contact with microelectrodes, simulating the local activation resulting from contact with surrounding electroactive tissue. Cells stimulated with a charged balanced voltage-controlled current source for up to 4 days were analyzed for cardiac and ES cell gene expression using real-time PCR, immunofluorescent imaging, and genome microarray analysis. Results varied between ES cells from three progressive differentiation stages and stimulation amplitudes (nine conditions), indicating a high sensitivity to electrical pacing. Conditions that maximally encouraged cardiomyocyte differentiation were found with Day 7 EBs stimulated at 30 µA. The resulting gene expression included a sixfold increase in troponin-T and a twofold increase in β-MHCwithout increasing ES cell proliferation marker Nanog. Subsequent genome microarray analysis revealed broad transcriptome changes after pacing. Concurrent to upregulation of mature gene programs including cardiovascular, neurological, and musculoskeletal systems is the apparent downregulation of important self-renewal and pluripotency genes. Overall, a robust system capable of long-term stimulation of ES cells is demonstrated, and specific conditions are outlined that most encourage cardiomyocyte differentiation. PMID:20652088

Mitochondrial matrix pH controls oxidative phosphorylation and metabolism-secretion coupling in INS-1E clonal beta cells.

PubMed

Akhmedov, Dmitry; Braun, Matthias; Mataki, Chikage; Park, Kyu-Sang; Pozzan, Tullio; Schoonjans, Kristina; Rorsman, Patrik; Wollheim, Claes B; Wiederkehr, Andreas

2010-11-01

Glucose-evoked mitochondrial signals augment ATP synthesis in the pancreatic β cell. This activation of energy metabolism increases the cytosolic ATP/ADP ratio, which stimulates plasma membrane electrical activity and insulin granule exocytosis. We have recently demonstrated that matrix pH increases during nutrient stimulation of the pancreatic β cell. Here, we have tested whether mitochondrial matrix pH controls oxidative phosphorylation and metabolism-secretion coupling in the rat β-cell line INS-1E. Acidification of the mitochondrial matrix pH by nigericin blunted nutrient-dependent respiratory and ATP responses (continuously monitored in intact cells). Using electrophysiology and single cell imaging, we find that the associated defects in energy metabolism suppress glucose-stimulated plasma membrane electrical activity and cytosolic calcium transients. The same parameters were unaffected after direct stimulation of electrical activity with tolbutamide, which bypasses mitochondrial function. Furthermore, lowered matrix pH strongly inhibited sustained, but not first-phase, insulin secretion. Our results demonstrate that the matrix pH exerts a control function on oxidative phosphorylation in intact cells and that this mode of regulation is of physiological relevance for the generation of downstream signals leading to insulin granule exocytosis. We propose that matrix pH serves a novel signaling role in sustained cell activation.

Decellularized heart valve as a scaffold for in vivo recellularization: deleterious effects of granulocyte colony-stimulating factor.

PubMed

Juthier, Francis; Vincentelli, André; Gaudric, Julien; Corseaux, Delphine; Fouquet, Olivier; Calet, Christine; Le Tourneau, Thierry; Soenen, Valérie; Zawadzki, Christophe; Fabre, Olivier; Susen, Sophie; Prat, Alain; Jude, Brigitte

2006-04-01

Autologous recellularization of decellularized heart valve scaffolds is a promising challenge in the field of tissue-engineered heart valves and could be boosted by bone marrow progenitor cell mobilization. The aim of this study was to examine the spontaneous in vivo recolonization potential of xenogeneic decellularized heart valves in a lamb model and the effects of granulocyte colony-stimulating factor mobilization of bone marrow cells on this process. Decellularized porcine aortic valves were implanted in 12 lambs. Six lambs received granulocyte colony-stimulating factor (10 microg x kg(-1) x d(-1) for 7 days, granulocyte colony-stimulating factor group), and 6 received no granulocyte colony-stimulating factor (control group). Additionally, nondecellularized porcine valves were implanted in 5 lambs (xenograft group). Angiographic and histologic evaluation was performed at 3, 6, 8, and 16 weeks. Few macroscopic modifications of leaflets and the aortic wall were observed in the control group, whereas progressive shrinkage and thickening of the leaflets appeared in the granulocyte colony-stimulating factor and xenograft groups. In the 3 groups progressive ovine cell infiltration (fluorescence in situ hybridization) was observed in the leaflets and in the adventitia and the intima of the aortic wall but not in the media. Neointimal proliferation of alpha-actin-positive cells, inflammatory infiltration, adventitial neovascularization, and calcifications were more important in the xenograft and the granulocyte colony-stimulating factor groups than in the control group. Continuous re-endothelialization appeared only in the control group. Decellularized xenogeneic heart valve scaffolds allowed partial autologous recellularization. Granulocyte colony-stimulating factor led to accelerated heart valve deterioration similar to that observed in nondecellularized xenogeneic cardiac bioprostheses.

Control of proliferation rate of N27 dopaminergic neurons using Transcranial Magnetic Stimulation orientation

NASA Astrophysics Data System (ADS)

Meng, Yiwen; Hadimani, Ravi; Anantharam, Vellareddy; Kanthasamy, Anumantha; Jiles, David

2015-03-01

Transcranial magnetic stimulation (TMS) has been used to investigate possible treatments for a variety of neurological disorders. However, the effect that magnetic fields have on neurons has not been well documented in the literature. We have investigated the effect of different orientation of magnetic field generated by TMS coils with a monophasic stimulator on the proliferation rate of N27 neuronal cells cultured in flasks and multi-well plates. The proliferation rate of neurons would increase by exposed horizontally adherent N27 cells to a magnetic field pointing upward through the neuronal proliferation layer compared with the control group. On the other hand, proliferation rate would decrease in cells exposed to a magnetic field pointing downward through the neuronal growth layer compared with the control group. We confirmed results obtained from the Trypan-blue and automatic cell counting methods with those from the CyQuant and MTS cell viability assays. Our findings could have important implications for the preclinical development of TMS treatments of neurological disorders and represents a new method to control the proliferation rate of neuronal cells.

[Effect of G-CSF in vitro Stimulation on Distribution of Peripheral Lymphocyte Subsets in the Healthy Persons].

PubMed

Zhao, Sha-Sha; Fang, Shu; Zhu, Cheng-Ying; Wang, Li-Li; Gao, Chun-Ji

2018-02-01

To investigate the effect of granulocyte-colony stimulating factor (G-CSF) in vitro stimulation on the distribution of lymphocyte subset in healthy human. Peripheral blood mononuclear cells (PBMNCs) were collected from 8 healthy volunteers by density gradient centrifugation on Ficoll-Paque TM . In vitro 200 ng/ml G-CSF or 200 ng/ml G-CSF plus 10 µg/ml ConA directly act on PBMNCs, then the colleted cells were cultivated for 3 days. Lymphocyte subsets were stained with the corresponding fluoresce labeled antibodies and detected by flow cytometry. The levels of T cells in G-CSF group and G-CSF+ConA group were both higher than that in the control group (P<0.001, P<0.05). However, there were not significantly different in B cells and NK cells levels among the 3 groups. Furthermore, analysis of the effect of G-CSF on T cell subsets indicated that the levels of CD4 + T cells and CD8 + T cells in G-CSF group were both significantly higher than those in control group (P<0.01, P<0.05), Treg cells was not different between G-CSF and control group. Compared with the control group, the level of CD4 + T cells, CD8 + T cells and Treg cells in G-CSF+ConA group significantly increased (P<0.05, P<0.01, P<0.01). Analysis of G-CSF receptor (G-CSFR) expression showed that G-CSFR expression on T cells in G-CSF+ConA group dramatically increased, as compared with control group (P<0.01). The levels of CD4 + T cells and CD8 + T cells in healthy human peripheral blood can be increased by G-CSF stimulation. ConA can enhance the level of T cells and induce G-CSFR expression on T cells.

Induction of neural differentiation by electrically stimulated gene expression of NeuroD2.

PubMed

Mie, Masayasu; Endoh, Tamaki; Yanagida, Yasuko; Kobatake, Eiry; Aizawa, Masuo

2003-02-13

Regulation of cell differentiation is an important assignment for cellular engineering. One of the techniques for regulation is gene transfection into undifferentiated cells. Transient expression of NeuroD2, one of neural bHLH transcription factors, converted mouse N1E-115 neuroblastoma cells into differentiated neurons. The regulation of neural bHLH expression should be a novel strategy for cell differentiation. In this study, we tried to regulate neural differentiation by NeuroD2 gene inserted under the control of heat shock protein-70 (HSP) promoter, which can be activated by electrical stimulation. Mouse neuroblastoma cell line, N1E-115, was stably transfected with expression vector containing mouse NeuroD2 cDNA under HSP promoter. Transfected cells were cultured on the electrode surface and applied electrical stimulation. After stimulation, NeuroD2 expression was induced, and transfected cells adopt a neuronal morphology at 3 days after stimulation. These results suggest that neural differentiation can be induced by electrically stimulated gene expression of NeuroD2.

Memory T cells in organ transplantation: progress and challenges

PubMed Central

Espinosa, Jaclyn R.; Samy, Kannan P.; Kirk, Allan D.

2017-01-01

Antigen-experienced T cells, also known as memory T cells, are functionally and phenotypically distinct from naive T cells. Their enhanced expression of adhesion molecules and reduced requirement for co-stimulation enables them to mount potent and rapid recall responses to subsequent antigen encounters. Memory T cells generated in response to prior antigen exposures can cross-react with other nonidentical, but similar, antigens. This heterologous cross-reactivity not only enhances protective immune responses, but also engenders de novo alloimmunity. This latter characteristic is increasingly recognized as a potential barrier to allograft acceptance that is worthy of immunotherapeutic intervention, and several approaches have been investigated. Calcineurin inhibition effectively controls memory T-cell responses to allografts, but this benefit comes at the expense of increased infectious morbidity. Lymphocyte depletion eliminates allospecific T cells but spares memory T cells to some extent, such that patients do not completely lose protective immunity. Co-stimulation blockade is associated with reduced adverse-effect profiles and improved graft function relative to calcineurin inhibition, but lacks efficacy in controlling memory T-cell responses. Targeting the adhesion molecules that are upregulated on memory T cells might offer additional means to control co-stimulation-blockade-resistant memory T-cell responses. PMID:26923209

β-Cell Function Improvements in Grade I/II Obese Subjects With Type 2 Diabetes 1 Month After Biliopancreatic Diversion

PubMed Central

Junqueira Vasques, Ana Carolina; Pareja, José Carlos; de Oliveira, Maria da Saude; Satake Novaes, Fernanda; Miranda de Oliveira Lima, Marcelo; Chaim, Élinton A.; Piccinini, Francesca; Dalla Man, Chiara; Cobelli, Claudio; Geloneze, Bruno

2013-01-01

OBJECTIVE To investigate the effect of biliopancreatic diversion (BPD) surgery on β-cell function in grade I and II obese patients with type 2 diabetes using oral and intravenous glucose loads. RESEARCH DESIGN AND METHODS Sixty-eight women were divided into the following three groups: 19 lean-control (23.0 ± 2.2 kg/m2) and 18 obese-control (35.0 ± 4.8 kg/m2) subjects with normal glucose tolerance, and 31 obese patients with type 2 diabetes (36.3 ± 3.7 kg/m2). Of the 31 diabetic women, 64% underwent BPD (n = 20, BMI: 36.5 ± 3.7 kg/m2) and were reassessed 1 month after surgery. Oral glucose tolerance tests and hyperglycemic clamps were performed. Mathematical modeling was used to analyze basal and stimulated β-cell function, insulin sensitivity (IS), hepatic extraction (HE) of insulin, and delay time of β-cell response to a specific plasma glucose concentration. RESULTS After BPD, restoration of the basal disposition index (P < 0.001) and improvement of the stimulated disposition indices in oral and intravenous glucose stimulation of the β-cell were observed (P < 0.05). In both dynamic tests, there were no changes in the delay time of β-cell response. IS for oral glucose stimulation (ISoral) and intravenous clamp glucose stimulation (ISclamp) was completely normalized (P < 0.001). ISoral and ISclamp increased approximately 5.0-fold and 3.5-fold, respectively (P < 0.01). The HE of insulin increased in the basal (P < 0.05) and stimulated states (P < 0.01). CONCLUSIONS β-Cell function, IS, and HE of insulin improved after BPD, which improved glycemic control. PMID:24135388

Laser controlled singlet oxygen generation in mitochondria to promote mitochondrial DNA replication in vitro.

PubMed

Zhou, Xin; Wang, Yupei; Si, Jing; Zhou, Rong; Gan, Lu; Di, Cuixia; Xie, Yi; Zhang, Hong

2015-11-18

Reports have shown that a certain level of reactive oxygen species (ROS) can promote mitochondrial DNA (mtDNA) replication. However, it is unclear whether it is the mitochondrial ROS that stimulate mtDNA replication and this requires further investigation. Here we employed a photodynamic system to achieve controlled mitochondrial singlet oxygen ((1)O2) generation. HeLa cells incubated with 5-aminolevulinic acid (ALA) were exposed to laser irradiation to induce (1)O2 generation within mitochondria. Increased mtDNA copy number was detected after low doses of 630 nm laser light in ALA-treated cells. The stimulated mtDNA replication was directly linked to mitochondrial (1)O2 generation, as verified using specific ROS scavengers. The stimulated mtDNA replication was regulated by mitochondrial transcription factor A (TFAM) and mtDNA polymerase γ. MtDNA control region modifications were induced by (1)O2 generation in mitochondria. A marked increase in 8-Oxoguanine (8-oxoG) level was detected in ALA-treated cells after irradiation. HeLa cell growth stimulation and G1-S cell cycle transition were also observed after laser irradiation in ALA-treated cells. These cellular responses could be due to a second wave of ROS generation detected in mitochondria. In summary, we describe a controllable method of inducing mtDNA replication in vitro.

The cell-stretcher: A novel device for the mechanical stimulation of cell populations

NASA Astrophysics Data System (ADS)

Seriani, S.; Del Favero, G.; Mahaffey, J.; Marko, D.; Gallina, P.; Long, C. S.; Mestroni, L.; Sbaizero, O.

2016-08-01

Mechanical stimulation appears to be a critical modulator for many aspects of biology, both of living tissue and cells. The cell-stretcher, a novel device for the mechanical uniaxial stimulation of populations of cells, is described. The system is based on a variable stroke cam-lever-tappet mechanism which allows the delivery of cyclic stimuli with frequencies of up to 10 Hz and deformation between 1% and 20%. The kinematics is presented and a simulation of the dynamics of the system is shown, in order to compute the contact forces in the mechanism. The cells, following cultivation and preparation, are plated on an ad hoc polydimethylsiloxane membrane which is then loaded on the clamps of the cell-stretcher via force-adjustable magnetic couplings. In order to show the viability of the experimentation and biocompatibility of the cell-stretcher, a set of two in vitro tests were performed. Human epithelial carcinoma cell line A431 and Adult Mouse Ventricular Fibroblasts (AMVFs) from a dual reporter mouse were subject to 0.5 Hz, 24 h cyclic stretching at 15% strain, and to 48 h stimulation at 0.5 Hz and 15% strain, respectively. Visual analysis was performed on A431, showing definite morphological changes in the form of cellular extroflections in the direction of stimulation compared to an unstimulated control. A cytometric analysis was performed on the AMVF population. Results show a post-stimulation live-dead ratio deviance of less than 6% compared to control, which proves that the environment created by the cell-stretcher is suitable for in vitro experimentation.

Electrostimulation of rat callus cells and human lymphocytes in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aro, H.; Eerola, E.; Aho, A.J.

1984-01-01

Asymmetrical pulsing low voltage current was supplied via electrodes to cultured rat fracture callus cells and human peripheral blood lymphocytes. The (/sup 3/H)thymidine incorporation of the callus cells and 5-(/sup 125/I)iodo-2'-deoxyuridine incorporation of the lymphocytes were determined. The growth pattern of callus cells (estimated by cellular density) did not respond to electrical stimulation. However, the uptake of (/sup 3/H)thymidine was increased at the early phase of cell proliferation and inhibited at later phases of proliferation. The (/sup 3/H)thymidine uptake of confluent callus cell cultures did not respond to electrical stimulation. Lymphocytes reacted in a similar way; stimulated cells took upmore » more DNA precursor than control cells at the early phase of stimulation. During cell division, induced by the mitogens phytohemagglutinin and Concanavalin-A, the uptake of DNA precursor by stimulated cells was constantly inhibited. The results suggest that electrical stimuli affect the uptake mechanisms of cell membranes. The duality of the effect seems to be dependent on the cell cycle.« less

Ultrasound Stimulation of Insulin Release from Pancreatic Beta Cells

NASA Astrophysics Data System (ADS)

Suarez Castellanos, Ivan M.

Type 2 diabetes (T2D) mellitus is a complex metabolic disease that has reached epidemic proportions in the United States and around the world. Controlling T2D is often difficult as pharmacological management routinely requires complex therapy with multiple medications, and loses its effectiveness over time. The objective of this dissertation was to explore a novel, non-pharmacological approach that utilizes the application of ultrasound energy to stimulate insulin release. Our experiments have focused on determination of effectiveness and safety of ultrasound application in stimulation of insulin release from the pancreatic beta cells. Our results showed that ultrasound treatment, applied at frequencies of 800 kHz and 1 MHz and intensities of 0.5 W/cm2 and 1 W/cm2, did not produce any significant effects on cell viability compared to sham group as assessed with trypan blue dye exclusion test and MTT cytotoxicity assay. ELISA quantification of insulin release from beta cells resulting from ultrasound treatment showed clinically-significant amounts of released insulin as compared to sham-treated beta cells. Carbon fiber amperometry detection of secretory events from dopamine-loaded beta cells treated with ultrasound showed that release of secretory content could be temporally controlled by careful selection of ultrasound parameters. Both ELISA and amperometry experiments demonstrated that ultrasound-stimulated insulin release is a calcium-dependent process, potentially mediated by the mechanical effects of ultrasound. This study demonstrated that therapeutic ultrasound is a technique capable of stimulating the release of insulin from pancreatic beta cells in a safe, effective and controlled manner.

A Hybrid Robotic Control System Using Neuroblastoma Cultures

NASA Astrophysics Data System (ADS)

Ferrández, J. M.; Lorente, V.; Cuadra, J. M.; Delapaz, F.; Álvarez-Sánchez, José Ramón; Fernández, E.

The main objective of this work is to analyze the computing capabilities of human neuroblastoma cultured cells and to define connection schemes for controlling a robot behavior. Multielectrode Array (MEA) setups have been designed for direct culturing neural cells over silicon or glass substrates, providing the capability to stimulate and record simultaneously populations of neural cells. This paper describes the process of growing human neuroblastoma cells over MEA substrates and tries to modulate the natural physiologic responses of these cells by tetanic stimulation of the culture. We show that the large neuroblastoma networks developed in cultured MEAs are capable of learning: establishing numerous and dynamic connections, with modifiability induced by external stimuli and we propose an hybrid system for controlling a robot to avoid obstacles.

Enhancing adoptive cancer immunotherapy with Vγ2Vδ2 T cells through pulse zoledronate stimulation.

PubMed

Nada, Mohanad H; Wang, Hong; Workalemahu, Grefachew; Tanaka, Yoshimasa; Morita, Craig T

2017-01-01

Human γδ T cells expressing Vγ2Vδ2 T cell receptors monitor foreign- and self-prenyl pyrophosphate metabolites in isoprenoid biosynthesis to mediate immunity to microbes and tumors. Adoptive immunotherapy with Vγ2Vδ2 T cells has been used to treat cancer patients with partial and complete remissions. Most clinical trials and preclinical studies have used continuous zoledronate exposure to expand Vγ2Vδ2 cells where zoledronate is slowly diluted over the course of the culture. Zoledronate inhibits farnesyl diphosphate synthase (FDPS) in monocytes causing isopentenyl pyrophosphate to accumulate that then stimulates Vγ2Vδ2 cells. Because zoledronate inhibition of FDPS is also toxic for T cells, we hypothesized that a short period of exposure would reduce T cell toxicity but still be sufficient for monocytes uptake. Additionally, IL-15 increases the anti-tumor activity of murine αβ T cells in mice but its effect on the in vivo anti-tumor activity of human Vγ2Vδ2 cells has not been assessed. Human Vγ2Vδ2 T cells were expanded by pulse or continuous zoledronate stimulation with IL-2 or IL-15. Expanded Vγ2Vδ2 cells were tested for their expression of effector molecules and killing of tumor cells as well as their in vivo control of human prostate cancer tumors in immunodeficient NSG mice. Pulse zoledronate stimulation with either IL-2 or IL-15 resulted in more uniform expansion of Vγ2Vδ2 cells with higher purity and cell numbers as compared with continuous exposure. The Vγ2Vδ2 cells had higher levels of CD107a and perforin and increased tumor cytotoxicity. Adoptive immunotherapy with Vγ2Vδ2 cells derived by pulse stimulation controlled human PC-3 prostate cancer tumors in NSG mice significantly better than those derived by continuous stimulation, halting tumor growth. Although pulse zoledronate stimulation with IL-15 preserved early memory subsets, adoptive immunotherapy with IL-15-derived Vγ2Vδ2 cells equally inhibited PC-3 tumor growth as those derived with IL-2. Pulse zoledronate stimulation maximizes the purity, quantity, and quality of expanded Vγ2Vδ2 cells for adoptive immunotherapy but there is no advantage to using IL-15 over IL-2 in our humanized mouse model. Pulse zoledronate stimulation is a simple modification to existing protocols that will enhance the effectiveness of adoptively transferred Vγ2Vδ2 cells by increasing their numbers and anti-tumor activity.

Allergen-stimulated T lymphocytes from allergic patients induce vascular cell adhesion molecule-1 (VCAM-1) expression and IL-6 production by endothelial cells.

PubMed Central

Delneste, Y; Jeannin, P; Gosset, P; Lassalle, P; Cardot, E; Tillie-Leblond, I; Joseph, M; Pestel, J; Tonnel, A B

1995-01-01

Adhesion of inflammatory cells to endothelium is a critical step for their transvascular migration to inflammatory sites. To evaluate the relationship between T lymphocytes (TL) and vascular endothelium, supernatants from allergen-stimulated TL obtained from patients sensitive to Dermatophagoides pteronyssinus (Dpt) versus healthy subjects were added to endothelial cell (EC) cultures. TL were stimulated by autologous-activated antigen-presenting cells (APC) previously fixed in paraformaldehyde to prevent monokine secretion. Two parameters were measured: the expression of adhesion molecule and the production of IL-6. Related allergen-stimulated TL supernatants from allergic patients induced an increase of VCAM-1 and intercellular adhesion molecule-1 (ICAM-1) expression when supernatants of the control groups (TL exposed to an unrelated allergen or not stimulated or TL obtained from healthy subjects) did not. E-selectin expression was not modulated whatever the supernatant added to EC culture. IL-6 production by EC was significantly enhanced after activation with related allergen-stimulated TL supernatants from allergics compared with control supernatants. Induction of VCAM-1 expression was inhibited by adding neutralizing antibodies against IL-4, whereas IL-6 production and ICAM-1 expression were inhibited by anti-interferon-gamma (IFN-gamma) antibodies. Enhanced production of IL-4 and IFN-gamma was detected in related allergen-stimulated TL supernatants from allergic subjects compared with the different supernatants. These data suggest that allergen-specific TL present in the peripheral blood of allergic patients are of Th1 and Th2 subtypes. Their stimulation in allergic patients may lead to the activation of endothelial cells and thereby participate in leucocyte recruitment towards the inflammatory site. PMID:7542574

Auxin-Dependent Cell Division and Cell Elongation. 1-Naphthaleneacetic Acid and 2,4-Dichlorophenoxyacetic Acid Activate Different Pathways1

PubMed Central

Campanoni, Prisca; Nick, Peter

2005-01-01

During exponential phase, the tobacco (Nicotiana tabacum) cell line cv Virginia Bright Italia-0 divides axially to produce linear cell files of distinct polarity. This axial division is controlled by exogenous auxin. We used exponential tobacco cv Virginia Bright Italia-0 cells to dissect early auxin signaling, with cell division and cell elongation as physiological markers. Experiments with 1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) demonstrated that these 2 auxin species affect cell division and cell elongation differentially; NAA stimulates cell elongation at concentrations that are much lower than those required to stimulate cell division. In contrast, 2,4-D promotes cell division but not cell elongation. Pertussis toxin, a blocker of heterotrimeric G-proteins, inhibits the stimulation of cell division by 2,4-D but does not affect cell elongation. Aluminum tetrafluoride, an activator of the G-proteins, can induce cell division at NAA concentrations that are not permissive for division and even in the absence of any exogenous auxin. The data are discussed in a model where the two different auxins activate two different pathways for the control of cell division and cell elongation. PMID:15734918

Photothermal and mechanical stimulation of cells via dualfunctional nanohybrids

NASA Astrophysics Data System (ADS)

Chechetka, Svetlana A.; Doi, Motomichi; Pichon, Benoit P.; Bégin-Colin, Sylvie; Miyako, Eijiro

2016-11-01

Stimulating cells by light is an attractive technology to investigate cellular function and deliver innovative cell-based therapy. However, current techniques generally use poorly biopermeable light, which prevents broad applicability. Here, we show that a new type of composite nanomaterial, synthesized from multi-walled carbon nanotubes, magnetic iron nanoparticles, and polyglycerol, enables photothermal and mechanical control of Ca2+ influx into cells overexpressing transient receptor potential vanilloid type-2. The nanohybrid is simply operated by application of highly biotransparent near-infrared light and a magnetic field. The technology may revolutionize remote control of cellular function.

Efficient generation of smooth muscle cells from adipose-derived stromal cells by 3D mechanical stimulation can substitute the use of growth factors in vascular tissue engineering.

PubMed

Parvizi, Mojtaba; Bolhuis-Versteeg, Lydia A M; Poot, André A; Harmsen, Martin C

2016-07-01

Occluding artery disease causes a high demand for bioartificial replacement vessels. We investigated the combined use of biodegradable and creep-free poly (1,3-trimethylene carbonate) (PTMC) with smooth muscle cells (SMC) derived by biochemical or mechanical stimulation of adipose tissue-derived stromal cells (ASC) to engineer bioartificial arteries. Biochemical induction of cultured ASC to SMC was done with TGF-β1 for 7d. Phenotype and function were assessed by qRT-PCR, immunodetection and collagen contraction assays. The influence of mechanical stimulation on non-differentiated and pre-differentiated ASC, loaded in porous tubular PTMC scaffolds, was assessed after culturing under pulsatile flow for 14d. Assays included qRT-PCR, production of extracellular matrix and scanning electron microscopy. ASC adhesion and TGF-β1-driven differentiation to contractile SMC on PTMC did not differ from tissue culture polystyrene controls. Mesenchymal and SMC markers were increased compared to controls. Interestingly, pre-differentiated ASC had only marginal higher contractility than controls. Moreover, in 3D PTMC scaffolds, mechanical stimulation yielded well-aligned ASC-derived SMC which deposited ECM. Under the same conditions, pre-differentiated ASC-derived SMC maintained their SMC phenotype. Our results show that mechanical stimulation can replace TGF-β1 pre-stimulation to generate SMC from ASC and that pre-differentiated ASC keep their SMC phenotype with increased expression of SMC markers. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Electrical Stimulation Promotes Cardiac Differentiation of Human Induced Pluripotent Stem Cells

PubMed Central

Hernández, Damián; Millard, Rodney; Sivakumaran, Priyadharshini; Wong, Raymond C. B.; Crombie, Duncan E.; Hewitt, Alex W.; Liang, Helena; Hung, Sandy S. C.; Pébay, Alice; Shepherd, Robert K.; Dusting, Gregory J.; Lim, Shiang Y.

2016-01-01

Background. Human induced pluripotent stem cells (iPSCs) are an attractive source of cardiomyocytes for cardiac repair and regeneration. In this study, we aim to determine whether acute electrical stimulation of human iPSCs can promote their differentiation to cardiomyocytes. Methods. Human iPSCs were differentiated to cardiac cells by forming embryoid bodies (EBs) for 5 days. EBs were then subjected to brief electrical stimulation and plated down for 14 days. Results. In iPS(Foreskin)-2 cell line, brief electrical stimulation at 65 mV/mm or 200 mV/mm for 5 min significantly increased the percentage of beating EBs present by day 14 after plating. Acute electrical stimulation also significantly increased the cardiac gene expression of ACTC1, TNNT2, MYH7, and MYL7. However, the cardiogenic effect of electrical stimulation was not reproducible in another iPS cell line, CERA007c6. Beating EBs from control and electrically stimulated groups expressed various cardiac-specific transcription factors and contractile muscle markers. Beating EBs were also shown to cycle calcium and were responsive to the chronotropic agents, isoproterenol and carbamylcholine, in a concentration-dependent manner. Conclusions. Our results demonstrate that brief electrical stimulation can promote cardiac differentiation of human iPS cells. The cardiogenic effect of brief electrical stimulation is dependent on the cell line used. PMID:26788064

Hyperactivity and reactivity of peripheral blood neutrophils in chronic periodontitis.

PubMed

Matthews, J B; Wright, H J; Roberts, A; Cooper, P R; Chapple, I L C

2007-02-01

Some evidence exists that peripheral neutrophils from patients with chronic periodontitis generate higher levels of reactive oxygen species (ROS) after Fcgamma-receptor stimulation than those from healthy controls. We hypothesized that peripheral neutrophils in periodontitis also show both hyper-reactivity to plaque organisms and hyperactivity in terms of baseline, unstimulated generation and release of ROS. Peripheral neutrophils from chronic periodontitis patients and age/sex/smoking-matched healthy controls (18 pairs) were assayed for total ROS generation and extracellular ROS release, with and without stimulation (Fcgamma-receptor and Fusobacterium nucleatum), using luminol and isoluminol chemiluminescence. Assays were performed with and without priming with Escherichia coli lipopolysaccharide (LPS) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Phox gene expression (p22, p47, p67, gp91) was investigated using reverse transcription-polymerase chain reaction (RT-PCR). Neutrophils from patients produced higher mean levels of ROS in all assays. Total generation and extracellular release of ROS by patients' cells were significantly greater than those from controls after FcgammaR-stimulation, with (P = 0.023) and without (P < or = 0.023) priming with GM-CSF. Differences in unstimulated total ROS generation were not significant. By contrast, patients' cells demonstrated greater baseline, extracellular ROS release than those from controls (P = 0.004). This difference was maintained after priming with LPS (P = 0.028) but not GM-CSF (P = 0.217). Phox gene expression was similar in patient and control cells at baseline and stimulation with F. nucleatum (3 h) consistently reduced gp91(PHOX) transcripts. Our data demonstrate that peripheral neutrophils from periodontitis patients exhibit hyper-reactivity following stimulation (Fcgamma-receptor and F. nucleatum) and hyperactivity in terms of excess ROS release in the absence of exogenous stimulation. This hyperactive/-reactive neutrophil phenotype is not associated with elevated phox gene expression.

Neuron matters: electric activation of neuronal tissue is dependent on the interaction between the neuron and the electric field.

PubMed

Ye, Hui; Steiger, Amanda

2015-08-12

In laboratory research and clinical practice, externally-applied electric fields have been widely used to control neuronal activity. It is generally accepted that neuronal excitability is controlled by electric current that depolarizes or hyperpolarizes the excitable cell membrane. What determines the amount of polarization? Research on the mechanisms of electric stimulation focus on the optimal control of the field properties (frequency, amplitude, and direction of the electric currents) to improve stimulation outcomes. Emerging evidence from modeling and experimental studies support the existence of interactions between the targeted neurons and the externally-applied electric fields. With cell-field interaction, we suggest a two-way process. When a neuron is positioned inside an electric field, the electric field will induce a change in the resting membrane potential by superimposing an electrically-induced transmembrane potential (ITP). At the same time, the electric field can be perturbed and re-distributed by the cell. This cell-field interaction may play a significant role in the overall effects of stimulation. The redistributed field can cause secondary effects to neighboring cells by altering their geometrical pattern and amount of membrane polarization. Neurons excited by the externally-applied electric field can also affect neighboring cells by ephaptic interaction. Both aspects of the cell-field interaction depend on the biophysical properties of the neuronal tissue, including geometric (i.e., size, shape, orientation to the field) and electric (i.e., conductivity and dielectricity) attributes of the cells. The biophysical basis of the cell-field interaction can be explained by the electromagnetism theory. Further experimental and simulation studies on electric stimulation of neuronal tissue should consider the prospect of a cell-field interaction, and a better understanding of tissue inhomogeneity and anisotropy is needed to fully appreciate the neural basis of cell-field interaction as well as the biological effects of electric stimulation.

Boron nitride nanotube-mediated stimulation modulates F/G-actin ratio and mechanical properties of human dermal fibroblasts

NASA Astrophysics Data System (ADS)

Ricotti, Leonardo; das Neves, Ricardo Pires; Ciofani, Gianni; Canale, Claudio; Nitti, Simone; Mattoli, Virgilio; Mazzolai, Barbara; Ferreira, Lino; Menciassi, Arianna

2014-02-01

F/G-actin ratio modulation is known to have an important role in many cell functions and in the regulation of specific cell behaviors. Several attempts have been made in the latest decades to finely control actin production and polymerization, in order to promote certain cell responses. In this paper we demonstrate the possibility of modulating F/G-actin ratio and mechanical properties of normal human dermal fibroblasts by using boron nitride nanotubes dispersed in the culture medium and by stimulating them with ultrasound transducers. Increasing concentrations of nanotubes were tested with the cells, without any evidence of cytotoxicity up to 10 μg/ml concentration of nanoparticles. Cells treated with nanoparticles and ultrasound stimulation showed a significantly higher F/G-actin ratio in comparison with the controls, as well as a higher Young's modulus. Assessment of Cdc42 activity revealed that actin nucleation/polymerization pathways, involving Rho GTPases, are probably influenced by nanotube-mediated stimulation, but they do not play a primary role in the significant increase of F/G-actin ratio of treated cells, such effect being mainly due to actin overexpression.

Effect of electrical stimulation on beta-adrenergic receptor population and cyclic amp production in chicken and rat skeletal muscle cell cultures

NASA Technical Reports Server (NTRS)

Young, R. B.; Bridge, K. Y.; Strietzel, C. J.

2000-01-01

Expression of the beta-adrenergic receptor (betaAR) and its coupling to cyclic AMP (cAMP) synthesis are important components of the signaling system that controls muscle atrophy and hypertrophy, and the goal of this study was to determine if electrical stimulation in a pattern simulating slow muscle contraction would alter the betaAR response in primary cultures of avian and mammalian skeletal muscle cells. Specifically, chicken skeletal muscle cells and rat skeletal muscle cells that had been grown for 7 d in culture were subjected to electrical stimulation for an additional 2 d at a pulse frequency of 0.5 pulses/sec and a pulse duration of 200 msec. In chicken skeletal muscle cells, the betaAR population was not significantly affected by electrical stimulation; however, the ability of these cells to synthesize cyclic AMP was reduced by approximately one-half. In contrast, the betaAR population in rat muscle cells was increased slightly but not significantly by electrical stimulation, and the ability of these cells to synthesize cyclic AMP was increased by almost twofold. The basal levels of intracellular cyclic AMP in neither rat muscle cells nor chicken muscle cells were affected by electrical stimulation.

Effect of Electrical Stimulation on Beta-Adrenergic Receptor Population and Cyclic AMP Production in Chicken and Rat Skeletal Muscle Cell Cultures

NASA Technical Reports Server (NTRS)

Young, Ronald B.; Bridge, Kristin Y.; Strietzel, Catherine J.

2000-01-01

Expression of the beta-adrenergic receptor (PAR) and its coupling to Adenosine 3'5' Cyclic Monophosphate (cAMP) synthesis are important components of the signaling system that controls muscle atrophy and hypertrophy and the goal of this study was to determine if electrical stimulation in a pattern simulating slow muscle contraction would alter the PAR response in primary cultures of avian and mammalian skeletal muscle cells. Specifically chicken skeletal muscle cells and rat skeletal muscle cells that had been grown for 7 d in culture, were subjected to electrical stimulation for an additional 2 d at a pulse frequency of 0.5 pulses/sec and a pulse duration of 200 msec. In chicken skeletal muscle cells, the PAR population was not significantly affected by electrical stimulation; however, the ability, of these cells to synthesize cyclic AMP was reduced by approximately one-half. In contrast, the PAR population in rat muscle cells was increased slightly but not significantly by electrical stimulation, and the ability of these cells to synthesize cyclic AMP was increased by almost twofold. The basal levels of intracellular cyclic AMP in neither rat muscle cells nor chicken muscle cells were affected by electrical stimulation.

Perturbed atrial calcium handling in an ovine model of heart failure: Potential roles for reductions in the L-type calcium current

PubMed Central

Clarke, Jessica D.; Caldwell, Jessica L.; Horn, Margaux A.; Bode, Elizabeth F.; Richards, Mark A.; Hall, Mark C.S.; Graham, Helen K.; Briston, Sarah J.; Greensmith, David J.; Eisner, David A.; Dibb, Katharine M.; Trafford, Andrew W.

2015-01-01

Heart failure (HF) is commonly associated with reduced cardiac output and an increased risk of atrial arrhythmias particularly during β-adrenergic stimulation. The aim of the present study was to determine how HF alters systolic Ca2 + and the response to β-adrenergic (β-AR) stimulation in atrial myocytes. HF was induced in sheep by ventricular tachypacing and changes in intracellular Ca2 + concentration studied in single left atrial myocytes under voltage and current clamp conditions. The following were all reduced in HF atrial myocytes; Ca2 + transient amplitude (by 46% in current clamped and 28% in voltage clamped cells), SR dependent rate of Ca2 + removal (kSR, by 32%), L-type Ca2 + current density (by 36%) and action potential duration (APD90 by 22%). However, in HF SR Ca2 + content was increased (by 19%) when measured under voltage-clamp stimulation. Inhibiting the L-type Ca2 + current (ICa-L) in control cells reproduced both the decrease in Ca2 + transient amplitude and increase of SR Ca2 + content observed in voltage-clamped HF cells. During β-AR stimulation Ca2 + transient amplitude was the same in control and HF cells. However, ICa-L remained less in HF than control cells whilst SR Ca2 + content was highest in HF cells during β-AR stimulation. The decrease in ICa-L that occurs in HF atrial myocytes appears to underpin the decreased Ca2 + transient amplitude and increased SR Ca2 + content observed in voltage-clamped cells. PMID:25463272

Lab on chip microdevices for cellular mechanotransduction in urothelial cells

NASA Astrophysics Data System (ADS)

Maziz, A.; Guan, N.; Svennersten, K.; Hallén-Grufman, K.; Jager, Edwin W. H.

2016-04-01

Cellular mechanotransduction is crucial for physiological function in the lower urinary tract. The bladder is highly dependent on the ability to sense and process mechanical inputs, illustrated by the regulated filling and voiding of the bladder. However, the mechanisms by which the bladder integrates mechanical inputs, such as intravesicular pressure, and controls the smooth muscles, remain unknown. To date no tools exist that satisfactorily mimic in vitro the dynamic micromechanical events initiated e.g. by an emerging inflammatory process or a growing tumour mass in the urinary tract. More specifically, there is a need for tools to study these events on a single cell level or in a small population of cells. We have developed a micromechanical stimulation chip that can apply physiologically relevant mechanical stimuli to single cells to study mechanosensitive cells in the urinary tract. The chips comprise arrays of microactuators based on the electroactive polymer polypyrrole (PPy). PPy offers unique possibilities and is a good candidate to provide such physiological mechanical stimulation, since it is driven at low voltages, is biocompatible, and can be microfabricated. The PPy microactuators can provide mechanical stimulation at different strains and/or strain rates to single cells or clusters of cells, including controls, all integrated on one single chip, without the need to preprepare the cells. This paper reports initial results on the mechano-response of urothelial cells using the micromechanical stimulation chips. We show that urothelial cells are viable on our microdevices and do respond with intracellular Ca2+ increase when subjected to a micro-mechanical stimulation.

Focal adhesion kinase is involved in mechanosensing during fibroblast migration

NASA Technical Reports Server (NTRS)

Wang, H. B.; Dembo, M.; Hanks, S. K.; Wang, Y.

2001-01-01

Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase localized at focal adhesions and is believed to mediate adhesion-stimulated effects. Although ablation of FAK impairs cell movement, it is not clear whether FAK might be involved in the guidance of cell migration, a role consistent with its putative regulatory function. We have transfected FAK-null fibroblasts with FAK gene under the control of the tetracycline repression system. Cells were cultured on flexible polyacrylamide substrates for the detection of traction forces and the application of mechanical stimulation. Compared with control cells expressing wild-type FAK, FAK-null cells showed a decrease in migration speed and directional persistence. In addition, whereas FAK-expressing cells responded to exerted forces by reorienting their movements and forming prominent focal adhesions, FAK-null cells failed to show such responses. Furthermore, FAK-null cells showed impaired responses to decreases in substrate flexibility, which causes control cells to generate weaker traction forces and migrate away from soft substrates. Cells expressing Y397F FAK, which cannot be phosphorylated at a key tyrosine site, showed similar defects in migration pattern and force-induced reorientation as did FAK-null cells. However, other aspects of F397-FAK cells, including the responses to substrate flexibility and the amplification of focal adhesions upon mechanical stimulation, were similar to that of control cells. Our results suggest that FAK plays an important role in the response of migrating cells to mechanical input. In addition, phosphorylation at Tyr-397 is required for some, but not all, of the functions of FAK in cell migration.

Electrical stimulation of cardiac adipose tissue-derived progenitor cells modulates cell phenotype and genetic machinery.

PubMed

Llucià-Valldeperas, A; Sanchez, B; Soler-Botija, C; Gálvez-Montón, C; Prat-Vidal, C; Roura, S; Rosell-Ferrer, J; Bragos, R; Bayes-Genis, A

2015-11-01

A major challenge of cardiac tissue engineering is directing cells to establish the physiological structure and function of the myocardium being replaced. Our aim was to examine the effect of electrical stimulation on the cardiodifferentiation potential of cardiac adipose tissue-derived progenitor cells (cardiac ATDPCs). Three different electrical stimulation protocols were tested; the selected protocol consisted of 2 ms monophasic square-wave pulses of 50 mV/cm at 1 Hz over 14 days. Cardiac and subcutaneous ATDPCs were grown on biocompatible patterned surfaces. Cardiomyogenic differentiation was examined by real-time PCR and immunocytofluorescence. In cardiac ATDPCs, MEF2A and GATA-4 were significantly upregulated at day 14 after stimulation, while subcutaneous ATDPCs only exhibited increased Cx43 expression. In response to electrical stimulation, cardiac ATDPCs elongated, and both cardiac and subcutaneous ATDPCs became aligned following the linear surface pattern of the construct. Cardiac ATDPC length increased by 11.3%, while subcutaneous ATDPC length diminished by 11.2% (p = 0.013 and p = 0.030 vs unstimulated controls, respectively). Compared to controls, electrostimulated cells became aligned better to the patterned surfaces when the pattern was perpendicular to the electric field (89.71 ± 28.47º for cardiac ATDPCs and 92.15 ± 15.21º for subcutaneous ATDPCs). Electrical stimulation of cardiac ATDPCs caused changes in cell phenotype and genetic machinery, making them more suitable for cardiac regeneration approaches. Thus, it seems advisable to use electrical cell training before delivery as a cell suspension or within engineered tissue. Copyright © 2013 John Wiley & Sons, Ltd.

Impaired Upregulation of the Costimulatory Molecules, CD27 and CD28, on CD4+ T Cells from HIV Patients Receiving ART Is Associated with Poor Proliferative Responses.

PubMed

Tanaskovic, Sara; Price, Patricia; French, Martyn A; Fernandez, Sonia

2017-02-01

HIV patients beginning antiretroviral therapy (ART) with advanced immunodeficiency often retain low CD4 + T cell counts despite virological control. We examined proliferative responses and upregulation of costimulatory molecules, following anti-CD3 stimulation, in HIV patients with persistent CD4 + T cell deficiency on ART. Aviremic HIV patients with nadir CD4 + T cell counts <100 cells/μL and who had received ART for a median time of 7 (range 1-11) years were categorized into those achieving low (<350 cells/μL; n = 13) or normal (>500 cells/μL; n = 20) CD4 + T cell counts. Ten healthy controls were also recruited. CD4 + T cell proliferation (Ki67) and upregulation of costimulatory molecules (CD27 and CD28) after anti-CD3 stimulation were assessed by flow cytometry. Results were related to proportions of CD4 + T cells expressing markers of T cell senescence (CD57), activation (HLA-DR), and apoptotic potential (Fas). Expression of CD27 and/or CD28 on uncultured CD4 + T cells was similar in patients with normal CD4 + T cell counts and healthy controls, but lower in patients with low CD4 + T cell counts. Proportions of CD4 + T cells expressing CD27 and/or CD28 correlated inversely with CD4 + T cell expression of CD57, HLA-DR, and Fas. After anti-CD3 stimulation, induction of CD27 hi CD28 hi expression was independent of CD4 + T cell counts, but lower in HIV patients than in healthy controls. Induction of CD27 hi CD28 hi expression correlated with induction of Ki67 expression in total, naïve, and CD31 + naïve CD4 + T cells from patients. In HIV patients responding to ART, impaired induction of CD27 and CD28 on CD4 + T cells after stimulation with anti-CD3 is associated with poor proliferative responses as well as greater CD4 + T cell activation and immunosenescence.

Simultaneous recording of mouse retinal ganglion cells during epiretinal or subretinal stimulation

PubMed Central

Sim, S.L.; Szalewski, R.J.; Johnson, L.J.; Akah, L.E.; Shoemaker, L.E.; Thoreson, W.B.; Margalit, E.

2015-01-01

We compared response patterns and electrical receptive fields (ERF) of retinal ganglion cells (RGCs) during epiretinal and subretinal electrical stimulation of isolated mouse retina. Retinas were stimulated with an array of 3200 independently controllable electrodes. Four response patterns were observed: a burst of activity immediately after stimulation (Type I cells, Vision Research (2008), 48, 1562–1568), delayed bursts beginning >25 ms after stimulation (Type II), a combination of both (Type III), and inhibition of ongoing spike activity. Type I responses were produced more often by epiretinal than subretinal stimulation whereas delayed and inhibitory responses were evoked more frequently by subretinal stimulation. Response latencies were significantly shorter with epiretinal than subretinal stimulation. These data suggest that subretinal stimulation is more effective at activating intraretinal circuits than epiretinal stimulation. There was no significant difference in charge threshold between subretinal and epiretinal configurations. ERFs were defined by the stimulating array surface area that successfully stimulated spikes in an RGC. ERFs were complex in shape, similar to receptive fields mapped with light. ERF areas were significantly smaller with subretinal than epiretinal stimulation. This may reflect the greater distance between stimulating electrodes and RGCs in the subretinal configuration. ERFs for immediate and delayed responses mapped within the same Type III cells differed in shape and size, consistent with different sites and mechanisms for generating these two response types. PMID:24863584

Pretreatment with oleic acid accelerates the entrance into the mitotic cycle of EGF-stimulated fibroblasts.

PubMed

Zugaza, J L; Casabiell, X A; Bokser, L; Eiras, A; Beiras, A; Casanueva, F F

1995-07-01

We have previously demonstrated that pretreatment of several cell lines with cis-unsaturated fatty acids, like oleic acid, blocks epidermal growth factor (EGF)-induced early ionic signals, and in particular the [Ca2+]i rise. In the present work we show that this blockade does not alter EGF-stimulated cellular proliferation evaluated by direct cell counting, but induces a powerful enhancement in the pulsed thymidine incorporation assay. The lack of effect of oleic acid on EGF-stimulated cellular proliferation was confirmed by repeated cell counts, cumulative thymidine incorporation, and protein synthesis, but a clear synergistic effect between oleic acid and EGF was again obtained by means of time course experiments with pulsed thymidine. Combined flow cytometry analysis and cell counts at earlier times in EGF-stimulated cells showed that oleic acids accelerates the entrance of cells into the replicative cycle leading to an earlier cell division. Afterward, these oleic acid-pretreated cells became delayed by an unknown compensatory mechanism in such a way that at 48 h post-EGF, the cell count in control and oleic acid-pretreated cells was equal. In conclusion (a) oleic acid accelerates or enhances the EGF mitogenic action and (b) in the long term cells compensate the initial perturbation with respect to untreated cells. As a side observation, the widely employed pulsed thymidine incorporation method as a measure of cell division could be extremely misleading unless experimental conditions are well controlled.

Timing is everything: Rac1 controls Net1A localization to regulate cell adhesion.

PubMed

Carr, Heather S; Frost, Jeffrey A

2013-01-01

Cell adhesion to the extracellular matrix elicits a temporal reorganization of the actin cytoskeleton that is regulated first by Rac1 and later by RhoA. The signaling mechanisms controlling late stage RhoA activation are incompletely understood. Net1A is a RhoA/RhoB-specific guanine nucleotide exchange factor that is required for cancer cell motility. The ability of Net1A to stimulate RhoA activation is negatively regulated by nuclear sequestration. However, mechanisms controlling the plasma membrane localization of Net1A had not previously been reported. Recently we have shown that Rac1 activation stimulates plasma membrane relocalization and activation of Net1A. Net1A relocalization is independent of its catalytic activity and does not require its C-terminal pleckstrin homology or PDZ interacting domains. Rac1 activation during cell adhesion stimulates a transient relocalization of Net1A that is terminated by proteasomal degradation of Net1A. Importantly, plasma membrane localization of Net1A is required for efficient myosin light chain phosphorylation, focal adhesion maturation, and cell spreading. These data show for the first time a physiological mechanism controlling Net1A relocalization from the nucleus. They also demonstrate a previously unrecognized role for Net1A in controlling actomyosin contractility and focal adhesion dynamics during cell adhesion.

A Pdx-1-Regulated Soluble Factor Activates Rat and Human Islet Cell Proliferation

PubMed Central

Hayes, Heather L.; Zhang, Lu; Becker, Thomas C.; Haldeman, Jonathan M.; Stephens, Samuel B.; Arlotto, Michelle; Moss, Larry G.; Newgard, Christopher B.

2016-01-01

The homeodomain transcription factor Pdx-1 has important roles in pancreas and islet development as well as in β-cell function and survival. We previously reported that Pdx-1 overexpression stimulates islet cell proliferation, but the mechanism remains unclear. Here, we demonstrate that overexpression of Pdx-1 triggers proliferation largely by a non-cell-autonomous mechanism mediated by soluble factors. Consistent with this idea, overexpression of Pdx-1 under the control of a β-cell-specific promoter (rat insulin promoter [RIP]) stimulates proliferation of both α and β cells, and overexpression of Pdx-1 in islets separated by a Transwell membrane from islets lacking Pdx-1 overexpression activates proliferation in the untreated islets. Microarray and gene ontology (GO) analysis identified inhibin beta-B (Inhbb), an activin subunit and member of the transforming growth factor β (TGF-β) superfamily, as a Pdx-1-responsive gene. Overexpression of Inhbb or addition of activin B stimulates rat islet cell and β-cell proliferation, and the activin receptors RIIA and RIIB are required for the full proliferative effects of Pdx-1 in rat islets. In human islets, Inhbb overexpression stimulates total islet cell proliferation and potentiates Pdx-1-stimulated proliferation of total islet cells and β cells. In sum, this study identifies a mechanism by which Pdx-1 induces a soluble factor that is sufficient to stimulate both rat and human islet cell proliferation. PMID:27620967

Paracellular transport through healthy and cystic fibrosis bronchial epithelial cell lines--do we have a proper model?

PubMed

Molenda, Natalia; Urbanova, Katarina; Weiser, Nelly; Kusche-Vihrog, Kristina; Günzel, Dorothee; Schillers, Hermann

2014-01-01

It has been reported recently that the cystic fibrosis transmembrane conductance regulator (CFTR) besides transcellular chloride transport, also controls the paracellular permeability of bronchial epithelium. The aim of this study was to test whether overexpressing wtCFTR solely regulates paracellular permeability of cell monolayers. To answer this question we used a CFBE41o- cell line transfected with wtCFTR or mutant F508del-CFTR and compered them with parental line and healthy 16HBE14o- cells. Transepithelial electrical resistance (TER) and paracellular fluorescein flux were measured under control and CFTR-stimulating conditions. CFTR stimulation significant decreased TER in 16HBE14o- and also in CFBE41o- cells transfected with wtCFTR. In contrast, TER increased upon stimulation in CFBE41o- cells and CFBE41o- cells transfected with F508del-CFTR. Under non-stimulated conditions, all four cell lines had similar paracellular fluorescein flux. Stimulation increased only the paracellular permeability of the 16HBE14o- cell monolayers. We observed that 16HBE14o- cells were significantly smaller and showed a different structure of cell-cell contacts than CFBE41o- and its overexpressing clones. Consequently, 16HBE14o- cells have about 80% more cell-cell contacts through which electrical current and solutes can leak. Also tight junction protein composition is different in 'healthy' 16HBE14o- cells compared to 'cystic fibrosis' CFBE41o- cells. We found that claudin-3 expression was considerably stronger in 16HBE14o- cells than in the three CFBE41o- cell clones and thus independent of the presence of functional CFTR. Together, CFBE41o- cell line transfection with wtCFTR modifies transcellular conductance, but not the paracellular permeability. We conclude that CFTR overexpression is not sufficient to fully reconstitute transport in CF bronchial epithelium. Hence, it is not recommended to use those cell lines to study CFTR-dependent epithelial transport.

Timing of pulsed electromagnetic field stimulation does not affect the promotion of bone cell development.

PubMed

Hannay, Gwynne; Leavesley, David; Pearcy, Mark

2005-12-01

Pulsed electromagnetic field (PEMF) devices have been used clinically to promote the healing of surgically resistant fractures in vivo. However, there is a sparsity of data on how the timing of an applied PEMF effects the osteogenic cells that would be present within the fracture gap. The purpose of this study was to examine the response of osteoblast-like cells to a PEMF stimulus, mimicking that of a clinically available device, using four protocols for the timing of the stimulus. The PEMF signal consisted of a 5 ms pulse burst (containing 20 pulses) repeated at 15 Hz. Cultures of a human osteosarcoma cell line, SaOS-2, were exposed to the four timing protocols, each conducted over 3 days. Protocol one stimulated the cells for 8 h each day, protocol two stimulated the cells for 24 h on the first day, protocol three stimulated the cells for 24 h on the second day, and protocol four stimulated the cells for 24 h on the third day. Cells were seeded with either 25,000 or 50,000 cells/well (24-well cell culture plates). All assays showed reduced proliferation and increased differentiation (alkaline phosphatase activity) in the PEMF stimulated cultures compared with the control cultures, except for protocol four alkaline phosphatase measurements. No clear trend was observed between the four protocols; however this may be due to cell density. The results indicated that an osteoblast-like cell line is responsive to a 15 Hz PEMF stimulus, which will stimulate the cell line to into an increasing state of maturity. Bioelectromagnetics (c) 2005 Wiley-Liss, Inc

Uroepithelial cells are part of a mucosal cytokine network.

PubMed Central

Hedges, S; Agace, W; Svensson, M; Sjögren, A C; Ceska, M; Svanborg, C

1994-01-01

This study compared the cytokine production of uroepithelial cell lines in response to gram-negative bacteria and inflammatory cytokines. Human kidney (A498) and bladder (J82) epithelial cell lines were stimulated with either Escherichia coli Hu734, interleukin 1 alpha (IL-1 alpha), or tumor necrosis factor alpha (TNF-alpha). Supernatant samples were removed, and the RNA was extracted from cells at 0, 2, 6, and 24 h. The secreted cytokine levels were determined by bioassay or immunoassay; mRNA was examined by reverse transcription-PCR. The two cell lines secreted IL-6 and IL-8 constitutively. IL-6 and IL-8 mRNA were constitutively produced in both cell lines; IL-1 beta mRNA was detected in J82 cells. IL-1 alpha induced significantly higher levels of IL-6 secretion than did E. coli Hu734 or TNF-alpha. IL-1 alpha and TNF-alpha induced significantly higher levels of IL-8 secretion than did E. coli Hu734. Secreted IL-1 beta was not detected; IL-1 alpha and TNF-alpha were not detected above the levels used for stimulation. IL-1 alpha, IL-1 beta, IL-6, and IL-8 mRNAs were detected in both cell lines after exposure to the stimulants. TNF-alpha mRNA was occasionally detected in the J82 cell line after TNF-alpha stimulation. Cytokine (IL-6 and IL-8) and control (glyceraldehyde 3-phosphate dehydrogenase [G3PDH] and beta-actin) mRNA concentrations were quantitated with internal PCR standards. Cytokine mRNA levels relative to beta-actin mRNA levels were the highest in E. coli-stimulated cells. In comparison, the cytokine mRNA levels relative to G3PDH mRNA levels were the highest in IL-1 alpha-stimulated cells. beta-Actin mRNA levels decreased after bacterial stimulation but not after cytokine stimulation, while G3PDH mRNA levels increased in response to all of the stimulants tested. These results suggested that E. coli Hu734 lowered the beta-actin mRNA levels in uroepithelial cells, thus distorting the IL-6 and IL-8 mRNA levels relative to this control. In summary, E. coli IL-1 alpha and TNF-alpha were found to activate the de novo synthesis and secretion of IL-6 and IL-8 in uroepithelial cells. These results emphasize the role of epithelial cells in cytokine-mediated responses during the early stages of infection. Images PMID:8188354

Cell-to-cell stimulation of movement in nonmotile mutants of Myxococcus

PubMed Central

Hodgkin, Jonathan; Kaiser, Dale

1977-01-01

A large number of nonmotile mutants of the gliding bacterium Myxococcus xanthus have been isolated and partly characterized. About [unk] of these mutants are conditional mutants of a novel kind: mutant cells become transiently motile after contact with nonmutant cells or with cells of a different mutant type. These “stimulatable” mutants fall into five phenotypic classes (types B, C, D, E, and F). Most mutants are nonstimulatable (type A) and never become motile, but type A cells (and wild-type cells) can stimulate cells of any of the other five types. Stimulatable mutants of different types are capable of stimulating each other. For example, in a mixture of B and C cells, both become motile. Linkage analysis using a generalized transducing phage has shown that each of types B, C, D, E, and F corresponds to a single distinct genetic locus. Type A mutants, by contrast, belong to at least 17 different loci. Stimulation depends on close apposition of interacting cells, because stimulation does not occur when contact between cells is prevented. It is possible that the stimulatable mutants are defective in components of the gliding mechanism that can be exchanged between cells. Alternatively, they may be defective in a system of cell communication controlling the coordinated cell movements observed in Myxococcus. Images PMID:16592422

Strigolactones Stimulate Internode Elongation Independently of Gibberellins1[C][W

PubMed Central

de Saint Germain, Alexandre; Ligerot, Yasmine; Dun, Elizabeth A.; Pillot, Jean-Paul; Ross, John J.; Beveridge, Christine A.; Rameau, Catherine

2013-01-01

Strigolactone (SL) mutants in diverse species show reduced stature in addition to their extensive branching. Here, we show that this dwarfism in pea (Pisum sativum) is not attributable to the strong branching of the mutants. The continuous supply of the synthetic SL GR24 via the root system using hydroponics can restore internode length of the SL-deficient rms1 mutant but not of the SL-response rms4 mutant, indicating that SLs stimulate internode elongation via RMS4. Cytological analysis of internode epidermal cells indicates that SLs control cell number but not cell length, suggesting that SL may affect stem elongation by stimulating cell division. Consequently, SLs can repress (in axillary buds) or promote (in the stem) cell division in a tissue-dependent manner. Because gibberellins (GAs) increase internode length by affecting both cell division and cell length, we tested if SLs stimulate internode elongation by affecting GA metabolism or signaling. Genetic analyses using SL-deficient and GA-deficient or DELLA-deficient double mutants, together with molecular and physiological approaches, suggest that SLs act independently from GAs to stimulate internode elongation. PMID:23943865

Optogenetic control of ATP release

NASA Astrophysics Data System (ADS)

Lewis, Matthew A.; Joshi, Bipin; Gu, Ling; Feranchak, Andrew; Mohanty, Samarendra K.

2013-03-01

Controlled release of ATP can be used for understanding extracellular purinergic signaling. While coarse mechanical forces and hypotonic stimulation have been utilized in the past to initiate ATP release from cells, these methods are neither spatially accurate nor temporally precise. Further, these methods cannot be utilized in a highly effective cell-specific manner. To mitigate the uncertainties regarding cellular-specificity and spatio-temporal release of ATP, we herein demonstrate use of optogenetics for ATP release. ATP release in response to optogenetic stimulation was monitored by Luciferin-Luciferase assay (North American firefly, photinus pyralis) using luminometer as well as mesoscopic bioluminescence imaging. Our result demonstrates repetitive release of ATP subsequent to optogenetic stimulation. It is thus feasible that purinergic signaling can be directly detected via imaging if the stimulus can be confined to single cell or in a spatially-defined group of cells. This study opens up new avenue to interrogate the mechanisms of purinergic signaling.

Production of macrophage inflammatory protein (MIP)-1alpha and MIP-1beta by human polymorphonuclear neutrophils stimulated with Porphyromonas endodontalis lipopolysaccharide.

PubMed

Ko, Hyun Jung; Lim, Sung Sam

2002-11-01

This study was undertaken to investigate the capacity of polymorphonuclear neutrophils (PMNs) to secrete Macrophage Inflammatory Protein (MIP)-1alpha and MIP-1beta after stimulation with Porphyromonas endodontalis lipopolysaccharide (LPS). Escherichia coli LPS was used as a positive control. Venous blood was collected and PMNs were isolated from healthy volunteers. Cells were cultured with various concentrations of LPS for different periods of time. Cell supernatants were assayed by enzyme-linked immunosorbent assay. The levels of chemokine secretion in PMNs stimulated with each LPS were found to be significantly higher than in the unstimulated control cells (p < 0.05), and this expression occurred in a time- and dose-dependent manner. E. coli LPS induced higher levels of cytokines than P. endodontalis LPS. These findings demonstrated that P. endodontalis LPS is capable of stimulating PMNs to produce chemotactic cytokines and suggested that PMNs stimulated with P. endodontalis LPS may play a crucial role in the inflammatory and immunopathological reactions of pulpal and periapical diseases.

Conductive polymers for controlled release and treatment of central nervous system injury

NASA Astrophysics Data System (ADS)

Saigal, Rajiv

As one of the most devastating forms of neurotrauma, spinal cord injury remains a challenging clinical problem. The difficulties in treatment could potentially be resolved by better technologies for therapeutic delivery. In order to develop new approaches to treating central nervous system injury, this dissertation focused on using electrically-conductive polymers, controlled drug release, and stem cell transplantation. We first sought to enhance the therapeutic potential of neural stem cells by electrically increasing their production of neurotrophic factors (NTFs), important molecules for neuronal cell survival, differentiation, synaptic development, plasticity, and growth. We fabricated a new cell culture device for growing neural stem cells on a biocompatible, conductive polymer. Electrical stimulation via the polymer led to upregulation of NTF production by neural stem cells. This approach has the potential to enhance stem cell function while avoiding the pitfalls of genetic manipulation, possibly making stem cells more viable as a clinical therapy. Seeing the therapeutic potential of conductive polymers, we extended our studies to an in vivo model of spinal cord injury (SCI). Using a novel fabrication and extraction technique, a conductive polymer was fabricated to fit to the characteristic pathology that follows contusive SCI. Assessed via quantitative analysis of MR images, the conductive polymer significantly reduced compression of the injured spinal cord. Further characterizing astroglial and neuronal response of injured host tissue, we found significant neuronal sparing as a result of this treatment. The in vivo studies also demonstrated improved locomotor recovery mediated by a conductive polymer scaffold over a non-conductive control. We next sought to take advantage of conductive polymers for local, electronically-controlled release of drugs. Seeking to overcome reported limitations in drug delivery via polypyrrole, we first embedded drugs in poly[(D,L-lactide-co-glycolide)-co-polyethylene glycol] (PLGA-PEG) nanoparticles and then demonstrated scalable incorporation and controlled release. In a functional application, electronically-controlled release of minocycline nanoparticles was used to rescue primary spinal cord neurons from an excitotoxic environment in vitro. This approach offers a wide range of therapeutic possibilities, especially for treating traumatic lesions of the central nervous system. Finally, we explored use of conductive polymers for directed differentiation of progenitor cells. Retinal progenitors were seeded on custom polypyrrole cell culture devices and subjected to a biomimetic pattern of electrical stimulation. Stimulated cells showed phenotypic changes, increased neurite outgrowth, increased immunocytochemical expression of cone rod homeobox (CRX) and protein kinase C (PK-C), and decreased expression of glial fibrillary acidic protein (GFAP). Biomimetic stimulation thus led cells towards early photoreceptor and bipolar cell fates, and away from an astrocytic cell fate. Electrical stimulation via a conductive polymer offers a novel approach for directing differentiation of progenitor cells.

Ghrelin restoration of function in vitro in somatotropes from male mice lacking the Janus kinase (JAK)-binding site of the leptin receptor.

PubMed

Syed, Mohsin; Cozart, Michael; Haney, Anessa C; Akhter, Noor; Odle, Angela K; Allensworth-James, Melody; Crane, Christopher; Syed, Farhan M; Childs, Gwen V

2013-04-01

Deletion of the signaling domain of leptin receptors selectively in somatotropes, with Cre-loxP technology, reduced the percentage of immunolabeled GH cells and serum GH. We hypothesized that the deficit occurred when leptin's postnatal surge failed to stimulate an expansion in the cell population. To learn more about the deficiency in GH cells, we tested their expression of GHRH receptors and GH mRNA and the restorative potential of secretagogue stimulation in vitro. In freshly plated dissociated pituitary cells from control male mice, GHRH alone (0.3 nM) increased the percentage of immunolabeled GH cells from 27 ± 0.05% (vehicle) to 42 ± 1.8% (P < .002) and the secretion of GH 1.8-3×. Deletion mutant pituitary cells showed a 40% reduction in percentages of immunolabeled GH cells (16.7 ± 0.4%), which correlated with a 47% reduction in basal GH levels (50 ng/mL control; 26.7 ng/mL mutants P = .01). A 50% reduction in the percentage of mutant cells expressing GHRH receptors (to 12%) correlated with no or reduced responses to GHRH. Ghrelin alone (10 nM) stimulated more GH cells in mutants (from 16.7-23%). When added with 1-3 nM GHRH, ghrelin restored GH cell percentages and GH secretion to levels similar to those of stimulated controls. Counts of somatotropes labeled for GH mRNA confirmed normal percentages of somatotropes in the population. These discoveries suggest that leptin may optimize somatotrope function by facilitating expression of membrane GHRH receptors and the production or maintenance of GH stores.

Intracellular ion concentrations and cell volume during cholinergic stimulation of eccrine secretory coil cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takemura, T.; Sato, F.; Saga, K.

Methacholine (MCh)-induced changes in intracellular concentrations of Na, K, and Cl (( Na)i, (K)i, and (Cl)i, respectively) and in cellular dry mass (a measure of cell shrinkage) were examined in isolated monkey eccrine sweat secretory coils by electron probe X-ray microanalysis using the peripheral standard method. To further confirm the occurrence of cell shrinkage during MCh stimulation, the change in cell volume of dissociated clear and dark cells were directly determined under a light microscope equipped with differential interference contrast (DIC) optics. X-ray microanalysis revealed a biphasic increase in cellular dry mass in clear cells during continuous MCh stimulation; anmore » initial increase of dry mass to 158% (of control) followed by a plateau at 140%, which correspond to the decrease in cell volume of 37 and 29%, respectively. The latter agrees with the MCh-induced cell shrinkage of 29% in dissociated clear cells. The MCh-induced increase in dry mass in myoepithelial cells was less than half that of clear cells. During the steady state of MCh stimulation, both (K+)i and (Cl)i of clear cells decreased by about 45%, whereas (Na)i increased in such a way to maintain the sum of (Na) i + (K)i constant. There was a small (12-15 mM) increase in (Na)i and a decrease in (K)i in myoepithelial cells during stimulation with MCh. Dissociated dark cells failed to significantly shrink during MCh stimulation. The decrease in (Cl)i in the face of constant (Na)i + (K)i suggests the accumulation of unknown anion(s) inside the clear cell during MCh stimulation.« less

Holographically generated structured illumination for cell stimulation in optogenetics

NASA Astrophysics Data System (ADS)

Schmieder, Felix; Büttner, Lars; Czarske, Jürgen; Torres, Maria Leilani; Heisterkamp, Alexander; Klapper, Simon; Busskamp, Volker

2017-06-01

In Optogenetics, cells, e.g. neurons or cardiac cells, are genetically altered to produce for example the lightsensitive protein Channelrhodopsin-2. Illuminating these cells induces action potentials or contractions and therefore allows to control electrical activity. Thus, light-induced cell stimulation can be used to gain insight to various biological processes. Many optogenetics studies, however, use only full field illumination and thus gain no local information about their specimen. But using modern spatial light modulators (SLM) in conjunction with computer-generated holograms (CGH), cells may be stimulated locally, thus enabling the research of the foundations of cell networks and cell communications. In our contribution, we present a digital holographic system for the patterned, spatially resolved stimulation of cell networks. We employ a fast ferroelectric liquid crystal on silicon SLM to display CGH at up to 1.7 kHz. With an effective working distance of 33 mm, we achieve a focus of 10 μm at a positioning accuracy of the individual foci of about 8 μm. We utilized our setup for the optogenetic stimulation of clusters of cardiac cells derived from induced pluripotent stem cells and were able to observe contractions correlated to both temporal frequency and spatial power distribution of the light incident on the cell clusters.

Importance of Thickness in Human Cardiomyocyte Network for Effective Electrophysiological Stimulation Using On-Chip Extracellular Microelectrodes

NASA Astrophysics Data System (ADS)

Hamada, Tomoyo; Nomura, Fumimasa; Kaneko, Tomoyuki; Yasuda, Kenji

2012-06-01

We have developed a three-dimensionally controlled in vitro human cardiomyocyte network assay for the measurements of drug-induced conductivity changes and the appearance of fatal arrhythmia such as ventricular tachycardia/fibrillation for more precise in vitro predictive cardiotoxicity. To construct an artificial conductance propagation model of a human cardiomyocyte network, first, we examined the cell concentration dependence of the cell network heights and found the existence of a height limit of cell networks, which was double-layer height, whereas the cardiomyocytes were effectively and homogeneously cultivated within the microchamber maintaining their spatial distribution constant and their electrophysiological conductance and propagation were successfully recorded using a microelectrode array set on the bottom of the microchamber. The pacing ability of a cardiomyocyte's electrophysiological response has been evaluated using microelectrode extracellular stimulation, and the stimulation for pacing also successfully regulated the beating frequencies of two-layered cardiomyocyte networks, whereas monolayered cardiomyocyte networks were hardly stimulated by the external electrodes using the two-layered cardiomyocyte stimulation condition. The stability of the lined-up shape of human cardiomyocytes within the rectangularly arranged agarose microchambers was limited for a two-layered cardiomyocyte network because their stronger force generation shrunk those cells after peeling off the substrate. The results indicate the importance of fabrication technology of thickness control of cellular networks for effective extracellular stimulation and the potential concerning thick cardiomyocyte networks for long-term cultivation.

Role of effector cells (CCR7(-)CD27(-)) and effector-memory cells (CCR7(-)CD27(+)) in drug-induced maculopapular exanthema.

PubMed

Fernandez, T D; Torres, M J; Lopez, S; Antunez, C; Gomez, E; Del Prado, M F; Canto, G; Blanca, M; Mayorga, C

2010-01-01

Maculopapular exanthema (MPE) induced by drugs is a T-cell mediated reaction and effector cells may play an important role in its development. We assessed the effector and cutaneous homing phenotype in peripheral blood cells from allergic patients after drug stimulation. This study included 10 patients and 10 controls. The effector phenotype (CCR7(-)CD27(+/-)), chemokine receptors (CCR4 and CCR10), and activation (CD25(low)) and regulatory markers (CD25(high)) were measured by flow cytometry in both peripheral blood mononuclear cells (PBMCs) and CD4-T-lymphocytes. Proliferation was determined by 5-(-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) assay and the migratory capacity by a chemotaxis assay using CCL17 and CCL27. Compared to controls, CCR7(-)CD27(-) cells were increased in patients without (p=0.003) and with drug stimulation (p less than 0.001) and had significantly higher proliferation (p=0.010). CCR10 expression was increased in patients after drug stimulation in total and memory CD27(+) T-cells. Lymphocyte migration with CCL27 was higher in patients with drug stimulation (p=0.048), with a decrease in CCR7(-)CD27(-) (p less than 0.0001) and an increase in CCR7(-)CD27(+) (p=0.017). In patients, CD4-T-lymphocytes were significantly activated after drug stimulation (p less than 0.001). In conclusion, we show that effector memory CD4(+) T-cells (CCR7(-)CD27(+)) respond specifically to the drug responsible for MPE and confirm previous data about the involvement of CCR10 in cell trafficking to the skin.

CacyBP/SIP nuclear translocation induced by gastrin promotes gastric cancer cell proliferation

PubMed Central

Zhai, Hui-Hong; Meng, Juan; Wang, Jing-Bo; Liu, Zhen-Xiong; Li, Yuan-Fei; Feng, Shan-Shan

2014-01-01

AIM: To investigate the role of nuclear translocation of calcyclin binding protein, also called Siah-1 interacting protein (CacyBP/SIP), in gastric carcinogenesis. METHODS: The expression of CacyBP/SIP protein in gastric cancer cell lines was detected by Western blot. Immunofluorescence experiments were performed on gastric cancer cell lines that had been either unstimulated or stimulated with gastrin. To confirm the immunofluorescence findings, the relative abundance of CacyBP/SIP in nuclear and cytoplasmic compartments was assessed by Western blot. The effect of nuclear translocation of CacyBP/SIP on cell proliferation was examined using MTT assay. The colony formation assay was used to measure clonogenic cell survival. The effect of CacyBP/SIP nuclear translocation on cell cycle progression was investigated. Two CacyBP/SIP-specific siRNA vectors were designed and constructed to inhibit CacyBP/SIP expression in order to reduce the nuclear translocation of CacyBP/SIP, and the expression of CacyBP/SIP in stably transfected cells was determined by Western blot. The effect of inhibiting CacyBP/SIP nuclear translocation on cell proliferation was then assessed. RESULTS: CacyBP/SIP protein was present in most of gastric cancer cell lines. In unstimulated cells, CacyBP/SIP was distributed throughout the cytoplasm; while in stimulated cells, CacyBP/SIP was found mainly in the perinuclear region. CacyBP/SIP nuclear translocation generated a growth-stimulatory effect on cells. The number of colonies in the CacyBP/SIP nuclear translocation group was significantly higher than that in the control group. The percentage of stimulated cells in G1 phase was significantly lower than that of control cells (69.70% ± 0.46% and 65.80% ± 0.60%, control cells and gastrin-treated SGC7901 cells, P = 0.008; 72.99% ± 0.46% and 69.36% ± 0.51%, control cells and gastrin-treated MKN45 cells, P = 0.022). CacyBP/SIPsi1 effectively down-regulated the expression of CacyBP/SIP, and cells stably transfected by CacyBP/SIPsi1 were then chosen for further cellular assays. In CacyBP/SIPsi1 stably transfected cells, CacyBP/SIP was shown to be distributed throughout the cytoplasm, irregardless of whether they were stimulated or not. After CacyBP/SIP nuclear translocation was reduced, there had no major effect on cell proliferation, as shown by MTT assay. There had no enhanced anchorage-dependent growth upon stimulation, as indicated by colony formation in flat plates. No changes appeared in the percentage of cells in G0-G1 phase in either cell line (71.09% ± 0.16% and 70.86% ± 0.25%, control cells and gastrin-treated SGC7901-CacyBP/SIPsi1 cells, P = 0.101; 74.17% ± 1.04% and 73.07% ± 1.00%, control cells and gastrin-treated MKN45-CacyBP/SIPsi1 cells, P = 0.225). CONCLUSION: CacyBP/SIP nuclear translocation promotes the proliferation and cell cycle progression of gastric cancer cells. PMID:25110433

CacyBP/SIP nuclear translocation induced by gastrin promotes gastric cancer cell proliferation.

PubMed

Zhai, Hui-Hong; Meng, Juan; Wang, Jing-Bo; Liu, Zhen-Xiong; Li, Yuan-Fei; Feng, Shan-Shan

2014-08-07

To investigate the role of nuclear translocation of calcyclin binding protein, also called Siah-1 interacting protein (CacyBP/SIP), in gastric carcinogenesis. The expression of CacyBP/SIP protein in gastric cancer cell lines was detected by Western blot. Immunofluorescence experiments were performed on gastric cancer cell lines that had been either unstimulated or stimulated with gastrin. To confirm the immunofluorescence findings, the relative abundance of CacyBP/SIP in nuclear and cytoplasmic compartments was assessed by Western blot. The effect of nuclear translocation of CacyBP/SIP on cell proliferation was examined using MTT assay. The colony formation assay was used to measure clonogenic cell survival. The effect of CacyBP/SIP nuclear translocation on cell cycle progression was investigated. Two CacyBP/SIP-specific siRNA vectors were designed and constructed to inhibit CacyBP/SIP expression in order to reduce the nuclear translocation of CacyBP/SIP, and the expression of CacyBP/SIP in stably transfected cells was determined by Western blot. The effect of inhibiting CacyBP/SIP nuclear translocation on cell proliferation was then assessed. CacyBP/SIP protein was present in most of gastric cancer cell lines. In unstimulated cells, CacyBP/SIP was distributed throughout the cytoplasm; while in stimulated cells, CacyBP/SIP was found mainly in the perinuclear region. CacyBP/SIP nuclear translocation generated a growth-stimulatory effect on cells. The number of colonies in the CacyBP/SIP nuclear translocation group was significantly higher than that in the control group. The percentage of stimulated cells in G1 phase was significantly lower than that of control cells (69.70% ± 0.46% and 65.80% ± 0.60%, control cells and gastrin-treated SGC7901 cells, P = 0.008; 72.99% ± 0.46% and 69.36% ± 0.51%, control cells and gastrin-treated MKN45 cells, P = 0.022). CacyBP/SIPsi1 effectively down-regulated the expression of CacyBP/SIP, and cells stably transfected by CacyBP/SIPsi1 were then chosen for further cellular assays. In CacyBP/SIPsi1 stably transfected cells, CacyBP/SIP was shown to be distributed throughout the cytoplasm, irregardless of whether they were stimulated or not. After CacyBP/SIP nuclear translocation was reduced, there had no major effect on cell proliferation, as shown by MTT assay. There had no enhanced anchorage-dependent growth upon stimulation, as indicated by colony formation in flat plates. No changes appeared in the percentage of cells in G0-G1 phase in either cell line (71.09% ± 0.16% and 70.86% ± 0.25%, control cells and gastrin-treated SGC7901-CacyBP/SIPsi1 cells, P = 0.101; 74.17% ± 1.04% and 73.07% ± 1.00%, control cells and gastrin-treated MKN45-CacyBP/SIPsi1 cells, P = 0.225). CacyBP/SIP nuclear translocation promotes the proliferation and cell cycle progression of gastric cancer cells.

Rapid Electrical Stimulation Increased Cardiac Apoptosis Through Disturbance of Calcium Homeostasis and Mitochondrial Dysfunction in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

PubMed

Geng, Le; Wang, Zidun; Cui, Chang; Zhu, Yue; Shi, Jiaojiao; Wang, Jiaxian; Chen, Minglong

2018-06-15

Heart failure induced by tachycardia, the most common arrhythmia, is frequently observed in clinical practice. This study was designed to investigate the underlying mechanisms. Rapid electrical stimulation (RES) at a frequency of 3 Hz was applied on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for 7 days, with 8 h/day and 24 h/day set to represent short-term and long-term tachycardia, respectively. Age-matched hiPSC-CMs without electrical stimulation or with slow electrical stimulation (1 Hz) were set as no electrical stimulation (NES) control or low-frequency electrical stimulation (LES) control. Following stimulation, JC-1 staining flow cytometry analysis was performed to examine mitochondrial conditions. Apoptosis in hiPSC-CMs was evaluated using Hoechst staining and Annexin V/propidium iodide (AV/PI) staining flow cytometry analysis. Calcium transients and L-type calcium currents were recorded to evaluate calcium homeostasis. Western blotting and qPCR were performed to evaluate the protein and mRNA expression levels of apoptosis-related genes and calcium homeostasis-regulated genes. Compared to the controls, hiPSC-CMs following RES presented mitochondrial dysfunction and an increased apoptotic percentage. Amplitudes of calcium transients and L-type calcium currents were significantly decreased in hiPSC-CMs with RES. Molecular analysis demonstrated upregulated expression of Caspase3 and increased Bax/Bcl-2 ratio. Genes related to calcium re-sequence were downregulated, while phosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII) was significantly upregulated following RES. There was no significant difference between the NES control and LES control groups in these aspects. Inhibition of CaMKII with 1 µM KN93 partly reversed these adverse effects of RES. RES on hiPSC-CMs disturbed calcium homeostasis, which led to mitochondrial stress, promoted cell apoptosis and caused electrophysiological remodeling in a time-dependent manner. CaMKII played a central role in the damages induced by RES, pharmacological inhibition of CaMKII activity partly reversed the adverse effects of RES on both structural and electrophysiological properties of cells. © 2018 The Author(s). Published by S. Karger AG, Basel.

Effects of combined mechanical stimulation on the proliferation and differentiation of pre-osteoblasts

PubMed Central

Kang, Kyung Shin; Lee, Seung-Jae; Lee, Haksue; Moon, Wonkyu

2011-01-01

We observed how combined mechanical stimuli affect the proliferation and differentiation of pre-osteoblasts. For this research, a bioreactor system was developed that can simultaneously stimulate cells with cyclic strain and ultrasound, each of which is known to effectively stimulate bone tissue regeneration. MC3T3-E1 pre-osteoblasts were chosen for bone tissue engineering due to their osteoblast-like characteristics. 3-D scaffolds were fabricated with polycaprolactone and poly-L-lactic acid using the salt leaching method. The cells were stimulated by the bioreactor with cyclic strain and ultrasound. The bioreactor was set at a frequency of 1.0 Hz and 10% strain for cyclic strain and 1.0 MHz and 30 mW/cm2 for ultrasound. Three experimental groups (ultrasound, cyclic strain, and combined stimulation) and a control group were examined. Each group was stimulated for 20 min/day. Mechanical stimuli did not affect MC3T3-E1 cell proliferation significantly up to 10 days when measured with the cell counting kit-8. However, gene expression analysis of collagen type-I, osteocalcin, RUNX2, and osterix revealed that the combined mechanical stimulation accelerated the matrix maturation of MC3T3-E1 cells. These results indicate that the combined mechanical stimulation can enhance the differentiation of pre-osteoblasts more efficiently than simple stimuli, in spite of no effect on cell proliferation. PMID:21532314

Intracellular IL-4, IL-5, and IFN-γ as the main characteristic of CD4+CD30+ T cells after allergen stimulation in patients with vernal keratoconjunctivitis

PubMed Central

Magaña, Diana; Aguilar, Gustavo; Linares, Marisela; Ayala-Balboa, Julio; Santacruz, Concepción; Chávez, Raúl; Estrada-Parra, Sergio; Garfias, Yonathan; Lascurain, Ricardo; Jiménez-Martínez, Maria C.

2015-01-01

Background Vernal keratoconjunctivitis (VKC) is a severe form of allergic conjunctivitis, in which inflammatory infiltrates of the conjunctiva are characterized by CD3+ and CD30+ cells. Until today, the functional involvement of CD30+ T cells in VKC was unclear. Our aim was to evaluate the functional characteristics of CD30+ T cells after allergen stimulation in peripheral blood mononuclear cells obtained from patients with VKC. Methods Seventeen consecutive patients at the Institute of Ophthalmology with active forms of VKC were included. Results After allergen stimulation, we observed the frequency of CD30+ T cells increased compared with non-stimulated cells (p<0.0001). The CD30+ T cells responded to the specific allergen-inducing expression of intracellular interleukin-4 (IL-4), IL-5, and interferon-gamma (IFN-γ) compared with the CD30- T cells (p<0.0001). Increased early secretion of soluble CD30 was observed in the supernatant of the cultured cells from patients with keratoconjunctivitis, compared with healthy controls (p=0.03). Blockage with IL-4 significantly diminished CD30 frequency in the allergen-stimulated cells. Conclusions Our results suggest that after allergenic stimulation, CD4+CD30+ cells are the most important source of IL-4, IL-5, and IFN-γ. IL-4 acts as an activation loop that increases CD30 expression on T cells after specific stimulation. These findings suggest that CD4+CD30+ T cells are effector cells and play a significant role in the immune pathogenic response in patients with vernal keratoconjunctivitis. PMID:25999672

Neural control of colonic cell proliferation.

PubMed

Tutton, P J; Barkla, D H

1980-03-15

The mitotic rate in rat colonic crypts and in dimethylhydrazine-induced colonic carcinomas was measured using a stathmokinetic technique. In sympathectomized animals cell proliferation was retarded in the crypts but not in the tumors, whereas in animals treated with Metaraminol, a drug which releases norepinephrine from nerve terminals, crypt cell but not tumor cell proliferation was accelerated. Blockade of alpha-adrenoceptors also inhibited crypt cell proliferation. However, stimulation of beta-adrenoceptors inhibited and blockade of beta-adrenoceptors accelerated tumor cell proliferation without influencing crypt cell proliferation. Injection of either serotonin or histamine stimulated tumor but not crypt cell proliferation and blockade or serotonin receptors or histamine H2-receptors inhibited tumor cell proliferation. It is postulated that cell proliferation in the colonic crypts, like that in the jejunal crypts, is under both endocrine and autonomic neural control whereas colonic tumor cell division is subject to endocrine regulation alone.

Optogenetic stimulation of multiwell MEA plates for neural and cardiac applications

NASA Astrophysics Data System (ADS)

Clements, Isaac P.; Millard, Daniel C.; Nicolini, Anthony M.; Preyer, Amanda J.; Grier, Robert; Heckerling, Andrew; Blum, Richard A.; Tyler, Phillip; McSweeney, K. M.; Lu, Yi-Fan; Hall, Diana; Ross, James D.

2016-03-01

Microelectrode array (MEA) technology enables advanced drug screening and "disease-in-a-dish" modeling by measuring the electrical activity of cultured networks of neural or cardiac cells. Recent developments in human stem cell technologies, advancements in genetic models, and regulatory initiatives for drug screening have increased the demand for MEA-based assays. In response, Axion Biosystems previously developed a multiwell MEA platform, providing up to 96 MEA culture wells arrayed into a standard microplate format. Multiwell MEA-based assays would be further enhanced by optogenetic stimulation, which enables selective excitation and inhibition of targeted cell types. This capability for selective control over cell culture states would allow finer pacing and probing of cell networks for more reliable and complete characterization of complex network dynamics. Here we describe a system for independent optogenetic stimulation of each well of a 48-well MEA plate. The system enables finely graded control of light delivery during simultaneous recording of network activity in each well. Using human induced pluripotent stem cell (hiPSC) derived cardiomyocytes and rodent primary neuronal cultures, we demonstrate high channel-count light-based excitation and suppression in several proof-of-concept experimental models. Our findings demonstrate advantages of combining multiwell optical stimulation and MEA recording for applications including cardiac safety screening, neural toxicity assessment, and advanced characterization of complex neuronal diseases.

The Production of Nitric Oxide, IL-6, and TNF-Alpha in Palmitate-Stimulated PBMNCs Is Enhanced through Hyperglycemia in Diabetes

PubMed Central

Volpe, Caroline Maria Oliveira; Abreu, Luana Farnese Machado; Gomes, Pollyanna Stephanie; Gonzaga, Raquel Miranda; Veloso, Clara Araújo; Nogueira-Machado, José Augusto

2014-01-01

We examined nitric oxide (NO), IL-6, and TNF-α secretion from cultured palmitate-stimulated PBMNCs or in the plasma from type 2 diabetes mellitus (T2MD) patients or nondiabetic (ND) controls. Free fatty acids (FFA) have been suggested to induce chronic low-grade inflammation, activate the innate immune system, and cause deleterious effects on vascular cells and other tissues through inflammatory processes. The levels of NO, IL-6, TNF-α, and MDA were higher in supernatant of palmitate stimulated blood cells (PBMNC) or from plasma from patients. The results obtained in the present study demonstrated that hyperglycemia in diabetes exacerbates in vitro inflammatory responses in PBMNCs stimulated with high levels of SFA (palmitate). These results suggest that hyperglycemia primes PBMNCs for NO, IL-6, and TNF-alpha secretion under in vitro FFA stimulation are associated with the secretion of inflammatory biomarkers in diabetes. A combined therapy targeting signaling pathways activated by hyperglycemia in conjunction with simultaneous control of hyperglycemia and hypertriglyceridemia would be suggested for controlling the progress of diabetic complications. PMID:24803982

Hormonal control of growth in the wing imaginal disks of Junonia coenia: the relative contributions of insulin and ecdysone.

PubMed

Nijhout, H Frederik; Laub, Emily; Grunert, Laura W

2018-03-19

The wing imaginal disks of Lepidoptera can be grown in tissue culture, but require both insulin and ecdysone to grow normally. Here, we investigate the contributions the two hormones make to growth. Ecdysone is required to maintain mitoses, whereas in the presence of insulin alone mitoses stop. Both ecdysone and insulin stimulate protein synthesis, but only ecdysone stimulates DNA synthesis. Insulin stimulates primarily cytoplasmic growth and an increase in cell size, whereas ecdysone, by virtue of its stimulation of DNA synthesis and mitosis, stimulates growth by an increase in cell number. Although both hormones stimulate protein synthesis, they do so in different spatial patterns. Both hormones stimulate protein synthesis in the inter-vein regions, but ecdysone stimulates synthesis more strongly in the veins and in the margin of the wing disk. We propose that the balance of insulin and ecdysone signaling must be regulated to maintain normal growth, and when growth appears to be due primarily to an increase in cell number, or an increase in cell size, this may indicate growth occurred under conditions that favored a stronger role for ecdysone, or insulin, respectively. © 2018. Published by The Company of Biologists Ltd.

Timing is everything

PubMed Central

Carr, Heather S; Frost, Jeffrey A

2013-01-01

Cell adhesion to the extracellular matrix elicits a temporal reorganization of the actin cytoskeleton that is regulated first by Rac1 and later by RhoA. The signaling mechanisms controlling late stage RhoA activation are incompletely understood. Net1A is a RhoA/RhoB-specific guanine nucleotide exchange factor that is required for cancer cell motility. The ability of Net1A to stimulate RhoA activation is negatively regulated by nuclear sequestration. However, mechanisms controlling the plasma membrane localization of Net1A had not previously been reported. Recently we have shown that Rac1 activation stimulates plasma membrane relocalization and activation of Net1A. Net1A relocalization is independent of its catalytic activity and does not require its C-terminal pleckstrin homology or PDZ interacting domains. Rac1 activation during cell adhesion stimulates a transient relocalization of Net1A that is terminated by proteasomal degradation of Net1A. Importantly, plasma membrane localization of Net1A is required for efficient myosin light chain phosphorylation, focal adhesion maturation, and cell spreading. These data show for the first time a physiological mechanism controlling Net1A relocalization from the nucleus. They also demonstrate a previously unrecognized role for Net1A in controlling actomyosin contractility and focal adhesion dynamics during cell adhesion. PMID:23792411

Creation of an In vivo cytosensor using engineered mesangial cells. Automatic sensing of glomerular inflammation controls transgene activity.

PubMed

Kitamura, M; Kawachi, H

1997-09-15

Automatic control over exogenous gene expression in response to the activity of disease is a crucial hurdle for gene transfer-based therapies. Towards achieving this goal, we created a "cytosensor" that perceives local inflammatory states and subsequently regulates foreign gene expression. alpha-Smooth muscle actin is known to be expressed in glomerular mesangial cells exclusively in pathologic situations. CArG box element, the crucial regulatory sequence of the alpha-smooth muscle actin promoter, was used as a sensor for glomerular inflammation. Rat mesangial cells were stably transfected with an expression plasmid that introduces a beta-galactosidase gene under the control of CArG box elements. In vitro, the established cells expressed beta-galactosidase exclusively after stimulation with serum. To examine whether the cells are able to automatically control transgene activity in vivo, serum-stimulated or unstimulated cells were transferred into normal rat glomeruli or glomeruli subjected to anti-Thy 1 glomerulonephritis. When stimulated cells were transferred into the normal glomeruli, beta-galactosidase expression was switched off in vivo within 3 d. In contrast, when unstimulated cells were transferred into the nephritic glomeruli, transgene expression was substantially induced. These data indicate the feasibility of using the CArG box element as a molecular sensor for glomerular injury. In the context of advanced forms of gene therapy, this approach provides a novel concept for automatic regulation of local transgene expression where the transgene is required to be activated during inflammation and deactivated when the inflammation has subsided.

Quantitative impedimetric monitoring of cell migration under the stimulation of cytokine or anti-cancer drug in a microfluidic chip

PubMed Central

Xiao, Xia; Lei, Kin Fong; Huang, Chia-Hao

2015-01-01

Cell migration is a cellular response and results in various biological processes such as cancer metastasis, that is, the primary cause of death for cancer patients. Quantitative investigation of the correlation between cell migration and extracellular stimulation is essential for developing effective therapeutic strategies for controlling invasive cancer cells. The conventional method to determine cell migration rate based on comparison of successive images may not be an objective approach. In this work, a microfluidic chip embedded with measurement electrodes has been developed to quantitatively monitor the cell migration activity based on the impedimetric measurement technique. A no-damage wound was constructed by microfluidic phenomenon and cell migration activity under the stimulation of cytokine and an anti-cancer drug, i.e., interleukin-6 and doxorubicin, were, respectively, investigated. Impedance measurement was concurrently performed during the cell migration process. The impedance change was directly correlated to the cell migration activity; therefore, the migration rate could be calculated. In addition, a good match was found between impedance measurement and conventional imaging analysis. But the impedimetric measurement technique provides an objective and quantitative measurement. Based on our technique, cell migration rates were calculated to be 8.5, 19.1, and 34.9 μm/h under the stimulation of cytokine at concentrations of 0 (control), 5, and 10 ng/ml. This technique has high potential to be developed into a powerful analytical platform for cancer research. PMID:26180566

Experiment K-6-23. Effect of spaceflight on levels and function of immune cells

NASA Technical Reports Server (NTRS)

Mandel, A. D.; Sonnenfeld, G.; Berry, W.; Taylor, G.; Wellhausen, S. R.; Konstantinova, I.; Lesnyak, A.; Fuchs, B.

1990-01-01

Two different immunology experiments were performed on samples received from rats flown on Cosmos 1887. In the first experiment, rat bone marrow cells were examined in Moscow for their response to colony stimulating factor-M. In the second experiment, rat spleen and bone marrow cells were stained in Moscow with a variety of antibodies directed against cell surface antigenic markers. These cells were preserved and shipped to the United States where they were subjected to analysis on a flow cytometer. The results of the studies indicate that bone marrow cells from flown rats showed a decreased response to colony stimulating factor than did bone marrow cells from control rats. There was a higher percentage of spleen cells from flown rats staining positively for pan-T-cell, suppressor-T-cell and innate interleukin-2 receptor antigens than from control animals. In addition, a higher percentage of cells that appeared to be part of the myelogenous population of bone marrow cells from flown rats stained positively for surface immunoglobulin than did equivalent cells from control rats.

Endothelial cells of extremely premature infants display impaired immune response after proinflammatory stimulation.

PubMed

Wisgrill, Lukas; Muck, Martina; Wessely, Isabelle; Berger, Angelika; Spittler, Andreas; Förster-Waldl, Elisabeth; Sadeghi, Kambis

2018-01-01

BackgroundEndothelial cells (ECs) exert immunological functions such as production of proinflammatory cytokines/chemokines as well as facilitation of extravasation of immune cells into infected tissue. Limited data are available on the functionality of ECs from extremely preterm neonates during infection. Accordingly, the aim of our study was to investigate the immune response of premature ECs after proinflammatory stimulation.MethodsCell adhesion receptors' expression and function, nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NFκB) signaling, and chemokine production were analyzed in umbilical cord ECs from extremely preterm and term neonates after proinflammatory stimulation.ResultsP-selectin and E-selectin surface expression as well as NFκB signaling were lower after lipopolysaccharide (LPS) stimulation in premature ECs. Preterm ECs exhibited lower, but significant, cell-adhesive functions after LPS stimulation compared with term ECs. CCL2/CXCL8 chemokine secretion was significantly upregulated after proinflammatory stimulation in both groups. CXCL10 production was significantly increased in term but not in preterm ECs upon stimulation with tumor necrosis factor compared with unstimulated ECs.ConclusionExtremely premature ECs showed partly reduced expression levels and function of cell adhesion molecules. Both NFκB signaling and chemokine/cytokine production were reduced in premature ECs. The diminished endothelial proinflammatory immune response might result in impaired infection control of preterm newborns rendering them prone to severe infection.

Controlled-rate freezer cryopreservation of highly concentrated peripheral blood mononuclear cells results in higher cell yields and superior autologous T-cell stimulation for dendritic cell-based immunotherapy.

PubMed

Buhl, Timo; Legler, Tobias J; Rosenberger, Albert; Schardt, Anke; Schön, Michael P; Haenssle, Holger A

2012-11-01

Availability of large quantities of functionally effective dendritic cells (DC) represents one of the major challenges for immunotherapeutic trials against infectious or malignant diseases. Low numbers or insufficient T-cell activation of DC may result in premature termination of treatment and unsatisfying immune responses in clinical trials. Based on the notion that cryopreservation of monocytes is superior to cryopreservation of immature or mature DC in terms of resulting DC quantity and immuno-stimulatory capacity, we aimed to establish an optimized protocol for the cryopreservation of highly concentrated peripheral blood mononuclear cells (PBMC) for DC-based immunotherapy. Cryopreserved cell preparations were analyzed regarding quantitative recovery, viability, phenotype, and functional properties. In contrast to standard isopropyl alcohol (IPA) freezing, PBMC cryopreservation in an automated controlled-rate freezer (CRF) with subsequent thawing and differentiation resulted in significantly higher cell yields of immature and mature DC. Immature DC yields and total protein content after using CRF were comparable with results obtained with freshly prepared PBMC and exceeded results of standard IPA freezing by approximately 50 %. While differentiation markers, allogeneic T-cell stimulation, viability, and cytokine profiles were similar to DC from standard freezing procedures, DC generated from CRF-cryopreserved PBMC induced a significantly higher antigen-specific IFN-γ release from autologous effector T cells. In summary, automated controlled-rate freezing of highly concentrated PBMC represents an improved method for increasing DC yields and autologous T-cell stimulation.

Hyperactivity and reactivity of peripheral blood neutrophils in chronic periodontitis

PubMed Central

Matthews, J B; Wright, H J; Roberts, A; Cooper, P R; Chapple, I L C

2007-01-01

Some evidence exists that peripheral neutrophils from patients with chronic periodontitis generate higher levels of reactive oxygen species (ROS) after Fcγ-receptor stimulation than those from healthy controls. We hypothesized that peripheral neutrophils in periodontitis also show both hyper-reactivity to plaque organisms and hyperactivity in terms of baseline, unstimulated generation and release of ROS. Peripheral neutrophils from chronic periodontitis patients and age/sex/smoking-matched healthy controls (18 pairs) were assayed for total ROS generation and extracellular ROS release, with and without stimulation (Fcγ-receptor and Fusobacterium nucleatum), using luminol and isoluminol chemiluminescence. Assays were performed with and without priming with Escherichia coli lipopolysaccharide (LPS) and granulocyte–macrophage colony-stimulating factor (GM-CSF). Phox gene expression (p22, p47, p67, gp91) was investigated using reverse transcription–polymerase chain reaction (RT–PCR). Neutrophils from patients produced higher mean levels of ROS in all assays. Total generation and extracellular release of ROS by patients' cells were significantly greater than those from controls after FcγR-stimulation, with (P = 0·023) and without (P ≤ 0·023) priming with GM-CSF. Differences in unstimulated total ROS generation were not significant. By contrast, patients' cells demonstrated greater baseline, extracellular ROS release than those from controls (P = 0·004). This difference was maintained after priming with LPS (P = 0·028) but not GM-CSF (P = 0·217). Phox gene expression was similar in patient and control cells at baseline and stimulation with F. nucleatum (3 h) consistently reduced gp91PHOX transcripts. Our data demonstrate that peripheral neutrophils from periodontitis patients exhibit hyper-reactivity following stimulation (Fcγ-receptor and F. nucleatum) and hyperactivity in terms of excess ROS release in the absence of exogenous stimulation. This hyperactive/-reactive neutrophil phenotype is not associated with elevated phox gene expression. PMID:17223966

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taguchi, M.; Field, J.B.

Thyrotropin (TSH) and carbachol stimulated in a dose-dependent manner the accumulation of 3H-glycerophosphoinositol (GPI), 3H-inositol monophosphate (IP1), 3H-inositol bisphosphate (IP2) and 3H-inositol trisphosphate (IP3) in primary cultures of dog thyroid cells prelabeled with myo-(2-3H)inositol. TSH, 250 mU/mL, stimulated 3H-IP3 level after a 10-minute incubation while 10 mU/mL TSH increased it during a 60-minute incubation. The effect of carbachol was more rapid and greater than that of TSH. Carbachol, 100 mumol/L, elevated 3H-IP3 after a 2-minute incubation and 3H-IP3 formation was increased by as little as 1 mumol/L carbachol. TSH stimulation was observed only if the cells were deprived of TSHmore » for 5 days before being labeled with 3H-inositol. Prolongation of the labeling period or addition of TSH, (Bu)2cAMP or carbachol during the labeling increased 3H-inositol incorporation into polyphoinositides (PIPs). When the cells were labeled without any other addition, control and TSH-stimulated 3H-IP3 levels increased in parallel with 3H-PIP levels. However, TSH or carbachol-stimulated 3H-IP3 levels did not increase in proportion to 3H-PIPs level when the cells were labeled with TSH or (Bu)2cAMP. Thus, the ratio of 3H-IP3/3H-PIPs (both control and TSH or carbachol-stimulated) decreased in the cells labeled with TSH or (Bu)2cAMP, which might reflect TSH stimulation of 3H-inositol incorporation into PIPs pool(s) that do not participate in hormone-induced hydrolysis of PIPs.« less

Direct stimulation of immediate-early genes by intranuclear insulin in trypsin-treated H35 hepatoma cells.

PubMed Central

Lin, Y J; Harada, S; Loten, E G; Smith, R M; Jarett, L

1992-01-01

H35 hepatoma cells were treated with trypsin to abolish insulin binding and insulin-stimulated receptor kinase activity. Insulin was, however, internalized by fluid-phase endocytosis in trypsin-treated cells. Furthermore, nuclear accumulation of insulin was similar in control and trypsin-treated hepatoma cells. Northern blot analysis revealed insulin increased g33 and c-fos mRNA concentrations identically in control and trypsin-treated cells but had no effect on beta 2-microglobulin mRNA. Actinomycin D treatment prior to or after insulin addition demonstrated that insulin increased gene transcription and had no effect on mRNA degradation. These studies suggest that the accumulation of intact insulin in cell nuclei may be directly involved in the increased transcription of immediate-early genes. Images PMID:1409684

Cell-stimulation therapy of lateral epicondylitis with frequency-modulated low-intensity electric current.

PubMed

Aliyev, R M; Geiger, G

2012-03-01

In addition to the routine therapy, the patients with lateral epicondylitis included into experimental group were subjected to a 12-week cell-stimulation therapy with low-intensity frequency-modulated electric current. The control group received the same routine therapy and sham stimulation (the therapeutic apparatus was not energized). The efficiency of this microcurrent therapy was estimated by comparing medical indices before therapy and at the end of a 12-week therapeutic course using a 10-point pain severity numeric rating scale (NRS) and Roles-Maudsley pain score. The study revealed high therapeutic efficiency of cell-stimulation with low-intensity electric current resulting probably from up-regulation of intracellular transmitters, interleukins, and prostaglandins playing the key role in the regulation of inflammation.

Studies on the mechanism of endogenous pyrogen production. II. Role of cell products in the regulation of pyrogen release from blood leukocytes.

PubMed

Bodel, P

1974-09-01

Some characteristics of the process by which endogenous pyrogen (EP), the mediator of fever, is released from cells were examined by using human blood leukocytes incubated in vitro. Studies were designed to examine a possible role for leukocyte products, including EP, in the induction, augmentation, or suppression of pyrogen release by blood leukocytes. Products of stimulated leukocytes, including a partially purified preparation of EP, did not induce significant activation of nonstimulated cells. Also, no evidence was obtained that stimulated cell products either augment or inhibit pyrogen production by other stimulated cells. A feedback control of EP production was thus not observed. A crude preparation of EP, containing other products of activated cells, maintained its pyrogenicity when incubated at pH 7.4 but not at pH 5.0. These studies thus provide no support for hypothesized control mechanisms regulating production of EP by blood leukocytes. By contrast, local inactivation of EP at inflammatory sites may modify the amount of EP entering the blood, and hence fever.

Studies on the Mechanism of Endogenous Pyrogen Production II. Role of Cell Products in the Regulation of Pyrogen Release from Blood Leukocytes

PubMed Central

Bodel, Phyllis

1974-01-01

Some characteristics of the process by which endogenous pyrogen (EP), the mediator of fever, is released from cells were examined by using human blood leukocytes incubated in vitro. Studies were designed to examine a possible role for leukocyte products, including EP, in the induction, augmentation, or suppression of pyrogen release by blood leukocytes. Products of stimulated leukocytes, including a partially purified preparation of EP, did not induce significant activation of nonstimulated cells. Also, no evidence was obtained that stimulated cell products either augment or inhibit pyrogen production by other stimulated cells. A feedback control of EP production was thus not observed. A crude preparation of EP, containing other products of activated cells, maintained its pyrogenicity when incubated at pH 7.4 but not at pH 5.0. These studies thus provide no support for hypothesized control mechanisms regulating production of EP by blood leukocytes. By contrast, local inactivation of EP at inflammatory sites may modify the amount of EP entering the blood, and hence fever. PMID:4426696

A possible signal-coupling role for cyclic AMP during endocytosis in Amoeba proteus.

PubMed

Prusch, R D; Roscoe, J C

1993-01-01

Cytoplasmic levels of cAMP in Amoeba proteus were measured utilizing radioimmunoassays under control conditions and when stimulated by inducers of either pinocytosis or phagocytosis. In control cells, cytoplasmic cAMP levels were approximately 0.39 pM/mg cells. When exposed to either chemotactic peptide or mannose which stimulate phagocytosis in the amoeba, there is a rapid doubling of the cAMP level within 45 sec of stimulation which then returns to the control level within 3-5 min. Theophylline prolongs the elevation of cytoplasmic cAMP in stimulated cells and is also capable of eliciting food vacuole formation in the amoeba. In addition isoproterenol also causes food vacuole formation in the amoeba as well as a large and prolonged increase in cytoplasmic cAMP levels. Inducers of pinocytosis (BSA and Na Cl) also elicit changes in cytoplasmic cAMP in the amoeba, but the response appears to differ from that elicited by inducers of phagocytosis in that the peak cAMP levels are broader and biphasic. It is concluded that cAMP plays a signal-coupling role during the early phases of both forms of endocytosis in Amoeba proteus.

Identification of a Novel, Putative Rho-specific GDP/GTP Exchange Factor and a RhoA-binding Protein: Control of Neuronal Morphology

PubMed Central

Gebbink, Martijn F.B.G.; Kranenburg, Onno; Poland, Mieke; van Horck, Francis P.G.; Houssa, Brahim; Moolenaar, Wouter H.

1997-01-01

The small GTP-binding protein Rho has been implicated in the control of neuronal morphology. In N1E-115 neuronal cells, the Rho-inactivating C3 toxin stimulates neurite outgrowth and prevents actomyosin-based neurite retraction and cell rounding induced by lysophosphatidic acid (LPA), sphingosine-1-phosphate, or thrombin acting on their cognate G protein–coupled receptors. We have identified a novel putative GDP/GTP exchange factor, RhoGEF (190 kD), that interacts with both wild-type and activated RhoA, but not with Rac or Cdc42. RhoGEF, like activated RhoA, mimics receptor stimulation in inducing cell rounding and in preventing neurite outgrowth. Furthermore, we have identified a 116-kD protein, p116Rip, that interacts with both the GDP- and GTP-bound forms of RhoA in N1E-115 cells. Overexpression of p116Rip stimulates cell flattening and neurite outgrowth in a similar way to dominant-negative RhoA and C3 toxin. Cells overexpressing p116Rip fail to change their shape in response to LPA, as is observed after Rho inactivation. Our results indicate that (a) RhoGEF may link G protein–coupled receptors to RhoA activation and ensuing neurite retraction and cell rounding; and (b) p116Rip inhibits RhoA-stimulated contractility and promotes neurite outgrowth. PMID:9199174

An In-situ glucose-stimulated insulin secretion assay under perfusion bioreactor conditions.

PubMed

Sharp, Jamie; Vermette, Patrick

2017-03-01

Perfusion bioreactors, unlike traditional in vitro cell culture systems, offer stringent control of physiological parameters such as pH, flow, temperature, and dissolved oxygen concentration which have been shown to have an impact on cellular behaviour and viability. Due to the relative infancy and the growing interest in these in vitro culture systems, detection methods to monitor cell function under dynamic perfusion bioreactor conditions remains one of the main challenges. In this study, INS-1 cells, a cell line which exhibit glucose-stimulated insulin secretion, were embedded in fibrin and cultured under perfusion bioreactor conditions for 48 h and then exposed to either a high-, or low-glucose concentration for 24 h. These cultures were compared to non-bioreacted controls. Bioreacted cultures exposed to a high-glucose concentration showed the highest glucose-stimulated insulin secretion when compared to those in a low-glucose environment. The stimulation index, a marker for insulin secretion functionality, increased over time. A lower incidence of apoptotic cells was observed in the bioreacted cultures when compared to non-bioreacted ones, as evaluated by a TUNEL assay. Immunofluorescence staining of Ki67 and insulin was performed and showed no differences in the incidence of proliferative cells between conditions (bioreacted and non-bioreacted), where all cells stained positive for insulin. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:454-462, 2017. © 2016 American Institute of Chemical Engineers.

Cell Signaling Experiments Driven by Optical Manipulation

PubMed Central

Difato, Francesco; Pinato, Giulietta; Cojoc, Dan

2013-01-01

Cell signaling involves complex transduction mechanisms in which information released by nearby cells or extracellular cues are transmitted to the cell, regulating fundamental cellular activities. Understanding such mechanisms requires cell stimulation with precise control of low numbers of active molecules at high spatial and temporal resolution under physiological conditions. Optical manipulation techniques, such as optical tweezing, mechanical stress probing or nano-ablation, allow handling of probes and sub-cellular elements with nanometric and millisecond resolution. PicoNewton forces, such as those involved in cell motility or intracellular activity, can be measured with femtoNewton sensitivity while controlling the biochemical environment. Recent technical achievements in optical manipulation have new potentials, such as exploring the actions of individual molecules within living cells. Here, we review the progress in optical manipulation techniques for single-cell experiments, with a focus on force probing, cell mechanical stimulation and the local delivery of active molecules using optically manipulated micro-vectors and laser dissection. PMID:23698758

Rapid alterations of cell cycle control proteins in human T lymphocytes in microgravity

PubMed Central

2012-01-01

In our study we aimed to identify rapidly reacting gravity-responsive mechanisms in mammalian cells in order to understand if and how altered gravity is translated into a cellular response. In a combination of experiments using "functional weightlessness" provided by 2D-clinostats and real microgravity provided by several parabolic flight campaigns and compared to in-flight-1g-controls, we identified rapid gravity-responsive reactions inside the cell cycle regulatory machinery of human T lymphocytes. In response to 2D clinorotation, we detected an enhanced expression of p21 Waf1/Cip1 protein within minutes, less cdc25C protein expression and enhanced Ser147-phosphorylation of cyclinB1 after CD3/CD28 stimulation. Additionally, during 2D clinorotation, Tyr-15-phosphorylation occurred later and was shorter than in the 1 g controls. In CD3/CD28-stimulated primary human T cells, mRNA expression of the cell cycle arrest protein p21 increased 4.1-fold after 20s real microgravity in primary CD4+ T cells and 2.9-fold in Jurkat T cells, compared to 1 g in-flight controls after CD3/CD28 stimulation. The histone acetyltransferase (HAT) inhibitor curcumin was able to abrogate microgravity-induced p21 mRNA expression, whereas expression was enhanced by a histone deacetylase (HDAC) inhibitor. Therefore, we suppose that cell cycle progression in human T lymphocytes requires Earth gravity and that the disturbed expression of cell cycle regulatory proteins could contribute to the breakdown of the human immune system in space. PMID:22273506

FGFR and PTEN signaling interact during lens development to regulate cell survival

PubMed Central

Chaffee, Blake R.; Hoang, Thanh V.; Leonard, Melissa R.; Bruney, Devin G.; Wagner, Brad D.; Dowd, Joseph Richard; Leone, Gustavo; Ostrowski, Michael C.; Robinson, Michael L.

2016-01-01

Lens epithelial cells express many receptor tyrosine kinases (RTKs) that stimulate PI3K-AKT and RAS-RAF-MEK-ERK intracellular signaling pathways. These pathways ultimately activate the phosphorylation of key cellular transcription factors and other proteins that control proliferation, survival, metabolism, and differentiation in virtually all cells. Among RTKs in the lens, only stimulation of fibroblast growth factor receptors (FGFRs) elicits a lens epithelial cell to fiber cell differentiation response in mammals. Moreover, although the lens expresses three different Fgfr genes, the isolated removal of Fgfr2 at the lens placode stage inhibits both lens cell survival and fiber cell differentiation. Phosphatase and tensin homolog (PTEN), commonly known as a tumor suppressor, inhibits ERK and AKT activation and initiates both apoptotic pathways, and cell cycle arrest. Here, we show that the combined deletion of Fgfr2 and Pten rescues the cell death phenotype associated with Fgfr2 loss alone. Additionally, Pten removal increased AKT and ERK activation, above the levels of controls, in the presence or absence of Fgfr2. However, isolated deletion of Pten failed to stimulate ectopic fiber cell differentiation, and the combined deletion of Pten and Fgfr2 failed to restore differentiation-specific Aquaporin0 and DnaseIIβ expression in the lens fiber cells. PMID:26764128

The role of nitric oxide synthase in reduced vasocontractile responsiveness induced by prolonged α1-adrenergic receptor stimulation in rat thoracic aorta

PubMed Central

Gürdal, Hakan; Can, Alp; Uğur, Mehmet

2005-01-01

Prolonged exposure (6–12 h) of rat aorta to alpha1-adrenergic receptor (α1AR) agonist phenylephrine (Phe) leads to a decrease in α1AR-mediated vasoconstriction. This reduced responsiveness to α1AR stimulation was strongly dependent on the intactness of the endothelium. We examined the effect of Phe on nitric oxide synthase (NOS) activity by measuring the conversion of [3H]L-arginine to [3H]L-citrulline in rat aorta or in endothelial cells isolated from rat aorta. Phe stimulation increased NOS activity in control aortas. This response was antagonized by prazosin. However, Phe increased neither the activity of NOS nor intracellular Ca2+ in the isolated endothelial cells from the control aortas, whereas acetylcholine (Ach) was able to stimulate both responses in these cells. This result suggests that Phe stimulates α1AR on vascular smooth muscle cells and has an indirect influence on endothelial cells to increase NOS activity. In Phe-exposed aortic rings, basal NOS activity was found to have increased compared to vehicle-exposed control rings. Stimulation with Phe or Ach caused a small increase over basal NOS activity in these preparations. Prolonged exposure to Phe also caused an enhancement of Ach-mediated vasorelaxation in rat aorta. Immunoblot and reverse transcription–polymerase chain reaction experiments showed that prolonged exposure of rat aorta to Phe resulted in an increased expression of eNOS, but not iNOS. This increase was antagonized by nonselective antagonist prazosin. Immunohistochemical staining experiments also showed that expression of eNOS increased in endothelial cells after Phe exposure of the aortas. These results, all together, showed that prolonged exposure of rat aorta to α1AR agonist Phe enhanced the expression of eNOS and basal NOS activity, which probably causes a decreased vasocontractile response to Phe or to other agonists such as 5HT (5-hydroxytryptamine) in rat aorta. This phenomenon can be considered more as a functional antagonism of vasocontractile response to agonists mediated by endothelium than a specific desensitization of α1AR-mediated signalling in vascular smooth muscle cells. PMID:15753950

Electrical Stimulation Decreases Coupling Efficiency Between Beta-Adrenergic Receptors and Cyclic AMP Production in Cultured Muscle Cells

NASA Technical Reports Server (NTRS)

Young, R. B.; Bridge, K. Y.

1999-01-01

Electrical stimulation of skeletal muscle cells in culture is an effective way to simulate the effects of muscle contraction and its effects on gene expression in muscle cells. Expression of the beta-adrenergic receptor and its coupling to cyclic AMP synthesis are important components of the signaling system that controls muscle atrophy and hypertrophy, and the goal of this project was to determine if electrical stimulation altered the beta-adrenergic response in muscle cells. Chicken skeletal muscle cells that had been grown for seven days in culture were subjected to electrical stimulation for an additional two days at a pulse frequency of 0.5 pulses/sec and a pulse duration of 200 msec. At the end of this two-day stimulation period, beta-adrenergic receptor population was measured by the binding of tritium-labeled CGP-12177 to muscle cells, and coupling to cAMP synthesis was measured by Radioimmunoassay (RIA) after treating the cells for 10 min with the potent (beta)AR agonist, isoproterenol. The number of beta adrenergic receptors and the basal levels of intracellular cyclic AMP were not affected by electrical stimulation. However, the ability of these cells to synthesize cyclic AMP was reduced by approximately 50%. Thus, an enhanced level of contraction reduces the coupling efficiency of beta-adrenergic receptors for cyclic AMP production.

Intravenous Glutamine Administration Modulates TNF-α/IL-10 Ratio and Attenuates NFkB Phosphorylation in a Protein Malnutrition Model.

PubMed

Santos, Andressa Cristina Antunes; Correia, Carolina Argondizo; de Oliveira, Dalila Cunha; Nogueira-Pedro, Amanda; Borelli, Primavera; Fock, Ricardo Ambrosio

2016-12-01

Protein malnutrition (PM) is a major public health problem in developing countries, affecting the inflammatory response and increasing susceptibility to opportunistic infections. For this reason, an adequate nutritional intervention can improve the quality of life of patients. Glutamine (GLN) is a nonessential amino acid, but can be considered "conditionally essential" for macrophage function in stress situations, in which it plays a role in the improvement of the inflammatory response. Concerning this issue, in the current study, it was of interest to evaluate some biological aspects of peritoneal cells from a protein malnutrition (PM) mouse model challenged with lipopolysaccharide (LPS) and treated intravenously with GLN. Two-month-old male Balb/c mice were subjected to a low-protein diet (2 % protein) and stimulated intravenously with LPS 1 h prior to the injection of 0.75 mg/kg GLN. Malnourished animals showed a reduced number of total peritoneal cells. Malnourished animals stimulated with LPS or LPS plus GLN did not show differences in peritoneal cell counts; however, the control group showed increased cellularity after LPS stimulus, which was reversed after GLN injection. Further, in the animals from both groups stimulated with LPS, GLN decreased the circulating levels of TNF-α and the levels of TNF-α produced by peritoneal cells; additionally, GLN decreased the IL-10 circulating levels in the malnourished animals stimulated with LPS. In addition, peritoneal cells of the control and malnourished groups stimulated with LPS showed a negative modulation of the NFkB signaling pathway after GLN injection. In conclusion, this study shows that GLN has the capacity to reduce TNF-α synthesis as well as to act as a negative regulator of NFkB phosphorylation, leading to a positive outcome in the control of TNF-α production.

On How Fas Apoptosis-Independent Pathways Drive T Cell Hyperproliferation and Lymphadenopathy in lpr Mice.

PubMed

Balomenos, Dimitrios; Shokri, Rahman; Daszkiewicz, Lidia; Vázquez-Mateo, Cristina; Martínez-A, Carlos

2017-01-01

Fas induces massive apoptosis in T cells after repeated in vitro T cell receptor (TCR) stimulation and is critical for lymphocyte homeostasis in Fas-deficient ( lpr ) mice. Although the in vitro Fas apoptotic mechanism has been defined, there is a large conceptual gap between this in vitro phenomenon and the pathway that leads to in vivo development of lymphadenopathy and autoimmunity. A striking abnormality in lpr mice is the excessive proliferation of CD4 + and CD8 + T cells, and more so of the double-negative TCR + CD4 - CD8 - B220 + T cells. The basis of lpr T cell hyperproliferation remains elusive, as it cannot be explained by Fas-deficient apoptosis. T cell-directed p21 overexpression reduces hyperactivation/hyperproliferation of all lpr T cell subtypes and lymphadenopathy in lpr mice. p21 controls expansion of repeatedly stimulated T cells without affecting apoptosis. These results confirm a direct link between hyperactivation/hyperproliferation, autoreactivity, and lymphadenopathy in lpr mice and, with earlier studies, suggest that Fas apoptosis-independent pathways control lpr T cell hyperproliferation. lpr T cell hyperproliferation could be an indirect result of the defective apoptosis of repeatedly stimulated lpr T cells. Nonetheless, in this perspective, we argue for an alternative setting, in which lack of Fas would directly cause lpr T cell hyperactivation/hyperproliferation in vivo . We propose that Fas/Fas ligand (FasL) acts as an activation inhibitor of recurrently stimulated T cells, and that its disruption causes overexpansion of T cells in lpr mice. Research to define the underlying mechanism of this Fas/FasL effect could resolve the phenotype of lpr mice and lead to therapeutics for related human syndromes.

Retinal ganglion cell responses to voltage and current stimulation in wild-type and rd1 mouse retinas

NASA Astrophysics Data System (ADS)

Goo, Yong Sook; Ye, Jang Hee; Lee, Seokyoung; Nam, Yoonkey; Ryu, Sang Baek; Kim, Kyung Hwan

2011-06-01

Retinal prostheses are being developed to restore vision for those with retinal diseases such as retinitis pigmentosa or age-related macular degeneration. Since neural prostheses depend upon electrical stimulation to control neural activity, optimal stimulation parameters for successful encoding of visual information are one of the most important requirements to enable visual perception. In this paper, we focused on retinal ganglion cell (RGC) responses to different stimulation parameters and compared threshold charge densities in wild-type and rd1 mice. For this purpose, we used in vitro retinal preparations of wild-type and rd1 mice. When the neural network was stimulated with voltage- and current-controlled pulses, RGCs from both wild-type and rd1 mice responded; however the temporal pattern of RGC response is very different. In wild-type RGCs, a single peak within 100 ms appears, while multiple peaks (approximately four peaks) with ~10 Hz rhythm within 400 ms appear in RGCs in the degenerated retina of rd1 mice. We find that an anodic phase-first biphasic voltage-controlled pulse is more efficient for stimulation than a biphasic current-controlled pulse based on lower threshold charge density. The threshold charge densities for activation of RGCs both with voltage- and current-controlled pulses are overall more elevated for the rd1 mouse than the wild-type mouse. Here, we propose the stimulus range for wild-type and rd1 retinas when the optimal modulation of a RGC response is possible.

Systemic administration of thrombin peptide TP508 enhances VEGF-stimulated angiogenesis and attenuates effects of chronic hypoxia

PubMed Central

Olszewska-Pazdrak, Barbara; Carney, Darrell H.

2015-01-01

Revascularization of chronic wounds and ischemic tissue is attenuated by endothelial dysfunction and the inability of angiogenic factors to stimulate angiogenesis. We recently showed that TP508, a nonproteolytic thrombin peptide, increases perfusion and NO-dependent vasodilation in hearts with chronic ischemia and stimulates NO production by endothelial cells. In this study, we investigated systemic in vivo effects of TP508 on VEGF-stimulated angiogenesis in vitro using aortic explants in normoxic and hypoxic conditions. Mice were injected with saline or TP508 and 24h later aortas were removed and cultured to quantify endothelial sprouting. TP508 injection increased endothelial sprouting and potentiated the in vitro response to VEGF. Exposure of control explants to hypoxia inhibited basal and VEGF-stimulated endothelial cell sprouting. This effect of hypoxia was significantly prevented by TP508 injection. Thus, TP508 systemic administration increases responsiveness of aortic endothelial cells to VEGF and diminishes the effect of chronic hypoxia on endothelial cell sprouting. Studies using human endothelial cells in culture suggest that protective effects of TP508 during hypoxia may involve stimulation of endothelial cell NO production. These data suggest potential clinical benefit of using a combination of systemic TP508 and local VEGF as a therapy for revascularization of ischemic tissue. PMID:23594718

Electrical conditioning of adipose-derived stem cells in a multi-chamber culture platform.

PubMed

Pavesi, A; Soncini, M; Zamperone, A; Pietronave, S; Medico, E; Redaelli, A; Prat, M; Fiore, G B

2014-07-01

In tissue engineering, several factors play key roles in providing adequate stimuli for cells differentiation, in particular biochemical and physical stimuli, which try to mimic the physiological microenvironments. Since electrical stimuli are important in the developing heart, we have developed an easy-to-use, cost-effective cell culture platform, able to provide controlled electrical stimulation aimed at investigating the influence of the electric field in the stem cell differentiation process. This bioreactor consists of an electrical stimulator and 12 independent, petri-like culture chambers and a 3-D computational model was used to characterize the distribution and the intensity of the electric field generated in the cell culture volume. We explored the effects of monophasic and biphasic square wave pulse stimulation on a mouse adipose-derived stem cell line (m17.ASC) comparing cell viability, proliferation, protein, and gene expression. Both monophasic (8 V, 2 ms, 1 Hz) and biphasic (+4 V, 1 ms and -4 V, 1 ms; 1 Hz) stimulation were compatible with cell survival and proliferation. Biphasic stimulation induced the expression of Connexin 43, which was found to localize also at the cell membrane, which is its recognized functional mediating intercellular electrical coupling. Electrically stimulated cells showed an induced transcriptional profile more closely related to that of neonatal cadiomyocytes, particularly for biphasic stimulation. The developed platform thus allowed to set-up precise conditions to drive adult stem cells toward a myocardial phenotype solely by physical stimuli, in the absence of exogenously added expensive bioactive molecules, and can thus represent a valuable tool for translational applications for heart tissue engineering and regeneration. © 2014 Wiley Periodicals, Inc.

Alveolar macrophage release of tumor necrosis factor-alpha in chronic alcoholics without liver disease.

PubMed

Omidvari, K; Casey, R; Nelson, S; Olariu, R; Shellito, J E

1998-05-01

Alcohol is an immunosuppressive drug, and chronic abuse has been associated with increased susceptibility to a variety of infections, including bacterial pneumonia and tuberculosis. Alveolar macrophages are the resident phagocytes of the lung and play a central role in lung host defenses against infection ranging from direct antibacterial activity to the release of proinflammatory cytokines such as tumor necrosis factor-alpha (TNFalpha). TNFalpha, in particular, plays a key role in the development of the early inflammatory response. In this study, we investigated the effects of chronic alcohol consumption on alveolar macrophage release of TNFalpha in vitro. We prospectively studied lipopolysaccharide (LPS)-stimulated release of TNFalpha from alveolar macrophages obtained from bronchoalveolar lavage fluid (BALF) in 22 alcoholic (18 smokers, 4 nonsmokers) and 7 nondrinking healthy volunteers (3 smokers, 4 nonsmokers). The total number of cells recovered by bronchoalveolar lavage (BAL) and their differential distribution were not significantly different in alcoholics versus controls (43 +/- 8 x 10(6) and 39 +/- 13 x 10(6), respectively). However, the total number of cells recovered from BALF was significantly higher in smokers (51 +/- 8 x 10(6)) than in nonsmokers (19 +/- 5 x 10(6)). Spontaneous (basal) release of TNFalpha by alveolar macrophages was the same in alcoholics and controls. In contrast, LPS-stimulated release of TNFalpha was significantly suppressed in alcoholics compared with that of controls (1343 +/- 271 vs. 3806 +/- 926 U TNF/ml/10(6) cells, respectively, p < 0.015). When controlled for smoking, LPS-stimulated TNFalpha production was suppressed in alcoholic nonsmokers (563 +/- 413 U TNF/ml/10(6)) compared with control nonsmokers (5113 +/- 1264 U TNF/ml/10(6)). LPS-stimulated TNFalpha production was also less in control smokers (2063 +/- 386 U TNF/ml/10(6) cells) than in control nonsmokers (5113 +/- 1264 U TNF/ml/10(6) cells). There was no difference in TNFalpha production between smoking alcoholics and smoking control subjects. We conclude that chronic alcohol consumption significantly suppresses LPS-stimulated alveolar macrophage production of TNFalpha. This effect is obscured if the subject also smokes. Because TNFalpha production is an important element in host defense, this may explain, in part, the susceptibility of chronic alcohol abusers to a variety of infections.

Modulation of the innate immune-related genes expression in H9N2 avian influenza virus-infected chicken macrophage-like cells (HD11) in response to Escherichia coli LPS stimulation.

PubMed

Qi, Xuefeng; Liu, Caihong; Li, Ruiqiao; Zhang, Huizhu; Xu, Xingang; Wang, Jingyu

2017-04-01

Macrophages play important roles in mediating virus-induced innate immune responses and are thought to be involved in the pathogenesis of bacterial superinfections. The innate immune response initiated by both low pathogenicity AIV and bacterial superinfection in their avian host is not fully understood. We therefore determine the transcripts of innate immune-related genes following avian H9N2 AIV virus infection and E. coli LPS co-stimulation of avian macrophage-like cell line HD11 cells. More pronounced expression of pro-inflammatory cytokines (IL-6 and IL-1β) as well as the inflammatory chemokines (CXCLi1 and CXCLi2) was observed in virus infected plus LPS treated HD11 cells compared to H9N2 virus solely infected control. For two superinfection groups, the levels of genes examined in a prior H9N2 virus infection before secondary LPS treatment group were significantly higher as compared with simultaneous virus infection plus LPS stimulation group. Interestingly, similar high levels of IL-6 gene were observed between LPS sole stimulation group and two superinfection groups. Moreover, IL-10 and TGF-β3 mRNA levels in both superinfection groups were moderately upregulated compared to sole LPS stimulation group or virus alone infection group. Although TLR4 and MDA5 levels in virus alone infection group were significantly lower compared to that in both superinfection groups, TLR4 upregulation respond more rapid to virus sole infection compared to LPS plus virus superinfection. Collectively, innate immune-related genes respond more pronounced in LPS stimulation plus H9N2 virus infection HD11 cells compared to sole virus infection or LPS alone stimulation control cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

Role of mast cells in bronchial contraction in nonallergic obstructive lung pathology.

PubMed

Kuzubova, Nataliya A; Lebedeva, Elena S; Titova, Olga N; Fedin, Anatoliy N; Dvorakovskaya, Ivetta V

2017-01-01

The role of mast cells in contractile bronchial smooth muscle activity has been evaluated in a model of chronic obstructive pulmonary disease induced in rats that were intermittently exposed to nitrogen dioxide (NO 2 ) for 60 days. Starting from the 31st day, one group of rats inhaled sodium cromoglycate before exposure to NO 2 to stabilize mast cell membranes. The second group (control) was not treated. Isometric smooth muscle contraction was analysed in isolated bronchial samples in response to nerve and smooth muscle stimulation. Histological analysis revealed large numbers of mast cells in lung tissue of COPD model rats. The inhibition of mast cell degranulation by sodium cromoglycate prevented the development of nerve-stimulated bronchial smooth muscle hyperactivity in COPD model rats. Histamine or adenosine-induced hyperactivity on nerve stimulation was also inhibited by sodium cromoglycate in bronchial smooth muscle in both control and COPD model rats. This suggests that the mechanism of contractile activity enhancement of bronchial wall smooth muscle cells may be mediated through the activation of resident mast cells transmembrane adenosine receptors resulting in their partial degranulation, with the released histamine acting upon histamine H1-receptors which trigger reflex pathways via intramural ganglion neurons.

Role of mast cells in bronchial contraction in nonallergic obstructive lung pathology

PubMed Central

Kuzubova, Nataliya A.; Lebedeva, Elena S.; Titova, Olga N.; Fedin, Anatoliy N.; Dvorakovskaya, Ivetta V.

2017-01-01

Abstract The role of mast cells in contractile bronchial smooth muscle activity has been evaluated in a model of chronic obstructive pulmonary disease induced in rats that were intermittently exposed to nitrogen dioxide (NO2) for 60 days. Starting from the 31st day, one group of rats inhaled sodium cromoglycate before exposure to NO2 to stabilize mast cell membranes. The second group (control) was not treated. Isometric smooth muscle contraction was analysed in isolated bronchial samples in response to nerve and smooth muscle stimulation. Histological analysis revealed large numbers of mast cells in lung tissue of COPD model rats. The inhibition of mast cell degranulation by sodium cromoglycate prevented the development of nerve-stimulated bronchial smooth muscle hyperactivity in COPD model rats. Histamine or adenosine-induced hyperactivity on nerve stimulation was also inhibited by sodium cromoglycate in bronchial smooth muscle in both control and COPD model rats. This suggests that the mechanism of contractile activity enhancement of bronchial wall smooth muscle cells may be mediated through the activation of resident mast cells transmembrane adenosine receptors resulting in their partial degranulation, with the released histamine acting upon histamine H1-receptors which trigger reflex pathways via intramural ganglion neurons. PMID:28867718

CCL18 synergises with high concentrations of glucose in stimulating fibronectin production in human renal tubuloepithelial cells.

PubMed

Montero, Rosa M; Bhangal, Gurjeet; Pusey, Charles D; Frankel, Andrew H; Tam, Frederick W K

2016-09-29

Diabetic nephropathy is the leading cause of end stage kidney disease worldwide. The pathogenesis of this disease remains elusive and multiple factors have been implicated. These include the effects of hyperglycaemia, haemodynamic and metabolic factors, and an inflammatory process that stimulates cellular signalling pathways leading to disease progression and severe fibrosis. Fibronectin (Fn) is an important protein of the extracellular matrix that is essential in fibrosis and its presence in increased amounts has been identified in the kidney in diabetic nephropathy. Proximal tubuloepithelial (HK-2) cells were stimulated with high glucose (30 mM D-glucose) or glycated albumin (500 μg/mmol) + 4 mM D-glucose or their controls, Mannitol (26 mM + 4 mM D-glucose) and 4 mM D-glucose, respectively. Following 48 h of stimulation the supernatant was collected and MTT [3-(4,5-dimethylthiazole-2,5-diphenyltetrazolium bromide] assay performed to assess cell viability. HK-2 cells were also stimulated in the above environments with recombinant CCL18 (rCCL18) or MCP-1 (rMCP-1) for 48 h with quantification of Fn levels using ELISA. Co-stimulation of HK-2 cells with high concentrations of glucose and rCCL18 significantly increased Fn (p < 0.001), in comparison to high concentrations of glucose alone. HK-2 cells stimulated with glycated albumin consistently produced Fn and this did not alter following co-stimulation with rCCL18 or rMCP-1. This study demonstrates how stimulation with a specific chemokine CCL18 in high glucose upregulates the production of Fn from proximal tubuloepithelial cells. This may be relevant to the development of renal fibrosis in diabetic nephropathy.

Post-Spaceflight (STS-135) Mouse Splenocytes Demonstrate Altered Activation Properties and Surface Molecule Expression

PubMed Central

Crucian, Brian; Sams, Clarence

2015-01-01

Alterations in immune function have been documented during or post-spaceflight and in ground based models of microgravity. Identification of immune parameters that are dysregulated during spaceflight is an important step in mitigating crew health risks during deep space missions. The in vitro analysis of leukocyte activity post-spaceflight in both human and animal species is primarily focused on lymphocytic function. This report completes a broader spectrum analysis of mouse lymphocyte and monocyte changes post 13 days orbital flight (mission STS-135). Analysis includes an examination in surface markers for cell activation, and antigen presentation and co-stimulatory molecules. Cytokine production was measured after stimulation with T-cell mitogen or TLR-2, TLR-4, or TLR-5 agonists. Splenocyte surface marker analysis immediate post-spaceflight and after in vitro culture demonstrated unique changes in phenotypic populations between the flight mice and matched treatment ground controls. Post-spaceflight splenocytes (flight splenocytes) had lower expression intensity of CD4+CD25+ and CD8+CD25+ cells, lower percentage of CD11c+MHC II+ cells, and higher percentage of CD11c+MHC I+ populations compared to ground controls. The flight splenocytes demonstrated an increase in phagocytic activity. Stimulation with ConA led to decrease in CD4+ population but increased CD4+CD25+ cells compared to ground controls. Culturing with TLR agonists led to a decrease in CD11c+ population in splenocytes isolated from flight mice compared to ground controls. Consequently, flight splenocytes with or without TLR-agonist stimulation showed a decrease in CD11c+MHC I+, CD11c+MHC II+, and CD11c+CD86+ cells compared to ground controls. Production of IFN-γ was decreased and IL-2 was increased from ConA stimulated flight splenocytes. This study demonstrated that expression of surface molecules can be affected by conditions of spaceflight and impaired responsiveness persists under culture conditions in vitro. PMID:25970640

Non-synaptic signaling from cerebellar climbing fibers modulates Golgi cell activity.

PubMed

Nietz, Angela K; Vaden, Jada H; Coddington, Luke T; Overstreet-Wadiche, Linda; Wadiche, Jacques I

2017-10-13

Golgi cells are the principal inhibitory neurons at the input stage of the cerebellum, providing feedforward and feedback inhibition through mossy fiber and parallel fiber synapses. In vivo studies have shown that Golgi cell activity is regulated by climbing fiber stimulation, yet there is little functional or anatomical evidence for synapses between climbing fibers and Golgi cells. Here, we show that glutamate released from climbing fibers activates ionotropic and metabotropic receptors on Golgi cells through spillover-mediated transmission. The interplay of excitatory and inhibitory conductances provides flexible control over Golgi cell spiking, allowing either excitation or a biphasic sequence of excitation and inhibition following single climbing fiber stimulation. Together with prior studies of spillover transmission to molecular layer interneurons, these results reveal that climbing fibers exert control over inhibition at both the input and output layers of the cerebellar cortex.

Stress-activated MAPKs and CRM1 regulate the subcellular localization of Net1A to control cell motility and invasion.

PubMed

Ulu, Arzu; Oh, Wonkyung; Zuo, Yan; Frost, Jeffrey A

2018-02-01

The neuroepithelial cell transforming gene 1A (Net1A, an isoform of Net1) is a RhoA subfamily guanine nucleotide exchange factor (GEF) that localizes to the nucleus in the absence of stimulation, preventing it from activating RhoA. Once relocalized in the cytosol, Net1A stimulates cell motility and extracellular matrix invasion. In the present work, we investigated mechanisms responsible for the cytosolic relocalization of Net1A. We demonstrate that inhibition of MAPK pathways blocks Net1A relocalization, with cells being most sensitive to JNK pathway inhibition. Moreover, activation of the JNK or p38 MAPK family pathway is sufficient to elicit Net1A cytosolic localization. Net1A relocalization stimulated by EGF or JNK activation requires nuclear export mediated by CRM1. JNK1 (also known as MAPK8) phosphorylates Net1A on serine 52, and alanine substitution at this site prevents Net1A relocalization caused by EGF or JNK activation. Glutamic acid substitution at this site is sufficient for Net1A relocalization and results in elevated RhoA signaling to stimulate myosin light chain 2 (MLC2, also known as MYL2) phosphorylation and F-actin accumulation. Net1A S52E expression stimulates cell motility, enables Matrigel invasion and promotes invadopodia formation. These data highlight a novel mechanism for controlling the subcellular localization of Net1A to regulate RhoA activation, cell motility, and invasion. © 2018. Published by The Company of Biologists Ltd.

High levels of type 2 cytokine-producing cells in chronic fatigue syndrome.

PubMed

Skowera, A; Cleare, A; Blair, D; Bevis, L; Wessely, S C; Peakman, M

2004-02-01

The aetiology of chronic fatigue syndrome (CFS) is not known. However, it has been suggested that CFS may be associated with underlying immune activation resulting in a Th2-type response. We measured intracellular production of interferon (IFN)-gamma and interleukin (IL)-2; type 1 cytokines), IL-4 (type 2) and IL-10 (regulatory) by both polyclonally stimulated and non-stimulated CD4 and CD8 lymphocytes from patients with CFS and control subjects by flow cytometry. After polyclonal activation we found evidence of a significant bias towards Th2- and Tc2-type immune responses in CFS compared to controls. In contrast, levels of IFN-gamma, IL-2 and IL-10-producing cells were similar in both study groups. Non-stimulated cultures revealed significantly higher levels of T cells producing IFN-gamma or IL-4 in CFS patients. Concluding, we show evidence for an effector memory cell bias towards type 2 responsiveness in patients with CFS, as well as ongoing type 0 immune activation in unstimulated cultures of peripheral blood cells.

High levels of type 2 cytokine-producing cells in chronic fatigue syndrome

PubMed Central

SKOWERA, A; CLEARE, A; BLAIR, D; BEVIS, L; WESSELY, S C; PEAKMAN, M

2004-01-01

The aetiology of chronic fatigue syndrome (CFS) is not known. However, it has been suggested that CFS may be associated with underlying immune activation resulting in a Th2-type response. We measured intracellular production of interferon (IFN)-γ and interleukin (IL)-2; type 1 cytokines), IL-4 (type 2) and IL-10 (regulatory) by both polyclonally stimulated and non-stimulated CD4 and CD8 lymphocytes from patients with CFS and control subjects by flow cytometry. After polyclonal activation we found evidence of a significant bias towards Th2- and Tc2-type immune responses in CFS compared to controls. In contrast, levels of IFN-γ, IL-2 and IL-10-producing cells were similar in both study groups. Non-stimulated cultures revealed significantly higher levels of T cells producing IFN-γ or IL-4 in CFS patients. Concluding, we show evidence for an effector memory cell bias towards type 2 responsiveness in patients with CFS, as well as ongoing type 0 immune activation in unstimulated cultures of peripheral blood cells. PMID:14738459

RSK is a principal effector of the RAS-ERK pathway for eliciting a coordinate, pro-motile/invasive gene program and phenotype in epithelial cells

PubMed Central

Doehn, Ulrik; Hauge, Camilla; Frank, Scott R.; Jensen, Claus J.; Duda, Katarzyna; Nielsen, Jakob V.; Cohen, Michael S.; Johansen, Jens V.; Winther, Benny R.; Lund, Leif R.; Winther, Ole; Taunton, Jack; Hansen, Steen H.; Frödin, Morten

2013-01-01

SUMMARY The RAS-stimulated RAF-MEK-ERK pathway confers epithelial cells with critical motile and invasive capacities during embryonic development, tissue regeneration and carcinoma progression. Yet many mechanisms by which ERK exerts this control remain elusive. Here, we demonstrate that the ERK-activated kinase RSK is necessary to induce motility and invasive capacities in non-transformed epithelial cells and carcinoma cells. RSK is moreover sufficient to induce certain motile responses. Expression profiling analysis revealed that a primary role of RSK is to induce transcription of potent pro-motile/invasive gene program by FRA1-dependent and independent mechanisms. Strikingly, the program enables RSK to coordinately modulate the extracellular environment, the intracellular motility apparatus, and receptors mediating communication between these compartments to stimulate motility and invasion. These findings uncover a general mechanism whereby the RAS-ERK pathway controls epithelial cell motility by identifying RSK as a key effector, from which emanates multiple highly coordinate transcription-dependent mechanisms for stimulation of motility and invasive properties. PMID:19716794

Naturally occurring tolerance acquisition to foods in previously allergic children is characterized by antigen specificity and associated with increased subsets of regulatory T cells.

PubMed

Qamar, N; Fishbein, A B; Erickson, K A; Cai, M; Szychlinski, C; Bryce, P J; Schleimer, R P; Fuleihan, R L; Singh, A M

2015-11-01

Food allergy affects approximately 6-8% of children, and increasing in prevalence. Some children naturally outgrow their food allergy without intervention, but the mechanisms by which this occurs remain poorly understood. We sought to investigate the role of regulatory T cells in the development of naturally acquired tolerance. Fifty-eight children (1-18 years) with either egg or peanut allergy, recent acquisition of natural tolerance to egg or peanut, or no food allergy were studied. Peripheral blood mononuclear cells (PBMC) from these groups were stimulated with relevant antigen for 48 h and flow cytometry performed to characterize both surface (CD3, CD4, CD25, CD14, CD19, and CD127) and intracellular markers (IL-10, Foxp3, and IL-5). Resting PBMC from naturally tolerant patients had significantly increased CD3+CD4+CD25+CD127loFoxp3+ cells, when compared to allergic or control patients (mean 6.36 vs. 2.37 vs. 2.62%, respectively, P < 0.05). Upon stimulation with relevant antigen, naturally tolerant patients also had increased IL-10-expressing CD25+CD127lo cells (6.33 vs. 1.65 vs. 0.7, P < 0.01), Foxp3+ cells (mean 12.6 vs. 5.42 vs. 3%, P < 0.01), and CD4+ cells (mean 4.48 vs. 1.59 vs. 0.87%, P < 0.01); the increase was not observed in PBMCs from allergic or control patients. Additionally, this upregulation was only seen with relevant antigen stimulation and not upon stimulation with unrelated antigen. The increased CD3+CD4+CD25+CD127lo cells at baseline and upon stimulation and increased induction of IL-10-producing cells of several types, including Tr1 cells, from naturally tolerant patients suggests an important role for regulatory T cell subsets in the acquisition of natural tolerance. © 2015 John Wiley & Sons Ltd.

Naturally Occurring Tolerance Acquisition to Foods in Previously Allergic Children is Characterized by Antigen Specificity and Associated with Increased Subsets of Regulatory T cells

PubMed Central

Qamar, Nashmia; Fishbein, Anna B.; Erickson, Kristin A.; Cai, Miao; Szychlinski, Christine; Bryce, Paul J.; Schleimer, Robert P.; Fuleihan, Ramsay L.; Singh, Anne Marie

2015-01-01

Background Food allergy affects approximately 6–8% of children, and increasing in prevalence. Some children naturally outgrow their food allergy without intervention but the mechanisms by which this occurs remain poorly understood. We sought to investigate the role of regulatory T cells in the development of naturally acquired tolerance. Methods Fifty-eight children (1 to 18 years) with either egg or peanut allergy, recent acquisition of natural tolerance to egg or peanut, or no food allergy were studied. Peripheral blood mononuclear cells (PBMC) from these groups were stimulated with relevant antigen for 48 hours and flow cytometry performed to characterize both surface (CD3, CD4, CD25, CD14, CD19, CD127) and intracellular markers (IL-10, Foxp3, and IL-5). Results Resting PBMC from naturally tolerant patients had significantly increased CD3+CD4+CD25+CD127loFoxp3+ cells, when compared to allergic or control patients [mean 6.36 vs 2.37 vs 2.62%, respectively, p<0.05]. Upon stimulation with relevant antigen, naturally tolerant patients also had increased IL-10-expressing CD25+CD127lo cells [6.33 vs 1.65 vs 0.7, p<0.01], Foxp3+ cells [mean 12.6 vs 5.42 vs 3%, p<0.01] and CD4+ cells [mean 4.48 vs 1.59 vs 0.87%, p<0.01]; the increase was not observed in PBMCs from allergic or control patients. Additionally, this upregulation was only seen with relevant antigen stimulation and not upon stimulation with unrelated antigen. Conclusion The increased CD3+CD4+CD25+CD127lo cells at baseline and upon stimulation and increased induction of IL-10-producing cells of several types, including Tr1 cells, from naturally tolerant patients suggests an important role for regulatory T cell subsets in the acquisition of natural tolerance. PMID:25989379

Foxp3+ regulatory T cells impede the priming of protective CD8+ T cells

PubMed Central

Ertelt, James M.; Rowe, Jared H.; Mysz, Margaret A.; Singh, Charanjeet; Roychowdhury, Monika; Aguilera, Marijo N.; Way, Sing Sing

2011-01-01

T cell activation is controlled by incompletely defined opposing stimulation and suppression signals that together sustain the balance between optimal host defense against infection and peripheral tolerance. Herein, we explored the impacts of Foxp3+ regulatory T cell (Treg) suppression in priming antigen-specific T cell activation under non-infection and infection conditions. We find the transient ablation of Foxp3+ Tregs unleashes the robust expansion and activation of peptide stimulated CD8+ T cells that provide protection against Listeria monocytogenes (Lm) infection in an antigen-specific fashion. By contrast, Treg-ablation had non-significant impacts on the CD8+ T cell response primed by infection with recombinant Lm. Similarly, non-recombinant Lm administered with peptide stimulated the expansion and activation of CD8+ T cells that paralleled the response primed by Treg-ablation. Interestingly, these adjuvant properties of Lm did not require CD8+ T cell stimulation by IL-12 produced in response to infection, but instead were associated with sharp reductions in Foxp3+ Treg suppressive potency. Therefore, Foxp3+ Tregs impose critical barriers that when overcome naturally during infection or artificially with ablation allows the priming of protective antigen-specific CD8+ T cells. PMID:21810602

Biomimetic perfusion and electrical stimulation applied in concert improved the assembly of engineered cardiac tissue

PubMed Central

Lee, Eun Jung; Luo, Jianwen; Duan, Yi; Yeager, Keith; Konofagou, Elisa; Vunjak-Novakovic, Gordana

2012-01-01

Maintenance of normal myocardial function depends intimately on synchronous tissue contraction driven by electrical activation and on adequate nutrient perfusion in support thereof. Bioreactors have been used to mimic aspects of these factors in vitro to engineer cardiac tissue, but due to design limitations, previous bioreactor systems have yet to simultaneously support nutrient perfusion, electrical stimulation, and unconstrained (i.e., not isometric) tissue contraction. To the best of our knowledge, the bioreactor system described herein is the first to integrate in concert these three key factors. We present the design of our bioreactor and characterize its capability in integrated experimental and mathematical modeling studies. We then culture cardiac cells obtained from neonatal rats in porous, channeled elastomer scaffolds with the simultaneous application of perfusion and electrical stimulation, with controls excluding either one or both of these two conditions. After eight days of culture, constructs grown with the simultaneous perfusion and electrical stimulation exhibited substantially improved functional properties, as evidenced by a significant increase in contraction amplitude (0.23±0.10% vs. 0.14±0.05, 0.13±0.08, or 0.09±0.02% in control constructs grown without stimulation, without perfusion, or either stimulation or perfusion, respectively). Consistently, these constructs had significantly improved DNA contents, cell distribution throughout the scaffold thickness, cardiac protein expression, cell morphology and overall tissue organization than either control group. Thus, the simultaneous application of medium perfusion and electrical conditioning enabled by the use of the novel bioreactor system may accelerate the generation of fully functional, clinically sized cardiac tissue constructs. PMID:22170772

Hydrogen peroxide stimulates cell motile activity through LPA receptor-3 in liver epithelial WB-F344 cells.

PubMed

Shibata, Ayano; Tanabe, Eriko; Inoue, Serina; Kitayoshi, Misaho; Okimoto, Souta; Hirane, Miku; Araki, Mutsumi; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

2013-04-12

Hydrogen peroxide which is one of reactive oxygen species (ROS) mediates a variety of biological responses, including cell proliferation and migration. In the present study, we investigated whether lysophosphatidic acid (LPA) signaling is involved in cell motile activity stimulated by hydrogen peroxide. The rat liver epithelial WB-F344 cells were treated with hydrogen peroxide at 0.1 or 1 μM for 48 h. In cell motility assays, hydrogen peroxide treated cells showed significantly high cell motile activity, compared with untreated cells. To measure the expression levels of LPA receptor genes, quantitative real time RT-PCR analysis was performed. The expressions of LPA receptor-3 (Lpar3) in hydrogen peroxide treated cells were significantly higher than those in control cells, but not Lpar1 and Lpar2 genes. Next, to assess the effect of LPA3 on cell motile activity, the Lpar3 knockdown cells from WB-F344 cells were also treated with hydrogen peroxide. The cell motile activity of the knockdown cells was not stimulated by hydrogen peroxide. Moreover, in liver cancer cells, hydrogen peroxide significantly activated cell motility of Lpar3-expressing cells, but not Lpar3-unexpressing cells. These results suggest that LPA signaling via LPA3 may be mainly involved in cell motile activity of WB-F344 cells stimulated by hydrogen peroxide. Copyright © 2013 Elsevier Inc. All rights reserved.

Metaphase yields from staphylococcal enterotoxin A stimulated peripheral blood lymphocytes of unirradiated and irradiated aged rhesus monkeys

NASA Technical Reports Server (NTRS)

Hill, F. S.; Cox, A. B.; Salmon, Y. L.; Cantu, A. O.; Lucas, J. N.

1994-01-01

The mitogen phytohemagglutinin (PHA) works well in both human and cynomolgus monkey (Macaca fascicularis) lymphocyte cultures to stimulate T cell proliferation. T cells from rhesus monkeys (Macaca mulatta) are less responsive than human cells, producing few metaphases when thousands are required, e.g. in biological dosimetry studies. We show that staphylococcal enterotoxin A (SEA), one of the most potent mitogens known, at a concentration of 0.5 microgram/ml stimulated peripheral lymphocytes to grow with a mitotic index (MI) averaging 0.13 metaphases/cell in old, irradiated rhesus macaques. This was significantly greater (p < 0.001) than that produced by PHA (MI < 0.01) in lymphocytes from the same animals. Whole blood was cultured for 96, 120 and 144 h for five irradiated individuals and for two controls. All cells cultured with SEA produced a high MI with a peak response at 120 h whereas the same cultures showed low MI for each PHA stimulated culture.

Local and Systemic CD4+ T Cell Exhaustion Reverses with Clinical Resolution of Pulmonary Sarcoidosis

PubMed Central

Hawkins, Charlene; Shaginurova, Guzel; Shelton, D. Auriel; Herazo-Maya, Jose D.; Oswald-Richter, Kyra A.; Young, Anjuli; Celada, Lindsay J.; Kaminski, Naftali; Sevin, Carla

2017-01-01

Investigation of the Th1 immune response in sarcoidosis CD4+ T cells has revealed reduced proliferative capacity and cytokine expression upon TCR stimulation. In other disease models, such cellular dysfunction has been associated with a step-wise, progressive loss of T cell function that results from chronic antigenic stimulation. T cell exhaustion is defined by decreased cytokine production upon TCR activation, decreased proliferation, increased expression of inhibitory cell surface receptors, and increased susceptibility to apoptosis. We characterized sarcoidosis CD4+ T cell immune function in systemic and local environments among subjects undergoing disease progression compared to those experiencing disease resolution. Spontaneous and TCR-stimulated Th1 cytokine expression and proliferation assays were performed in 53 sarcoidosis subjects and 30 healthy controls. PD-1 expression and apoptosis were assessed by flow cytometry. Compared to healthy controls, sarcoidosis CD4+ T cells demonstrated reductions in Th1 cytokine expression, proliferative capacity (p < 0.05), enhanced apoptosis (p < 0.01), and increased PD-1 expression (p < 0.001). BAL-derived CD4+ T cells also demonstrated multiple facets of T cell exhaustion (p < 0.05). Reversal of CD4+ T cell exhaustion was observed in subjects undergoing spontaneous resolution (p < 0.05). Sarcoidosis CD4+ T cells exhibit loss of cellular function during progressive disease that follows the archetype of T cell exhaustion. PMID:29234685

Peripheral T-Cell Reactivity to Heat Shock Protein 70 and Its Cofactor GrpE from Tropheryma whipplei Is Reduced in Patients with Classical Whipple's Disease

PubMed Central

Trotta, Lucia; Weigt, Kathleen; Schinnerling, Katina; Geelhaar-Karsch, Anika; Oelkers, Gerrit; Biagi, Federico; Corazza, Gino Roberto; Allers, Kristina; Schneider, Thomas; Erben, Ulrike

2017-01-01

ABSTRACT Classical Whipple's disease (CWD) is characterized by the lack of specific Th1 response toward Tropheryma whipplei in genetically predisposed individuals. The cofactor GrpE of heat shock protein 70 (Hsp70) from T. whipplei was previously identified as a B-cell antigen. We tested the capacity of Hsp70 and GrpE to elicit specific proinflammatory T-cell responses. Peripheral mononuclear cells from CWD patients and healthy donors were stimulated with T. whipplei lysate or recombinant GrpE or Hsp70 before levels of CD40L, CD69, perforin, granzyme B, CD107a, and gamma interferon (IFN-γ) were determined in T cells by flow cytometry. Upon stimulation with total bacterial lysate or recombinant GrpE or Hsp70 of T. whipplei, the proportions of activated effector CD4+ T cells, determined as CD40L+ IFN-γ+, were significantly lower in patients with CWD than in healthy controls; CD8+ T cells of untreated CWD patients revealed an enhanced activation toward unspecific stimulation and T. whipplei-specific degranulation, although CD69+ IFN-γ+ CD8+ T cells were reduced upon stimulation with T. whipplei lysate and recombinant T. whipplei-derived proteins. Hsp70 and its cofactor GrpE are immunogenic in healthy individuals, eliciting effective responses against T. whipplei to control bacterial spreading. The lack of specific T-cell responses against these T. whipplei-derived proteins may contribute to the pathogenesis of CWD. PMID:28559404

Peripheral T-Cell Reactivity to Heat Shock Protein 70 and Its Cofactor GrpE from Tropheryma whipplei Is Reduced in Patients with Classical Whipple's Disease.

PubMed

Trotta, Lucia; Weigt, Kathleen; Schinnerling, Katina; Geelhaar-Karsch, Anika; Oelkers, Gerrit; Biagi, Federico; Corazza, Gino Roberto; Allers, Kristina; Schneider, Thomas; Erben, Ulrike; Moos, Verena

2017-08-01

Classical Whipple's disease (CWD) is characterized by the lack of specific Th1 response toward Tropheryma whipplei in genetically predisposed individuals. The cofactor GrpE of heat shock protein 70 (Hsp70) from T. whipplei was previously identified as a B-cell antigen. We tested the capacity of Hsp70 and GrpE to elicit specific proinflammatory T-cell responses. Peripheral mononuclear cells from CWD patients and healthy donors were stimulated with T. whipplei lysate or recombinant GrpE or Hsp70 before levels of CD40L, CD69, perforin, granzyme B, CD107a, and gamma interferon (IFN-γ) were determined in T cells by flow cytometry. Upon stimulation with total bacterial lysate or recombinant GrpE or Hsp70 of T. whipplei , the proportions of activated effector CD4 + T cells, determined as CD40L + IFN-γ + , were significantly lower in patients with CWD than in healthy controls; CD8 + T cells of untreated CWD patients revealed an enhanced activation toward unspecific stimulation and T. whipplei -specific degranulation, although CD69 + IFN-γ + CD8 + T cells were reduced upon stimulation with T. whipplei lysate and recombinant T. whipplei -derived proteins. Hsp70 and its cofactor GrpE are immunogenic in healthy individuals, eliciting effective responses against T. whipplei to control bacterial spreading. The lack of specific T-cell responses against these T. whipplei -derived proteins may contribute to the pathogenesis of CWD. Copyright © 2017 American Society for Microbiology.

Hydrogen peroxide stimulates cell motile activity through LPA receptor-3 in liver epithelial WB-F344 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shibata, Ayano; Tanabe, Eriko; Inoue, Serina

2013-04-12

Highlights: •Hydrogen peroxide stimulates cell motility of WB-F344 cells. •LPA{sub 3} is induced by hydrogen peroxide in WB-F344 cells. •Cell motility by hydrogen peroxide is inhibited in LPA{sub 3} knockdown cells. •LPA signaling is involved in cell migration by hydrogen peroxide. -- Abstract: Hydrogen peroxide which is one of reactive oxygen species (ROS) mediates a variety of biological responses, including cell proliferation and migration. In the present study, we investigated whether lysophosphatidic acid (LPA) signaling is involved in cell motile activity stimulated by hydrogen peroxide. The rat liver epithelial WB-F344 cells were treated with hydrogen peroxide at 0.1 or 1more » μM for 48 h. In cell motility assays, hydrogen peroxide treated cells showed significantly high cell motile activity, compared with untreated cells. To measure the expression levels of LPA receptor genes, quantitative real time RT-PCR analysis was performed. The expressions of LPA receptor-3 (Lpar3) in hydrogen peroxide treated cells were significantly higher than those in control cells, but not Lpar1 and Lpar2 genes. Next, to assess the effect of LPA{sub 3} on cell motile activity, the Lpar3 knockdown cells from WB-F344 cells were also treated with hydrogen peroxide. The cell motile activity of the knockdown cells was not stimulated by hydrogen peroxide. Moreover, in liver cancer cells, hydrogen peroxide significantly activated cell motility of Lpar3-expressing cells, but not Lpar3-unexpressing cells. These results suggest that LPA signaling via LPA{sub 3} may be mainly involved in cell motile activity of WB-F344 cells stimulated by hydrogen peroxide.« less

An Evaluation of LH-Stimulated Testosterone Production by ...

EPA Pesticide Factsheets

An Evaluation of LH-Stimulated Testosterone Production by Highly Purified Rat Leydig Cells: A Complementary Screen for Steroidogenesis in the Testis. 1Botteri, N., 2Suarez, J., 2Laws, S., 2Klinefelter, G.1Oak Ridge Institute for Science and Education, Oak Ridge, TN, 2 U.S. Environmental Protection Agency, ORD, NHEERL, TAD, RTP, NCThe H295R steroidogenesis assay uses an adrenocarcinoma cell line which fails to elicit LH mediated responses. This limits the assay’s ability to detect chemicals which disrupt LH-mediated Leydig cell responses in the testis. This study evaluated whether LH-stimulated T production by purified rat Leydig cells would be altered after exposure to chemicals that failed to decrease T production in the ToxCast H295R screen. Ten chemicals negative for T inhibition in the H295R screen, were selected based on alterations in upstream substrates (deoxycorticosterone, hydroxyprogesterone) expected to result in a decrease in T. Based on earlier work, simvastatin served as our positive control. Each chemical was tested over 6 concentrations ranging from 0.1 µM to 100 µM. Leydig cells were cultured overnight under maximal LH stimulation. A minimum of 3 replicate experiments were conducted for each format (24 and 96 well) and chemical tested; cell viability was assessed using a live/dead cytotoxicity kit. T data were excluded if viability was less than 80% of control. Initial evaluation using a 24-well Leydig cell assay confir

Equine chorionic gonadotropin alters luteal cell morphologic features related to progesterone synthesis.

PubMed

Rigoglio, Nathia N; Fátima, Luciana A; Hanassaka, Jaqueline Y; Pinto, Gizélia L; Machado, Alex S D; Gimenes, Lindsay U; Baruselli, Pietro S; Rennó, Francisco P; Moura, Carlos E B; Watanabe, Il-Sei; Papa, Paula C

2013-03-01

Exogenous eCG for stimulation of a single dominant follicle or for superovulation are common strategies to improve reproductive efficiency by increasing pregnancy rates and embryo production, respectively. Morphofunctional changes in the CL of eCG-treated cattle include increases in CL volume and plasma progesterone concentrations. Therefore, we tested the hypothesis that eCG alters the content of luteal cells and mitochondria related to hormone production. Twelve crossbred beef cows were synchronized and then allocated into three groups (four cows per group) and received no further treatment (control) or were given eCG either before or after follicular deviation (superovulation and stimulation of the dominant follicle, respectively). Six days after ovulation, cows were slaughtered and CL collected for morphohistologic and ultrastructural analysis. Mitochondrial volume per CL was highest in superovulated followed by stimulated and then control cows (18,500 ± 2630, 12,300 ± 2640, and 7670 ± 3400 μm(3); P < 0.001), and the density of spherical mitochondria and the total number of large luteal cells were increased (P < 0.05) in stimulated cows compared with the other two groups (110.32 ± 14.22, 72.26 ± 8.77, and 70.46 ± 9.58 mitochondria per μm(3) and 678 ± 147, 245 ± 199, and 346 ± 38 × 10(6) cells, respectively. However, the largest diameters of the large luteal cells were increased in superovulated and control cows versus stimulated ones (32.32 ± 0.06, 31.59 ± 0.81, and 29.44 ± 0.77 μm; P < 0.0001). In contrast, the total number of small luteal cells was increased in superovulated cows (1456 ± 268, 492 ± 181, and 822 ± 461 × 10(6), P < 0.05). In conclusion, there were indications of cellular changes related to increased hormonal production (stimulatory treatment) and increased CL volume (superovulatory treatment). Copyright © 2013 Elsevier Inc. All rights reserved.

Endothelial Progenitor Cells as Shuttle of Anticancer Agents.

PubMed

Laurenzana, Anna; Margheri, Francesca; Chillà, Anastasia; Biagioni, Alessio; Margheri, Giancarlo; Calorini, Lido; Fibbi, Gabriella; Del Rosso, Mario

2016-10-01

Cell therapies are treatments in which stem or progenitor cells are stimulated to differentiate into specialized cells able to home to and repair damaged tissues. After their discovery, endothelial progenitor cells (EPCs) stimulated worldwide interest as possible vehicles to perform autologous cell therapy of tumors. Taking into account the tumor-homing properties of EPCs, two different approaches to control cancer progression have been pursued by combining cell-based therapy with gene therapy or with nanomedicine. The first approach is based on the possibility of engineering EPCs to express different transgenes, and the second is based on the capacity of EPCs to take up nanomaterials. Here we review the most important progress covering the following issues: the characterization of bona fide endothelial progenitor cells, their role in tumor vascularization and metastasis, and preclinical data about their use in cell-based tumor therapy, considering antiangiogenic, suicide, immune-stimulating, and oncolytic virus gene therapy. The mixed approach of EPC cell therapy and nanomedicine is discussed in terms of plasmonic-dependent thermoablation and molecular imaging.

Experiment K-7-23: Effect of Spaceflight on Level and Function of Immune Cells. Part 2; Proliferation and Cytokines

NASA Technical Reports Server (NTRS)

Nash, P. V.; Konstantinova, I. V.; Fuchs, B. B.; Rakhmilevich, A. L.; Lesnyak, A. T.; Mastro, A. M.

1994-01-01

Lymphocytes from the superficial inguinal lymph nodes of rats flown on the Cosmos 2044 space mission were tested for proliferation in response to polyclonal activators. Cells were cultured with T or B cell mitogens, phorbol ester and calcium ionophore, or T cell mitogen and the lymphokines interleukin-1 (IL-1) or interleukin-2 (IL-2), and assayed for DNA synthesis by (3)H-thymidine incorporation. Lymphocytes also were incubated with concanavalin A (Con A), a T cell mitogen, and tested for IL-2 production. Mitogen-stimulated proliferation of lymphocytes from rats exposed to microgravity was not significantly different from synchronous or vivarium controls. Responses to Con A and IL-2, and Con A and IL-1 likewise were unaffected by space flight. Lymphocytes from all of these groups responded well to phorbol ester and calcium ionophore stimulation. Furthermore, lymph node cells (LNC) from control rats and rats flown on Cosmos 2044 produced similar amounts of IL-2. The results obtained using hindlimb suspended rats were notably different from those of flight and control animals. LNC from suspended rats generally had greater proliferative responses to T cell mitogens than did lymphocytes from other groups. Responsiveness to a B cell mitogen was not enhanced. Con A-stimulated LNC from hindlimb suspended rats also produced more IL-2 than did lymphocytes from the other groups. This difference was statistically significant at both IL-2 induction times tested.

Fiber-array based optogenetic prosthetic system for stimulation therapy

NASA Astrophysics Data System (ADS)

Gu, Ling; Cote, Chris; Tejeda, Hector; Mohanty, Samarendra

2012-02-01

Recent advent of optogenetics has enabled activation of genetically-targeted neuronal cells using low intensity blue light with high temporal precision. Since blue light is attenuated rapidly due to scattering and absorption in neural tissue, optogenetic treatment of neurological disorders may require stimulation of specific cell types in multiple regions of the brain. Further, restoration of certain neural functions (vision, and auditory etc) requires accurate spatio-temporal stimulation patterns rather than just precise temporal stimulation. In order to activate multiple regions of the central nervous system in 3D, here, we report development of an optogenetic prosthetic comprising of array of fibers coupled to independently-controllable LEDs. This design avoids direct contact of LEDs with the brain tissue and thus does not require electrical and heat isolation, which can non-specifically stimulate and damage the local brain regions. The intensity, frequency, and duty cycle of light pulses from each fiber in the array was controlled independently using an inhouse developed LabView based program interfaced with a microcontroller driving the individual LEDs. While the temporal profile of the light pulses was controlled by varying the current driving the LED, the beam profile emanating from each fiber tip could be sculpted by microfabrication of the fiber tip. The fiber array was used to stimulate neurons, expressing channelrhodopsin-2, in different locations within the brain or retina. Control of neural activity in the mice cortex, using the fiber-array based prosthetic, is evaluated from recordings made with multi-electrode array (MEA). We also report construction of a μLED array based prosthetic for spatio-temporal stimulation of cortex.

Origin of tumor-promoter released fibronectin in fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burrous, B.A.; Wolf, G.

1986-05-01

Previous work from the laboratory showed that the chemical tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated release of the cell surface glycoprotein, fibronectin (FN) from human lung fibroblasts (HLF), leading to depletion of cell surface FN, while FN synthesis is not altered by TPA. To further investigate the mechanism(s) by which TPA stimulates FN release, two types of experiments were performed. In the first, HLF were pulsed with /sup 35/S-methionine-labeled medium with or without TPA. In the second, cell-surface proteins were labeled by iodination (/sup 125/I) and then incubated in unlabeled medium with or without TPA. In both cases, the fate ofmore » labeled FN was followed over 12 hr. The /sup 35/S-meth-labeled HLF showed a rapid loss of labeled FN, first into a small, highly-labeled pool of cell surface FN (1 hr), later into the medium (4 hr or longer). Specific activities showed that this small pool in the cell surface turned over rapidly. TPA treatment resulted in more rapid movement of /sup 35/S-meth pulse-labeled FN to the cell surface and into the medium than in control cells. TPA thus affected the fate of intracellular FN. TPA treatment of HLF also resulted in more rapid removal of /sup 125/I-cell surface-labeled FN into the medium than in control cells. Thus, TPA affects the fate of preexisting cell surface FN in HLF. From these results, they hypothesize that TPA has two separate effects: it stimulates depletion of preexisting intracellular FN during the first hr of treatment, and it stimulates release of preexisting cell surface FN over all treatment times.« less

Altered Gene Expression and Function of Peripheral Blood Natural Killer Cells in Children with Autism

PubMed Central

Enstrom, A M; Lit, L; Onore, C E; Gregg, J P; Hansen, R; Pessah, I N; Hertz-Picciotto, I; Van de Water, J A; Sharp, F R; Ashwood, P

2009-01-01

Immune related abnormalities have repeatedly been reported in autism spectrum disorders (ASD), including evidence of immune dysregulation and autoimmune phenomena. NK cells may play an important role in neurodevelopmental disorders such as ASD. Here we performed a gene expression screen and cellular functional analysis on peripheral blood obtained from 52 children with ASD and 27 typically developing control children enrolled in the case-control CHARGE study. RNA expression of NK cell receptors and effector molecules were significantly upregulated in ASD. Flow cytometric analysis of NK cells demonstrated increased production of perforin, granzyme B, and interferon gamma (IFNγ) under resting conditions in children with ASD (p<0.01). Following NK cell stimulation in the presence of K562 target cells, the cytotoxicity of NK cells was significantly reduced in ASD compared with controls (p<0.02). Furthermore, under similar stimulation conditions the presence of perforin, granzyme B, and IFNγ in NK cells from ASD children was significantly lower compared with controls (p<0.001). These findings suggest possible dysfunction of NK cells in children with ASD. Abnormalities in NK cells may represent a susceptibility factor in ASD and may predispose to the development of autoimmunity and/or adverse neuroimmune interactions during critical periods of development. PMID:18762240

Production of interleukin-2 (IL-2) and expression of IL-2 receptor in patients with IgA nephropathy.

PubMed

Lee, T W; Kim, M J

1992-01-01

IL-2 production has been measured in several disease including type I diabetes mellitus, systemic lupus erythematosus, acquired immunodeficiency syndrome and active pulmonary sarcoidosis and its pathogenetic role was suggested. In IgA nephropathy, altered T cell subsets were reported to be associated with increased synthesis of IgA. The altered IL-2 production and the expression of IL-2 receptor might be involved in the pathogenesis of IgA nephropathy. To investigate the role of T cell mediated immunity in the pathogenesis of IgA nephropathy, the immune parameters such as T cell subsets, NK cell activity, interleukin-2 (IL-2) production and IL-2 receptor expression on peripheral blood mononuclear cells (PBMC) were measured before and/or after phytohemagglutinin (PHA) stimulation in 15 patients with IgA nephropathy. Age and sex matched 15 healthy controls and the correlations between the IL-2 production and immune parameters were evaluated. The mean percentages of T helper/inducer cells (CD4), T suppressor/cytotoxic cells (CD8) and the CD4/CD8 ratio of the patients were not different from those of controls and the proportions of CD8 CD11b cell in the patients (21.0 +/- 3.6%) were significantly lower than those in controls (30.5 +/- 5.3%) (p < 0.005). The production of IL-2 by fresh PBMC of both patients and controls was in undetectable ranges. The production of IL-2 by PHA stimulated PBMC of patients was significantly higher than that of controls (140.03 +/- 43.2 U/ml vs 106.5 +/- 42.1 U/ml, p < 0.05). The proportions of lymphocytes expressing the IL-2 receptor (CD25) before the stimulation with PHA in patients were 1.22 +/- 1.00 percent and were not different from those in controls (1.12 +/- 0.78 percent). The correlations between the production of IL-2 and the concentrations of serum IgA, the degrees of histologic alterations and the proportions of CD8 and CD8CD11b cells were not significant. There was a weak tendency of a positive correlation (p < 0.1) between the production of IL-2 and the proportions of CD4 cells, and the CD4/CD8 ratio showed a significant correlation with the production of IL-2 (p < 0.05). After PHA stimulation, the mean percentages of lymphocytes expressing the IL-2 receptors in patients were increased to 47.6 +/- 8.9 percents which is higher than those (40.4 +/- 9.9%) in controls (p < 0.05). The NK cell activity of the patients was higher than that of controls (75.6 +/- 19.6% vs 56.1 +/- 16.2%, p < 0.005), and was well correlated with the production of IL-2 by PBMC (r = 0.89, p < 0.05). It seemed that patients with IgA nephropathy have an 'latent' cellular immunoregulatory dysfunction that becomes apparent on the stimulation of extrinsic antigens or mitogens.

Regulation of Cell Cytoskeleton and Membrane Mechanics by Electric Field: Role of Linker Proteins

PubMed Central

Titushkin, Igor; Cho, Michael

2009-01-01

Abstract Cellular mechanics is known to play an important role in the cell homeostasis including proliferation, motility, and differentiation. Significant variation in the mechanical properties between different cell types suggests that control of the cell metabolism is feasible through manipulation of the cell mechanical parameters using external physical stimuli. We investigated the electrocoupling mechanisms of cellular biomechanics modulation by an electrical stimulation in two mechanically distinct cell types—human mesenchymal stem cells and osteoblasts. Application of a 2 V/cm direct current electric field resulted in approximately a twofold decrease in the cell elasticity and depleted intracellular ATP. Reduction in the ATP level led to inhibition of the linker proteins that are known to physically couple the cell membrane and cytoskeleton. The membrane separation from the cytoskeleton was confirmed by up to a twofold increase in the membrane tether length that was extracted from the cell membrane after an electrical stimulation. In comparison to human mesenchymal stem cells, the membrane-cytoskeleton attachment in osteoblasts was much stronger but, in response to the same electrical stimulation, the membrane detachment from the cytoskeleton was found to be more pronounced. The observed effects mediated by an electric field are cell type- and serum-dependent and can potentially be used for electrically assisted cell manipulation. An in-depth understanding and control of the mechanisms to regulate cell mechanics by external physical stimulus (e.g., electric field) may have great implications for stem cell-based tissue engineering and regenerative medicine. PMID:19167316

T cell activation and proliferation following acute exercise in human subjects is altered by storage conditions and mitogen selection.

PubMed

Siedlik, Jacob A; Deckert, Jake A; Benedict, Stephen H; Bhatta, Anuja; Dunbar, Amanda J; Vardiman, John P; Gallagher, Philip M

2017-07-01

Recent work investigating exercise induced changes in immunocompetence suggests that some of the ambiguity in the literature is resultant from different cell isolation protocols and mitogen selection. To understand this effect, we compared post-exercise measures of T cell activation and proliferation using two different stimulation methods (costimulation through CD28 or stimulation with phytohaemagglutinin [PHA]). Further, we investigated whether exercise induced changes are maintained when T cell isolation from whole blood is delayed overnight in either a room temperature or chilled (4°C) environment. As expected, an increased proliferation response was observed post-exercise in T cells isolated from whole blood of previously trained individuals immediately after blood collection. Also, cells stimulated with PHA after resting overnight in whole blood were not adversely impacted by the storage conditions. In contrast, allowing cells to rest overnight in whole blood prior to stimulation through CD28, lessened the proliferation observed by cells following exercise rendering both the room temperature and chilled samples closer to the results seen in the control condition. Changes in early markers of activation (CD25), followed a similar pattern, with activation in PHA stimulated cells remaining fairly robust after overnight storage; whereas cell activation following stimulation through CD3+CD28 was disproportionately decreased by the influence of overnight storage. These findings indicate that decisions regarding cell stimulation methods need to be paired with the timeline for T cell isolation from whole blood. These considerations will be especially important for field based studies of immunocompetence where there is a delay in getting whole blood samples to a lab for processing as well as clinical applications where a failure to isolate T cells in a timely manner may result in loss of the response of interest. Copyright © 2017 Elsevier B.V. All rights reserved.

Idiotypic Cascades in Cancer Patients Treated with Monoclonal Antibody CO17-1A

NASA Astrophysics Data System (ADS)

Wettendorff, Martine; Iliopoulos, Dimitrios; Tempero, Margaret; Kay, David; Defreitas, Elaine; Koprowski, Hilary; Herlyn, Dorothee

1989-05-01

We have previously shown that gastrointestinal cancer patients treated with monoclonal antibody CO17-1A (Ab1) developed anti-idiotypic antibodies (Ab2) to the Ab1. We now demonstrate that patients produce anti-anti-idiotypic antibodies (Ab3) to their autologous Ab2. Ab3 were demonstrated in culture supernatants of peripheral blood mononuclear cells from five Ab1-treated patients after stimulation of the cells with heterologous Ab2 that functionally mimicked the tumor antigen (Ag) defined by Ab1 and immunologically cross reacted with the patients' Ab2. Ab3 shared idiotopes with Ab1 and were Ab1-like in their binding specificities to tumor cells, Ag, and Ab2. Such antibodies were also elicited by stimulating cells with Ag. However, they were not produced by stimulating posttreatment mononuclear cells with control proteins or by stimulating pretreatment cells with either Ag or Ab2. Our results demonstrate idiotypic cascades in cancer patients treated with monoclonal antibody. Ag-specific Ab3 responses may underlie delayed clinical responses often observed in cancer patients treated with monoclonal antibodies of various specificities.

Stimulus-secretion coupling in chromaffin cells isolated from bovine adrenal medulla

PubMed Central

Schneider, Allan S.; Herz, Ruth; Rosenheck, Kurt

1977-01-01

Bovine adrenal chromaffin cells were isolated by removal of the cortex and sequential collagenase digestion of the medulla. The catecholamine secretory function of these cells was characterized with respect to acetylcholine stimulation, cation requirements, and cytoskeletal elements. The dose-response curve for stimulated release had its half-maximum value at 10-5 M acetylcholine, and maximum secretion was on the average 7 times that of control basal secretion. The differential release of epinephrine versus norepinephrine after stimulation with 0.1 mM acetylcholine occurred in proportion to their distribution in the cell suspension. The cholinergic receptors were found to be predominantly nicotinic. The kinetics of catecholamine release were rapid, with significant secretion occurring in less than 60 sec and 85% of maximum secretion within 5 min. A critical requirement for calcium in the extracellular medium was demonstrated, and 80% of maximum secretion was achieved at physiologic calcium concentrations. Stimulation by excess potassium (65 mM KCl) also induced catecholamine secretion which differed from acetylcholine stimulation in being less potent, in having a different dependence on calcium concentration, and in its response to the local anesthetic tetracaine. Tetracaine, which is thought to inhibit membrane cation permeability, was able to block acetylcholine-stimulated but not KCl-stimulated secretion. The microtubule disrupting agent vinblastine was able to block catecholamine release whereas the microfilament disrupter cytochalasin B had little effect. The results show the isolated bovine chromaffin cells to be viable, functioning, and available in large quantity. These cells now provide an excellent system for studying cell surface regulation of hormone and neurotransmitter release. PMID:270738

Cellular localization of the activated EGFR determines its effect on cell growth in MDA-MB-468 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hyatt, Dustin C.; Ceresa, Brian P.

2008-11-01

The epidermal growth factor (EGF) receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase that regulates diverse cell functions that are dependent upon cell type, the presence of downstream effectors, and receptor density. In addition to activating biochemical pathways, ligand stimulation causes the EGFR to enter the cell via clathrin-coated pits. Endocytic trafficking influences receptor signaling by controlling the duration of EGFR phosphorylation and coordinating the receptor's association with downstream effectors. To better understand the individual contributions of cell surface and cytosolic EGFRs on cell physiology, we used EGF that was conjugated to 900 nm polystyrene beads (EGF-beads). EGF-beads canmore » stimulate the EGFR and retain the activated receptor at the plasma membrane. In MDA-MB-468 cells, a breast cancer cell line that over-expresses the EGFR, only internalized, activated EGFRs stimulate caspase-3 and induce cell death. Conversely, signaling cascades triggered from activated EGFR retained at the cell surface inhibit caspase-3 and promote cell proliferation. Thus, through endocytosis, the activated EGFR can differentially regulate cell growth in MDA-MB-468 cells.« less

Phorbol esters alter adenylate cyclase responses to vasoactive intestinal peptide and forskolin in the GH cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Summers, S.; Florio, T.; Cronin, M.

1986-05-01

Activation of protein kinase C with phorbol ester modifies cyclic AMP production in several anterior pituitary cell systems. In the GH cell line from a rat pituitary tumor, exposure to phorbol 12-myristate 13-acetate (PMA: 100 nM) for 30 minutes significantly reduces vasoactive intestinal peptide (VIP: 100 nM) stimulated adenylate cyclase (AC) activity in subsequent membrane preparations to 62 + 4% of control (n = 6 independent studies). In contrast, these same membrane preparations respond to forskolin (1 ..mu..M) with significantly more activity, 130 +/- 6% of controls (n = 6 independent studies). Finally, phorbol ester does not block an inhibitorymore » hormone input into the AC system; somatostatin (100 nM) reduction of VIP-stimulated AC activity is not significantly different in membrane preparations from PMA treated and control cells (n = 3 independent studies). These other findings lead the authors to propose that protein kinase C can modify several sites in the AC complex in anterior pituitary cells.« less

Extracellular formation and uptake of adenosine during skeletal muscle contraction in the rat: role of adenosine transporters

PubMed Central

Lynge, J; Juel, C; Hellsten, Y

2001-01-01

The existence of adenosine transporters in plasma membrane giant vesicles from rat skeletal muscles and in primary skeletal muscle cell cultures was investigated. In addition, the contribution of intracellularly or extracellularly formed adenosine to the overall extracellular adenosine concentration during muscle contraction was determined in primary skeletal muscle cell cultures. In plasma membrane giant vesicles, the carrier-mediated adenosine transport demonstrated saturation kinetics with Km= 177 ± 36 μm and Vmax= 1.9 ± 0.2 nmol ml−1 s−1 (0.7 nmol (mg protein)−1 s−1). The existence of an adenosine transporter was further evidenced by the inhibition of the carrier-mediated adenosine transport in the presence of NBMPR (nitrobenzylthioinosine; 72 % inhibition) or dipyridamol (64 % inhibition; P < 0.05). In primary skeletal muscle cells, the rate of extracellular adenosine accumulation was 5-fold greater (P < 0.05) with electrical stimulation than without electrical stimulation. Addition of the adenosine transporter inhibitor NBMPR led to a 57 % larger (P < 0.05) rate of extracellular adenosine accumulation in the electro-stimulated muscle cells compared with control cells, demonstrating that adenosine is taken up by the skeletal muscle cells during contractions. Inhibition of ecto-5′-nucleotidase with AOPCP in electro-stimulated cells resulted in a 70 % lower (P < 0.05) rate of extracellular adenosine accumulation compared with control cells, indicating that adenosine to a large extent is formed in the extracellular space during contraction. The present study provides evidence for the existence of an NBMPR-sensitive adenosine transporter in rat skeletal muscle. Our data furthermore demonstrate that the increase in extracellular adenosine observed during electro-stimulation of skeletal muscle is due to production of adenosine in the extracellular space of skeletal muscle and that adenosine is taken up rather than released by the skeletal muscle cells during contraction. PMID:11731589

Heat shock protein 22 (HSPB8) limits TGF-β-stimulated migration of osteoblasts.

PubMed

Yamamoto, Naohiro; Tokuda, Haruhiko; Kuroyanagi, Gen; Kainuma, Shingo; Matsushima-Nishiwaki, Rie; Fujita, Kazuhiko; Kozawa, Osamu; Otsuka, Takanobu

2016-11-15

Heat shock proteins (HSPs) are induced in response to various physiological and environmental conditions such as chemical and heat stress, and recognized to function as molecular chaperones. HSP22 (HSPB8), a low-molecular weight HSP, is ubiquitously expressed in many cell types. However, the precise role of HSP22 in bone metabolism remains to be clarified. In the present study, we investigated whether HSP22 is implicated in the transforming growth factor-β (TGF-β)-stimulated migration of osteoblast-like MC3T3-E1 cells. Although protein levels of HSP22 were clearly detected in unstimulated MC3T3-E1 cells, TGF-β failed to induce the protein levels. The TGF-β-stimulated migration was significantly up-regulated by knockdown of HSP22 expression. The cell migration stimulated by platelet-derived growth factor-BB was also enhanced by HSP22 knockdown. SB203580, an inhibitor of p38 mitogen-activated protein kinase, PD98059, an inhibitor of MEK1/2, or SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase had no effects on the TGF-β-induced migration. SIS3, a specific inhibitor of TGF-β-dependent Smad3 phosphorylation, significantly reduced the migration with or without TGF-β stimulation. Smad2, Smad3, Smad4 or Smad7 was not coimmunoprecipitated with HSP22. On the other hand, the TGF-β-induced Smad2 phosphorylation was enhanced by HSP22 down-regulation. The protein levels of TGF-β type II receptor (TGF-β RII) but not TGF-β type I receptor (TGF-β RI) was significantly up-regulated in HSP22 knockdown cells compared with those in the control cells. However, the levels of TGF-β RII mRNA in HSP22 knockdown cells were little different from those of the control cells. Neither TGF-β RI nor TGF-β RII was coimmunoprecipitated with HSP22. SIS3 reduced the amplification by HSP22 knockdown of the TGF-β-stimulated cell migration almost to the basal level. Our results strongly suggest that HSP22 functions as a negative regulator in the TGF-β-stimulated migration of osteoblasts via suppression of the Smad-dependent pathway, resulting from modulating the protein levels of TGF-β RII. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Antitumor Responses Stimulated by Dendritic Cells Are Improved by Triiodothyronine Binding to the Thyroid Hormone Receptor β.

PubMed

Alamino, Vanina A; Mascanfroni, Iván D; Montesinos, María M; Gigena, Nicolás; Donadio, Ana C; Blidner, Ada G; Milotich, Sonia I; Cheng, Sheue-Yann; Masini-Repiso, Ana M; Rabinovich, Gabriel A; Pellizas, Claudia G

2015-04-01

Bidirectional cross-talk between the neuroendocrine and immune systems orchestrates immune responses in both physiologic and pathologic settings. In this study, we provide in vivo evidence of a critical role for the thyroid hormone triiodothyronine (T3) in controlling the maturation and antitumor functions of dendritic cells (DC). We used a thyroid hormone receptor (TR) β mutant mouse (TRβPV) to establish the relevance of the T3-TRβ system in vivo. In this model, TRβ signaling endowed DCs with the ability to stimulate antigen-specific cytotoxic T-cell responses during tumor development. T3 binding to TRβ increased DC viability and augmented DC migration to lymph nodes. Moreover, T3 stimulated the ability of DCs to cross-present antigens and to stimulate cytotoxic T-cell responses. In a B16-OVA mouse model of melanoma, vaccination with T3-stimulated DCs inhibited tumor growth and prolonged host survival, in part by promoting the generation of IFNγ-producing CD8(+) T cells. Overall, our results establish an adjuvant effect of T3-TRβ signaling in DCs, suggesting an immediately translatable method to empower DC vaccination approaches for cancer immunotherapy. ©2015 American Association for Cancer Research.

Apparatus and methods for manipulation and optimization of biological systems

NASA Technical Reports Server (NTRS)

Sun, Ren (Inventor); Ho, Chih-Ming (Inventor); Wong, Pak Kin (Inventor); Yu, Fuqu (Inventor)

2012-01-01

The invention provides systems and methods for manipulating, e.g., optimizing and controlling, biological systems, e.g., for eliciting a more desired biological response of biological sample, such as a tissue, organ, and/or a cell. In one aspect, systems and methods of the invention operate by efficiently searching through a large parametric space of stimuli and system parameters to manipulate, control, and optimize the response of biological samples sustained in the system, e.g., a bioreactor. In alternative aspects, systems include a device for sustaining cells or tissue samples, one or more actuators for stimulating the samples via biochemical, electromagnetic, thermal, mechanical, and/or optical stimulation, one or more sensors for measuring a biological response signal of the samples resulting from the stimulation of the sample. In one aspect, the systems and methods of the invention use at least one optimization algorithm to modify the actuator's control inputs for stimulation, responsive to the sensor's output of response signals. The compositions and methods of the invention can be used, e.g., to for systems optimization of any biological manufacturing or experimental system, e.g., bioreactors for proteins, e.g., therapeutic proteins, polypeptides or peptides for vaccines, and the like, small molecules (e.g., antibiotics), polysaccharides, lipids, and the like. Another use of the apparatus and methods includes combination drug therapy, e.g. optimal drug cocktail, directed cell proliferations and differentiations, e.g. in tissue engineering, e.g. neural progenitor cells differentiation, and discovery of key parameters in complex biological systems.

Function of beta 2-adrenergic receptors in chronic localized myalgia.

PubMed

Maekawa, Kenji; Kuboki, Takuo; Inoue, Eitoku; Inoue-Minakuchi, Mami; Suzuki, Koji; Yatani, Hirofumi; Clark, Glenn T

2003-01-01

To investigate alteration of beta 2-adrenergic receptor (beta 2 AR) function in chronic localized myalgia subjects by evaluating levels of the beta 2 AR second messenger, cyclic adenosine monophosphate (cAMP), in mononuclear cells after beta AR-agonist stimulation. Eleven chronic localized myalgia subjects and 21 matched healthy controls participated in this study. Peripheral blood (30 cc) was drawn from the subjects' anterocubital vein. Mononuclear cells were isolated from the total blood by using the Ficoll-Hypaque gradient technique. Basal and stimulated intracellular cAMP levels were determined by enzyme immunoassay using a commercially available kit. Aliquots of 5 x 10(6) cells were incubated with or without stimulation of the beta AR-agonist isoproterenol for 5 minutes. Five different concentrations of isoproterenol (10(-3) M to 10(-7) M) were utilized. cAMP levels in both groups were tested statistically by a 2-way repeated-measures ANOVA with 2 predictors, group difference and isoproterenol concentration difference. As with isoproterenol stimulation, the cAMP responses to forskolin, which activates adenylyl cyclase directly and produces cAMP, bypassing the cell surface receptors were also measured. The basal cAMP levels in both groups (myalgia: 0.33 +/- 0.02 pmol/5 x 10(6) cells; control: 0.43 +/- 0.10 pmol/5 x 10(6) cells) were almost identical, and isoproterenol-produced cAMP levels increased dose-dependently in both groups. No significant differences in the mean cAMP levels were observed between the groups (P = .909). Significant increases were observed according to the isoproterenol concentration increase (P < .0001). The cAMP responses to forskolin stimulation also showed no significant group difference (P = .971). These results suggest that beta 2 AR function is not different between localized myalgia subjects and healthy individuals.

High PD-L1/CD86 MFI ratio and IL-10 secretion characterize human regulatory dendritic cells generated for clinical testing in organ transplantation.

PubMed

Zahorchak, Alan F; Macedo, Camila; Hamm, David E; Butterfield, Lisa H; Metes, Diana M; Thomson, Angus W

2018-01-01

Human regulatory dendritic cells (DCreg) were generated from CD14 immunobead-purified or elutriated monocytes in the presence of vitamin D3 and IL-10. They exhibited similar, low levels of costimulatory CD80 and CD86, but comparatively high levels of co-inhibitory programed death ligand-1 (PD-L1) and IL-10 production compared to control immature DC (iDC). Following Toll-like receptor 4 ligation, unlike control iDC, DCreg resisted phenotypic and functional maturation and further upregulated PD-L1:CD86 expression. Whereas LPS-stimulated control iDC (mature DC; matDC) secreted pro-inflammatory tumor necrosis factor but no IL-10, the converse was observed for LPS-stimulated DCreg. DCreg weakly stimulated naïve and memory allogeneic CD4 + and CD8 + T cell proliferation and IFNγ, IL-17A and perforin/granzyme B production in MLR. Their stimulatory function was enhanced however, by blocking PD-1 ligation. High-throughput T cell receptor (TCR) sequencing revealed that, among circulating T cell subsets, memory CD8 + T cells contained the most alloreactive TCR clonotypes and that, while matDC expanded these alloreactive memory CD8 TCR clonotypes, DCreg induced more attenuated responses. These findings demonstrate the feasibility of generating highly-purified GMP-grade DCreg for systemic infusion, their influence on the alloreactive T cell response, and a key mechanistic role of the PD1 pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

Contractile activity of human skeletal muscle cells prevents insulin resistance by inhibiting pro-inflammatory signalling pathways.

PubMed

Lambernd, S; Taube, A; Schober, A; Platzbecker, B; Görgens, S W; Schlich, R; Jeruschke, K; Weiss, J; Eckardt, K; Eckel, J

2012-04-01

Obesity is closely associated with muscle insulin resistance and is a major risk factor for the pathogenesis of type 2 diabetes. Regular physical activity not only prevents obesity, but also considerably improves insulin sensitivity and skeletal muscle metabolism. We sought to establish and characterise an in vitro model of human skeletal muscle contraction, with a view to directly studying the signalling pathways and mechanisms that are involved in the beneficial effects of muscle activity. Contracting human skeletal muscle cell cultures were established by applying electrical pulse stimulation. To induce insulin resistance, skeletal muscle cells were incubated with human adipocyte-derived conditioned medium, monocyte chemotactic protein (MCP)-1 and chemerin. Similarly to in exercising skeletal muscle in vivo, electrical pulse stimulation induced contractile activity in human skeletal muscle cells, combined with the formation of sarcomeres, activation of AMP-activated protein kinase (AMPK) and increased IL-6 secretion. Insulin-stimulated glucose uptake was substantially elevated in contracting cells compared with control. The incubation of skeletal muscle cells with adipocyte-conditioned media, chemerin and MCP-1 significantly reduced the insulin-stimulated phosphorylation of Akt. This effect was abrogated by concomitant pulse stimulation of the cells. Additionally, pro-inflammatory signalling by adipocyte-derived factors was completely prevented by electrical pulse stimulation of the myotubes. We showed that the effects of electrical pulse stimulation on skeletal muscle cells were similar to the effect of exercise on skeletal muscle in vivo in terms of enhanced AMPK activation and IL-6 secretion. In our model, muscle contractile activity eliminates insulin resistance by blocking pro-inflammatory signalling pathways. This novel model therefore provides a unique tool for investigating the molecular mechanisms that mediate the beneficial effects of muscle contraction.

Protein kinase D2 is a digital amplifier of T cell receptor–stimulated diacylglycerol signaling in naïve CD8+ T cells

PubMed Central

Navarro, María N.; Feijoo-Carnero, Carmen; Arandilla, Alba Gonzalez; Trost, Matthias; Cantrell, Doreen A.

2016-01-01

Protein kinase D2 (PKD2) is a serine and threonine kinase that is activated in T cells by diacylglycerol and protein kinase C in response to stimulation of the T cell receptor (TCR) by antigen. We quantified the activation of PKD2 at the single-cell level and found that this kinase acts as a sensitive digital amplifier of TCR engagement, enabling CD8+ T cells to match the production of inflammatory cytokines to the quality and quantity of TCR ligands. There was a digital response pattern of PKD2 activation in response to TCR engagement, such that increasing the concentration and potency of TCR ligands increased the number of cells that exhibited activated PKD2. However, for each cell that responded to TCR stimulation, the entire cellular pool of PKD2 (~400,000 molecules) was activated. Moreover, PKD2 acted as an amplification checkpoint for antigen-stimulated digital cytokine responses and translated the differential strength of TCR signaling to determine the number of naïve CD8+ T cells that became effector cells. Together, these results provide insights into PKD family kinases and how they act digitally to amplify signaling networks controlled by the TCR. PMID:25336615

Control of cell behaviour through nanovibrational stimulation: nanokicking

NASA Astrophysics Data System (ADS)

Robertson, Shaun N.; Campsie, Paul; Childs, Peter G.; Madsen, Fiona; Donnelly, Hannah; Henriquez, Fiona L.; Mackay, William G.; Salmerón-Sánchez, Manuel; Tsimbouri, Monica P.; Williams, Craig; Dalby, Matthew J.; Reid, Stuart

2018-05-01

Mechanical signals are ubiquitous in our everyday life and the process of converting these mechanical signals into a biological signalling response is known as mechanotransduction. Our understanding of mechanotransduction, and its contribution to vital cellular responses, is a rapidly expanding field of research involving complex processes that are still not clearly understood. The use of mechanical vibration as a stimulus of mechanotransduction, including variation of frequency and amplitude, allows an alternative method to control specific cell behaviour without chemical stimulation (e.g. growth factors). Chemical-independent control of cell behaviour could be highly advantageous for fields including drug discovery and clinical tissue engineering. In this review, a novel technique is described based on nanoscale sinusoidal vibration. Using finite-element analysis in conjunction with laser interferometry, techniques that are used within the field of gravitational wave detection, optimization of apparatus design and calibration of vibration application have been performed. We further discuss the application of nanovibrational stimulation, or `nanokicking', to eukaryotic and prokaryotic cells including the differentiation of mesenchymal stem cells towards an osteoblast cell lineage. Mechanotransductive mechanisms are discussed including mediation through the Rho-A kinase signalling pathway. Optimization of this technique was first performed in two-dimensional culture using a simple vibration platform with an optimal frequency and amplitude of 1 kHz and 22 nm. A novel bioreactor was developed to scale up cell production, with recent research demonstrating that mesenchymal stem cell differentiation can be efficiently triggered in soft gel constructs. This important step provides first evidence that clinically relevant (three-dimensional) volumes of osteoblasts can be produced for the purpose of bone grafting, without complex scaffolds and/or chemical induction. Initial findings have shown that nanovibrational stimulation can also reduce biofilm formation in a number of clinically relevant bacteria. This demonstrates additional utility of the bioreactor to investigate mechanotransduction in other fields of research. This article is part of a discussion meeting issue `The promises of gravitational-wave astronomy'.

Control of cell behaviour through nanovibrational stimulation: nanokicking.

PubMed

Robertson, Shaun N; Campsie, Paul; Childs, Peter G; Madsen, Fiona; Donnelly, Hannah; Henriquez, Fiona L; Mackay, William G; Salmerón-Sánchez, Manuel; Tsimbouri, Monica P; Williams, Craig; Dalby, Matthew J; Reid, Stuart

2018-05-28

Mechanical signals are ubiquitous in our everyday life and the process of converting these mechanical signals into a biological signalling response is known as mechanotransduction. Our understanding of mechanotransduction, and its contribution to vital cellular responses, is a rapidly expanding field of research involving complex processes that are still not clearly understood. The use of mechanical vibration as a stimulus of mechanotransduction, including variation of frequency and amplitude, allows an alternative method to control specific cell behaviour without chemical stimulation (e.g. growth factors). Chemical-independent control of cell behaviour could be highly advantageous for fields including drug discovery and clinical tissue engineering. In this review, a novel technique is described based on nanoscale sinusoidal vibration. Using finite-element analysis in conjunction with laser interferometry, techniques that are used within the field of gravitational wave detection, optimization of apparatus design and calibration of vibration application have been performed. We further discuss the application of nanovibrational stimulation, or 'nanokicking', to eukaryotic and prokaryotic cells including the differentiation of mesenchymal stem cells towards an osteoblast cell lineage. Mechanotransductive mechanisms are discussed including mediation through the Rho-A kinase signalling pathway. Optimization of this technique was first performed in two-dimensional culture using a simple vibration platform with an optimal frequency and amplitude of 1 kHz and 22 nm. A novel bioreactor was developed to scale up cell production, with recent research demonstrating that mesenchymal stem cell differentiation can be efficiently triggered in soft gel constructs. This important step provides first evidence that clinically relevant (three-dimensional) volumes of osteoblasts can be produced for the purpose of bone grafting, without complex scaffolds and/or chemical induction. Initial findings have shown that nanovibrational stimulation can also reduce biofilm formation in a number of clinically relevant bacteria. This demonstrates additional utility of the bioreactor to investigate mechanotransduction in other fields of research.This article is part of a discussion meeting issue 'The promises of gravitational-wave astronomy'. © 2018 The Author(s).

Nitric oxide donors, sodium nitroprusside and S-nitroso-N-acetylpencillamine, stimulate myoblast proliferation in vitro

NASA Technical Reports Server (NTRS)

Ulibarri, J. A.; Mozdziak, P. E.; Schultz, E.; Cook, C.; Best, T. M.

1999-01-01

Nitric oxide (NO) is an inter- and intracellular messenger involved in a variety of physiologic and pathophysiologic conditions. The effect of two NO donors, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) and their effect on myoblast proliferation was examined. Both donors stimulated an increase in myoblast cell number over a range (1-10 microM) of donor concentrations. However, 50 microM SNAP inhibited myoblast proliferation. Cell numbers from cultures treated with degraded 10 microM SNAP were equivalent to the control. Therefore, it appears NO can stimulate as well as inhibit myoblast proliferation.

Biomimetic perfusion and electrical stimulation applied in concert improved the assembly of engineered cardiac tissue.

PubMed

Maidhof, Robert; Tandon, Nina; Lee, Eun Jung; Luo, Jianwen; Duan, Yi; Yeager, Keith; Konofagou, Elisa; Vunjak-Novakovic, Gordana

2012-11-01

Maintenance of normal myocardial function depends intimately on synchronous tissue contraction, driven by electrical activation and on adequate nutrient perfusion in support thereof. Bioreactors have been used to mimic aspects of these factors in vitro to engineer cardiac tissue but, due to design limitations, previous bioreactor systems have yet to simultaneously support nutrient perfusion, electrical stimulation and unconstrained (i.e. not isometric) tissue contraction. To the best of our knowledge, the bioreactor system described herein is the first to integrate these three key factors in concert. We present the design of our bioreactor and characterize its capability in integrated experimental and mathematical modelling studies. We then cultured cardiac cells obtained from neonatal rats in porous, channelled elastomer scaffolds with the simultaneous application of perfusion and electrical stimulation, with controls excluding either one or both of these two conditions. After 8 days of culture, constructs grown with simultaneous perfusion and electrical stimulation exhibited substantially improved functional properties, as evidenced by a significant increase in contraction amplitude (0.23 ± 0.10% vs 0.14 ± 0.05%, 0.13 ± 0.08% or 0.09 ± 0.02% in control constructs grown without stimulation, without perfusion, or either stimulation or perfusion, respectively). Consistently, these constructs had significantly improved DNA contents, cell distribution throughout the scaffold thickness, cardiac protein expression, cell morphology and overall tissue organization compared to control groups. Thus, the simultaneous application of medium perfusion and electrical conditioning enabled by the use of the novel bioreactor system may accelerate the generation of fully functional, clinically sized cardiac tissue constructs. Copyright © 2011 John Wiley & Sons, Ltd.

PFKFB3 Control of Cancer Growth by Responding to Circadian Clock Outputs

PubMed Central

Chen, Lili; Zhao, Jiajia; Tang, Qingming; Li, Honggui; Zhang, Chenguang; Yu, Ran; Zhao, Yan; Huo, Yuqing; Wu, Chaodong

2016-01-01

Circadian clock dysregulation promotes cancer growth. Here we show that PFKFB3, the gene that encodes for inducible 6-phosphofructo-2-kinase as an essential supporting enzyme of cancer cell survival through stimulating glycolysis, mediates circadian control of carcinogenesis. In patients with tongue cancers, PFKFB3 expression in both cancers and its surrounding tissues was increased significantly compared with that in the control, and was accompanied with dys-regulated expression of core circadian genes. In the in vitro systems, SCC9 tongue cancer cells displayed rhythmic expression of PFKFB3 and CLOCK that was distinct from control KC cells. Furthermore, PFKFB3 expression in SCC9 cells was stimulated by CLOCK through binding and enhancing the transcription activity of PFKFB3 promoter. Inhibition of PFKFB3 at zeitgeber time 7 (ZT7), but not at ZT19 caused significant decreases in lactate production and in cell proliferation. Consistently, PFKFB3 inhibition in mice at circadian time (CT) 7, but not CT19 significantly reduced the growth of implanted neoplasms. Taken together, these findings demonstrate PFKFB3 as a mediator of circadian control of cancer growth, thereby highlighting the importance of time-based PFKFB3 inhibition in cancer treatment. PMID:27079271

Folate-conjugated immunoglobulin targets melanoma tumor cells for NK cell effector functions

PubMed Central

Skinner, Cassandra C.; McMichael, Elizabeth L.; Jaime-Ramirez, Alena C.; Abrams, Zachary B.; Lee, Robert J.; Carson, William E.

2016-01-01

The folate receptor (FR) is over-expressed on the vascular side of cancerous cells including those of the breast, ovaries, testes, and cervix. We hypothesized that a folate-conjugated immunoglobulin (F-IgG) would bind to the FR that is over-expressed on melanoma tumor cells to target these cells for lysis by natural killer (NK) cells. Folate receptor expression was confirmed in the Mel-39 (human melanoma) cell line by flow cytometry and immunoblot analysis, using KB (human oral epithelial) and F01 (human melanoma) as a positive and negative control, respectively. FR-positive and negative cell lines were treated with F-IgG or control immunoglobulin G (C-IgG) in the presence or absence of cytokines in order to determine NK cell ability to lyse FR-positive cell lines. NK cell activation was significantly upregulated and lysis of Mel 39 tumor cells enhanced following treatment with F-IgG, as compared to C-IgG at all effector:target (E:T) ratios (p<0.01). This trend was further enhanced by NK cell stimulation with the activating cytokine interleukin-12 (IL-12). NK cell production of cytokines such as interferon-gamma (IFN-γ), macrophage inflammatory protein 1 alpha (MIP-1α), and regulated on activation normal T-cell expressed and secreted (RANTES) were also significantly increased in response to co-stimulation with IL-12 stimulation and F-IgG-coated Mel 39 target cells, as compared to controls (p<0.01). In contrast, F-IgG did not bind to the FR-negative cell line F01 and had no significant effect on NK cell lysis or cytokine production. This research indicates the potential use of F-IgG for its ability to induce an immune response from NK cells against FR-positive melanoma tumor cells which can be further enhanced by the addition of cytokines. PMID:27035691

IL-6 as a corneal wound healing mediator in an in vitro scratch assay.

PubMed

Arranz-Valsero, Isabel; Soriano-Romaní, Laura; García-Posadas, Laura; López-García, Antonio; Diebold, Yolanda

2014-08-01

Corneal healing process under inflammatory conditions is not fully understood. We aimed at determining the effect of an inflammatory (presence of IL-6) or anti-inflammatory (presence of IL-10) environment and a mixture of both in the expression of IL-6 signaling pathway mediators, and on corneal wound healing in an in vitro scratch assay. For that purpose, human corneal epithelial cells were cultured until confluence. The effect of IL-6 (10 ng/ml), IL-10 (20 ng/ml) or IL-6 + IL-10 exposure on the expression of IL-6R, gp130, and STAT3 was determined by Western blotting and quantitative PCR, at different time points. The monolayer was mechanically wounded using a sterile 10 μl pipette tip. Wound healing rate in the presence or absence of these cytokines was measured immediately after cytokine exposure and after 4, 8, and 24 h. The effect of mitomycin C on wound healing rate, in control and IL-6-stimulated cells, was also evaluated. Detection of proliferative cells was performed with an EdU imaging kit. For the visualization of migrating cells, cold methanol-fixed cells were incubated with an α-actinin antibody. For the statistical analysis a two-factor design of experiment method was applied. Levene test was used to contrast equality of variances. If variances were equal, ANOVA was performed to test the equality of means. If variances were not equal, a Mood's median test was performed. We observed that IL-6 and IL-10 stimulation, and their combination, increased gp130 production at different time points. STAT3 production was increased in IL-6-stimulated cells, at 72 h. An increase in pSTAT3 production was found in IL-6- and IL-10-stimulated cells, that was sustained in time in IL-6 + IL-10 co-stimulated cultures. Scraped areas had an initial width of 570.57 ± 75.82 μm. In IL-6-exposed cells wound healing closure was faster than in control cells or IL-10-exposed cells. After 8 h, wound width in IL-10-exposed cells, was also significantly smaller than that of control cells. Cells exposed to IL-6 + IL-10 had the slowest wound healing rate, similar to control cells. Wounds were closed after 24 h regardless the experimental condition. Mitomycin C exposure increased the wound closure rate in every experimental condition. No significant differences in the percentage of proliferative cells at the edge of the scratch and in distant areas of the monolayer were found. At the edge of the scratch, some actin filaments of non-proliferative cells were directed through the cell-free area, independently of the stimulating condition. In conclusion, the presence of IL-10 and, most importantly, of IL-6, increased the wound healing rate in an in vitro corneal wound healing model. The combination of both cytokines did not have a synergistic action in wound healing. In our model, wound closure was the result of the combination of cell proliferation and cell migration. Copyright © 2014 Elsevier Ltd. All rights reserved.

Kinetics and clonality of immunological memory in humans.

PubMed

Beverley, Peter C L

2004-10-01

T-cell immunological memory consists largely of clones of proliferating lymphocytes maintained by antigenic stimulation and the survival and proliferative effects of cytokines. The duration of survival of memory clones in humans is determine by the Hayflick limit on the number of cell divisions, the rate of cycling of memory cells and factors that control erosion of telomeres, including mechanisms that control telomerase.

Altered macrophage arachidonic acid metabolism induced by endotoxin tolerance: characterization and mechanisms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rogers, T.S.

Altered macrophage arachidonic acid (AA) metabolism may play a role in endotoxic shock and the phenomenon of endotoxin tolerance induced by repeated injections of endotoxin. Studies were initiated to characterize both lipoxygenase and cyclooxygenase metabolite formation by endotoxin tolerant and non-tolerant macrophages in response to 4 different stimuli, i.e., endotoxin, glucan, zymosan, and the calcium ionophore A23187. In contrast to previous reports of decreased prostaglandin synthesis by tolerant macrophages, A23187-stimulated immunoreactive (i) leukotriene (LT) C/sub 4/D/sub 4/ and prostaglandin (PG) E/sub 2/ production by tolerant cells was greater than that by non-tolerant controls (p <0.001). However, A23187-stimulated i6-keto PGF/sub 1a/more » levels were lower in tolerant macrophages compared to controls (P < 0.05). iL TC/sub 4/D/sub 4/ production was not significantly stimulated by endotoxin or glucan, but was stimulated by zymosan in non-tolerant cells. Synthesis of iLTB/sub 4/ by control macrophages was stimulated by endotoxin (p <0.01). The effect of tolerance on factors that affect AA release was investigated by measuring /sup 14/C-AA incorporation and release and phospholipase A/sub 2/ activity« less

Impact of tobacco smoke on upper airway dendritic cell accumulation and regulation by sinonasal epithelial cells.

PubMed

Mulligan, Jennifer K; O'Connell, Brendan P; Pasquini, Whitney; Mulligan, Ryan M; Smith, Sarah; Soler, Zachary M; Atkinson, Carl; Schlosser, Rodney J

2017-08-01

In these studies we examined the impact of environmental tobacco smoke (ETS) and active smoking on sinonasal dendritic cell (DC) subsets in controls or patients with chronic rhinosinusitis with nasal polyps (CRSwNP). In subsequent in-vitro investigations, we examined the influence of cigarette smoke extract (CSE) on human sinonasal epithelial cells' (HSNECs) ability to regulate DC functions. Sinonasal tissue, blood, and hair were collected from patients undergoing sinus surgery. Smoking status and ETS exposure were determined by hair nicotine. DC subsets were examined by flow cytometric analysis. Monocyte-derived dendritic cells (moDCs) were treated with conditioned medium from non-smoked-exposed HSNECs (NS-HSNECs) or cigarette-smoke-extract-exposed HSNECs (CSE-HSNECs) to assess the impact of CSE exposure on HSNEC regulation of moDC functions. Control subjects who were active smokers displayed increased sinonasal moDC and myeloid dendritic 1 (mDC1) cells and reduced mDC2 cells, whereas, in CRSwNP patients, only moDC and mDC2 cells were altered. ETS was found to increase only moDCs in the CRSwNP patients. In vitro, CSE stimulated HSNEC secretion of the moDC regulatory products chemokine (C-C motif) ligand 20, prostaglandin E 2 , and granulocyte-macrophage colony-stimulating factor. CSE exposure also promoted HSNECs to stimulate monocyte and moDC migration. moDCs treated with CSE-HSNEC media stimulated an increase in antigen uptake and expression of CD80 and CD86. Last, CSE-HSNEC-treated moDCs secreted increased levels of interleukin-10, interferon-γ, and thymic stromal lymphopoietin. Active smoking, and to a lesser degree ETS, alters the sinonasal composition of DCs. A potential mechanism to account for this is that cigarette smoke stimulates HSNECs to induce moDC migration, maturation, and activation. © 2017 ARS-AAOA, LLC.

Feeding a Mixture of Choline Forms during Lactation Improves Offspring Growth and Maternal Lymphocyte Response to Ex Vivo Immune Challenges.

PubMed

Lewis, Erin D; Richard, Caroline; Goruk, Susan; Wadge, Emily; Curtis, Jonathan M; Jacobs, René L; Field, Catherine J

2017-07-07

Study objectives were to examine the impact of feeding a mixture of choline forms, or a diet high in glycerophosphocholine (GPC) on maternal immune function and offspring growth during lactation. Lactating Sprague-Dawley rat dams ( n = 6/diet) were randomized to one of three diets, providing 1 g/kg total choline: Control (100% free choline (FC)), Mixed Choline (MC; 50% phosphatidylcholine (PC), 25% FC, 25% GPC), or High GPC (HGPC; 75% GPC, 12.5% PC, 12.5% FC). At 3 weeks, cell phenotypes and cytokine production with Concanavalin A (ConA)-or lipopolysaccharide (LPS)-stimulated splenocytes and mesenteric lymphocytes were measured. Feeding MC or HGPC diets improved pups' growth compared to Control (+22% body weight, p < 0.05). In spleen, MC-and HGPC-fed dams had higher proportions of cytotoxic (CD8+) T cells expressing CD27, CD71 and CD127, total B cells (CD45RA+) and dendritic cells (OX6+OX62+), and produced less IL-6 and IFN-γ after ConA than Control-fed dams ( p < 0.05). MC and HGPC LPS-stimulated splenocytes produced less IL-1β and IL-6 than Control. ConA-stimulated mesenteric lymphocytes from MC and HGPC dams produced more IL-2 and IFN-γ than Control ( p < 0.05). In summary, feeding a mixture of choline forms during lactation improved offspring growth and resulted in a more efficient maternal immune response following mitogenic immune challenge.

Feeding a Mixture of Choline Forms during Lactation Improves Offspring Growth and Maternal Lymphocyte Response to Ex Vivo Immune Challenges

PubMed Central

Lewis, Erin D.; Goruk, Susan; Wadge, Emily; Curtis, Jonathan M.; Field, Catherine J.

2017-01-01

Study objectives were to examine the impact of feeding a mixture of choline forms, or a diet high in glycerophosphocholine (GPC) on maternal immune function and offspring growth during lactation. Lactating Sprague-Dawley rat dams (n = 6/diet) were randomized to one of three diets, providing 1 g/kg total choline: Control (100% free choline (FC)), Mixed Choline (MC; 50% phosphatidylcholine (PC), 25% FC, 25% GPC), or High GPC (HGPC; 75% GPC, 12.5% PC, 12.5% FC). At 3 weeks, cell phenotypes and cytokine production with Concanavalin A (ConA)-or lipopolysaccharide (LPS)-stimulated splenocytes and mesenteric lymphocytes were measured. Feeding MC or HGPC diets improved pups’ growth compared to Control (+22% body weight, p < 0.05). In spleen, MC-and HGPC-fed dams had higher proportions of cytotoxic (CD8+) T cells expressing CD27, CD71 and CD127, total B cells (CD45RA+) and dendritic cells (OX6+OX62+), and produced less IL-6 and IFN-γ after ConA than Control-fed dams (p < 0.05). MC and HGPC LPS-stimulated splenocytes produced less IL-1β and IL-6 than Control. ConA-stimulated mesenteric lymphocytes from MC and HGPC dams produced more IL-2 and IFN-γ than Control (p < 0.05). In summary, feeding a mixture of choline forms during lactation improved offspring growth and resulted in a more efficient maternal immune response following mitogenic immune challenge. PMID:28686201

Boron nitride nanotubes as vehicles for intracellular delivery of fluorescent drugs and probes.

PubMed

Niskanen, Jukka; Zhang, Issan; Xue, Yanming; Golberg, Dmitri; Maysinger, Dusica; Winnik, Françoise M

2016-01-01

To evaluate the response of cells to boron nitride nanotubes (BNNTs) carrying fluorescent probes or drugs in their inner channel by assessment of the cellular localization of the fluorescent cargo, evaluation of the in vitro release and biological activity of a drug (curcumin) loaded in BNNTs. Cells treated with curcumin-loaded BNNTs and stimulated with lipopolysaccharide were assessed for nitric oxide release and stimulation of IL-6 and TNF-α. The cellular trafficking of two cell-permeant dyes and a non-cell-permeant dye loaded within BNNTs was imaged. BNNTs loaded with up to 13 wt% fluorophores were internalized by cells and controlled release of curcumin triggered cellular pathways associated with the known anti-inflammatory effects of the drug. The overall findings indicate that BNNTs can function as nanocarriers of biologically relevant probes/drugs allowing one to examine/control their local intracellular localization and biochemical effects, leading the way to applications as intracellular nanosensors.

Ethylene is an endogenous stimulator of cell division in the cambial meristem of Populus

PubMed Central

Love, Jonathan; Björklund, Simon; Vahala, Jorma; Hertzberg, Magnus; Kangasjärvi, Jaakko; Sundberg, Björn

2009-01-01

The plant hormone ethylene is an important signal in plant growth responses to environmental cues. In vegetative growth, ethylene is generally considered as a regulator of cell expansion, but a role in the control of meristem growth has also been suggested based on pharmacological experiments and ethylene-overproducing mutants. In this study, we used transgenic ethylene-insensitive and ethylene-overproducing hybrid aspen (Populus tremula × tremuloides) in combination with experiments using an ethylene perception inhibitor [1-methylcyclopropene (1-MCP)] to demonstrate that endogenous ethylene produced in response to leaning stimulates cell division in the cambial meristem. This ethylene-controlled growth gives rise to the eccentricity of Populus stems that is formed in association with tension wood. PMID:19293381

The effects of acoustic vibration on fibroblast cell migration.

PubMed

Mohammed, Taybia; Murphy, Mark F; Lilley, Francis; Burton, David R; Bezombes, Frederic

2016-12-01

Cells are known to interact and respond to external mechanical cues and recent work has shown that application of mechanical stimulation, delivered via acoustic vibration, can be used to control complex cell behaviours. Fibroblast cells are known to respond to physical cues generated in the extracellular matrix and it is thought that such cues are important regulators of the wound healing process. Many conditions are associated with poor wound healing, so there is need for treatments/interventions, which can help accelerate the wound healing process. The primary aim of this research was to investigate the effects of mechanical stimulation upon the migratory and morphological properties of two different fibroblast cells namely; human lung fibroblast cells (LL24) and subcutaneous areolar/adipose mouse fibroblast cells (L929). Using a speaker-based system, the effects of mechanical stimulation (0-1600Hz for 5min) on the mean cell migration distance (μm) and actin organisation was investigated. The results show that 100Hz acoustic vibration enhanced cell migration for both cell lines whereas acoustic vibration above 100Hz was found to decrease cell migration in a frequency dependent manner. Mechanical stimulation was also found to promote changes to the morphology of both cell lines, particularly the formation of lamellipodia and filopodia. Overall lamellipodia was the most prominent actin structure displayed by the lung cell (LL24), whereas filopodia was the most prominent actin feature displayed by the fibroblast derived from subcutaneous areolar/adipose tissue. Mechanical stimulation at all the frequencies used here was found not to affect cell viability. These results suggest that low-frequency acoustic vibration may be used as a tool to manipulate the mechanosensitivity of cells to promote cell migration. Copyright © 2016 Elsevier B.V. All rights reserved.

Phosphoinositide 3–kinase γ participates in T cell receptor–induced T cell activation

PubMed Central

Alcázar, Isabela; Marqués, Miriam; Kumar, Amit; Hirsch, Emilio; Wymann, Matthias; Carrera, Ana C.; Barber, Domingo F.

2007-01-01

Class I phosphoinositide 3–kinases (PI3Ks) constitute a family of enzymes that generates 3-phosphorylated polyphosphoinositides at the cell membrane after stimulation of protein tyrosine (Tyr) kinase–associated receptors or G protein–coupled receptors (GPCRs). The class I PI3Ks are divided into two types: class IA p85/p110 heterodimers, which are activated by Tyr kinases, and the class IB p110γ isoform, which is activated by GPCR. Although the T cell receptor (TCR) is a protein Tyr kinase–associated receptor, p110γ deletion affects TCR-induced T cell stimulation. We examined whether the TCR activates p110γ, as well as the consequences of interfering with p110γ expression or function for T cell activation. We found that after TCR ligation, p110γ interacts with Gαq/11, lymphocyte-specific Tyr kinase, and ζ-associated protein. TCR stimulation activates p110γ, which affects 3-phosphorylated polyphosphoinositide levels at the immunological synapse. We show that TCR-stimulated p110γ controls RAS-related C3 botulinum substrate 1 activity, F-actin polarization, and the interaction between T cells and antigen-presenting cells, illustrating a crucial role for p110γ in TCR-induced T cell activation. PMID:17998387

The influence of hydro-ethanolic extract of Portulaca oleracea L. on Th1/Th2 balance in isolated human lymphocytes.

PubMed

Askari, Vahid Reza; Rezaee, Seyed Abdolrahim; Abnous, Khalil; Iranshahi, Mehrdad; Boskabady, Mohammad Hossein

2016-12-24

The anti-inflammatory and anti-oxidants activity of Portulaca oleracea L. (P. oleracea) were mentioned in traditional texts. In previous studies, different anti-inflammatory and anti-oxidant effects of P. oleracea were demonstrated. However, the mechanism of action and immunomodulatory property of this plant are greatly unknown. In the present study, the effect of the extract of this plant on IL-4, IL10, IFN-γ and T helper (h)1/Th2 balance in non-stimulated and stimulated human lymphocytes was examined. The effect of three concentrations (160, 40 and 10µg/ml) of P. oleracea or dexamethasone were evaluated on percentage of cell proliferation and nitric oxide (NO) production as well as secretion of cytokines (IL-4, IL10 and IFN-γ) in PHA-stimulated and non-stimulated lymphocytes, and compared to control and dexamethasone as positive control (n=15 for each group). In stimulated cells, dexamethasone significantly inhibited the percentage of cell proliferation, NO production, and secretion of cytokines in comparison to control group (P<0.001 for all cases). The percentage of cell proliferation, NO production, and secretion of cytokines were significantly decreased while Th1/Th2 (IFN-γ/IL-4) and Treg/Th2 (IL-10/IL-4) balances significantly enhanced in treated groups with all three concentrations of extract compared to control group (P<0.001 for all cases). The effect of all concentrations of the extract on cell proliferation, NO production and secretion of cytokines as well as Treg/Th2 balance were significantly lower than dexamethasone (P<0.001 for all cases), but Th1/Th2 ratio obtained in the presence of only low extract concentration was lower than dexamethasone (P<0.01). Different concentrations of extract promoted Th1/Th2 and Treg/Th2 balances which may suggest the therapeutic value of the plant in inflammatory disease associated with decreased Th1/Th2 balance such as asthma or cancers. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Mechanical Stimulation Protocols of Human Derived Cells in Articular Cartilage Tissue Engineering - A Systematic Review.

PubMed

Khozoee, Baktash; Mafi, Pouya; Mafi, Reza; Khan, Wasim S

2017-01-01

Mechanical stimulation is a key factor in articular cartilage generation and maintenance. Bioreactor systems have been designed and built in order to deliver specific types of mechanical stimulation. The focus has been twofold, applying a type of preconditioning in order to stimulate cell differentiation, and to simulate in vivo conditions in order to gain further insight into how cells respond to different stimulatory patterns. Due to the complex forces at work within joints, it is difficult to simulate mechanical conditions using a bioreactor. The aim of this review is to gain a deeper understanding of the complexities of mechanical stimulation protocols by comparing those employed in bioreactors in the context of tissue engineering for articular cartilage, and to consider their effects on cultured cells. Allied and Complementary Medicine 1985 to 2016, Ovid MEDLINE[R] 1946 to 2016, and Embase 1974 to 2016 were searched using key terms. Results were subject to inclusion and exclusion criteria, key findings summarised into a table and subsequently discussed. Based on this review it is overwhelmingly clear that mechanical stimulation leads to increased chondrogenic properties in the context of bioreactor articular cartilage tissue engineering using human cells. However, given the variability and lack of controlled factors between research articles, results are difficult to compare, and a standardised method of evaluating stimulation protocols proved challenging. With improved standardisation in mechanical stimulation protocol reporting, bioreactor design and building processes, along with a better understanding of joint behaviours, we hope to perform a meta-analysis on stimulation protocols and methods. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Expanding and characterizing esophageal epithelial cells obtained from children with eosinophilic esophagitis.

PubMed

Sayej, Wael N; Foster, Christopher; Jensen, Todd; Chatfield, Sydney; Finck, Christine

2018-06-12

The role of epithelial cells in eosinophilic esophagitis (EoE) is not well understood. In this study, our aim was to isolate, culture, and expand esophageal epithelial cells obtained from patients with or without EoE and characterize differences observed over time in culture. Biopsies were obtained at the time of endoscopy from children with EoE or suspected to have EoE. We established patient-derived esophageal epithelial cell (PDEEC) lines utilizing conditional reprogramming methods. We determined integrin profiles, gene expression, MHC class II expression, and reactivity to antigen stimulation. The PDEECs were found to maintain their phenotype over several passages. There were differences in integrin profiles and gene expression levels in EoE-Active compared to normal controls and EoE-Remission patients. Once stimulated with antigens, PDEECs express MHC class II molecules on their surface, and when co-cultured with autologous T-cells, there is increased IL-6 and TNF-α secretion in EoE-Active patients vs. controls. We are able to isolate, culture, and expand esophageal epithelial cells from pediatric patients with and without EoE. Once stimulated with antigens, these cells express MHC class II molecules and behave as non-professional antigen-presenting cells. This method will help us in developing an ex vivo, individualized, patient-specific model for diagnostic testing for causative antigens.

Phosphatidic acid inhibits ceramide 1-phosphate-stimulated macrophage migration.

PubMed

Ouro, Alberto; Arana, Lide; Rivera, Io-Guané; Ordoñez, Marta; Gomez-Larrauri, Ana; Presa, Natalia; Simón, Jorge; Trueba, Miguel; Gangoiti, Patricia; Bittman, Robert; Gomez-Muñoz, Antonio

2014-12-15

Ceramide 1-phosphate (C1P) was recently demonstrated to potently induce cell migration. This action could only be observed when C1P was applied exogenously to cells in culture, and was inhibited by pertussis toxin. However, the mechanisms involved in this process are poorly understood. In this work, we found that phosphatidic acid (PA), which is structurally related to C1P, displaced radiolabeled C1P from its membrane-binding site and inhibited C1P-stimulated macrophage migration. This effect was independent of the saturated fatty acid chain length or the presence of a double bond in each of the fatty acyl chains of PA. Treatment of RAW264.7 macrophages with exogenous phospholipase D (PLD), an enzyme that produces PA from membrane phospholipids, also inhibited C1P-stimulated cell migration. Likewise, PA or exogenous PLD inhibited C1P-stimulated extracellularly regulated kinases (ERK) 1 and 2 phosphorylation, leading to inhibition of cell migration. However, PA did not inhibit C1P-stimulated Akt phosphorylation. It is concluded that PA is a physiological regulator of C1P-stimulated macrophage migration. These actions of PA may have important implications in the control of pathophysiological functions that are regulated by C1P, including inflammation and various cellular processes associated with cell migration such as organogenesis or tumor metastasis. Copyright © 2014 Elsevier Inc. All rights reserved.

[Influence of macrophages on some biological features of endothelial cells].

PubMed

Liu, Liang; Wang, Ying; Ziiang, Xiao-Qi; Liu, Xu-Sheng

2008-02-01

To establish the co-culture model of human macrophage cell line (U937) with human vein umbilical cell line (ECV304), and to explore the feasibility of using concanavalin A (ConA) as U937 cell stimulator in regulating angiogenesis. ECV304 cells were cultured in vitro, and to which were respectively added U937 cells (1 x 10(5)), 25 microg/mL ConA, and U937 cell (1 x 10(5)) + ConA (25 microg/mL) after cell fusion rate reaching 60%, and then co-cultured for 48 hours. ECV 304 cells in conventional culture were used as controls. 3H-TdR incorporation test was employed to determine the DNA synthesis of vascular endothelial cells. Flow cytometry was used to determine the changes in the cell cycle, and RT-PCR was adopted to determine the expression of homeobox (HOXB2) mRNA. After conA stimulation to ECV 304 co-cultured with U937 cells, the percentage of cells in S phase (48.860 +/- 2.290), the DNA synthesis [(5694 +/- 917) min(-1)], and the expression of HOXB2 mRNA (0.947 +/- 0.003) were obviously higher than those in control group [41.590 +/- 2.590 vs (2498 +/- 1109) min(-1) vs 0.646 +/- 0.004, P > 0.01]. There was no obvious difference in apoptosis among above stimulation methods (P >0.05). U937 cells activated by ConA can promote the proliferation of ECV304 cells and further regulate angiogenesis. HOXB2 gene is closely related to the endothelial proliferation.

Cell stimulus and lysis in a microfluidic device with segmented gas-liquid flow.

PubMed

El-Ali, Jamil; Gaudet, Suzanne; Günther, Axel; Sorger, Peter K; Jensen, Klavs F

2005-06-01

We describe a microfluidic device with rapid stimulus and lysis of mammalian cells for resolving fast transient responses in cell signaling networks. The device uses segmented gas-liquid flow to enhance mixing and has integrated thermoelectric heaters and coolers to control the temperature during cell stimulus and lysis. Potential negative effects of segmented flow on cell responses are investigated in three different cell types, with no morphological changes and no activation of the cell stress-sensitive mitogen activated protein kinases observed. Jurkat E6-1 cells are stimulated in the device using alpha-CD3, and the resulting activations of ERK and JNK are presented for different time points. Stimulation of cells performed on chip results in pathway activation identical to that of conventionally treated cells under the same conditions.

Interferon-gamma and T-bet expression in a patient with toxoplasmic lymphadenopathy.

PubMed

Jöhrens, Korinna; Moos, Verena; Schneider, Thomas; Stein, Harald; Anagnostopoulos, Ioannis

2010-04-01

Infection with Toxoplasma gondii (TG) presents in some individuals as a self-limited disease with a predominant lymphadenopathy characterized by prominent B-cell activation. As this is in contrast to the in vitro based concept of a T(h)1-immune response against TG, we investigated native lymphoid tissue and peripheral blood of a patient with serologic evidence of toxoplasmosis to verify which cells show T(h)1-response features. High-level expression of T-bet in monocytoid B-cells, in germinal center B-cells, and in a lesser amount in T cells could be demonstrated by immunohistochemistry. In vitro stimulation of lymph node cells with either TG, staphylococcus enterotoxin B, or phorbol 12-myristate 13-acetate/ionomycin revealed an interferon-gamma expression in T-bet(+) B cells only in the patient and not in controls. Similar results were found for T-bet(+) T cells which were also present in controls. CD4(+) peripheral blood cells stimulated with TG antigens showed a TG-specific but attenuated T(h)1-reactivity in the patient associated with a reduced expression of IL-2 when compared with controls. We conclude that the pathogenesis and course of toxoplasmic lymphadenopathy is based on a T(h)1-cell defect, which becomes compensated by the B cells mounting a T(h)1-like immune response.

Applied electric field enhances DRG neurite growth: influence of stimulation media, surface coating and growth supplements

NASA Astrophysics Data System (ADS)

Wood, Matthew D.; Willits, Rebecca Kuntz

2009-08-01

Electrical therapies have been found to aid repair of nerve injuries and have been shown to increase and direct neurite outgrowth during stimulation. This enhanced neural growth existed even after the electric field (EF) or stimulation was removed, but the factors that may influence the enhanced growth, such as stimulation media or surface coating, have not been fully investigated. This study characterized neurite outgrowth and branching under various conditions: EF magnitude and application time, ECM surface coating, medium during EF application and growth supplements. A uniform, low-magnitude EF (24 or 44 V m-1) was applied to dissociated chick embryo dorsal root ganglia seeded on collagen or laminin-coated surfaces. During the growth period, cells were either exposed to NGF or N2, and during stimulation cells were exposed to either unsupplemented media (Ca2+) or PBS (no Ca2+). Parallel controls for each experiment included cells exposed to the chamber with no stimulation and cells remaining outside the chamber. After brief electrical stimulation (10 min), neurite length significantly increased 24 h after application for all conditions studied. Of particular interest, increased stimulation time (10-100 min) further enhanced neurite length on laminin but not on collagen surfaces. Neurite branching was not affected by stimulation on any surface, and no preferential growth of neurites was noted after stimulation. Overall, the results of this report suggest that short-duration electric stimulation is sufficient to enhance neurite length under a variety of conditions. While further data are needed to fully elucidate a mechanism for this increased growth, these data suggest that one focus of those investigations should be the interaction between the growth cone and the substrata.

Role of Akt/PKB and PFKFB isoenzymes in the control of glycolysis, cell proliferation and protein synthesis in mitogen-stimulated thymocytes.

PubMed

Houddane, Amina; Bultot, Laurent; Novellasdemunt, Laura; Johanns, Manuel; Gueuning, Marie-Agnès; Vertommen, Didier; Coulie, Pierre G; Bartrons, Ramon; Hue, Louis; Rider, Mark H

2017-06-01

Proliferating cells depend on glycolysis mainly to supply precursors for macromolecular synthesis. Fructose 2,6-bisphosphate (Fru-2,6-P 2 ) is the most potent positive allosteric effector of 6-phosphofructo-1-kinase (PFK-1), and hence of glycolysis. Mitogen stimulation of rat thymocytes with concanavalin A (ConA) led to time-dependent increases in lactate accumulation (6-fold), Fru-2,6-P 2 content (4-fold), 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase isoenzyme 3 and 4 (PFKFB3 and PFKFB4) protein levels (~2-fold and ~15-fold, respectively) and rates of cell proliferation (~40-fold) and protein synthesis (10-fold) after 68h of incubation compared with resting cells. After 54h of ConA stimulation, PFKFB3 mRNA levels were 45-fold higher than those of PFKFB4 mRNA. Although PFKFB3 could be phosphorylated at Ser461 by protein kinase B (PKB) in vitro leading to PFK-2 activation, PFKFB3 Ser461 phosphorylation was barely detectable in resting cells and only increased slightly in ConA-stimulated cells. On the other hand, PFKFB3 and PFKFB4 mRNA levels were decreased (90% and 70%, respectively) by exposure of ConA-stimulated cells to low doses of PKB inhibitor (MK-2206), suggesting control of expression of the two PFKFB isoenzymes by PKB. Incubation of thymocytes with ConA resulted in increased expression and phosphorylation of the translation factors eukaryotic initiation factor-4E-binding protein-1 (4E-BP1) and ribosomal protein S6 (rpS6). Treatment of ConA-stimulated thymocytes with PFK-2 inhibitor (3PO) or MK-2206 led to significant decreases in Fru-2,6-P 2 content, medium lactate accumulation and rates of cell proliferation and protein synthesis. These data were confirmed by using siRNA knockdown of PFKFB3, PFKFB4 and PKB α/β in the more easily transfectable Jurkat E6-1 cell line. The findings suggest that increased PFKFB3 and PFKFB4 expression, but not increased PFKFB3 Ser461 phosphorylation, plays a role in increasing glycolysis in mitogen-stimulated thymocytes and implicate PKB in the upregulation of PFKFB3 and PFKFB4. The results also support a role for Fru-2,6-P 2 in coupling glycolysis to cell proliferation and protein synthesis in this model. Copyright © 2017 Elsevier Inc. All rights reserved.

Protective Capacity of Memory CD8+ T Cells is Dictated by Antigen Exposure History and Nature of the Infection

PubMed Central

Nolz, Jeffrey C.; Harty, John T.

2011-01-01

SUMMARY Infection or vaccination confers heightened resistance to pathogen re-challenge due to quantitative and qualitative differences between naïve and primary memory T cells. Herein, we show that secondary (boosted) memory CD8+ T cells were better than primary memory CD8+ T cells in controlling some, but not all acute infections with diverse pathogens. However, secondary memory CD8+ T cells were less efficient than an equal number of primary memory cells at preventing chronic LCMV infection and are more susceptible to functional exhaustion. Importantly, localization of memory CD8+ T cells within lymph nodes, which is reduced by antigen re-stimulation, was critical for both viral control in lymph nodes and for the sustained CD8+ T cell response required to prevent chronic LCMV infection. Thus, repeated antigen-stimulation shapes memory CD8+ T cell populations to either enhance or decrease per cell protective immunity in a pathogen-specific manner, a concept of importance in vaccine design against specific diseases. PMID:21549619

CHEMOKINE RECEPTOR 7 (CCR7)-EXPRESSION AND IFNγ PRODUCTION DEFINE VACCINE-SPECIFIC CANINE T CELL SUBSETS

PubMed Central

Hartley, Ashley N.; Tarleton, Rick L.

2015-01-01

Canines suffer from and serve as strong translational animals models for many immunological disorders and infectious diseases. Routine vaccination has been a mainstay of protecting dogs through the stimulation of robust antibody responses and expansion of memory T cell populations. Commercially available reagents and described techniques are limited for identifying and characterizing canine T cell subsets and evaluating T cell-specific effector function. To define reagents for delineating naïve versus activated T cells and identify antigen-specific T cells, we tested anti-human and anti-bovine T-cell specific cell surface marker reagents for cross-reactivity with canine peripheral blood mononuclear cells (PBMCs. Both CD4+ and CD8+ T cells from healthy canine donors showed reactivity to CCL19-Ig, a CCR7 ligand, and coexpression with CD62L. An in vitro stimulation with concanavalin A validated downregulation of CCR7 and CD62L expression on stimulated healthy control PBMCs, consistent with an activated T cell phenotype. Anti-IFNγ antibodies identified antigen-specific IFNγ-producing CD4+ and CD8+ T cells upon in vitro vaccine antigen PBMC stimulation. PBMC isolation within 24 hours of sample collection allowed for efficient cell recovery and accurate T cell effector function characterization. These data provide a reagent and techniques platform via flow cytometry for identifying canine T cell subsets and characterizing circulating antigen-specific canine T cells for potential use in diagnostic and field settings. PMID:25758065

Zebrafish heart as a model to study the integrative autonomic control of pacemaker function

PubMed Central

Stoyek, Matthew R.; Quinn, T. Alexander; Croll, Roger P.

2016-01-01

The cardiac pacemaker sets the heart's primary rate, with pacemaker discharge controlled by the autonomic nervous system through intracardiac ganglia. A fundamental issue in understanding the relationship between neural activity and cardiac chronotropy is the identification of neuronal populations that control pacemaker cells. To date, most studies of neurocardiac control have been done in mammalian species, where neurons are embedded in and distributed throughout the heart, so they are largely inaccessible for whole-organ, integrative studies. Here, we establish the isolated, innervated zebrafish heart as a novel alternative model for studies of autonomic control of heart rate. Stimulation of individual cardiac vagosympathetic nerve trunks evoked bradycardia (parasympathetic activation) and tachycardia (sympathetic activation). Simultaneous stimulation of both vagosympathetic nerve trunks evoked a summative effect. Effects of nerve stimulation were mimicked by direct application of cholinergic and adrenergic agents. Optical mapping of electrical activity confirmed the sinoatrial region as the site of origin of normal pacemaker activity and identified a secondary pacemaker in the atrioventricular region. Strong vagosympathetic nerve stimulation resulted in a shift in the origin of initial excitation from the sinoatrial pacemaker to the atrioventricular pacemaker. Putative pacemaker cells in the sinoatrial and atrioventricular regions expressed adrenergic β2 and cholinergic muscarinic type 2 receptors. Collectively, we have demonstrated that the zebrafish heart contains the accepted hallmarks of vertebrate cardiac control, establishing this preparation as a viable model for studies of integrative physiological control of cardiac function by intracardiac neurons. PMID:27342878

Stimulation of Chronic Lymphocytic Leukemia (CLL) Cells with CpG Oligodeoxynucleotide (ODN) Gives Consistent Karyotypic Results among Laboratories: a CLL Research Consortium (CRC)h Study

PubMed Central

Heerema, Nyla A.; Byrd, John C.; Cin, Paola Dal; Dell’ Aquila, Marie L.; Koduru, Prasad; Aviram, Ayala; Smoley, Stephanie; Rassenti, Laura Z.; Greaves, Andrew W.; Brown, Jennifer R.; Rai, Kanti R.; Kipps, Thomas J.; Kay, Neil E.; van Dyke, Daniel

2010-01-01

Cytogenetic abnormalities in CLL are important prognostic indicators. Historically, only interphase cytogenetics was clinically useful in CLL because traditional mitogens are not effective mitotic stimulants. Recently, CpG-oligodeoxynucleotide (ODN) stimulation has shown effectiveness in CLL. The CLL Research Consortium (CRC) tested the effectiveness and reproducibility of CpG-ODN stimulation to detect chromosomally abnormal clones by five laboratories. More clonal abnormalities were observed after culture of CLL cells with CpG-ODN than with pokeweed mitogen (PWM)+12-O-tetradecanoyl-phorobol-13-acetate (TPA). All clonal abnormalities in PWM+TPA cultures were observed in CpG-ODN cultures, whereas CpG-ODN identified some clones not found by PWM+TPA. CpG-ODN stimulation of one normal control and 12 CLL samples showed that excepting clones of del(13q) in low frequencies and one translocation, results in all five laboratories were consistent, and all abnormalities were concordant with FISH. Thus, abnormal clones in CLL are more readily detected with CpG-ODN stimulation than with traditional B-cell mitogens. After CpG-ODN stimulation, abnormalities were reproducible among cytogenetic laboratories. CpG-ODN did not appear to induce aberrations in cell culture and enhanced detection of abnormalities and complexity in CLL. Since karyotypic complexity is prognostic and is not detectable by standard FISH analyses, stimulation with CpG-ODN is useful to identify this additional prognostic factor in CLL. PMID:21156225

An emerging role for gasotransmitters in the control of breathing and ionic regulation in fish.

PubMed

Perry, Steve; Kumai, Y; Porteus, C S; Tzaneva, V; Kwong, R W M

2016-02-01

Three gases comprising nitric oxide, carbon monoxide and hydrogen sulphide, collectively are termed gasotransmitters. The gasotransmitters control several physiological functions in fish by acting as intracellular signaling molecules. Hydrogen sulphide, first implicated in vasomotor control in fish, plays a critical role in oxygen chemoreception owing to its production and downstream effects within the oxygen chemosensory cells, the neuroepithelial cells. Indeed, there is emerging evidence that hydrogen sulphide may contribute to oxygen sensing in both fish and mammals by promoting membrane depolarization of the chemosensory cells. Unlike hydrogen sulphide which stimulates breathing in zebrafish, carbon monoxide inhibits ventilation in goldfish and zebrafish whereas nitric oxide stimulates breathing in zebrafish larvae while inhibiting breathing in adults. Gasotransmitters also modulate ionic uptake in zebrafish. Though nothing is known about the role of CO, reduced activities of branchial Na(+)/K(+)-ATPase and H(+)-ATPase activities in the presence of NO donors suggest an inhibitory role of NO in fish osmoregulation. Hydrogen sulphide inhibits Na(+) uptake in zebrafish larvae and contributes to lowering Na(+) uptake capacity in fish acclimated to Na(+)-enriched water whereas it stimulates Ca(2+) uptake in larvae exposed to Ca(2+)-poor water.

Resveratrol Increases Osteoblast Differentiation In Vitro Independently of Inflammation.

PubMed

Ornstrup, Marie Juul; Harsløf, Torben; Sørensen, Lotte; Stenkjær, Liselotte; Langdahl, Bente Lomholt; Pedersen, Steen Bønløkke

2016-08-01

Low-grade inflammation negatively affects bone. Resveratrol is a natural compound proven to possess both anti-inflammatory and bone protective properties. However, it is uncertain if the bone effects are mediated though anti-inflammatory effects. Firstly, we investigated if resveratrol affects proliferation and differentiation of human bone marrow-derived mesenchymal stem cells. Secondly, we investigated if inflammation negatively affects proliferation and differentiation, and if resveratrol counteracts this through anti-inflammatory effects. Mesenchymal stem cells were obtained from bone marrow aspiration in 13 healthy individuals and cultured towards the osteoblast cell lineage. The cells were stimulated with resveratrol, lipopolysaccharide (LPS), LPS + resveratrol, or vehicle (control) for 21 days. Compared to control, resveratrol decreased cell number by 35 % (p < 0.05) and induced differentiation (a 3-fold increase in alkaline phosphatase (p < 0.002), while P1NP and OPG showed similar trends). LPS induced inflammation with a 44-fold increase in interleukin-6 (p < 0.05) and an extremely prominent increase in interleukin-8 production (p < 0.05) relative to control. In addition, LPS increased cell count (p < 0.05) and decreased differentiation (a reduction in P1NP production (p < 0.02)). Co-stimulation with LPS + resveratrol did not reduce interleukin-6 or interleukin-8, but nonetheless, cell count was reduced (p < 0.05) and alkaline phosphatase, P1NP, and OPG increased (p < 0.05 for all). Thus, resveratrol stimulates osteoblast differentiation independently of inflammation.

Spiral ganglion cell site of excitation I: comparison of scala tympani and intrameatal electrode responses.

PubMed

Cartee, Lianne A; Miller, Charles A; van den Honert, Chris

2006-05-01

To determine the site of excitation on the spiral ganglion cell in response to electrical stimulation similar to that from a cochlear implant, single-fiber responses to electrical stimuli delivered by an electrode positioned in the scala tympani were compared to responses from stimuli delivered by an electrode placed in the internal auditory meatus. The response to intrameatal stimulation provided a control set of data with a known excitation site, the central axon of the spiral ganglion cell. For both intrameatal and scala tympani stimuli, the responses to single-pulse, summation, and refractory stimulus protocols were recorded. The data demonstrated that summation pulses, as opposed to single pulses, are likely to give the most insightful measures for determination of the site of excitation. Single-fiber summation data for both scala tympani and intrameatally stimulated fibers were analyzed with a clustering algorithm. Combining cluster analysis and additional numerical modeling data, it was hypothesized that the scala tympani responses corresponded to central excitation, peripheral excitation adjacent to the cell body, and peripheral excitation at a site distant from the cell body. Fibers stimulated by an intrameatal electrode demonstrated the greatest range of jitter measurements indicating that greater fiber independence may be achieved with intrameatal stimulation.

African swine fever virus IAP-like protein induces the activation of nuclear factor kappa B.

PubMed

Rodríguez, Clara I; Nogal, María L; Carrascosa, Angel L; Salas, María L; Fresno, Manuel; Revilla, Yolanda

2002-04-01

African swine fever virus (ASFV) encodes a homologue of the inhibitor of apoptosis (IAP) that promotes cell survival by controlling the activity of caspase-3. Here we show that ASFV IAP is also able to activate the transcription factor NF-kappaB. Thus, transient transfection of the viral IAP increases the activity of an NF-kappaB reporter gene in a dose-responsive manner in Jurkat cells. Similarly, stably transfected cells expressing ASFV IAP have elevated basal levels of c-rel, an NF-kappaB-dependent gene. NF-kappaB complexes in the nucleus were increased in A224L-expressing cells compared with control cells upon stimulation with phorbol myristate acetate (PMA) plus ionomycin. This resulted in greater NF-kappaB-dependent promoter activity in ASFV IAP-expressing than in control cells, both in basal conditions and after PMA plus ionophore stimulation. The elevated NF-kappaB activity seems to be the consequence of higher IkappaB kinase (IKK) basal activity in these cells. The NF-kappaB-inducing activity of ASFV IAP was abrogated by an IKK-2 dominant negative mutant and enhanced by expression of tumor necrosis factor receptor-associated factor 2.

African Swine Fever Virus IAP-Like Protein Induces the Activation of Nuclear Factor Kappa B

PubMed Central

Rodríguez, Clara I.; Nogal, María L.; Carrascosa, Angel L.; Salas, María L.; Fresno, Manuel; Revilla, Yolanda

2002-01-01

African swine fever virus (ASFV) encodes a homologue of the inhibitor of apoptosis (IAP) that promotes cell survival by controlling the activity of caspase-3. Here we show that ASFV IAP is also able to activate the transcription factor NF-κB. Thus, transient transfection of the viral IAP increases the activity of an NF-κB reporter gene in a dose-responsive manner in Jurkat cells. Similarly, stably transfected cells expressing ASFV IAP have elevated basal levels of c-rel, an NF-κB-dependent gene. NF-κB complexes in the nucleus were increased in A224L-expressing cells compared with control cells upon stimulation with phorbol myristate acetate (PMA) plus ionomycin. This resulted in greater NF-κB-dependent promoter activity in ASFV IAP-expressing than in control cells, both in basal conditions and after PMA plus ionophore stimulation. The elevated NF-κB activity seems to be the consequence of higher IκB kinase (IKK) basal activity in these cells. The NF-κB-inducing activity of ASFV IAP was abrogated by an IKK-2 dominant negative mutant and enhanced by expression of tumor necrosis factor receptor-associated factor 2. PMID:11907233

IL-4 Modulates CCL11 and CCL20 Productions from IL-1β-Stimulated Human Periodontal Ligament Cells.

PubMed

Hosokawa, Yoshitaka; Hosokawa, Ikuko; Shindo, Satoru; Ozaki, Kazumi; Matsuo, Takashi

2016-01-01

IL-4 is a multifunctional cytokine that is related with the pathological conditions of periodontal disease. However, it is uncertain whether IL-4 could control T cells migration in periodontal lesions. The aim of this study was to examine the effects of IL-4 on CCL11, which is a Th2-type chemokine, and CCL20, which is related with Th17 cells migration, productions from human periodontal ligament cells (HPDLCs). CCL20 and CCL11 productions from HPDLCs were monitored by ELISA. Western blot analysis was performed to detect phosphorylations of signal transduction molecules in HPDLCs. IL-1β could induce both CCL11 and CCL20 productions in HPDLCs. IL-4 enhanced CCL11 productions from IL-1β-stimulated HPDLCs, though IL-4 inhibited CCL20 production. Western blot analysis showed that protein kinase B (Akt) and signal transducer and activator of transcription (STAT)6 pathways were highly activated in IL-4/IL-1β-stimulated HPDLCs. Akt and STAT6 inhibitors decreased CCL11 production, but enhanced CCL20 production in HPDLCs stimulated with IL-4 and IL-1β. These results mean that IL-4 enhanced Th2 cells migration in periodontal lesion to induce CCL11 production from HPDLCs. On the other hand, IL-4 inhibits Th17 cells accumulation in periodontally diseased tissues to inhibit CCL20 production. Therefore, IL-4 is positively related with the pathogenesis of periodontal disease to control chemokine productions in periodontal lesions. © 2016 The Author(s) Published by S. Karger AG, Basel.

Reduced response to Epstein–Barr virus antigens by T-cells in systemic lupus erythematosus patients

PubMed Central

Draborg, Anette Holck; Jacobsen, Søren; Westergaard, Marie; Mortensen, Shila; Larsen, Janni Lisander; Houen, Gunnar; Duus, Karen

2014-01-01

Objective Epstein–Barr virus (EBV) has for long been associated with systemic lupus erythematosus (SLE). In this study, we investigated the levels of latent and lytic antigen EBV-specific T-cells and antibodies in SLE patients. Methods T cells were analyzed by flow cytometry and antibodies were analyzed by enzyme-linked immunosorbent assay. Results SLE patients showed a significantly reduced number of activated (CD69) T-cells upon ex vivo stimulation with EBV nuclear antigen (EBNA) 1 or EBV early antigen diffuse (EBV-EA/D) in whole blood samples compared with healthy controls. Also, a reduced number of T-cells from SLE patients were found to produce interferon-γ upon stimulation with these antigens. Importantly, responses to a superantigen were normal in SLE patients. Compared with healthy controls, SLE patients had fewer EBV-specific T-cells but higher titres of antibodies against EBV. Furthermore, an inverse correlation was revealed between the number of lytic antigen EBV-specific T-cells and disease activity of the SLE patients, with high-activity SLE patients having fewer T-cells than low-activity SLE patients. Conclusions These results indicate a limited or a defective EBV-specific T-cell response in SLE patients, which may suggest poor control of EBV infection in SLE with an immune reaction shift towards a humoral response in an attempt to control viral reactivation. A role for decreased control of EBV as a contributing agent in the development or exacerbation of SLE is proposed. PMID:25396062

Stimulation of osteogenic differentiation in human osteoprogenitor cells by pulsed electromagnetic fields: an in vitro study

PubMed Central

2010-01-01

Background Although pulsed electromagnetic field (PEMF) stimulation may be clinically beneficial during fracture healing and for a wide range of bone disorders, there is still debate on its working mechanism. Mesenchymal stem cells are likely mediators facilitating the observed clinical effects of PEMF. Here, we performed in vitro experiments to investigate the effect of PEMF stimulation on human bone marrow-derived stromal cell (BMSC) metabolism and, specifically, whether PEMF can stimulate their osteogenic differentiation. Methods BMSCs derived from four different donors were cultured in osteogenic medium, with the PEMF treated group being continuously exposed to a 15 Hz, 1 Gauss EM field, consisting of 5-millisecond bursts with 5-microsecond pulses. On culture day 1, 5, 9, and 14, cells were collected for biochemical analysis (DNA amount, alkaline phosphatase activity, calcium deposition), expression of various osteoblast-relevant genes and activation of extracellular signal-regulated kinase (ERK) signaling. Differences between treated and control groups were analyzed using the Wilcoxon signed rank test, and considered significant when p < 0.05. Results Biochemical analysis revealed significant, differentiation stage-dependent, PEMF-induced differences: PEMF increased mineralization at day 9 and 14, without altering alkaline phosphatase activity. Cell proliferation, as measured by DNA amounts, was not affected by PEMF until day 14. Here, DNA content stagnated in PEMF treated group, resulting in less DNA compared to control. Quantitative RT-PCR revealed that during early culture, up to day 9, PEMF treatment increased mRNA levels of bone morphogenetic protein 2, transforming growth factor-beta 1, osteoprotegerin, matrix metalloproteinase-1 and -3, osteocalcin, and bone sialoprotein. In contrast, receptor activator of NF-κB ligand expression was primarily stimulated on day 14. ERK1/2 phosphorylation was not affected by PEMF stimulation. Conclusions PEMF exposure of differentiating human BMSCs enhanced mineralization and seemed to induce differentiation at the expense of proliferation. The osteogenic stimulus of PEMF was confirmed by the up-regulation of several osteogenic marker genes in the PEMF treated group, which preceded the deposition of mineral itself. These findings indicate that PEMF can directly stimulate osteoprogenitor cells towards osteogenic differentiation. This supports the theory that PEMF treatment may recruit these cells to facilitate an osteogenic response in vivo. PMID:20731873

The median preoptic nucleus reciprocally modulates activity of arousal-related and sleep-related neurons in the perifornical lateral hypothalamus.

PubMed

Suntsova, Natalia; Guzman-Marin, Ruben; Kumar, Sunil; Alam, Md Noor; Szymusiak, Ronald; McGinty, Dennis

2007-02-14

The perifornical-lateral hypothalamic area (PF/LH) contains neuronal groups playing an important role in control of waking and sleep. Among the brain regions that regulate behavioral states, one of the strongest sources of projections to the PF/LH is the median preoptic nucleus (MnPN) containing a sleep-active neuronal population. To evaluate the role of MnPN afferents in the control of PF/LH neuronal activity, we studied the responses of PF/LH cells to electrical stimulation or local chemical manipulation of the MnPN in freely moving rats. Single-pulse electrical stimulation evoked responses in 79% of recorded PF/LH neurons. No cells were activated antidromically. Direct and indirect transsynaptic effects depended on sleep-wake discharge pattern of PF/LH cells. The majority of arousal-related neurons, that is, cells discharging at maximal rates during active waking (AW) or during AW and rapid eye movement (REM) sleep, exhibited exclusively or initially inhibitory responses to stimulation. Sleep-related neurons, the cells with elevated discharge during non-REM and REM sleep or selectively active in REM sleep, exhibited exclusively or initially excitatory responses. Activation of the MnPN via microdialytic application of L-glutamate or bicuculline resulted in reduced discharge of arousal-related and in excitation of sleep-related PF/LH neurons. Deactivation of the MnPN with muscimol caused opposite effects. The results indicate that the MnPN contains subset(s) of neurons, which exert inhibitory control over arousal-related and excitatory control over sleep-related PF/LH neurons. We hypothesize that MnPN sleep-active neuronal group has both inhibitory and excitatory outputs that participate in the inhibitory control of arousal-promoting PF/LH mechanisms.

IFN-α and CD46 stimulation are associated with active lupus and skew natural T regulatory cell differentiation to type 1 regulatory T (Tr1) cells

PubMed Central

Le Buanec, Hélène; Gougeon, Marie-Lise; Mathian, Alexis; Lebon, Pierre; Dupont, Jean-Michel; Peltre, Gabriel; Hemon, Patrice; Schmid, Michel; Bizzini, Bernard; Künding, Thomas; Burny, Arsène; Bensussan, Armand; Amoura, Zahir; Gallo, Robert C.; Zagury, Daniel

2011-01-01

Immune suppressive activities exerted by regulatory T-cell subsets have several specific functions, including self-tolerance and regulation of adaptive immune reactions, and their dysfunction can lead to autoimmune diseases and contribute to AIDS and cancer. Two functionally distinct regulatory T-cell subsets are currently identified in peripheral tissues: thymus-developed natural T regulatory cells (nTregs) controlling self-tolerance and antiinflammatory IL-10–secreting type 1 regulatory T cells (Tr1) derived from Ag-stimulated T cells, which regulate inflammation-dependent adaptive immunity and minimize immunopathology. We establish herein that cell contact-mediated nTreg regulatory function is inhibited by inflammation, especially in the presence of the complement C3b receptor (CD46). Instead, as with other T-cell subsets, the latter inflammatory conditions of stimulation skew nTreg differentiation to Tr1 cells secreting IL-10, an effect potentiated by IFN-α. The clinical relevance of these findings was verified in a study of 152 lupus patients, in which we showed that lupus nTreg dysfunction is not due to intrinsic defects but is rather induced by C3b stimulation of CD46 and IFN-α and that these immune components of inflammation are directly associated with active lupus. These results provide a rationale for using anti–IFN-α Ab immunotherapy in lupus patients. PMID:22065791

Impairment of the humoral and CD4+ T cell responses in HTLV-1-infected individuals immunized with tetanus toxoid

PubMed Central

Souza, Anselmo; Santos, Silvane; Carvalho, Lucas P.; Grassi, Maria Fernanda R.; Carvalho, Edgar M.

2016-01-01

T cells from HTLV-1-infected individuals have a decreased ability to proliferate after stimulation with recall antigens. This abnormality may be due to the production of regulatory cytokine or a dysfunctional antigen presentation. The aims of this study were to evaluate the antibody production and cytokine expression by lymphocytes before and after immunization with tetanus toxoid (TT) and to evaluate the immune response of monocytes after stimulation with TT and frequency of dendritic cells (DC) subsets. HTLV-1 carriers (HC) and uninfected controls with negative serology for TT were immunized with TT, and the antibody titers were determined by ELISA as well as the cell activation markers expression by monocytes. The frequencies of DC subsets were determined by flow cytometry. Following immunization, the IgG anti-TT titers and the frequency of CD4+ T cells expressing IFN-γ, TNF and IL-10 in response to TT were lower in the (HC) than in the controls. Additionally, monocytes from HC did not exhibit increased HLA-DR expression after stimulation with TT, and presented low numbers of DC subsets, therefore, it’s necessary to perform functional studies with antigen-presenting cells. Collectively, our finding suggests that HC present an impairment of the humoral and CD4+ T cell immune responses after vaccination. PMID:27282836

Non-Faradaic Electrochemical Detection of Exocytosis from Mast and Chromaffin Cells Using Floating-Gate MOS Transistors.

PubMed

Jayant, Krishna; Singhai, Amit; Cao, Yingqiu; Phelps, Joshua B; Lindau, Manfred; Holowka, David A; Baird, Barbara A; Kan, Edwin C

2015-12-21

We present non-faradaic electrochemical recordings of exocytosis from populations of mast and chromaffin cells using chemoreceptive neuron MOS (CνMOS) transistors. In comparison to previous cell-FET-biosensors, the CνMOS features control (CG), sensing (SG) and floating gates (FG), allows the quiescent point to be independently controlled, is CMOS compatible and physically isolates the transistor channel from the electrolyte for stable long-term recordings. We measured exocytosis from RBL-2H3 mast cells sensitized by IgE (bound to high-affinity surface receptors FcεRI) and stimulated using the antigen DNP-BSA. Quasi-static I-V measurements reflected a slow shift in surface potential () which was dependent on extracellular calcium ([Ca]o) and buffer strength, which suggests sensitivity to protons released during exocytosis. Fluorescent imaging of dextran-labeled vesicle release showed evidence of a similar time course, while un-sensitized cells showed no response to stimulation. Transient recordings revealed fluctuations with a rapid rise and slow decay. Chromaffin cells stimulated with high KCl showed both slow shifts and extracellular action potentials exhibiting biphasic and inverted capacitive waveforms, indicative of varying ion-channel distributions across the cell-transistor junction. Our approach presents a facile method to simultaneously monitor exocytosis and ion channel activity with high temporal sensitivity without the need for redox chemistry.

Non-Faradaic Electrochemical Detection of Exocytosis from Mast and Chromaffin Cells Using Floating-Gate MOS Transistors

PubMed Central

Jayant, Krishna; Singhai, Amit; Cao, Yingqiu; Phelps, Joshua B.; Lindau, Manfred; Holowka, David A.; Baird, Barbara A.; Kan, Edwin C.

2015-01-01

We present non-faradaic electrochemical recordings of exocytosis from populations of mast and chromaffin cells using chemoreceptive neuron MOS (CνMOS) transistors. In comparison to previous cell-FET-biosensors, the CνMOS features control (CG), sensing (SG) and floating gates (FG), allows the quiescent point to be independently controlled, is CMOS compatible and physically isolates the transistor channel from the electrolyte for stable long-term recordings. We measured exocytosis from RBL-2H3 mast cells sensitized by IgE (bound to high-affinity surface receptors FcεRI) and stimulated using the antigen DNP-BSA. Quasi-static I-V measurements reflected a slow shift in surface potential () which was dependent on extracellular calcium ([Ca]o) and buffer strength, which suggests sensitivity to protons released during exocytosis. Fluorescent imaging of dextran-labeled vesicle release showed evidence of a similar time course, while un-sensitized cells showed no response to stimulation. Transient recordings revealed fluctuations with a rapid rise and slow decay. Chromaffin cells stimulated with high KCl showed both slow shifts and extracellular action potentials exhibiting biphasic and inverted capacitive waveforms, indicative of varying ion-channel distributions across the cell-transistor junction. Our approach presents a facile method to simultaneously monitor exocytosis and ion channel activity with high temporal sensitivity without the need for redox chemistry. PMID:26686301

Myogenic Maturation by Optical-Training in Cultured Skeletal Muscle Cells.

PubMed

Asano, Toshifumi; Ishizuka, Toru; Yawo, Hiromu

2017-01-01

Optogenetic techniques are powerful tools for manipulating biological processes in identified cells using light under high temporal and spatial resolutions. Here, we describe an optogenetic training strategy to promote morphological maturation and functional development of skeletal muscle cells in vitro. Optical stimulation with a rhythmical frequency facilitates specific structural alignment of sarcomeric proteins. Optical stimulation also depolarizes the membrane potential, and induces contractile responses in synchrony with the given pattern of light pulses. These results suggest that optogenetic techniques can be employed to manipulate activity-dependent processes during myogenic development and control contraction of photosensitive skeletal muscle cells with high temporal and special precision.

Active skin as new haptic interface

NASA Astrophysics Data System (ADS)

Vuong, Nguyen Huu Lam; Kwon, Hyeok Yong; Chuc, Nguyen Huu; Kim, Duksang; An, Kuangjun; Phuc, Vuong Hong; Moon, Hyungpil; Koo, Jachoon; Lee, Youngkwan; Nam, Jae-Do; Choi, Hyouk Ryeol

2010-04-01

In this paper, we present a new haptic interface, called "active skin", which is configured with a tactile sensor and a tactile stimulator in single haptic cell, and multiple haptic cells are embedded in a dielectric elastomer. The active skin generates a wide variety of haptic feel in response to the touch by synchronizing the sensor and the stimulator. In this paper, the design of the haptic cell is derived via iterative analysis and design procedures. A fabrication method dedicated to the proposed device is investigated and a controller to drive multiple haptic cells is developed. In addition, several experiments are performed to evaluate the performance of the active skin.

Evidence of functional cell-mediated immune responses to nontypeable Haemophilus influenzae in otitis-prone children

PubMed Central

Seppanen, Elke; Tan, Dino; Corscadden, Karli J.; Currie, Andrew J.; Richmond, Peter C.; Thornton, Ruth B.

2018-01-01

Otitis media (OM) remains a common paediatric disease, despite advances in vaccinology. Susceptibility to recurrent acute OM (rAOM) has been postulated to involve defective cell-mediated immune responses to common otopathogenic bacteria. We compared the composition of peripheral blood mononuclear cells (PBMC) from 20 children with a history of rAOM (otitis-prone) and 20 healthy non-otitis-prone controls, and assessed innate and cell-mediated immune responses to the major otopathogen nontypeable Haemophilus influenzae (NTHi). NTHi was a potent stimulator of inflammatory cytokine secretion from PBMC within 4 hours, with no difference in cytokine levels produced between PBMC from cases or controls. In the absence of antigen stimulation, otitis-prone children had more circulating Natural Killer (NK) cells (p<0.01), particularly NKdim (CD56lo) cells (p<0.01), but fewer CD4+ T cells (p<0.01) than healthy controls. NTHi challenge significantly increased the proportion of activated (CD107a+) NK cells in otitis-prone and non-otitis-prone children (p<0.01), suggesting that NK cells from otitis-prone children are functional and respond to NTHi. CD8+ T cells and NK cells from both cases and controls produced IFNγ in response to polyclonal stimulus (Staphylococcal enterotoxin B; SEB), with more IFNγ+ CD8+ T cells present in cases than controls (p<0.05) but similar proportions of IFNγ+ NK cells. Otitis-prone children had more circulating IFNγ-producing NK cells (p<0.05) and more IFNγ-producing CD4+ (p<0.01) or CD8+ T-cells (p<0.05) than healthy controls. In response to SEB, more CD107a-expressing CD8+ T cells were present in cases than controls (p<0.01). Despite differences in PBMC composition, PBMC from otitis-prone children mounted innate and T cell-mediated responses to NTHi challenge that were comparable to healthy children. These data provide evidence that otitis-prone children do not have impaired functional cell mediated immunity. PMID:29621281

Deletion of admB gene encoding a fungal ADAM affects cell wall construction in Aspergillus oryzae.

PubMed

Kobayashi, Takuji; Maeda, Hiroshi; Takeuchi, Michio; Yamagata, Youhei

2017-05-01

Mammals possess a unique signaling system based on the proteolytic mechanism of a disintegrin and metalloproteinases (ADAMs) on the cell surface. We found two genes encoding ADAMs in Aspergillus oryzae and named them admA and admB. We produced admA and admB deletion strains to elucidate their biological function and clarify whether fungal ADAMs play a similar role as in mammals. The ∆admA∆admB and ∆admB strains were sensitive to cell wall-perturbing agents, congo red, and calcofluor white. Moreover, the two strains showed significantly increased weights of total alkali-soluble fractions from the mycelial cell wall compared to the control strain. Furthermore, ∆admB showed MpkA phosphorylation at lower concentration of congo red stimulation than the control strain. However, the MpkA phosphorylation level was not different between ∆admB and the control strain without the stimulation. The results indicated that A. oryzae AdmB involved in the cell wall integrity without going through the MpkA pathway.

HER4 selectively coregulates estrogen stimulated genes associated with breast tumor cell proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Wen; Jones, Frank E., E-mail: fjones3@tulane.edu

2014-01-10

Highlights: •HER4/4ICD is an obligate coactivator for 37% of estrogen regulated genes. •HER4/4ICD coactivated genes selectively regulate estrogen stimulated proliferation. •Estrogen stimulated tumor cell migration occurs independent of HER4/4ICD. •Disrupting HER4/4ICD and ER coactivated gene expression may suppress breast cancer. -- Abstract: The EGFR-family member HER4 undergoes regulated intramembrane proteolysis (RIP) to generate an intracellular domain (4ICD) that functions as a transcriptional coactivator. Accordingly, 4ICD coactivates the estrogen receptor (ER) and associates with ER at target gene promoters in breast tumor cells. However, the extent of 4ICD coactivation of ER and the functional significance of the 4ICD/ER transcriptional complex ismore » unclear. To identify 4ICD coactivated genes we performed a microarray gene expression analysis of β-estradiol treated cells comparing control MCF-7 breast cancer cells to MCF-7 cells where HER4 expression was stably suppressed using a shRNA. In the MCF-7 cell line, β-estradiol significantly stimulated or repressed by 2-fold or more 726 or 53 genes, respectively. Significantly, HER4/4ICD was an obligate coactivator for 277 or 38% of the β-estradiol stimulated genes. Ingenuity Pathway Analysis of β-estradiol regulated genes identified significant associations with multiple cellular functions regulating cellular growth and proliferation, cell cycle progression, cancer metastasis, decreased hypoplasia, tumor cell migration, apoptotic resistance of tumor cells, and increased transcription. Genes coactivated by 4ICD displayed functional specificity by only significantly contributing to cellular growth and proliferation, cell cycle progression, and decreased hypoplasia. In direct concordance with these in situ results we show that HER4 knockdown in MCF-7 cells results in a loss of estrogen stimulated tumor cell proliferation and cell cycle progression, whereas, estrogen stimulated tumor cell migration was unaffected by loss of HER4 expression. In summary, we demonstrate for the first time that a cell surface receptor functions as an obligate ER coactivator with functional specificity associated with breast tumor cell proliferation and cell cycle progression. Nearly 90% of ER positive tumors coexpress HER4, therefore we predict that the majority of breast cancer patients would benefit from a strategy to therapeutic disengage ER/4ICD coregulated tumor cell proliferation.« less

Mechanical stimulation of cyclic tensile strain induces reduction of pluripotent related gene expressions via activation of Rho/ROCK and subsequent decreasing of AKT phosphorylation in human induced pluripotent stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Teramura, Takeshi, E-mail: teramura@med.kindai.ac.jp; Takehara, Toshiyuki; Onodera, Yuta

2012-01-13

Highlights: Black-Right-Pointing-Pointer Mechanical stimulation is an important factor for regulation of stem cell fate. Black-Right-Pointing-Pointer Cyclic stretch to human induced pluripotent stem cells activated small GTPase Rho. Black-Right-Pointing-Pointer Rho-kinase activation attenuated pluripotency via inhibition of AKT activation. Black-Right-Pointing-Pointer This reaction could be reproduced only by transfection of dominant active Rho. Black-Right-Pointing-Pointer Rho/ROCK are important molecules in mechanotransduction and control of stemness. -- Abstract: Mechanical stimulation has been shown to regulate the proliferation and differentiation of stem cells. However, the effects of the mechanical stress on the stemness or related molecular mechanisms have not been well determined. Pluripotent stem cells suchmore » as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are used as good materials for cell transplantation therapy and research of mammalian development, since they can self-renew infinitely and differentiate into various cell lineages. Here we demonstrated that the mechanical stimulation to human iPS cells altered alignment of actin fibers and expressions of the pluripotent related genes Nanog, POU5f1 and Sox2. In the mechanically stimulated iPS cells, small GTPase Rho was activated and interestingly, AKT phosphorylation was decreased. Inhibition of Rho-associated kinase ROCK recovered the AKT phosphorylation and the gene expressions. These results clearly suggested that the Rho/ROCK is a potent primary effector of mechanical stress in the pluripotent stem cells and it participates to pluripotency-related signaling cascades as an upper stream regulator.« less

Calbindin-D(28k) controls [Ca(2+)](i) and insulin release. Evidence obtained from calbindin-d(28k) knockout mice and beta cell lines

NASA Technical Reports Server (NTRS)

Sooy, K.; Schermerhorn, T.; Noda, M.; Surana, M.; Rhoten, W. B.; Meyer, M.; Fleischer, N.; Sharp, G. W.; Christakos, S.

1999-01-01

The role of the calcium-binding protein, calbindin-D(28k) in potassium/depolarization-stimulated increases in the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and insulin release was investigated in pancreatic islets from calbindin-D(28k) nullmutant mice (knockouts; KO) or wild type mice and beta cell lines stably transfected and overexpressing calbindin. Using single islets from KO mice and stimulation with 45 mM KCl, the peak of [Ca(2+)](i) was 3.5-fold greater in islets from KO mice compared with wild type islets (p < 0.01) and [Ca(2+)](i) remained higher during the plateau phase. In addition to the increase in [Ca(2+)](i) in response to KCl there was also a significant increase in insulin release in islets isolated from KO mice. Evidence for modulation by calbindin of [Ca(2+)](i) and insulin release was also noted using beta cell lines. Rat calbindin was stably expressed in betaTC-3 and betaHC-13 cells. In response to depolarizing concentrations of K(+), insulin release was decreased by 45-47% in calbindin expressing betaTC cells and was decreased by 70-80% in calbindin expressing betaHC cells compared with insulin release from vector transfected betaTC or betaHC cells (p < 0.01). In addition, the K(+)-stimulated intracellular calcium peak was markedly inhibited in calbindin expressing betaHC cells compared with vector transfected cells (225 nM versus 1,100 nM, respectively). Buffering of the depolarization-induced rise in [Ca(2+)](i) was also observed in calbindin expressing betaTC cells. In summary, our findings, using both isolated islets from calbindin-D(28k) KO mice and beta cell lines, establish a role for calbindin in the modulation of depolarization-stimulated insulin release and suggest that calbindin can control the rate of insulin release via regulation of [Ca(2+)](i).

The Identification of Novel Protein-Protein Interactions in Liver that Affect Glucagon Receptor Activity

PubMed Central

Froese, Sean; Dai, Feihan F.; Robitaille, Mélanie; Bhattacharjee, Alpana; Huang, Xinyi; Jia, Weiping; Angers, Stéphane; Wheeler, Michael B.; Wei, Li

2015-01-01

Glucagon regulates glucose homeostasis by controlling glycogenolysis and gluconeogenesis in the liver. Exaggerated and dysregulated glucagon secretion can exacerbate hyperglycemia contributing to type 2 diabetes (T2D). Thus, it is important to understand how glucagon receptor (GCGR) activity and signaling is controlled in hepatocytes. To better understand this, we sought to identify proteins that interact with the GCGR to affect ligand-dependent receptor activation. A Flag-tagged human GCGR was recombinantly expressed in Chinese hamster ovary (CHO) cells, and GCGR complexes were isolated by affinity purification (AP). Complexes were then analyzed by mass spectrometry (MS), and protein-GCGR interactions were validated by co-immunoprecipitation (Co-IP) and Western blot. This was followed by studies in primary hepatocytes to assess the effects of each interactor on glucagon-dependent glucose production and intracellular cAMP accumulation, and then in immortalized CHO and liver cell lines to further examine cell signaling. Thirty-three unique interactors were identified from the AP-MS screening of GCGR expressing CHO cells in both glucagon liganded and unliganded states. These studies revealed a particularly robust interaction between GCGR and 5 proteins, further validated by Co-IP, Western blot and qPCR. Overexpression of selected interactors in mouse hepatocytes indicated that two interactors, LDLR and TMED2, significantly enhanced glucagon-stimulated glucose production, while YWHAB inhibited glucose production. This was mirrored with glucagon-stimulated cAMP production, with LDLR and TMED2 enhancing and YWHAB inhibiting cAMP accumulation. To further link these interactors to glucose production, key gluconeogenic genes were assessed. Both LDLR and TMED2 stimulated while YWHAB inhibited PEPCK and G6Pase gene expression. In the present study, we have probed the GCGR interactome and found three novel GCGR interactors that control glucagon-stimulated glucose production by modulating cAMP accumulation and genes that control gluconeogenesis. These interactors may be useful targets to control glucose homeostasis in T2D. PMID:26075596

Heavy isotope labeling study of the turnover of forskolin-stimulated adenylate cyclase in BC/sup 3/H1 cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bouhelal, R.; Bockaert, J.; Mermet-Bouvier, R.

1987-06-25

We have used the method of heavy isotope labeling to study the metabolic turnover of adenylate cyclase in a nonfusing muscle cell line, the BC/sup 3/H1 cells. These cells contains an adenylate cyclase coupled to beta-adrenergic receptors and highly stimulated by forskolin, a potent activator of the enzyme. After transfer of the cells from normal medium to heavy medium (a medium containing heavy labeled amino acids, /sup 3/H, /sup 13/C, /sup 15/N), heavy isotope-labeled adenylate cyclase molecules progressively replace pre-existing light molecules. In sucrose gradient differential sedimentation, after a 5-day switch in heavy medium, the enzyme exhibited a higher massmore » (s = 8.40 +/- 0.03 S, n = 13) compared to the control enzyme. Indeed, the increase in the sedimentation coefficient of the heavy molecules was due to the synthesis of new molecules of adenylate cyclase labeled with heavy isotope amino acids since in the presence of cycloheximide, an inhibitor of protein synthesis, no change in the sedimentation pattern of the forskolin-stimulated adenylate cyclase occurred. After incorporation of heavy isotope amino acids in the adenylate cyclase molecules, the kinetics parameters of the enzyme did not change. However, adenylate cyclase from cells incubated with heavy medium exhibits an activity about 2-fold lower than control. After switching the cells to the heavy medium, the decrease of the activity of the enzyme occurred during the first 24 h and thereafter remained at a steady state for at least 4 days. In contrast, 24 h after the switch, the sedimentation coefficient of forskolin-stimulated adenylate cyclase was progressively shifted to a higher value.« less

Vasoactive amines modulate actin cables (stress fibers) and surface area in cultured bovine endothelium.

PubMed

Welles, S L; Shepro, D; Hechtman, H B

1985-06-01

Cultured bovine aortic endothelial cells were fixed and stained with NBD-phallicidin and quantitated with a digital image analyzer for changes in actin cables and surface area. Serotonin (5-HT), norepinephrine (NE), dopamine and histamine (all at 10(-4)M concentrations) were tested for their ability to induce cytoskeletal changes. Only 5-HT and NE increased actin cables significantly (p less than 0.01), 80.7% and 97.9%, respectively. Dopamine and histamine treated cells showed a 67.4% and 80.8% decrease in actin cables respectively (p less than 0.01). Stimulated increases of actin cables by 5-HT were inhibited by Ketanserin, and propranolol inhibited NE stimulation of actin cables. Treatment of cells with these blockers alone also decreased actin cables below control values (p less than 0.01). Pretreatment of cells with diphenhydramine, but not cimetidine, inhibited histamine-induced decreases in actin cables. Stimulation of surface area by 5-HT and NE was also observed, with 40.8% and 80.7% increases respectively, when compared with controls (p less than 0.01). The increases in actin cables were associated with a lack of ruffled edges that are indicative of motile cells. In contrast, induced decreases in actin cables resulted in cells with ruffled edges. Exogenous 5-HT and NE have been shown to prevent the increased permeability visible as extravasation of red blood cells from postcapillary venules in thrombocytopenic animals. The present data suggest that 5-HT and NE may be involved in maintaining the endothelial barrier function by a receptor-mediated stimulation of actin cables. Also, histamine-induced decreases in actin cables may be correlated with the amine's action in vivo as a mediator of increased inflammatory permeability.

Antimicrobial activity and regulation of CXCL9 and CXCL10 in oral keratinocytes.

PubMed

Marshall, Alison; Celentano, Antonio; Cirillo, Nicola; Mignogna, Michele D; McCullough, Michael; Porter, Stephen

2016-10-01

Chemokine (C-X-C motif) ligand (CXCL)9 and CXCL10 are dysregulated in oral inflammatory conditions, and it is not known if these chemokines target microorganisms that form oral biofilm. The aim of this study was to investigate the antimicrobial activity of CXCL9 and CXCL10 on oral microflora and their expression profiles in oral keratinocytes following exposure to inflammatory and infectious stimuli. Streptococcus sanguinis was used as a model and Escherichia coli as a positive control. The antimicrobial effect of CXCL9/CXCL10 was tested using a radial diffusion assay. mRNA transcripts were isolated from lipopolysaccharide (LPS)-treated and untreated (control) oral keratinocyte cell lines at 2-, 4-, 6-, and 8-h time-points of culture. The CXCL9/10 expression profile in the presence or absence of interferon-γ (IFN-γ) was assessed using semiquantitative PCR. Although both chemokines demonstrated antimicrobial activity, CXCL9 was the most effective chemokine against both S. sanguinis and E coli. mRNA for CXCL10 was expressed in control cells and its production was enhanced at all time-points following stimulation with LPS. Conversely, CXCL9 mRNA was not expressed in control or LPS-stimulated cells. Finally, stimulation with IFN-γ enhanced basal expression of both CXCL9 and CXCL10 in oral keratinocytes. Chemokines derived from oral epithelium, particularly CXCL9, demonstrate antimicrobial properties. Bacterial and inflammatory-stimulated up-regulation of CXCL9/10 could represent a key element in oral bacterial colonization homeostasis and host-defense mechanisms. © 2016 Eur J Oral Sci.

The significance of measuring monocyte tissue factor activity in patients with breast and colorectal cancer

PubMed Central

Lwaleed, B A; Chisholm, M; Francis, J L

1999-01-01

Monocytes express tissue factor (mTF) in several conditions including cancer where levels may be valuable in assessing tumour presence and progression. Using a two-stage kinetic chromogenic assay (KCA), mTF levels were measured in controls [normal subjects (n = 60) and patients undergoing hernia repair or cholecystectomy (n = 60)], in patients with benign and malignant disease of the breast (n = 83) and of the large bowel (n = 62). This was performed under fresh (resting) conditions and after incubation for 6 h without (unstimulated) and with (stimulated) Escherichia coli endotoxin. The malignant groups showed higher mTF levels than each of the three controls for resting (P < 0.05 breast, P < 0.05 colorectal) unstimulated (P < 0.05 breast, P < 0.05 colorectal) and stimulated cells (P < 0.001 breast, P < 0.01 colorectal). Similarly, the benign inflammatory groups had higher mTF levels than controls for resting (P < 0.05 colorectal), unstimulated (P < 0.05 colorectal) and stimulated cells (P < 0.01 breast, P < 0.01 colorectal). There was no significant difference between malignant and benign inflammatory groups in each organ. mTF levels showed an increase corresponding to that of histological tumour progression and were higher in non-surviving patients. In conclusion, mTF levels are raised in malignant and inflammatory disease compared to controls and patients with non-inflammatory conditions. Stimulated cells give better discrimination between the groups and may be of value in identifying high risk individuals. mTF levels showed an association with tumour grade or stage and the patients' survival time. © 1999 Cancer Research Campaign PMID:10390009

Effects of umbilical cord blood stem cells on healing factors for diabetic foot injuries.

PubMed

Çil, N; Oğuz, E O; Mete, E; Çetinkaya, A; Mete, G A

2017-01-01

The use of stem or progenitor cells from bone marrow, or peripheral or umbilical cord blood is becoming more common for treatment of diabetic foot problems. These cells promote neovascularization by angiogenic factors and they promote epithelium formation by stimulating cell replication and migration under certain pathological conditions. We investigated the role of CD34 + stem cells from human umbilical cord blood in wound healing using a rat model. Rats were randomly divided into a control group and two groups with diabetes induced by a single dose of 55 mg/kg intraperitoneal streptozocin. Scarred areas 5 mm in diameter were created on the feet of all rats. The diabetic rats constituted the diabetes control group and a diabetes + stem cell group with local injection into the wound site of 0.5 × 106 CD34 + stem cells from human umbilical cord blood. The newly formed skin in the foot wounds following CD34 + stem cell treatment showed significantly improvement by immunohistochemistry and TUNEL staining, and were closer to the wound healing of the control group than the untreated diabetic animals. The increase in FGF expression that accompanied the local injection of CD34 + stem cells indicates that FGF stimulation helped prevent apoptosis. Our findings suggest a promising new treatment approach to diabetic wound healing.

T cell immunoregulation in active ocular toxoplasmosis.

PubMed

Cordeiro, Cynthia A; Vieira, Erica L M; Castro, Vinicius M; Dutra, Walderez O; Costa, Rogerio A; Orefice, Juliana L; Campos, Wesley R; Orefice, Fernando; Young, Lucy H; Teixeira, Antonio Lucio

2017-04-01

Toxoplasma gondii infection is an important cause of infectious ocular disease. The physiopathology of retinochoroidal lesions associated with this infection is not completely understood. The present study was undertaken to investigate cytokine production by T cells from individuals with active toxoplasmic retinochoroiditis (TR) comparing with controls. Eighteen patients with active TR and 15 healthy controls (6 controls IgG + to Toxoplasma and 9 negative controls) were included in the study. Peripheral blood mononuclear cells were incubated in the presence or absence of T. gondii antigen (STAg), and stained against CD4, CD8, TNF, IL-10 and IFN-γ. Baseline expression of cytokines was higher in TR/IgG + patients in comparison with controls. Cytokine expression was not increased by STAg in vitro stimulation in controls. After stimulation, TR/IgG + patients' lymphocytes increased cytokine as compared to cultures from both controls. While T cells were the main source of IL-10, but also IFN-γ and TNF, other lymphocyte populations were relevant source of inflammatory cytokines. Interestingly, it was observed a negative correlation between ocular lesion size and IL-10 expression by CD4 + lymphocytes. This study showed that T cells are the main lymphocyte populations expressing IL-10 in patients with TR. Moreover, expression of IL-10 plays a protective role in active TR. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Highly Human Immunodeficiency Virus-Exposed Seronegative Men Have Lower Mucosal Innate Immune Reactivity.

PubMed

Fulcher, Jennifer A; Romas, Laura; Hoffman, Jennifer C; Elliott, Julie; Saunders, Terry; Burgener, Adam D; Anton, Peter A; Yang, Otto O

2017-08-01

Risk of HIV acquisition varies, and some individuals are highly HIV-1-exposed, yet, persistently seronegative (HESN). The immunologic mechanisms contributing to this phenomenon are an area of intense interest. As immune activation and inflammation facilitate disease progression in HIV-1-infected persons and gastrointestinal-associated lymphoid tissue is a highly susceptible site for transmission, we hypothesized that reduced gut mucosal immune reactivity may contribute to reduced HIV-1 susceptibility in HESN men with a history of numerous rectal sexual exposures. To test this, we used ex vivo mucosal explants from freshly acquired colorectal biopsies from healthy control and HESN subjects who were stimulated with specific innate immune ligands and inactivated whole pathogens. Immune reactivity was then assessed via cytokine arrays and proteomic analysis. Mucosal immune cell compositions were quantified via immunohistochemistry. We found that explants from HESN subjects produced less proinflammatory cytokines compared with controls following innate immune stimulation; while noninflammatory cytokines were similar between groups. Proteomic analysis identified several immune response proteins to be differentially expressed between HIV-1-stimulated HESN and control explants. Immunohistochemical examination of colorectal mucosa showed similar amounts of T cells, macrophages, and dendritic cells between groups. The results of this pilot study suggest that mucosal innate immune reactivity is dampened in HESN versus control groups, despite presence of similar densities of immune cells in the colorectal mucosa. This observed modulation of the rectal mucosal immune response may contribute to lower risk of mucosal HIV-1 transmission in these individuals.

Control of lipopolysaccharide biosynthesis and release by Escherichia coli and Salmonella typhimurium.

PubMed Central

Ishiguro, E E; Vanderwel, D; Kusser, W

1986-01-01

The influence of the relA gene on lipopolysaccharide (LPS) biosynthesis and release by Escherichia coli and Salmonella typhimurium was investigated. Similar results were obtained with both species. The incorporation of [3H]galactose into LPS by galE mutants was inhibited by at least 50% (as compared with normal growing controls) during amino acid deprivation of relA+ strains. This inhibition could be prevented by the treatment of the amino acid-deprived relA+ bacteria with chloramphenicol, a known antagonist of the stringent control mechanism. Furthermore, LPS biosynthesis was not inhibited during amino acid deprivation of isogenic relA mutant strains. These results indicate that LPS synthesis is regulated by the stringent control mechanism. Normal growing cells of both relA+ and relA strains released LPS into the culture fluid at low rates. Amino acid deprivation stimulated the rate of LPS release by relA mutants but not by relA+ bacteria. Chloramphenicol treatment markedly stimulated the release of cell-bound LPS by amino acid-deprived relA+ cells. Thus, a low rate of LPS release was characteristic of normal growth and could be increased in nongrowing cells by relaxing the control of LPS synthesis. Images PMID:3531174

A novel anti-inflammatory drug, SDZ ASM 981, for the treatment of skin diseases: in vitro pharmacology.

PubMed

Grassberger, M; Baumruker, T; Enz, A; Hiestand, P; Hultsch, T; Kalthoff, F; Schuler, W; Schulz, M; Werner, F J; Winiski, A; Wolff, B; Zenke, G

1999-08-01

SDZ ASM 981, a novel ascomycin macrolactam derivative, has high anti-inflammatory activity in animal models of allergic contact dermatitis and shows clinical efficacy in atopic dermatitis, allergic contact dermatitis and psoriasis, after topical application. Here we report on the in vitro activities of this promising new drug. SDZ ASM 981 inhibits the proliferation of human T cells after antigen-specific or non-specific stimulation. It downregulates the production of Th1 [interleukin (IL)-2, interferon-gamma] and Th2 (IL-4, IL-10) type cytokines after antigen-specific stimulation of a human T-helper cell clone isolated from the skin of an atopic dermatitis patient. SDZ ASM 981 inhibits the phorbol myristate acetate/phytohaemagglutinin-stimulated transcription of a reporter gene coupled to the human IL-2 promoter in the human T-cell line Jurkat and the IgE/antigen-mediated transcription of a reporter gene coupled to the human tumour necrosis factor (TNF)-alpha promoter in the murine mast-cell line CPII. It does not, however, affect the human TNF-alpha promoter controlled transcription of a reporter gene in a murine dendritic cell line (DC18 RGA) after stimulation via the FcgammaRIII receptor. SDZ ASM 981 also prevents the release of preformed pro-inflammatory mediators from mast cells, as shown in the murine cell line CPII after stimulation with IgE/antigen. In summary, these results demonstrate that SDZ ASM 981 is a specific inhibitor of the production of pro-inflammatory cytokines from T cells and mast cells in vitro.

Altered Innate and Lymphocytic Immune Responses in Mouse Splenocytes Post-Flight

NASA Technical Reports Server (NTRS)

Hwang, ShenAn; Crucian, Brian E.; Sams, Clarence F.; Actor, Jeffrey K.

2011-01-01

Space flight is known to affect immune responses of astronauts and animals, decreasing lymphocytic responses to mitogenic stimuli, delayed typed hypersensitivity reactions, and T-cell activation. Despite changes in immune suppression, there are no reports of consistent adverse clinical events post flight. To further investigate the spectrum of affected immune responses, murine splenocytes were stimulated immediately post-shuttle flight (14 days on STS-135) with T-cell stimulators or toll-like receptor agonists. Comparisons were made to ground control splenocytes from age-matched mice. Cell phenotypes were assessed, as well as activation markers and associated cytokine production. The CD4+ population decreased with no concurrent decrease in CD8+ cells from shuttle mice post flight compared to ground controls. Regarding antigen presenting cell populations, the number of CD11c+ cells were slightly elevated post flight, compared to ground controls, with increased MHC Class I expression (I-A(sup b)) and no change in Class II expression (H-2K(sup b)). CD86+ populations were also significantly diminished. However, the decreased markers did not correlate with activity. Stimulation of splenocytes post flight showed significant increase in bead uptake, increased Class I expression, increased TNF-alpha and IL-6 production in response to TLR-2 (zymosan) and TLR-4 (LPS) agonists. While most activated (ConA or anti-CD3/anti-CD28) CD4+ cells showed markedly diminished responses (reduced IL-2 production), non-specific T cell responses to superantigen (SEA/SEB) increased post flight as determined by expression of early activation markers. Production of additional cytokines was also dysregulated postflight. Overall, persistent immune changes during space flight could represent unique clinical risks for exploration class missions. The consequences of pathogenic encounter remain an important concern that should be addressed.

Interleukin-10 Gene-Modified Dendritic Cell-Induced Type 1 Regulatory T Cells Induce Transplant-Tolerance and Impede Graft Versus Host Disease After Allogeneic Stem Cell Transplantation.

PubMed

Wan, Jiangbo; Huang, Fang; Hao, Siguo; Hu, Weiwei; Liu, Chuanxu; Zhang, Wenhao; Deng, Xiaohui; Chen, Linjun; Ma, Liyuan; Tao, Rong

2017-01-01

Tr1 cells can induce peripheral tolerance to self- and foreign antigens, and have been developed as a therapeutic tool for the induction of tolerance to transplanted tissue. We explored the feasibility of generating Tr1 cells by using IL-10 gene-modified recipient DCs (DCLV-IL-10) to stimulate donor naive CD4+ T cells. We also investigated some biological properties of Tr1 cells. DCLV-IL-10 were generated through DCs transduced with a lentivirus vector carrying the IL-10 gene, and Tr1 cells were produced by using DCLV-IL-10 to stimulate naive CD4+ T cells. The effects of Tr1 cells on T-cell proliferation and the occurrence of graft versus host disease (GVHD) following allogeneic stem-cell transplantation (allo-HSCT) were investigated. The DCLV-IL-10-induced Tr1 cells co-expressed LAG-3 and CD49b. Moreover, they also expressed CD4, CD25, and IL-10, but not Foxp3, and secreted significantly higher levels of IL-10 (1,729.36 ± 185.79 pg/mL; P < 0.001) and INF-γ (1,524.48 ± 168.65 pg/mL; P < 0.01) than the control T cells upon the stimulation by allogeneic DCs. Tr1 cells markedly suppressed T-lymphocyte proliferation and the mixed lymphocytic response (MLR) in vitro. The mice used in the allo-HSCT model had longer survival times and lower clinical and pathological GVHD scores than the control mice. IL-10 gene-modified DC-induced Tr1 cells may be used as a potent cellular therapy for the prevention of GVHD after allo-HSCT. © 2017 The Author(s). Published by S. Karger AG, Basel.

Shock Wave-Stimulated Periosteum for Cartilage Repair

DTIC Science & Technology

2013-12-01

were added to the Gtn-HPA prior to the gelation 6 process, at a cell density of 1×105 cells/ml. In the control groups, cells received no treatment...Mesenchymal Stem Cell Viability Viability test was performed 24 hours post- gelation using the Live/Dead assay. Viability/cytotoxicity kit was used (Molecular

[Cell surface peroxidase--generator of superoxide anion in wheat root cells under wound stress].

PubMed

Chasov, A V; Gordon, L Kh; Kolesnikov, O P; Minibaeva, F V

2002-01-01

Development of wound stress in excised wheat roots is known to be accompanied with an increase in reactive oxygen species (ROS) production, fall of membrane potential, release of K+ from cells, alkalization of extracellular solution, changes in respiration and metabolism of structural lipids. Dynamics of superoxide release correlates with changes in other physiological parameters, indicating the cross-reaction of these processes. Activity of peroxidase in extracellular solution after a 1 h incubation and removal of roots was shown to be stimulated by the range of organic acids, detergents, metals, and to be inhibited by cyanide. Superoxide production was sensitive to the addition of Mn2+ and H2O2. Increase in superoxide production correlates with the enhancement of peroxidase activity at the application of organic acids and detergents. The results obtained indicate that cell surface peroxidase is one of the main generators of superoxide in wounded wheat root cells. Different ways of stimulation of the ROS producing activity in root cells is supposed. By controlling superoxide and hydrogen peroxide formation, the cell surface peroxidase can control the adaptation processes in stressed plant cells.

Regulatory T cells generated during cytomegalovirus in vitro stimulation of mononuclear cells from HIV-infected individuals on HAART correlate with decreased lymphocyte proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jesser, Renee D.; Li, Shaobing; Weinberg, Adriana

2006-09-01

HIV-infected patients fail to fully recover cell-mediated immunity despite HAART. To identify regulatory factors, we studied the phenotype and function of in vitro cytomegalovirus (CMV)-stimulated T cells from HAART recipients. CFSE-measured proliferation showed CD4{sup +} and CD8{sup +} cells dividing in CMV-stimulated cultures. Compared with healthy controls, CMV-stimulated lymphocytes from HAART recipients had lower {sup 3}H-thymidine incorporation; lower IFN{gamma} and TNF{alpha} production; higher CD4{sup +}CD27{sup -}CD28{sup -} and CD8{sup +}CD27{sup -}CD28{sup -} frequencies; lower CD4{sup +}CD25{sup hi}; and higher FoxP3 expression in CD8{sup +}CD25{sup hi} cells. CMV-specific proliferation correlated with higher IFN{gamma}, TNF{alpha} and IL10 levels and higher CD4{sup +}perforin{supmore » +} and CD8{sup +}perforin{sup +} frequencies. Decreased proliferation correlated with higher CD4{sup +}CD27{sup -}CD28{sup -} frequencies and TGF{beta}1 production, which also correlated with each other. Anti-TGF{beta}1 neutralizing antibodies restored CMV-specific proliferation in a dose-dependent fashion. In HIV-infected subjects, decreased proliferation correlated with higher CMV-stimulated CD8{sup +}CD25{sup hi} frequencies and their FoxP3 expression. These data indicate that FoxP3- and TGF{beta}1-expressing regulatory T cells contribute to decreased immunity in HAART recipients.« less

Phosphatidylinositol 3,4,5-trisphosphate modulation in SHIP2-deficient mouse embryonic fibroblasts.

PubMed

Blero, Daniel; Zhang, Jing; Pesesse, Xavier; Payrastre, Bernard; Dumont, Jacques E; Schurmans, Stéphane; Erneux, Christophe

2005-05-01

SHIP2, the ubiquitous SH2 domain containing inositol 5-phosphatase, includes a series of protein interacting domains and has the ability to dephosphorylate phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)]in vitro. The present study, which was undertaken to evaluate the impact of SHIP2 on PtdIns(3,4,5)P(3) levels, was performed in a mouse embryonic fibroblast (MEF) model using SHIP2 deficient (-/-) MEF cells derived from knockout mice. PtdIns(3,4,5)P(3) was upregulated in serum stimulated -/- MEF cells as compared to +/+ MEF cells. Although the absence of SHIP2 had no effect on basal PtdIns(3,4,5)P(3) levels, we show here that this lipid was significantly upregulated in SHIP2 -/- cells but only after short-term (i.e. 5-10 min) incubation with serum. The difference in PtdIns(3,4,5)P(3) levels in heterozygous fibroblast cells was intermediate between the +/+ and the -/- cells. In our model, insulin-like growth factor-1 stimulation did not show this upregulation. Serum stimulated phosphoinositide 3-kinase (PI 3-kinase) activity appeared to be comparable between +/+ and -/- cells. Moreover, protein kinase B, but not mitogen activated protein kinase activity, was also potentiated in SHIP2 deficient cells stimulated by serum. The upregulation of protein kinase B activity in serum stimulated cells was totally reversed in the presence of the PI 3-kinase inhibitor LY-294002, in both +/+ and -/- cells. Altogether, these data establish a link between SHIP2 and the acute control of PtdIns(3,4,5)P(3) levels in intact cells.

Lithium stimulates the recovery of granulopoiesis following acute radiation injury.

PubMed

Gallicchio, V S; Chen, M G; Watts, T D; Gamba-Vitalo, C

1983-07-01

Lithium (Li) is a known stimulator of steady-state granulopoiesis, influencing both pluripotential (CFUS) and granulocyte-macrophage committed stem cell (CFUGM) populations. Li has therefore been suggested to be an effective agent to reduce the neutropenia that often is seen after either cytotoxic chemotherapy or radiotherapy protocols. In this report, we have examined bone marrow and spleen cells for their recovery patterns of CFUS, CFUGM, CFUE, BFUE and 59Fe-incorporation, along with the usual peripheral blood indices (packed red cell volume, WBC and differential) from mice administered Li after receiving 200 rad whole body irradiation. Li increased granulopoietic recovery as measured by significant elevations in both marrow and spleen derived CFUGM compared to those values obtained from radiation controls. Significant elevation in the WBC, consisting mainly of neutrophils, was also observed. Bone marrow and splenic derived erythroid stem cells (CFUE, BFUE) and % 59Fe-incorporation measured from peripheral blood, femur and spleen were all slightly reduced, but not to a significant degree to alter the packed red cell volume. The CFUS populations from both irradiated groups (control and Li-treated) were depressed when compared to normal non-irr controls and this degree of suppression was greater in the Li-treated group. These results document the ability of Li to stimulate the recovery of granulopoiesis after radiation-induced hematopoietic injury and suggest Li may be useful in ameliorating the neutropenia that can often develop after routine radiotherapy protocols.

Effects of lactoferrin on the production of interferon-λ by the human intestinal epithelial cell line HT-29.

PubMed

Shin, Kouichirou; Oda, Hirotsugu; Wakabayashi, Hiroyuki; Yamauchi, Koji; Abe, Fumiaki

2017-02-01

We examined the in-vitro effects of bovine lactoferrin (LF) on the production of interferon-λ (IFN-λ), an antiviral cytokine important for the defense of enterocytes, using the human intestinal epithelial cell line HT-29. HT-29 cell cultures were treated with LF for 1 h, and the cultures were stimulated with polyinosinic-polycytidylic acid (poly I:C). LF increased the concentration of IFN-λ in the culture supernatant after stimulation in a dose-dependent manner. A similar increase in the concentration of IFN-λ was observed in the supernatant of cells washed between treatment with LF and stimulation with poly I:C. At 6 and 24 h after stimulation with poly I:C (early and late phases, respectively) treated cultures contained significantly higher concentrations of IFN-λ1 in the culture supernatant, and significantly higher IFN-λ1 and IFN-λ2 mRNA levels, than controls. These results suggest that LF activates the innate cellular immunity of the enterocytes to double-stranded RNA and increases the production of IFN-λ.

Defective quorum sensing of acute lymphoblastic leukemic cells: evidence of collective behavior of leukemic populations as semi-autonomous aberrant ecosystems

PubMed Central

Patel, Sapan J; Dao, Su; Darie, Costel C; Clarkson, Bayard D

2016-01-01

Quorum sensing (QS) is a generic term used to describe cell-cell communication and collective decision making by bacterial and social insects to regulate the expression of specific genes in controlling cell density and other properties of the populations in response to nutrient supply or changes in the environment. QS mechanisms also have a role in higher organisms in maintaining homeostasis, regulation of the immune system and collective behavior of cancer cell populations. In the present study, we used a p190BCR-ABL driven pre-B acute lymphoblastic leukemia (ALL3) cell line derived from the pleural fluid of a terminally ill patient with ALL to test the QS hypothesis in leukemia. ALL3 cells don’t grow at low density (LD) in liquid media but grow progressively faster at increasingly high cell densities (HD) in contrast to other established leukemic cell lines that grow well at very low starting cell densities. The ALL3 cells at LD are poised to grow but shortly die without additional stimulation. Supernates of ALL3 cells (HDSN) and some other primary cells grown at HD stimulate the growth of the LD ALL3 cells without which they won’t survive. To get further insight into the activation processes we performed microarray analysis of the LD ALL3 cells after stimulation with ALL3 HDSN at days 1, 3, and 6. This screen identified several candidate genes, and we linked them to signaling networks and their functions. We observed that genes involved in lipid, cholesterol, fatty acid metabolism, and B cell activation are most up- or down-regulated upon stimulation of the LD ALL3 cells using HDSN. We also discuss other pathways that are differentially expressed upon stimulation of the LD ALL3 cells. Our findings suggest that the Ph+ ALL population achieves dominance by functioning as a collective aberrant ecosystem subject to defective quorum-sensing regulatory mechanisms. PMID:27429840

Dual effect of cell-cell contact disruption on cytosolic calcium and insulin secretion.

PubMed

Jaques, Fabienne; Jousset, Hélène; Tomas, Alejandra; Prost, Anne-Lise; Wollheim, Claes B; Irminger, Jean-Claude; Demaurex, Nicolas; Halban, Philippe A

2008-05-01

Cell-to-cell interactions play an important role in insulin secretion. Compared with intact islets, dispersed pancreatic beta-cells show increased basal and decreased glucose-stimulated insulin secretion. In this study, we used mouse MIN6B1 cells to investigate the mechanisms that control insulin secretion when cells are in contact with each other or not. RNAi-mediated silencing of the adhesion molecule E-cadherin in confluent cells reduced glucose-stimulated secretion to the levels observed in isolated cells but had no impact on basal secretion. Dispersed cells presented high cytosolic Ca(2+) activity, depolymerized cytoskeleton and ERK1/2 activation in low glucose conditions. Both the increased basal secretion and the spontaneous Ca(2+) activity were corrected by transient removal of Ca(2+) or prolonged incubation of cells in low glucose, a procedure that restored the ability of dispersed cells to respond to glucose (11-fold stimulation). In conclusion, we show that dispersed pancreatic beta-cells can respond robustly to glucose once their elevated basal secretion has been corrected. The increased basal insulin secretion of dispersed cells is due to spontaneous Ca(2+) transients that activate downstream Ca(2+) effectors, whereas engagement of cell adhesion molecules including E-cadherin contributes to the greater secretory response to glucose seen in cells with normal intercellular contacts.

A Dual-Mode Bioreactor System for Tissue Engineered Vascular Models.

PubMed

Bono, N; Meghezi, S; Soncini, M; Piola, M; Mantovani, D; Fiore, Gianfranco Beniamino

2017-06-01

In the past decades, vascular tissue engineering has made great strides towards bringing engineered vascular tissues to the clinics and, in parallel, obtaining in-lab tools for basic research. Herein, we propose the design of a novel dual-mode bioreactor, useful for the fabrication (construct mode) and in vitro stimulation (culture mode) of collagen-based tubular constructs. Collagen-based gels laden with smooth muscle cells (SMCs) were molded directly within the bioreactor culture chamber. Based on a systematic characterization of the bioreactor culture mode, constructs were subjected to 10% cyclic strain at 0.5 Hz for 5 days. The effects of cyclic stimulation on matrix re-arrangement and biomechanical/viscoelastic properties were examined and compared vs. statically cultured constructs. A thorough comparison of cell response in terms of cell localization and expression of contractile phenotypic markers was carried out as well. We found that cyclic stimulation promoted cell-driven collagen matrix bi-axial compaction, enhancing the mechanical strength of strained samples with respect to static controls. Moreover, cyclic strain positively affected SMC behavior: cells maintained their contractile phenotype and spread uniformly throughout the whole wall thickness. Conversely, static culture induced a noticeable polarization of cell distribution to the outer rim of the constructs and a sharp reduction in total cell density. Overall, coupling the use of a novel dual-mode bioreactor with engineered collagen-gel-based tubular constructs demonstrated to be an interesting technology to investigate the modulation of cell and tissue behavior under controlled mechanically conditioned in vitro maturation.

[Bifidobacterium DNA upregulates Th1 type response of umbilical cord blood mononuclear cell].

PubMed

Zhao, Hui; Wang, Xiao-chuan; Wang, Jing-yi; Yu, Ye-heng; Wang, Chuan-qing; Yang, Yi

2006-06-01

To study the effect of bifidobacterium genomic DNA on umbilical cord blood mononuclear cell (CBMC), and investigate the immunoregulation of bifidobacterium DNA and explore possible mechanisms by which bifidobacterium acts against allergic reaction. Bifidobacterium genomic DNA (bDNA) and human DNA (hDNA) were extracted with phenol/chloroform/isoamyl alcohol and stored at -20 degrees C for later use. Parts of bDNA were completely digested with DNaseI (d-bDNA) at 37 degrees C. CBMCs were isolated with Ficoll from umbilical cord blood and incubated at 37 degrees C in a 5% CO2 humidified incubator. These cells were divided into four groups, control group: without any stimulant; bDNA group: stimulated with 25 microg/ml bDNA; d-bDNA group: stimulated with 25 microg/ml d-bDNA; hDNA group: stimulated with 25 microg/ml hDNA. The cells were stimulated with different stimulants in vitro, at the end of incubation culture supernatant and cells were collected. IL-12 and IL-10 levels in the culture supernatant were measured by enzyme linked immuno sorbent assay (ELISA); cells secreting IL-4 and IFN-gamma were counted by enzyme linked immunospot (ELISPOT) assay; and total RNA was isolated from the cells to assay T-bet and GATA3 mRNA expression levels by reverse transcription polymerase chain reaction (RT-PCR). Six hours after stimulation there was no significant difference in IL-12 level in supernatant among the four groups; 12 hours after stimulation, IL-12 level in supernatant of bDNA treated group was significantly higher than that of each of the other groups, so were the results obtained at 24 hours and 48 hours after stimulation (P < 0.05). No significant difference could be detected in IL-12 level in supernatant among the other 3 groups. On the other hand, 6 hours after stimulation there was no significant difference in IL-10 level in supernatant among the four groups. But 12 and 24 hours after stimulation IL-10 level in supernatant of bDNA treated group was lower than that of each of the other groups, but the difference was not statistically significant. The count of IFN-gamma secreting cells of bDNA treated group was higher than that of the other groups, while IL-4 secteting cells of bDNA treated group were lower than that of the other groups. After bDNA stimulation, nuclear factor T-box expressed in T cells (T-bet) mRNA expression was conspicuously enhanced as compared to the other three groups (P < 0.05). GATA3 mRNA transcription in CBMC had no significant change after bDNA stimulation. bDNA could promote secretion of Th1 type cytokine IL-12, while Th2 type cytokine IL-10 level of cell supernatant was decreased. bDNA could stimulate secretion of IFN-gamma by CBMC and inhibit secretion of IL-4. T-bet mRNA expression was highly enhanced after bDNA stimulation. bDNA could upregulate Th1 type response, which may be one of important mechanisms by which bifidobacterium inhibit allergic response.

Phenotypic Changes and Impaired Function of Peripheral γδ T Cells in Patients With Sepsis.

PubMed

Liao, Xue-Lian; Feng, Ting; Zhang, Jiang-Qian; Cao, Xing; Wu, Qi-Hong; Xie, Zhi-Chao; Kang, Yan; Li, Hong

2017-09-01

Recent studies demonstrated the significant loss of gamma delta T (γδ T) cells in patients with sepsis. Given the distinct functions of γδ T cells in human anti-infection immunity, we are interested in evaluating the phenotype and function of peripheral γδ T cells in septic patients and determining their prognostic implication. This prospective study has been conducted in three intensive care units of a university hospital. During the period from October 2014 to June 2015, we enrolled 107 patients who were consecutively admitted and diagnosed with severe sepsis or septic shock (excluding previous immunosuppression) and 45 healthy controls. Using flow cytometry, we analyzed the in vivo percentage of γδ T cells in cluster of differentiation (CD)3 cells from peripheral blood mononuclear cells as well as their expression of surface markers (CD69, natural-killer group 2 member D [NKG2D], programmed death receptor 1 [PD-1]) and intracellular cytokines (interferon-γ [IFN-γ], interleukin [IL]-17, IL-10, transforming growth factor-β [TGF-β]). Then we further evaluated the different responses of γδ T cells after the antigen stimulation ex vivo by measuring CD69 and IFN-γ expression. Lastly, we conducted the multiple logistic regressions to analyze the risk factor for prognosis. Compared with control group, γδ T cells in septic patients displayed a decrease in percentage, increase in CD69, decrease in NKG2D, and increase in cytokine expression (pro-inflammatory IFN-γ, IL-17, anti-inflammatory IL-10, TGF-β) in vivo. After the antigen stimulation ex vivo, both CD69 and IFN-γ expression in γδ T cells were significantly lower in septic patients than control group. Importantly, the decrease in CD69 and IFN-γ expression was more pronounced in non-survivors than survivors. Multiple logistic regression analysis revealed that lower expression of IFN-γ after stimulation is a dependent risk factor that associated with patient 28-day death in septic patients (OR: 0.908 [95% CI: 0.853-0.966]). Septic patients showed altered phenotype and function of γδ T cells. The impaired IFN-γ expression by γδ T cells after the antigen stimulation is associated with mortality in septic patients.

Presence of Epstein-Barr virus-infected B lymphocytes with thyrotropin receptor antibodies on their surface in Graves' disease patients and in healthy individuals.

PubMed

Nagata, Keiko; Higaki, Katsumi; Nakayama, Yuji; Miyauchi, Hiromi; Kiritani, Yui; Kanai, Kyosuke; Matsushita, Michiko; Iwasaki, Takeshi; Sugihara, Hirotsugu; Kuwamoto, Satoshi; Kato, Masako; Murakami, Ichiro; Nanba, Eiji; Kimura, Hiroshi; Hayashi, Kazuhiko

2014-05-01

Graves' disease is an autoimmune hyperthyroidism caused by thyrotropin receptor antibodies (TRAbs). Because Epstein-Barr virus (EBV) persists in B cells and is occasionally reactivated, we hypothesized that EBV contributes to TRAbs production in Graves' disease patients by stimulating the TRAbs-producing B cells. In order for EBV to stimulate antibody-producing cells, EBV must be present in those cells but that have not yet been observed. We examined whether EBV-infected (EBV(+)) B cells with TRAbs on their surface (TRAbs(+)) as membrane immunoglobulin were present in peripheral blood of Graves' disease patients. We analyzed cultured or non-cultured peripheral blood mononuclear cells (PBMCs) from 13 patients and 11 healthy controls by flow-cytometry and confocal laser microscopy, and confirmed all cultured PBMCs from 8 patients really had TRAbs(+) EBV(+) double positive cells. We unexpectedly detected TRAbs(+) cells in all healthy controls, and TRAbs(+) EBV(+) double positive cells in all cultured PBMC from eight healthy controls. The frequency of TRAbs(+) cells in cultured PBMCs was significantly higher in patients than in controls (p = 0.021). In this study, we indicated the presence of EBV-infected B lymphocytes with TRAbs on their surface, a possible player of the production of excessive TRAbs, the causative autoantibody for Graves' disease. This is a basic evidence for our hypothesis that EBV contributes to TRAbs production in Graves' disease patients. Our results further suggest that healthy controls have the potential for TRAbs production. This gives us an important insight into the pathogenesis of Graves' disease.

Presence of Epstein–Barr virus-infected B lymphocytes with thyrotropin receptor antibodies on their surface in Graves’ disease patients and in healthy individuals

PubMed Central

Nagata, Keiko; Higaki, Katsumi; Nakayama, Yuji; Miyauchi, Hiromi; Kiritani, Yui; Kanai, Kyosuke; Matsushita, Michiko; Iwasaki, Takeshi; Sugihara, Hirotsugu; Kuwamoto, Satoshi; Kato, Masako; Murakami, Ichiro; Nanba, Eiji; Kimura, Hiroshi; Hayashi, Kazuhiko

2014-01-01

Abstract Graves’ disease is an autoimmune hyperthyroidism caused by thyrotropin receptor antibodies (TRAbs). Because Epstein–Barr virus (EBV) persists in B cells and is occasionally reactivated, we hypothesized that EBV contributes to TRAbs production in Graves’ disease patients by stimulating the TRAbs-producing B cells. In order for EBV to stimulate antibody-producing cells, EBV must be present in those cells but that have not yet been observed. We examined whether EBV-infected (EBV(+)) B cells with TRAbs on their surface (TRAbs(+)) as membrane immunoglobulin were present in peripheral blood of Graves’ disease patients. We analyzed cultured or non-cultured peripheral blood mononuclear cells (PBMCs) from 13 patients and 11 healthy controls by flow-cytometry and confocal laser microscopy, and confirmed all cultured PBMCs from 8 patients really had TRAbs(+) EBV(+) double positive cells. We unexpectedly detected TRAbs(+) cells in all healthy controls, and TRAbs(+) EBV(+) double positive cells in all cultured PBMC from eight healthy controls. The frequency of TRAbs(+) cells in cultured PBMCs was significantly higher in patients than in controls (p = 0.021). In this study, we indicated the presence of EBV-infected B lymphocytes with TRAbs on their surface, a possible player of the production of excessive TRAbs, the causative autoantibody for Graves’ disease. This is a basic evidence for our hypothesis that EBV contributes to TRAbs production in Graves’ disease patients. Our results further suggest that healthy controls have the potential for TRAbs production. This gives us an important insight into the pathogenesis of Graves’ disease. PMID:24467196

Receptor-Targeted, Magneto-Mechanical Stimulation of Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

PubMed Central

Hu, Bin; El Haj, Alicia J; Dobson, Jon

2013-01-01

Mechanical cues are employed to promote stem cell differentiation and functional tissue formation in tissue engineering and regenerative medicine. We have developed a Magnetic Force Bioreactor (MFB) that delivers highly targeted local forces to cells at a pico-newton level, utilizing magnetic micro- and nano-particles to target cell surface receptors. In this study, we investigated the effects of magnetically targeting and actuating specific two mechanical-sensitive cell membrane receptors—platelet-derived growth factor receptor α (PDGFRα) and integrin ανβ3. It was found that a higher mineral-to-matrix ratio was obtained after three weeks of magneto-mechanical stimulation coupled with osteogenic medium culture by initially targeting PDGFRα compared with targeting integrin ανβ3 and non-treated controls. Moreover, different initiation sites caused a differentiated response profile when using a 2-day-lagged magneto-mechanical stimulation over culture periods of 7 and 12 days). However, both resulted in statistically higher osteogenic marker genes expression compared with immediate magneto-mechanical stimulation. These results provide insights into important parameters for designing appropriate protocols for ex vivo induced bone formation via magneto-mechanical actuation. PMID:24065106

Stimulation of progesterone production by phorbol-12-myristate 13-acetate (PMA) in cultured Leydig tumor cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chaudhary, L.R.; Raju, V.S.; Stocco, D.M.

1987-05-01

It has been shown that addition of hCG or c-AMP to cultured Leydig tumor cells (MA-10) increases synthesis of progesterone as the major steroid. To investigate the possible involvement of protein kinase C (PK-C) in the regulation of steroid synthesis, the authors have studied the effect of PMA, an activator of PK-C, on progesterone production in MA-10 cells. The addition of PMA (100 ng/ml) stimulated steroid production whereas 4 -phorbol-12,13-didecanoate, an inactive phorbol ester, did not have any effects. Like hCG and c-AMP, PMA-stimulated progesterone production was inhibited by cycloheximide. hCG-stimulated steroid synthesis was inhibited by PMA. The addition ofmore » PMA to MA-10 Leydig cells further increased the c-AMP-stimulated progesterone production. To determine whether c-AMP has a obligatory role in the regulation of steroid production, the effect of adenylate cyclase inhibitor, 9-(tetrahydro-2-furyl)adenine (TFA), was studied on progesterone production in the presence of hCG. At lower dose (17 ng/ml) hCG-stimulated intracellular c-AMP levels and steroid production were inhibited by TFA (300 M). At higher dose of hCG (34 ng/ml) TFA did not inhibit the hCG-stimulated intracellular c-AMP levels, however, progesterone production was inhibited. Results suggest that the action of hCG, c-AMP and PMA in controlling steroidogenesis might be regulated by similar but different mechanisms.« less

Comparative effects of sub-stimulating concentrations of non-human versus human Luteinizing Hormones (LH) or chorionic gonadotropins (CG) on adenylate cyclase activation by forskolin in MLTC cells.

PubMed

Nguyen, Thi-Mong Diep; Filliatreau, Laura; Klett, Danièle; Combarnous, Yves

2018-05-15

We have compared various Luteinizing Hormone (LH) and Chorionic Gonadotropin (CG) preparations from non-human and human species in their ability to synergize with 10 µM forskolin (FSK) for cyclic AMP intracellular accumulation, in MLTC cells. LH from rat pituitary as well as various isoforms of pituitary ovine, bovine, porcine, equine and human LHs and equine and human CG were studied. In addition, recombinant human LH and CG were also compared with the natural human and non-human hormones. Sub-stimulating concentrations of all LHs and CGs (2-100 pM) were found to stimulate cyclic AMP accumulation in MLTC cells in the presence of an also non-stimulating FSK concentration (10 µM). Like rat LH, the most homologous available hormone for mouse MLTC cells, all non-human LHs and CG exhibit a strong potentiating effect on FSK response. The human, natural and recombinant hLH and hCG also do so but in addition, they were found to elicit a permissive effect on FSK stimulation. Indeed, when incubated alone with MLTC cells at non-stimulating concentrations (2-70 pM) hLH and hCG permit, after being removed, a dose-dependent cyclic AMP accumulation with 10 µM FSK. Our data show a clearcut difference between human LH and CG compared to their non-human counterparts on MLTC cells adenylate cyclase activity control. This points out the risk of using hCG as a reference ligand for LHR in studies using non-human cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Role of Protein Kinase C in Endothelin Converting Enzyme-1 trafficking and shedding from endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuruppu, Sanjaya, E-mail: Sanjaya.Kuruppu@med.monash.edu.au; Tochon-Danguy, Natalie; Ian Smith, A.

2010-07-23

Research highlights: {yields} PKC activation increases the trafficking of ECE-1 to the cell surface. {yields} This in turn leads to an increase in the amount of ECE-1 shed. {yields} Only the catalytically active C-terminal region is shed from the cell surface. -- Abstract: This study aimed to determine the consequences of Protein Kinase C (PKC) mediated Endothelin Converting Enzyme-1 (ECE-1) phosphorylation and its relationship to ECE-1 expression and shedding. The proteins on the surface of EA.hy926 cells were labelled with EZ-Link NHS-SS-Biotin both prior to (control) and following stimulation by 2 {mu}M phorbol 12-myristate 13-acetate (PMA) which activates PKC. Themore » biotinylated proteins were isolated using neutravidin beads, resolved by gel electrophoresis and analysed by western blotting using anti-ECE-1 antibodies. Significant increase in ECE-1 expression at the cell surface was observed following stimulation by PMA, compared to unstimulated control cells (170 {+-} 32.3% of control, n = 5). The ECE-1 activity (expressed as {mu}M substrate cleaved/min) was determined by monitoring the cleavage of a quenched fluorescent substrate. The specificity of cleavage was confirmed using the ECE-1 inhibitor (CGS35066). The stimulation of cells by PMA (1 {mu}M, 6 h) significantly increased the ECE-1 activity (0.28 {+-} 0.02; n = 3) compared to the control (0.07 {+-} 0.02; n = 3). This increase was prevented by prior incubation with the PKC inhibitor bisindolymaleimide (BIM; 2 {mu}M for 1 h; 0.10 {+-} 0.01; n = 3). Treatment with PMA also increased the activity of ECE-1 in the media (0.18 {+-} 0.01; n = 3) compared to control (0.08 {+-} 0.01; n = 3). In addition, this study confirmed by western immunoblotting that only the extracellular region of ECE-1 is released from the cell surface. These data indicate for the first time that PKC activation induces the trafficking and shedding of ECE to and from the cell surface, respectively.« less

Immunoregulatory mechanisms in Chagas disease: modulation of apoptosis in T-cell mediated immune responses.

PubMed

Chaves, Ana Thereza; de Assis Silva Gomes Estanislau, Juliana; Fiuza, Jacqueline Araújo; Carvalho, Andréa Teixeira; Ferreira, Karine Silvestre; Fares, Rafaelle Christine Gomes; Guimarães, Pedro Henrique Gazzinelli; de Souza Fagundes, Elaine Maria; Morato, Maria José; Fujiwara, Ricardo Toshio; da Costa Rocha, Manoel Otávio; Correa-Oliveira, Rodrigo

2016-04-30

Chronic Chagas disease presents different clinical manifestations ranging from asymptomatic (namely indeterminate) to severe cardiac and/or digestive. Previous results have shown that the immune response plays an important role, although no all mechanisms are understood. Immunoregulatory mechanisms such as apoptosis are important for the control of Chagas disease, possibly affecting the morbidity in chronic clinical forms. Apoptosis has been suggested to be an important mechanism of cellular response during T. cruzi infection. We aimed to further understand the putative role of apoptosis in Chagas disease and its relation to the clinical forms of the disease. Apoptosis of lymphocytes, under antigenic stimuli (soluble T. cruzi antigens - TcAg) where compared to that of non-stimulated cells. Apoptosis was evaluated using the expression of annexin and caspase 3(+) by T cells and the percentage of cells positive evaluated by flow cytometry. In addition activation and T cell markers were used for the identification of TCD4(+) and TCD8(+) subpopulations. The presence of intracellular and plasma cytokines were also evaluated. Analysis of the activation status of the peripheral blood cells showed that patients with Chagas disease presented higher levels of activation determined by the expression of activation markers, after TcAg stimulation. PCR array were used to evaluate the contribution of this mechanism in specific cell populations from patients with different clinical forms of human Chagas disease. Our results showed a reduced proliferative response associated a high expression of T CD4(+)CD62L(-) cells in CARD patients when compared with IND group and NI individuals. We also observed that both groups of patients presented a significant increase of CD4(+) and CD8(+) T cell subsets in undergoing apoptosis after in vitro stimulation with T. cruzi antigens. In CARD patients, both CD4(+) and CD8(+) T cells expressing TNF-α were highly susceptible to undergo apoptosis after in vitro stimulation. Interestingly, the in vitro TcAg stimulation increased considerably the expression of cell death TNF/TNFR superfamily and Caspase family receptors genes in CARD patients. Taken together, our results suggest that apoptosis may be an important mechanism for the control of morbidity in T. cruzi infection by modulating the expression of apoptosis genes, the cytokine environment and/or killing of effector cells.

Magnetically stimulated ciprofloxacin release from polymeric microspheres entrapping iron oxide nanoparticles

PubMed Central

Sirivisoot, Sirinrath; Harrison, Benjamin S

2015-01-01

To extend the external control capability of drug release, iron oxide nanoparticles (NPs) encapsulated into polymeric microspheres were used as magnetic media to stimulate drug release using an alternating magnetic field. Chemically synthesized iron oxide NPs, maghemite or hematite, and the antibiotic ciprofloxacin were encapsulated together within polycaprolactone microspheres. The polycaprolactone microspheres entrapping ciprofloxacin and magnetic NPs could be triggered for immediate drug release by magnetic stimulation at a maximum value of 40%. Moreover, the microspheres were cytocompatible with fibroblasts in vitro with a cell viability percentage of more than 100% relative to a nontreated control after 24 hours of culture. Macrophage cell cultures showed no signs of increased inflammatory responses after in vitro incubation for 56 hours. Treatment of Staphylococcus aureus with the magnetic microspheres under an alternating (isolating) magnetic field increased bacterial inhibition further after 2 days and 5 days in a broth inhibition assay. The findings of the present study indicate that iron oxide NPs, maghemite and hematite, can be used as media for stimulation by an external magnetic energy to activate immediate drug release. PMID:26185446

Gold nanoparticle-assisted all optical localized stimulation and monitoring of Ca2+ signaling in neurons

PubMed Central

Lavoie-Cardinal, Flavie; Salesse, Charleen; Bergeron, Éric; Meunier, Michel; De Koninck, Paul

2016-01-01

Light-assisted manipulation of cells to control membrane activity or intracellular signaling has become a major avenue in life sciences. However, the ability to perform subcellular light stimulation to investigate localized signaling has been limited. Here, we introduce an all optical method for the stimulation and the monitoring of localized Ca2+ signaling in neurons that takes advantage of plasmonic excitation of gold nanoparticles (AuNPs). We show with confocal microscopy that 800 nm laser pulse application onto a neuron decorated with a few AuNPs triggers a transient increase in free Ca2+, measured optically with GCaMP6s. We show that action potentials, measured electrophysiologically, can be induced with this approach. We demonstrate activation of local Ca2+ transients and Ca2+ signaling via CaMKII in dendritic domains, by illuminating a single or few functionalized AuNPs specifically targeting genetically-modified neurons. This NP-Assisted Localized Optical Stimulation (NALOS) provides a new complement to light-dependent methods for controlling neuronal activity and cell signaling. PMID:26857748

Characterization of MUDENG, a novel anti-apoptotic protein

PubMed Central

Choi, J-H; Lim, J-B; Wickramanayake, D D; Wagley, Y; Kim, J; Lee, H-C; Seo, H G; Kim, T-H; Oh, J-W

2016-01-01

MUDENG (Mu-2-related death-inducing gene, MuD) is revealed to be involved in cell death signaling. Astrocytes, the major glial cell type in the central nervous system, are a source of brain tumors. In this study, we examined MuD expression and function in human astroglioma cells. Stimulation of U251-MG cells with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resulted in a 40% decrease in cell viability and a 33% decrease in MuD protein levels, although not in MuD mRNA levels. To study the functional relevance of MuD expression, stable transfectants expressing high levels of MuD were generated. Stimulation of these transfectants with TRAIL resulted in enhanced cell survival (77% for stable and 46% for control transfectants). Depletion of MuD led to a marked reduction upon TRAIL stimulation in cell viability (22% in MuD-depleted cells and 54% in control cells). In addition, we observed that MuD depletion increased the susceptibility of the cells to TRAIL by enhancing the cleavage of caspase-3/-9 and BH3-interacting domain death agonist (Bid). A unique 25-kDa fragment of B-cell lymphoma 2 (Bcl-2) lacking BH4 was observed 60–180 min post TRAIL treatment in MuD-depleted cells, suggesting that Bcl-2 is converted from its anti-apoptotic form to the truncated pro-apoptotic form. Importantly, the TRAIL-mediated decrease in cell viability in MuD-depleted cells was abrogated upon Bid depletion, indicating that the role of MuD in apoptotic signaling takes place at the Bid and Bcl-2 junction. MuD localizes predominantly in the endoplasmic reticulum and partly in the mitochondria and its amounts are reduced 6 h post TRAIL stimulation, presumably via caspase-3-mediated MuD cleavage. Collectively, these results suggest that MuD, a novel signaling protein, not only possesses an anti-apoptotic function but may also constitute an important target for the design of ideal candidates for combinatorial treatment strategies for glioma cells. PMID:27136675

Mechanical Coupling of Smooth Muscle Cells Using Microengineered Substrates and Local Stimulation

NASA Astrophysics Data System (ADS)

Copeland, Craig; Hunter, David; Tung, Leslie; Chen, Christopher; Reich, Daniel

2013-03-01

Mechanical stresses directly affect many cellular processes, including signal transduction, growth, differentiation, and survival. Cells can themselves generate such stresses by activating myosin to contract the actin cytoskeleton, which in turn can regulate both cell-substrate and cell-cell interactions. We are studying mechanical forces at cell-cell and cell-substrate interactions using arrays of selectively patterned flexible PDMS microposts combined with the ability to apply local chemical stimulation. Micropipette ``spritzing'', a laminar flow technique, uses glass micropipettes mounted on a microscope stage to deliver drugs to controlled regions within a cellular construct while cell traction forces are recorded via the micropost array. The pipettes are controlled by micromanipulators allowing for rapid and precise movement across the array and the ability to treat multiple constructs within a sample. This technique allows for observing the propagation of a chemically induced mechanical stimulus through cell-cell and cell-substrate interactions. We have used this system to administer the acto-myosin inhibitors Blebbistatin and Y-27632 to single cells and observed the subsequent decrease in cell traction forces. Experiments using trypsin-EDTA have shown this system to be capable of single cell manipulation through removal of one cell within a pair configuration while leaving the other cell unaffected. This project is supported in part by NIH grant HL090747

[Natural killer cell cytotoxic activity in critical pediatric patients with suspected hemophagocytic syndrome].

PubMed

Martínez, I; Fernández, L; Valentín, J; Castillo, C; Chamorro, C; Pérez-Martínez, A

2015-05-01

To determine the role of natural killer (NK) cytotoxic activity in patients with suspected hemophagocytic lymphohistiocytosis syndrome (HLH). A prospective study was conducted from September 2008 to February 2014. The study was carried out in the Hematological Oncology Laboratory of Hospital Infantil Universitario Niño Jesús, Madrid (Spain). We analyzed 30 peripheral blood samples from intensive care patients with suspected HLH. There were 18 males and 12 females, with a mean age of 4.7 years (range 0.2-22). NK cell cytotoxicity was compared with healthy controls according to age and sex. In vitro NK cell cytotoxicity against the K562 cell line was determined by time-resolved fluorescence (Europium-TDA) under resting conditions, after interleukin 15 stimulation, and following block with Fas ligand antibody. NK cell cytotoxicity. A total of 20 patients showed a significant decrease of NK cell activity compared with controls (P=.001). Nine of these patients were diagnosed with primary HLH. A total of 10 patients were diagnosed with secondary HLH. Cytotoxic activity was normal in 10 subjects. None of them were diagnosed with HLH. Interleukin 15 stimulation increased NK cell cytotoxicity in secondary HLH, and blocking Fas ligand on NK cells decreased cytotoxic activity in primary HLH patients (P=.001). In our experience, NK cell cytotoxic activity measured by time-resolved fluorescence is a simple and useful clinical diagnostic test for HLH. Interleukin 15 stimulation and Fas ligand blocking on NK cells could help differentiate between primary and secondary HLH. Copyright © 2014 Elsevier España, S.L.U. and SEMICYUC. All rights reserved.

Neural feedback for instantaneous spatiotemporal modulation of afferent pathways in bi-directional brain-machine interfaces.

PubMed

Liu, Jianbo; Khalil, Hassan K; Oweiss, Karim G

2011-10-01

In bi-directional brain-machine interfaces (BMIs), precisely controlling the delivery of microstimulation, both in space and in time, is critical to continuously modulate the neural activity patterns that carry information about the state of the brain-actuated device to sensory areas in the brain. In this paper, we investigate the use of neural feedback to control the spatiotemporal firing patterns of neural ensembles in a model of the thalamocortical pathway. Control of pyramidal (PY) cells in the primary somatosensory cortex (S1) is achieved based on microstimulation of thalamic relay cells through multiple-input multiple-output (MIMO) feedback controllers. This closed loop feedback control mechanism is achieved by simultaneously varying the stimulation parameters across multiple stimulation electrodes in the thalamic circuit based on continuous monitoring of the difference between reference patterns and the evoked responses of the cortical PY cells. We demonstrate that it is feasible to achieve a desired level of performance by controlling the firing activity pattern of a few "key" neural elements in the network. Our results suggest that neural feedback could be an effective method to facilitate the delivery of information to the cortex to substitute lost sensory inputs in cortically controlled BMIs.

Heat-killed cell preparation of Corynebacterium glutamicum stimulates the immune activity and improves survival of mice against enterohemorrhagic Escherichia coli.

PubMed

Ebisawa, Makoto; Tsukahara, Takamitsu; Fudou, Ryosuke; Ohta, Yasuhiro; Tokura, Mitsunori; Onishi, Norimasa; Fujieda, Takeshi

2017-05-01

Fermentation by Corynebacterium glutamicum is used by various industries to produce L-Glutamate, and the heat-killed cell preparation of this bacterium (HCCG) is a by-product of the fermentation process. In present study, we evaluated the immunostimulating and survival effects against enterohemorrhagic Escherichia coli (STEC) infection of HCCG. HCCG significantly stimulated in vitro IgA and interleukin-12 p70 production in murine Peyer's patch cells and peritoneal macrophages, respectively. Oral administration of 10 mg/kg body weight (BW) of HCCG for seven consecutive days stimulated IgA concentration in murine cecal digesta. Mice were orally administered HCCG for 17 consecutive days (d0-d17), and challenged with STEC on d4 to d6. Survival of mice tended to improve by 100 mg/kg BW of HCCG administration compared with those in control group. In conclusion, HCCG supplementation was found to prevent STEC infection in mice, and thus it may have the potential to stimulate the immune status of mammals.

In vitro stimulation of peripheral blood mononuclear cells (PBMC) from HIV- and HIV+ chancroid patients by Haemophilus ducreyi antigens.

PubMed Central

Van Laer, L; Vingerhoets, J; Vanham, G; Kestens, L; Bwayo, J; Otido, J; Piot, P; Roggen, E

1995-01-01

The cellular immune responses to fractionated Haemophilus ducreyi antigens, coated on latex beads, were assessed in patients with chancroid and in controls, using an in vitro lymphocyte proliferation assay. Several fractions of H. ducreyi antigen revealed stimulating activity. However, only the molecular size ranges 91-78 kD, 59-29 kD, and 25-21 kD induced proliferation that may be specifically related to H. ducreyi infection. Lymphocytes from four HIV- patients, successfully treated for chancroid, were not stimulated by H. ducreyi antigen. In general, lymphocytes from HIV+ chancroid patients were less responsive to H. ducreyi antigen compared with those from HIV- chancroid patients. However, two HIV-infected patients showed exceptionally strong responses to high molecular weight fractions. To our knowledge this is the first report demonstrating that H. ducreyi contains specific T cell-stimulating antigens. Based on this work, further identification and purification of the T cell antigens is feasible. PMID:7586673

In vitro stimulation of peripheral blood mononuclear cells (PBMC) from HIV- and HIV+ chancroid patients by Haemophilus ducreyi antigens.

PubMed

Van Laer, L; Vingerhoets, J; Vanham, G; Kestens, L; Bwayo, J; Otido, J; Piot, P; Roggen, E

1995-11-01

The cellular immune responses to fractionated Haemophilus ducreyi antigens, coated on latex beads, were assessed in patients with chancroid and in controls, using an in vitro lymphocyte proliferation assay. Several fractions of H. ducreyi antigen revealed stimulating activity. However, only the molecular size ranges 91-78 kD, 59-29 kD, and 25-21 kD induced proliferation that may be specifically related to H. ducreyi infection. Lymphocytes from four HIV- patients, successfully treated for chancroid, were not stimulated by H. ducreyi antigen. In general, lymphocytes from HIV+ chancroid patients were less responsive to H. ducreyi antigen compared with those from HIV- chancroid patients. However, two HIV-infected patients showed exceptionally strong responses to high molecular weight fractions. To our knowledge this is the first report demonstrating that H. ducreyi contains specific T cell-stimulating antigens. Based on this work, further identification and purification of the T cell antigens is feasible.

A caspase 8-based suicide switch induces apoptosis in nanobody-directed chimeric receptor expressing T cells.

PubMed

Khaleghi, Sepideh; Rahbarizadeh, Fatemeh; Ahmadvand, Davoud; Rasaee, Mohammad J; Pognonec, Philippe

2012-04-01

In accordance with the two-step hypothesis of T cell activation and the observation that stimulation through the T cell receptor (TCR) alone may lead to anergy, we focused on the introduction of co-stimulatory signaling to this type of receptors to achieve optimal activation. Enhanced mRNA and cell surface receptor expression via the co-stimulatory gene fragment (OX40) was confirmed by RT-PCR and flow cytometry. Inclusion of the OX40 co-stimulatory signaling region in series with the TCR led to enhanced antigen-induced IL-2 production after stimulation by MUC1-expressing cancer cell lines as compared to the chimeric receptor without OX40. Moreover, with the aim of maintaining high efficiency, while providing a means of controlling any possible unwanted proliferation in vivo, a regulation system was used. This controls the dimerization of a membrane-bound caspase 8 protein. Toward that goal, pFKC8 and CAR constructs were co-transfected into Jurkat cells, and the level of apoptosis was measured. 24 h after addition of the dimerizer, a 91% decrease in transfected cells was observed.

A microfluidic platform for controlled biochemical stimulation of twin neuronal networks.

PubMed

Biffi, Emilia; Piraino, Francesco; Pedrocchi, Alessandra; Fiore, Gianfranco B; Ferrigno, Giancarlo; Redaelli, Alberto; Menegon, Andrea; Rasponi, Marco

2012-06-01

Spatially and temporally resolved delivery of soluble factors is a key feature for pharmacological applications. In this framework, microfluidics coupled to multisite electrophysiology offers great advantages in neuropharmacology and toxicology. In this work, a microfluidic device for biochemical stimulation of neuronal networks was developed. A micro-chamber for cell culturing, previously developed and tested for long term neuronal growth by our group, was provided with a thin wall, which partially divided the cell culture region in two sub-compartments. The device was reversibly coupled to a flat micro electrode array and used to culture primary neurons in the same microenvironment. We demonstrated that the two fluidically connected compartments were able to originate two parallel neuronal networks with similar electrophysiological activity but functionally independent. Furthermore, the device allowed to connect the outlet port to a syringe pump and to transform the static culture chamber in a perfused one. At 14 days invitro, sub-networks were independently stimulated with a test molecule, tetrodotoxin, a neurotoxin known to block action potentials, by means of continuous delivery. Electrical activity recordings proved the ability of the device configuration to selectively stimulate each neuronal network individually. The proposed microfluidic approach represents an innovative methodology to perform biological, pharmacological, and electrophysiological experiments on neuronal networks. Indeed, it allows for controlled delivery of substances to cells, and it overcomes the limitations due to standard drug stimulation techniques. Finally, the twin network configuration reduces biological variability, which has important outcomes on pharmacological and drug screening.

The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder

NASA Astrophysics Data System (ADS)

Dalby, Matthew J.; Gadegaard, Nikolaj; Tare, Rahul; Andar, Abhay; Riehle, Mathis O.; Herzyk, Pawel; Wilkinson, Chris D. W.; Oreffo, Richard O. C.

2007-12-01

A key tenet of bone tissue engineering is the development of scaffold materials that can stimulate stem cell differentiation in the absence of chemical treatment to become osteoblasts without compromising material properties. At present, conventional implant materials fail owing to encapsulation by soft tissue, rather than direct bone bonding. Here, we demonstrate the use of nanoscale disorder to stimulate human mesenchymal stem cells (MSCs) to produce bone mineral in vitro, in the absence of osteogenic supplements. This approach has similar efficiency to that of cells cultured with osteogenic media. In addition, the current studies show that topographically treated MSCs have a distinct differentiation profile compared with those treated with osteogenic media, which has implications for cell therapies.

Computational optogenetics: A novel continuum framework for the photoelectrochemistry of living systems

NASA Astrophysics Data System (ADS)

Wong, Jonathan; Abilez, Oscar J.; Kuhl, Ellen

2012-06-01

Electrical stimulation is currently the gold standard treatment for heart rhythm disorders. However, electrical pacing is associated with technical limitations and unavoidable potential complications. Recent developments now enable the stimulation of mammalian cells with light using a novel technology known as optogenetics. The optical stimulation of genetically engineered cells has significantly changed our understanding of electrically excitable tissues, paving the way towards controlling heart rhythm disorders by means of photostimulation. Controlling these disorders, in turn, restores coordinated force generation to avoid sudden cardiac death. Here, we report a novel continuum framework for the photoelectrochemistry of living systems that allows us to decipher the mechanisms by which this technology regulates the electrical and mechanical function of the heart. Using a modular multiscale approach, we introduce a non-selective cation channel, channelrhodopsin-2, into a conventional cardiac muscle cell model via an additional photocurrent governed by a light-sensitive gating variable. Upon optical stimulation, this channel opens and allows sodium ions to enter the cell, inducing electrical activation. In side-by-side comparisons with conventional heart muscle cells, we show that photostimulation directly increases the sodium concentration, which indirectly decreases the potassium concentration in the cell, while all other characteristics of the cell remain virtually unchanged. We integrate our model cells into a continuum model for excitable tissue using a nonlinear parabolic second-order partial differential equation, which we discretize in time using finite differences and in space using finite elements. To illustrate the potential of this computational model, we virtually inject our photosensitive cells into different locations of a human heart, and explore its activation sequences upon photostimulation. Our computational optogenetics tool box allows us to virtually probe landscapes of process parameters, and to identify optimal photostimulation sequences with the goal to pace human hearts with light and, ultimately, to restore mechanical function.

Computational Optogenetics: A Novel Continuum Framework for the Photoelectrochemistry of Living Systems.

PubMed

Wong, Jonathan; Abilez, Oscar J; Kuhl, Ellen

2012-06-01

Electrical stimulation is currently the gold standard treatment for heart rhythm disorders. However, electrical pacing is associated with technical limitations and unavoidable potential complications. Recent developments now enable the stimulation of mammalian cells with light using a novel technology known as optogenetics. The optical stimulation of genetically engineered cells has significantly changed our understanding of electrically excitable tissues, paving the way towards controlling heart rhythm disorders by means of photostimulation. Controlling these disorders, in turn, restores coordinated force generation to avoid sudden cardiac death. Here, we report a novel continuum framework for the photoelectrochemistry of living systems that allows us to decipher the mechanisms by which this technology regulates the electrical and mechanical function of the heart. Using a modular multiscale approach, we introduce a non-selective cation channel, channelrhodopsin-2, into a conventional cardiac muscle cell model via an additional photocurrent governed by a light-sensitive gating variable. Upon optical stimulation, this channel opens and allows sodium ions to enter the cell, inducing electrical activation. In side-by-side comparisons with conventional heart muscle cells, we show that photostimulation directly increases the sodium concentration, which indirectly decreases the potassium concentration in the cell, while all other characteristics of the cell remain virtually unchanged. We integrate our model cells into a continuum model for excitable tissue using a nonlinear parabolic second order partial differential equation, which we discretize in time using finite differences and in space using finite elements. To illustrate the potential of this computational model, we virtually inject our photosensitive cells into different locations of a human heart, and explore its activation sequences upon photostimulation. Our computational optogenetics tool box allows us to virtually probe landscapes of process parameters, and to identify optimal photostimulation sequences with the goal to pace human hearts with light and, ultimately, to restore mechanical function.

Altered Regulation of Airway Epithelial Cell Chloride Channels in Cystic Fibrosis

NASA Astrophysics Data System (ADS)

Frizzell, Raymond A.; Rechkemmer, Gerhard; Shoemaker, Richard L.

1986-08-01

In many epithelial cells the chloride conductance of the apical membrane increases during the stimulation of electrolyte secretion. Single-channel recordings from human airway epithelial cells showed that β -adrenergic stimulation evoked apical membrane chloride channel activity, but this response was absent in cells from patients with cystic fibrosis (CF). However, when membrane patches were excised from CF cells into media containing sufficient free calcium (approximately 180 nanomolar), chloride channels were activated. The chloride channels of CF cells were similar to those of normal cells as judged by their current-voltage relations, ion selectivity, and kinetic behavior. These findings demonstrate the presence of chloride channels in the apical membranes of CF airway cells. Their regulation by calcium appears to be intact, but cyclic adenosine monophosphate (cAMP)-dependent control of their activity is defective.

Sarcoidosis Th17 Cells are ESAT-6 Antigen Specific but Demonstrate Reduced IFN-γ Expression

PubMed Central

Richmond, Bradley W.; Ploetze, Kristen; Isom, Joan; Chambers-Harris, Isfahan; Braun, Nicole A.; Taylor, Thyneice; Abraham, Susamma; Mageto, Yolanda; Culver, Dan A.; Oswald-Richter, Kyra A.; Drake, Wonder P.

2013-01-01

Rationale Sarcoidosis is a granulomatous disease of unknown etiology. Many patients with sarcoidosis demonstrate antigen-specific immunity to mycobacterial virulence factors. Th-17 cells are crucial to the immune response in granulomatous inflammation, and have recently been shown to be present in greater numbers in the peripheral blood and bronchoalveolar lavage (BAL) fluid (BALF) of sarcoidosis patients than healthy controls. It is unclear whether Th-17 cells in sarcoidosis are specific for mycobacterial antigens, or whether they have similar functionality to control Th-17 cells. Methods Flow cytometry was used to determine the numbers of Th-17 cells present in the peripheral blood and BALF of patients with sarcoidosis, the percentage of Th-17 cells that were specific to the mycobacterial virulence factor ESAT-6, and as well as to assess IFN-γ expression in Th-17 cells following polyclonal stimulation. Results Patients with sarcoidosis had greater numbers of Th-17 cells in the peripheral blood and BALF than controls and produced significantly more extracellular IL-17A (p=0.03 and p=0.02, respectively). ESAT-6 specific Th-17 cells were present in both peripheral blood and BALF of sarcoidosis patients (p<0.001 and p=0.03, respectively). After polyclonal stimulation, Th-17 cells from sarcoidosis patients produced less IFN-γ than healthy controls. Conclusions Patients with sarcoidosis have mycobacterial antigen-specific Th-17 cells peripherally and in sites of active sarcoidosis involvement. Despite the Th1 immunophenotype of sarcoidosis immunology, the Th-17 cells have reduced IFN-γ expression, compared to healthy controls. This reduction in immunity may contribute to sarcoidosis pathogenesis. PMID:23073617

Estrogen sulfotransferases in breast and endometrial cancers.

PubMed

Pasqualini, Jorge Raul

2009-02-01

Estrogen sulfotransferase is significantly more active in the normal breast cell (e.g., Human 7) than in the cancer cell (e.g., MCF-7). The data suggest that in breast cancer sulfoconjugated activity is carried out by another enzyme, the SULT1A, which acts at high concentration of the substrates. In breast cancer cells sulfotransferase (SULT) activity can be stimulated by various progestins: medrogestone, promegestone, and nomegestrol acetate, as well as by tibolone and its metabolites. SULT activities can also be controlled by other substances including phytoestrogens, celecoxib, flavonoids (e.g., quercetin, resveratrol), and isoflavones. SULT expression was localized in breast cancer cells, which can be stimulated by promegestone and correlated with the increase of the enzyme activity. The estrogen sulfotransferase (SULT1E1), which acts at nanomolar concentration of estradiol, can inactivate most of this hormone present in the normal breast; however, in the breast cancer cells, the sulfotransferase denoted as SULT1A1 is mainly present, and this acts at micromolar concentrations of E(2). A correlation was postulated among breast cancer cell proliferation, the effect of various progestins, and sulfotransferase stimulation. In conclusion, it is suggested that factors involved in the stimulation of the estrogen sulfotransferases could provide new possibilities for the treatment of patients with hormone-dependent breast and endometrial cancers.

[Response of peripheral blood mononuclear leukocyte to nickel stimulation in patients with systemic and contact allergy to nickel].

PubMed

Czarnobilska, Ewa; Thor, Piotr; Kaszuba-Zwoinska, Jolanta; Słodowska-Hajduk, Zofia; Stobiecki, Marcin; Dyga, Wojciech; Wsołek, Katarzyna; Obtułowicz, Krystyna

2006-01-01

Nickel is knows as the most common cause of allergic contact dermatitis, as well as diffuse eczema, allergic rhinitis and bronchial asthma. The mechanism of contact allergy to nickel is well known. In spite of numerous investigations, the mechanism of systemic allergy to nickel is still not clear. 22 patients with positive patch tests to nickel were analyzed. On basis of clinical symptoms the patients were divided into two groups: 1. with contact allergy dermatitis to nickel--8 patients 2. with systemic allergy to nickel (allergic rhinitis and/or diffuse eczema--14 patients. The control group included non-atopic patients with negative patch test to nickel--6 patients. 10 ml of blood were taken from each patient and peripheral mononuclear blood cells (PMBC) were isolated. In PBMC culture, NiSO4 and PHA were stimulated. The control group was non-stimulated cells. The supernatants were collected after 3 and 6 days of culture and the levels of cytokines IL-5, 4 and IFNgamma were measured (ELISA). The concentration of IFNgamma in supernatants from stimulated as well as non-stimulated cells from patients with contact allergy to nickel was higher in comparison to the control group. The concentration of IL-5 in this group was low. There was an increase in the production of IFNgamma and IL-5 after NiSO4 stimulation in patients with systemic allergy to nickel. The higher concentration of IFNgamma in the same groups of patients investigated was in supernatants from the third day of PBMC culture were compared to the sixth day. After 3 and 6 days of culture, the concentration of IL-4 (ELISA) was below detection level in all supernatants analyzed. IFNgamma plays an essential role in the mechanism of developing of contact allergy to nickel; and IFNgamma as well as IL-5 play a role in the mechanism of developing systemic allergy to nickel. The third day of PBMC culture is more reliable for IFNgamma estimation.

Biomarker-guided screening of Juzen-taiho-to, an oriental herbal formulation for immunostimulation.

PubMed

Takaoka, Anna; Iacovidou, Maria; Hasson, Tal H; Montenegro, Diego; Li, Xiangming; Tsuji, Moriya; Kawamura, Akira

2014-03-01

Juzen-taiho-to is an immunostimulatory herbal formulation that is clinically used in East Asia for cancer patients undergoing chemotherapy and radiation. The formulation stimulates various leukocytes, including T, B, and NK cells and macrophages. Although Juzen-taiho-to is known to contain numerous compounds with various pharmacological activities, it is not clear which compounds are responsible for the stimulation of individual cell types. Here, we conducted what we call "biomarker-guided screening" to purify compounds responsible for the macrophages stimulatory activity. To this end, gene expression was analyzed by a DNA array for macrophages treated with Juzen-taiho-to and DMSO (vehicle control), which identified intercellular adhesion molecule 1 as a biomarker of macrophage stimulation by Juzen-taiho-to. A quantitative reverse transcription polymerase chain reaction assay of intercellular adhesion molecule 1 was then used to guide the purification of active compounds. The screening resulted in the purification of a glycolipid mixture, containing β-glucosylceramides. The glycolipid mixture potently stimulated intercellular adhesion molecule 1 expression in primary dendritic cells as well as in primary CD14+ (macrophages) cells. The identification of this glycolipid mixture opens up an opportunity for further studies to understand how plant-derived glycolipids stimulate macrophages and dendritic cells in a safe and effective manner as demonstrated by Juzen-taiho-to. Georg Thieme Verlag KG Stuttgart · New York.

Interferon beta 2/B-cell stimulatory factor type 2 shares identity with monocyte-derived hepatocyte-stimulating factor and regulates the major acute phase protein response in liver cells.

PubMed Central

Gauldie, J; Richards, C; Harnish, D; Lansdorp, P; Baumann, H

1987-01-01

One of the oldest and most preserved of the homeostatic responses of the body to injury is the acute phase protein response associated with inflammation. The liver responds to hormone-like mediators by the increased synthesis of a series of plasma proteins called acute phase reactants. In these studies, we examined the relationship of hepatocyte-stimulating factor derived from peripheral blood monocytes to interferon beta 2 (IFN-beta 2), which has been cloned. Antibodies raised against fibroblast-derived IFN-beta having neutralizing activity against both IFN-beta 1 and -beta 2 inhibited the major hepatocyte-stimulating activity derived from monocytes. Fibroblast-derived mediator elicited the identical stimulated response in human HepG2 cells and primary rat hepatocytes as the monocyte cytokine. Finally, recombinant-derived human B-cell stimulatory factor type 2 (IFN-beta 2) from Escherichia coli induced the synthesis of all major acute phase proteins studied in human hepatoma HepG2 and primary rat hepatocyte cultures. These data demonstrate that monocyte-derived hepatocyte-stimulating factor and IFN-beta 2 share immunological and functional identity and that IFN-beta 2, also known as B-cell stimulatory factor and hybridoma plasmacytoma growth factor, has the hepatocyte as a major physiologic target and thereby is essential in controlling the hepatic acute phase response. Images PMID:2444978

Pulsed electromagnetic field affects intrinsic and endoplasmatic reticulum apoptosis induction pathways in MonoMac6 cell line culture.

PubMed

Kaszuba-Zwoinska, J; Chorobik, P; Juszczak, K; Zaraska, W; Thor, P J

2012-10-01

Current studies were aimed to elucidate influence of pulsed electromagnetic field stimulation on cell viability and apoptosis induction pathways. For the experimental model we have chosen monocytic cell line MonoMac6 and several apoptosis inducers with different mechanism of death induction like puromycin, colchicine, cyclophosphamide, minocycline and hydrogen peroxide. MonoMac6 cell line was grown at density 1x10(5) cells/well in 96-well culture plates. To induce cell death cell cultures were treated with different apoptosis inducers like puromycin, colchicine, cyclophosphamide, minocycline, hydrogen peroxide and at the same time with pulsed electromagnetic field 50 Hz, 45±5 mT (PEMF) for 4 hour per each stimulation, three times, in 24 hours intervals. Afterwards, cells were harvested for flow cytometry analysis of cell viability measured by annexin V-APC labeled and propidium iodide staining. Expression of apoptosis related genes was evaluated by semi quantitative reverse transcription (RT)-PCR assay. NuPAGE Novex Western blot analysis was carried out for apoptosis inducing factor (AIF) abundance in cytosolic and nuclear extracts of MonoMac6 cells. Puromycin, colchicine and minocycline activated cells and simultaneously treated with PEMF have shown out diminished percentage of annexinV positive (AnV+) cells comparing to controls without PEMF stimulation. MonaMac6 cells puromycin/colchicyne and PEMF treated were to a higher extent double stained (AnV+,PI+), which means increased late apoptotic as well as necrotic (PI+) cells, than non-stimulated controls. On the other hand, minocycline activated cells prior to PEMF treatment showed diminished amount of apoptotic and necrotic (annexin V, annexin V and propidium iodide, propidium iodide positive staining) cells. The opposite effect of PEMF on the percentage of annexin V positively stained cells has been achieved after treatment of MonoMac6 culture with cyclophoshamide and hydrogen peroxide. PEMF enhanced early phase of apoptosis induced by both apoptosis inducing agents. The analysis of expression of the apoptosis related genes in MonoMac6 cultures treated with puromycin and exposed to PEMF performed in reverse transcription of polymerase chain reaction (PCR) assay has shown changes in mRNA of genes engaged in intrinsic apoptotic pathway and pathway with AIF abundance. The most influenced was expression of gene belonging to pro-apoptotic family of Bcl-2 and AIF agent. Examination of immunoblots developed with anti-AIF antibody showed that cytosol content of AIF protein was diminished after puromycin and PEMF treatment of MonoMac6 cells. The obtained results indicate that PEMF affects induction of apoptosis in MonoMac6 cells stimulated to death with inducing agents to a different extent. Main finding of the current results is that, PEMF stimulation of MonoMac6 cells simultaneously treated with puromycin caused changes in the Bcl-family genes expression as well as in caspase independent pathway of apoptosis inducing factor (AIF).

Fecal bacteria from treatment-naive Crohn's disease patients can skew helper T cell responses.

PubMed

Ma, Fei; Zhang, Yi; Xing, Junjie; Song, Xiaoling; Huang, Ling; Weng, Hao; Wu, Xiangsong; Walker, Emma; Wang, Zhongchuan

2017-12-01

Many studies have demonstrated that the inflamed mucosa of Crohn's disease (CD) patients presented a disturbed gut commensal community, and the shift in microbial composition and species variety is associated with disease severity. To establish a link between changes in the intestinal bacterial composition and the alteration of inflammation, we obtained fecal bacteria from CD patients and non-CD controls. The bacteria were then used to stimulate the peripheral blood mononuclear cells (PBMCs) from one non-CD individual. We found that the frequency of IFN-γ- and IL-17-expressing CD4 T cells was significantly higher after stimulation with CD bacteria than with non-CD bacteria, while the frequency of IL-4- and IL-10-expressing CD4 T cells was significantly decreased after stimulation with CD bacteria. A similar trend was observed in the level of cytokine expression and transcription expression. However, this difference was not clear-cut, as overlapping regions were observed between the two groups. With longer stimulation using CD bacteria, the skewing toward Th1/Th17 responses were further increased. This increase depended on the presence of monocytes/macrophages. Interestingly, we also found that B cells presented an inhibitory effect in CD bacteria-mediated skewing toward Th1/Th17 cells and promoted IL-10 secretion in CD bacteria-stimulated PBMCs. Together, our results demonstrated that CD bacteria could promote Th1/Th17 inflammation in a host factor-independent fashion. Copyright © 2017 Elsevier Inc. All rights reserved.

Potent Cell-Intrinsic Immune Responses in Dendritic Cells Facilitate HIV-1-Specific T Cell Immunity in HIV-1 Elite Controllers.

PubMed

Martin-Gayo, Enrique; Buzon, Maria Jose; Ouyang, Zhengyu; Hickman, Taylor; Cronin, Jacqueline; Pimenova, Dina; Walker, Bruce D; Lichterfeld, Mathias; Yu, Xu G

2015-06-01

The majority of HIV-1 elite controllers (EC) restrict HIV-1 replication through highly functional HIV-1-specific T cell responses, but mechanisms supporting the evolution of effective HIV-1-specific T cell immunity in these patients remain undefined. Cytosolic immune recognition of HIV-1 in conventional dendritic cells (cDC) can facilitate priming and expansion of HIV-1-specific T cells; however, HIV-1 seems to be able to avoid intracellular immune recognition in cDCs in most infected individuals. Here, we show that exposure of cDCs from EC to HIV-1 leads to a rapid and sustained production of type I interferons and upregulation of several interferon-stimulated effector genes. Emergence of these cell-intrinsic immune responses was associated with a reduced induction of SAMHD1 and LEDGF/p75, and an accumulation of viral reverse transcripts, but inhibited by pharmacological blockade of viral reverse transcription or siRNA-mediated silencing of the cytosolic DNA sensor cGAS. Importantly, improved cell-intrinsic immune recognition of HIV-1 in cDCs from elite controllers translated into stronger abilities to stimulate and expand HIV-1-specific CD8 T cell responses. These data suggest an important role of cell-intrinsic type I interferon secretion in dendritic cells for the induction of effective HIV-1-specific CD8 T cells, and may be helpful for eliciting functional T cell immunity against HIV-1 for preventative or therapeutic clinical purposes.

Biomarker-guided screening of Juzen-taiho-to, an Oriental herbal formulation for immmunostimulation

PubMed Central

Takaoka, Anna; Iacovidou, Maria; Hasson, Tal H.; Montenegro, Diego; Li, Xiangming; Tsuji, Moriya; Kawamura, Akira

2014-01-01

Juzen-taiho-to (JTT) is an immunostimulatory herbal formulation that is clinically used in East Asia for cancer patients undergoing chemotherapy and radiation. The formulation stimulates various leukocytes, including T, B, and NK cells and macrophages (MΦ). Although JTT is known to contain numerous compounds with various pharmacological activities, it is not clear which compounds are responsible for the stimulation of individual cell types. Here, we conducted what we call, “biomarker-guided screening,” to purify compounds responsible for the MΦ stimulatory activity. To this end, gene expression was analyzed by a DNA array for MΦ treated with JTT and DMSO (vehicle control), which identified intercellular adhesion molecule 1 (ICAM-1) as a biomarker of MΦ-stimulation by JTT. A qRT-PCR assay of ICAM-1 was then used to guide the purification of active compounds. The screening resulted in the purification of a glycolipid mixture, containing β-glucosylceramides. The glycolipid mixture potently stimulated ICAM-1 expression in primary dendritic cells (DC) as well as in primary CD14+ (MΦ) cells. Identification of this glycolipid mixture opens an opportunity for further studies to understand how plant-derived glycolipids stimulate MΦ and DC in a safe and effective manner as demonstrated by JTT. PMID:24549928

Activation of adenosine A(3) receptors supports hematopoiesis-stimulating effects of granulocyte colony-stimulating factor in sublethally irradiated mice.

PubMed

Hofer, Michal; Pospísil, Milan; Sefc, Ludek; Dusek, Ladislav; Vacek, Antonín; Holá, Jirina; Hoferová, Zuzana; Streitová, Denisa

2010-08-01

Research areas of 'post-exposure treatment' and 'cytokines and growth factors' have top priority among studies aimed at radiological nuclear threat countermeasures. The experiments were aimed at testing the ability of N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA), an adenosine A(3) receptor agonist, to modulate hematopoiesis in sublethally irradiated mice, when administered alone or in a combination with granulocyte colony-stimulating factor (G-CSF) in a two-day post-irradiation treatment regimen. A complete analysis of hematopoiesis including determination of numbers of bone marrow hematopoietic progenitor and precursor cells, as well as of numbers of peripheral blood cells, was performed. The outcomes of the treatment were assessed at days 3 to 22 after irradiation. IB-MECA alone has been found to induce a significant elevation of numbers of bone marrow granulocyte-macrophage progenitor cells (GM-CFC) and peripheral blood neutrophils. IB-MECA given concomitantly with G-CSF increased significantly bone marrow GM-CFC and erythroid progenitor cells (BFU-E) in comparison with the controls and with animals administered each of the drugs alone. The findings suggest the ability of IB-MECA to stimulate hematopoiesis and to support the hematopoiesis-stimulating effects of G-CSF in sublethally irradiated mice.

The control of fatty acid metabolism in liver cells from fed and starved sheep.

PubMed Central

Lomax, M A; Donaldson, I A; Pogson, C I

1983-01-01

Isolated liver cells prepared from starved sheep converted palmitate into ketone bodies at twice the rate seen with cells from fed animals. Carnitine stimulated palmitate oxidation only in liver cells from fed sheep, and completely abolished the difference between fed and starved animals in palmitate oxidation. The rates of palmitate oxidation to CO2 and of octanoate oxidation to ketone bodies and CO2 were not affected by starvation or carnitine. Neither starvation nor carnitine altered the ratio of 3-hydroxybutyrate to acetoacetate or the rate of esterification of [1-14C]palmitate. Propionate, lactate, pyruvate and fructose inhibited ketogenesis from palmitate in cells from fed sheep. Starvation or the addition of carnitine decreased the antiketogenic effectiveness of gluconeogenic precursors. Propionate was the most potent inhibitor of ketogenesis, 0.8 mM producing 50% inhibition. Propionate, lactate, fructose and glycerol increased palmitate esterification under all conditions examined. Lactate, pyruvate and fructose stimulated oxidation of palmitate and octanoate to CO2. Starvation and the addition of gluconeogenic precursors stimulated apparent palmitate utilization by cells. Propionate, lactate and pyruvate decreased cellular long-chain acylcarnitine concentrations. Propionate decreased cell contents of CoA and acyl-CoA. It is suggested that propionate may control hepatic ketogenesis by acting at some point in the beta-oxidation sequence. The results are discussed in relation to the differences in the regulation of hepatic fatty acid metabolism between sheep and rats. PMID:6615480

Temporal dynamics of contrast gain in single cells of the cat striate cortex.

PubMed

Bonds, A B

1991-03-01

The response amplitude of cat striate cortical cells is usually reduced after exposure to high-contrast stimuli. The temporal characteristics and contrast sensitivity of this phenomenon were explored by stimulating cortical cells with drifting gratings in which contrast sequentially incremented and decremented in stepwise fashion over time. All responses showed a clear hysteresis, in which contrast gain dropped on average 0.36 log unit and then returned to baseline values within 60 s. Noticeable gain adjustments were seen in as little as 3 s and with peak contrasts as low as 3%. Contrast adaptation was absent in responses from LGN cells. Adaptation was found to depend on temporal frequency of stimulation, with greater and more rapid adaptation at higher temporal frequencies. Two different tests showed that the mechanism controlling response reduction was influenced primarily by stimulus contrast rather than response amplitude. These results support the existence of a rapid and sensitive cortically based system that normalizes the output of cortical cells as a function of local mean contrast. Control of the adaptation appears to arise at least in part across a population of cells, which is consistent with the idea that the gain control serves to limit the information converging from many cells onto subsequent processing areas.

Innate control of adaptive immunity: Beyond the three-signal paradigm

PubMed Central

Jain, Aakanksha; Pasare, Chandrashekhar

2017-01-01

Activation of cells in the adaptive immune system is a highly orchestrated process dictated by multiples cues from the innate immune system. Although the fundamental principles of innate control of adaptive immunity are well established, it is not fully understood how innate cells integrate qualitative pathogenic information in order to generate tailored protective adaptive immune responses. In this review, we discuss complexities involved in the innate control of adaptive immunity that extend beyond T cell receptor engagement, co-stimulation and priming cytokine production but are critical for generation of protective T cell immunity. PMID:28483987

Single cell RNA Seq reveals dynamic paracrine control of cellular variation

PubMed Central

Shalek, Alex K.; Satija, Rahul; Shuga, Joe; Trombetta, John J.; Gennert, Dave; Lu, Diana; Chen, Peilin; Gertner, Rona S.; Gaublomme, Jellert T.; Yosef, Nir; Schwartz, Schraga; Fowler, Brian; Weaver, Suzanne; Wang, Jing; Wang, Xiaohui; Ding, Ruihua; Raychowdhury, Raktima; Friedman, Nir; Hacohen, Nir; Park, Hongkun; May, Andrew P.; Regev, Aviv

2014-01-01

High-throughput single-cell transcriptomics offers an unbiased approach for understanding the extent, basis, and function of gene expression variation between seemingly identical cells. Here, we sequence single-cell RNA-Seq libraries prepared from over 1,700 primary mouse bone marrow derived dendritic cells (DCs) spanning several experimental conditions. We find substantial variation between identically stimulated DCs, in both the fraction of cells detectably expressing a given mRNA and the transcript’s level within expressing cells. Distinct gene modules are characterized by different temporal heterogeneity profiles. In particular, a “core” module of antiviral genes is expressed very early by a few “precocious” cells, but is later activated in all cells. By stimulating cells individually in sealed microfluidic chambers, analyzing DCs from knockout mice, and modulating secretion and extracellular signaling, we show that this response is coordinated via interferon-mediated paracrine signaling. Surprisingly, preventing cell-to-cell communication also substantially reduces variability in the expression of an early-induced “peaked” inflammatory module, suggesting that paracrine signaling additionally represses part of the inflammatory program. Our study highlights the importance of cell-to-cell communication in controlling cellular heterogeneity and reveals general strategies that multicellular populations use to establish complex dynamic responses. PMID:24919153

Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dong, Yan; Hirane, Miku; Araki, Mutsumi

2014-04-04

Highlights: • LPA{sub 5} inhibits the cell growth and motile activities of 3T3 cells. • LPA{sub 5} suppresses the cell motile activities stimulated by hydrogen peroxide in 3T3 cells. • Enhancement of LPA{sub 5} on the cell motile activities inhibited by LPA{sub 1} in 3T3 cells. • The expression and activation of Mmp-9 were inhibited by LPA{sub 5} in 3T3 cells. • LPA signaling via LPA{sub 5} acts as a negative regulator of cellular responses in 3T3 cells. - Abstract: Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA{sub 1}–LPA{sub 6}) mediates a variety of biological functions, including cellmore » migration. Recently, we have reported that LPA{sub 1} inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA{sub 5} in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA{sub 1} and LPA{sub 5} on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA{sub 5} may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA{sub 1}.« less

Borrelia burgdorferi basic membrane protein A could induce chemokine production in murine microglia cell line BV2.

PubMed

Zhao, Hua; Liu, Aihua; Cui, Yuhui; Liang, Zhang; Li, Bingxue; Bao, Fukai

2017-10-01

Lyme neuroborreliosis is a nervous system infectious disease caused by Borrelia burgdorferi (B. burgdorferi). It has been demonstrated that cytokines induced by B. burgdorferi are related to Lyme neuroborreliosis. Microglia is known as a key player in the immune responses that occur within the central nervous system. In response to inflammation, it will be activated and generate cytokines and chemokines. Experiments in vitro cells have showed that B. Burgdorferi membrane protein A (BmpA), a major immunogen of B. Burgdorferi, could induce Lyme arthritis and stimulate human and murine lymphocytes to produce inflammatory cytokines. In our study, the murine microglia BV2 cell line was used as a cell model to explore the stimulating effects of recombinant BmpA (rBmpA); Chemokine chip, ELISA and QPCR technology were used to measure the production of chemokines from microglial cells stimulated by rBmpA. Compared with the negative control group, CXCL2, CCL22, and CCL5 concentrations in the cell supernatant increased significantly after the rBmpA stimulation; the concentration of these chemokines increased with rBmpA concentration increasing; the mRNA expression levels of chemokines (CXCL2, CCL22, and CCL5) in murine BV2 cells increased significantly with 10 μg/mL and 20 μg/mL rBmpA stimulation; CXCL13 was not change after the rBmpA stimulation. Our study shows that chemokines, such as CXCL2, CCL22, and CCL5 were up-regulated by the rBmpA in the BV2 cells. The production of chemokines in Lyme neuroborreliosis may be mainly from microglia cells and the rBmpA may be closely related with the development of Lyme neuroborreliosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Phosphoproteomics Reveals Regulatory T Cell-Mediated DEF6 Dephosphorylation That Affects Cytokine Expression in Human Conventional T Cells

PubMed Central

Joshi, Rubin N.; Binai, Nadine A.; Marabita, Francesco; Sui, Zhenhua; Altman, Amnon; Heck, Albert J. R.; Tegnér, Jesper; Schmidt, Angelika

2017-01-01

Regulatory T cells (Tregs) control key events of immune tolerance, primarily by suppression of effector T cells. We previously revealed that Tregs rapidly suppress T cell receptor (TCR)-induced calcium store depletion in conventional CD4+CD25− T cells (Tcons) independently of IP3 levels, consequently inhibiting NFAT signaling and effector cytokine expression. Here, we study Treg suppression mechanisms through unbiased phosphoproteomics of primary human Tcons upon TCR stimulation and Treg-mediated suppression, respectively. Tregs induced a state of overall decreased phosphorylation as opposed to TCR stimulation. We discovered novel phosphosites (T595_S597) in the DEF6 (SLAT) protein that were phosphorylated upon TCR stimulation and conversely dephosphorylated upon coculture with Tregs. Mutation of these DEF6 phosphosites abrogated interaction of DEF6 with the IP3 receptor and affected NFAT activation and cytokine transcription in primary Tcons. This novel mechanism and phosphoproteomics data resource may aid in modifying sensitivity of Tcons to Treg-mediated suppression in autoimmune disease or cancer. PMID:28993769

Aging increases cell-to-cell transcriptional variability upon immune stimulation.

PubMed

Martinez-Jimenez, Celia Pilar; Eling, Nils; Chen, Hung-Chang; Vallejos, Catalina A; Kolodziejczyk, Aleksandra A; Connor, Frances; Stojic, Lovorka; Rayner, Timothy F; Stubbington, Michael J T; Teichmann, Sarah A; de la Roche, Maike; Marioni, John C; Odom, Duncan T

2017-03-31

Aging is characterized by progressive loss of physiological and cellular functions, but the molecular basis of this decline remains unclear. We explored how aging affects transcriptional dynamics using single-cell RNA sequencing of unstimulated and stimulated naïve and effector memory CD4 + T cells from young and old mice from two divergent species. In young animals, immunological activation drives a conserved transcriptomic switch, resulting in tightly controlled gene expression characterized by a strong up-regulation of a core activation program, coupled with a decrease in cell-to-cell variability. Aging perturbed the activation of this core program and increased expression heterogeneity across populations of cells in both species. These discoveries suggest that increased cell-to-cell transcriptional variability will be a hallmark feature of aging across most, if not all, mammalian tissues. Copyright © 2017, American Association for the Advancement of Science.

TRPV2 mediates adrenomedullin stimulation of prostate and urothelial cancer cell adhesion, migration and invasion.

PubMed

Oulidi, Agathe; Bokhobza, Alexandre; Gkika, Dimitra; Vanden Abeele, Fabien; Lehen'kyi, V'yacheslav; Ouafik, L'houcine; Mauroy, Brigitte; Prevarskaya, Natalia

2013-01-01

Adrenomedullin (AM) is a 52-amino acid peptide initially isolated from human pheochromocytoma. AM is expressed in a variety of malignant tissues and cancer cell lines and was shown to be a mitogenic factor capable of stimulating growth of several cancer cell types. In addition, AM is a survival factor for certain cancer cells. Some data suggest that AM might be involved in the progression cancer metastasis via angiogenesis and cell migration and invasion control. The Transient Receptor Potential channel TRPV2 is known to promote in prostate cancer cell migration and invasive phenotype and is correlated with the stage and grade of bladder cancer. In this work we show that AM induces prostate and urothelial cancer cell migration and invasion through TRPV2 translocation to plasma membrane and the subsequent increase in resting calcium level.

Effect of OPC-12759 on EGF receptor activation, p44/p42 MAPK activity, and secretion in conjunctival goblet cells.

PubMed

Ríos, J David; Shatos, Marie A; Urashima, Hiroki; Dartt, Darlene A

2008-04-01

The purpose of the study was to determine if OPC-12759 stimulates secretion from conjunctival goblet cells in culture and if it activates the EGF receptor (EGFR) and p44/p42 mitogen-activated protein kinase (MAPK) to cause mucin secretion. Conjunctival goblet cells were cultured from pieces of male rat conjunctiva. OPC-12759 was added at increasing concentrations and for varying times to the cultured cells. The cholinergic agonist carbachol was used as a positive control. In selected experiments an inhibitor of the EGFR, AG1478, or an inhibitor of the kinase that activates MAPK, U0126, were added before OPC-12759. Goblet cell secretion of high molecular weight glycoconjugates was measured by an enzyme-linked lectin assay using the lectin UEA-1. Activation of the EGFR and MAPK were determined with Western blotting analysis using antibodies specific to the phosphorylated and the total amounts of these proteins. We found that OPC-12759 induced goblet cell secretion in a time- and concentration-dependent manner. Inhibition of the EGFR with AG1478 blocked secretion stimulated by OPC-12759. Inhibition of MAPK with U0126 also blocked secretion stimulated by OPC-12759. OPC-12759 increased the phosphorylation of the EGFR and MAPK in a time-dependent manner. We concluded that OPC-12759 stimulates secretion from cultured conjunctival goblet cells by activating the EGFR, which then induces MAPK activity.

Neurite outgrowth is significantly increased by the simultaneous presentation of Schwann cells and moderate exogenous electric fields

NASA Astrophysics Data System (ADS)

Koppes, Abigail N.; Seggio, Angela M.; Thompson, Deanna M.

2011-08-01

Axonal extension is influenced by a variety of external guidance cues; therefore, the development and optimization of a multi-faceted approach is probably necessary to address the intricacy of functional regeneration following nerve injury. In this study, primary dissociated neonatal rat dorsal root ganglia neurons and Schwann cells were examined in response to an 8 h dc electrical stimulation (0-100 mV mm-1). Stimulated samples were then fixed immediately, immunostained, imaged and analyzed to determine Schwann cell orientation and characterize neurite outgrowth relative to electric field strength and direction. Results indicate that Schwann cells are viable following electrical stimulation with 10-100 mV mm-1, and retain a normal morphology relative to unstimulated cells; however, no directional bias is observed. Neurite outgrowth was significantly enhanced by twofold following exposure to either a 50 mV mm-1 electric field (EF) or co-culture with unstimulated Schwann cells by comparison to neurons cultured alone. Neurite outgrowth was further increased in the presence of simultaneously applied cues (Schwann cells + 50 mV mm-1 dc EF), exhibiting a 3.2-fold increase over unstimulated control neurons, and a 1.2-fold increase over either neurons cultured with unstimulated Schwann cells or the electrical stimulus alone. These results indicate that dc electric stimulation in combination with Schwann cells may provide synergistic guidance cues for improved axonal growth relevant to nerve injuries in the peripheral nervous system.

Cleaved CD147 shed from the surface of malignant melanoma cells activates MMP2 produced by fibroblasts.

PubMed

Hatanaka, Miho; Higashi, Yuko; Fukushige, Tomoko; Baba, Naoko; Kawai, Kazuhiro; Hashiguchi, Teruto; Su, Juan; Zeng, Weiqi; Chen, Xiang; Kanekura, Takuro

2014-12-01

Cluster of differentiation 147 (CD147)/basigin on the malignant tumor cell surface is critical for tumor proliferation, invasiveness, metastasis, and angiogenesis. CD147 expressed on malignant melanoma cells can induce tumor cell invasion by stimulating the production of matrix metalloproteinases (MMPs) by surrounding fibroblasts. Membrane vesicles, microvesicles and exosomes have attracted attention, as vehicles of functional molecules and their association with CD147 has been reported. Cleaved CD147 fragments released from tumor cells were reported to interact with fibroblasts. We investigated the intercellular mechanisms by which CD147 stimulates fibroblasts to induce MMP2 activity. CD147 was knocked-down using short hairpin RNA (shRNA). The stimulatory effect of CD147 in cell culture supernatants, microvesicles, and exosomes on the enzymatic activity of MMP2 was examined by gelatin zymography. Supernatants from A375 control cells induced increased enzymatic activity of fibroblasts; such activity was significantly lower in CD147 knock-down cells. Cleaved CD147 plays a pivotal role in stimulating fibroblasts to induce MMP2 activity. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Unique regulation of Na-glutamine cotransporter SN2/SNAT5 in rabbit intestinal crypt cells during chronic enteritis.

PubMed

Singh, Soudamani; Arthur, Subha; Sundaram, Uma

2018-03-01

The only Na-nutrient cotransporter described in mammalian small intestinal crypt cells is SN2/SNAT5, which facilitates glutamine uptake. In a rabbit model of chronic intestinal inflammation, SN2 stimulation is secondary to an increase in affinity of the cotransporter for glutamine. However, the immune regulation of SN2 in the crypt cells during chronic intestinal inflammation is unknown. We sought to determine the mechanism of regulation of Na-nutrient cotransporter SN2 by arachidonic acid metabolites in crypt cells. The small intestines of New Zealand white male rabbits were inflamed via inoculation with Eimeria magna oocytes. After 2-week incubation, control and inflamed rabbits were subjected to intramuscular injections of arachidonyl trifluoromethyl ketone (ATK), piroxicam and MK886 for 48 hrs. After injections, the rabbits were euthanized and crypt cells from small intestines were harvested and used. Treatment of rabbits with ATK prevented the release of AA and reversed stimulation of SN2. Inhibition of cyclooxygenase (COX) with piroxicam did not affect stimulation of SN2. However, inhibition of lipoxygenase (LOX) with MK886, thus reducing leukotriene formation during chronic enteritis, reversed the stimulation of SN2. Kinetic studies showed that the mechanism of restoration of SN2 by ATK or MK886 was secondary to the restoration of the affinity of the cotransporter for glutamine. For all treatment conditions, Western blot analysis revealed no change in SN2 protein levels. COX inhibition proved ineffective at reversing the stimulation of SN2. Thus, this study provides evidence that SN2 stimulation in crypt cells is mediated by the leukotriene pathway during chronic intestinal inflammation. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Kidney tubular-cell secretion of osteoblast growth factor is increased by kaempferol: a scientific basis for "the kidney controlling the bone" theory of Chinese medicine.

PubMed

Long, Mian; Li, Shun-xiang; Xiao, Jiang-feng; Wang, Jian; Lozanoff, Scott; Zhang, Zhi-guang; Luft, Benjamin J; Johnson, Francis

2014-09-01

To study, at the cytological level, the basic concept of Chinese medicine that "the Kidney (Shen) controls the bone". Kaempferol was isolated form Rhizoma Drynariae (Gu Sui Bu, GSB) and at several concentrations was incubated with opossum kidney (OK) cells, osteoblasts (MC3T3 E1) and human fibroblasts (HF) at cell concentrations of 2×10(4)/mL. Opossum kidney cell-conditioned culture media with kaempferol at 70 nmol/L (70kaeOKM) and without kaempferol (0OKM) were used to stimulate MC3T3 E1 and HF proliferation. The bone morphological protein receptors I and II (BMPR I and II) in OK cells were identified by immune-fluorescence staining and Western blot analysis. Kaempferol was found to increase OK cell growth (P<0.05), but alone did not promote MC3T3 E1 or HF cell proliferation. However, although OKM by itself increased MC3T3 E1 growth by 198% (P<0.01), the 70kaeOKM further increased the growth of these cells by an additional 127% (P<0.01). It indicates that the kidney cell generates a previously unknown osteoblast growth factor (OGF) and kaempferol increases kidney cell secretion of OGF. Neither of these media had any significant effect on HF growth. Kaempferol also was found to increase the level of the BMPR II in OK cells. This lends strong support to the original idea that the Kidney has a significant influence over bone-formation, as suggested by some long-standing Chinese medical beliefs, kaempferol may also serve to stimulate kidney repair and indirectly stimulate bone formation.

Prolonged recurrence-free survival following OK432-stimulated dendritic cell transfer into hepatocellular carcinoma during transarterial embolization

PubMed Central

Nakamoto, Y; Mizukoshi, E; Kitahara, M; Arihara, F; Sakai, Y; Kakinoki, K; Fujita, Y; Marukawa, Y; Arai, K; Yamashita, T; Mukaida, N; Matsushima, K; Matsui, O; Kaneko, S

2011-01-01

Despite curative locoregional treatments for hepatocellular carcinoma (HCC), tumour recurrence rates remain high. The current study was designed to assess the safety and bioactivity of infusion of dendritic cells (DCs) stimulated with OK432, a streptococcus-derived anti-cancer immunotherapeutic agent, into tumour tissues following transcatheter hepatic arterial embolization (TAE) treatment in patients with HCC. DCs were derived from peripheral blood monocytes of patients with hepatitis C virus-related cirrhosis and HCC in the presence of interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor and stimulated with 0·1 KE/ml OK432 for 2 days. Thirteen patients were administered with 5 × 106 of DCs through arterial catheter during the procedures of TAE treatment on day 7. The immunomodulatory effects and clinical responses were evaluated in comparison with a group of 22 historical controls treated with TAE but without DC transfer. OK432 stimulation of immature DCs promoted their maturation towards cells with activated phenotypes, high expression of a homing receptor, fairly well-preserved phagocytic capacity, greatly enhanced cytokine production and effective tumoricidal activity. Administration of OK432-stimulated DCs to patients was found to be feasible and safe. Kaplan–Meier analysis revealed prolonged recurrence-free survival of patients treated in this manner compared with the historical controls (P = 0·046, log-rank test). The bioactivity of the transferred DCs was reflected in higher serum concentrations of the cytokines IL-9, IL-15 and tumour necrosis factor-α and the chemokines CCL4 and CCL11. Collectively, this study suggests that a DC-based, active immunotherapeutic strategy in combination with locoregional treatments exerts beneficial anti-tumour effects against liver cancer. PMID:21087443

Influence of extremely low frequency, low energy electromagnetic fields and combined mechanical stimulation on chondrocytes in 3-D constructs for cartilage tissue engineering.

PubMed

Hilz, Florian M; Ahrens, Philipp; Grad, Sibylle; Stoddart, Martin J; Dahmani, Chiheb; Wilken, Frauke L; Sauerschnig, Martin; Niemeyer, Philipp; Zwingmann, Jörn; Burgkart, Rainer; von Eisenhart-Rothe, Rüdiger; Südkamp, Norbert P; Weyh, Thomas; Imhoff, Andreas B; Alini, Mauro; Salzmann, Gian M

2014-02-01

Articular cartilage, once damaged, has very low regenerative potential. Various experimental approaches have been conducted to enhance chondrogenesis and cartilage maturation. Among those, non-invasive electromagnetic fields have shown their beneficial influence for cartilage regeneration and are widely used for the treatment of non-unions, fractures, avascular necrosis and osteoarthritis. One very well accepted way to promote cartilage maturation is physical stimulation through bioreactors. The aim of this study was the investigation of combined mechanical and electromagnetic stress affecting cartilage cells in vitro. Primary articular chondrocytes from bovine fetlock joints were seeded into three-dimensional (3-D) polyurethane scaffolds and distributed into seven stimulated experimental groups. They either underwent mechanical or electromagnetic stimulation (sinusoidal electromagnetic field of 1 mT, 2 mT, or 3 mT; 60 Hz) or both within a joint-specific bioreactor and a coil system. The scaffold-cell constructs were analyzed for glycosaminoglycan (GAG) and DNA content, histology, and gene expression of collagen-1, collagen-2, aggrecan, cartilage oligomeric matrix protein (COMP), Sox9, proteoglycan-4 (PRG-4), and matrix metalloproteinases (MMP-3 and -13). There were statistically significant differences in GAG/DNA content between the stimulated versus the control group with highest levels in the combined stimulation group. Gene expression was significantly higher for combined stimulation groups versus static control for collagen 2/collagen 1 ratio and lower for MMP-13. Amongst other genes, a more chondrogenic phenotype was noticed in expression patterns for the stimulated groups. To conclude, there is an effect of electromagnetic and mechanical stimulation on chondrocytes seeded in a 3-D scaffold, resulting in improved extracellular matrix production. © 2013 Wiley Periodicals, Inc.

Regional acidosis locally inhibits but remotely stimulates Ca2+ waves in ventricular myocytes

PubMed Central

Ford, Kerrie L.; Moorhouse, Emma L.; Bortolozzi, Mario; Richards, Mark A.; Swietach, Pawel; Vaughan-Jones, Richard D.

2017-01-01

Abstract Aims Spontaneous Ca2+ waves in cardiomyocytes are potentially arrhythmogenic. A powerful controller of Ca2+ waves is the cytoplasmic H+ concentration ([H+]i), which fluctuates spatially and temporally in conditions such as myocardial ischaemia/reperfusion. H+-control of Ca2+ waves is poorly understood. We have therefore investigated how [H+]i co-ordinates their initiation and frequency. Methods and results Spontaneous Ca2+ waves were imaged (fluo-3) in rat isolated ventricular myocytes, subjected to modest Ca2+-overload. Whole-cell intracellular acidosis (induced by acetate-superfusion) stimulated wave frequency. Pharmacologically blocking sarcolemmal Na+/H+ exchange (NHE1) prevented this stimulation, unveiling inhibition by H+. Acidosis also increased Ca2+ wave velocity. Restricting acidosis to one end of a myocyte, using a microfluidic device, inhibited Ca2+ waves in the acidic zone (consistent with ryanodine receptor inhibition), but stimulated wave emergence elsewhere in the cell. This remote stimulation was absent when NHE1 was selectively inhibited in the acidic zone. Remote stimulation depended on a locally evoked, NHE1-driven rise of [Na+]i that spread rapidly downstream. Conclusion Acidosis influences Ca2+ waves via inhibitory Hi+ and stimulatory Nai+ signals (the latter facilitating intracellular Ca2+-loading through modulation of sarcolemmal Na+/Ca2+ exchange activity). During spatial [H+]i-heterogeneity, Hi+-inhibition dominates in acidic regions, while rapid Nai+ diffusion stimulates waves in downstream, non-acidic regions. Local acidosis thus simultaneously inhibits and stimulates arrhythmogenic Ca2+-signalling in the same myocyte. If the principle of remote H+-stimulation of Ca2+ waves also applies in multicellular myocardium, it raises the possibility of electrical disturbances being driven remotely by adjacent ischaemic areas, which are known to be intensely acidic. PMID:28339694

Regional acidosis locally inhibits but remotely stimulates Ca2+ waves in ventricular myocytes.

PubMed

Ford, Kerrie L; Moorhouse, Emma L; Bortolozzi, Mario; Richards, Mark A; Swietach, Pawel; Vaughan-Jones, Richard D

2017-07-01

Spontaneous Ca2+ waves in cardiomyocytes are potentially arrhythmogenic. A powerful controller of Ca2+ waves is the cytoplasmic H+ concentration ([H+]i), which fluctuates spatially and temporally in conditions such as myocardial ischaemia/reperfusion. H+-control of Ca2+ waves is poorly understood. We have therefore investigated how [H+]i co-ordinates their initiation and frequency. Spontaneous Ca2+ waves were imaged (fluo-3) in rat isolated ventricular myocytes, subjected to modest Ca2+-overload. Whole-cell intracellular acidosis (induced by acetate-superfusion) stimulated wave frequency. Pharmacologically blocking sarcolemmal Na+/H+ exchange (NHE1) prevented this stimulation, unveiling inhibition by H+. Acidosis also increased Ca2+ wave velocity. Restricting acidosis to one end of a myocyte, using a microfluidic device, inhibited Ca2+ waves in the acidic zone (consistent with ryanodine receptor inhibition), but stimulated wave emergence elsewhere in the cell. This remote stimulation was absent when NHE1 was selectively inhibited in the acidic zone. Remote stimulation depended on a locally evoked, NHE1-driven rise of [Na+]i that spread rapidly downstream. Acidosis influences Ca2+ waves via inhibitory Hi+ and stimulatory Nai+ signals (the latter facilitating intracellular Ca2+-loading through modulation of sarcolemmal Na+/Ca2+ exchange activity). During spatial [H+]i-heterogeneity, Hi+-inhibition dominates in acidic regions, while rapid Nai+ diffusion stimulates waves in downstream, non-acidic regions. Local acidosis thus simultaneously inhibits and stimulates arrhythmogenic Ca2+-signalling in the same myocyte. If the principle of remote H+-stimulation of Ca2+ waves also applies in multicellular myocardium, it raises the possibility of electrical disturbances being driven remotely by adjacent ischaemic areas, which are known to be intensely acidic. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Cardiology.

[Secretion of growth hormone in hyperthyroidism].

PubMed

Hervás, F; Morreale de Escobar, G; Escobar Del Rey, F; Pozuelo, V

1976-01-01

The authors studied growth hormone (GH) secretion in a group of adult controls and another group of hyperthyroid patients after stimulation with intravenous insulin-induced (0,1 IU/kg) hypoglycemia, aiming to clear out the problem of discrepancies in literature concerning GH secretion in hyperthyroidism. They concluded that in this syndrome, GH levels are significantly higher than those of controls. The GH releasing response is normal, though it could be expected to be decreased due to decreased pituitary GH contents as a result of permanent somatotrophic cell stimulation.

S-Equol Activates cAMP Signaling at the Plasma Membrane of INS-1 Pancreatic β-Cells and Protects against Streptozotocin-Induced Hyperglycemia by Increasing β-Cell Function in Male Mice.

PubMed

Horiuchi, Hiroko; Usami, Atsuko; Shirai, Rie; Harada, Naoki; Ikushiro, Shinichi; Sakaki, Toshiyuki; Nakano, Yoshihisa; Inui, Hiroshi; Yamaji, Ryoichi

2017-09-01

Background: S -equol, which is enantioselectively produced from daidzein by gut microbiota, has been suggested as a chemopreventive agent against type 2 diabetes mellitus (T2DM), but the underlying mechanisms remain unclear. Objective: We investigated the effects of S -equol on pancreatic β-cell function. Methods: β-Cell growth and insulin secretion were evaluated with male Institute of Cancer Research mice and isolated pancreatic islets from the mice, respectively. The mechanisms by which S -equol stimulated β-cell response were examined in INS-1 β-cells. The effect of S -equol treatment on β-cell function was assessed in low-dose streptozotocin-treated mice. S -equol was used at 10 μmol/L for in vitro and ex vivo studies and was administered by oral gavage (20 mg/kg, 2 times/d throughout the experimental period) for in vivo studies. Results: S -equol administration for 7 d increased Ki67-positive β-cells by 27% ( P < 0.01) in mice. S -equol enantioselectively enhanced glucose-stimulated insulin secretion in mouse pancreatic islets by 41% ( P < 0.001). In INS-1 cells, S -equol exerted stronger effects than daidzein on cell growth, insulin secretion, and cAMP-response element (CRE)-mediated transcription. These S -equol effects were diminished by inhibiting protein kinase A. The effective concentration of S -equol for stimulating cAMP production at the plasma membrane was lower than that for phosphodiesterase inhibition. S -equol-stimulated CRE activation was negatively controlled by the knockdown of G-protein α subunit group S (stimulatory) and positively controlled by that of G-protein-coupled receptor kinase-3 and -6. Compared with vehicle-treated controls, S -equol gavage treatment resulted in an increase in β-cell mass of 104% ( P < 0.05), a trend toward high plasma insulin concentrations (by 118%; P = 0.06), and resistance to hyperglycemia after streptozotocin treatment (78% of AUC after glucose challenge; P < 0.01). S -equol administration significantly increased the number of Ki67-positive proliferating β-cells by 62% ( P < 0.01) and decreased that of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive apoptotic β-cells by 75% ( P < 0.05). Conclusions: Our results show that S -equol boosts β-cell function and prevents hypoglycemia in mice, suggesting its potential for T2DM prevention. © 2017 American Society for Nutrition.

Descending antinociception induced by secondary somatosensory cortex stimulation in experimental neuropathy: role of the medullospinal serotonergic pathway

PubMed Central

Sagalajev, Boriss; Viisanen, Hanna; Wei, Hong

2017-01-01

Stimulation of the secondary somatosensory cortex (S2) has attenuated pain in humans and inflammatory nociception in animals. Here we studied S2 stimulation-induced antinociception and its underlying mechanisms in an experimental animal model of neuropathy induced by spinal nerve ligation (SNL). Effect of S2 stimulation on heat-evoked limb withdrawal latency was assessed in lightly anesthetized rats that were divided into three groups based on prior surgery and monofilament testing before induction of anesthesia: 1) sham-operated group and 2) hypersensitive and 3) nonhypersensitive (mechanically) SNL groups. In a group of hypersensitive SNL animals, a 5-HT1A receptor agonist was microinjected into the rostroventromedial medulla (RVM) to assess whether autoinhibition of serotonergic cell bodies blocks antinociception. Additionally, effect of S2 stimulation on pronociceptive ON-cells and antinociceptive OFF-cells in the RVM or nociceptive spinal wide dynamic range (WDR) neurons were assessed in anesthetized hypersensitive SNL animals. S2 stimulation induced antinociception in hypersensitive but not in nonhypersensitive SNL or sham-operated animals. Antinociception was prevented by a 5-HT1A receptor agonist in the RVM. Antinociception was associated with decreased duration of heat-evoked response in RVM ON-cells. In spinal WDR neurons, heat-evoked discharge was delayed by S2 stimulation, and this antinociceptive effect was prevented by blocking spinal 5-HT1A receptors. The results indicate that S2 stimulation suppresses nociception in SNL animals if SNL is associated with tactile allodynia-like hypersensitivity. In hypersensitive SNL animals, S2 stimulation induces antinociception mediated by medullospinal serotonergic pathways acting on the spinal 5-HT1A receptor, and partly through reduction of the RVM ON-cell discharge. NEW & NOTEWORTHY Stimulation of S2 cortex, but not that of an adjacent cortical area, induced descending heat antinociception in rats with the spinal nerve ligation-induced model of neuropathy. Antinociception was bilateral, and it involved suppression of pronociceptive medullary cells and activation of serotonergic pathways that act on the spinal 5-HT1A receptor. S2 stimulation failed to induce descending antinociceptive effect in sham-operated controls or in nerve-ligated animals that had not developed mechanical hypersensitivity. PMID:28053243

Insulin stimulates synthesis and release of human chorionic gonadotropin by choriocarcinoma cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ren, S.G.; Braunstein, G.D.

1991-03-01

Recent studies have shown that insulin regulates placental lactogen, progesterone, and estrogen production from human trophoblast cells. This study was performed to examine whether insulin also regulates the production of hCG by this type of cell. After 24-36 h of preincubation, JEG-3 and JAR cells (2-3 x 10(5) cells/ml.well) or human term trophoblast cells (1 x 10(6) cells/ml.well) were exposed to the test hormone in serum-free Dulbecco's Modified Eagle's Medium for 24-96 h. Secretion of hCG from JEG-3 cells was stimulated by human insulin, human proinsulin, or porcine insulin in a dose-dependent manner, with lowest effective doses of 6.7, 96,more » and 53 mg/L, respectively. Time-course studies showed that hCG secretion peaked at 72-96 h with insulin exposure; in contrast, no decernable peak was seen without insulin in serum-free media. Exposure of JEG-3 cells for 24 h to 209 mg/liter insulin stimulated hCG synthesis, with 40 +/- 3% more immunoreactive intracellular hCG (P less than 0.05). Cells grown in the presence of insulin and (35S)methionine had 47 +/- 21% more labeled intracellular hCG and 56 +/- 13% more immunoprecipitable (35S)methionine-hCG secreted into the medium than the control cultures (P less than 0.05). During this time period, human placental lactogen release and total trichloroacetice acid-precipitable (35S)methionine protein were not increased. The insulin-induced stimulation of hCG synthesis was inhibited by cycloheximide. Additionally, insulin did not significantly affect total intracellular protein during 24-96 h of incubation. Insulin also increased hCG release from JAR cells, but not from human term trophoblast cells. A mouse monoclonal antibody to the IGF-I receptor inhibited the stimulation of insulin in JEG-3 cells.« less

Plasmids encoding PKI(1-31), a specific inhibitor of cAMP-stimulated gene expression, inhibit the basal transcriptional activity of some but not all cAMP-regulated DNA response elements in JEG-3 cells.

PubMed

Grove, J R; Deutsch, P J; Price, D J; Habener, J F; Avruch, J

1989-11-25

Plasmids that encode a bioactive amino-terminal fragment of the heat-stable inhibitor of the cAMP-dependent protein kinase, PKI(1-31), were employed to characterize the role of this protein kinase in the control of transcriptional activity mediated by three DNA regulatory elements in the JEG-3 human placental cell line. The 5'-flanking sequence of the human collagenase gene contains the heptameric sequence, 5'-TGAGTCA-3', previously identified as a "phorbol ester" response element. Reporter genes containing either the intact 1.2-kilobase 5'-flanking sequence from the human collagenase gene or just the 7-base pair (bp) response element, when coupled to an enhancerless promoter, each exhibit both cAMP and phorbol ester-stimulated expression in JEG-3 cells. Cotransfection of either construct with plasmids encoding PKI(1-31) inhibits cAMP-stimulated but not basal- or phorbol ester-stimulated expression. Pretreatment of cells with phorbol ester for 1 or 2 days abrogates completely the response to rechallenge with phorbol ester but does not alter the basal expression of either construct; cAMP-stimulated expression, while modestly inhibited, remains vigorous. The 5'-flanking sequence of the human chorionic gonadotropin-alpha subunit (HCG alpha) gene has two copies of the sequence, 5'-TGACGTCA-3', contained in directly adjacent identical 18-bp segments, previously identified as a cAMP-response element. Reporter genes containing either the intact 1.5 kilobase of 5'-flanking sequence from the HCG alpha gene, or just the 36-bp tandem repeat cAMP response element, when coupled to an enhancerless promoter, both exhibit a vigorous cAMP stimulation of expression but no response to phorbol ester in JEG-3 cells. Cotransfection with plasmids encoding PKI(1-31) inhibits both basal and cAMP-stimulated expression in a parallel fashion. The 5'-flanking sequence of the human enkephalin gene mediates cAMP-stimulated expression of reporter genes in both JEG-3 and CV-1 cells. Plasmids encoding PKI(1-31) inhibit the expression that is stimulated by the addition of cAMP analogs in both cell lines; basal expression, however, is inhibited by PKI(1-31) only in the JEG-3 cell line and not in the CV-1 cells. These observations indicate that, in JEG-3 cells, PKI(1-31) is a specific inhibitor of kinase A-mediated gene transcription, but it does not modify kinase C-directed transcription.(ABSTRACT TRUNCATED AT 400 WORDS)

Suboptimal T-cell receptor signaling compromises protein translation, ribosome biogenesis, and proliferation of mouse CD8 T cells.

PubMed

Tan, Thomas C J; Knight, John; Sbarrato, Thomas; Dudek, Kate; Willis, Anne E; Zamoyska, Rose

2017-07-25

Global transcriptomic and proteomic analyses of T cells have been rich sources of unbiased data for understanding T-cell activation. Lack of full concordance of these datasets has illustrated that important facets of T-cell activation are controlled at the level of translation. We undertook translatome analysis of CD8 T-cell activation, combining polysome profiling and microarray analysis. We revealed that altering T-cell receptor stimulation influenced recruitment of mRNAs to heavy polysomes and translation of subsets of genes. A major pathway that was compromised, when TCR signaling was suboptimal, was linked to ribosome biogenesis, a rate-limiting factor in both cell growth and proliferation. Defective TCR signaling affected transcription and processing of ribosomal RNA precursors, as well as the translation of specific ribosomal proteins and translation factors. Mechanistically, IL-2 production was compromised in weakly stimulated T cells, affecting the abundance of Myc protein, a known regulator of ribosome biogenesis. Consequently, weakly activated T cells showed impaired production of ribosomes and a failure to maintain proliferative capacity after stimulation. We demonstrate that primary T cells respond to various environmental cues by regulating ribosome biogenesis and mRNA translation at multiple levels to sustain proliferation and differentiation.

Scaffold Architecture Controls Insulinoma Clustering, Viability, and Insulin Production

PubMed Central

Blackstone, Britani N.; Palmer, Andre F.; Rilo, Horacio R.

2014-01-01

Recently, in vitro diagnostic tools have shifted focus toward personalized medicine by incorporating patient cells into traditional test beds. These cell-based platforms commonly utilize two-dimensional substrates that lack the ability to support three-dimensional cell structures seen in vivo. As monolayer cell cultures have previously been shown to function differently than cells in vivo, the results of such in vitro tests may not accurately reflect cell response in vivo. It is therefore of interest to determine the relationships between substrate architecture, cell structure, and cell function in 3D cell-based platforms. To investigate the effect of substrate architecture on insulinoma organization and function, insulinomas were seeded onto 2D gelatin substrates and 3D fibrous gelatin scaffolds with three distinct fiber diameters and fiber densities. Cell viability and clustering was assessed at culture days 3, 5, and 7 with baseline insulin secretion and glucose-stimulated insulin production measured at day 7. Small, closely spaced gelatin fibers promoted the formation of large, rounded insulinoma clusters, whereas monolayer organization and large fibers prevented cell clustering and reduced glucose-stimulated insulin production. Taken together, these data show that scaffold properties can be used to control the organization and function of insulin-producing cells and may be useful as a 3D test bed for diabetes drug development. PMID:24410263

NFATc1 affects mouse splenic B cell function by controlling the calcineurin–NFAT signaling network

PubMed Central

Bhattacharyya, Sankar; Deb, Jolly; Patra, Amiya K.; Thuy Pham, Duong Anh; Chen, Wen; Vaeth, Martin; Berberich-Siebelt, Friederike; Klein-Hessling, Stefan; Lamperti, Edward D.; Reifenberg, Kurt; Jellusova, Julia; Schweizer, Astrid; Nitschke, Lars; Leich, Ellen; Rosenwald, Andreas; Brunner, Cornelia; Engelmann, Swen; Bommhardt, Ursula; Avots, Andris; Müller, Martin R.; Kondo, Eisaku

2011-01-01

By studying mice in which the Nfatc1 gene was inactivated in bone marrow, spleen, or germinal center B cells, we show that NFATc1 supports the proliferation and suppresses the activation-induced cell death of splenic B cells upon B cell receptor (BCR) stimulation. BCR triggering leads to expression of NFATc1/αA, a short isoform of NFATc1, in splenic B cells. NFATc1 ablation impaired Ig class switch to IgG3 induced by T cell–independent type II antigens, as well as IgG3+ plasmablast formation. Mice bearing NFATc1−/− B cells harbor twofold more interleukin 10–producing B cells. NFATc1−/− B cells suppress the synthesis of interferon-γ by T cells in vitro, and these mice exhibit a mild clinical course of experimental autoimmune encephalomyelitis. In large part, the defective functions of NFATc1−/− B cells are caused by decreased BCR-induced Ca2+ flux and calcineurin (Cn) activation. By affecting CD22, Rcan1, CnA, and NFATc1/αA expression, NFATc1 controls the Ca2+-dependent Cn–NFAT signaling network and, thereby, the fate of splenic B cells upon BCR stimulation. PMID:21464221

Anti-Inflammatory Effect of Ginsenoside Rg5 in Lipopolysaccharide-Stimulated BV2 Microglial Cells

PubMed Central

Lee, Yu Young; Park, Jin-Sun; Jung, Ji-Sun; Kim, Dong-Hyun; Kim, Hee-Sun

2013-01-01

Microglia are resident immune cells in the central nervous system. They play a role in normal brain development and neuronal recovery. However, overactivation of microglia causes neuronal death, which is associated with neurodegenerative diseases, such as Parkinson’s disease and Alzheimer’s disease. Therefore, controlling microglial activation has been suggested as an important target for treatment of neurodegenerative diseases. In the present study, we investigated the anti-inflammatory effect of ginsenoside Rg5 in lipopolysaccharide (LPS)-stimulated BV2 microglial cells and rat primary microglia. The data showed that Rg5 suppressed LPS-induced nitric oxide (NO) production and proinflammatory TNF-α secretion. In addition, Rg5 inhibited the mRNA expressions of iNOS, TNF-α, IL-1β, COX-2 and MMP-9 induced by LPS. Further mechanistic studies revealed that Rg5 inhibited the phophorylations of PI3K/Akt and MAPKs and the DNA binding activities of NF-κB and AP-1, which are upstream molecules controlling inflammatory reactions. Moreover, Rg5 suppressed ROS production with upregulation of hemeoxygenase-1 (HO-1) expression in LPS-stimulated BV2 cells. Overall, microglial inactivation by ginsenoside Rg5 may provide a therapeutic potential for various neuroinflammatory disorders. PMID:23698769

Electrically Stimulated Adipose Stem Cells on Polypyrrole-Coated Scaffolds for Smooth Muscle Tissue Engineering.

PubMed

Björninen, Miina; Gilmore, Kerry; Pelto, Jani; Seppänen-Kaijansinkko, Riitta; Kellomäki, Minna; Miettinen, Susanna; Wallace, Gordon; Grijpma, Dirk; Haimi, Suvi

2017-04-01

We investigated the use of polypyrrole (PPy)-coated polymer scaffolds and electrical stimulation (ES) to differentiate adipose stem cells (ASCs) towards smooth muscle cells (SMCs). Since tissue engineering lacks robust and reusable 3D ES devices we developed a device that can deliver ES in a reliable, repeatable, and cost-efficient way in a 3D environment. Long pulse (1 ms) or short pulse (0.25 ms) biphasic electric current at a frequency of 10 Hz was applied to ASCs to study the effects of ES on ASC viability and differentiation towards SMCs on the PPy-coated scaffolds. PPy-coated scaffolds promoted proliferation and induced stronger calponin, myosin heavy chain (MHC) and smooth muscle actin (SMA) expression in ASCs compared to uncoated scaffolds. ES with 1 ms pulse width increased the number of viable cells by day 7 compared to controls and remained at similar levels to controls by day 14, whereas shorter pulses significantly decreased viability compared to the other groups. Both ES protocols supported smooth muscle expression markers. Our results indicate that electrical stimulation on PPy-coated scaffolds applied through the novel 3D ES device is a valid approach for vascular smooth muscle tissue engineering.

Holographic Photolysis for Multiple Cell Stimulation in Mouse Hippocampal Slices

PubMed Central

Papagiakoumou, Eirini; Ventalon, Cathie; Angulo, María Cecilia; Emiliani, Valentina

2010-01-01

Background Advanced light microscopy offers sensitive and non-invasive means to image neural activity and to control signaling with photolysable molecules and, recently, light-gated channels. These approaches require precise and yet flexible light excitation patterns. For synchronous stimulation of subsets of cells, they also require large excitation areas with millisecond and micrometric resolution. We have recently developed a new method for such optical control using a phase holographic modulation of optical wave-fronts, which minimizes power loss, enables rapid switching between excitation patterns, and allows a true 3D sculpting of the excitation volumes. In previous studies we have used holographic photololysis to control glutamate uncaging on single neuronal cells. Here, we extend the use of holographic photolysis for the excitation of multiple neurons and of glial cells. Methods/Principal Findings The system combines a liquid crystal device for holographic patterned photostimulation, high-resolution optical imaging, the HiLo microscopy, to define the stimulated regions and a conventional Ca2+ imaging system to detect neural activity. By means of electrophysiological recordings and calcium imaging in acute hippocampal slices, we show that the use of excitation patterns precisely tailored to the shape of multiple neuronal somata represents a very efficient way for the simultaneous excitation of a group of neurons. In addition, we demonstrate that fast shaped illumination patterns also induce reliable responses in single glial cells. Conclusions/Significance We show that the main advantage of holographic illumination is that it allows for an efficient excitation of multiple cells with a spatiotemporal resolution unachievable with other existing approaches. Although this paper focuses on the photoactivation of caged molecules, our approach will surely prove very efficient for other probes, such as light-gated channels, genetically encoded photoactivatable proteins, photoactivatable fluorescent proteins, and voltage-sensitive dyes. PMID:20195547

Increased proliferation of endothelial cells with overexpression of soluble TNF-alpha receptor I gene.

PubMed

Sugano, Masahiro; Tsuchida, Keiko; Tomita, Hideharu; Makino, Naoki

2002-05-01

Vascular endothelial growth factor (VEGF) can overcome a potential anti-angiogenic effect of TNF-alpha by inhibiting endothelial apoptosis induced by this cytokine. Soluble TNF-alpha receptor I (sTNFRI) is an extracellular domain of TNFRI and antagonizes the activity of TNF-alpha. Here we report that sTNFRI is able to stimulate the growth of endothelial cells not by antagonizing TNF-alpha. Exogenously added recombinant human sTNFRI stimulated significantly more cell growth of human umbilical venous endothelial cells (HUVEC) with a low dose (50-200 pg/ml) compared with smooth muscle cells. In contrast, monoclonal antibody against TNF-alpha did not stimulate growth of human HUVEC. The sTNFRI expression plasmid (pcDNA3.1 plasmid) was introduced into the cell culture using OPTI-MEM, lipofectin and transferrin. Growth of HUVEC transfected with sTNFRI vector also increased significantly compared with those transfected with control vector. HUVEC transfected with sTNFRI vector increased the extracellular domain of TNFRI mRNA levels, but did not affect the intracellular domain of TNFRI mRNA levels. Accumulation of sTNFRI significantly increased in conditioned medium from HUVEC transfected with sTNFRI vector compared with those transfected with control vector. HUVEC transfected with sTNFRI vector not only increased sTNFRI but also prevented shedding of sTNFRI from TNFRI. The TNF-alpha -induced internucleosomic fragmentation was also significantly prevented in HUVEC transfected with sTNFRI vector compared with those transfected with control vector. These results suggest that instead of growth factors such as VEGF, local transfection of the sTNFRI gene may have potential therapeutic value in vascular diseases in which TNF-alpha is also usually highly expressed.

The Ras-related Protein, Rap1A, Mediates Thrombin-stimulated, Integrin-dependent Glioblastoma Cell Proliferation and Tumor Growth*

PubMed Central

Sayyah, Jacqueline; Bartakova, Alena; Nogal, Nekeisha; Quilliam, Lawrence A.; Stupack, Dwayne G.; Brown, Joan Heller

2014-01-01

Rap1 is a Ras family GTPase with a well documented role in ERK/MAP kinase signaling and integrin activation. Stimulation of the G-protein-coupled receptor PAR-1 with thrombin in human 1321N1 glioblastoma cells led to a robust increase in Rap1 activation. This response was sustained for up to 6 h and mediated through RhoA and phospholipase D (PLD). Thrombin treatment also induced a 5-fold increase in cell adhesion to fibronectin, which was blocked by down-regulating PLD or Rap1A or by treatment with a β1 integrin neutralizing antibody. In addition, thrombin treatment led to increases in phospho-focal adhesion kinase (tyrosine 397), ERK1/2 phosphorylation and cell proliferation, which were significantly inhibited in cells treated with β1 integrin antibody or Rap1A siRNA. To assess the role of Rap1A in tumor formation in vivo, we compared growth of 1321N1 cells stably expressing control, Rap1A or Rap1B shRNA in a mouse xenograft model. Deletion of Rap1A, but not of Rap1B, reduced tumor mass by >70% relative to control. Similar observations were made with U373MG glioblastoma cells in which Rap1A was down-regulated. Collectively, these findings implicate a Rap1A/β1 integrin pathway, activated downstream of G-protein-coupled receptor stimulation and RhoA, in glioblastoma cell proliferation. Moreover, our data demonstrate a critical role for Rap1A in glioblastoma tumor growth in vivo. PMID:24790104

Progranulin Deficiency Reduces CDK4/6/pRb Activation and Survival of Human Neuroblastoma SH-SY5Y Cells.

PubMed

de la Encarnación, Ana; Alquézar, Carolina; Esteras, Noemí; Martín-Requero, Ángeles

2015-12-01

Null mutations in GRN are associated with frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP). However, the influence of progranulin (PGRN) deficiency in neurodegeneration is largely unknown. In neuroblastoma cells, silencing of GRN gene causes significantly reduced cell survival after serum withdrawal. The following observations suggest that alterations of the CDK4/6/retinoblastoma protein (pRb) pathway, secondary to changes in PI3K/Akt and ERK1/2 activation induced by PGRN deficiency, are involved in the control of serum deprivation-induced apoptosis: (i) inhibiting CDK4/6 levels or their associated kinase activity by sodium butyrate or PD332991 sensitized control SH-SY5Y cells to serum deprivation-induced apoptosis without affecting survival of PGRN-deficient cells; (ii) CDK4/6/pRb seems to be downstream of the PI3K/Akt and ERK1/2 signaling pathways since their specific inhibitors, LY294002 and PD98059, were able to decrease CDK6-associated kinase activity and induce death of control SH-SY5Y cells; (iii) PGRN-deficient cells show reduced stimulation of PI3K/Akt, ERK1/2, and CDK4/6 activities compared with control cells in the absence of serum; and (iv) supplementation of recombinant human PGRN was able to rescue survival of PGRN-deficient cells. These observations highlight the important role of PGRN-mediated stimulation of the PI3K/Akt-ERK1/2/CDK4/6/pRb pathway in determining the cell fate survival/death under serum deprivation.

Beta cell chromogranin B is partially segregated in distinct granules and can be released separately from insulin in response to stimulation.

PubMed

Giordano, T; Brigatti, C; Podini, P; Bonifacio, E; Meldolesi, J; Malosio, M L

2008-06-01

We investigated, in three beta cell lines (INS-1E, RIN-5AH, betaTC3) and in human and rodent primary beta cells, the storage and release of chromogranin B, a secretory protein expressed in beta cells and postulated to play an autocrine role. We asked whether chromogranin B is stored together with and discharged in constant ratio to insulin upon various stimuli. The intracellular distribution of insulin and chromogranin B was revealed by immunofluorescence followed by three-dimensional image reconstruction and by immunoelectron microscopy; their stimulated discharge was measured by ELISA and immunoblot analysis of homogenates and incubation media. Insulin and chromogranin B, co-localised in the Golgi complex/trans-Golgi network, appeared largely segregated from each other in the secretory granule compartment. In INS-1E cells, the percentage of granules positive only for insulin or chromogranin B and of those positive for both was 66, 7 and 27%, respectively. In resting cells, both insulin and chromogranin B were concentrated in the granule cores; upon stimulation, chromogranin B (but not insulin) was largely redistributed to the core periphery and the surrounding halo. Strong stimulation with a secretagogue mixture induced parallel release of insulin and chromogranin B, whereas with 3-isobutyl-1-methylxantine and forskolin +/- high glucose release of chromogranin B predominated. Weak, Ca(2+)-dependent stimulation with ionomycin or carbachol induced exclusive release of chromogranin B, suggesting a higher Ca(2+) sensitivity of the specific granules. The unexpected complexity of the beta cell granule population in terms of heterogeneity, molecular plasticity and the differential discharge, could play an important role in physiological control of insulin release and possibly also in beta cell pathology.

Insulin like growth factor 2 regulation of aryl hydrocarbon receptor in MCF-7 breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tomblin, Justin K.; Salisbury, Travis B., E-mail: salisburyt@marshall.edu

2014-01-17

Highlights: •IGF-2 stimulates concurrent increases in AHR and CCND1 expression. •IGF-2 promotes the binding of AHR to the endogenous cyclin D1 promoter. •AHR knockdown inhibits IGF-2 stimulated increases in CCND1 mRNA and protein. •AHR knockdown inhibits IGF-2 stimulated increases in MCF-7 proliferation. -- Abstract: Insulin like growth factor (IGF)-1 and IGF-2 stimulate normal growth, development and breast cancer cell proliferation. Cyclin D1 (CCND1) promotes cell cycle by inhibiting retinoblastoma protein (RB1). The aryl hydrocarbon receptor (AHR) is a major xenobiotic receptor that also regulates cell cycle. The purpose of this study was to investigate whether IGF-2 promotes MCF-7 breast cancermore » proliferation by inducing AHR. Western blot and quantitative real time PCR (Q-PCR) analysis revealed that IGF-2 induced an approximately 2-fold increase (P < .001) in the expression of AHR and CCND1. Chromatin immunoprecipitation (ChIP), followed by Q-PCR indicated that IGF-2 promoted (P < .001) a 7-fold increase in AHR binding on the CCND1 promoter. AHR knockdown significantly (P < .001) inhibited IGF-2 stimulated increases in CCND1 mRNA and protein. AHR knockdown cells were less (P < .001) responsive to the proliferative effects of IGF-2 than control cells. Collectively, our findings have revealed a new regulatory mechanism by which IGF-2 induction of AHR promotes the expression of CCND1 and the proliferation of MCF-7 cells. This previously uncharacterized pathway could be important for the proliferation of IGF responsive cancer cells that also express AHR.« less

Temporal gradients in shear stimulate osteoblastic proliferation via ERK1/2 and retinoblastoma protein

NASA Technical Reports Server (NTRS)

Jiang, Guang-Liang; White, Charles R.; Stevens, Hazel Y.; Frangos, John A.

2002-01-01

Bone cells are subject to interstitial fluid flow (IFF) driven by venous pressure and mechanical loading. Rapid dynamic changes in mechanical loading cause transient gradients in IFF. The effects of pulsatile flow (temporal gradients in fluid shear) on rat UMR106 cells and rat primary osteoblastic cells were studied. Pulsatile flow induced a 95% increase in S-phase UMR106 cells compared with static controls. In contrast, ramped steady flow stimulated only a 3% increase. Similar patterns of S-phase induction were also observed in rat primary osteoblastic cells. Pulsatile flow significantly increased relative UMR106 cell number by 37 and 62% at 1.5 and 24 h, respectively. Pulsatile flow also significantly increased extracellular signal-regulated kinase (ERK1/2) phosphorylation by 418%, whereas ramped steady flow reduced ERK1/2 activation to 17% of control. Correspondingly, retinoblastoma protein was significantly phosphorylated by pulsatile fluid flow. Inhibition of mitogen-activated protein (MAP)/ERK kinase (MEK)1/2 by U0126 (a specific MEK1/2 inhibitor) reduced shear-induced ERK1/2 phosphorylation and cell proliferation. These findings suggest that temporal gradients in fluid shear stress are potent stimuli of bone cell proliferation.

The two pore channel TPC2 is dispensable in pancreatic β-cells for normal Ca2+ dynamics and insulin secretion

PubMed Central

Cane, Matthew C.; Parrington, John; Rorsman, Patrik; Galione, Antony; Rutter, Guy A.

2016-01-01

Ca2+ signals are central to the stimulation of insulin secretion from pancreatic β-cells by glucose and other agents, including glucagon-like peptide-1 (GLP-1). Whilst Ca2+ influx through voltage-gated Ca2+ channels on the plasma membrane is a key trigger for glucose-stimulated secretion, mobilisation of Ca2+ from acidic stores has been implicated in the control of more localised Ca2+ changes and membrane potential. Nicotinic acid adenine dinucleotide phosphate (NAADP), generated in β-cells in response to high glucose, is a potent mobiliser of these stores, and has been proposed to act through two pore channels (TPC1 and TPC2, murine gene names Tpcn1 and Tpcn2). Whilst the role of TPC1 in the control of Ca2+ mobilisation and insulin secretion was recently confirmed, conflicting data exist for TPC2. Here, we used the selective and efficient deleter strain, Ins1Cre to achieve β-cell selective deletion of the Tpcn2 gene in mice. βTpcn2 KO mice displayed normal intraperitoneal and oral glucose tolerance, and glucose-stimulated Ca2+ dynamics and insulin secretion from islets were similarly normal. GLP-1-induced Ca2+ increases involved an increase in oscillation frequency from 4.35 to 4.84 per minute (p = 0.04) at 8 mM glucose, and this increase was unaffected by the absence of Tpcn2. The current data thus indicate that TPC2 is not absolutely required for normal glucose- or incretin-stimulated insulin secretion from the β-cell. Our findings suggest that TPC1, whose expression tended to increase in Tpcn2 null islets, might be sufficient to support normal Ca2+ dynamics in response to stimulation by nutrients or incretins. PMID:26769314

Anti-inflammatory effects of probiotic yogurt in inflammatory bowel disease patients

PubMed Central

Lorea Baroja, M; Kirjavainen, P V; Hekmat, S; Reid, G

2007-01-01

Our aim was to assess anti-inflammatory effects on the peripheral blood of subjects with inflammatory bowel disease (IBD) who consumed probiotic yogurt for 1 month. We studied 20 healthy controls and 20 subjects with IBD, 15 of whom had Crohn's disease and five with ulcerative colitis. All the subjects consumed Lactobacillus rhamnosus GR-1 and L. reuteri RC-14 supplemented yogurt for 30 days. The presence of putative regulatory T (Treg) cells (CD4+ CD25high) and cytokines in T cells, monocytes and dendritic cells (DC) was determined by flow cytometry from peripheral blood before and after treatment, with or without ex vivo stimulation. Serum and faecal cytokine concentrations were determined by enzyme-linked immunosorbent assays. The proportion of CD4+ CD25high T cells increased significantly (P = 0.007) in IBD patients, mean (95% confidence interval: CI) 0.84% (95% CI 0.55–1.12) before and 1.25% (95% CI 0.97–1.54) after treatment, but non-significantly in controls. The basal proportion of tumour necrosis factor (TNF)-α+/interleukin (IL)-12+ monocytes and myeloid DC decreased in both subject groups, but of stimulated cells only in IBD patients. Also serum IL-12 concentrations and proportions of IL-2+ and CD69+ T cells from stimulated cells decreased in IBD patients. The increase in CD4+ CD25high T cells correlated with the decrease in the percentage of TNF-α- or IL-12-producing monocytes and DC. The effect of the probiotic yogurt was confirmed by a follow-up study in which subjects consumed the yogurt without the probiotic organisms. Probiotic yogurt intake was associated with significant anti-inflammatory effects that paralleled the expansion of peripheral pool of putative Treg cells in IBD patients and with few effects in controls. PMID:17590176

Progranulin and its biological effects in cancer.

PubMed

Arechavaleta-Velasco, Fabian; Perez-Juarez, Carlos Eduardo; Gerton, George L; Diaz-Cueto, Laura

2017-11-07

Cancer cells have defects in regulatory mechanisms that usually control cell proliferation and homeostasis. Different cancer cells share crucial alterations in cell physiology, which lead to malignant growth. Tumorigenesis or tumor growth requires a series of events that include constant cell proliferation, promotion of metastasis and invasion, stimulation of angiogenesis, evasion of tumor suppressor factors, and avoidance of cell death pathways. All these events in tumor progression may be regulated by growth factors produced by normal or malignant cells. The growth factor progranulin has significant biological effects in different types of cancer. This protein is a regulator of tumorigenesis because it stimulates cell proliferation, migration, invasion, angiogenesis, malignant transformation, resistance to anticancer drugs, and immune evasion. This review focuses on the biological effects of progranulin in several cancer models and provides evidence that this growth factor should be considered as a potential biomarker and target in cancer treatment.

Optogenetic control of contractile function in skeletal muscle

PubMed Central

Bruegmann, Tobias; van Bremen, Tobias; Vogt, Christoph C.; Send, Thorsten; Fleischmann, Bernd K.; Sasse, Philipp

2015-01-01

Optogenetic stimulation allows activation of cells with high spatial and temporal precision. Here we show direct optogenetic stimulation of skeletal muscle from transgenic mice expressing the light-sensitive channel Channelrhodopsin-2 (ChR2). Largest tetanic contractions are observed with 5-ms light pulses at 30 Hz, resulting in 84% of the maximal force induced by electrical stimulation. We demonstrate the utility of this approach by selectively stimulating with a light guide individual intralaryngeal muscles in explanted larynges from ChR2-transgenic mice, which enables selective opening and closing of the vocal cords. Furthermore, systemic injection of adeno-associated virus into wild-type mice provides sufficient ChR2 expression for optogenetic opening of the vocal cords. Thus, direct optogenetic stimulation of skeletal muscle generates large force and provides the distinct advantage of localized and cell-type-specific activation. This technology could be useful for therapeutic purposes, such as restoring the mobility of the vocal cords in patients suffering from laryngeal paralysis. PMID:26035411

Minoxidil upregulates the expression of vascular endothelial growth factor in human hair dermal papilla cells.

PubMed

Lachgar, S; Charveron, M; Gall, Y; Bonafe, J L

1998-03-01

The hair follicle dermal papilla which controls hair growth, is characterized in the anagen phase by a highly developed vascular network. We have demonstrated in a previous study that the expression of an angiogenic growth factor called vascular endothelial growth factor (VEGF) mRNA varied during the hair cycle. VEGF mRNA is strongly expressed in dermal papilla cells (DPC) in the anagen phase, but during the catagen and telogen phases. VEGF mRNA is less strongly expressed. This involvement of VEGF during the hair cycle allowed us to determine whether VEGF mRNA expression by DPC was regulated by minoxidil. In addition, the effect of minoxidil on VEGF protein synthesis in both cell extracts and DPC-conditioned medium, was investigated immunoenzymatically. Both VEGF mRNA and protein were significantly elevated in treated DPC compared with controls. DPC incubated with increasing minoxidil concentrations (0.2, 2, 6, 12 and 24 mumol/L) induced a dose-dependent expression of VEGF mRNA. Quantification of transcripts showed that DPC stimulated with 24 mumol/L minoxidil express six times more VEGF mRNA than controls. Similarly, VEGF protein production increases in cell extracts and conditioned media following minoxidil stimulation. These studies strongly support the likely involvement of minoxidil in the development of dermal papilla vascularization via a stimulation of VEGF expression, and support the hypothesis that minoxidil has a physiological role in maintaining a good vascularization of hair follicles in androgenetic alopecia.

Characterisation of the immune response to type I collagen in scleroderma

PubMed Central

Warrington, Kenneth J; Nair, Usha; Carbone, Laura D; Kang, Andrew H; Postlethwaite, Arnold E

2006-01-01

This study was conducted to examine the frequency, phenotype, and functional profile of T lymphocytes that proliferate in response to type I collagen (CI) in patients with scleroderma (SSc). Peripheral blood mononuclear cells (PBMCs) from SSc patients, healthy controls, and rheumatoid arthritis disease controls were labeled with carboxy-fluorescein diacetate, succinimidyl ester (CFSE), cultured with or without antigen (bovine CI) for 14 days, and analysed by flow cytometry. Surface markers of proliferating cells were identified by multi-color flow cytometry. T-cell lines were derived after sorting for proliferating T cells (CFSElow). Cytokine expression in CI-responsive T cells was detected by intracellular staining/flow cytometry and by multiplex cytokine bead assay (Bio-Plex). A T-cell proliferative response to CI was detected in 8 of 25 (32%) SSc patients, but was infrequent in healthy or disease controls (3.6%; p = 0.009). The proliferating T cells expressed a CD4+, activated (CD25+), memory (CD45RO+) phenotype. Proliferation to CI did not correlate with disease duration or extent of skin involvement. T-cell lines were generated using in vitro CI stimulation to study the functional profile of these cells. Following activation of CI-reactive T cells, we detected intracellular interferon (IFN)-γ but not interleukin (IL)-4 by flow cytometry. Supernatants from the T-cell lines generated in vitro contained IL-2, IFN-γ, GM-CSF (granulocyte macrophage-colony-stimulating factor), and tumour necrosis factor-α, but little or no IL-4 and IL-10, suggesting that CI-responsive T cells express a predominantly Th1 cytokine pattern. In conclusion, circulating memory CD4 T cells that proliferate to CI are present in a subset of patients with SSc, but are infrequent in healthy or disease controls. PMID:16879746

Increased expression of both insulin receptor substrates 1 and 2 confers increased sensitivity to IGF-1 stimulated cell migration.

PubMed

de Blaquière, Gail E; May, Felicity E B; Westley, Bruce R

2009-06-01

Insulin-like growth factors (IGFs) are thought to promote tumour progression and metastasis in part by stimulating cell migration. Insulin receptor substrate-1 (IRS-1) and IRS-2 are multisite docking proteins positioned immediately downstream from the type I IGF and insulin receptors. IRS-2 but not IRS-1 has been reported to be involved in the migratory response of breast cancer cells to IGFs. The purpose of this investigation was to determine if IRS-1 is involved in, and to assess the contributions of IRS-1 and IRS-2 to, the migratory response of breast cancer cells to IGFs. The expression of IRS-1 and IRS-2 varied considerably between ten breast cancer cell lines. Oestrogen increases expression of the type I IGF receptor, IRS-1 and IRS-2 in MCF-7 and ZR-75 cells. Oestrogens may control the sensitivity of breast cancer cells to IGFs by regulating the expression of components of the IGF signal transduction pathway. The migratory response to a range of IGF-1 concentrations was measured in MCF-7 and MDA-MB-231 breast cancer cells in which IRS-1 and IRS-2 levels were modulated using a doxycycline-inducible expression system. Induction of both IRS-1 and IRS-2 expression increased the sensitivity of the migratory response to IGF-1 but did not increase the magnitude of the response stimulated at higher concentrations of IGF-1. Knockdown of IRS-1, IRS-2 and the type I IGF receptor in MCF-7 and MDA-MB-2231 cells decreased sensitivity to IGF-1. We conclude that both IRS-1 and IRS-2 control the migratory response of breast cancer cells to IGF-1 and may, therefore, be key molecules in determining breast cancer spread.

Application of cytoplasmic Ca2+ fluorescence imaging techniques to study the molecular mechanisms of exercise-induced fatigue eliminated by Chinese medicine ginseng extract

NASA Astrophysics Data System (ADS)

Liu, Yi; Zhao, Yanping; Zhang, Heming; Liu, Songhao

2009-11-01

The exercise-induced fatigue eliminated by Chinese medicine offers advantages including good efficiency and smaller side-effects, however, the exact mechanisms have not been classified. A lot of literatures indicated the cytosolic free Ca2+ concentrations of skeletal muscle cells increased significantly during exercise-induced fatigue. This study is aimed to establish a rat skeletal muscle cell model of exercise-induced fatigue. We applied cytoplasmic Ca2+ fluorescence imaging techniques to study the molecular mechanisms of exercise-induced fatigue eliminated by Chinese medicine ginseng extract. In our research, the muscle tissues from the newborn 3 days rats were taken out and digested into cells. The cells were randomly divided into the ginseng extract group and the control group. The cells from the two groups were cultured in the medium respectively added 2mg/ml ginseng extract and 2mg/ml D-hanks solution. After differentiating into myotubes, the two groups of cells treated with a fluorescent probe Fluo-3 AM were put on the confocal microscope and the fluorescence intensity of cells pre- and post- stimulation with dexamethasone were detected. It was found that cytoplasmic Ca2+ concentrations of the two groups of cells both increased post-stimulation, however, the increasing amplitude of fluorescence intensity of the ginseng extract group was significantly lower than that of the control group. In conclusion, stimulating the cells with dexamethasone is a kind of workable cell models of exercise-induced fatigue, and the molecular mechanisms of exercise-induced fatigue eliminated by ginseng extract may be connected to regulatating cytosolic free Ca2+ concentrations.

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays.

PubMed

Gertz, Monica L; Baker, Zachary; Jose, Sharon; Peixoto, Nathalia

2017-05-29

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Nisin production of Lactococcus lactis N8 with hemin-stimulated cell respiration in fed-batch fermentation system.

PubMed

Kördikanlıoğlu, Burcu; Şimşek, Ömer; Saris, Per E J

2015-01-01

In this study, nisin production of Lactococcus lactis N8 was optimized by independent variables of glucose, hemin and oxygen concentrations in fed-batch fermentation in which respiration of cells was stimulated with hemin. Response surface model was able to explain the changes of the nisin production of L. lactis N8 in fed-batch fermentation system with high fidelity (R(2) 98%) and insignificant lack of fit. Accordingly, the equation developed indicated the optimum parameters for glucose, hemin, and dissolved oxygen were 8 g L(-1) h(-1) , 3 μg mL(-1) and 40%, respectively. While 1711 IU mL(-1) nisin was produced by L. lactis N8 in control fed-batch fermentation, 5410 IU mL(-1) nisin production was achieved within the relevant optimum parameters where the respiration of cell was stimulated with hemin. Accordingly, nisin production was enhanced 3.1 fold in fed-batch fermentation using hemin. In conclusion the nisin production of L. lactis N8 was enhanced extensively as a result of increasing the biomass by stimulating the cell respiration with adding the hemin in the fed-batch fermentation. © 2015 American Institute of Chemical Engineers.

Intervertebral Disc Cells Produce Interleukins Found in Patients with Back Pain.

PubMed

Zhang, Yejia; Chee, Ana; Shi, Peng; Adams, Sherrill L; Markova, Dessislava Z; Anderson, David Greg; Smith, Harvey E; Deng, Youping; Plastaras, Christopher T; An, Howard S

2016-06-01

To examine the link between cytokines in intervertebral disc (IVD) tissues and axial back pain. In vitro study with human IVD cells cultured from cadaveric donors and annulus fibrosus (AF) tissues from patients. Cultured nucleus pulposus (NP) and AF cells were stimulated with interleukin (IL)-1β. IL-8 and IL-7 gene expression was analyzed using real-time polymerase chain reaction. IL-8 protein was quantified by enzyme-linked immunosorbent assay. After IL-1β stimulation, IL-8 gene expression increased 26,541 fold in NP cells and 22,429 fold in AF cells, whereas protein released by the NP and AF cells increased 2,389- and 1,784-fold, respectively. IL-7 gene expression increased 3.3-fold in NP cells (P < 0.05).Cytokine profiles in AF tissues collected from patients undergoing surgery for back pain (painful group) or scoliosis (controls) were compared by cytokine array. IL-8 protein in the AF tissues from patients with back pain was 1.81-fold of that in controls. IL-7 and IL-10 in AF tissues from the painful group were 6.87 and 4.63 times greater than the corresponding values in controls, respectively (P < 0.05). Inflammatory mediators found in AF tissues from patients with discogenic back pain are likely produced by IVD cells and may play a key role in back pain.

GENETIC CONTROL OF THE IMMUNE RESPONSE

PubMed Central

Lonai, Peter; McDevitt, Hugh O.

1974-01-01

In vitro antigen-induced tritiated thymidine uptake has been used to study the response of sensitized lymphocytes to (T,G)-A--L, (H,G)-A--L, and (Phe,G)-A--L in responder and nonresponder strains of mice. The reaction is T-cell and macrophage dependent. Highly purified T cells (91% Thy 1.2 positive) are also responsive, suggesting that this in vitro lymphocyte transformation system is not B-cell dependent. Lymphocytes from high and low responder mice stimulated in vitro react as responders and nonresponders in a pattern identical to that seen with in vivo immunization. Stimulation occurs only if soluble antigen is added at physiological temperatures; antigen exposure at 4°C followed by washing and incubation at 37°C fails to induce lymphocyte transformation. Stimulation is specific for the immunizing antigen and does not exhibit the serologic cross-reactivity which is characteristic of these three antigens and their respective antisera. The reaction can be inhibited by anti-H-2 sera but not by anti-immunoglobulin sera. The anti-immunoglobulin sera did, however, inhibit lipopolysaccharide or pokeweed mitogen stimulation. These results suggest that the Ir-1A gene(s) are expressed in T cells, and that there are fundamental physiologic differences between T- and B-cell antigen recognition. PMID:4547782

Rac-mediated Stimulation of Phospholipase Cγ2 Amplifies B Cell Receptor-induced Calcium Signaling*♦

PubMed Central

Walliser, Claudia; Tron, Kyrylo; Clauss, Karen; Gutman, Orit; Kobitski, Andrei Yu.; Retlich, Michael; Schade, Anja; Röcker, Carlheinz; Henis, Yoav I.; Nienhaus, G. Ulrich; Gierschik, Peter

2015-01-01

The Rho GTPase Rac is crucially involved in controlling multiple B cell functions, including those regulated by the B cell receptor (BCR) through increased cytosolic Ca2+. The underlying molecular mechanisms and their relevance to the functions of intact B cells have thus far remained unknown. We have previously shown that the activity of phospholipase Cγ2 (PLCγ2), a key constituent of the BCR signalosome, is stimulated by activated Rac through direct protein-protein interaction. Here, we use a Rac-resistant mutant of PLCγ2 to functionally reconstitute cultured PLCγ2-deficient DT40 B cells and to examine the effects of the Rac-PLCγ2 interaction on BCR-mediated changes of intracellular Ca2+ and regulation of Ca2+-regulated and nuclear-factor-of-activated-T-cell-regulated gene transcription at the level of single, intact B cells. The results show that the functional Rac-PLCγ2 interaction causes marked increases in the following: (i) sensitivity of B cells to BCR ligation; (ii) BCR-mediated Ca2+ release from intracellular stores; (iii) Ca2+ entry from the extracellular compartment; and (iv) nuclear translocation of the Ca2+-regulated nuclear factor of activated T cells. Hence, Rac-mediated stimulation of PLCγ2 activity serves to amplify B cell receptor-induced Ca2+ signaling. PMID:25903139

Mechanical stretch triggers rapid epithelial cell division through Piezo1.

PubMed

Gudipaty, S A; Lindblom, J; Loftus, P D; Redd, M J; Edes, K; Davey, C F; Krishnegowda, V; Rosenblatt, J

2017-03-02

Despite acting as a barrier for the organs they encase, epithelial cells turn over at some of the fastest rates in the body. However, epithelial cell division must be tightly linked to cell death to preserve barrier function and prevent tumour formation. How does the number of dying cells match those dividing to maintain constant numbers? When epithelial cells become too crowded, they activate the stretch-activated channel Piezo1 to trigger extrusion of cells that later die. However, it is unclear how epithelial cell division is controlled to balance cell death at the steady state. Here we show that mammalian epithelial cell division occurs in regions of low cell density where cells are stretched. By experimentally stretching epithelia, we find that mechanical stretch itself rapidly stimulates cell division through activation of the Piezo1 channel. To stimulate cell division, stretch triggers cells that are paused in early G2 phase to activate calcium-dependent phosphorylation of ERK1/2, thereby activating the cyclin B transcription that is necessary to drive cells into mitosis. Although both epithelial cell division and cell extrusion require Piezo1 at the steady state, the type of mechanical force controls the outcome: stretch induces cell division, whereas crowding induces extrusion. How Piezo1-dependent calcium transients activate two opposing processes may depend on where and how Piezo1 is activated, as it accumulates in different subcellular sites with increasing cell density. In sparse epithelial regions in which cells divide, Piezo1 localizes to the plasma membrane and cytoplasm, whereas in dense regions in which cells extrude, it forms large cytoplasmic aggregates. Because Piezo1 senses both mechanical crowding and stretch, it may act as a homeostatic sensor to control epithelial cell numbers, triggering extrusion and apoptosis in crowded regions and cell division in sparse regions.

Mifepristone enhances insulin-stimulated Akt phosphorylation and glucose uptake in skeletal muscle cells.

PubMed

Bernal-Sore, Izela; Navarro-Marquez, Mario; Osorio-Fuentealba, César; Díaz-Castro, Francisco; Del Campo, Andrea; Donoso-Barraza, Camila; Porras, Omar; Lavandero, Sergio; Troncoso, Rodrigo

2018-02-05

Mifepristone is the only FDA-approved drug for glycaemia control in patients with Cushing's syndrome and type 2 diabetes. Mifepristone also has beneficial effects in animal models of diabetes and patients with antipsychotic treatment-induced obesity. However, the mechanisms through which Mifepristone produces its beneficial effects are not completely elucidated. To determine the effects of mifepristone on insulin-stimulated glucose uptake on a model of L6 rat-derived skeletal muscle cells. Mifepristone enhanced insulin-dependent glucose uptake, GLUT4 translocation to the plasma membrane and Akt Ser 473 phosphorylation in L6 myotubes. In addition, mifepristone reduced oxygen consumption and ATP levels and increased AMPK Thr 172 phosphorylation. The knockdown of AMPK prevented the effects of mifepristone on insulin response. Mifepristone enhanced insulin-stimulated glucose uptake through a mechanism that involves a decrease in mitochondrial function and AMPK activation in skeletal muscle cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Light-regulated leaf expansion in two Populus species: dependence on developmentally controlled ion transport.

PubMed

Stiles, Kari A; Van Volkenburgh, Elizabeth

2002-07-01

Leaf growth responses to light have been compared in two species of Populus, P. deltoides and P. trichocarpa. These species differ markedly in morphology, anatomy, and dependence on light during leaf expansion. Light stimulates the growth rate and acidification of cell walls in P. trichocarpa but not in P. deltoides, whereas leaves of P. deltoides maintain growth in the dark. Light-induced growth is promoted in P. deltoides when cells are provided 50-100 mM KCl. In both species, light initially depolarizes, then hyperpolarizes mesophyll plasma membranes. However, in the dark, the resting E(m) of mesophyll cells in P. deltoides, but not in P. trichocarpa, is relatively insensitive to decade changes in external [K+]. Results suggest that light-stimulated leaf growth depends on developmentally regulated cellular mechanisms controlling ion fluxes across the plasma membrane. These developmental differences underlie species-level differences in growth and physiological responses to the photoenvironment.

Correspondence between visual and electrical input filters of ON and OFF mouse retinal ganglion cells

NASA Astrophysics Data System (ADS)

Sekhar, S.; Jalligampala, A.; Zrenner, E.; Rathbun, D. L.

2017-08-01

Objective. Over the past two decades retinal prostheses have made major strides in restoring functional vision to patients blinded by diseases such as retinitis pigmentosa. Presently, implants use single pulses to activate the retina. Though this stimulation paradigm has proved beneficial to patients, an unresolved problem is the inability to selectively stimulate the on and off visual pathways. To this end our goal was to test, using white noise, voltage-controlled, cathodic, monophasic pulse stimulation, whether different retinal ganglion cell (RGC) types in the wild type retina have different electrical input filters. This is an important precursor to addressing pathway-selective stimulation. Approach. Using full-field visual flash and electrical and visual Gaussian noise stimulation, combined with the technique of spike-triggered averaging (STA), we calculate the electrical and visual input filters for different types of RGCs (classified as on, off or on-off based on their response to the flash stimuli). Main results. Examining the STAs, we found that the spiking activity of on cells during electrical stimulation correlates with a decrease in the voltage magnitude preceding a spike, while the spiking activity of off cells correlates with an increase in the voltage preceding a spike. No electrical preference was found for on-off cells. Comparing STAs of wild type and rd10 mice revealed narrower electrical STA deflections with shorter latencies in rd10. Significance. This study is the first comparison of visual cell types and their corresponding temporal electrical input filters in the retina. The altered input filters in degenerated rd10 retinas are consistent with photoreceptor stimulation underlying visual type-specific electrical STA shapes in wild type retina. It is therefore conceivable that existing implants could target partially degenerated photoreceptors that have only lost their outer segments, but not somas, to selectively activate the on and off visual pathways.

1,2-Diacylglycerols, but not phorbol esters, activate a potential inhibitory pathway for protein kinase C in GH3 pituitary cells. Evidence for involvement of a sphingomyelinase.

PubMed

Kolesnick, R N; Clegg, S

1988-05-15

It has been suggested that sphingoid bases may serve as physiologic inhibitors of protein kinase C. Because 1,2-diacylglycerols, but not phorbol esters, enhance sphingomyelin degradation via a sphingomyelinase in GH3 pituitary cells (Kolesnick, R. N. (1987) J. Biol. Chem. 262, 16759-16762), the effects of phorbol esters, 1,2-diacylglycerols, and sphingomyelinase on protein kinase C activation were assessed. Under basal conditions, the inactive cytosolic form of protein kinase C predominated. 1,2-Diacylglycerols stimulated transient protein kinase C redistribution to the membrane. 1,2-Dioctanoylglycerol (200 micrograms/ml) reduced cytosolic protein kinase C activity to 67% of control from 72 to 48 pmol.min-1.10(6) cells-1 and enhanced membrane-bound activity to 430% of control from 6 to 25 pmol.min-1.10(6) cells-1 after 4 min of stimulation. Thereafter, protein kinase C activity returned to the cytosol. In contrast, the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulated redistribution to the membrane without return to the cytosol. Exogenous sphingomyelinase reduced membrane-bound protein kinase C activity to 30% of control, yet did not alter cytosolic activity. Sphingomyelinase, added after phorbol ester-induced redistribution was completed, restored activity to the cytosol. In these studies, TPA (10(-8) M) reduced cytosolic activity to 62% of control and elevated membrane-bound protein kinase C activity to 650% of control. Sphingomyelinase restored cytosolic activity to 84% of control and reduced membrane-bound activity to 297% of control. Similarly, the free sphingoid bases, sphingosine, sphinganine, and phytosphingosine, reversed phorbol ester-induced protein kinase C redistribution. Since 1,2-diacylglycerols activate a sphingomyelinase and sphingomyelinase action can reverse protein kinase C activation, these studies suggest that a pathway involving a sphingomyelinase might comprise a physiologic negative effector system for protein kinase C. Further, the failure of phorbol esters to activate this system might account for some differences between these agents.

T cell resistance to activation by dendritic cells requires long-term culture in simulated microgravity

NASA Astrophysics Data System (ADS)

Bradley, Jillian H.; Stein, Rachel; Randolph, Brad; Molina, Emily; Arnold, Jennifer P.; Gregg, Randal K.

2017-11-01

Immune impairment mediated by microgravity threatens the success of space exploration requiring long-duration spaceflight. The cells of most concern, T lymphocytes, coordinate the host response against microbial and cancerous challenges leading to elimination and long-term protection. T cells are activated upon recognition of specific microbial peptides bound on the surface of antigen presenting cells, such as dendritic cells (DC). Subsequently, this engagement results in T cell proliferation and differentiation into effector T cells driven by autocrine interleukin-2 (IL-2) and other cytokines. Finally, the effector T cells acquire the weaponry needed to destroy microbial invaders and tumors. Studies conducted on T cells during spaceflight, or using Earth-based culture systems, have shown reduced production of cytokines, proliferation and effector functions as compared to controls. This may account for the cases of viral reactivation events and opportunistic infections associated with astronauts of numerous missions. This work has largely been based upon the outcome of T cell activation by stimulatory factors that target select T cell signaling pathways rather than the complex, signaling events related to the natural process of antigen presentation by DC. This study tested the response of an ovalbumin peptide-specific T cell line, OT-II TCH, to activation by DC when the T cells were cultured 24-120 h in a simulated microgravity (SMG) environment generated by a rotary cell culture system. Following 72 h culture of T cells in SMG (SMG-T) or control static (Static-T) conditions, IL-2 production by the T cells was reduced in SMG-T cells compared to Static-T cells upon stimulation by phorbol 12-myristate 13-acetate (PMA) and ionomycin. However, when the SMG-T cells were stimulated with DC and peptide, IL-2 was significantly increased compared to Static-T cells. Such enhanced IL-2 production by SMG-T cells peaked at 72 h SMG culture time and decreased thereafter. When activation of SMG-T cells occurred in SMG, the T cells produced less IL-2 than control T cell cultures upon incubation with PMA and ionomycin. Short-term (24 h) SMG culture and activation of T cells by DC resulted in enhanced IL-2 production compared to Static-T cells, however, when culture was extended to 120 h, SMG-T cells secreted significantly less IL-2 than Static-T cells. SMG-T cell IL-2 doubled upon stimulation of the DC prior to addition to the T cell culture but remained less than control. SMG-T cell resistance to activation appeared comparable to the phenomenon of T cell exhaustion observed in patients with chronic diseases or persistent tumors. That is, long-term culture of T cells in SMG resulted in increased expression of the inhibitory receptor, CTLA-4. Blockade of CTLA-4 interaction with DC ligands resulted in improved T cell IL-2 production. Overall, this is the first study to determine the efficacy of DC in activating peptide-specific T cells. Furthermore, the findings suggests that countermeasures to restore T cell responsiveness in astronauts during long-term spaceflight or those living in microgravity environments should target possible inhibitory pathways that arise on activated T cells following stimulation.

T cell resistance to activation by dendritic cells requires long-term culture in simulated microgravity.

PubMed

Bradley, Jillian H; Stein, Rachel; Randolph, Brad; Molina, Emily; Arnold, Jennifer P; Gregg, Randal K

2017-11-01

Immune impairment mediated by microgravity threatens the success of space exploration requiring long-duration spaceflight. The cells of most concern, T lymphocytes, coordinate the host response against microbial and cancerous challenges leading to elimination and long-term protection. T cells are activated upon recognition of specific microbial peptides bound on the surface of antigen presenting cells, such as dendritic cells (DC). Subsequently, this engagement results in T cell proliferation and differentiation into effector T cells driven by autocrine interleukin-2 (IL-2) and other cytokines. Finally, the effector T cells acquire the weaponry needed to destroy microbial invaders and tumors. Studies conducted on T cells during spaceflight, or using Earth-based culture systems, have shown reduced production of cytokines, proliferation and effector functions as compared to controls. This may account for the cases of viral reactivation events and opportunistic infections associated with astronauts of numerous missions. This work has largely been based upon the outcome of T cell activation by stimulatory factors that target select T cell signaling pathways rather than the complex, signaling events related to the natural process of antigen presentation by DC. This study tested the response of an ovalbumin peptide-specific T cell line, OT-II TCH, to activation by DC when the T cells were cultured 24-120 h in a simulated microgravity (SMG) environment generated by a rotary cell culture system. Following 72 h culture of T cells in SMG (SMG-T) or control static (Static-T) conditions, IL-2 production by the T cells was reduced in SMG-T cells compared to Static-T cells upon stimulation by phorbol 12-myristate 13-acetate (PMA) and ionomycin. However, when the SMG-T cells were stimulated with DC and peptide, IL-2 was significantly increased compared to Static-T cells. Such enhanced IL-2 production by SMG-T cells peaked at 72 h SMG culture time and decreased thereafter. When activation of SMG-T cells occurred in SMG, the T cells produced less IL-2 than control T cell cultures upon incubation with PMA and ionomycin. Short-term (24 h) SMG culture and activation of T cells by DC resulted in enhanced IL-2 production compared to Static-T cells, however, when culture was extended to 120 h, SMG-T cells secreted significantly less IL-2 than Static-T cells. SMG-T cell IL-2 doubled upon stimulation of the DC prior to addition to the T cell culture but remained less than control. SMG-T cell resistance to activation appeared comparable to the phenomenon of T cell exhaustion observed in patients with chronic diseases or persistent tumors. That is, long-term culture of T cells in SMG resulted in increased expression of the inhibitory receptor, CTLA-4. Blockade of CTLA-4 interaction with DC ligands resulted in improved T cell IL-2 production. Overall, this is the first study to determine the efficacy of DC in activating peptide-specific T cells. Furthermore, the findings suggests that countermeasures to restore T cell responsiveness in astronauts during long-term spaceflight or those living in microgravity environments should target possible inhibitory pathways that arise on activated T cells following stimulation. Copyright © 2017 The Committee on Space Research (COSPAR). Published by Elsevier Ltd. All rights reserved.

Oral feeding with polyunsaturated fatty acids fosters hematopoiesis and thrombopoiesis in healthy and bone marrow-transplanted mice.

PubMed

Limbkar, Kedar; Dhenge, Ankita; Jadhav, Dipesh D; Thulasiram, Hirekodathakallu V; Kale, Vaijayanti; Limaye, Lalita

2017-09-01

Hematopoietic stem cells play the vital role of maintaining appropriate levels of cells in blood. Therefore, regulation of their fate is essential for their effective therapeutic use. Here we report the role of polyunsaturated fatty acids (PUFAs) in regulating hematopoiesis which has not been explored well so far. Mice were fed daily for 10 days with n-6/n-3 PUFAs, viz. linoleic acid (LA), arachidonic acid (AA), alpha-linolenic acid and docosahexanoic acid (DHA) in four separate test groups with phosphate-buffered saline fed mice as control set. The bone marrow cells of PUFA-fed mice showed a significantly higher hematopoiesis as assessed using side population, Lin-Sca-1 + ckit+, colony-forming unit (CFU), long-term culture, CFU-spleen assay and engraftment potential as compared to the control set. Thrombopoiesis was also stimulated in PUFA-fed mice. A combination of DHA and AA was found to be more effective than when either was fed individually. Higher incorporation of PUFAs as well as products of their metabolism was observed in the bone marrow cells of PUFA-fed mice. A stimulation of the Wnt, CXCR4 and Notch1 pathways was observed in PUFA-fed mice. The clinical relevance of this study was evident when bone marrow-transplanted recipient mice, which were fed with PUFAs, showed higher engraftment of donor cells, suggesting that the bone marrow microenvironment may also be stimulated by feeding with PUFAs. These data indicate that oral administration of PUFAs in mice stimulates hematopoiesis and thrombopoiesis and could serve as a valuable supplemental therapy in situations of hematopoietic failure. Copyright © 2017 Elsevier Inc. All rights reserved.

Hematopoietic growth factors and human acute leukemia.

PubMed

Löwenberg, B; Touw, I

1988-10-22

The study of myelopoietic maturation arrest in acute myeloblastic leukemia (AML) has been eased by availability of the human recombinant hemopoietic growth factors, macrophage colony stimulating factor (M-CSF), granulocyte-(G-CSF), granulocyte-macrophage-(GM-CSF) and multilineage stimulating factor (IL-3). Nonphysiological expansion of the leukemic population is not due to escape from control by these factors. Proliferation in vitro of AML cells is dependent on the presence of one or several factors in most cases. The pattern of factor-dependency does not correlate with morphological criteria in individual cases, and may thus offer a new tool for classification of AML. Overproduction of undifferentiated cells is not due to abnormal expression of receptors for the stimulating factors acting at an immature level. Rather, autocrine secretion of early acting lymphokines maintains proliferation of the leukemic clone. When looking at causes of leukemic dysregulation, yet undefined inhibitors of differentiation probably are of equal importance as dysequilibrated stimulation by lymphokines.

An Arg-Gly-Asp peptide stimulates Ca2+ efflux from osteoclast precursors through a novel mechanism

NASA Technical Reports Server (NTRS)

Yamakawa, K.; Duncan, R.; Hruska, K. A.

1994-01-01

We examined the effect of a peptide containing the Arg-Gly-Asp (RGD) sequence on 45Ca2+ efflux from osteoclast precursors. 45Ca(2+)-loaded osteoclast precursors were treated with GRGDSP (170 microM) for 10 min after 30 min of basal perfusion with a bicarbonate-containing buffer. GRGDSP significantly increased fractional efflux of Ca2+ from treated cells compared with vehicle-treated cells (P < 0.01) or cells treated with up to 200 micrograms/ml of a control peptide containing GRGESP. The effect of RGD was sustained for 15 min after the peptide was removed from the perfusate, but control levels of Ca2+ efflux returned by 1 h. The Ca2+ efflux effect of GRGDSP was most likely due to activation of the plasma membrane Ca(2+)-adenosinetriphosphatase (Ca(2+)-ATPase) pump, as indicated by its inhibition with vanadate and a calmodulin antagonist, N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide, and the absence of an effect of Na+/Ca2+ exchange inhibition. An inhibitor of cyclic nucleotide-dependent protein kinases, N-[2-(methylamino)ethyl]-5-isoquinoline-sulfonamide (0.1 mM), failed to inhibit GRGDSP-stimulated Ca2+ efflux. However, genistein and herbimycin A, inhibitors of protein-tyrosine kinases, blocked Ca2+ efflux stimulated by GRGDSP. The results indicate that RGD sequences of matrix proteins may stimulate Ca2+ efflux from osteoclasts through activation of protein-tyrosine kinases and suggest that GRGDSP-stimulated Ca2+ efflux is mediated via the plasma membrane Ca(2+)-ATPase.

A 70% Ethanol Extract of Mistletoe Rich in Betulin, Betulinic Acid, and Oleanolic Acid Potentiated β-Cell Function and Mass and Enhanced Hepatic Insulin Sensitivity

PubMed Central

Ko, Byoung-Seob; Kang, Suna; Moon, Bo Reum; Ryuk, Jin Ah; Park, Sunmin

2016-01-01

We investigated that the long-term consumption of the water (KME-W) and 70% ethanol (KME-E) mistletoe extracts had antidiabetic activities in partial pancreatectomized (Px) rats. Px rats were provided with a high-fat diet containing 0.6% KME-E, 0.6% KME-W, and 0.6% dextrin (control) for 8 weeks. As normal-control, Sham-operated rats were provided with 0.6% dextrin. In cell-based studies, the effects of its main terpenoids (betulin, betulinic acid, and oleanolic acid) on glucose metabolism were measured. Both KME-W and KME-E decreased epididymal fat mass by increasing fat oxidation in diabetic rats. KME-E but not KME-W exhibited greater potentiation of first-phase insulin secretion than the Px-control in a hyperglycemic clamp. KME-E also made β-cell mass greater than the control by increasing β-cell proliferation and decreasing its apoptosis. In a euglycemic-hyperinsulinemic clamp, whole-body glucose infusion rate and hepatic glucose output increased with potentiating hepatic insulin signaling in the following order: Px-control, KME-W, KME-E, and normal-control. Betulin potentiated insulin-stimulated glucose uptake via increased PPAR-γ activity and insulin signaling in 3T3-L1 adipocytes, whereas oleanolic acid enhanced glucose-stimulated insulin secretion and cell proliferation in insulinoma cells. In conclusion, KME-E prevented the deterioration of glucose metabolism in diabetic rats more effectively than KME-W and KME-E can be a better therapeutic agent for type 2 diabetes than KME-W. PMID:26884795

Outcome From a Randomized Controlled Clinical Trial　- Improvement of Peripheral Arterial Disease by Granulocyte Colony-Stimulating Factor-Mobilized Autologous Peripheral-Blood-Mononuclear Cell Transplantation (IMPACT).

PubMed

Horie, Takashi; Yamazaki, Seiji; Hanada, Sayaka; Kobayashi, Shuzo; Tsukamoto, Tatsuo; Haruna, Tetsuya; Sakaguchi, Katsuhiko; Sakai, Ken; Obara, Hideaki; Morishita, Kiyofumi; Saigo, Kenichi; Shintani, Yoshiaki; Kubo, Kohmei; Hoshino, Junichi; Oda, Teiji; Kaneko, Eiji; Nishikido, Masaharu; Ioji, Tetsuya; Kaneda, Hideaki; Fukushima, Masanori

2018-06-07

The clinical usefulness of peripheral blood (PB) mononuclear cell (MNC) transplantation in patients with peripheral arterial disease (PAD), especially in those with mild-to-moderate severity, has not been fully clarified.Methods and Results:A randomized clinical trial was conducted to evaluate the efficacy and safety of granulocyte colony-stimulating factor (G-CSF)-mobilized PBMNC transplantation in patients with PAD (Fontaine stage II-IV and Rutherford category 1-5) caused by arteriosclerosis obliterans or Buerger's disease. The primary endpoint was progression-free survival (PFS). In total, 107 subjects were enrolled. At baseline, Fontaine stage was II/III in 82 patients and IV in 21, and 54 patients were on hemodialysis. A total of 50 patients had intramuscular transplantation of PBMNC combined with standard of care (SOC) (cell therapy group), and 53 received SOC only (control group). PFS tended to be improved in the cell therapy group than in the control group (P=0.07). PFS in Fontaine stage II/III subgroup was significantly better in the cell therapy group than in the control group. Cell therapy-related adverse events were transient and not serious. In this first randomized, large-scale clinical trial of G-CSF-mobilized PBMNC transplantation, the cell therapy was tolerated by a variety of PAD patients. The PBMNC therapy was significantly effective for inhibiting disease progression in mild-to-moderate PAD.

Adenylate cyclase-stimulating, bone-resorbing and B TGF-like activities in canine apocrine cell adenocarcinoma of the anal sac.

PubMed

Weir, E C; Centrella, M; Matus, R E; Brooks, M L; Wu, T; Insogna, K L

1988-12-01

Canine apocrine cell adenocarcinoma of the anal sac (APO-AS) is a spontaneously occurring tumor that causes humorally mediated hypercalcemia in 90% of cases. To further define the nature of the responsible mediator in APO-AS, we examined tumor extracts from five APO-AS and four control tumors for adenylate cyclase-stimulating activity (ACSA). All extracts from APO-AS contained potent ACSA, whereas the four control tumors did not. The ACSA extracted from one tumor demonstrated a dose response curve parallel to that of synthetic bovinePTH-(1-34) and was 80% inhibited by Nle8,18,Tyr34 bPTH-(3-34)amide at a concentration of 10(-5) M. Extracts from three APO-AS and three control tumors were also examined for in vitro bone-resorbing activity (BRA). All APO-AS contained significant BRA, stimulating resorption 1.47 to 2.13-fold over basal, whereas none of the control tumors stimulated resorption. Purification of one extract using C18 reverse-phase high pressure liquid chromatography (RP-HPLC) resulted in a single sharp peak of ACSA which was 400-fold purified compared with the initial extract. This pool also contained significant bone-resorbing activity, whereas none of the adjacent pools did. Purification of a second extract using sequential CN and C18 RP-HPLC followed by size exclusion HPLC resulted in material that was at least 10,000-fold purified, and showed co-purification of ACSA and B TGF-like activity.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aerts-Kaya, Fatima S.F.; Visser, Trudi P.; Arshad, Shazia

Purpose: 5-Androstene-3{beta},17{beta}-diol (5-AED) stimulates recovery of hematopoiesis after exposure to radiation. To elucidate its cellular targets, the effects of 5-AED alone and in combination with (pegylated) granulocyte colony-stimulating factor and thrombopoietin (TPO) on immature hematopoietic progenitor cells were evaluated following total body irradiation. Methods and Materials: BALB/c mice were exposed to radiation delivered as a single or as a fractionated dose, and recovery of bone marrow progenitors and peripheral blood parameters was assessed. Results: BALB/c mice treated with 5-AED displayed accelerated multilineage blood cell recovery and elevated bone marrow (BM) cellularity and numbers of progenitor cells. The spleen colony-forming unitmore » (CFU-S) assay, representing the life-saving short-term repopulating cells in BM of irradiated donor mice revealed that combined treatment with 5-AED plus TPO resulted in a 20.1-fold increase in CFU-S relative to that of placebo controls, and a 3.7 and 3.1-fold increase in comparison to 5-AED and TPO, whereas no effect was seen of Peg-G-CSF with or without 5-AED. Contrary to TPO, 5-AED also stimulated reconstitution of the more immature marrow repopulating (MRA) cells. Conclusions: 5-AED potently counteracts the hematopoietic effects of radiation-induced myelosuppression and promotes multilineage reconstitution by stimulating immature bone marrow cells in a pattern distinct from, but synergistic with TPO.« less

Serum elevation of B lymphocyte stimulator does not increase regulatory B cells in glioblastoma patients undergoing immunotherapy.

PubMed

Saraswathula, Anirudh; Reap, Elizabeth A; Choi, Bryan D; Schmittling, Robert J; Norberg, Pamela K; Sayour, Elias J; Herndon, James E; Healy, Patrick; Congdon, Kendra L; Archer, Gerald E; Sanchez-Perez, Luis; Sampson, John H

2016-02-01

Regulatory B cells that secrete IL-10 (IL-10(+) Bregs) represent a suppressive subset of the B cell compartment with prominent anti-inflammatory capacity, capable of suppressing cellular and humoral responses to cancer and vaccines. B lymphocyte stimulator (BLyS) is a key regulatory molecule in IL-10(+) Breg biology with tightly controlled serum levels. However, BLyS levels can be drastically altered upon chemotherapeutic intervention. We have previously shown that serum BLyS levels are elevated, and directly associated, with increased antigen-specific antibody titers in patients with glioblastoma (GBM) undergoing lymphodepletive temozolomide chemotherapy and vaccination. In this study, we examined corresponding IL-10(+) Breg responses within this patient population and demonstrate that the IL-10(+) Breg compartment remains constant before and after administration of the vaccine, despite elevated BLyS levels in circulation. IL-10(+) Breg frequencies were not associated with serum BLyS levels, and ex vivo stimulation with a physiologically relevant concentration of BLyS did not increase IL-10(+) Breg frequency. However, BLyS stimulation did increase the frequency of the overall B cell compartment and promoted B cell proliferation upon B cell receptor engagement. Therefore, using BLyS as an adjuvant with therapeutic peptide vaccination could promote humoral immunity with no increase in immunosuppressive IL-10(+) Bregs. These results have implications for modulating humoral responses in human peptide vaccine trials in patients with GBM.

Kupffer cells facilitate the acute effects of leptin on hepatic lipid metabolism.

PubMed

Metlakunta, Anantha; Huang, Wan; Stefanovic-Racic, Maja; Dedousis, Nikolaos; Sipula, Ian; O'Doherty, Robert M

2017-01-01

Leptin has potent effects on lipid metabolism in a number of peripheral tissues. In liver, an acute leptin infusion (~120 min) stimulates hepatic fatty acid oxidation (~30%) and reduces triglycerides (TG, ~40%), effects that are dependent on phosphoinositol-3-kinase (PI3K) activity. In the current study we addressed the hypothesis that leptin actions on liver-resident immune cells are required for these metabolic effects. Myeloid cell-specific deletion of the leptin receptor (ObR) in mice or depletion of liver Kupffer cells (KC) in rats in vivo prevented the acute effects of leptin on liver lipid metabolism, while the metabolic effects of leptin were maintained in mice lacking ObR in hepatocytes. Notably, liver TG were elevated in both lean and obese myeloid cell ObR, but the degree of obesity and insulin resistance induced by a high-fat diet was similar to control mice. In isolated primary hepatocytes (HEP), leptin had no effects on HEP lipid metabolism and only weakly stimulated PI3K. However, the coculture of KC with HEP restored leptin action on HEP fatty acid metabolism and stimulation of HEP PI3K. Notably, leptin stimulated the release from KC of a number of cytokines. However, the exposure of HEP to these cytokines individually [granulocyte macrophage colony-stimulating factor, IL-1α, IL-1β, IL-6, IL-10, and IL-18] or in combination had no effects on HEP lipid metabolism. Together, these data demonstrate a role for liver mononuclear cells in the regulation of liver lipid metabolism by leptin. Copyright © 2017 the American Physiological Society.

Effects of the pulsed electromagnetic field PST® on human tendon stem cells: a controlled laboratory study.

PubMed

Randelli, Pietro; Menon, Alessandra; Ragone, Vincenza; Creo, Pasquale; Alfieri Montrasio, Umberto; Perucca Orfei, Carlotta; Banfi, Giuseppe; Cabitza, Paolo; Tettamanti, Guido; Anastasia, Luigi

2016-08-18

Current clinical procedures for rotator cuff tears need to be improved, as a high rate of failure is still observed. Therefore, new approaches have been attempted to stimulate self-regeneration, including biophysical stimulation modalities, such as low-frequency pulsed electromagnetic fields, which are alternative and non-invasive methods that seem to produce satisfying therapeutic effects. While little is known about their mechanism of action, it has been speculated that they may act on resident stem cells. Thus, the purpose of this study was to evaluate the effects of a pulsed electromagnetic field (PST®) on human tendon stem cells (hTSCs) in order to elucidate the possible mechanism of the observed therapeutic effects. hTSCs from the rotator cuff were isolated from tendon biopsies and cultured in vitro. Then, cells were exposed to a 1-h PST® treatment and compared to control untreated cells in terms of cell morphology, proliferation, viability, migration, and stem cell marker expression. Exposure of hTSCs to PST® did not cause any significant changes in proliferation, viability, migration, and morphology. Instead, while stem cell marker expression significantly decreased in control cells during cell culturing, PST®-treated cells did not have a significant reduction of the same markers. While PST® did not have significant effects on hTSCs proliferation, the treatment had beneficial effects on stem cell marker expression, as treated cells maintained a higher expression of these markers during culturing. These results support the notion that PST® treatment may increase the patient stem cell regenerative potential.

Adrenal Demedullation and Oxygen Supplementation Independently Increase Glucose-Stimulated Insulin Concentrations in Fetal Sheep With Intrauterine Growth Restriction

PubMed Central

Macko, Antoni R.; Yates, Dustin T.; Chen, Xiaochuan; Shelton, Leslie A.; Kelly, Amy C.; Davis, Melissa A.; Camacho, Leticia E.; Anderson, Miranda J.

2016-01-01

In pregnancies complicated by placental insufficiency and intrauterine growth restriction (IUGR), fetal glucose and oxygen concentrations are reduced, whereas plasma norepinephrine and epinephrine concentrations are elevated throughout the final third of gestation. Here we study the effects of chronic hypoxemia and hypercatecholaminemia on β-cell function in fetal sheep with placental insufficiency-induced IUGR that is produced by maternal hyperthermia. IUGR and control fetuses underwent a sham (intact) or bilateral adrenal demedullation (AD) surgical procedure at 0.65 gestation. As expected, AD-IUGR fetuses had lower norepinephrine concentrations than intact-IUGR fetuses despite being hypoxemic and hypoglycemic. Placental insufficiency reduced fetal weights, but the severity of IUGR was less with AD. Although basal plasma insulin concentrations were lower in intact-IUGR and AD-IUGR fetuses compared with intact-controls, glucose-stimulated insulin concentrations were greater in AD-IUGR fetuses compared with intact-IUGR fetuses. Interestingly, AD-controls had lower glucose- and arginine-stimulated insulin concentrations than intact-controls, but AD-IUGR and AD-control insulin responses were not different. To investigate chronic hypoxemia in the IUGR fetus, arterial oxygen tension was increased to normal levels by increasing the maternal inspired oxygen fraction. Oxygenation of IUGR fetuses enhanced glucose-stimulated insulin concentrations 3.3-fold in intact-IUGR and 1.7-fold in AD-IUGR fetuses but did not lower norepinephrine and epinephrine concentrations. Together these findings show that chronic hypoxemia and hypercatecholaminemia have distinct but complementary roles in the suppression of β-cell responsiveness in IUGR fetuses. PMID:26937714

TRPV2 Mediates Adrenomedullin Stimulation of Prostate and Urothelial Cancer Cell Adhesion, Migration and Invasion

PubMed Central

Vanden Abeele, Fabien; Lehen’kyi, V’yacheslav; Ouafik, L’Houcine; Mauroy, Brigitte; Prevarskaya, Natalia

2013-01-01

Adrenomedullin (AM) is a 52-amino acid peptide initially isolated from human pheochromocytoma. AM is expressed in a variety of malignant tissues and cancer cell lines and was shown to be a mitogenic factor capable of stimulating growth of several cancer cell types. In addition, AM is a survival factor for certain cancer cells. Some data suggest that AM might be involved in the progression cancer metastasis via angiogenesis and cell migration and invasion control. The Transient Receptor Potential channel TRPV2 is known to promote in prostate cancer cell migration and invasive phenotype and is correlated with the stage and grade of bladder cancer. In this work we show that AM induces prostate and urothelial cancer cell migration and invasion through TRPV2 translocation to plasma membrane and the subsequent increase in resting calcium level. PMID:23741410

Synthetic gene network restoring endogenous pituitary–thyroid feedback control in experimental Graves’ disease

PubMed Central

Saxena, Pratik; Charpin-El Hamri, Ghislaine; Folcher, Marc; Zulewski, Henryk; Fussenegger, Martin

2016-01-01

Graves’ disease is an autoimmune disorder that causes hyperthyroidism because of autoantibodies that bind to the thyroid-stimulating hormone receptor (TSHR) on the thyroid gland, triggering thyroid hormone release. The physiological control of thyroid hormone homeostasis by the feedback loops involving the hypothalamus–pituitary–thyroid axis is disrupted by these stimulating autoantibodies. To reset the endogenous thyrotrophic feedback control, we designed a synthetic mammalian gene circuit that maintains thyroid hormone homeostasis by monitoring thyroid hormone levels and coordinating the expression of a thyroid-stimulating hormone receptor antagonist (TSHAntag), which competitively inhibits the binding of thyroid-stimulating hormone or the human autoantibody to TSHR. This synthetic control device consists of a synthetic thyroid-sensing receptor (TSR), a yeast Gal4 protein/human thyroid receptor-α fusion, which reversibly triggers expression of the TSHAntag gene from TSR-dependent promoters. In hyperthyroid mice, this synthetic circuit sensed pathological thyroid hormone levels and restored the thyrotrophic feedback control of the hypothalamus–pituitary–thyroid axis to euthyroid hormone levels. Therapeutic plug and play gene circuits that restore physiological feedback control in metabolic disorders foster advanced gene- and cell-based therapies. PMID:26787873

TLR-mediated stimulation of APC: Distinct cytokine responses of B cells and dendritic cells

PubMed Central

Barr, Tom A; Brown, Sheila; Ryan, Gemma; Zhao, Jiexin; Gray, David

2007-01-01

In addition to their role in humoral immunity, B lymphocytes are important antigen-presenting cells (APC). In the same way as other APC, B cells make cytokines upon activation and have the potential to modulate T cell responses. In this study, we investigated which mouse B cell subsets are the most potent cytokine producers, and examined the role of Toll-like receptors (TLR) in the control of secretion of IL-6, IL-10, IL-12 and IFN-γ by B cells. Production of some cytokines was restricted to particular subsets. Marginal zone and B1 cells were the predominant source of B cell IL-10 in the spleen. Conversely, follicular B cells were found to express IFN-γ mRNA directly ex vivo. The nature of the activating stimulus dramatically influenced the cytokine made by B cells. Thus, in response to combined TLR stimulation, or via phorbol esters, IFN-γ was secreted. IL-10 was elicited by T-dependent activation or stimulation through TLR2, 4 or 9. This pattern of cytokine expression contrasts with that elicited from dendritic cells. QRT-PCR array data indicate that this may be due to differential expression of TLR signalling molecules, effectors and adaptors. Our data highlight the potentially unique nature of immune modulation when B cells act as APC. PMID:17918201

The metabolism of N-acetylcysteine by human endothelial cells.

PubMed

Cotgreave, I; Moldéus, P; Schuppe, I

1991-06-21

When human umbilical endothelial cells were depleted of their glutathione by incubation in a sulfur amino acid-free medium, subsequent incubation of the cells with this deficient medium supplemented with N-acetylcysteine resulted in a dose-dependent stimulation of the synthesis of cellular glutathione. Similarly, the inclusion of N-acetylcysteine in the medium during the period of depletion of glutathione caused a dose-dependent retardation of the depletion kinetics. In contrast, the incubation of control cells in normal medium supplemented with N-acetylcysteine did not increase cellular glutathione levels above controls. These observations indicate the presence of an N-deacetylase in/on the cells with specificity for N-acetylcysteine. Due to the large surface area of the endothelium in the vasculature it seems likely that endothelial cell N-deacetylation plays a role in the metabolic disposition of N-acetylcysteine, particularly when administered intravenously. N-Acetylcysteine is, however, a relatively poor precursor to glutathione biosynthesis in comparison to cystine. Thus, any cytoprotective, antioxidant effect exerted by N-acetylcysteine on the human endothelium is likely to be due to direct scavenging of reactive intermediates rather than by stimulated glutathione synthesis in the endothelial cells themselves.

Differences In Early T-Cell Signaling In Cultures Grown In a Rotating Clinostat vs. Static Controls

NASA Technical Reports Server (NTRS)

Alexamder. M.; Nelman-Gonzales, M.; Penkala, J.; Sams, C.

1999-01-01

Altered gravity has previously been demonstrated to be a stress that can influence components of the immune system. Specifically, T-cell activation has been shown to be affected by changes in gravity, exhibiting a decrease in proliferative response to in vitro stimulation in microgravity. Subsequent ground based studies utilizing a rotating clinostat to model some of the effects of microgravity have been consistent with earlier flight based experiments. These ground and flight experiments have examined T-cell activation by measuring various responses including production of cytokines, DNA synthesis and the production of various cell surface activation markers. These indicators of T-cell activation were measured anywhere from 4 to 72 hours after stimulation. Prior to the work described here, the initial signaling events in T-cell activation had not been directly examined. The goal of this project was to determine how the process of early signal transduction was affected by growth in a rotating clinostat. Here we directly show a defect in signaling from TCR to MAPK in purified peripheral T-cells activated in the clinostat by OKT3/antiCD28 coated microbeads as compared to static controls.

Peptides of a major histocompatibility complex class I (Kb) molecule cause prolongation of skin graft survival and induce specific down-regulatory T cells demonstrable in the mixed lymphocyte reaction.

PubMed Central

Brondz, B D; Kazansky, D B; Chernyshova, A D; Ivanov, V S

1995-01-01

Six individual peptides of the major histocompatibility complex (MHC) class I molecule H-2Kb were synthesized. Intravenous injection of peptide 6 into mice prolonged the survival of Kb (BL/6 or B10.MBR) skin grafts on allogeneic R101 and B10.AKM mice, respectively. This was specific, as control skin grafts from Kk (B10.BR) or Kd (DBA/2) donors, respectively, were rejected at the same time in both control and peptide-treated mice. The optimal doses for peptide 6, which is from the alpha 2 domain, were defined. The test system was the inhibition of proliferation in vitro of naive lymph node cells by syngeneic mitomycin c-treated spleen cells from R101 mice preimmunized with irradiated stimulator splenocytes of Kb (BL/6) origin. Down-regulation was specific, as proliferation in response to third-party allogeneic stimulator Kk (B10.BR) splenocytes was not inhibited. Of the six peptides of H-2Kb tested, potent down-regulatory cells were induced by peptides 2 (alpha 1 domain) and 5 and 6 (alpha 2 domain). The greatest down-regulatory activity was obtained by giving peptide 2 to mice that had already been immunized against H-2Kb by injecting EL4 cells. Under the same conditions, injecting peptide 2 did not induce any cytotoxic T cells. In contrast, specific cytotoxic lymphocytes (CTL) were induced when cells from primed mice were incubated for 4 days with heated stimulator cells from BL/6 mice. The data suggest that peptides from MHC class I molecules activate precursors of down-regulatory T cells, but not of CTL, and this may explain their ability to prolong skin allograft survival. PMID:7490121

Cross-talk between an activator of nuclear receptors-mediated transcription and the D1 dopamine receptor signaling pathway.

PubMed

Schmidt, Azriel; Vogel, Robert; Rutledge, Su Jane; Opas, Evan E; Rodan, Gideon A; Friedman, Eitan

2005-03-01

Nuclear receptors are transcription factors that usually interact, in a ligand-dependent manner, with specific DNA sequences located within promoters of target genes. The nuclear receptors can also be controlled in a ligand-independent manner via the action of membrane receptors and cellular signaling pathways. 5-Tetradecyloxy-2-furancarboxylic acid (TOFA) was shown to stimulate transcription from the MMTV promoter via chimeric receptors that consist of the DNA binding domain of GR and the ligand binding regions of the PPARbeta or LXRbeta nuclear receptors (GR/PPARbeta and GR/LXRbeta). TOFA and hydroxycholesterols also modulate transcription from NF-kappaB- and AP-1-controlled reporter genes and induce neurite differentiation in PC12 cells. In CV-1 cells that express D(1) dopamine receptors, D(1) dopamine receptor stimulation was found to inhibit TOFA-stimulated transcription from the MMTV promoter that is under the control of chimeric GR/PPARbeta and GR/LXRbeta receptors. Treatment with the D(1) dopamine receptor antagonist, SCH23390, prevented dopamine-mediated suppression of transcription, and by itself increased transcription controlled by GR/LXRbeta. Furthermore, combined treatment of CV-1 cells with TOFA and SCH23390 increased transcription controlled by the GR/LXRbeta chimeric receptor synergistically. The significance of this in vitro synergy was demonstrated in vivo, by the observation that SCH23390 (but not haloperidol)-mediated catalepsy in rats was potentiated by TOFA, thus showing that an agent that mimics the in vitro activities of compounds that activate members of the LXR and PPAR receptor families can influence D1 dopamine receptor elicited responses.

Ultrasonic and electromagnetic enhancement of a culture of human SAOS-2 osteoblasts seeded onto a titanium plasma-spray surface.

PubMed

Fassina, Lorenzo; Saino, Enrica; Sbarra, Maria Sonia; Visai, Livia; Cusella De Angelis, Maria Gabriella; Mazzini, Giuliano; Benazzo, Francesco; Magenes, Giovanni

2009-06-01

Several studies suggest that the surface coating of titanium could play an important role in bone tissue engineering. In the present study, we have followed a particular biomimetic strategy where ultrasonically or electromagnetically stimulated SAOS-2 human osteoblasts proliferated and built their extracellular matrix on a titanium plasma-spray surface. In comparison with control conditions, the ultrasonic stimulation (average power, 149 mW; frequency, 1.5 MHz) and the electromagnetic stimulation (magnetic field intensity, 2 mT; frequency, 75 Hz) caused higher cell proliferation, and increased surface coating with decorin, osteocalcin, osteopontin, and type I collagen together with higher incorporation of calcium and phosphorus inside the extracellular matrix. The immunofluorescence related to the preceding bone matrix proteins showed their colocalization in the cell-rich areas. The use of the two physical stimulations aimed at obtaining the coating of the rough titanium plasma-spray surface in terms of cell colonization and deposition of extracellular matrix. The superficially cultured biomaterial could be theoretically used, in clinical applications, as an implant for bone repair.

Immunostimulatory CpG-oligonucleotides induce functional high affinity IL-2 receptors on B-CLL cells: costimulation with IL-2 results in a highly immunogenic phenotype.

PubMed

Decker, T; Schneller, F; Kronschnabl, M; Dechow, T; Lipford, G B; Wagner, H; Peschel, C

2000-05-01

CpG-oligodeoxynucleotides (CpG-ODN) have been shown to induce proliferation, cytokine production, and surface molecule regulation in normal and malignant human B cells. In the present study, we investigated the potential of CpG-ODN to induce functional high-affinity receptors in leukemic and normal B cells and the effects of costimulation with IL-2 on proliferation, cytokine secretion, and surface molecule regulation. Highly purified B cells from B-CLL patients and normal controls were stimulated with CpG-ODN with or without IL-2. Expression of CD25 was determined using FACS, and the presence of high-affinity IL-2 receptors was determined by scatchard analysis. Costimulatory effects of IL-2 and CpG-ODN were investigated using proliferation assays, ELISA (IL-6, TNF-alpha), and FACS analysis (CD80, CD86 expression). Reactivity of autologous and allogeneic T cells toward activated B-CLL cells was determined in mixed lymphocyte reactions and Interferon-gamma Elispot assays. The CpG-ODN DSP30 caused a significantly stronger induction of the IL-2 receptor alpha chain in malignant as compared with normal B cells (p = 0.03). This resulted in the expression of functional high-affinity IL-2 receptors in B-CLL cells, but fewer numbers of receptors with less affinity were expressed in normal B cells. Although addition of IL-2 to CpG-ODN-stimulated cells augmented proliferation in both normal B cells and B-CLL cells, no costimulatory effect on cytokine production or surface molecule expression could be observed in normal B cells. In contrast, TNF-alpha and IL-6 production was increased in B-CLL cells, and the expression of CD80 and CD86 was further enhanced when IL-2 was used as a costimulus. Autologous and allogeneic immune recognition of B-CLL cells stimulated with CpG-ODN and IL-2 was increased compared with B-CLL cells stimulated with CpG-ODN alone. Stimulation of B-CLL cells with CpG-ODN and IL-2 might be an attractive strategy for potential immunotherapies for B-CLL patients.

Casticin, a flavonoid isolated from Vitex rotundifolia, inhibits prolactin release in vivo and in vitro

PubMed Central

Ye, Qi; Zhang, Qiao-yan; Zheng, Cheng-jian; Wang, Yang; Qin, Lu-ping

2010-01-01

Aim: To investigate the anti-hyperprolactinemia activity of casticin, a flavonoid isolated from Vitex rotundifolia, and elucidate its molecular mechanism. Methods: Hyperprolactinemia (MIHP) was induced by administration of metoclopramide dihydrochloride (50 mg/kg, tid, ip, for 10 d) in SD rats and the primary pituitary cells were prepared from the pituitary glands of the SD rats. Prolactin concentrations were measured using a radioimmunoassay. Cell viability was measured using an MTT assay. The mRNA expression of estrogen receptor alpha and beta in rat pituitary cells was measured using semi-quantitative RT-PCR analysis. Results: The level of serum prolactin in the MIHP model group was 2.1 fold higher than that in the untreated control group (P<0.01). Casticin (10, 20, and 40 mg/kg, ip, for 7 d) reduced serum prolactin levels by 33.9%, 54.3%, and 64.7%, respectively (P<0.01). The positive control drug bromocriptine 1 mg/kg decreased the serum prolactin concentration in MIHP rats by 44.9%. 17β-Estradiol (E2) significantly increased the proliferation of pituitary cells and casticin (1 and 10 μmol/L) markedly inhibited E2-induced pituitary cell proliferation by 27.7% and 42.1%, respectively. Stimulation of pituitary cells with E2 increased prolactin secretion into the cell culture supernatants, and casticin (0.1, 1, and 10 μmol/L) significantly inhibited the prolactin release stimulated by E2 in a concentration-dependent manner. Casticin (1 and 10 μmol/L) significantly inhibited ERα mRNA expression in pituitary cells stimulated with E2 (P<0.01) but increased ERβ mRNA expression at a concentration of 10 μmol/L (P<0.01). However, casticin had no effects on proliferation and prolectin release of the unstimulated primary pituitary cells in vitro. Conclusion: Casticin inhibited the release of prolactin from pituitary cells of SD rats stimulated with E2 in vivo and in vitro. These effects might be related with inhibiting the ERα mRNA expression and increasing the ERβ mRNA expression. PMID:21042288

Differential growth factor control of bone formation through osteoprogenitor differentiation.

PubMed

Chaudhary, L R; Hofmeister, A M; Hruska, K A

2004-03-01

The osteogenic factors bone morphogenetic protein (BMP-7), platelet-derived growth factor (PDGF)-BB, and fibroblast growth factor (FGF-2) regulate the recruitment of osteoprogenitor cells and their proliferation and differentiation into mature osteoblasts. However, their mechanisms of action on osteoprogenitor cell growth, differentiation, and bone mineralization remain unclear. Here, we tested the hypothesis that these osteogenic agents were capable of regulating osteoblast differentiation and bone formation in vitro. Normal human bone marrow stromal (HBMS) cells were treated with BMP-7 (40 ng ml(-1)), PDGF-BB (20 ng ml(-1)), FGF-2 (20 ng ml(-1)), or FGF-2 plus BMP-7 for 28 days in a serum-containing medium with 10 mM beta-glycerophosphate and 50 microg ml(-1) ascorbic acid. BMP-7 stimulated a morphological change to cuboidal-shaped cells, increased alkaline phosphatase (ALKP) activity, bone sialoprotein (BSP) gene expression, and alizarin red S positive nodule formation. Hydroxyapatite (HA) crystal deposition in the nodules was demonstrated by Fourier transform infrared (FTIR) spectroscopy only in BMP-7- and dexamethasone (DEX)-treated cells. DEX-treated cells appeared elongated and fibroblast-like compared to BMP-7-treated cells. FGF-2 did not stimulate ALKP, and cell morphology was dystrophic. PDGF-BB had little or no effect on ALKP activity and biomineralization. Alizarin Red S staining of cells and calcium assay indicated that BMP-7, DEX, and FGF-2 enhanced calcium mineral deposition, but FTIR spectroscopic analysis demonstrated no formation of HA similar to human bone in control, PDGF-BB-, and FGF-2-treated samples. Thus, FGF-2 stimulated amorphous octacalcium phosphate mineral deposition that failed to mature into HA. Interestingly, FGF-2 abrogated BMP-7-induced ALKP activity and HA formation. Results demonstrate that BMP-7 was competent as a sole factor in the differentiation of human bone marrow stromal cells to bone-forming osteoblasts confirmed by FTIR examination of mineralized matrix. Other growth factors, PDGF, and FGF-2 were incompetent as sole factors, and FGF-2 inhibited BMP-7-stimulated osteoblast differentiation.

Sulfasalazine and Mesalamine Modulate Beryllium-Specific Lymphocyte Proliferation and Inflammatory Cytokine Production

PubMed Central

Dobis, Dave R.; Sawyer, Richard T.; Gillespie, May M.; Newman, Lee S.; Maier, Lisa A.; Day, Brian J.

2010-01-01

Occupational exposure to beryllium (Be) results in Be sensitization (BeS) that can progress to pulmonary granulomatous inflammation associated with chronic Be disease (CBD). Be-specific lymphocytes are present in the blood of patients with BeS and in the blood and lungs of patients with CBD. Sulfasalazine and its active metabolite, mesalamine, are clinically used to ameliorate chronic inflammation associated with inflammatory bowel disease. We tested whether sulfasalazine or mesalamine could decrease Be-stimulated peripheral blood mononuclear cell (PBMC) proliferation in subjects with CBD and BeS and Be-induced cytokine production in CBD bronchoalveolar lavage (BAL) cells. CBD (n = 25), BeS (n = 12) and healthy normal control (n = 6) subjects were enrolled and ex vivo proliferation and cytokine production were assessed in the presence of Be and sulfasalazine or mesalamine. Be-stimulated PBMC proliferation was inhibited by treatment with either sulfasalazine or mesalamine. Be-stimulated CBD BAL cell IFN-γ and TNF-α cytokine production was decreased by treatment with sulfasalazine or mesalamine. Our data suggest that both sulfasalazine and mesalamine interfere with Be-stimulated PBMC proliferation in CBD and BeS and dampens Be-stimulated CBD BAL cell proinflammatory cytokine production. These studies demonstrate that sulfasalazine and mesalamine can disrupt inflammatory pathways critical to the pathogenesis of chronic granulomatous inflammation in CBD, and may serve as novel therapy for human granulomatous lung diseases. PMID:19901345

Sulfasalazine and mesalamine modulate beryllium-specific lymphocyte proliferation and inflammatory cytokine production.

PubMed

Dobis, Dave R; Sawyer, Richard T; Gillespie, May M; Newman, Lee S; Maier, Lisa A; Day, Brian J

2010-10-01

Occupational exposure to beryllium (Be) results in Be sensitization (BeS) that can progress to pulmonary granulomatous inflammation associated with chronic Be disease (CBD). Be-specific lymphocytes are present in the blood of patients with BeS and in the blood and lungs of patients with CBD. Sulfasalazine and its active metabolite, mesalamine, are clinically used to ameliorate chronic inflammation associated with inflammatory bowel disease. We tested whether sulfasalazine or mesalamine could decrease Be-stimulated peripheral blood mononuclear cell (PBMC) proliferation in subjects with CBD and BeS and Be-induced cytokine production in CBD bronchoalveolar lavage (BAL) cells. CBD (n = 25), BeS (n = 12) and healthy normal control (n = 6) subjects were enrolled and ex vivo proliferation and cytokine production were assessed in the presence of Be and sulfasalazine or mesalamine. Be-stimulated PBMC proliferation was inhibited by treatment with either sulfasalazine or mesalamine. Be-stimulated CBD BAL cell IFN-γ and TNF-α cytokine production was decreased by treatment with sulfasalazine or mesalamine. Our data suggest that both sulfasalazine and mesalamine interfere with Be-stimulated PBMC proliferation in CBD and BeS and dampens Be-stimulated CBD BAL cell proinflammatory cytokine production. These studies demonstrate that sulfasalazine and mesalamine can disrupt inflammatory pathways critical to the pathogenesis of chronic granulomatous inflammation in CBD, and may serve as novel therapy for human granulomatous lung diseases.

Comparison of transplantation of bone marrow stromal cells (BMSC) and stem cell mobilization by granulocyte colony stimulating factor after traumatic brain injury in rat.

PubMed

Bakhtiary, Mehrdad; Marzban, Mohsen; Mehdizadeh, Mehdi; Joghataei, Mohammad Taghi; Khoei, Samideh; Pirhajati Mahabadi, Vahid; Laribi, Bahareh; Tondar, Mahdi; Moshkforoush, Arash

2010-10-01

Recent clinical studies of treating traumatic brain injury (TBI) with autologous adult stem cells led us to compare effect of intravenous injection of bone marrow mesenchymal stem cells (BMSC) and bone marrow hematopoietic stem cell mobilization, induced by granulocyte colony stimulating factor (G-CSF), in rats with a cortical compact device. Forty adult male Wistar rats were injured with controlled cortical impact device and divided randomly into four groups. The treatment groups were injected with 2 × 106 intravenous bone marrow stromal stem cell (n = 10) and also with subcutaneous G-CSF (n = 10) and sham-operation group (n = 10) received PBS and "bromodeoxyuridine (Brdu)" alone, i.p. All injections were performed 1 day after injury into the tail veins of rats. All cells were labeled with Brdu before injection into the tail veins of rats. Functional neurological evaluation of animals was performed before and after injury using modified neurological severity scores (mNSS). Animals were sacrificed 42 days after TBI and brain sections were stained by Brdu immunohistochemistry. Statistically, significant improvement in functional outcome was observed in treatment groups compared with control group (P<0.01). mNSS showed no significant difference between the BMSC and G-CSF-treated groups during the study period (end of the trial). Histological analyses showed that Brdu-labeled (MSC) were present in the lesion boundary zone at 42nd day in all injected animals. In our study, we found that administration of a bone marrow-stimulating factor (G-CSF) and BMSC in a TBI model provides functional benefits.

Mechanical stimuli differentially control stem cell behavior: morphology, proliferation, and differentiation

PubMed Central

Maul, Timothy M.; Chew, Douglas W.; Nieponice, Alejandro

2011-01-01

Mesenchymal stem cell (MSC) therapy has demonstrated applications in vascular regenerative medicine. Although blood vessels exist in a mechanically dynamic environment, there has been no rigorous, systematic analysis of mechanical stimulation on stem cell differentiation. We hypothesize that mechanical stimuli, relevant to the vasculature, can differentiate MSCs toward smooth muscle (SMCs) and endothelial cells (ECs). This was tested using a unique experimental platform to differentially apply various mechanical stimuli in parallel. Three forces, cyclic stretch, cyclic pressure, and laminar shear stress, were applied independently to mimic several vascular physiologic conditions. Experiments were conducted using subconfluent MSCs for 5 days and demonstrated significant effects on morphology and proliferation depending upon the type, magnitude, frequency, and duration of applied stimulation. We have defined thresholds of cyclic stretch that potentiate SMC protein expression, but did not find EC protein expression under any condition tested. However, a second set of experiments performed at confluence and aimed to elicit the temporal gene expression response of a select magnitude of each stimulus revealed that EC gene expression can be increased with cyclic pressure and shear stress in a cell-contact-dependent manner. Further, these MSCs also appear to express genes from multiple lineages simultaneously which may warrant further investigation into post-transcriptional mechanisms for controlling protein expression. To our knowledge, this is the first systematic examination of the effects of mechanical stimulation on MSCs and has implications for the understanding of stem cell biology, as well as potential bioreactor designs for tissue engineering and cell therapy applications. PMID:21253809

Expression of human choline kinase in NIH 3T3 fibroblasts increases the mitogenic potential of insulin and insulin-like growth factor I.

PubMed

Chung, T; Huang, J S; Mukherjee, J J; Crilly, K S; Kiss, Z

2000-05-01

In mammalian cells, growth factors, oncogenes, and carcinogens stimulate phosphocholine (PCho) synthesis by choline kinase (CK), suggesting that PCho may regulate cell growth. To validate the role of PCho in mitogenesis, we determined the effects of insulin, insulin-like growth factor I (IGF-I), and other growth factors on DNA synthesis in NIH 3T3 fibroblast sublines highly expressing human choline kinase (CK) without increasing phosphatidylcholine synthesis. In serum-starved CK expressor cells, insulin and IGF-I stimulated DNA synthesis, p70 S6 kinase (p70 S6K) activity, phosphatidylinositol 3-kinase (PI3K) activity, and activating phosphorylation of p42/p44 mitogen-activated protein kinases (MAPK) to greater extents than in the corresponding vector control cells. Furthermore, the CK inhibitor hemicholinium-3 (HC-3) inhibited insulin- and IGF-I-induced DNA synthesis in the CK overexpressors, but not in the vector control cells. The results indicate that high cellular levels of PCho potentiate insulin- and IGF-I-induced DNA synthesis by MAPK- and p70 S6K-regulated mechanisms.

Repulsive guidance molecule a blockade exerts the immunoregulatory function in DCs stimulated with ABP and LPS

PubMed Central

Xu, Xuxu; Gao, Yan; Zhai, Zhiyong; Zhang, Shuo; Shan, Fengping; Feng, Juan

2016-01-01

ABSTRACT Repulsive guidance molecule a (RGMa) is an axonal guidance molecule that has recently found to exert function in immune system. This study evaluated the function of RGMa in modulation of dendritic cells (DCs) function stimulated with Achyranthes bidentata polysaccharide (ABP) and lipopolysaccharide (LPS) using a RGMa-neutralizing antibody. Compared with the Control-IgG/ABP and Control-IgG/LPS groups, DCs in the Anti-RGMa/ABP and Anti-RGMa/LPS groups 1) showed small, round cells with a few cell processes and organelles, and many pinocytotic vesicles; 2) had decreased MHC II, CD86, CD80, and CD40 expression; 3) displayed the decreased IL-12p70, IL-1β and TNF-α levels and increased IL-10 secretion; 4) had a high percentage of FITC-dextran uptake; and 5) displayed a reduced ability to drive T cell proliferation and reinforced T cell polarization toward a Th2 cytokine pattern. We conclude that DCs treated with RGMa-neutralizing antibodies present with tolerogenic and immunoregulatory characteristics, which provides new insights into further understanding of the function of RGMa. PMID:26986456

Repulsive guidance molecule a blockade exerts the immunoregulatory function in DCs stimulated with ABP and LPS.

PubMed

Xu, Xuxu; Gao, Yan; Zhai, Zhiyong; Zhang, Shuo; Shan, Fengping; Feng, Juan

2016-08-02

Repulsive guidance molecule a (RGMa) is an axonal guidance molecule that has recently found to exert function in immune system. This study evaluated the function of RGMa in modulation of dendritic cells (DCs) function stimulated with Achyranthes bidentata polysaccharide (ABP) and lipopolysaccharide (LPS) using a RGMa-neutralizing antibody. Compared with the Control-IgG/ABP and Control-IgG/LPS groups, DCs in the Anti-RGMa/ABP and Anti-RGMa/LPS groups 1) showed small, round cells with a few cell processes and organelles, and many pinocytotic vesicles; 2) had decreased MHC II, CD86, CD80, and CD40 expression; 3) displayed the decreased IL-12p70, IL-1β and TNF-α levels and increased IL-10 secretion; 4) had a high percentage of FITC-dextran uptake; and 5) displayed a reduced ability to drive T cell proliferation and reinforced T cell polarization toward a Th2 cytokine pattern. We conclude that DCs treated with RGMa-neutralizing antibodies present with tolerogenic and immunoregulatory characteristics, which provides new insights into further understanding of the function of RGMa.

Organization and mobility of CD11b/CD18 and targeting of superoxide on the surface of degranulated human neutrophils.

PubMed

Mukherjee, G; Rasmusson, B; Linner, J G; Quinn, M T; Parkos, C A; Magnusson, K E; Jesaitis, A J

1998-09-01

A monoclonal IgM, specifically recognizing both CD11b and CD18 of human neutrophils, was used to examine the organization and mobility of CD11b/CD18 in the plasma membrane of human neutrophils degranulated by dihydrocytochalasin B (dhCB) treatment and fMet-Leu-Phe (fMLF) stimulation. Subcellular fractionation analysis of untreated or dhCB-treated control neutrophils indicated that 20% of CD11b/CD18 cosedimented with plasma membrane and the remainder with specific granules. In contrast, fMLF stimulation of dhCB-treated cells caused a major reorganization of CD11b/CD18, in which 60-70% of CD11b/CD18 sedimented in dense plasma membrane fractions that were also enriched in superoxide-generating NADPH oxidase activity. Similarly pretreated neutrophils were fixed, immunogold labeled, and examined by scanning electron microscopy. Immunogold particles were distributed uniformly over the symmetrically ruffled surface of unstimulated neutrophils. On dhCB-treated cells, immunogold was mostly uniformly distributed on a smooth membrane with a small percentage of particles lining up into linear arrays. After fMLF + dhCB stimulation, CD11b/CD18 gold label was more abundant on the cell surface and formed large aggregates on polarized membrane protrusions. However, when cells were adhered to an albumin-coated quartz surface and stimulated with fMLF in the presence of dhCB, immunogold was excluded on the articulated and rounded cell body but concentrated on the periphery of adherent lamellae. Fluorescence photobleaching recovery indicated that in unstimulated cells 38 +/- 3% of CD11b/CD18 was mobile (R) with a diffusion constant D of 3.1 +/- 0.3 x 10(-10) cm2/s. Treatment with dhCB raised R and D 24 and 74%, respectively. Stimulation using 1 microM fMLF with dhCB lowered D and R to near control levels. Since NADPH oxidase and CD11b/CD18 cosediment in high-density plasma membrane domains after fMLF + dhCB stimulation, we speculate that a stimulus-induced reorganization of CD11b/CD18 and NADPH oxidase to common membrane domains may occur in fMLF + dhCB-degranulated neutrophils. Copyright 1998 Academic Press.

Reorganization of the raccoon cuneate nucleus after peripheral denervation.

PubMed

Rasmusson, D D; Northgrave, S A

1997-12-01

The effects of peripheral nerve transection on the cuneate nucleus were studied in anesthetized raccoons using extracellular, single-unit recordings. The somatotopic organization of the cuneate nucleus first was examined in intact, control animals. The cuneate nucleus in the raccoon is organized with the digits represented in separate cell clusters. The dorsal cap region of the cuneate nucleus contains a representation of the claws and hairy skin of the digits. Within the representation of the glabrous skin, neurons with rapidly adapting properties tended to be segregated from those with slowly adapting properties. The representations of the distal and proximal pads on a digit also were segregated. Electrical stimulation of two adjacent digits provided a detailed description of the responses originating from the digit that contains the tactile receptive field (the on-focus digit) and from the adjacent (off-focus) digit. Stimulation of the on-focus digit produced a short latency excitation in all 99 neurons tested, with a mean of 10.5 ms. These responses had a low threshold (426 microA). Stimulation of an off-focus digit activated 65% of these neurons. These responses had a significantly longer latency (15.3 ms) than on-focus responses and the threshold was more than twice as large. Two to five months after amputation of digit 4, 97 cells were tested with stimulation of digits 3 and 5. A total of 44 were in the intact regions of the cuneate nucleus. They had small receptive fields on intact digits and their responses to electrical stimulation did not differ from the control neurons. The remaining 53 neurons were judged to be deafferented and in the fourth digit region on the basis of their location with respect to intact neurons. All but two of these cells had receptive fields that were much larger than normal, often including more than one digit and part of the palm. When compared with the off-focus control neurons, their responses to electrical stimulation had lower thresholds and an increased response probability and magnitude. The latencies of these cells did not decrease, however, and were the same as the off-focus control values. The enhanced responses of the deafferented neurons to adjacent digit stimulation indicate that there is a strengthening of synapses that were previously ineffective. The increased proportion of neurons that could be activated after amputation suggests that there is also a growth of new connections. This experiment demonstrates that reorganization in the adult somatotopic system does occur at the level of the dorsal column nuclei. As a consequence, many of the changes reported at the cortex and thalamus may be due to the changes occurring at this first synapse in the somatosensory pathway.

Truncated Hormone Inhibits Breast Tumor Blood Vessel Formation, Not Tumor Growth | Center for Cancer Research

Cancer.gov

The hormone prolactin (PRL) plays a critical role in normal breast development by stimulating the proliferation of mammary cells, the production of milk proteins, and the formation of new mammary blood vessels. Unfortunately, the same cell and vessel growth pathways controlled by PRL in normal cells also operate in breast cancer cells, and elevated plasma PRL is a risk factor

Tax-dependent stimulation of G1 phase-specific cyclin-dependent kinases and increased expression of signal transduction genes characterize HTLV type 1-transformed T cells.

PubMed

Haller, K; Ruckes, T; Schmitt, I; Saul, D; Derow, E; Grassmann, R

2000-11-01

Human T cell leukemia virus protein induces T cells to permanent IL-2-dependent growth. These cells occasionally convert to factor independence. The viral oncoprotein Tax acts as an essential growth factor of transformed lymphocytes and stimulates the cell cycle in the G(1) phase. In T cells and fibroblasts Tax enhances the activity of the cyclin-dependent kinases (CDK) CDK4 and CDK6. These kinases, which require binding to cyclin D isotypes for their activity, control the G(1) phase. Coimmunoprecipitation from these cells revealed that Tax associates with cyclin D3/CDK6, suggesting a direct activation of this kinase. The CDK stimulation may account in part for the mitogenic Tax effect, which causes IL-2-dependent T cell growth by Tax. To address the conversion to IL-2-independent proliferation and to identify overexpressed genes, which contribute to the transformed growth, the gene expression patterns of HTLV-1-transformed T cells were compared with that of peripheral blood lymphocytes. Potentially overexpressed cDNAs were cloned, sequenced, and used to determine the RNA expression. Genes found to be up-regulated are involved in signal transduction (STAT5a, cyclin G(1), c-fgr, hPGT) and also glycoprotein synthesis (LDLC, ribophorin). Many of these are also activated during T cell activation and implicated in the regulation of growth and apoptosis. The transcription factor STAT5a, which is involved in IL-2 signaling, was strongly up-regulated only in IL-2-independent cells, thus suggesting that it contributes to factor-independent growth. Thus, the differentially expressed genes could cooperate with the Tax-induced cell cycle stimulation in the maintenance of IL-2-dependent and IL-2-independent growth of HTLV-transformed lymphocytes.

Quantitative, Phenotypical, and Functional Characterization of Cellular Immunity in Children and Adolescents With Down Syndrome.

PubMed

Schoch, Justine; Rohrer, Tilman R; Kaestner, Michael; Abdul-Khaliq, Hashim; Gortner, Ludwig; Sester, Urban; Sester, Martina; Schmidt, Tina

2017-05-15

Infections and autoimmune disorders are more frequent in Down syndrome, suggesting abnormality of adaptive immunity. Although the role of B cells and antibodies is well characterized, knowledge regarding T cells is limited. Lymphocyte subpopulations of 40 children and adolescents with Down syndrome and 51 controls were quantified, and phenotype and functionality of antigen-specific effector T cells were analyzed with flow cytometry after polyclonal and pathogen-specific stimulation (with varicella-zoster virus [VZV] and cytomegalovirus [CMV]). Results were correlated with immunoglobulin (Ig) G responses. Apart from general alterations in the percentage of lymphocytes, regulatory T cells, and T-helper 1 and 17 cells, all major T-cell subpopulations showed higher expression of the inhibitory receptor PD-1. Polyclonally stimulated effector CD4+ T-cell frequencies were significantly higher in subjects with Down syndrome, whereas their inhibitory receptor expression (programmed cell death 1 [PD-1] and cytotoxic T-lymphocyte antigen 4 [CTLA-4]) was similar to that of controls and cytokine expression profiles were only marginally altered. Pathogen-specific immunity showed age-appropriate levels of endemic infection, with correlation of CMV-specific cellular and humoral immunity in all subjects. Among VZV IgG-positive individuals, a higher percentage of VZV-specific T-cell-positive subjects was seen in those with Down syndrome. Despite alterations in lymphocyte subpopulations, individuals with Down syndrome can mount effector T-cell responses with similar phenotype and functionality as controls but may require higher effector T-cell frequencies to ensure pathogen control. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Physiological stimuli evoke two forms of endocytosis in bovine chromaffin cells.

PubMed

Chan, S A; Smith, C

2001-12-15

1. Exocytosis and endocytosis were measured following single, or trains of, simulated action potentials (sAP) in bovine adrenal chromaffin cells. Catecholamine secretion was measured by oxidative amperometry and cell membrane turnover was measured by voltage clamp cell capacitance measurements. 2. The sAPs evoked inward Na(+) and Ca(2+) currents that were statistically identical to those evoked by native action potential waveforms. On average, a single secretory granule underwent fusion following sAP stimulation. An equivalent amount of membrane was then quickly internalised (tau = 560 ms). 3. Stimulation with sAP trains revealed a biphasic relationship between cell firing rate and endocytic activity. At basal stimulus frequencies (single to 0.5 Hz) cells exhibited a robust membrane internalisation that then diminished as firing increased to intermediate levels (1.9 and 6 Hz). However at the higher stimulation rates (10 and 16 Hz) endocytic activity rebounded and was again able to effectively maintain cell surface near pre-stimulus levels. 4. Treatment with cyclosporin A and FK506, inhibitors of the phosphatase calcineurin, left endocytosis characteristics unaltered at the lower basal stimulus levels, but blocked the resurgence in endocytosis seen in control cells at higher sAP frequencies. 5. Based on these findings we propose that, under physiological electrical stimulation, chromaffin cells internalise membrane via two distinct pathways that are separable. One is prevalent at basal stimulus frequencies, is lessened with increased firing, and is insensitive to cyclosporin A and FK506. A second endocytic form is activated by increased firing frequencies, and is selectively blocked by cyclosporin A and FK506.

Physiological stimuli evoke two forms of endocytosis in bovine chromaffin cells

PubMed Central

Chan, Shyue-An; Smith, Corey

2001-01-01

Exocytosis and endocytosis were measured following single, or trains of, simulated action potentials (sAP) in bovine adrenal chromaffin cells. Catecholamine secretion was measured by oxidative amperometry and cell membrane turnover was measured by voltage clamp cell capacitance measurements. The sAPs evoked inward Na+ and Ca2+ currents that were statistically identical to those evoked by native action potential waveforms. On average, a single secretory granule underwent fusion following sAP stimulation. An equivalent amount of membrane was then quickly internalised (τ = 560 ms). Stimulation with sAP trains revealed a biphasic relationship between cell firing rate and endocytic activity. At basal stimulus frequencies (single to 0.5 Hz) cells exhibited a robust membrane internalisation that then diminished as firing increased to intermediate levels (1.9 and 6 Hz). However at the higher stimulation rates (10 and 16 Hz) endocytic activity rebounded and was again able to effectively maintain cell surface near pre-stimulus levels. Treatment with cyclosporin A and FK506, inhibitors of the phosphatase calcineurin, left endocytosis characteristics unaltered at the lower basal stimulus levels, but blocked the resurgence in endocytosis seen in control cells at higher sAP frequencies. Based on these findings we propose that, under physiological electrical stimulation, chromaffin cells internalise membrane via two distinct pathways that are separable. One is prevalent at basal stimulus frequencies, is lessened with increased firing, and is insensitive to cyclosporin A and FK506. A second endocytic form is activated by increased firing frequencies, and is selectively blocked by cyclosporin A and FK506. PMID:11744761

Peripheral Blood Cells from Patients with Autoimmune Addison's Disease Poorly Respond to Interferons In Vitro, Despite Elevated Serum Levels of Interferon-Inducible Chemokines.

PubMed

Edvardsen, Kine; Bjånesøy, Trine; Hellesen, Alexander; Breivik, Lars; Bakke, Marit; Husebye, Eystein S; Bratland, Eirik

2015-10-01

Autoimmune Addison's disease (AAD) is a disorder caused by an immunological attack on the adrenal cortex. The interferon (IFN)-inducible chemokine CXCL10 is elevated in serum of AAD patients, suggesting a peripheral IFN signature. However, CXCL10 can also be induced in adrenocortical cells stimulated with IFNs, cytokines, or microbial components. We therefore investigated whether peripheral blood mononuclear cells (PBMCs) from AAD patients display an enhanced propensity to produce CXCL10 and the related chemokine CXCL9, after stimulation with type I or II IFNs or the IFN inducer poly (I:C). Although serum levels of CXCL10 and CXCL9 were significantly elevated in patients compared with controls, IFN stimulated patient PBMC produced significantly less CXCL10/CXCL9 than control PBMC. Low CXCL10 production was not significantly associated with medication, disease duration, or comorbidities, but the low production of poly (I:C)-induced CXCL10 among patients was associated with an AAD risk allele in the phosphatase nonreceptor type 22 (PTPN22) gene. PBMC levels of total STAT1 and -2, and IFN-induced phosphorylated STAT1 and -2, were not significantly different between patients and controls. We conclude that PBMC from patients with AAD are deficient in their response to IFNs, and that the adrenal cortex itself may be responsible for the increased serum levels of CXCL10.

Production and action of cytokines in space

NASA Technical Reports Server (NTRS)

Chapes, Stephen K.; Morrison, Dennis R.; Guikema, James A.; Lewis, Marian L.; Spooner, Brian S.

1994-01-01

B6MP102 cells, a continuously cultured murine bone marrow macrophage cell line, were tested for secretion of tumor necrosis factor-alpha and Interleukin-1 during space flight. We found that B6MP102 cells secreted more tumor necrosis factor-alpha and interleukin-1 when stimulated in space with lipopolysaccharide than controls similarly stimulated on earth. This compared to increased secretion of interferon-beta and -gamma by lymphocytes that was measured on the same shuttle flights. Although space flight enhanced B6MP102 secretion of tumor necrosis factor-alpha, an experiment on a subsequent space flight (STS-50) found that cellular cytotoxicity, mediated by tumor necrosis factor-alpha, was inhibited.

Functional subcellular distribution of β1- and β2-adrenergic receptors in rat ventricular cardiac myocytes

PubMed Central

Cros, Caroline; Brette, Fabien

2013-01-01

β-adrenergic stimulation is a key regulator of cardiac function. The localization of major cardiac adrenergic receptors (β1 and β2) has been investigated using biochemical and biophysical approaches and has led to contradictory results. This study investigates the functional subcellular localization of β1- and β2-adrenergic receptors in rat ventricular myocytes using a physiological approach. Ventricular myocytes were isolated from the hearts of rat and detubulated using formamide. Physiological cardiac function was measured as Ca2+ transient using Fura-2-AM and cell shortening. Selective activation of β1- and β2-adrenergic receptors was induced with isoproterenol (0.1 μmol/L) and ICI-118,551 (0.1 μmol/L); and with salbutamol (10 μmol/L) and atenolol (1 μmol/L), respectively. β1- and β2-adrenergic stimulations induced a significant increase in Ca2+ transient amplitude and cell shortening in intact rat ventricular myocytes (i.e., surface sarcolemma and t-tubules) and in detubulated cells (depleted from t-tubules, surface sarcolemma only). Both β1- and β2-adrenergic receptors stimulation caused a greater effect on Ca2+ transient and cell shortening in detubulated myocytes than in control myocytes. Quantitative analysis indicates that β1-adrenergic stimulation is ∼3 times more effective at surface sarcolemma compared to t-tubules, whereas β2- adrenergic stimulation occurs almost exclusively at surface sarcolemma (∼100 times more effective). These physiological data demonstrate that in rat ventricular myocytes, β1-adrenergic receptors are functionally present at surface sarcolemma and t-tubules, while β2-adrenergic receptors stimulation occurs only at surface sarcolemma of cardiac cells. PMID:24303124

Tilapia Hepcidin 2-3 Peptide Modulates Lipopolysaccharide-induced Cytokines and Inhibits Tumor Necrosis Factor-α through Cyclooxygenase-2 and Phosphodiesterase 4D*

PubMed Central

Rajanbabu, Venugopal; Pan, Chieh-Yu; Lee, Shang-Chun; Lin, Wei-Ju; Lin, Ching-Chun; Li, Chung-Leung; Chen, Jyh-Yih

2010-01-01

The antimicrobial peptide, tilapia hepcidin (TH) 2-3, belongs to the hepcidin family, and its antibacterial function has been reported. Here, we examined the TH2-3-mediated regulation of proinflammatory cytokines in bacterial endotoxin lipopolysaccharide (LPS)-stimulated mouse macrophages. The presence of TH2-3 in LPS-stimulated cells reduced the amount of tumor necrosis factor (TNF)-α secretion. From a microarray, real-time polymerase chain reaction (PCR), and cytokine array studies, we showed down-regulation of the proinflammatory cytokines TNF-α, interleukin (IL)-1α, IL-1β, IL-6, and the prostaglandin synthesis gene, cyclooxygenase (COX)-2, by TH2-3. Studies with the COX-2-specific inhibitor, melaxicam, and with COX-2-overexpressing cells demonstrated the positive regulation of TNF-α and negative regulation of cAMP degradation-specific phosphodiesterase (PDE) 4D by COX-2. In LPS-stimulated cells, TH2-3 acts like melaxicam and down-regulates COX-2 and up-regulates PDE4D. The reduction in intracellular cAMP by TH2-3 or melaxicam in LPS-stimulated cells supports the negative regulation of PDE4D by COX-2 and TH2-3. This demonstrates that the inhibition of COX-2 is among the mechanisms through which TH2-3 controls TNF-α release. At 1 h after treatment, the presence of TH2-3 in LPS-stimulated cells had suppressed the induction of pERK1/2 and prevented the LPS-stimulated nuclear accumulation of NF-κB family proteins of p65, NF-κB2, and c-Rel. In conclusion, TH2-3 inhibits TNF-α and other proinflammatory cytokines through COX-2-, PDE4D-, and pERK1/2-dependent mechanisms. PMID:20675368

Granulocyte macrophage-colony stimulating factor and interleukin-3 increase expression of type II tumour necrosis factor receptor, increasing susceptibility to tumour necrosis factor-induced apoptosis. Control of leukaemia cell life/death switching.

PubMed

Rae, C; MacEwan, D J

2004-12-01

Tumour necrosis factor (TNF) induces apoptosis in a range of cell types via its two receptors, TNFR1 and TNFR2. Here, we demonstrate that proliferation and TNFR2 expression was increased in human leukaemic TF-1 cells by granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-3 (IL-3), with TNFR1 expression unaffected. Consequently, they switch from a proliferative to a TNF-induced apoptotic phenotype. Raised TNFR2 expression and susceptibility to TNF-induced apoptosis was not a general effect of proliferation as IL-1beta and IFN-gamma both proliferated TF-1 cells with no effect on TNFR expression or apoptosis. Although raised TNFR2 expression correlated with the apoptotic phenotype, stimulation of apoptosis in GM-CSF-pretreated cells was mediated by TNFR1, with stimulation of TNFR2 alone insufficient to initiate cell death. However, TNFR2 did play a role in apoptotic and proliferative responses as they were blocked by the presence of an antagonistic TNFR2 antibody. Additionally, coincubation with cycloheximide blocked the mitotic effects of GM-CSF or IL-3, allowing only the apoptotic responses of TNF to persist. TNF life/death was also observed in K562, but not MOLT-4 and HL-60 human leukaemic cell types. These findings show a cooperative role of TNFR2 in the TNF life/death switching phenomenon.

Direct, rapid effects of 25-hydroxyvitamin D3 on isolated intestinal cells.

PubMed

Phadnis, Ruta; Nemere, Ilka

2003-10-01

Scattered reports in the literature have suggested that the metabolite 25-hydroxyvitamin D(3) [25(OH)D(3)] has biological activity. In the present work, perfusion of isolated duodenal loops of normal chickens with 100 nM 25(OH)D(3) resulted in enhanced transport of (45)Ca within 2 min relative to the vehicle controls. We then tested the effect of a range of 25(OH)D(3) concentrations on (45)Ca handling by isolated intestinal cells in time course studies. Following a basal uptake period, cell suspensions from 7-week old chicks were treated either with 25, 100, or 300 nM 25(OH)D(3), or the vehicle ethanol (0.01%, final concentration). Both 25 and 100 nM 25(OH)D(3) resulted in a significant (P < 0.05) reduction in (45)Ca levels, relative to controls, between 1-10 min after treatment, while 300 nM 25(OH)D(3) resulted in a significant increase in (45)Ca levels, relative to controls, after 10 min of incubation. The effect of 100 nM 25(OH)D(3) (a physiological level) on cell calcium was abolished by the presence of 6.5 nM 24,25-dihydroxyvitamin D(3). In cell preparations from 14- or 28-week old birds 100nM 25(OH)D(3) had no effect, relative to vehicle controls. Incubation of cells with 2 microM BAY K8644, a calcium channel activator, stimulated (45)Ca uptake within 3 min relative to vehicle controls (P < 0.05), while addition of either 20 microM forskolin or 100 nM phorbol ester (stimulators of the PKA and PKC pathways, respectively) resulted in enhanced radionuclide levels after 10 min of incubation (P < 0.05, relative to corresponding controls). Finally, cells were treated with 100 nM 25(OH)D(3) or vehicle and samples taken at various times for analyses of protein kinase C and A activities. No effect of 25(OH)D(3) on protein kinase C activity was observed, while protein kinase A activity was stimulated to nearly 200% of controls at 1 min after 25(OH)D(3) addition (P < 0.05, relative to corresponding controls) and began declining at 3 min, returning to control levels 5 min after additions. We conclude that 25(OH)D(3) has a direct effect on calcium handling in enterocytes of young animals that may in part be mediated by the protein kinase A signal transduction pathway. Copyright 2003 Wiley-Liss, Inc.

Physiologically based microenvironment for in vitro neural differentiation of adipose-derived stem cells.

PubMed

Graziano, Adriana Carol Eleonora; Avola, Rosanna; Perciavalle, Vincenzo; Nicoletti, Ferdinando; Cicala, Gianluca; Coco, Marinella; Cardile, Venera

2018-03-26

The limited capacity of nervous system to promote a spontaneous regeneration and the high rate of neurodegenerative diseases appearance are keys factors that stimulate researches both for defining the molecular mechanisms of pathophysiology and for evaluating putative strategies to induce neural tissue regeneration. In this latter aspect, the application of stem cells seems to be a promising approach, even if the control of their differentiation and the maintaining of a safe state of proliferation should be troubled. Here, we focus on adipose tissue-derived stem cells and we seek out the recent advances on the promotion of their neural differentiation, performing a critical integration of the basic biology and physiology of adipose tissue-derived stem cells with the functional modifications that the biophysical, biomechanical and biochemical microenvironment induces to cell phenotype. The pre-clinical studies showed that the neural differentiation by cell stimulation with growth factors benefits from the integration with biomaterials and biophysical interaction like microgravity. All these elements have been reported as furnisher of microenvironments with desirable biological, physical and mechanical properties. A critical review of current knowledge is here proposed, underscoring that a real advance toward a stable, safe and controllable adipose stem cells clinical application will derive from a synergic multidisciplinary approach that involves material engineer, basic cell biology, cell and tissue physiology.

Transient stimulation expands superior antitumor T cells for adoptive therapy.

PubMed

Kagoya, Yuki; Nakatsugawa, Munehide; Ochi, Toshiki; Cen, Yuchen; Guo, Tingxi; Anczurowski, Mark; Saso, Kayoko; Butler, Marcus O; Hirano, Naoto

2017-01-26

Adoptive cell therapy is a potentially curative therapeutic approach for patients with cancer. In this treatment modality, antitumor T cells are exponentially expanded in vitro prior to infusion. Importantly, the results of recent clinical trials suggest that the quality of expanded T cells critically affects their therapeutic efficacy. Although anti-CD3 mAb-based stimulation is widely used to expand T cells in vitro, a protocol to generate T cell grafts for optimal adoptive therapy has yet to be established. In this study, we investigated the differences between T cell stimulation mediated by anti-CD3/CD28 mAb-coated beads and cell-based artificial antigen-presenting cells (aAPCs) expressing CD3/CD28 counter-receptors. We found that transient stimulation with cell-based aAPCs, but not prolonged stimulation with beads, resulted in the superior expansion of CD8 + T cells. Transiently stimulated CD8 + T cells maintained a stem cell-like memory phenotype and were capable of secreting multiple cytokines significantly more efficiently than chronically stimulated T cells. Importantly, the chimeric antigen receptor-engineered antitumor CD8 + T cells expanded via transient stimulation demonstrated superior persistence and antitumor responses in adoptive immunotherapy mouse models. These results suggest that restrained stimulation is critical for generating T cell grafts for optimal adoptive immunotherapy for cancer.

The Mitochondrial 2-Oxoglutarate Carrier Is Part of a Metabolic Pathway That Mediates Glucose- and Glutamine-stimulated Insulin Secretion*

PubMed Central

Odegaard, Matthew L.; Joseph, Jamie W.; Jensen, Mette V.; Lu, Danhong; Ilkayeva, Olga; Ronnebaum, Sarah M.; Becker, Thomas C.; Newgard, Christopher B.

2010-01-01

Glucose-stimulated insulin secretion from pancreatic islet β-cells is dependent in part on pyruvate cycling through the pyruvate/isocitrate pathway, which generates cytosolic α-ketoglutarate, also known as 2-oxoglutarate (2OG). Here, we have investigated if mitochondrial transport of 2OG through the 2-oxoglutarate carrier (OGC) participates in control of nutrient-stimulated insulin secretion. Suppression of OGC in clonal pancreatic β-cells (832/13 cells) and isolated rat islets by adenovirus-mediated delivery of small interfering RNA significantly decreased glucose-stimulated insulin secretion. OGC suppression also reduced insulin secretion in response to glutamine plus the glutamate dehydrogenase activator 2-amino-2-norbornane carboxylic acid. Nutrient-stimulated increases in glucose usage, glucose oxidation, glutamine oxidation, or ATP:ADP ratio were not affected by OGC knockdown, whereas suppression of OGC resulted in a significant decrease in the NADPH:NADP+ ratio during stimulation with glucose but not glutamine + 2-amino-2-norbornane carboxylic acid. Finally, OGC suppression reduced insulin secretion in response to a membrane-permeant 2OG analog, dimethyl-2OG. These data reveal that the OGC is part of a mechanism of fuel-stimulated insulin secretion that is common to glucose, amino acid, and organic acid secretagogues, involving flux through the pyruvate/isocitrate cycling pathway. Although the components of this pathway must remain intact for appropriate stimulus-secretion coupling, production of NADPH does not appear to be the universal second messenger signal generated by these reactions. PMID:20356834

[INHIBITORS OF MAP-KINASE PATHWAY U0126 AND PD98059 DIFFERENTLY AFFECT ORGANIZATION OF TUBULIN CYTOSKELETON AFTER STIMULATION OF EGF RECEPTOR ENDOCYTOSIS].

PubMed

Zlobina, M V; Steblyanko, Yu Yu; Shklyaeva, M A; Kharchenko, V V; Salova, A V; Kornilova, E S

2015-01-01

To confirm the hypothesis about the involvement of EGF-stimulated MAP-kinase ERK1/2 in the regulation of microtubule (MT) system, the influence of two widely used ERK1/2 inhibitors, U0126 and PD98059, on the organization of tubulin cytoskeleton in interphase HeLa cells during EGF receptor endocytosis has been investigated. We have found that addition of U0126 or PD98059 to not-stimulated with EGF ells for 30 min has no effect on radially organized MT system. However, in the case of U0126 addition before EGF endocytosis stimulation, the number of MT per cell decreased within 15 min after such stimulation and was followed by complete MT depolymerization by 60-90 min. Stimulation of EGF endocytosis in the presence of PD98059 resulted only in insignificant depolymerization of MT and it could be detected mainly from their minus-ends. At the same time, MT regions close to plasma membrane became stabilized, which was proved by increase in tubulin acetylation level. This situation was characteristic for all period of the experiment. It has been also found that the inhibitors affect endocytosis dynamics of EGF-receptor complexes. Quantitative analysis demonstrated that the stimulation of endocytosis in the presence of U0126 generated a greater number of endosomes compared to control cells, and their number did not change significantly during the experiment. All these endosomes were localized peripherally. Effect of PD98059 resulted in the formation of lower number of endosomes that in control, but they demonstrated very slow clusterization despite the presence of some intact MT. Both inhibitors decreased EGFR colocolization with early endosomal marker EEA1, which indicated a delay in endosome fusions and maturation. The inhibitors were also shown to affect differently phospho-ERK 1 and 2 forms: U0126 completely inhibited phospho-ERK1 and 2, white, in the presence of PD98059, the two ERK forms demonstrated sharp transient activation in 15 min after stimulation, but only phospho-ERK2 could be detected after 60 min of endocytosis. In both cases, MAP-kinase activation dynamics was significantly different from the control. Our results suggest involvement of EGF-stimulated MAP-kinase pathway in cytoskeleton regulation. At the same time, they demonstrate that the two studied and widely used inhibitors are not equivalent with respect to not only the effect on MAP-kinase activity but also to such interdependent processes such as changes in cytoskeleton organization and signaling receptor' endocytosis.

[Inhibition of antisense-endothelin converting enzyme RNA on interleukin-5 released from dust mite-challenged peripheral blood mononuclear cells in patients with allergic asthma].

PubMed

Li, Li; Xu, Ju; Zhong, Nan-shan

2003-09-01

To investigate the effect of antisense endothelin converting enzyme (ECE) RNA on levels of cytokines released from CD(4)(+) lymphocytes in patients with allergic diseases responsive to house dust mites. Peripheral blood mononuclear cells (PBMCs) were separated from 21 patients who were sensitive to dust mites. PBMCs from those patients were divided into two groups. No stimulation group (A group) induded A(1) group (anti-ECE epithelial cells + PBMCs) and A(2) group (control cells + PBMCs). Stimulation group (B group) included B(1) group (anti-ECE epithelial cells + PBMCs + dust mites extract) and B(2) group (control cells + PBMCs + dust mites extract). House dust mite extract (20 microg/ml) was added to the culture of stimulation group as described above. After 72 hours, supernatants from both groups were collected and the levels of IL-5 and IFN-ggr; released into the supernatants were detected by enzyme-linked immunoabsorbent assay. IL-5 levels were increased significantly after treatment with dust mite in twelve of 21 cases. No significant differences of IL-5 were found between the groups of A(1)[(6.0 +/- 1.3) x 10(-9) g/L] and A(2) [(7.5 +/- 1.1) x 10(-9) g/L] before house dust mite stimulation in the 12 cases (P > 0.05), and no significant differences in IFN-ggr; were found between the groups of A(1) [(63 +/- 26) x 10(-9) g/L] and A(2) [(70 +/- 52) x 10(-9) g/L] before house dust mite stimulation (P > 0.05). IL-5 level was increased in both groups after stimulation but it was significantly lower in the B(1) group [(8.2 +/- 1.6) x 10(-9) g/L] than that in the B(2) [(12.0 +/- 1.8) x 10(-9) g/L] (P = 0.047). It seemed that increased IFN-ggr; level after stimulation was higher in B(2) [(153 +/- 71) x 10(-9) g/L] than that in the B(1) group (100 +/- 41) x 10(-9) g/L], but there was no statistic significance (P > 0.05). In addition, our results also showed that the release of IL-5 was significantly increased in those cases with asthma, or asthma plus allergic rhinitis after dust mites stimulation [(44 +/- 15)%] compared with that in those with urticaria [(7 +/- 4)%] (P = 0.047). Antisense-ECE downregulated the IL-5 secretion from Th2 lymphocytes in patients with allergic asthma after being challenged with dust mites. It is indicated that ET-1 is an important cytokine involved with allergic airway inflammation. Antisense-ECE RNA management in airways may be of value in treating allergic asthma.

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

PubMed

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.

The upregulation of receptor activator NF-kappaB ligand expression by interleukin-1alpha and Porphyromonas endodontalis in human osteoblastic cells.

PubMed

Chen, S-C; Huang, F-M; Lee, S-S; Li, M-Z; Chang, Y-C

2009-04-01

To investigate the receptor activator of nuclear factor-kappa B (NF-kappaB) ligand (RANKL) in osteoblastic cells stimulated with inflammatory mediators. The expression of RANKL in human osteoblastic cell line U2OS stimulated by pro-inflammatory cytokine interleukin (IL)-1alpha and black-pigmented bacteria Porphyromonas endodontalis was investigated by Western blot and enzyme-linked immunosorbent assay (ELISA). The significance of the results obtained from control and treated groups was statistically analysed by the paired Student's t-test. IL-1alpha was found to upregulate RANKL production in U2OS cells (P < 0.05). Investigations of the time dependence of RANKL expression in IL-1alpha-treated cells revealed a rapid accumulation of RANKL protein after 1 h of exposure; it remained elevated throughout the 24-h incubation period shown by Western blot and ELISA. In addition, P. endodontalis also increased RANKL expression in U2OS cells after 4-h incubation period demonstrated by Western blot and ELISA (P < 0.05). IL-1alpha and P. endodontalis may be involved in developing apical periodontitis through the stimulation of RANKL production.

Interferon-γ and interleukin-17 production from PPD-stimulated PBMCss of patients with pulmonary tuberculosis.

PubMed

Nunnari, Giuseppe; Pinzone, Marilia R; Vancheri, Carlo; Palermo, Filippo; Cacopardo, Bruno

2013-04-01

The purpose of this study was to evaluate Interferon (IFN)-γ and Interleukin(IL)-17 profiles in patients with different clinical presentations of pulmonary tuberculosis (TB) and to compare them with those of tuberculin-negative and tuberculin-reactive healthy controls Peripheral blood mononuclear cells (PBMCss), isolated from patients (n=52) and controls (n=30), were stimulated ex vivo with purified protein derivative (PPD) and IFN-γ and IL-17 levels in the supernatant were measured. At baseline, PBMCss from patients with TB released a significantly lower amount of IL-17 (p=0.043) than PBMCss from healthy controls, whereas IFN-γ levels were similar in the two groups. After PPD stimulation, a significant rise in IL-17 levels was found only among healthy controls (p=0.02). This rise in IL-17 levels was similar between tuberculin-reactive and tuberculin-negative subjects. After PPD stimulation, patients with infiltrative TB secreted higher levels of IL-17 and IFN-γ than those affected with chronic, miliary and cavitary TB (p < 0.01). IFN-γ production from patients with infiltrative TB was even higher than for healthy controls (p < 0.01). PBMCss from tuberculin-reactive patients released higher levels of IFN-γ than tuberculin-negative subjects after PPD stimulation (p < 0.01). Ex vivo PPD stimulation of PBMCs from patients with pulmonary TB does not significantly stimulate IL-17 release; however, higher IL-17 and IFN-γ levels are found in patients with infiltrative disease, in comparison with those affected with miliary, cavitary and chronic TB.

GARP is regulated by miRNAs and controls latent TGF-β1 production by human regulatory T cells.

PubMed

Gauthy, Emilie; Cuende, Julia; Stockis, Julie; Huygens, Caroline; Lethé, Bernard; Collet, Jean-François; Bommer, Guido; Coulie, Pierre G; Lucas, Sophie

2013-01-01

GARP is a transmembrane protein present on stimulated human regulatory T lymphocytes (Tregs), but not on other T lymphocytes (Th cells). It presents the latent form of TGF-β1 on the Treg surface. We report here that GARP favors the cleavage of the pro-TGF-β1 precursor and increases the amount of secreted latent TGF-β1. Stimulated Tregs, which naturally express GARP, and Th cells transfected with GARP secrete a previously unknown form of latent TGF-β1 that is disulfide-linked to GARP. These GARP/TGF-β1 complexes are possibly shed from the T cell surface. Secretion of GARP/TGF-β1 complexes was not observed with transfected 293 cells and may thus be restricted to the T cell lineage. We conclude that in stimulated human Tregs, GARP not only displays latent TGF-β1 at the cell surface, but also increases its secretion by forming soluble disulfide-linked complexes. Moreover, we identified six microRNAs (miRNAs) that are expressed at lower levels in Treg than in Th clones and that target a short region of the GARP 3' UTR. In transfected Th cells, the presence of this region decreased GARP levels, cleavage of pro-TGF-β1, and secretion of latent TGF-β1.

Induction of CXC chemokines in human mesenchymal stem cells by stimulation with secreted frizzled-related proteins through non-canonical Wnt signaling.

PubMed

Bischoff, David S; Zhu, Jian-Hua; Makhijani, Nalini S; Yamaguchi, Dean T

2015-12-26

To investigate the effect of secreted frizzled-related proteins (sFRPs) on CXC chemokine expression in human mesenchymal stem cells (hMSCs). CXC chemokines such as CXCL5 and CXCL8 are induced in hMSCs during differentiation with osteogenic differentiation medium (OGM) and may be involved in angiogenic stimulation during bone repair. hMSCs were treated with conditioned medium (CM) from L-cells expressing non-canonical Wnt5a protein, or with control CM from wild type L-cells, or directly with sFRPs for up to 10 d in culture. mRNA expression levels of both CXCL5 and CXCL8 were quantitated by real-time reverse transcriptase-polymerase chain reaction and secreted protein levels of these proteins determined by ELISA. Dose- (0-500 ng/mL) and time-response curves were generated for treatment with sFRP1. Signal transduction pathways were explored by western blot analysis with pan- or phosphorylation-specific antibodies, through use of specific pathway inhibitors, and through use of siRNAs targeting specific frizzled receptors (Fzd)-2 and 5 or the receptor tyrosine kinase-like orphan receptor-2 (RoR2) prior to treatment with sFRPs. CM from L-cells expressing Wnt5a, a non-canonical Wnt, stimulated an increase in CXCL5 mRNA expression and protein secretion in comparison to control L-cell CM. sFRP1, which should inhibit both canonical and non-canonical Wnt signaling, surprisingly enhanced the expression of CXCL5 at 7 and 10 d. Dickkopf1, an inhibitor of canonical Wnt signaling prevented the sFRP-stimulated induction of CXCL5 and actually inhibited basal levels of CXCL5 expression at 7 but not at 10 d post treatment. In addition, all four sFRPs isoforms induced CXCL8 expression in a dose- and time-dependent manner with maximum expression at 7 d with treatment at 150 ng/mL. The largest increases in CXCL5 expression were seen from stimulation with sFRP1 or sFRP2. Analysis of mitogen-activated protein kinase signaling pathways in the presence of OGM showed sFRP1-induced phosphorylation of extracellular signal-regulated kinase (ERK) (p44/42) maximally at 5 min after sFRP1 addition, earlier than that found in OGM alone. Addition of a phospholipase C (PLC) inhibitor also prevented sFRP-stimulated increases in CXCL8 mRNA. siRNA technology targeting the Fzd-2 and 5 and the non-canonical Fzd co-receptor RoR2 also significantly decreased sFRP1/2-stimulated CXCL8 mRNA levels. CXC chemokine expression in hMSCs is controlled in part by sFRPs signaling through non-canonical Wnt involving Fzd2/5 and the ERK and PLC pathways.

Induction of CXC chemokines in human mesenchymal stem cells by stimulation with secreted frizzled-related proteins through non-canonical Wnt signaling

PubMed Central

Bischoff, David S; Zhu, Jian-Hua; Makhijani, Nalini S; Yamaguchi, Dean T

2015-01-01

AIM: To investigate the effect of secreted frizzled-related proteins (sFRPs) on CXC chemokine expression in human mesenchymal stem cells (hMSCs). METHODS: CXC chemokines such as CXCL5 and CXCL8 are induced in hMSCs during differentiation with osteogenic differentiation medium (OGM) and may be involved in angiogenic stimulation during bone repair. hMSCs were treated with conditioned medium (CM) from L-cells expressing non-canonical Wnt5a protein, or with control CM from wild type L-cells, or directly with sFRPs for up to 10 d in culture. mRNA expression levels of both CXCL5 and CXCL8 were quantitated by real-time reverse transcriptase-polymerase chain reaction and secreted protein levels of these proteins determined by ELISA. Dose- (0-500 ng/mL) and time-response curves were generated for treatment with sFRP1. Signal transduction pathways were explored by western blot analysis with pan- or phosphorylation-specific antibodies, through use of specific pathway inhibitors, and through use of siRNAs targeting specific frizzled receptors (Fzd)-2 and 5 or the receptor tyrosine kinase-like orphan receptor-2 (RoR2) prior to treatment with sFRPs. RESULTS: CM from L-cells expressing Wnt5a, a non-canonical Wnt, stimulated an increase in CXCL5 mRNA expression and protein secretion in comparison to control L-cell CM. sFRP1, which should inhibit both canonical and non-canonical Wnt signaling, surprisingly enhanced the expression of CXCL5 at 7 and 10 d. Dickkopf1, an inhibitor of canonical Wnt signaling prevented the sFRP-stimulated induction of CXCL5 and actually inhibited basal levels of CXCL5 expression at 7 but not at 10 d post treatment. In addition, all four sFRPs isoforms induced CXCL8 expression in a dose- and time-dependent manner with maximum expression at 7 d with treatment at 150 ng/mL. The largest increases in CXCL5 expression were seen from stimulation with sFRP1 or sFRP2. Analysis of mitogen-activated protein kinase signaling pathways in the presence of OGM showed sFRP1-induced phosphorylation of extracellular signal-regulated kinase (ERK) (p44/42) maximally at 5 min after sFRP1 addition, earlier than that found in OGM alone. Addition of a phospholipase C (PLC) inhibitor also prevented sFRP-stimulated increases in CXCL8 mRNA. siRNA technology targeting the Fzd-2 and 5 and the non-canonical Fzd co-receptor RoR2 also significantly decreased sFRP1/2-stimulated CXCL8 mRNA levels. CONCLUSION: CXC chemokine expression in hMSCs is controlled in part by sFRPs signaling through non-canonical Wnt involving Fzd2/5 and the ERK and PLC pathways. PMID:26730270

[Effect of lipopolysaccharides from Porphyromonas endodontalis on the expression of macrophage colony stimulating factor in mouse osteoblasts].

PubMed

Yu, Yaqiong; Qiu, Lihong; Guo, Jiajie; Qu, Liu; Xu, Liya; Zhong, Ming

2014-09-01

To investigate the effects of lipopolysaccharides (LPS) extracted from Porphyromonas endodontalis (Pe) on the expression of macrophage colony stimulating factor (M-CSF) mRNA and protein in MC3T3-E1 cells and the role of nucler factor-κB (NF-κB) in the process. MC3T3-E1 cells were treated with different concentrations of Pe-LPS (0-50 mg/L) and 10 mg/L Pe-LPS for different hours (0-24 h). The expression of M-CSF mRNA and protein was detected by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunoadsordent assay (ELISA). The cells untreated by Pe-LPS served as control. The expression of M- CSF mRNA and protein was also detected in 10 mg/L Pe- LPS treated MC3T3-E1 cells after pretreated with BAY 11-7082 for 1 h, a special NF-κB inhibitor. The groups were divided as follows, control group, BAY group (10 µmol/L BAY 11-7082 treated alone MC3T3-E1 cells), Pe-LPS group (10 mg/L Pe-LPS stimulated MC3T3-E1 cells for 6 h), BAY combine with Pe-LPS group (10 µmol/L BAY 11-7082 pretreated cells for 1 h and 10 mg/L of Pe-LPS stimulated MC3T3-E1 cells for 6 h). The level of M- CSF mRNA and protein increased significantly after treatment with different concentrations of Pe-LPS (0-50 mg/L), which indicated that Pe-LPS induced osteoblasts to express M-CSF mRNA and protein in dose dependent manners. The expression of M-CSF protein increased from (35 ± 2) ng/L (control group) to (170 ± 8) ng/L (50 mg/L group). Maximal induction of M-CSF mRNA expression was found in the MC3T3- E1 cells treated with 10 mg/L Pe-LPS for 6 h. After 6 h, the expression of M-CSF mRNA decreased gradually. The expression of M-CSF protein also increased with the treatment of 10 mg/L Pe-LPS for 10 h [(122 ± 4) ng/L]. After 10 h, the expression of M-CSF protein decreased gradually. The mRNA and proteins of M-CSF decreased significantly after pretreatment with 10 µmol/L BAY 11-7082 for 1 h. There was no significant difference between BAY group and the control. Pe-LPS may induce the expression of M-CSF mRNA and protein in MC3T3-E1 cells through the signaling of NF-κB.

Kinetic analysis of F-actin depolymerization in polymorphonuclear leukocyte lysates indicates that chemoattractant stimulation increases actin filament number without altering the filament length distribution

PubMed Central

1991-01-01

The rate of filamentous actin (F-actin) depolymerization is proportional to the number of filaments depolarizing and changes in the rate are proportional to changes in filament number. To determine the number and length of actin filaments in polymorphonuclear leukocytes and the change in filament number and length that occurs during the increase in F-actin upon chemoattractant stimulation, the time course of cellular F-actin depolymerization in lysates of control and peptide- stimulated cells was examined. F-actin was quantified by the TRITC- labeled phalloidin staining of pelletable actin. Lysis in 1.2 M KCl and 10 microM DNase I minimized the effects of F-actin binding proteins and G-actin, respectively, on the kinetics of depolymerization. To determine filament number and length from a depolymerization time course, depolymerization kinetics must be limited by the actin monomer dissociation rate. Comparison of time courses of depolymerization in the presence (pointed ends free) or absence (barbed and pointed ends free) of cytochalasin suggested depolymerization occurred from both ends of the filament and that monomer dissociation was rate limiting. Control cells had 1.7 +/- 0.4 x 10(5) filaments with an average length of 0.29 +/- 0.09 microns. Chemo-attractant stimulation for 90 s at room temperature with 0.02 microM N-formylnorleucylleucylphenylalanine caused a twofold increase in F-actin and about a two-fold increase in the total number of actin filaments to 4.0 +/- 0.5 x 10(5) filaments with an average length of 0.27 +/- 0.07 microns. In both cases, most (approximately 80%) of the filaments were quite short (less than or equal to 0.18 micron). The length distributions of actin filaments in stimulated and control cells were similar. PMID:1918158

The Vagal Nerve Stimulates Activation of the Hepatic Progenitor Cell Compartment via Muscarinic Acetylcholine Receptor Type 3

PubMed Central

Cassiman, David; Libbrecht, Louis; Sinelli, Nicoletta; Desmet, Valeer; Denef, Carl; Roskams, Tania

2002-01-01

In the rat the hepatic branch of the nervus vagus stimulates proliferation of hepatocytes after partial hepatectomy and growth of bile duct epithelial cells after bile duct ligation. We studied the effect of hepatic vagotomy on the activation of the hepatic progenitor cell compartment in human and rat liver. The number of hepatic progenitor cells and atypical reactive ductular cells in transplanted (denervated) human livers with hepatitis was significantly lower than in innervated matched control livers and the number of oval cells in vagotomized rat livers with galactosamine hepatitis was significantly lower than in livers of sham-operated rats with galactosamine hepatitis. The expression of muscarinic acetylcholine receptors (M1-M5 receptor) was studied by immunohistochemistry and reverse transcriptase-polymerase chain reaction. In human liver, immunoreactivity for M3 receptor was observed in hepatic progenitor cells, atypical reactive ductules, intermediate hepatocyte-like cells, and bile duct epithelial cells. mRNA for the M1-M3 and the M5 receptor, but not the M4 receptor, was detected in human liver homogenates. In conclusion, the hepatic vagus branch stimulates activation of the hepatic progenitor cell compartment in diseased liver, most likely through binding of acetylcholine to the M3 receptor expressed on these cells. These findings may be of clinical importance for patients with a transplant liver. PMID:12163377

Transient stimulation expands superior antitumor T cells for adoptive therapy

PubMed Central

Kagoya, Yuki; Nakatsugawa, Munehide; Ochi, Toshiki; Guo, Tingxi; Anczurowski, Mark; Saso, Kayoko; Butler, Marcus O.

2017-01-01

Adoptive cell therapy is a potentially curative therapeutic approach for patients with cancer. In this treatment modality, antitumor T cells are exponentially expanded in vitro prior to infusion. Importantly, the results of recent clinical trials suggest that the quality of expanded T cells critically affects their therapeutic efficacy. Although anti-CD3 mAb-based stimulation is widely used to expand T cells in vitro, a protocol to generate T cell grafts for optimal adoptive therapy has yet to be established. In this study, we investigated the differences between T cell stimulation mediated by anti–CD3/CD28 mAb–coated beads and cell-based artificial antigen-presenting cells (aAPCs) expressing CD3/CD28 counter-receptors. We found that transient stimulation with cell-based aAPCs, but not prolonged stimulation with beads, resulted in the superior expansion of CD8+ T cells. Transiently stimulated CD8+ T cells maintained a stem cell–like memory phenotype and were capable of secreting multiple cytokines significantly more efficiently than chronically stimulated T cells. Importantly, the chimeric antigen receptor–engineered antitumor CD8+ T cells expanded via transient stimulation demonstrated superior persistence and antitumor responses in adoptive immunotherapy mouse models. These results suggest that restrained stimulation is critical for generating T cell grafts for optimal adoptive immunotherapy for cancer. PMID:28138559

The Ras-related protein, Rap1A, mediates thrombin-stimulated, integrin-dependent glioblastoma cell proliferation and tumor growth.

PubMed

Sayyah, Jacqueline; Bartakova, Alena; Nogal, Nekeisha; Quilliam, Lawrence A; Stupack, Dwayne G; Brown, Joan Heller

2014-06-20

Rap1 is a Ras family GTPase with a well documented role in ERK/MAP kinase signaling and integrin activation. Stimulation of the G-protein-coupled receptor PAR-1 with thrombin in human 1321N1 glioblastoma cells led to a robust increase in Rap1 activation. This response was sustained for up to 6 h and mediated through RhoA and phospholipase D (PLD). Thrombin treatment also induced a 5-fold increase in cell adhesion to fibronectin, which was blocked by down-regulating PLD or Rap1A or by treatment with a β1 integrin neutralizing antibody. In addition, thrombin treatment led to increases in phospho-focal adhesion kinase (tyrosine 397), ERK1/2 phosphorylation and cell proliferation, which were significantly inhibited in cells treated with β1 integrin antibody or Rap1A siRNA. To assess the role of Rap1A in tumor formation in vivo, we compared growth of 1321N1 cells stably expressing control, Rap1A or Rap1B shRNA in a mouse xenograft model. Deletion of Rap1A, but not of Rap1B, reduced tumor mass by >70% relative to control. Similar observations were made with U373MG glioblastoma cells in which Rap1A was down-regulated. Collectively, these findings implicate a Rap1A/β1 integrin pathway, activated downstream of G-protein-coupled receptor stimulation and RhoA, in glioblastoma cell proliferation. Moreover, our data demonstrate a critical role for Rap1A in glioblastoma tumor growth in vivo. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

PIEZO channel protein naturally expressed in human breast cancer cell MDA-MB-231 as probed by atomic force microscopy

NASA Astrophysics Data System (ADS)

Weng, Yuanqi; Yan, Fei; Chen, Runkang; Qian, Ming; Ou, Yun; Xie, Shuhong; Zheng, Hairong; Li, Jiangyu

2018-05-01

Mechanical stimuli drives many physiological processes through mechanically activated channels, and the recent discovery of PIEZO channel has generated great interests in its mechanotransduction. Many previous researches investigated PIEZO proteins by transcribing them in cells that originally have no response to mechanical stimulation, or by forming PIEZO-combined complexes in vitro, and few studied PIEZO protein's natural characteristics in cells. In this study we show that MDA-MB-231, a malignant cell in human breast cancer cell line, expresses the mechanosensitive behavior of PIEZO in nature without extra treatment, and we report its characteristics in response to localized mechanical stimulation under an atomic force microscope, wherein a correlation between the force magnitude applied and the channel opening probability is observed. The results on PIEZO of MDA-MB-231 can help establish a basis of preventing and controlling of human breast cancer cell via mechanical forces.

Enhancement of UV-induced nucleotide excision repair activity upon forskolin treatment is cell growth-dependent.

PubMed

Lee, Jeong-Min; Park, Jeong-Min; Kang, Tae-Hong

2016-10-01

Forskolin (FSK), an adenylyl cyclase activator, has recently been shown to enhance nucleotide excision repair (NER) upon UV exposure. However, our study revealed that this effect was detected in human skin epithelial ARPE19 cells only in growing cells, but not in non-cycling cells. When the cells were grown at low density (70% confluence), FSK was capable of stimulating cAMP responsive element binding (CREB) phosphorylation, a marker for FSK-stimulated PKA activation, and resulted in a significant increase of NER activity compared to control treatment. However, cells grown under 100% confluent conditions showed neither FSK-induced CREB phosphorylation nor the resulting NER enhancement. These findings indicate that cellular growth is critical for FSK-induced NER enhancement and suggest that cellular growth conditions should be considered as a variable while evaluating a reagent's pharmacotherapeutic efficacy. [BMB Reports 2016; 49(10): 566-571].

T Cell Inactivation by Poxviral B22 Family Proteins Increases Viral Virulence

PubMed Central

Alzhanova, Dina; Hammarlund, Erika; Reed, Jason; Meermeier, Erin; Rawlings, Stephanie; Ray, Caroline A.; Edwards, David M.; Bimber, Ben; Legasse, Alfred; Planer, Shannon; Sprague, Jerald; Axthelm, Michael K.; Pickup, David J.; Lewinsohn, David M.; Gold, Marielle C.; Wong, Scott W.; Sacha, Jonah B.; Slifka, Mark K.; Früh, Klaus

2014-01-01

Infections with monkeypox, cowpox and weaponized variola virus remain a threat to the increasingly unvaccinated human population, but little is known about their mechanisms of virulence and immune evasion. We now demonstrate that B22 proteins, encoded by the largest genes of these viruses, render human T cells unresponsive to stimulation of the T cell receptor by MHC-dependent antigen presentation or by MHC-independent stimulation. In contrast, stimuli that bypass TCR-signaling are not inhibited. In a non-human primate model of monkeypox, virus lacking the B22R homologue (MPXVΔ197) caused only mild disease with lower viremia and cutaneous pox lesions compared to wild type MPXV which caused high viremia, morbidity and mortality. Since MPXVΔ197-infected animals displayed accelerated T cell responses and less T cell dysregulation than MPXV US2003, we conclude that B22 family proteins cause viral virulence by suppressing T cell control of viral dissemination. PMID:24832205

T cell inactivation by poxviral B22 family proteins increases viral virulence.

PubMed

Alzhanova, Dina; Hammarlund, Erika; Reed, Jason; Meermeier, Erin; Rawlings, Stephanie; Ray, Caroline A; Edwards, David M; Bimber, Ben; Legasse, Alfred; Planer, Shannon; Sprague, Jerald; Axthelm, Michael K; Pickup, David J; Lewinsohn, David M; Gold, Marielle C; Wong, Scott W; Sacha, Jonah B; Slifka, Mark K; Früh, Klaus

2014-05-01

Infections with monkeypox, cowpox and weaponized variola virus remain a threat to the increasingly unvaccinated human population, but little is known about their mechanisms of virulence and immune evasion. We now demonstrate that B22 proteins, encoded by the largest genes of these viruses, render human T cells unresponsive to stimulation of the T cell receptor by MHC-dependent antigen presentation or by MHC-independent stimulation. In contrast, stimuli that bypass TCR-signaling are not inhibited. In a non-human primate model of monkeypox, virus lacking the B22R homologue (MPXVΔ197) caused only mild disease with lower viremia and cutaneous pox lesions compared to wild type MPXV which caused high viremia, morbidity and mortality. Since MPXVΔ197-infected animals displayed accelerated T cell responses and less T cell dysregulation than MPXV US2003, we conclude that B22 family proteins cause viral virulence by suppressing T cell control of viral dissemination.

Evidence for a Regulatory Role of Calcium in Gravitropism

NASA Technical Reports Server (NTRS)

Roux, S. J.

1983-01-01

Experiments conducted to determine the cellular basis of gravitropism, the phenomenon of calcium migration following gravitropic stimulation, and the preferential accumulation of calcium in cells are described. Results of autoradiographic studies of cross sections of oat, and the pryoantimony precipitation of calcium in situ are discussed. It was found that the movement of calcium during gravimetric stimulation is a redistribution of calcium from the vacuolar regions into the cells walls. This movement requires precipitation of a calcium ATPase. The control of calcium ATPase by calmodulin and whether chlorpromazine is binding to calmodulin in plants are considered.

Metabolic alterations induced in cultured skeletal muscle by stretch-relaxation activity

NASA Technical Reports Server (NTRS)

Hatfaludy, Sophia; Shansky, Janet; Vandenburgh, Herman H.

1989-01-01

Muscle cells differentiated in vitro are repetitively stretched and relaxed in order to determine the presence of short- and long-term alterations occurring in glucose uptake and lactate efflux that are similar to the metabolic alterations occurring in stimulated organ-cultured muscle and in vivo skeletal muscle during the active state. It is observed that whereas mechanical stimulation increases these metabolic parameters within 4-6 h of starting activity, unstimulated basal rates in control cultures also increase during this period of time, and by 8 h, their rates have reached or exceeded the rates in continuously stimulated cells. Measurements of these parameters in media of different compositions show that activity-induced long-term alterations in the parameters occur independently of growth factors in serium and embryo extracts.

Beryllium-stimulated neopterin as a diagnostic adjunct in chronic beryllium disease.

PubMed

Maier, Lisa A; Kittle, Lori A; Mroz, Margaret M; Newman, Lee S

2003-06-01

The diagnosis of chronic beryllium disease (CBD) relies on the beryllium lymphocyte proliferation test (BeLPT) to demonstrate a Be specific immune response. This test has improved early diagnosis, but cannot discriminate beryllium sensitization (BeS) from CBD. We previously found high neopterin levels in CBD patients' serum and questioned whether Be-stimulated neopterin production by peripheral blood cells in vitro might be useful in the diagnosis of CBD. CBD, BeS, Be exposed workers without disease (Be-exp) normal controls and sarcoidosis subjects were enrolled. Peripheral blood mononuclear cells (PBMN) were cultured in the presence and absence of beryllium sulfate. Neopterin levels were determined from cell supernatants by enzyme linked immunosorbent assay (ELISA). Clinical evaluation of CBD subjects included chest radiography, pulmonary function testing, exercise testing, and the BeLPT. CBD patients produced higher levels of neopterin in both unstimulated and Be-stimulated conditions compared to all other subjects (P < 0.0001). Unstimulated neopterin mononuclear cell levels overlapped among groups, however, Be-stimulated neopterin levels in CBD showed little overlap. Using a neopterin concentration of 2.5 ng/ml as a cutoff, Be-stimulated neopterin had a sensitivity of 80% and specificity of 100% for CBD and was able to differentiate CBD from BeS. Be-stimulated neopterin was inversely related to measures of pulmonary function, exercise capacity, and gas exchange. Neopterin may be a useful diagnostic adjunct in the non-invasive assessment of CBD, differentiating CBD from BeS. Further studies will be required to determine how it performs in workplace screening. Copyright 2003 Wiley-Liss, Inc.

[Enhanced lymphocyte proliferation in the presence of epidermal cells of HIV-infected patients in vitro].

PubMed

Kappus, R P; Berger, S; Thomas, C A; Ottmann, O G; Ganser, A; Stille, W; Shah, P M

1992-07-01

Clinical observations show that the HIV infection is often associated with affections of the skin. In order to examine the involvement of the epidermal immune system in the HIV infection, we determined accessory cell function of epidermal cells from HIV-1-infected patients. For this we measured the proliferative response of enriched CD(4+)-T-lymphocytes from HIV-infected patients and noninfected controls to stimulation with anti-CD3 and IL-2 in the presence of epidermal cells; the enhancement of the response is dependent on the presence of functionally intact accessory cells. The capacity of epidermal cells to increase the anti-CD3-stimulated T-cell proliferative response was significantly enhanced in HIV patients (CDC III/IVA) as compared with noninfected donors. It is discussed, whether the increased activity of epidermal cells from HIV-infected patients may be responsible for several of the dermal lesions in the course of an HIV infection as due to an enhanced production and release of epidermal cell-derived cytokines.

Controlling fertilization and cAMP signaling in sperm by optogenetics.

PubMed

Jansen, Vera; Alvarez, Luis; Balbach, Melanie; Strünker, Timo; Hegemann, Peter; Kaupp, U Benjamin; Wachten, Dagmar

2015-01-20

Optogenetics is a powerful technique to control cellular activity by light. The light-gated Channelrhodopsin has been widely used to study and manipulate neuronal activity in vivo, whereas optogenetic control of second messengers in vivo has not been examined in depth. In this study, we present a transgenic mouse model expressing a photoactivated adenylyl cyclase (bPAC) in sperm. In transgenic sperm, bPAC mimics the action of the endogenous soluble adenylyl cyclase (SACY) that is required for motility and fertilization: light-stimulation rapidly elevates cAMP, accelerates the flagellar beat, and, thereby, changes swimming behavior of sperm. Furthermore, bPAC replaces endogenous adenylyl cyclase activity. In mutant sperm lacking the bicarbonate-stimulated SACY activity, bPAC restored motility after light-stimulation and, thereby, enabled sperm to fertilize oocytes in vitro. We show that optogenetic control of cAMP in vivo allows to non-invasively study cAMP signaling, to control behaviors of single cells, and to restore a fundamental biological process such as fertilization.

CD40-Mediated NF-κB Activation in B Cells Is Increased in Multiple Sclerosis and Modulated by Therapeutics.

PubMed

Chen, Ding; Ireland, Sara J; Remington, Gina; Alvarez, Enrique; Racke, Michael K; Greenberg, Benjamin; Frohman, Elliot M; Monson, Nancy L

2016-12-01

CD40 interacts with CD40L and plays an essential role in immune regulation and homeostasis. Recent research findings, however, support a pathogenic role of CD40 in a number of autoimmune diseases. We previously showed that memory B cells from relapsing-remitting multiple sclerosis (RRMS) patients exhibited enhanced proliferation with CD40 stimulation compared with healthy donors. In this study, we used a multiparameter phosflow approach to analyze the phosphorylation status of NF-κB and three major MAPKs (P38, ERK, and JNK), the essential components of signaling pathways downstream of CD40 engagement in B cells from MS patients. We found that memory and naive B cells from RRMS and secondary progressive MS patients exhibited a significantly elevated level of phosphorylated NF-κB (p-P65) following CD40 stimulation compared with healthy donor controls. Combination therapy with IFN-β-1a (Avonex) and mycophenolate mofetil (Cellcept) modulated the hyperphosphorylation of P65 in B cells of RRMS patients at levels similar to healthy donor controls. Lower disease activity after the combination therapy correlated with the reduced phosphorylation of P65 following CD40 stimulation in treated patients. Additionally, glatiramer acetate treatment also significantly reduced CD40-mediated P65 phosphorylation in RRMS patients, suggesting that reducing CD40-mediated p-P65 induction may be a general mechanism by which some current therapies modulate MS disease. Copyright © 2016 by The American Association of Immunologists, Inc.

Ionizing irradiation not only inactivates clonogenic potential in primary normal human diploid lens epithelial cells but also stimulates cell proliferation in a subset of this population.

PubMed

Fujimichi, Yuki; Hamada, Nobuyuki

2014-01-01

Over the past century, ionizing radiation has been known to induce cataracts in the crystalline lens of the eye, but its mechanistic underpinnings remain incompletely understood. This study is the first to report the clonogenic survival of irradiated primary normal human lens epithelial cells and stimulation of its proliferation. Here we used two primary normal human cell strains: HLEC1 lens epithelial cells and WI-38 lung fibroblasts. Both strains were diploid, and a replicative lifespan was shorter in HLEC1 cells. The colony formation assay demonstrated that the clonogenic survival of both strains decreases similarly with increasing doses of X-rays. A difference in the survival between two strains was actually insignificant, although HLEC1 cells had the lower plating efficiency. This indicates that the same dose inactivates the same fraction of clonogenic cells in both strains. Intriguingly, irradiation enlarged the size of clonogenic colonies arising from HLEC1 cells in marked contrast to those from WI-38 cells. Such enhanced proliferation of clonogenic HLEC1 cells was significant at ≥2 Gy, and manifested as increments of ≤2.6 population doublings besides sham-irradiated controls. These results suggest that irradiation of HLEC1 cells not only inactivates clonogenic potential but also stimulates proliferation of surviving uniactivated clonogenic cells. Given that the lens is a closed system, the stimulated proliferation of lens epithelial cells may not be a homeostatic mechanism to compensate for their cell loss, but rather should be regarded as abnormal. This is because these findings are consistent with the early in vivo evidence documenting that irradiation induces excessive proliferation of rabbit lens epithelial cells and that suppression of lens epithelial cell divisions inhibits radiation cataractogenesis in frogs and rats. Thus, our in vitro model will be useful to evaluate the excessive proliferation of primary normal human lens epithelial cells that may underlie radiation cataractogenesis, warranting further investigations.

IL-9 expression by human eosinophils: regulation by IL-1beta and TNF-alpha.

PubMed

Gounni, A S; Nutku, E; Koussih, L; Aris, F; Louahed, J; Levitt, R C; Nicolaides, N C; Hamid, Q

2000-09-01

IL-9 is a pleiotropic cytokine that exhibits biologic activity on cells of diverse hemopoietic lineage. IL-9 stimulates the proliferation of activated T cells, enhances the production of IgE from B cells, and promotes the proliferation and differentiation of mast cells and hematopoietic progenitors. In this study we evaluated the expression of IL-9 messenger (m)RNA and protein by human peripheral blood eosinophils. We also investigated the role of IL-1beta and TNF-alpha in the release of IL-9 from human peripheral blood eosinophils. RT-PCR, in situ hybridization, and immunocytochemistry were used to investigate the presence of IL-9 mRNA and protein in human peripheral blood eosinophils from asthmatic patients and normal control subjects. Furthermore, biologic assay was used to investigate the release of IL-9 protein from IL-1beta- or TNF-alpha-stimulated eosinophils in vitro. RT-PCR analysis showed the presence of IL-9 mRNA in human peripheral blood eosinophil RNA preparations from subjects with atopic asthma, as well as in the eosinophil-differentiated HL-60 cell line. By using in situ hybridization, a significant difference (P <.01) in IL-9 mRNA expression was detected in human peripheral blood eosinophils freshly isolated from asthmatic subjects compared with those isolated from normal control subjects. Furthermore, the percentage of IL-9 immunoreactive eosinophils from asthmatic patients was increased compared with that found in normal control subjects (P <.01). We also demonstrate that cultured human peripheral blood eosinophils from asthmatic subjects synthesize and release IL-9 protein, which is upregulated on stimulation with TNF-alpha and IL-1beta. Human eosinophils express biologically active IL-9, which suggests that these cells may influence the recruitment and activation of effector cells linked to the pathogenesis of allergic disease. These observations provide further evidence for the role of eosinophils in regulating airway immune responses.

Metabolic control of T-cell activation and death in SLE

PubMed Central

Fernandez, David; Perl, Andras

2009-01-01

Systemic lupus erythematosus (SLE) is characterized by abnormal T-cell activation and death, processes which are crucially dependent on the controlled production of reactive oxygen intermediates (ROI) and of ATP in mitochondria. The mitochondrial transmembrane potential (Δψm) has conclusively emerged as a critical checkpoint of ATP synthesis and cell death. Lupus T cells exhibit persistent elevation of Δψm or mitochondrial hyperpolarization (MHP) as well as depletion of ATP and glutathione which decrease activation-induced apoptosis and instead predispose T cells for necrosis, thus stimulating inflammation in SLE. NO-induced mitochondrial biogenesis in normal T cells accelerates the rapid phase and reduces the plateau of Ca2+ influx upon CD3/CD28 co-stimulation, thus mimicking the Ca2+ signaling profile of lupus T cells. Treatment of SLE patients with rapamycin improves disease activity, normalizes CD3/CD28-induced Ca2+ fluxing but fails to affect MHP, suggesting that altered Ca2+ fluxing is downstream or independent of mitochondrial dysfunction. Understanding the molecular basis and consequences of MHP is essential for controlling T-cell activation and death signaling in SLE. Lupus T cells exhibit mitochondrial dysfunctionMitochondrial hyperpolarization (MHP) and ATP depletion predispose lupus T cells to death by necrosis which is pro-inflammatoryMHP is caused by depletion of glutathione and exposure to nitric oxide (NO)NO-induced mitochondrial biogenesis regenerates the Ca2+ signaling profile of lupus T cellsRapamycin treatment normalizes Ca2+ fluxing but not MHP, suggesting that the mammalian target of rapamycin, acts as a sensor and effector of MHP in SLE PMID:18722557

Energy utilization and gluconeogenesis in isolated leech segmental ganglia: Quantitative studies on the control and cellular localization of endogenous glycogen.

PubMed

Pennington, A J; Pentreath, V W

1988-01-01

The isolated segmental ganglia of the horse leech Haemopis sanguisuga were used as a model system to study the utilization and control of glycogen stores within nervous tissue. The glycogen in the ganglia was extracted and assayed fluorimentrically and its cellular localization and turnover studied by autoradiography in conjunction with [(3)H]glucose. We measured the glycogen after various periods of electrical stimulation and after incubation with K(+), Ca(2+), ouabain and glucose. The results for each experimental ganglion were compared to a paired control ganglion and the results analysed by paired t-tests. Electrical stimulation caused sequential changes in glycogen levels: a reduction of up to 67% (5-10 min); followed by an increase of up to 124% (between 15-50 min); followed by a reduction of up to 63% (60-90 min). Values were calculated for glucose utilization (e.g. 0.53 ?mol glucose/gm wet weight/min after 90 min) and estimates derived for glucose consumption per action potential per neuron (e.g. 0.12 fmol at 90 min). Glucose (1.5-10 mM) increased the amount of glycogen (1.5 mM by 30% at 60 min) and attenuated the effects of electrical stimulation. Ouabain (1 mM) blocked the effect of 5 min electrical stimulation. Nine millimolar K(+) increased glycogen by 27% after 10 min and decreased glycogen by 34% after 60 min; 3 mM Ca(2+) had no effect after 10 or 20 min and decreased glycogen by 29% after 60 min. Other concentrations of K(+) and Ca(2+) reduced glycogen after 60 min. Autoradiographic analysis demonstrated that the effects of elevated K(+) were principally within the glial cells. We conclude that (i) the glycogen stores in the glial cells of leech segmental ganglia provide an endogenous energy source which can support sustained neuronal activity, (ii) both electrical stimulation and elevated K(+) can induce gluconeogenesis within the ganglia, (iii) that electrical activation of neurons produces changes in the glycogen in the glial cells which are controlled in part by changes in K(+).

Impairment of Natural Killer Cytotoxic Activity and Interferon γ Production in Ccaat/Enhancer Binding Protein γ–Deficient Mice

PubMed Central

Kaisho, Tsuneyasu; Tsutsui, Hiroko; Tanaka, Takashi; Tsujimura, Tohru; Takeda, Kiyoshi; Kawai, Taro; Yoshida, Nobuaki; Nakanishi, Kenji; Akira, Shizuo

1999-01-01

We have investigated in vivo roles of CCAAT/enhancer binding protein γ (C/EBPγ) by gene targeting. C/EBPγ-deficient (C/EBPγ2/−) mice showed a high mortality rate within 48 h after birth. To analyze the roles of C/EBPγ in lymphoid lineage cells, bone marrow chimeras were established. C/EBPγ2/− chimeras showed normal T and B cell development. However, cytolytic functions of their splenic natural killer (NK) cells after stimulation with cytokines such as interleukin (IL)-12, IL-18, and IL-2 were significantly reduced as compared with those of control chimera NK cells. In addition, the ability of C/EBPγ−/− chimera splenocytes to produce interferon (IFN)-γ in response to IL-12 and/or IL-18 was markedly impaired. NK cells could be generated in vitro with normal surface marker expression in the presence of IL-15 from C/EBPγ2/− newborn spleen cells. However, they also showed lower cytotoxic activity and IFN-γ production when stimulated with IL-12 plus IL-18 than control NK cells, as observed in C/EBPγ2/− chimera splenocytes. In conclusion, our study reveals that C/EBPγ is a critical transcription factor involved in the functional maturation of NK cells. PMID:10587348

Novel engineered tendon-fibrocartilage-bone composite with cyclic tension for rotator cuff repair.

PubMed

Liu, Qian; Hatta, Taku; Qi, Jun; Liu, Haoyu; Thoreson, Andrew R; Amadio, Peter C; Moran, Steven L; Steinmann, Scott P; Gingery, Anne; Zhao, Chunfeng

2018-05-15

Surgical repair of rotator cuff tears presents a significant clinical challenge with high failure rates and inferior functional outcomes. Graft augmentation improves repair outcomes, however currently available grafting materials have limitations. While cell-seeded decellularized tendon slices may facilitate cell infiltration, promote tendon incorporation and preserve original mechanical strength, the unique fibrocartilage zone is yet to be successfully reestablished. In this study, we investigated the biological and mechanical properties of an engineered tendon-fibrocartilage-bone composite (TFBC) with cyclic tension (3% strain, 0.2 Hz). Decellularized TFBCs seeded with bone marrow-derived mesenchymal stem cell (BMSCs) sheets and subjected to mechanical stimulation for up to 7 days, were characterized by histology, immunohistochemistry, scanning electron microscopy, mechanical testing, and transcriptional regulation. The decellularized TFBC maintained native enthesis structure and properties. Mechanically stimulated TFBC-BMSC constructs displayed increased cell migration after 7 days of culture compared to static groups. The seeded cell sheet not only integrated well with tendon scaffold but also distributed homogeneously and aligned to the direction of stretch under dynamic culture. Developmental genes were regulated including, scleraxis which was significantly upregulated with mechanical stimulation. The Young's modulus of the cell-seeded constructs was significantly higher compared to the non-cell-seeded controls. In conclusion, the results of this study reveal that the TFBC-BMSC composite provides an ideal multilayer construct for cell seeding and growth, with mechanical preconditioning further enhances cell penetration and differentiation. The BMSC cell sheet revitalized TFBC in conjunction with mechanical stimulation could serve as a novel and primed biological patch to improve rotator cuff repair. This article is protected by copyright. All rights reserved.

Responsiveness of blood and sputum inflammatory cells in Japanese COPD patients, non-COPD smoking controls, and non-COPD nonsmoking controls

PubMed Central

Kawayama, Tomotaka; Kinoshita, Takashi; Matsunaga, Kazuko; Kobayashi, Akihiro; Hayamizu, Tomoyuki; Johnson, Malcolm; Hoshino, Tomoaki

2016-01-01

Purpose To compare pulmonary and systemic inflammatory mediator release, pre- and poststimulation, ex vivo, in cells from Japanese patients with chronic obstructive pulmonary disease (COPD), non-COPD smoking controls, and non-COPD nonsmoking controls (NSC). Patients and methods This was a nontreatment study with ten subjects per group. Inflammatory biomarker release, including interleukin (IL)-6 and -8, matrix metalloproteinase-9, and tumor necrosis factor (TNF)-α, was measured in peripheral blood mononuclear cells (PBMC) and sputum cells with and without lipopolysaccharide or TNF-α stimulation. Results In PBMC, basal TNF-α release (mean ± standard deviation) was significantly different between COPD (81.6±111.4 pg/mL) and nonsmoking controls (9.5±5.2 pg/mL) (P<0.05). No other significant differences were observed. Poststimulation biomarker release tended to increase, with the greatest changes in the COPD group. The greatest mean increases were seen in the lipopolysaccharide-induced release of matrix metalloproteinase-9, TNF-α, and IL-6 from PBMC. Pre- and poststimulation data from sputum samples were more variable and less conclusive than from PBMC. In the COPD group, induced sputum neutrophil levels were higher and macrophage levels were lower than in either control group. Significant correlations were seen between the number of sputum cells (macrophages and neutrophils) and biomarker levels (IL-8, IL-6, and TNF-α). Conclusion This was the first study to compare cellular inflammatory mediator release before and after stimulation among Japanese COPD, smoking controls, and nonsmoking controls populations. Poststimulation levels tended to be higher in patients with COPD. The results suggest that PBMC are already preactivated in the circulation in COPD patients. This provides further evidence that COPD is a multicomponent disease, involving both airway and systemic inflammation. PMID:26929615

Responsiveness of blood and sputum inflammatory cells in Japanese COPD patients, non-COPD smoking controls, and non-COPD nonsmoking controls.

PubMed

Kawayama, Tomotaka; Kinoshita, Takashi; Matsunaga, Kazuko; Kobayashi, Akihiro; Hayamizu, Tomoyuki; Johnson, Malcolm; Hoshino, Tomoaki

2016-01-01

To compare pulmonary and systemic inflammatory mediator release, pre- and poststimulation, ex vivo, in cells from Japanese patients with chronic obstructive pulmonary disease (COPD), non-COPD smoking controls, and non-COPD nonsmoking controls (NSC). This was a nontreatment study with ten subjects per group. Inflammatory biomarker release, including interleukin (IL)-6 and -8, matrix metalloproteinase-9, and tumor necrosis factor (TNF)-α, was measured in peripheral blood mononuclear cells (PBMC) and sputum cells with and without lipopolysaccharide or TNF-α stimulation. In PBMC, basal TNF-α release (mean ± standard deviation) was significantly different between COPD (81.6±111.4 pg/mL) and nonsmoking controls (9.5±5.2 pg/mL) (P<0.05). No other significant differences were observed. Poststimulation biomarker release tended to increase, with the greatest changes in the COPD group. The greatest mean increases were seen in the lipopolysaccharide-induced release of matrix metalloproteinase-9, TNF-α, and IL-6 from PBMC. Pre- and poststimulation data from sputum samples were more variable and less conclusive than from PBMC. In the COPD group, induced sputum neutrophil levels were higher and macrophage levels were lower than in either control group. Significant correlations were seen between the number of sputum cells (macrophages and neutrophils) and biomarker levels (IL-8, IL-6, and TNF-α). This was the first study to compare cellular inflammatory mediator release before and after stimulation among Japanese COPD, smoking controls, and nonsmoking controls populations. Poststimulation levels tended to be higher in patients with COPD. The results suggest that PBMC are already preactivated in the circulation in COPD patients. This provides further evidence that COPD is a multicomponent disease, involving both airway and systemic inflammation.

Human Peripheral Blood Mononuclear Cells Cultured in Normal and Hyperglycemic Media in Simulated Microgravity Using NASA Bioreactors

NASA Technical Reports Server (NTRS)

Lawless, DeSales

2003-01-01

We sought answers to several questions this summer at NASA Johnson Space Center. Initial studies involved the in vitro culture of human peripheral blood mononuclear in cells in different conditioned culture media. Several human cancer clones were similarly studied to determine responses to aberrant glycosylation by the argon laser. The cells were grown at unit gravity in flasks and in simulated microgravity using NASA bioreactors. The cells in each instance were analyzed by flow cytometry. Cell cycle analysis was acquired by staining nuclear DNA with propidium iodide. Responses to the laser stimulation was measured by observing autofluorescence emitted in the green and red spectra after stimulation. Extent of glycosylation correlated with the intensity of the laser stimulated auto-fluorescence. Our particular study was to detect and monitor aberrant glycosylation and its role in etiopathogenesis. Comparisons were made between cells known to be neoplastic and normal cell controls using the same Laser Induced Autofluorescence technique. Studies were begun after extensive literature searches on using the antigen presenting potential of dendritic cells to induce proliferation of antigen specific cytotoxic T-cells. The Sendai virus served as the antigen. Our goal is to generate sufficient numbers of such cells in the simulated microgravity environment for use in autologous transplants of virally infected individuals including those positive for hepatitis and HIV.

Chronophin coordinates cell leading edge dynamics by controlling active cofilin levels

PubMed Central

Delorme-Walker, Violaine; Seo, Ji-Yeon; Gohla, Antje; Fowler, Bruce; Bohl, Ben; DerMardirossian, Céline

2015-01-01

Cofilin, a critical player of actin dynamics, is spatially and temporally regulated to control the direction and force of membrane extension required for cell locomotion. In carcinoma cells, although the signaling pathways regulating cofilin activity to control cell direction have been established, the molecular machinery required to generate the force of the protrusion remains unclear. We show that the cofilin phosphatase chronophin (CIN) spatiotemporally regulates cofilin activity at the cell edge to generate persistent membrane extension. We show that CIN translocates to the leading edge in a PI3-kinase–, Rac1-, and cofilin-dependent manner after EGF stimulation to activate cofilin, promotes actin free barbed end formation, accelerates actin turnover, and enhances membrane protrusion. In addition, we establish that CIN is crucial for the balance of protrusion/retraction events during cell migration. Thus, CIN coordinates the leading edge dynamics by controlling active cofilin levels to promote MTLn3 cell protrusion. PMID:26324884

Acute mental stress but not enforced muscle activity transiently increases natural cytotoxicity in spontaneously hypertensive rats.

PubMed

Jonsdottir, I H; Johansson, C; Asea, A; Hellstrand, K; Hoffmann, P

1996-08-01

The influence of acute mental stress and the effect of electrically induced skeletal muscle contractions on natural cytotoxicity in vivo was investigated in spontaneously hypertensive rats Natural cytotoxicity in vivo was measured as the clearance of injected 51Cr-labelled YAC-1 lymphoma cells from the lungs, which are specifically lysed by natural killer cells. The mental stress consisted of an air jet directed towards the animals in their cage for 25 min. During the mental stress there was a significant increase in natural cytotoxicity. Thus, retained radioactivity in the lungs was decreased to 74 +/- 6% of the control levels which was set to 100% (P < 0.01). This augmentation of YAC-1-cell clearance could be blocked with the beta-adrenergic receptor antagonist Timolol. Two hours after termination of the air stress, in vivo cytotoxicity had returned to control levels. In contrast, acute physical stress, consisting of electrically induced muscle contractions for 60 min, had no significant effects on in vivo cytotoxicity, either during the stimulation or 1, 2 or 24 h after the stimulation. Further, significantly increased plasma levels of adrenaline were seen after the air jet stress, but not after muscle stimulation. There were no significant changes in plasma noradrenaline levels either after air stress or muscle stimulation. These results indicate that changes in in vivo cytotoxicity after mild mental stress are dependent on increased plasma catecholamine levels while acute physical stress without changes in catecholamine levels, does not influence in vivo cytotoxicity.

Notch1 stimulation induces a vascularization switch with pericyte-like cell differentiation of glioblastoma stem cells.

PubMed

Guichet, Pierre-Olivier; Guelfi, Sophie; Teigell, Marisa; Hoppe, Liesa; Bakalara, Norbert; Bauchet, Luc; Duffau, Hugues; Lamszus, Katrin; Rothhut, Bernard; Hugnot, Jean-Philippe

2015-01-01

Glioblastoma multiforms (GBMs) are highly vascularized brain tumors containing a subpopulation of multipotent cancer stem cells. These cells closely interact with endothelial cells in neurovascular niches. In this study, we have uncovered a close link between the Notch1 pathway and the tumoral vascularization process of GBM stem cells. We observed that although the Notch1 receptor was activated, the typical target proteins (HES5, HEY1, and HEY2) were not or barely expressed in two explored GBM stem cell cultures. Notch1 signaling activation by expression of the intracellular form (NICD) in these cells was found to reduce their growth rate and migration, which was accompanied by the sharp reduction in neural stem cell transcription factor expression (ASCL1, OLIG2, and SOX2), while HEY1/2, KLF9, and SNAI2 transcription factors were upregulated. Expression of OLIG2 and growth were restored after termination of Notch1 stimulation. Remarkably, NICD expression induced the expression of pericyte cell markers (NG2, PDGFRβ, and α-smooth muscle actin [αSMA]) in GBM stem cells. This was paralleled with the induction of several angiogenesis-related factors most notably cytokines (heparin binding epidermal growth factor [HB-EGF], IL8, and PLGF), matrix metalloproteinases (MMP9), and adhesion proteins (vascular cell adhesion molecule 1 [VCAM1], intercellular adhesion molecule 1 [ICAM1], and integrin alpha 9 [ITGA9]). In xenotransplantation experiments, contrasting with the infiltrative and poorly vascularized tumors obtained with control GBM stem cells, Notch1 stimulation resulted in poorly disseminating but highly vascularized grafts containing large vessels with lumen. Notch1-stimulated GBM cells expressed pericyte cell markers and closely associated with endothelial cells. These results reveal an important role for the Notch1 pathway in regulating GBM stem cell plasticity and angiogenic properties. © 2014 AlphaMed Press.

Evaluation of dermal wound healing activity of synthetic peptide SVVYGLR.

PubMed

Uchinaka, Ayako; Kawaguchi, Naomasa; Ban, Tsuyoshi; Hamada, Yoshinosuke; Mori, Seiji; Maeno, Yoshitaka; Sawa, Yoshiki; Nagata, Kohzo; Yamamoto, Hirofumi

2017-09-23

SVVYGLR peptide (SV peptide) is a 7-amino-acid sequence with angiogenic properties that is derived from osteopontin in the extracellular matrix and promotes differentiation of fibroblasts to myofibroblast-like cells and the production of collagen type Ⅲ by cardiac fibroblasts. However, the effects of SV peptide on dermal cells and tissue are unknown. In this study, we evaluated the effects of this peptide in a rat model of dermal wound healing. The synthetic SV peptide was added to dermal fibroblasts or keratinocytes, and their cellular motility was evaluated. In an in vivo wound healing exeriment, male rats aged 8 weeks were randomly assigned to the SV peptide treatment, non-treated control, or phosphate-buffered saline (PBS) groups. Wound healing was assessed by its repair rate and histological features. Scratch assay and cell migration assays using the Chemotaxicell method showed that SV peptide significantly promoted the cell migration in both fibroblasts and keratinocytes. In contrast the proliferation potency of these cells was not affected by SV peptide. In the rat model, wound healing progressed faster in the SV peptide-treated group than in the control and PBS groups. The histopathological analyses showed that the SV peptide treatment stimulated the migration of fibroblasts to the wound area and increased the number of myofibroblasts. Immunohistochemical staining showed a marked increase of von Willebland factor-positive neomicrovessels in the SV peptide-treated group. In conclusion, SV peptide has a beneficial function to promote wound healing by stimulating granulation via stimulating angiogenesis, cell migration, and the myofibroblastic differentiation of fibroblasts. Copyright © 2017 Elsevier Inc. All rights reserved.

Myosin-II controls cellular branching morphogenesis and migration in 3D by minimizing cell surface curvature

PubMed Central

Elliott, Hunter; Fischer, Robert A.; Myers, Kenneth A.; Desai, Ravi A.; Gao, Lin; Chen, Christopher S.; Adelstein, Robert; Waterman, Clare M.; Danuser, Gaudenz

2014-01-01

In many cases cell function is intimately linked to cell shape control. We utilized endothelial cell branching morphogenesis as a model to understand the role of myosin-II in shape control of invasive cells migrating in 3D collagen gels. We applied principles of differential geometry and mathematical morphology to 3D image sets to parameterize cell branch structure and local cell surface curvature. We find that Rho/ROCK-stimulated myosin-II contractility minimizes cell-scale branching by recognizing and minimizing local cell surface curvature. Utilizing micro-fabrication to constrain cell shape identifies a positive feedback mechanism in which low curvature stabilizes myosin-II cortical association, where it acts to maintain minimal curvature. The feedback between myosin-II regulation by and control of curvature drives cycles of localized cortical myosin-II assembly and disassembly. These cycles in turn mediate alternating phases of directionally biased branch initiation and retraction to guide 3D cell migration. PMID:25621949

Cosmos: 1989 immunology studies

NASA Technical Reports Server (NTRS)

Sonnenfeld, Gerald

1991-01-01

The effects of flight on Cosmos mission 2044 on leukocyte subset distribution and the sensitivity of bone marrow cells to colony stimulating factor-GM were determined. A parallel study with antiorthostatic suspension was also carried out. The study involved repetition and expansion of studies performed on Cosmos 1887. Spleen and bone marrow cells were obtained from flown, vivarium control, synchronous control, and suspended rats. The cells were stained with a series of monoclonal antibodies directed against rat leukocyte cell surface antigens. Control cells were stained with a monoclonal antibody directed against an irrelevant species or were unstained. Cells were then analyzed for fluorescence using a FACSCAN flow cytometer. Bone marrow cells were placed in culture with GM-CSF in McCoy's 5a medium and incubated for 5 days. Cultures were then evaluated for the number of colonies of 50 cells or greater.

Co-administration of plasmid-encoded granulocyte-macrophage colony-stimulating factor increases human immunodeficiency virus-1 DNA vaccine-induced polyfunctional CD4+ T-cell responses

PubMed Central

Santana, Vinicius Canato; Almeida, Rafael Ribeiro; Ribeiro, Susan Pereira; Ferreira, Luís Carlos de Souza; Kalil, Jorge; Rosa, Daniela Santoro; Cunha-Neto, Edecio

2015-01-01

T-cell based vaccines against human immunodeficiency virus (HIV) generate specific responses that may limit both transmission and disease progression by controlling viral load. Broad, polyfunctional, and cytotoxic CD4+T-cell responses have been associated with control of simian immunodeficiency virus/HIV-1 replication, supporting the inclusion of CD4+ T-cell epitopes in vaccine formulations. Plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) co-administration has been shown to induce potent CD4+ T-cell responses and to promote accelerated priming and increased migration of antigen-specific CD4+ T-cells. However, no study has shown whether co-immunisation with pGM-CSF enhances the number of vaccine-induced polyfunctional CD4+ T-cells. Our group has previously developed a DNA vaccine encoding conserved, multiple human leukocyte antigen (HLA)-DR binding HIV-1 subtype B peptides, which elicited broad, polyfunctional and long-lived CD4+ T-cell responses. Here, we show that pGM-CSF co-immunisation improved both magnitude and quality of vaccine-induced T-cell responses, particularly by increasing proliferating CD4+ T-cells that produce simultaneously interferon-γ, tumour necrosis factor-α and interleukin-2. Thus, we believe that the use of pGM-CSF may be helpful for vaccine strategies focused on the activation of anti-HIV CD4+ T-cell immunity. PMID:26602876

Possible role of natural killer cells in pemphigus vulgaris − preliminary observations

PubMed Central

Stern, J N H; Keskin, D B; Barteneva, N; Zuniga, J; Yunis, E J; Ahmed, A R

2008-01-01

Pemphigus vulgaris (PV) is an autoimmune blistering disease that affects the skin and multiple mucous membranes, and is caused by antibodies to desmoglein (Dsg) 1 and 3. Natural killer (NK) cells have a role in autoimmunity, but their role in PV is not known. NK cells in the peripheral blood leucocytes (PBL) of 15 untreated Caucasian patients with active PV were studied and compared with healthy controls for the expression of major histocompatibility complex (MHC) class II and co-stimulatory molecules. CD56+ CD16- CD3- NK or CD56+ CD16+ CD3- NK cells from the PBL of PV patients co-express MHC class II and co-stimulatory molecule B7-H3 without exogenous stimulation. CD4+ T cells from the PBL and perilesional skin of PV patients were co-cultured with CD56+ CD3- NK cells from the PBL of the same patients; in the presence of Dsg3 peptides underwent statistically significant proliferation, indicating that NK cells functioned as antigen-presenting cells. Supernatants from these co-cultures and serum of the same patients with active PV had statistically significantly elevated levels of interleukin (IL)-6, IL-8 and interferon-γ, compared with controls indicating that the NK cells stimulated CD4+ T cells to produce proinflammatory cytokines. In these experiments, we present preliminary evidence that NK cells may play a role in the pathobiology of PV. PMID:18373702

Activation of histamine H4 receptor inhibits TNFα/IMD-0354-induced apoptosis in human salivary NS-SV-AC cells.

PubMed

Stegajev, Vasili; Kouri, Vesa-Petteri; Salem, Abdelhakim; Rozov, Stanislav; Stark, Holger; Nordström, Dan C E; Konttinen, Yrjö T

2014-12-01

Apoptosis is involved in the pathogenesis of Sjögren's syndrome (SS), an autoimmune disease affecting exocrine glands. Our recent studies revealed diminished histamine H4 receptor (H₄R) expression and impaired histamine transport in the salivary gland epithelial cells in SS. The aim was now to test if nanomolar histamine and high-affinity H₄R signaling affect apoptosis of human salivary gland epithelial cell. Simian virus 40-immortalized acinar NS-SV-AC cells were cultured in serum-free keratinocyte medium ± histamine H₄R agonist HST-10. Expression and internalization of H₄R were studied by immunofluorescence staining ± clathrin inhibitor methyl-β-cyclodextrin (MβCD). Apoptosis induced using tumor necrosis factor-α with nuclear factor-κB inhibitor IMD-0354 was studied using phase contrast microscopy, Western blot, flow cytometry and polymerase chain reaction (qRT-PCR). HST-10-stimulated H₄R internalization was inhibited by MβCD. Western blotting revealed diminished phosphorylated c-Jun N-terminal kinase JNK, but unchanged levels of phosphorylated extracellular signal regulated kinase pERK1/2 in H₄R-stimulated samples compared to controls. qRT-PCR showed up-regulated expression of anti-apoptotic B cell lymphoma-extra large/Bcl-xL mRNAs and proteins, whereas pro-apoptotic Bcl-2-associated X protein/BAX remained unchanged in H4R-stimulated samples. H₄R stimulation diminished cleavage of PARP and flow cytometry showed significant dose-dependent inhibitory effect of H₄R stimulation on apoptosis. As far as we know this is the first study showing inhibitory effect of H₄R activation on apoptosis of human salivary gland cells. Diminished H₄R-mediated activation may contribute to loss of immune tolerance in autoimmune diseases and in SS in particular.

A role for the thermal environment in defining co-stimulation requirements for CD4+ T cell activation

PubMed Central

Zynda, Evan R; Grimm, Melissa J; Yuan, Min; Zhong, Lingwen; Mace, Thomas A; Capitano, Maegan; Ostberg, Julie R; Lee, Kelvin P; Pralle, Arnd; Repasky, Elizabeth A

2015-01-01

Maintenance of normal core body temperature is vigorously defended by long conserved, neurovascular homeostatic mechanisms that assist in heat dissipation during prolonged, heat generating exercise or exposure to warm environments. Moreover, during febrile episodes, body temperature can be significantly elevated for at least several hours at a time. Thus, as blood cells circulate throughout the body, physiologically relevant variations in surrounding tissue temperature can occur; moreover, shifts in core temperature occur during daily circadian cycles. This study has addressed the fundamental question of whether the threshold of stimulation needed to activate lymphocytes is influenced by temperature increases associated with physiologically relevant increases in temperature. We report that the need for co-stimulation of CD4+ T cells via CD28 ligation for the production of IL-2 is significantly reduced when cells are exposed to fever-range temperature. Moreover, even in the presence of sufficient CD28 ligation, provision of extra heat further increases IL-2 production. Additional in vivo and in vitro data (using both thermal and chemical modulation of membrane fluidity) support the hypothesis that the mechanism by which temperature modulates co-stimulation is linked to increases in membrane fluidity and membrane macromolecular clustering in the plasma membrane. Thermally-regulated changes in plasma membrane organization in response to physiological increases in temperature may assist in the geographical control of lymphocyte activation, i.e., stimulating activation in lymph nodes rather than in cooler surface regions, and further, may temporarily and reversibly enable CD4+ T cells to become more quickly and easily activated during times of infection during fever. PMID:26131730

Increased expression of Fas receptor and Fas ligand in the culture of the peripheral blood mononuclear cells stimulated with Borrelia burgdorferi sensu lato.

PubMed

Grygorczuk, Sambor; Osada, Joanna; Moniuszko, Anna; Świerzbińska, Renata; Kondrusik, Maciej; Zajkowska, Joanna; Dunaj, Justyna; Dąbrowska, Milena; Pancewicz, Sławomir

2015-03-01

Apoptosis of the lymphocytes plays an essential role in the regulation of inflammatory/immune responses and its abnormalities may contribute to a chronic infection, persistent inflammation and autoimmunity. Its role in the pathogenesis of the late Lyme borreliosis manifestations has not been studied so far. We have measured Th lymphocyte apoptosis rate, membrane expression of pro-apoptotic Fas receptor, and supernatant concentrations of selected soluble pro- and anti-apoptotic mediators in cultures of peripheral blood mononuclear cells from 16 patients with disseminated Lyme borreliosis (6 with osteoarticular symptoms, 7 with neuroborreliosis and 3 with acrodermatitis chronica atrophicans) and 8 healthy controls. The cultures stimulated for 48h with live Borrelia burgdorferi sensu stricto, B. garinii or B. afzelii spirochetes. Fraction of the apoptotic Th (CD3+CD4+) lymphocytes and expression of Fas in this cell population was measured cytometrically and concentrations of soluble Fas, soluble Fas ligand, IL-10, IL-12 and TGF-β in culture supernatant with ELISA assays. The expression of IL-10, soluble and membrane Fas and soluble Fas ligand was increased under stimulation and higher in the presence of B. burgdorferi sensu stricto than the other species. Apoptosis rate was not affected. There was no difference between Lyme borreliosis patients and controls. IL-10 concentration correlated negatively with the membrane Fas expression and apoptosis under stimulation with B. afzelii and B. garinii. Expression of Fas/FasL system is up-regulated under stimulation with B. burgdorferi, but without corresponding increase in lymphocyte apoptosis. Variable responses observed with different B. burgdorferi species may reflect differences in the pathogenesis of the infection in vivo. Copyright © 2014 Elsevier GmbH. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Huang-Joe; Division of Cardiology, Department of Medicine, China Medical University Hospital, No. 2, Yuh-Der Road, Taichung 40447, Taiwan; Lo, Wan-Yu

Patients with paclitaxel-eluting stents are concerned with stent thrombosis caused by premature discontinuation of dual antiplatelet therapy or clopidogrel resistance. This study investigates the effect of (-)-epigallocatechin-3-gallate (EGCG) on the expression of thrombin/paclitaxel-induced endothelial tissue factor (TF) expressions in human aortic endothelial cells (HAECs). EGCG was nontoxic to HAECs at 6 h up to a concentration of 25 {mu}mol/L. At a concentration of 25 {mu}mol/L, EGCG pretreatment potently inhibited both thrombin-stimulated and thrombin/paclitaxel-stimulated endothelial TF protein expression. Thrombin and thrombin/paclitaxel-induced 2.6-fold and 2.9-fold increases in TF activity compared with the control. EGCG pretreatment caused a 29% and 38% decrease inmore » TF activity on thrombin and thrombin/paclitaxel treatment, respectively. Real-time polymerase chain reaction (PCR) showed that thrombin and thrombin/paclitaxel-induced 3.0-fold and 4.6-fold TF mRNA expressions compared with the control. EGCG pretreatment caused an 82% and 72% decrease in TF mRNA expression on thrombin and thrombin/paclitaxel treatment, respectively. The c-Jun terminal NH2 kinase (JNK) inhibitor SP600125 reduced thrombin/paclitaxel-induced TF expression. Furthermore, EGCG significantly inhibited the phosphorylation of JNK to 49% of thrombin/paclitaxel-stimulated HAECs at 60 min. Immunofluorescence assay did not show an inhibitory effect of EGCG on P65 NF-{kappa}B nuclear translocation in the thrombin/paclitaxel-stimulated endothelial cells. In conclusion, EGCG can inhibit TF expression in thrombin/paclitaxel-stimulated endothelial cells via the inhibition of JNK phosphorylation. The unique property of EGCG may be used to develop a new drug-eluting stent by co-coating EGCG and paclitaxel.« less

Aldosterone does not alter endothelin B receptor signaling in the inner medullary collecting duct.

PubMed

Ramkumar, Nirupama; Stuart, Deborah; Yang, Tianxin; Kohan, Donald E

2017-03-01

Recent studies suggest that aldosterone-mediated sulfenic acid modification of the endothelin B receptor (ETB) promotes renal injury in an ischemia/reperfusion model through reduced ETB-stimulated nitric oxide production. Similarly, aldosterone inactivation of ETB signaling promotes pulmonary artery hypertension. Consequently, we asked whether aldosterone inhibits collecting duct ETB signaling; this could promote fluid retention since CD ETB exerts natriuretic and diuretic effects. A mouse inner medullary collecting duct cell line (IMCD3) was treated with aldosterone for 48 h followed by sarafotoxin-6c, an ETB-selective agonist, and extracellular signal-related kinase 1/2 (ERK) phosphorylation assessed. S6c increased the phospho/total-ERK ratio similarly in control and aldosterone-treated cells (aldosterone alone increased phospho/total-ERK). Since cultured IMCD cell lines lack ETB inhibited AVP signaling, the effect of S6c on AVP-stimulated cAMP in acutely isolated IMCD was assessed. Rats (have much higher CD ETB expression than mice) were exposed to 3 days of a normal or low Na + diet, or low Na + diet + desoxycorticosterone acetate. S6c inhibited AVP-stimulated cAMP in rat IMCD by the same degree in the high mineralocorticoid groups compared to controls. Finally, S6c-stimulated cGMP accumulation in cultured IMCD, or S6c-stimulated nitric oxide or cGMP in acutely isolated IMCD, was not affected by prior aldosterone exposure. These findings provide evidence that aldosterone does not modify ETB effects on ERK phosphorylation, AVP-dependent cAMP inhibition, or NO/cGMP accumulation in the IMCD Thus, while aldosterone can inhibit endothelial cell ETB activity to promote hypertension and injury, this response does not appear to occur in the IMCD. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Triiodothyronine Acutely Stimulates Glucose Transport into L6 Muscle Cells Without Increasing Surface GLUT4, GLUT1, or GLUT3

PubMed Central

Teixeira, Silvania Silva; Tamrakar, Akhilesh K.; Goulart-Silva, Francemilson; Serrano-Nascimento, Caroline; Klip, Amira

2012-01-01

Background Thyroid hormones (THs) act genomically to stimulate glucose transport by elevating glucose transporter (Slc2a) expression and glucose utilization by cells. However, nongenomic effects of THs are now emerging. Here, we assess how triiodothyronine (T3) acutely affects glucose transport and the content of GLUT4, GLUT1, and GLUT3 at the surface of muscle cells, and possible interactions between T3 and insulin action. Methods Differentiated L6 myotubes transfected with myc-tagged Slc2a4 (L6-GLUT4myc) or Slc2a1 (L6-GLUT1myc) and wild-type L6 myotubes were studied in the following conditions: control, hypothyroid (Tx), Tx plus T3, Tx plus insulin, and Tx plus insulin and T3. Results Glucose uptake and GLUT4 content at the cell surface decreased in the Tx group relative to controls. T3 treatment for 30 minutes increased glucose transport into L6-GLUT4myc cells without altering surface GLUT4 content, which increased only thereafter. The total amount of GLUT4 protein remained unchanged among the groups studied. The surface GLUT1 content of L6-GLUT1myc cells also remained unaltered after T3 treatment; however, in these cells glucose transport was not stimulated by T3. In wild-type L6 cells, although T3 treatment increased the total amount of GLUT3, it did not change the surface GLUT3 content. Moreover, within 30 minutes, T3 stimulation of glucose uptake was additive to that of insulin in L6-GLUT4myc cells. As expected, insulin elevated surface GLUT4 content and glucose uptake. However, interestingly, surface GLUT4 content remained unchanged or even dropped with T3 plus insulin. Conclusions These data reveal that T3 rapidly increases glucose uptake in L6-GLUT4myc cells, which, at least for 30 minutes, did not depend on an increment in GLUT4 at the cell surface yet potentiates insulin action. We propose that this rapid T3 effect involves activation of GLUT4 transporters at the cell surface, but cannot discount the involvement of an unknown GLUT. PMID:22663547

Construction and application of a bovine immune-endocrine cDNA microarray.

PubMed

Tao, Wenjing; Mallard, Bonnie; Karrow, Niel; Bridle, Byram

2004-09-01

A variety of commercial DNA arrays specific for humans and rodents are widely available; however, microarrays containing well-characterized genes to study pathway-specific gene expression are not as accessible for domestic animals, such as cattle, sheep and pigs. Therefore, a small-scale application-targeted bovine immune-endocrine cDNA array was developed to evaluate genetic pathways involved in the immune-endocrine axis of cattle during periods of altered homeostasis provoked by physiological or environmental stressors, such as infection, vaccination or disease. For this purpose, 167 cDNA sequences corresponding to immune, endocrine and inflammatory response genes were collected and categorized. Positive controls included 5 housekeeping genes (glyceraldehydes-3-phosphate dehydrogenase, hypoxanthine phosphoribosyltransferase, ribosomal protein L19, beta-actin, beta2-microglobulin) and bovine genomic DNA. Negative controls were a bacterial gene (Rhodococcus equi 17-kDa virulence-associated protein) and a partial sequence of the plasmid pACYC177. In addition, RNA extracted from un-stimulated, as well as superantigen (Staphylococcus aureus enterotoxin-A, S. aureus Cowan Pansorbin Cells) and mitogen-stimulated (LPS, ConA) bovine blood leukocytes was mixed, reverse transcribed and PCR amplified using gene-specific primers. The endocrine-associated genes were amplified from cDNA derived from un-stimulated bovine hypothalamus, pituitary, adrenal and thyroid gland tissues. The array was constructed in 4 repeating grids of 180 duplicated spots by coupling the PCR amplified 213-630 bp gene fragments onto poly-l-lysine coated glass slides. The bovine immune-endocrine arrays were standardized and preliminary gene expression profiles generated using Cy3 and Cy5 labelled cDNA from un-stimulated and ConA (5 microg/ml) stimulated PBMC of 4 healthy Holstein cows (2-4 replicate arrays/cow) in a time course study. Mononuclear cell-derived cytokine and chemokine (IL-2, IL-1alpha, TNFalpha, IFN-gamma, TGFbeta-1, MCP-1, MCP-2 and MIP-3alpha) mRNA exhibited a repeatable and consistently low expression in un-stimulated cells and at least a two-fold increased expression following 6 and 24 h ConA stimulation as compared to 0 h un-stimulated controls. In contrast, expression of antigen presenting molecules, MHC-DR, MHC-DQ and MHC-DY, were consistently at least two-fold lower following 6 and 24 h ConA stimulation. The only endocrine gene with differential expression following ConA stimulation was prolactin. Additionally, due to the high level of genetic homology between ovine, swine and bovine genes, RNA similarly acquired from sheep and pigs was evaluated and similar gene expression patterns were noted. These data demonstrate that this application-targeted array containing a set of well characterized genes can be used to determine the relative gene expression corresponding to immune-endocrine responses of cattle and related species, sheep and pigs.

Iodine stimulates estrogen receptor singling and its systemic level is increased in surgical patients due to topical absorption.

PubMed

He, Shaohua; Wang, Bingchan; Lu, Xiyi; Miao, Suyu; Yang, Fengming; Zava, Theodore; Ding, Qiang; Zhang, Shijiang; Liu, Jiayin; Zava, David; Shi, Yuenian Eric

2018-01-02

Iodine is crucial for thyroid hormone production. However, recent epidemiologic studies have shown that breast cancer patients have an elevated risk of developing thyroid cancer and vice versa. A notable finding in this study is that iodine stimulated the transcriptional activity of estrogen receptor-α (ER-α) in breast cancer cells. Iodine stimulated expression of several ER-α regulated gene including PS2 , Cathepsin D , CyclinD1 , and PR both in vitro and in nude mice, which is consistent with its stimulation of both anchorage-dependent and -independent growth of ER-α positive breast cancer cells and the effect to dampen tumor shrinkage of MCF-7 xenograft in ovariectomized nude mice. Analyses of clinical urine samples from breast cancer patients undergoing surgery demonstrated that urinary iodine levels were significantly higher than that in controls; and this increased level is due to the antiseptic use of iodine during breast surgery. The present study indicates that excess iodine intake may be an unfavorable factor in breast cancer by stimulation of ER-α transcriptional activity.

Pancreatic islet cell therapy for type I diabetes: understanding the effects of glucose stimulation on islets in order to produce better islets for transplantation.

PubMed

Ren, Jiaqiang; Jin, Ping; Wang, Ena; Liu, Eric; Harlan, David M; Li, Xin; Stroncek, David F

2007-01-03

While insulin replacement remains the cornerstone treatment for type I diabetes mellitus (T1DM), the transplantation of pancreatic islets of Langerhans has the potential to become an important alternative. And yet, islet transplant therapy is limited by several factors, including far too few donor pancreases. Attempts to expand mature islets or to produce islets from stem cells are far from clinical application. The production and expansion of the insulin-producing cells within the islet (so called beta cells), or even creating cells that secrete insulin under appropriate physiological control, has proven difficult. The difficulty is explained, in part, because insulin synthesis and release is complex, unique, and not entirely characterized. Understanding beta-cell function at the molecular level will likely facilitate the development of techniques to manufacture beta-cells from stem cells. We will review islet transplantation, as well as the mechanisms underlying insulin transcription, translation and glucose stimulated insulin release.

Pancreatic islet cell therapy for type I diabetes: understanding the effects of glucose stimulation on islets in order to produce better islets for transplantation

PubMed Central

Ren, Jiaqiang; Jin, Ping; Wang, Ena; Liu, Eric; Harlan, David M; Li, Xin; Stroncek, David F

2007-01-01

While insulin replacement remains the cornerstone treatment for type I diabetes mellitus (T1DM), the transplantation of pancreatic islets of Langerhans has the potential to become an important alternative. And yet, islet transplant therapy is limited by several factors, including far too few donor pancreases. Attempts to expand mature islets or to produce islets from stem cells are far from clinical application. The production and expansion of the insulin-producing cells within the islet (so called β cells), or even creating cells that secrete insulin under appropriate physiological control, has proven difficult. The difficulty is explained, in part, because insulin synthesis and release is complex, unique, and not entirely characterized. Understanding β-cell function at the molecular level will likely facilitate the development of techniques to manufacture β-cells from stem cells. We will review islet transplantation, as well as the mechanisms underlying insulin transcription, translation and glucose stimulated insulin release. PMID:17201925

Simulation system of arrhythmia using ActiveX control.

PubMed

Takeuchi, Akihiro; Hirose, Minoru; Hamada, Atsushi; Ikeda, Noriaki

2005-07-01

A simulation system for arrhythmias has been developed using Windows-based software technology, ActiveX control. The cardiac module consists of six cells, the sinus, atrium, AV node, ventricle, and ectopic foci. The physiological properties of the cells, the automaticity and conduction delay, were modelled, respectively, by the phase response curve and the excitability recovery curve. Cell functions were implemented in the ActiveX control and incorporated into the cardiac module. The system draws the ECG sequence as a ladder diagram in real time. The system interactively shows diverse arrhythmias for various user settings of the cell function and bidirectional conduction between the cells. Users are able to experiment virtually by setting up a so-called electrophysiological stimulation. This system is useful for learning and for teaching the interaction between the cells and arrhythmias.

Closed-Loop Efficient Searching of Optimal Electrical Stimulation Parameters for Preferential Excitation of Retinal Ganglion Cells

PubMed Central

Guo, Tianruo; Yang, Chih Yu; Tsai, David; Muralidharan, Madhuvanthi; Suaning, Gregg J.; Morley, John W.; Dokos, Socrates; Lovell, Nigel H.

2018-01-01

The ability for visual prostheses to preferentially activate functionally-distinct retinal ganglion cells (RGCs) is important for improving visual perception. This study investigates the use of high frequency stimulation (HFS) to elicit RGC activation, using a closed-loop algorithm to search for optimal stimulation parameters for preferential ON and OFF RGC activation, resembling natural physiological neural encoding in response to visual stimuli. We evaluated the performance of a wide range of electrical stimulation amplitudes and frequencies on RGC responses in vitro using murine retinal preparations. It was possible to preferentially excite either ON or OFF RGCs by adjusting amplitudes and frequencies in HFS. ON RGCs can be preferentially activated at relatively higher stimulation amplitudes (>150 μA) and frequencies (2–6.25 kHz) while OFF RGCs are activated by lower stimulation amplitudes (40–90 μA) across all tested frequencies (1–6.25 kHz). These stimuli also showed great promise in eliciting RGC responses that parallel natural RGC encoding: ON RGCs exhibited an increase in spiking activity during electrical stimulation while OFF RGCs exhibited decreased spiking activity, given the same stimulation amplitude. In conjunction with the in vitro studies, in silico simulations indicated that optimal HFS parameters could be rapidly identified in practice, whilst sampling spiking activity of relevant neuronal subtypes. This closed-loop approach represents a step forward in modulating stimulation parameters to achieve appropriate neural encoding in retinal prostheses, advancing control over RGC subtypes activated by electrical stimulation. PMID:29615857

RNA sequencing demonstrates large-scale temporal dysregulation of gene expression in stimulated macrophages derived from MHC-defined chicken haplotypes.

PubMed

Irizarry, Kristopher J L; Downs, Eileen; Bryden, Randall; Clark, Jory; Griggs, Lisa; Kopulos, Renee; Boettger, Cynthia M; Carr, Thomas J; Keeler, Calvin L; Collisson, Ellen; Drechsler, Yvonne

2017-01-01

Discovering genetic biomarkers associated with disease resistance and enhanced immunity is critical to developing advanced strategies for controlling viral and bacterial infections in different species. Macrophages, important cells of innate immunity, are directly involved in cellular interactions with pathogens, the release of cytokines activating other immune cells and antigen presentation to cells of the adaptive immune response. IFNγ is a potent activator of macrophages and increased production has been associated with disease resistance in several species. This study characterizes the molecular basis for dramatically different nitric oxide production and immune function between the B2 and the B19 haplotype chicken macrophages.A large-scale RNA sequencing approach was employed to sequence the RNA of purified macrophages from each haplotype group (B2 vs. B19) during differentiation and after stimulation. Our results demonstrate that a large number of genes exhibit divergent expression between B2 and B19 haplotype cells both prior and after stimulation. These differences in gene expression appear to be regulated by complex epigenetic mechanisms that need further investigation.

Optogenetic stimulation of cortex to map evoked whisker movements in awake head-restrained mice.

PubMed

Auffret, Matthieu; Ravano, Veronica L; Rossi, Giulia M C; Hankov, Nicolas; Petersen, Merissa F A; Petersen, Carl C H

2018-01-01

Whisker movements are used by rodents to touch objects in order to extract spatial and textural tactile information about their immediate surroundings. To understand the mechanisms of such active sensorimotor processing it is important to investigate whisker motor control. The activity of neurons in the neocortex affects whisker movements, but many aspects of the organization of cortical whisker motor control remain unknown. Here, we filmed whisker movements evoked by sequential optogenetic stimulation of different locations across the left dorsal sensorimotor cortex of awake head-restrained mice. Whisker movements were evoked by optogenetic stimulation of many regions in the dorsal sensorimotor cortex. Optogenetic stimulation of whisker sensory barrel cortex evoked retraction of the contralateral whisker after a short latency, and a delayed rhythmic protraction of the ipsilateral whisker. Optogenetic stimulation of frontal cortex evoked rhythmic bilateral whisker protraction with a longer latency compared to stimulation of sensory cortex. Compared to frontal cortex stimulation, larger amplitude bilateral rhythmic whisking in a less protracted position was evoked at a similar latency by stimulating a cortical region posterior to Bregma and close to the midline. These data suggest that whisker motor control might be broadly distributed across the dorsal mouse sensorimotor cortex. Future experiments must investigate the complex neuronal circuits connecting specific cell-types in various cortical regions with the whisker motor neurons located in the facial nucleus. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Angiotensin II stimulates calcium-dependent activation of c-Jun N-terminal kinase.

PubMed Central

Zohn, I E; Yu, H; Li, X; Cox, A D; Earp, H S

1995-01-01

In GN4 rat liver epithelial cells, angiotensin II (Ang II) and other agonists which activate phospholipase C stimulate tyrosine kinase activity in a calcium-dependent, protein kinase C (PKC)-independent manner. Since Ang II also produces a proliferative response in these cells, we investigated downstream signaling elements traditionally linked to growth control by tyrosine kinases. First, Ang II, like epidermal growth factor (EGF), stimulated AP-1 binding activity in a PKC-independent manner. Because increases in AP-1 can reflect induction of c-Jun and c-Fos, we examined the activity of the mitogen-activated protein (MAP) kinase family members Erk-1 and -2 and the c-Jun N-terminal kinase (JNK), which are known to influence c-Jun and c-Fos transcription. Ang II stimulated MAP kinase (MAPK) activity but only approximately 50% as effectively as EGF; again, these effects were independent of PKC. Ang II also produced a 50- to 200-fold activation of JNK in a PKC-independent manner. Unlike its smaller effect on MAPK, Ang II was approximately four- to sixfold more potent in activating JNK than EGF was. Although others had reported a lack of calcium ionophore-stimulated JNK activity in lymphocytes and several other cell lines, we examined the role of calcium in GN4 cells. The following results suggest that JNK activation in rat liver epithelial cells is at least partially Ca(2+) dependent: (i) norepinephrine and vasopressin hormones that increase inositol 1,4,5-triphosphate stimulated JNK; (ii) both thapsigargin, a compound that produces an intracellular Ca(2+) signal, and Ca(2+) ionophores stimulated a dramatic increase in JNK activity (up to 200-fold); (iii) extracellular Ca(2+) chelation with ethylene glycol tetraacetic acid (EGTA) inhibited JNK activation by ionophore and intracellular chelation with 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl-ester (BAPTA-AM) partially inhibited JNK activation by Ang II or thapsigargin; and (iv) JNK activation by Ang II was inhibited by pretreatment of cells with thapsigargin and EGTA, a procedure which depletes intracellular Ca(2+) stores. JNK activation following Ang II stimulation did not involve calmodulin; either W-7 nor calmidizolium, in concentrations sufficient to inhibit Ca(2+)/calmodulin-dependent kinase II, blocked JNK activation by Ang II. In contrast, genistein, in concentrations sufficient to inhibit Ca(2+)-dependent tyrosine phosphorylation, prevented Ang II and thapsigargin-induced JNK activation. In summary, in GN4 rat liver epithelial cells, Ang II stimulates JNK via a novel Ca(2+)-dependent pathway. The inhibition by genistein suggest that Ca(2+)-dependent tyrosine phosphorylation may modulate the JNK pathway in a cell type-specific manner, particularly in cells with a readily detectable Ca(2+)-regulated tyrosine kinase. PMID:7565768

Doxorubicin resistance mediated by cytoplasmic macrophage colony-stimulating factor is associated with switch from apoptosis to autophagic cell death in MCF-7 breast cancer cells

PubMed Central

Zhang, Mengxia; Zhang, Hailiang; Tang, Fan; Wang, Yuhua; Mo, Zhongcheng; Lei, Xiaoyong

2016-01-01

Macrophage colony-stimulating factor is a vital factor in maintaining the biological function of monocyte–macrophage lineage. It is expressed in many tumor tissues and cancer cells. Recent findings indicate that macrophage colony-stimulating factor might contribute to chemoresistance, but the precise mechanisms are unclear. This study was to explore the effect of macrophage colony-stimulating factor on doxorubicin resistance in MCF-7 breast cancer cells and the possible mechanism. In the study, the human breast cancer cells, MCF-7, were transfected with macrophage colony-stimulating factor. We document that cytoplasmic macrophage colony-stimulating factor induces doxorubicin resistance and inhibits apoptosis in MCF-7 cells. Further studies demonstrated that cytoplasmic macrophage colony-stimulating factor-mediated apoptosis inhibition was dependent on the activation of PI3K/Akt/Survivin pathway. More importantly, we found that macrophage colony-stimulating factor-induced autophagic cell death in doxorubicin-treated MCF-7 cells. Taken together, we show for the first time that macrophage colony-stimulating factor-induced doxorubicin resistance is associated with the changes in cell death response with defective apoptosis and promotion of autophagic cell death. PMID:27439542

E-Cadherin-Dependent Stimulation of Traction Force at Focal Adhesions via the Src and PI3K Signaling Pathways

PubMed Central

Jasaitis, Audrius; Estevez, Maruxa; Heysch, Julie; Ladoux, Benoit; Dufour, Sylvie

2012-01-01

The interplay between cadherin- and integrin-dependent signals controls cell behavior, but the precise mechanisms that regulate the strength of adhesion to the extracellular matrix remains poorly understood. We deposited cells expressing a defined repertoire of cadherins and integrins on fibronectin (FN)-coated polyacrylamide gels (FN-PAG) and on FN-coated pillars used as a micro-force sensor array (μFSA), and analyzed the functional relationship between these adhesion receptors to determine how it regulates cell traction force. We found that cadherin-mediated adhesion stimulated cell spreading on FN-PAG, and this was modulated by the substrate stiffness. We compared S180 cells with cells stably expressing different cadherins on μFSA and found that traction forces were stronger in cells expressing cadherins than in parental cells. E-cadherin-mediated contact and mechanical coupling between cells are required for this increase in cell-FN traction force, which was not observed in isolated cells, and required Src and PI3K activities. Traction forces were stronger in cells expressing type I cadherins than in cells expressing type II cadherins, which correlates with our previous observation of a higher intercellular adhesion strength developed by type I compared with type II cadherins. Our results reveal one of the mechanisms whereby molecular cross talk between cadherins and integrins upregulates traction forces at cell-FN adhesion sites, and thus provide additional insight into the molecular control of cell behavior. PMID:22853894

Gene expression profiling of peripheral blood mononuclear cells (PBMC) from Mycobacterium bovis infected cattle after in vitro antigenic stimulation with purified protein derivative of tuberculin (PPD).

PubMed

Meade, Kieran G; Gormley, Eamonn; Park, Stephen D E; Fitzsimons, Tara; Rosa, Guilherme J M; Costello, Eamon; Keane, Joseph; Coussens, Paul M; MacHugh, David E

2006-09-15

Microarray analysis of messenger RNA (mRNA) abundance was used to investigate the gene expression program of peripheral blood mononuclear cells (PBMC) from cattle infected with Mycobacterium bovis, the causative agent of bovine tuberculosis. An immunospecific bovine microarray platform (BOTL-4) with spot features representing 1336 genes was used for transcriptional profiling of PBMC from six M. bovis-infected cattle stimulated in vitro with bovine purified protein derivative of tuberculin (PPD-bovine). Cells were harvested at four time points (3 h, 6 h, 12 h and 24 h post-stimulation) and a split-plot design with pooled samples was used for the microarray experiment to compare gene expression between PPD-bovine stimulated PBMC and unstimulated controls for each time point. Statistical analyses of these data revealed 224 genes (approximately 17% of transcripts on the array) differentially expressed between stimulated and unstimulated PBMC across the 24 h time course (P<0.05). Of the 224 genes, 87 genes were significantly upregulated and 137 genes were significantly downregulated in M. bovis-infected PBMC stimulated with PPD-bovine across the 24 h time course. However, perturbation of the PBMC transcriptome was most apparent at time points 3 h and 12 h post-stimulation, with 81 and 84 genes differentially expressed, respectively. In addition, a more stringent statistical threshold (P<0.01) revealed 35 genes (approximately 3%) that were differentially expressed across the time course. Real-time quantitative reverse transcription PCR (qRT-PCR) of selected genes validated the microarray results and demonstrated a wide range of differentially expressed genes in PPD-bovine-, PPD-avian- and Concanavalin A (ConA) stimulated PBMC, including the interferon-gamma gene (IFNG), which was upregulated in PBMC stimulated with PPD-bovine (40-fold), PPD-avian (10-fold) and ConA (8-fold) after in vitro culture for 12 h. The pattern of expression of these genes in PPD-bovine stimulated PBMC provides the first description of an M. bovis-specific signature of infection that may provide insights into the molecular basis of the host response to infection. Although the present study was carried out with mixed PBMC cell populations, it will guide future studies to dissect immune cell-specific gene expression patterns in response to M. bovis infection.

TRANSCUTANEOUS CERVICAL VAGUS NERVE STIMULATION AMELIORATES ACUTE ISCHEMIC INJURY IN RATS

PubMed Central

Ay, Ilknur; Nasser, Rena; Simon, Bruce; Ay, Hakan

2016-01-01

Background Direct stimulation of the vagus nerve in the neck via surgically implanted electrodes is protective in animal models of stroke. We sought to determine the safety and efficacy of a non-invasive cervical VNS (nVNS) method using surface electrodes applied to the skin overlying the vagus nerve in the neck in a model of middle cerebral artery occlusion (MCAO). Methods nVNS was initiated variable times after MCAO hour in rats (n=33). Control animals received sham stimulation (n=33). Infarct volume and functional outcome were assessed on day 7. Brains were processed by immunohistochemistry for microglial activation and cytokine levels. The ability of nVNS to activate the nucleus tractus solitarius (NTS) was assessed using c-Fos immunohistochemistry. Results Infarct volume was 43.15±3.36 percent of the contralateral hemisphere (PCH) in control and 28.75±4.22 PCH in nVNS-treated animals (p<0.05). The effect of nVNS on infarct size was consistent when stimulation was initiated up to 4 hours after MCAO. There was no difference in heart rate and blood pressure between control and nVNS-treated animals. The number of c-Fos positive cells was 32.4±10.6 and 6.2±6.3 in the ipsilateral NTS (p<0.05) and 30.4±11.2 and 5.8±4.3 in the contralateral NTS (p<0.05) in nVNS-treated and control animals, respectively. nVNS reduced the number of Iba-1, CD68, and TNF-α positive cells and increased the number of HMGB1 positive cells. Conclusions nVNS inhibits ischemia-induced immune activation and reduces the extent of tissue injury and functional deficit in rats without causing cardiac or hemodynamic adverse effects when initiated up to 4 hours after MCAO. PMID:26723020

In vitro effects of docosahexaenoic and eicosapentaenoic acid on human meibomian gland epithelial cells.

PubMed

Hampel, Ulrike; Krüger, Magret; Kunnen, Carolina; Garreis, Fabian; Willcox, Mark; Paulsen, Friedrich

2015-11-01

To investigate the effect of ω-3 fatty acids on human meibomian gland epithelial cells (HMGECs, cell line) in vitro. HMGECs were stimulated with docosahexaenoic acid (DHA) or combinations with eicosapentaenoic acid (EPA) and acetyl sialic acid (ASA). Sudan III fat staining, viability and proliferation assays, electric cell-substrate impedance sensing, real-time PCR for gene expression of cyclooxygenase-2 and 15-lipoxygenase and ELISAs for resolvin D1 (RvD1), IFNγ, TNFα and IL-6 were applied. Lipid droplet accumulation and viability was increased by 100 μM DHA in the presence or absence of EPA in serum cultured HMGECs. In contrast, HMGECs cultured with DHA and EPA under serum-free conditions showed minimal lipid accumulation, decreased proliferation and viability. Normalized impedance was significantly reduced in serum-free cultured HMGECs when stimulated with DHA and EPA. HMGECs cultured in serum containing medium showed increased normalized impedance under DHA and EPA stimulation compared to DHA or EPA alone or controls. IL-6 and IFNγ were downregulated in HMGECs treated for 72 h with DHA and EPA. In general, TNFα, IFNγ and IL-6 levels were decreased after 72 h compared to 24 h in serum containing medium with or without DHA or EPA. The concentration of RvD1 was elevated 2-fold after DHA treatment. Cyclooxygenase-2 gene expression decreased compared to controls during DHA stimulation after 72 h. Treatment with DHA and ASA revealed a decreased 15-lipoxygenase gene expression which was reduced after three days of DHA incubation. DHA and EPA supplementation affected HMGECs in vitro and supported anti-inflammatory effects by influencing cytokine levels, decreasing COX-2 expression and increasing the production of RvD1. Copyright © 2015 Elsevier Ltd. All rights reserved.

The role of alveolar epithelial cells in initiating and shaping pulmonary immune responses: communication between innate and adaptive immune systems.

PubMed

Chuquimia, Olga D; Petursdottir, Dagbjort H; Rahman, Muhammad J; Hartl, Katharina; Singh, Mahavir; Fernández, Carmen

2012-01-01

Macrophages and dendritic cells have been recognized as key players in the defense against mycobacterial infection. However, more recently, other cells in the lungs such as alveolar epithelial cells (AEC) have been found to play important roles in the defense and pathogenesis of infection. In the present study we first compared AEC with pulmonary macrophages (PuM) isolated from mice in their ability to internalize and control Bacillus Calmette-Guérin (BCG) growth and their capacity as APCs. AEC were able to internalize and control bacterial growth as well as present antigen to primed T cells. Secondly, we compared both cell types in their capacity to secrete cytokines and chemokines upon stimulation with various molecules including mycobacterial products. Activated PuM and AEC displayed different patterns of secretion. Finally, we analyzed the profile of response of AEC to diverse stimuli. AEC responded to both microbial and internal stimuli exemplified by TLR ligands and IFNs, respectively. The response included synthesis by AEC of several factors, known to have various effects in other cells. Interestingly, TNF could stimulate the production of CCL2/MCP-1. Since MCP-1 plays a role in the recruitment of monocytes and macrophages to sites of infection and macrophages are the main producers of TNF, we speculate that both cell types can stimulate each other. Also, another cell-cell interaction was suggested when IFNs (produced mainly by lymphocytes) were able to induce expression of chemokines (IP-10 and RANTES) by AEC involved in the recruitment of circulating lymphocytes to areas of injury, inflammation, or viral infection. In the current paper we confirm previous data on the capacity of AEC regarding internalization of mycobacteria and their role as APC, and extend the knowledge of AEC as a multifunctional cell type by assessing the secretion of a broad array of factors in response to several different types of stimuli.

Impact of pulmonary arterial endothelial cells on duroquinone redox status.

PubMed

Merker, Marilyn P; Bongard, Robert D; Krenz, Gary S; Zhao, Hongtao; Fernandes, Viola S; Kalyanaraman, Balaraman; Hogg, Neil; Audi, Said H

2004-07-01

The study objective was to use pulmonary arterial endothelial cells to examine kinetics and mechanisms contributing to the disposition of the quinone 2,3,5,6-tetramethyl-1,4-benzoquinone (duroquinone, DQ) observed during passage through the pulmonary circulation. The approach was to add DQ, durohydroquinone (DQH2), or DQ with the cell membrane-impermeant oxidizing agent, ferricyanide (Fe(CN)6(3)-), to the cell medium, and to measure the medium concentrations of substrates and products over time. Studies were carried out under control conditions and with dicumarol, to inhibit NAD(P)H:quinone oxidoreductase 1 (NQO1), or cyanide, to inhibit mitochondrial electron transport. In control cells, DQH2 appears in the extracellular medium of cells incubated with DQ, and DQ appears when the cells are incubated with DQH2. Dicumarol blocked the appearance of DQH2 when DQ was added to the cell medium, and cyanide blocked the appearance of DQ when DQH2 was added to the cell medium, suggesting that the two electron reductase NQO1 dominates DQ reduction and mitochondrial electron transport complex III is the predominant route of DQH2 oxidation. In the presence of cyanide, the addition of DQ also resulted in an increased rate of appearance of DQH2 and stimulation of cyanide-insensitive oxygen consumption. As DQH2 does not autoxidize-comproportionate over the study time course, these observations suggest a cyanide-stimulated one-electron DQ reduction and durosemiquinone (DQ*-) autoxidation. The latter processes are apparently confined to the cell interior, as the cell membrane impermeant oxidant, ferricyanide, did not inhibit the DQ-stimulated cyanide-insensitive oxygen consumption. Thus, regardless of whether DQ is reduced via a one- or two-electron reduction pathway, the net effect in the extracellular medium is the appearance of DQH2. These endothelial redox functions and their apposition to the vessel lumen are consistent with the pulmonary endothelium being an important site of DQ reduction to DQH2 observed in the lungs. Copyright 2004 Elsevier Inc.

Differential cytokine production in clonal macrophage and T-cell lines cultured with bifidobacteria.

PubMed

Marin, M L; Lee, J H; Murtha, J; Ustunol, Z; Pestka, J J

1997-11-01

When used in commercial fermented dairy products, bifidobacteria may enhance immunity by stimulating cytokine secretion by leukocytes. To assess whether interaction between bifidobacteria and leukocytes promote cytokine production, we cultured RAW 264.7 cells (macrophage model) and EL-4.IL-2 thymoma cells (helper T-cell model) in the presence of 14 representative strains of heat-killed bifidobacteria. In unstimulated RAW 264.7 cells, all bifidobacteria induced pronounced increases (up to several hundred-fold) in the production of tumor necrosis factor-alpha compared with that of controls. Interleukin-6 production by unstimulated cells also increased significantly, but less than did tumor necrosis factor-alpha. Upon concurrent stimulation of RAW 264.7 cells with lipopolysaccharide, production of tumor necrosis factor-alpha and interleukin-6 were both enhanced between 1.5- to 5.8-fold and 4.7- to 7.9-fold, respectively, when cultured with 10(8) bifidobacteria/ml. In unstimulated EL-4.IL-2 cells, bifidobacteria had no effect on the production of interleukin-2 or interleukin-5. Upon stimulation of EL-4.IL-2 with phorbol-12-myristate-13-acetate, there were variable increases in interleukin-2 secretion (up to 2.4-fold for 10(6) Bifidobacterium Bf-1/ml) and interleukin-5 secretion (up to 4.6-fold for 10(8) B. adolescentis M101-4). The results indicated that, even when variations among strains were considered, direct interaction of most bifidobacteria with macrophages enhanced cytokine production, but the effects on cytokine production by the T-cell model were less marked. Interestingly, the 4 bifidobacteria strains used commercially for diary foods showed the greatest capacity for cytokine stimulation. The in vitro approaches employed here should be useful in future characterization of the effects of bifidobacteria on gastrointestinal and systemic immunity.

An optical neural interface: in vivo control of rodent motor cortex with integrated fiberoptic and optogenetic technology

NASA Astrophysics Data System (ADS)

Aravanis, Alexander M.; Wang, Li-Ping; Zhang, Feng; Meltzer, Leslie A.; Mogri, Murtaza Z.; Schneider, M. Bret; Deisseroth, Karl

2007-09-01

Neural interface technology has made enormous strides in recent years but stimulating electrodes remain incapable of reliably targeting specific cell types (e.g. excitatory or inhibitory neurons) within neural tissue. This obstacle has major scientific and clinical implications. For example, there is intense debate among physicians, neuroengineers and neuroscientists regarding the relevant cell types recruited during deep brain stimulation (DBS); moreover, many debilitating side effects of DBS likely result from lack of cell-type specificity. We describe here a novel optical neural interface technology that will allow neuroengineers to optically address specific cell types in vivo with millisecond temporal precision. Channelrhodopsin-2 (ChR2), an algal light-activated ion channel we developed for use in mammals, can give rise to safe, light-driven stimulation of CNS neurons on a timescale of milliseconds. Because ChR2 is genetically targetable, specific populations of neurons even sparsely embedded within intact circuitry can be stimulated with high temporal precision. Here we report the first in vivo behavioral demonstration of a functional optical neural interface (ONI) in intact animals, involving integrated fiberoptic and optogenetic technology. We developed a solid-state laser diode system that can be pulsed with millisecond precision, outputs 20 mW of power at 473 nm, and is coupled to a lightweight, flexible multimode optical fiber, ~200 µm in diameter. To capitalize on the unique advantages of this system, we specifically targeted ChR2 to excitatory cells in vivo with the CaMKIIα promoter. Under these conditions, the intensity of light exiting the fiber (~380 mW mm-2) was sufficient to drive excitatory neurons in vivo and control motor cortex function with behavioral output in intact rodents. No exogenous chemical cofactor was needed at any point, a crucial finding for in vivo work in large mammals. Achieving modulation of behavior with optical control of neuronal subtypes may give rise to fundamental network-level insights complementary to what electrode methodologies have taught us, and the emerging optogenetic toolkit may find application across a broad range of neuroscience, neuroengineering and clinical questions.

Gene expression analysis of pig cumulus-oocyte complexes stimulated in vitro with follicle stimulating hormone or epidermal growth factor-like peptides.

PubMed

Blaha, Milan; Nemcova, Lucie; Kepkova, Katerina Vodickova; Vodicka, Petr; Prochazka, Radek

2015-10-06

The gonadotropin-induced resumption of oocyte meiosis in preovulatory follicles is preceded by expression of epidermal growth factor (EGF)-like peptides, amphiregulin (AREG) and epiregulin (EREG), in mural granulosa and cumulus cells. Both the gonadotropins and the EGF-like peptides possess the capacity to stimulate resumption of oocyte meiosis in vitro via activation of a broad signaling network in cumulus cells. To better understand the rapid genomic actions of gonadotropins (FSH) and EGF-like peptides, we analyzed transcriptomes of cumulus cells at 3 h after their stimulation. We hybridized aRNA from cumulus cells to a pig oligonucleotide microarray and compared the transcriptomes of FSH- and AREG/EREG-stimulated cumulus cells with untreated control cells and vice versa. The identified over- and underexpressed genes were subjected to functional genomic analysis according to their molecular and cellular functions. The expression pattern of 50 selected genes with a known or potential function in ovarian development was verified by real-time qRT-PCR. Both FSH and AREG/EREG increased the expression of genes associated with regulation of cell proliferation, cell migration, blood coagulation and extracellular matrix remodeling. FSH alone induced the expression of genes involved in inflammatory response and in the response to reactive oxygen species. Moreover, FSH stimulated the expression of genes closely related to some ovulatory events either exclusively or significantly more than AREG/EREG (AREG, ADAMTS1, HAS2, TNFAIP6, PLAUR, PLAT, and HSD17B7). In contrast to AREG/EREG, FSH also increased the expression of genes coding for key transcription factors (CEBPB, FOS, ID1/3, and NR5A2), which may contribute to the differing expression profiles of FSH- and AREG/EREG-treated cumulus cells. The impact of FSH on cumulus cell gene transcription was higher than the impact of EGF-like factors in terms of the number of cell functions affected as well as the number of over- and underexpressed genes. Both FSH and EGF-like factors overexpressed genes involved in the post-ovulatory switch in steroidogenesis and tissue remodelling. However, FSH was remarkably more efficient in the up-regulation of several specific genes essential for ovulation of matured oocytes and also genes that been reported to play an important role in maturation of cumulus-enclosed oocytes in vitro.

Characterization and identification of a chymotryptic hydrolysate of alpha-lactalbumin stimulating cholecystokinin release in STC-1 cells.

PubMed

Catiau, Lucie; Delval-Dubois, Véronique; Guillochon, Didier; Nedjar-Arroume, Naïma

2011-11-01

Alpha-lactalbumin hydrolysate is of significant interest, due to its potential application as a source of bioactive peptides in nutraceutical and pharmaceutical domains. This study was focused on the cholecystokinin (CCK) family compounds which are small peptides involved in the satiety control. The action of chymotryptic hydrolysate of alpha-lactalbumin on cholecystokinin release from intestinal endocrine STC-1 cells was investigated. We demonstrated for the first time that a chymotryptic hydrolysate of alpha-lactalbumin was able to highly stimulate CCK-releasing activity from STC-1 cells. The peptidic hydrolysate was characterized by LC/MS and MS/MS, thus highlighting the presence of 11 fractions containing 21 peptides, each potentially having the desired activity.

A Programmable Optical Stimulator for the Drosophila Eye.

PubMed

Chen, Xinping; Leon-Salas, Walter D; Zigon, Taylor; Ready, Donald F; Weake, Vikki M

2017-10-01

A programmable optical stimulator for Drosophila eyes is presented. The target application of the stimulator is to induce retinal degeneration in fly photoreceptor cells by exposing them to light in a controlled manner. The goal of this work is to obtain a reproducible system for studying age-related changes in susceptibility to environmental ocular stress. The stimulator uses light emitting diodes and an embedded computer to control illuminance, color (blue or red) and duration in two independent chambers. Further, the stimulator is equipped with per-chamber light and temperature sensors and a fan to monitor light intensity and to control temperature. An ON/OFF temperature control implemented on the embedded computer keeps the temperature from reaching levels that will induce the heat shock stress response in the flies. A custom enclosure was fabricated to house the electronic components of the stimulator. The enclosure provides a light-impermeable environment that allows air flow and lets users easily load and unload fly vials. Characterization results show that the fabricated stimulator can produce light at illuminances ranging from 0 to 16000 lux and power density levels from 0 to 7.2 mW/cm 2 for blue light. For red light the maximum illuminance is 8000 lux which corresponds to a power density of 3.54 mW/cm 2 . The fans and the ON/OFF temperature control are able to keep the temperature inside the chambers below 28.17°C. Experiments with white-eye male flies were performed to assess the ability of the fabricated simulator to induce blue light-dependent retinal degeneration. Retinal degeneration is observed in flies exposed to 8 hours of blue light at 7949 lux. Flies in a control experiment with no light exposure show no retinal degeneration. Flies exposed to red light for the similar duration and light intensity (8 hours and 7994 lux) do not show retinal degeneration either. Hence, the fabricated stimulator can be used to create environmental ocular stress using blue light.

[Effect of trichostatin A on the osteogenic differentiation potential of periodontal ligament stem cells in inflammatory microenvironment induced by tumor necrosis factor-α stimulation].

PubMed

Wang, H; Chen, Q; Liu, W J; Yang, Z H; Li, D; Jin, F

2016-04-09

To compare the expression of histone deacetylase(HDAC)1-11 of human periodontal ligament stem cells(PDLSC)in normal and inflammatory microenvironments, and to investigate the effect of histone deacetylase inhibitor trichostatin A(TSA)on the osteogenic differentiation potential of PDLSC in inflammatory microenvironment induced by tumor necrosis factor-α(TNF-α)stimulation. PDLSC were isolated from periodontal ligament tissues obtained from the surgically extracted human teeth and cultured by single-colony selection. The expression of HDAC1-11 in cells with or without TNF-α(10 μg/L)stimulation was evaluated by quantitative real time-PCR(RT-PCR). The effect of TSA on cell proliferation was investigated by methyl thiazolyl tetrazolium(MTT)assay. The influence of TSA on osteogenic differentiation of PDLSC in inflammatory microenvironment with TNF-α stimulation was assessed by alizarin red staining, quantitative RT-PCR and Western blotting, respectively. The expression of HDAC in PDLSC with TNF-α stimulation was significantly higher than that in normal PDLSC(P<0.05)(except HDAC7, P=0.243). TSA had no significant effect on PDLSC proliferation at the concentration of 50 nmol/L(P=0.232). The alizarin red staining showed that PDLSC in TNF-α group generated less mineralized nodule than the control group, while the cell matrix mineralization in TSA group was improved obviously. TNF-α had an inhibitory effect on the expression of osteogenesis related genes, runt-related transcription factor-2(RUNX2)and alkaline phosphatase(ALP), with relative gene expression ratio(experimental/control)decreased to 0.17 ± 0.02 and 0.32 ± 0.03, while TSA could significantly increase the genes' expression to 0.67±0.03 and 0.89±0.02(P<0.01). Western blotting test showed that in TNF-α group the expression of osteogenesis related proteins was obviously reduced, and compared with the TNF-α group, TSA could significantly promote the expression of proteinsin inflammatory microenvironment. PDLSC in inflammatory microenvironment by TNF-α stimulation had a higher expression of HDAC than that in normal conditions. TSA, as a histone deacetylase inhibitor, could significantly promote the osteogenic differentiation potential of PDLSC in inflammatory microenvironment by suppressing HDAC.

Prostaglandin E1 inhibits endocytosis in the β-cell endocytosis.

PubMed

Zhao, Ying; Fang, Qinghua; Straub, Susanne G; Lindau, Manfred; Sharp, Geoffrey W G

2016-06-01

Prostaglandins inhibit insulin secretion in a manner similar to that of norepinephrine (NE) and somatostatin. As NE inhibits endocytosis as well as exocytosis, we have now examined the modulation of endocytosis by prostaglandin E1 (PGE1). Endocytosis following exocytosis was recorded by whole-cell patch clamp capacitance measurements in INS-832/13 cells. Prolonged depolarizing pulses producing a high level of Ca(2+) influx were used to stimulate maximal exocytosis and to deplete the readily releasable pool (RRP) of granules. This high Ca(2+) influx eliminates the inhibitory effect of PGE1 on exocytosis and allows specific characterization of the inhibitory effect of PGE1 on the subsequent compensatory endocytosis. After stimulating exocytosis, endocytosis was apparent under control conditions but was inhibited by PGE1 in a Pertussis toxin-sensitive (PTX)-insensitive manner. Dialyzing a synthetic peptide mimicking the C-terminus of the α-subunit of the heterotrimeric G-protein Gz into the cells blocked the inhibition of endocytosis by PGE1, whereas a control-randomized peptide was without effect. These results demonstrate that PGE1 inhibits endocytosis and Gz mediates the inhibition. © 2016 Society for Endocrinology.

A 3D magnetic tissue stretcher for remote mechanical control of embryonic stem cell differentiation.

PubMed

Du, Vicard; Luciani, Nathalie; Richard, Sophie; Mary, Gaëtan; Gay, Cyprien; Mazuel, François; Reffay, Myriam; Menasché, Philippe; Agbulut, Onnik; Wilhelm, Claire

2017-09-12

The ability to create a 3D tissue structure from individual cells and then to stimulate it at will is a major goal for both the biophysics and regenerative medicine communities. Here we show an integrated set of magnetic techniques that meet this challenge using embryonic stem cells (ESCs). We assessed the impact of magnetic nanoparticles internalization on ESCs viability, proliferation, pluripotency and differentiation profiles. We developed magnetic attractors capable of aggregating the cells remotely into a 3D embryoid body. This magnetic approach to embryoid body formation has no discernible impact on ESC differentiation pathways, as compared to the hanging drop method. It is also the base of the final magnetic device, composed of opposing magnetic attractors in order to form embryoid bodies in situ, then stretch them, and mechanically stimulate them at will. These stretched and cyclic purely mechanical stimulations were sufficient to drive ESCs differentiation towards the mesodermal cardiac pathway.The development of embryoid bodies that are responsive to external stimuli is of great interest in tissue engineering. Here, the authors culture embryonic stem cells with magnetic nanoparticles and show that the presence of magnetic fields could affect their aggregation and differentiation.

Temporal sampling, resetting, and adaptation orchestrate gradient sensing in sperm

PubMed Central

Alvarez, Luis; Seifert, Reinhard; Gregor, Ingo; Jäckle, Oliver; Beyermann, Michael; Krause, Eberhard

2012-01-01

Sperm, navigating in a chemical gradient, are exposed to a periodic stream of chemoattractant molecules. The periodic stimulation entrains Ca2+ oscillations that control looping steering responses. It is not known how sperm sample chemoattractant molecules during periodic stimulation and adjust their sensitivity. We report that sea urchin sperm sampled molecules for 0.2–0.6 s before a Ca2+ response was produced. Additional molecules delivered during a Ca2+ response reset the cell by causing a pronounced Ca2+ drop that terminated the response; this reset was followed by a new Ca2+ rise. After stimulation, sperm adapted their sensitivity following the Weber–Fechner law. Taking into account the single-molecule sensitivity, we estimate that sperm can register a minimal gradient of 0.8 fM/µm and be attracted from as far away as 4.7 mm. Many microorganisms sense stimulus gradients along periodic paths to translate a spatial distribution of the stimulus into a temporal pattern of the cell response. Orchestration of temporal sampling, resetting, and adaptation might control gradient sensing in such organisms as well. PMID:22986497

Temporal sampling, resetting, and adaptation orchestrate gradient sensing in sperm.

PubMed

Kashikar, Nachiket D; Alvarez, Luis; Seifert, Reinhard; Gregor, Ingo; Jäckle, Oliver; Beyermann, Michael; Krause, Eberhard; Kaupp, U Benjamin

2012-09-17

Sperm, navigating in a chemical gradient, are exposed to a periodic stream of chemoattractant molecules. The periodic stimulation entrains Ca(2+) oscillations that control looping steering responses. It is not known how sperm sample chemoattractant molecules during periodic stimulation and adjust their sensitivity. We report that sea urchin sperm sampled molecules for 0.2-0.6 s before a Ca(2+) response was produced. Additional molecules delivered during a Ca(2+) response reset the cell by causing a pronounced Ca(2+) drop that terminated the response; this reset was followed by a new Ca(2+) rise. After stimulation, sperm adapted their sensitivity following the Weber-Fechner law. Taking into account the single-molecule sensitivity, we estimate that sperm can register a minimal gradient of 0.8 fM/µm and be attracted from as far away as 4.7 mm. Many microorganisms sense stimulus gradients along periodic paths to translate a spatial distribution of the stimulus into a temporal pattern of the cell response. Orchestration of temporal sampling, resetting, and adaptation might control gradient sensing in such organisms as well.

Mechanism and control of fluid secretion.

PubMed

Oschman, J L

1977-01-01

Fluid secretion and reabsorption by a variety of plant and animal tissues appear to be accomplished by osmotic coupling between solute transport and water movement. The local osmosis model suggests that active accumulation of solutes within narrow folds at the cell surface may produce the local gradients that generate water flow. Both micropuncture techniques and electron-probe X-ray microanalysis have established that local osmotic gradients occur in absorptive epithelia, but they have not as yet been detected in secretory tissues.Hormonal control of secretion involves stimulation of solute pumps and adjustments of permeability to non-transported solutes. Since hormone receptors and pumps are often located on opposite surfaces of the cell, intracellular second messengers convey the secretory signal through cytoplasm. Much has been learned by study of insect tissues that are anatomically simple and that function for long periods in vitro. Aspects of hormone-receptor interaction have been explored, including the action of halluninogenic molecules. In insect salivary glands cyclic AMP appears to stimulate cation transport, while calcium increases anion permeability. The various second messengers probably interact with each other in complex feedback loops that stabilize the system and make it quickly responsive to hormone. Cyclic AMP may stimulate release of calcium from mitochondria. Unresolved is the way second messengers alter properties of the cell surface.

Phenotypic and functional characterization of T cells from patients with myasthenia gravis.

PubMed Central

Mokhtarian, F; Pino, M; Ofosu-Appiah, W; Grob, D

1990-01-01

A study of cell surface phenotypes of PBL of myasthenia gravis (MG) patients showed that their T cells had a significantly higher percentage of 4B4+ T cells (the helper/inducer subset) than age- and sex-matched controls. The PBL of MG patients proliferated significantly higher than those of normal subjects (NS) in response to the purified alpha chain of the acetylcholine receptor (AChR). Anti-AChR antibody was present in sera of 88% of MG and none of the NS. The PBL B cells from MG only, when cultured with autologous T cells and stimulated with either pokeweed mitogen (69%), or AChR-alpha chain (38%), secreted antibody to AChR-alpha chain, whereas T and B cells alone secreted no antibody. T cells from PBL of MG patients were more readily cloned than T cells of NS, by limiting dilution, in the presence of recombinant IL-2 and in the absence of AChR-alpha chain. About 50% of T cell clones from MG patients, compared to none from NS, proliferated to AChR-alpha chain. This response was HLA-DR restricted. MG T cell clones did not display significant cytotoxic activity, as compared to control T cell clones. Our results indicate that in MG, 4B4+ regulatory T cells play their role in the pathogenesis of MG, not by cytotoxicity, but more likely by their ability to stimulate specific antibody production by B cells. Images PMID:1979338

Control of cell interaction using quasi-monochromatic light with varying spatiotemporal coherence

NASA Astrophysics Data System (ADS)

Budagovsky, A. V.; Maslova, M. V.; Budagovskaya, O. N.; Budagovsky, I. A.

2017-02-01

By the example of plants, fungi and bacteria, we consider the possibility of controlling the interaction of cells, being in competitive, antagonistic, or parasitic relations. For this aim we used short-time irradiation (a few seconds or minutes) with the red (633 nm) quasi-monochromatic light having different spatiotemporal coherence. It is shown that the functional activity is mostly increased in the cells whose size does not exceed the coherence length and the correlation radius of the light field. Thus, in the case of cells essentially differing in size, it is possible to increase the activity of smaller cells, avoiding the stimulation of larger ones. For example, the radiation having relatively low coherence (Lcoh, rcor <= 10 μm) facilitates mainly the damage of large-size plant cells by pathogen fungi, while the exposure to light with less statistical regularity (Lcoh = 4 μm, rcor = 5 μm) inhibits the growth of the Fusarium microcera fungus, infected by the bacterium of the Pseudomonas species. The quasi-monochromatic radiation with sufficiently high spatiotemporal coherence stimulated all interacting species (bacteria, fungi, plants). In the considered biocenosis, the equilibrium was shifted towards the favour of organisms having the highest rate of cell division or the ones better using their adaptation potential.

The effect of SIRT1 protein knock down on PGC-1α acetylation during skeletal muscle contraction.

PubMed

Park, Dae Ryoung; Kim, Jeong Seok; Kim, Chang Keun

2014-03-01

The purpose of this study was to investigate the effect of Sirtuin 1 (SIRT1) and General control nonderepressible 5 (GCN5) knock down on peroxisome proliferator- activated receptor gamma coactivator 1-alpha (PGC-1α) deacetylation during electrical stimulated skeletal muscle contraction. Skeletal muscle primary cell were isolated from C57BL/6 mice gastrocnemius and transfected lentiviral SIRT1 and GCN5 shRNA. Knock downed muscle cell were stimulated by electrical stimulation (1Hz, 3min) and collected for PGC-1α deceatylation assays. Immunoprecipitation performed for PGC-1α deacetylation, acetyl-lysine level was measured. Our resulted showed SIRT1 knock down not influenced to PGC-1α deacetylation during electrical stimulation induced muscle contraction while GCN5 knock down decreased PGC-1α deacetylation significantly (p<0.05). This study can be concluded that GCN5 is a critical factor for muscle contraction induced PGC-1α deacetylation.

Truncated Hormone Inhibits Breast Tumor Blood Vessel Formation, Not Tumor Growth | Center for Cancer Research

Cancer.gov

The hormone prolactin (PRL) plays a critical role in normal breast development by stimulating the proliferation of mammary cells, the production of milk proteins, and the formation of new mammary blood vessels. Unfortunately, the same cell and vessel growth pathways controlled by PRL in normal cells also operate in breast cancer cells, and elevated plasma PRL is a risk factor for breast cancer, especially in post-menopausal women.

The development and validation of a LIPUS system with preliminary observations of ultrasonic effects on human adult stem cells.

PubMed

Marvel, Skylar; Okrasinski, Stan; Bernacki, Susan H; Loboa, Elizabeth; Dayton, Paul A

2010-09-01

To study the potential effects of low-intensity pulsed ultrasound (LIPUS) on cell response in vitro, the ability to alter LIPUS parameters is required. However, commercial LIPUS systems have very little control over parameter selection. In this study, a custom LIPUS system was designed and validated by exploring the effects of using different pulse repetition frequency (PRF) parameters on human adipose derived adult stem cells (hASCs) and bone marrow derived mesenchymal stem cells (hMSCs), two common stem cell sources for creating bone constructs in vitro. Changing the PRF was found to affect cellular response to LIPUS stimulation for both cell types. Proliferation of LIPUS-stimulated cells was found to decrease for hASCs by d 7 for all three groups compared with unstimulated control cells (P = 0.008, 0.011, 0.014 for 1 Hz, 100 Hz and 1 kHz PRF, respectively) and for hMSCs by d 14 (donor 1: P = 0.0005, 0.0002, 0.0003; donor 2: P = 0.0003, 0.0002, 0.0001; for PRFs of 1 Hz, 100 Hz, and 1 kHz, respectively). Additionally, LIPUS was shown to strongly accelerate osteogenic differentiation of hASCs based on amount of calcium accretion normalized by total DNA (P = 0.003, 0.001, 0.003, and 0.032 between control/100 Hz, control/1 kHz, 1 Hz/1 kHz, and 100 Hz/1 kHz pulse repetition frequencies, respectively). These findings promote the study of using LIPUS to induce osteogenic differentiation and further encourage the exploration of LIPUS parameter optimization. The custom LIPUS system was successfully designed to allow extreme parameter variation, specifically PRF, and encourages further studies.

Similar disturbances in B cell activity and regulatory T cell function in Henoch-Schonlein purpura and systemic lupus erythematosus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beale, M.G.; Nash, G.S.; Bertovich, M.J.

1982-01-01

The immunoglobulin synthesizing activities of peripheral mononuclear cells (MNC) from five patients with Henoch-Schonlein purpura (HSP) and eight patients with active systemic lupus erythematosus (SLE) were compared. Cumulative amounts of IgM, IgG, and IgA synthesized and secreted by unstimulated and PWM-stimulated patient cells over a 12-day period were determied in a solid-phase radioimmunoassay. In unstimulated control cultures mean rates of IgM, IgG, and IgA synthesis were less than 250 ng/ml. The synthetic activities of patient MNC were markedly increased. In HSP cultures IgA was the major immunoglobulin class produced (2810 x/divide 1.33 ng/ml) followed by IgG (1754 x/divide 1.32 ng/ml)more » and IgM (404 x/divide 1.16 ng/ml). In SLE cultures IgA and IgG syntheses were equally elevated (4427 x/divide 1.20 and 4438 x/divide 1.49 ng/ml, respectively) whereas IgM synthesis averaged 967 x/divide 1.66 ng/ml. PWM stimulation of pateient MNC caused a sharp decline in the synthesis of all three immunoglobulin classes. After T cell depletion B cell-enriched fractions from HSP and SLE patients maintained high levels of IgA and IgG synthesis that were inhibited by PWM and by normal allogeneic but not autologous T cells. In PWM-stimulted co-cultures, patient T cells nonspecifically suppressed the synthetic activities of autologous and control B cells. in contrast patient B cells achieved normal levels of immunoglobulin synthesis when cultured with control T cells plus PWM. In longitudinal studies patient B and T cell disturbances persisted despite clinical improvement.« less

Peripheral Blood Cells from Patients with Autoimmune Addison's Disease Poorly Respond to Interferons In Vitro, Despite Elevated Serum Levels of Interferon-Inducible Chemokines

PubMed Central

Bjånesøy, Trine; Hellesen, Alexander; Breivik, Lars; Bakke, Marit; Husebye, Eystein S.; Bratland, Eirik

2015-01-01

Autoimmune Addison's disease (AAD) is a disorder caused by an immunological attack on the adrenal cortex. The interferon (IFN)-inducible chemokine CXCL10 is elevated in serum of AAD patients, suggesting a peripheral IFN signature. However, CXCL10 can also be induced in adrenocortical cells stimulated with IFNs, cytokines, or microbial components. We therefore investigated whether peripheral blood mononuclear cells (PBMCs) from AAD patients display an enhanced propensity to produce CXCL10 and the related chemokine CXCL9, after stimulation with type I or II IFNs or the IFN inducer poly (I:C). Although serum levels of CXCL10 and CXCL9 were significantly elevated in patients compared with controls, IFN stimulated patient PBMC produced significantly less CXCL10/CXCL9 than control PBMC. Low CXCL10 production was not significantly associated with medication, disease duration, or comorbidities, but the low production of poly (I:C)-induced CXCL10 among patients was associated with an AAD risk allele in the phosphatase nonreceptor type 22 (PTPN22) gene. PBMC levels of total STAT1 and -2, and IFN-induced phosphorylated STAT1 and -2, were not significantly different between patients and controls. We conclude that PBMC from patients with AAD are deficient in their response to IFNs, and that the adrenal cortex itself may be responsible for the increased serum levels of CXCL10. PMID:25978633

Spaceflight alters immune cell function and distribution

NASA Technical Reports Server (NTRS)

Sonnenfeld, Gerald; Mandel, Adrian D.; Konstantinova, Irina V.; Berry, Wallace D.; Taylor, Gerald R.; Lesniak, A. T.; Fuchs, Boris B.; Rakhmilevich, Alexander L.

1992-01-01

Experiments are described which were performed onboard Cosmos 2044 to determine spaceflight effects on immunologically important cell function and distribution. Results indicate that bone marrow cells from flown and suspended rats exhibited a decreased response to a granulocyte/monocyte colony-stimulating factor compared with the bone marrow cells from control rats. Bone marrow cells showed an increase in the percentage of cells expressing markers for helper T-cells in the myelogenous population and increased percentages of anti-asialo granulocyte/monocyte-1-bearing interleulin-2 receptor bearing pan T- and helper T-cells in the lymphocytic population.

Immune modulation by genetic modification of dendritic cells with lentiviral vectors.

PubMed

Liechtenstein, Therese; Perez-Janices, Noemi; Bricogne, Christopher; Lanna, Alessio; Dufait, Inès; Goyvaerts, Cleo; Laranga, Roberta; Padella, Antonella; Arce, Frederick; Baratchian, Mehdi; Ramirez, Natalia; Lopez, Natalia; Kochan, Grazyna; Blanco-Luquin, Idoia; Guerrero-Setas, David; Breckpot, Karine; Escors, David

2013-09-01

Our work over the past eight years has focused on the use of HIV-1 lentiviral vectors (lentivectors) for the genetic modification of dendritic cells (DCs) to control their functions in immune modulation. DCs are key professional antigen presenting cells which regulate the activity of most effector immune cells, including T, B and NK cells. Their genetic modification provides the means for the development of targeted therapies towards cancer and autoimmune disease. We have been modulating with lentivectors the activity of intracellular signalling pathways and co-stimulation during antigen presentation to T cells, to fine-tune the type and strength of the immune response. In the course of our research, we have found unexpected results such as the surprising immunosuppressive role of anti-viral signalling pathways, and the close link between negative co-stimulation in the immunological synapse and T cell receptor trafficking. Here we review our major findings and put them into context with other published work. Copyright © 2013 Elsevier B.V. All rights reserved.

IL-27 Modulates Chemokine Production in TNF-α -Stimulated Human Oral Epithelial Cells.

PubMed

Hosokawa, Yoshitaka; Hosokawa, Ikuko; Ozaki, Kazumi; Matsuo, Takashi

2017-01-01

Interleukin-27 (IL-27) is a cytokine which belongs to the IL-12 family. However, the role of IL-27 in the pathogenesis of periodontal disease is uncertain. The aim of this study was to examine the effect of IL-27 on chemokine production in TNF-α-stimulated human oral epithelial cells (TR146). We measured chemokine production in TR146 by ELISA. We used western blot analysis to detect the phosphorylation levels of signal transduction molecules, including STAT1 and STAT3 in TR146. We used inhibitors to examine the role of STAT1 and STAT3 activation. IL-27 increased CXCR3 ligands production in TNF-α-stimulated TR146. Meanwhile, IL-27 suppressed IL-8 and CCL20 production induced by TNF-α. STAT1 phosphorylation level in IL-27 and TNF-α-stimulated TR146 was enhanced in comparison to TNF-α-stimulated TR146. STAT3 phosphorylation level in IL-27-treated TR146 did not change by TNF-α. Both STAT1 inhibitor and STAT3 inhibitor decreased CXCR3 ligands production. STAT1 inhibitor overrode the inhibitory effect of IL-27 on IL-8 and CCL20 production in TNF-α-stimulated TR146. Meanwhile, STAT3 inhibitor did not modulate IL-8 and CCL20 production. IL-27 might control leukocyte migration in periodontal lesion by modulating chemokine production from epithelial cells. © 2017 The Author(s). Published by S. Karger AG, Basel.

Thyrotropin Receptor Antibody (TRAb)-IgM Levels Are Markedly Higher Than TRAb-IgG Levels in Graves' Disease Patients and Controls, and TRAb-IgM Production Is Related to Epstein-Barr Virus Reactivation.

PubMed

Kumata, Keisuke; Nagata, Keiko; Matsushita, Michiko; Kuwamoto, Satoshi; Kato, Masako; Murakami, Ichiro; Fukata, Shuji; Hayashi, Kazuhiko

2016-10-01

Graves' disease is an autoimmune thyroid disorder that mainly presents as hyperthyroidism and is caused by thyrotropin receptor antibodies (TRAbs) that stimulate thyroid-stimulating hormone receptors. We previously reported that Graves' disease patients and healthy controls both had Epstein-Barr virus (EBV)-infected TRAb-positive B cells and the EBV-reactivated induction of these B cells in cultures may induce the production of TRAbs. In the present study, we quantified serum TRAb-IgG and TRAb-IgM levels in 34 Graves' disease patients and 15 controls using ELISA to elucidate the mechanisms underlying EBV-related antibody production. As expected, TRAb-IgG and TRAb-IgM levels were higher in Graves' disease patients than in controls; however, TRAb-IgM levels were significantly higher than those of TRAb-IgG levels, whereas total IgM levels were lower than total IgG levels. On the other hand, the enhanced production of TRAb-IgM was frequently observed in patients with EBV reactivation. These results are consistent with the fact that the percentage of autoreactive IgM B cells are higher than that of autoreactive IgG B cells, and support the EBV-related polyclonal B cell activation. It is necessary to clarify the biological characteristics of TRAb-IgM and the relationship between TRAb isotypes and the biology of Graves' disease.

Exercising Spatiotemporal Control of Cell Attachment with Optically Transparent Microelectrodes

PubMed Central

Shah, Sunny S.; Lee, Ji Youn; Verkhoturov, Stanislav; Tuleuova, Nazgul; Schweikert, Emile A.; Ramanculov, Erlan; Revzin, Alexander

2013-01-01

This paper describes a novel approach of controlling cell-surface interactions through an electrochemical “switching” of biointerfacial properties of optically transparent microelectrodes. The indium tin oxide (ITO) microelectrodes, fabricated on glass substrates, were modified with poly(ethylene glycol) (PEG) silane to make glass and ITO regions resistant to protein and cell adhesion. Cyclic voltammetry, with potassium ferricyanide serving as a redox reporter molecule, was used to monitor electron transfer across the electrolyte–ITO interface. PEG silane modification of ITO correlated with diminished electron transfer, judged by the disappearance of ferricyanide redox activity. Importantly, application of reductive potential (−1.4 V vs Ag/AgCl reference) corresponded with reappearance of typical ferricyanide redox peaks, thus pointing to desorption of an insulating PEG silane layer. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) characterization of the silanized ITO surfaces after electrical stimulation indicated complete removal of the silane layer. Significantly, electrical stimulation allowed to “switch” chosen electrodes from nonfouling to protein-adhesive while leaving other ITO and glass regions protected by a nonfouling PEG silane layer. The spatial and temporal control of biointerfacial properties afforded by our approach was utilized to micropattern proteins and cells and to construct micropatterned co-cultures. In the future, control of the biointerfacial properties afforded by this novel approach may allow the organization of multiple cell types into precise geometric configurations in order to create better in vitro mimics of cellular complexity of the native tissues. PMID:18512875

A single-cell pedigree analysis of alternative stochastic lymphocyte fates

PubMed Central

Hawkins, E. D.; Markham, J. F.; McGuinness, L. P.; Hodgkin, P. D.

2009-01-01

In contrast to most stimulated lymphocytes, B cells exposed to Toll-like receptor 9 ligands are nonself-adherent, allowing individual cells and families to be followed in vitro for up to 5 days. These B cells undergo phases typical of an adaptive response, dividing up to 6 times before losing the impetus for further growth and division and eventually dying by apoptosis. Using long-term microscopic imaging, accurate histories of individual lymphocyte fates were collected. Quantitative analysis of family relationships revealed that times to divide of siblings were strongly related but these correlations were progressively lost through consecutive divisions. A weaker, but significant, correlation was also found for death times among siblings. Division cessation is characterized by a loss of cell growth and the division in which this occurs is strongly inherited from the original founder cell and is related to the size this cell reaches before its first division. Thus, simple division-based dilution of factors synthesized during the first division may control the maximum division reached by stimulated cells. The stochastic distributions of times to divide, times to die, and divisions reached are also measured. Together, these results highlight the internal cellular mechanisms that control immune responses and provide a foundation for the development of new mathematical models that are correct at both single-cell and population levels. PMID:19633185

Reactivity of thyroid papillary carcinoma cells to thyroid stimulating hormone-dominated endocrine therapy

PubMed Central

Ma, Yuqin; Zhang, Xia; Wang, Yutao

2017-01-01

This study investigated the effect of thyroid stimulating hormone (TSH) on the proliferation of papillary thyroid carcinoma (PTC) cells and the therapeutic effect of levothyroxine sodium (TH). PTC cells (TPC-1) were cultured using 0.1, 1.0 and 10 U/l TSH and 10−2, 10−4 and 10−6 mol/l TH. After the appropriate concentration was screened, TPC-1 cells were further divided into control group, TSH group, TH group and TSH+TH group. The cell proliferation was detected via methyl thiazolyl tetrazolium (MTT) method, TPC-1 cell cycle was detected via flow cytometer, and the mRNA and protein expression of cyclin D1 were detected via real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Compared with control group, TSH significantly promoted the proliferation of TPC-1 cells (P<0.05 or P<0.01), obviously promoted the transition of TPC-1 cells from G1 phase to S phase (P<0.01) and remarkably increased the mRNA and protein expression of cyclin D1 (P<0.01); but TH had a significant inhibitory effect on these results of TSH (P<0.05 or P<0.01). TSH can promote the proliferation of PTC cells, and the appropriate complement of TH can inhibit its proliferation. PMID:29250166

Physiologically based microenvironment for in vitro neural differentiation of adipose-derived stem cells

PubMed Central

Graziano, Adriana Carol Eleonora; Avola, Rosanna; Perciavalle, Vincenzo; Nicoletti, Ferdinando; Cicala, Gianluca; Coco, Marinella; Cardile, Venera

2018-01-01

The limited capacity of nervous system to promote a spontaneous regeneration and the high rate of neurodegenerative diseases appearance are keys factors that stimulate researches both for defining the molecular mechanisms of pathophysiology and for evaluating putative strategies to induce neural tissue regeneration. In this latter aspect, the application of stem cells seems to be a promising approach, even if the control of their differentiation and the maintaining of a safe state of proliferation should be troubled. Here, we focus on adipose tissue-derived stem cells and we seek out the recent advances on the promotion of their neural differentiation, performing a critical integration of the basic biology and physiology of adipose tissue-derived stem cells with the functional modifications that the biophysical, biomechanical and biochemical microenvironment induces to cell phenotype. The pre-clinical studies showed that the neural differentiation by cell stimulation with growth factors benefits from the integration with biomaterials and biophysical interaction like microgravity. All these elements have been reported as furnisher of microenvironments with desirable biological, physical and mechanical properties. A critical review of current knowledge is here proposed, underscoring that a real advance toward a stable, safe and controllable adipose stem cells clinical application will derive from a synergic multidisciplinary approach that involves material engineer, basic cell biology, cell and tissue physiology. PMID:29588808

The exhausted CD4+CXCR5+ T cells involve the pathogenesis of human tuberculosis disease.

PubMed

Bosco, Munyemana Jean; Wei, Ming; Hou, Hongyan; Yu, Jing; Lin, Qun; Luo, Ying; Sun, Ziyong; Wang, Feng

2018-06-21

The CD4 + CXCR5 + T cells have been previously established. However, their decreased frequency during tuberculosis (TB) disease is partially understood. The aim of this study was to explore the depletion of CD4 + CXCR5 + T cells in human TB. The frequency and function of CD4 + CXCR5 + T cells were evaluated in active TB (ATB) patients and healthy control (HC) individuals. The function of CD4 + CXCR5 + T cells was determined after blockade of inhibitory receptors. The frequency of CD4 + CXCR5 + T cells was decreased in ATB patients. The expression of activation markers (HLA-DR and ICOS) and inhibitory receptors (Tim-3 and PD-1) on CD4 + CXCR5 + T cells was increased in ATB group. TB-specific antigen stimulation induced higher expression of inhibitory receptors than phytohemagglutinin stimulation in ATB group. In contrast, TB antigen stimulation did not induce a significantly increased expression of IL-21 and Ki-67 on CD4 + CXCR5 + T cells. However, blockade of inhibitory receptors Tim-3 and PD-1 not only increased the frequency of CD4 + CXCR5 + T cells, but also restored their proliferation and cytokine secretion potential. An increased expression of inhibitory receptors involves the depletion of CD4 + CXCR5 + T cells, and blockade of inhibitory receptors can restore the function of CD4 + CXCR5 + T cells in ATB patients. Copyright © 2018. Published by Elsevier Ltd.

L1 stimulation of human glioma cell motility correlates with FAK activation

PubMed Central

Yang, Muhua; Li, Yupei; Chilukuri, Kalyani; Brady, Owen A.; Boulos, Magdy I.; Kappes, John C.

2011-01-01

The neural adhesion/recognition protein L1 (L1CAM; CD171) has been shown or implicated to function in stimulation of cell motility in several cancer types, including high-grade gliomas. Our previous work demonstrated the expression and function of L1 protein in stimulation of cell motility in rat glioma cells. However, the mechanism of this stimulation is still unclear. This study further investigated the function of L1 and L1 proteolysis in human glioblastoma multiforme (GBM) cell migration and invasion, as well as the mechanism of this stimulation. L1 mRNA was found to be present in human T98G GBM cell line but not in U-118 MG grade III human glioma cell line. L1 protein expression, proteolysis, and release were found in T98G cells and human surgical GBM cells by Western blotting. Exosome-like vesicles released by T98G cells were purified and contained full-length L1. In a scratch assay, T98G cells that migrated into the denuded scratch area exhibited upregulation of ADAM10 protease expression coincident with loss of surface L1. GBM surgical specimen cells exhibited a similar loss of cell surface L1 when xenografted into the chick embryo brain. When lentivirally introduced shRNA was used to attenuate L1 expression, such T98G/shL1 cells exhibited significantly decreased cell motility by time lapse microscopy in our quantitative Super Scratch assay. These cells also showed a decrease in FAK activity and exhibited increased focal complexes. L1 binding integrins which activate FAK were found in T98G and U-118 MG cells. Addition of L1 ectodomain-containing media (1) rescued the decreased cell motility of T98G/shL1 cells and (2) increased cell motility of U-118 MG cells but (3) did not further increase T98G cell motility. Injection of L1-attenuated T98G/shL1 cells into embryonic chick brains resulted in the absence of detectable invasion compared to control cells which invaded brain tissue. These studies support a mechanism where glioma cells at the edge of a cell mass upregulate ADAM10 to proteolyze surface L1 and the resultant ectodomain increases human glioma cell migration and invasion by binding to integrin receptors, activating FAK, and increasing turnover of focal complexes. PMID:21373966

L1 stimulation of human glioma cell motility correlates with FAK activation.

PubMed

Yang, Muhua; Li, Yupei; Chilukuri, Kalyani; Brady, Owen A; Boulos, Magdy I; Kappes, John C; Galileo, Deni S

2011-10-01

The neural adhesion/recognition protein L1 (L1CAM; CD171) has been shown or implicated to function in stimulation of cell motility in several cancer types, including high-grade gliomas. Our previous work demonstrated the expression and function of L1 protein in stimulation of cell motility in rat glioma cells. However, the mechanism of this stimulation is still unclear. This study further investigated the function of L1 and L1 proteolysis in human glioblastoma multiforme (GBM) cell migration and invasion, as well as the mechanism of this stimulation. L1 mRNA was found to be present in human T98G GBM cell line but not in U-118 MG grade III human glioma cell line. L1 protein expression, proteolysis, and release were found in T98G cells and human surgical GBM cells by Western blotting. Exosome-like vesicles released by T98G cells were purified and contained full-length L1. In a scratch assay, T98G cells that migrated into the denuded scratch area exhibited upregulation of ADAM10 protease expression coincident with loss of surface L1. GBM surgical specimen cells exhibited a similar loss of cell surface L1 when xenografted into the chick embryo brain. When lentivirally introduced shRNA was used to attenuate L1 expression, such T98G/shL1 cells exhibited significantly decreased cell motility by time lapse microscopy in our quantitative Super Scratch assay. These cells also showed a decrease in FAK activity and exhibited increased focal complexes. L1 binding integrins which activate FAK were found in T98G and U-118 MG cells. Addition of L1 ectodomain-containing media (1) rescued the decreased cell motility of T98G/shL1 cells and (2) increased cell motility of U-118 MG cells but (3) did not further increase T98G cell motility. Injection of L1-attenuated T98G/shL1 cells into embryonic chick brains resulted in the absence of detectable invasion compared to control cells which invaded brain tissue. These studies support a mechanism where glioma cells at the edge of a cell mass upregulate ADAM10 to proteolyze surface L1 and the resultant ectodomain increases human glioma cell migration and invasion by binding to integrin receptors, activating FAK, and increasing turnover of focal complexes.

Fluorescence-Activated Cell Sorting-Based Analysis Reveals an Asymmetric Induction of Interferon-Stimulated Genes in Response to Seasonal Influenza A Virus

PubMed Central

von Recum-Knepper, Jessica; Sadewasser, Anne; Weinheimer, Viola K.

2015-01-01

ABSTRACT Influenza A virus (IAV) infection provokes an antiviral response involving the expression of type I and III interferons (IFN) and IFN-stimulated genes (ISGs) in infected cell cultures. However, the spatiotemporal dynamics of the IFN reaction are incompletely understood, as previous studies investigated mainly the population responses of virus-infected cultures, although substantial cell-to-cell variability has been documented. We devised a fluorescence-activated cell sorting-based assay to simultaneously quantify expression of viral antigens and ISGs, such as ISG15, MxA, and IFIT1, in IAV-infected cell cultures at the single-cell level. This approach revealed that seasonal IAV triggers an unexpected asymmetric response, as the major cell populations expressed either viral antigen or ISG, but rarely both. Further investigations identified a role of the viral NS1 protein in blocking ISG expression in infected cells, which surprisingly did not reduce paracrine IFN signaling to noninfected cells. Interestingly, viral ISG control was impaired in cultures infected with avian-origin IAV, including the H7N9 virus from eastern China. This phenotype was traced back to polymorphic NS1 amino acids known to be important for stable binding of the polyadenylation factor CPSF30 and concomitant suppression of host cell gene expression. Most significantly, mutation of two amino acids within the CPSF30 attachment site of NS1 from seasonal IAV diminished the strict control of ISG expression in infected cells and substantially attenuated virus replication. In conclusion, our approach revealed an asymmetric, NS1-dependent ISG induction in cultures infected with seasonal IAV, which appears to be essential for efficient virus propagation. IMPORTANCE Interferons are expressed by infected cells in response to IAV infection and play important roles in the antiviral immune response by inducing hundreds of interferon-stimulated genes (ISGs). Unlike many previous studies, we investigated the ISG response at the single-cell level, enabling novel insights into this virus-host interaction. Hence, cell cultures infected with seasonal IAV displayed an asymmetric ISG induction that was confined almost exclusively to noninfected cells. In comparison, ISG expression was observed in larger cell populations infected with avian-origin IAV, suggesting a more resolute antiviral response to these strains. Strict control of ISG expression by seasonal IAV was explained by the binding of the viral NS1 protein to the polyadenylation factor CPSF30, which reduces host cell gene expression. Mutational disruption of CPSF30 binding within NS1 concomitantly attenuated ISG control and replication of seasonal IAV, illustrating the importance of maintaining an asymmetric ISG response for efficient virus propagation. PMID:25903337

Embryonic stem cells in scaffold-free three-dimensional cell culture: osteogenic differentiation and bone generation.

PubMed

Handschel, Jörg; Naujoks, Christian; Depprich, Rita; Lammers, Lydia; Kübler, Norbert; Meyer, Ulrich; Wiesmann, Hans-Peter

2011-07-14

Extracorporeal formation of mineralized bone-like tissue is still an unsolved challenge in tissue engineering. Embryonic stem cells may open up new therapeutic options for the future and should be an interesting model for the analysis of fetal organogenesis. Here we describe a technique for culturing embryonic stem cells (ESCs) in the absence of artificial scaffolds which generated mineralized miromasses. Embryonic stem cells were harvested and osteogenic differentiation was stimulated by the addition of dexamethasone, ascorbic acid, and ß-glycerolphosphate (DAG). After three days of cultivation microspheres were formed. These spherical three-dimensional cell units showed a peripheral zone consisting of densely packed cell layers surrounded by minerals that were embedded in the extracellular matrix. Alizarine red staining confirmed evidence of mineralization after 10 days of DAG stimulation in the stimulated but not in the control group. Transmission electron microscopy demonstrated scorching crystallites and collagenous fibrils as early indication of bone formation. These extracellular structures resembled hydroxyl apatite-like crystals as demonstrated by distinct diffraction patterns using electron diffraction analysis. The micromass culture technique is an appropriate model to form three-dimensional bone-like micro-units without the need for an underlying scaffold. Further studies will have to show whether the technique is applicable also to pluripotent stem cells of different origin. © 2011 Handschel et al; licensee BioMed Central Ltd.

Ex Vivo Induction of Multiple Myeloma-specific Immune Responses by Monocyte-derived Dendritic Cells Following Stimulation by Whole-tumor Antigen of Autologous Myeloma Cells.

PubMed

Vasileiou, Spyridoula; Baltadakis, Ioannis; Delimpasi, Sosanna; Karatza, Maria-Helena; Liapis, Konstantinos; Garofalaki, Maria; Tziotziou, Eirini; Poulopoulou, Zoe; Karakasis, Dimitri; Harhalakis, Nicholas

2017-09-01

The introduction of novel agents has significantly expanded treatment options for multiple myeloma (MM), albeit long-term disease control cannot be achieved in the majority of patients. Vaccination with MM antigen-loaded dendritic cells (DCs) represents an alternative strategy that is currently being explored. The aim of this study was to assess the immunogenic potential of ex vivo-generated monocyte-derived DCs (moDCs), following stimulation with the whole-antigen array of autologous myeloma cells (AMC). MoDCs were loaded with antigens of myeloma cells by 2 different methods: phagocytosis of apoptotic bodies from γ-irradiated AMC, or transfection with AMC total RNA by square-wave electroporation. Twenty patients with MM were enrolled in the study. Following stimulation and maturation, moDCs were tested for their capacity to induce T-helper 1 and cytotoxic T lymphocyte responses in vitro. Both strategies were effective in the induction of myeloma-specific cytotoxic T lymphocyte and T-helper 1 cells, as demonstrated by cytotoxicity and ELISpot assays. On the whole, T-cell responses were observed in 18 cases by either method of DC pulsing. We conclude that both whole-tumor antigen approaches are efficient in priming autologous antimyeloma T-cell responses and warrant further study aiming at the development of individualized DC vaccines for MM patients.

Miniaturized neural sensing and optogenetic stimulation system for behavioral studies in the rat

NASA Astrophysics Data System (ADS)

Kim, Min Hyuck; Nam, Ilho; Ryu, Youngki; Wellman, Laurie W.; Sanford, Larry D.; Yoon, Hargsoon

2015-04-01

Real time sensing of localized electrophysiological and neurochemical signals associated with spontaneous and evoked neural activity is critically important for understanding neural networks in the brain. Our goal is to enhance the functionality and flexibility of a neural sensing and stimulation system for the observation of brain activity that will enable better understanding from the level of individual cells to that of global structures. We have thus developed a miniaturized electronic system for in-vivo neurotransmitter sensing and optogenetic stimulation amenable to behavioral studies in the rat. The system contains a potentiostat, a data acquisition unit, a control unit, and a wireless data transfer unit. For the potentiostat, we applied embedded op-amps to build single potential amperometry for electrochemical sensing of dopamine. A light emitting diode is controlled by a microcontroller and pulse width modulation utilized to control optogenetic stimulation within a sub-millisecond level. In addition, this proto-typed electronic system contains a Bluetooth module for wireless data communication. In the future, an application-specific integrated circuit (ASIC) will be designed for further miniaturization of the system.

Pulsed DC Electric Field–Induced Differentiation of Cortical Neural Precursor Cells

PubMed Central

Chang, Hui-Fang; Lee, Ying-Shan; Tang, Tang K.; Cheng, Ji-Yen

2016-01-01

We report the differentiation of neural stem and progenitor cells solely induced by direct current (DC) pulses stimulation. Neural stem and progenitor cells in the adult mammalian brain are promising candidates for the development of therapeutic neuroregeneration strategies. The differentiation of neural stem and progenitor cells depends on various in vivo environmental factors, such as nerve growth factor and endogenous EF. In this study, we demonstrated that the morphologic and phenotypic changes of mouse neural stem and progenitor cells (mNPCs) could be induced solely by exposure to square-wave DC pulses (magnitude 300 mV/mm at frequency of 100-Hz). The DC pulse stimulation was conducted for 48 h, and the morphologic changes of mNPCs were monitored continuously. The length of primary processes and the amount of branching significantly increased after stimulation by DC pulses for 48 h. After DC pulse treatment, the mNPCs differentiated into neurons, astrocytes, and oligodendrocytes simultaneously in stem cell maintenance medium. Our results suggest that simple DC pulse treatment could control the fate of NPCs. With further studies, DC pulses may be applied to manipulate NPC differentiation and may be used for the development of therapeutic strategies that employ NPCs to treat nervous system disorders. PMID:27352251

Adrenergic-agonist-induced Ca2+ fluxes in rat parotid cells are not Na+-dependent.

PubMed Central

Helman, J; Roth, G S; Baum, B J

1985-01-01

We investigated the hypothesis that extracellular Na+ is required for the rapid mobilization of Ca2+ by rat parotid cells after adrenergic stimulation. When Na+ salts in the media were osmotically replaced with either choline chloride (+atropine) or sucrose, efflux of 45Ca2+ from preloaded cells, caused by 10 microM-(-)-adrenaline, was unchanged. Similarly adrenaline stimulated 45Ca2+ uptake into cells under nonsteady-state conditions in the presence or absence of Na+. Monensin, a Na+ ionophore, was able to elicit a modest increase in 45Ca2+ efflux, compared with controls. Studies of net 45Ca2+ flux, performed under near-steady-state conditions, showed that adrenaline caused net 45Ca2+ accumulation, whereas monensin caused net 45Ca2+ release. The effect of monensin required the presence of Na+ in the incubation medium. Both 1 mM-LaCl3 and 0.1 mM-D-600 prevented adrenaline-stimulated 45Ca2+ uptake into cells, but had no effect on monensin-induced changes. We conclude that (1) the rapid mobilization of Ca2+ by adrenergic agonists seen in rat parotid cells does not require a Na+out greater than Na+in gradient and (2) the nature of the monensin effect is quite different from the adrenergic-agonist-induced response. PMID:2413840

Activation of human B cells by phosphorothioate oligodeoxynucleotides.

PubMed Central

Liang, H; Nishioka, Y; Reich, C F; Pisetsky, D S; Lipsky, P E

1996-01-01

To investigate the potential of DNA to elicit immune responses in man, we examined the capacity of a variety of oligodeoxynucleotides (ODNs) to stimulate highly purified T cell-depleted human peripheral blood B cells. Among 47 ODNs of various sequences tested, 12 phosphorothioate oligodeoxynucleotides (sODNs) induced marked B cell proliferation and Ig production. IL-2 augmented both proliferation and production of IgM, IgG, and IgA, as well as IgM anti-DNA antibodies, but was not necessary for B cell stimulation. Similarly, T cells enhanced stimulation, but were not necessary for B cell activation. After stimulation with the active sODNs, more than 95% of B cells expressed CD25 and CD86. In addition, B cells stimulated with sODNs expressed all six of the major immunoglobulin VH gene families. These results indicate that the human B cell response to sODN is polyclonal. Active sODN coupled to Sepharose beads stimulated B cells as effectively as the free sODN, suggesting that stimulation resulted from engagement of surface receptors. These data indicate that sODNs can directly induce polyclonal activation of human B cells in a T cell-independent manner by engaging as yet unknown B cell surface receptors. PMID:8787674

Hypergravity Stimulates the Extracellular Matrix/Integrin-Signaling Axis and Proliferation in Primary Osteoblasts

NASA Technical Reports Server (NTRS)

Parra, M.; Vercoutere, W.; Roden, C.; Banerjee, I.; Krauser, W.; Holton, E.; Searby, N.; Globus, R.; Almeida, E.

2003-01-01

We set out to determine the molecular mechanisms involved in the proliferative response of primary rat osteoblasts to mechanical stimulation using cell culture centrifugation as a model for hypergravity. We hypothesized that this proliferative response is mediated by specific integrin/Extracellular Matrix (ECM) interactions. To investigate this question we developed a cell culture centrifuge and an automated system that performs cell fixation during hypergravity loading. We generated expression vectors for various focal adhesion and cytoskeletal proteins fused to GFP or dsRed and visualized these structures in transfected (or infected) osteoblasts. The actin cytoskeleton was also visualized using rhodamine-phalloidin staining and Focal Adhesion Kinase (FAK) levels were assessed biochemically. We observed that a 24 hour exposure to 50-g stimulated proliferation compared to the 1-g control when cells were plated on fibronectin, collagen Type I , and collagen Type IV, but not on uncoated tissue culture plastic surfaces. This proliferative response was greatest for osteoblasts grown on fibronectin (2-fold increase over 1-g control) and collagen Type I (1.4 fold increase over 1-g control), suggesting that specific matrices and integrins are involved in the signaling pathways required for proliferation. Exposing osteoblasts grown on different matrices to 10-g or 25-g showed that effects on proliferation depended on both matrix type and loading level. We found that osteoblasts exposed to a short pulse of hypergravity during adhesion spread further and had more GFP-FAK containing focal adhesions compared to their 1-g controls. While overall levels of FAK did not change, more FAK was in the active (phosphorylated) form under hypergravity than in the 1-g controls. Cytoskeletal F-actin organization into filaments was also more prominent after brief exposures to hypergravity during the first five minutes of adhesion. These results suggest that specific integrins sense hypergravity and activate distinct matrix-dependent FAK signaling pathways that can enhance proliferation. Our results also imply that brief exposures to hypergravity accelerate cell adhesion and spreading processes via the focal adhesion-signaling axis. These results support the role of the ECM/integrin-signaling axis in osteoblast response to hypergravity loading.

Co-incubation of PMN and CaCo-2 cells modulates inflammatory potential.

PubMed

Schaefer, M B; Schaefer, C A; Hecker, M; Morty, R E; Witzenrath, M; Seeger, W; Mayer, K

2017-05-20

Polymorphonuclear granulocytes (PMN) are activated in inflammatory reactions. Intestinal epithelial cells are relevant for maintaining the intestinal barrier. We examined interactions of PMN and intestinal epithelial cell-like CaCo-2 cells to elucidate their regulation of inflammatory signalling and the impact of cyclooxygenase (COX), nitric oxide (NO) and platelet-activating factor (PAF). Human PMN and CaCo-2 cells, separately and in co-incubation, were stimulated with the calcium ionophore A23187 or with N-Formyl-methionyl-leucyl-phenylalanin (fMLP) that activates PMN only. Human neutrophil elastase (HNE) and respiratory Burst were measured. To evaluate the modulation of inflammatory crosstalk we applied inhibitors of COX (acetyl salicylic acid; ASA), NO-synthase (N-monomethyl-L-arginin; L-NMMA), and the PAF-receptor (WEB2086). Unstimulated, co-incubation of CaCo-2 cells and PMN led to significantly reduced Burst and elevated HNE as compared to PMN. After stimulation with A23187, co-incubation resulted in an inhibition of Burst and HNE. Using fMLP co-incubation failed to modulate Burst but increased HNE. Without stimulation, all three inhibitors abolished the effect of co-incubation on Burst but did not change HNE.  ASA partly prevented modulation of Burst L-NMMA and WEB2086 did not change Burst but abolished mitigation of HNE. Without stimulation, co-incubation reduced Burst and elevated HNE. Activation of PMN and CaCo-2 cells by fMLP as compared to A23187 resulted in a completely different pattern of Burst and HNE, possibly due to single vs. dual cell activation. Anti-inflammatory effect of co-incubation might in part be due to due to COX-signalling governing Burst whereas NO- and PAF-dependent signalling seemed to control HNE release.

Rapid and Localized Mechanical Stimulation and Adhesion Assay: TRPM7 Involvement in Calcium Signaling and Cell Adhesion

PubMed Central

Nishitani, Wagner Shin; Alencar, Adriano Mesquita; Wang, Yingxiao

2015-01-01

A cell mechanical stimulation equipment, based on cell substrate deformation, and a more sensitive method for measuring adhesion of cells were developed. A probe, precisely positioned close to the cell, was capable of a vertical localized mechanical stimulation with a temporal frequency of 207 Hz, and strain magnitude of 50%. This setup was characterized and used to probe the response of Human Umbilical Endothelial Vein Cells (HUVECs) in terms of calcium signaling. The intracellular calcium ion concentration was measured by the genetically encoded Cameleon biosensor, with the Transient Receptor Potential cation channel, subfamily M, member 7 (TRPM7) expression inhibited. As TRPM7 expression also regulates adhesion, a relatively simple method for measuring adhesion of cells was also developed, tested and used to study the effect of adhesion alone. Three adhesion conditions of HUVECs on polyacrylamide gel dishes were compared. In the first condition, the substrate is fully treated with Sulfo-SANPAH crosslinking and fibronectin. The other two conditions had increasingly reduced adhesion: partially treated (only coated with fibronectin, with no use of Sulfo-SANPAH, at 5% of the normal amount) and non-treated polyacrylamide gels. The cells showed adhesion and calcium response to the mechanical stimulation correlated to the degree of gel treatment: highest for fully treated gels and lowest for non-treated ones. TRPM7 inhibition by siRNA on HUVECs caused an increase in adhesion relative to control (no siRNA treatment) and non-targeting siRNA, but a decrease to 80% of calcium response relative to non-targeting siRNA which confirms the important role of TRPM7 in mechanotransduction despite the increase in adhesion. PMID:25946314

Anti-fibrotic characteristics of Vγ9+ γδ T cells in systemic sclerosis.

PubMed

Markovits, Noa; Bendersky, Anna; Loebstein, Ronen; Brusel, Marina; Kessler, Efrat; Bank, Ilan

2016-01-01

γδ T cells of the Vγ9Vδ2 subtype secrete anti-fibrotic cytokines upon isopentenyl pyrophosphate (IPP) stimulation. In this study, we sought to compare IPP and Zoledronate, an up-regulator of IPP, effects on proliferation and cytokine secretion of Vγ9+ T cells from systemic sclerosis (SSc) patients and healthy controls (HCs). We also examined the effect of IPP-triggered peripheral blood mononuclear cells (PBMC) on fibroblast procolla- gen secretion. PBMC from SSc patients and HCs were stimulated by increasing concentrations of Zoledronate, with or without IPP, and Vγ9+ T cell percentages were calculated using FACScan analysis. Subsequently, PBMC were cultured with IPP or toxic shock syndrome toxin-1 (TSST-1), and contents of the anti-fibrotic cytokines tumour necrosis factor (TNF)-α and interferon (IFN)-γ were measured by ELISA kits. Finally, supernatants of IPP-triggered Vγ9+ T cells from SSc patients were added to fibroblast cultures, and relative intensities of procollagen α1 chains were determined by densinometry. Higher concentrations of Zoledronate were required for maximal proliferation of Vγ9+ T cells in 9 SSc patients compared to 9 HCs, irrespective of exogenous IPP. When compared to stimulation by TSST-1, a non-Vγ9+ selective reagent, secretion of the anti-fibrotic cytokines TNF-α and IFN-γ in response to IPP was relatively diminished in SSc but not in HCs. Reduction of procollagen secretion by fibroblasts cultured with supernatants of IPP-stimulated PBMC was observed only in some SSc patients. Activated Vγ9+ T cells could act as anti-fibrotic mediators in SSc, although decreased responsiveness to IPP may play a role in the pathological fibrosis of this disease.

Caffeine, dibutyryl cyclic-AMP and heparin affect the chemotactic and phagocytotic activities of neutrophils for boar sperm in vitro.

PubMed

Li, J-C; Yamaguchi, S; Kondo, Y; Funahashi, H

2011-04-15

The objective was to examine the effects of caffeine, dibutyryl cyclic AMP, and heparin on the chemotaxis and/or phagocytosis of PMNs for porcine sperm. The chemotactic activity of PMNs, determined in a blind well chamber, increased (P < 0.05) when fresh serum was added to the medium (control containing BSA, 1109.5 cells/mm(2) vs serum, 1226.3 cells/mm(2)), regardless of the presence of sperm (control, 1121.1 cells/mm(2) vs serum, 1245.8 cells/mm(2)), whereas heat-inactivated serum did not affect activity (without sperm, 1099.4 cells/mm(2) and with sperm, 1132.6 cells/mm(2)). Regardless of live and dead sperm and of the origin of PMNs (boars vs sows), the phagocytotic activity of PMNs, as determined by co-culture of PMNs with sperm for 60 min, increased (P < 0.05) in the presence of fresh serum containing active complement (46.7 and 43.0%, respectively), but stimulation was decreased (P < 0.05) when 1 mM or higher concentrations of caffeine was added to the medium (from 40.7 to 20.8-31.6%). The origin of PMNs (sows vs boars) did not significantly affect phagocytotic activity. The percentage of PMNs that phagocytized polystyrene latex beads decreased when 2 mM caffeine was added to the medium containing porcine serum (from 43.7 to 21.5%). Serum-stimulated chemotactic activity of PMNs (1089.9 cells/mm(2)) was also reduced (P < 0.05) with 2 mM caffeine (942.5 cells/mm(2)). Furthermore, dibutyryl cAMP at ≥ 0.1 mM or heparin at ≥ 100 μg/mL decreased phagocytotic activity, in a concentration-dependent manner (P < 0.05). Supplementation of PMNs with heparin at 100 or 500 μg/mL decreased (P < 0.05) chemotactic activity in the presence of serum (from 1137.1 cells/mm(2) to 1008.8-1026.3 cells/mm(2)). We inferred that opsonization in the presence of active complement stimulated phagocytotic and chemotactic activities of PMNs, whereas supplementation with caffeine and dibutyryl cAMP (which could be associated with the intracellular cAMP level of PMNs) or adding heparin decreased serum-stimulated phagocytotic and chemotactic activities. Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.

Betacellulin ameliorates hyperglycemia in obese diabetic db/db mice.

PubMed

Oh, Yoon Sin; Shin, Seungjin; Li, Hui Ying; Park, Eun-Young; Lee, Song Mi; Choi, Cheol Soo; Lim, Yong; Jung, Hye Seung; Jun, Hee-Sook

2015-11-01

We found that administration of a recombinant adenovirus (rAd) expressing betacellulin (BTC) into obese diabetic db/db mice ameliorated hyperglycemia. Exogenous glucose clearance was significantly improved, and serum insulin levels were significantly higher in rAd-BTC-treated mice than rAd-β-gal-treated control mice. rAd-BTC treatment increased insulin/bromodeoxyuridine double-positive cells in the islets, and islets from rAd-BTC-treated mice exhibited a significant increase in the level of G1-S phase-related cyclins as compared with control mice. In addition, BTC treatment increased messenger RNA (mRNA) and protein levels of these cyclins and cyclin-dependent kinases in MIN-6 cells. BTC treatment induced intracellular Ca(2+) levels through phospholipase C-γ1 activation, and upregulated calcineurin B (CnB1) levels as well as calcineurin activity. Upregulation of CnB1 by BTC treatment was observed in isolated islet cells from db/db mice. When treated with CnB1 small interfering RNA (siRNA) in MIN-6 cells and isolated islets, induction of cell cycle regulators by BTC treatment was blocked and consequently reduced BTC-induced cell viability. As well as BTC's effects on cell survival and insulin secretion, our findings demonstrate a novel pathway by which BTC controls beta-cell regeneration in the obese diabetic condition by regulating G1-S phase cell cycle expression through Ca(2+) signaling pathways. Administration of BTC to db/db mice results in amelioration of hyperglycemia. BTC stimulates beta-cell proliferation in db/db mice. Ca(2+) signaling was involved in BTC-induced beta-cell proliferation. BTC has an anti-apoptotic effect and potentiates glucose-stimulated insulin secretion.

Electrical stimulation as a biomimicry tool for regulating muscle cell behavior

PubMed Central

Ahadian, Samad; Ostrovidov, Serge; Hosseini, Vahid; Kaji, Hirokazu; Ramalingam, Murugan; Bae, Hojae; Khademhosseini, Ali

2013-01-01

There is a growing need to understand muscle cell behaviors and to engineer muscle tissues to replace defective tissues in the body. Despite a long history of the clinical use of electric fields for muscle tissues in vivo, electrical stimulation (ES) has recently gained significant attention as a powerful tool for regulating muscle cell behaviors in vitro. ES aims to mimic the electrical environment of electroactive muscle cells (e.g., cardiac or skeletal muscle cells) by helping to regulate cell-cell and cell-extracellular matrix (ECM) interactions. As a result, it can be used to enhance the alignment and differentiation of skeletal or cardiac muscle cells and to aid in engineering of functional muscle tissues. Additionally, ES can be used to control and monitor force generation and electrophysiological activity of muscle tissues for bio-actuation and drug-screening applications in a simple, high-throughput, and reproducible manner. In this review paper, we briefly describe the importance of ES in regulating muscle cell behaviors in vitro, as well as the major challenges and prospective potential associated with ES in the context of muscle tissue engineering. PMID:23823664

Electrical stimulation as a biomimicry tool for regulating muscle cell behavior.

PubMed

Ahadian, Samad; Ostrovidov, Serge; Hosseini, Vahid; Kaji, Hirokazu; Ramalingam, Murugan; Bae, Hojae; Khademhosseini, Ali

2013-01-01

There is a growing need to understand muscle cell behaviors and to engineer muscle tissues to replace defective tissues in the body. Despite a long history of the clinical use of electric fields for muscle tissues in vivo, electrical stimulation (ES) has recently gained significant attention as a powerful tool for regulating muscle cell behaviors in vitro. ES aims to mimic the electrical environment of electroactive muscle cells (e.g., cardiac or skeletal muscle cells) by helping to regulate cell-cell and cell-extracellular matrix (ECM) interactions. As a result, it can be used to enhance the alignment and differentiation of skeletal or cardiac muscle cells and to aid in engineering of functional muscle tissues. Additionally, ES can be used to control and monitor force generation and electrophysiological activity of muscle tissues for bio-actuation and drug-screening applications in a simple, high-throughput, and reproducible manner. In this review paper, we briefly describe the importance of ES in regulating muscle cell behaviors in vitro, as well as the major challenges and prospective potential associated with ES in the context of muscle tissue engineering.

T cell exit from quiescence and differentiation into Th2 cells depend on Raptor-mTORC1-mediated metabolic programming

PubMed Central

Yang, Kai; Shrestha, Sharad; Zeng, Hu; Karmaus, Peer W.F.; Neale, Geoffrey; Vogel, Peter; Guertin, David A.; Lamb, Richard F.; Chi, Hongbo

2014-01-01

SUMMARY Naïve T cells respond to antigen stimulation by exiting from quiescence and initiating clonal expansion and functional differentiation, but the control mechanism is elusive. Here we describe that Raptor-mTORC1-dependent metabolic programming is a central determinant of this transitional process. Loss of Raptor abrogated T cell priming and Th2 cell differentiation, although Raptor function is less important for continuous proliferation of actively cycling cells. mTORC1 coordinated multiple metabolic programs in T cells including glycolysis, lipid synthesis and oxidative phosphorylation to mediate antigen-triggered exit from quiescence. mTORC1 further linked glucose metabolism to the initiation of Th2 cell differentiation by orchestrating cytokine receptor expression and cytokine responsiveness. Activation of Raptor-mTORC1 integrated T cell receptor and CD28 co-stimulatory signals in antigen-stimulated T cells. Our studies identify a Raptor-mTORC1-dependent pathway linking signal-dependent metabolic reprogramming to quiescence exit, and this in turn coordinates lymphocyte activation and fate decisions in adaptive immunity. PMID:24315998

The non-classical MAP kinase ERK3 controls T cell activation.

PubMed

Marquis, Miriam; Boulet, Salix; Mathien, Simon; Rousseau, Justine; Thébault, Paméla; Daudelin, Jean-François; Rooney, Julie; Turgeon, Benjamin; Beauchamp, Claudine; Meloche, Sylvain; Labrecque, Nathalie

2014-01-01

The classical mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 are activated upon stimulation of cells with a broad range of extracellular signals (including antigens) allowing cellular responses to occur. ERK3 is an atypical member of the MAPK family with highest homology to ERK1/2. Therefore, we evaluated the role of ERK3 in mature T cell response. Mouse resting T cells do not transcribe ERK3 but its expression is induced in both CD4⁺ and CD8⁺ T cells following T cell receptor (TCR)-induced T cell activation. This induction of ERK3 expression in T lymphocytes requires activation of the classical MAPK ERK1 and ERK2. Moreover, ERK3 protein is phosphorylated and associates with MK5 in activated primary T cells. We show that ERK3-deficient T cells have a decreased proliferation rate and are impaired in cytokine secretion following in vitro stimulation with low dose of anti-CD3 antibodies. Our findings identify the atypical MAPK ERK3 as a new and important regulator of TCR-induced T cell activation.

The Non-Classical MAP Kinase ERK3 Controls T Cell Activation

PubMed Central

Mathien, Simon; Rousseau, Justine; Thébault, Paméla; Daudelin, Jean-François; Rooney, Julie; Turgeon, Benjamin; Beauchamp, Claudine; Meloche, Sylvain; Labrecque, Nathalie

2014-01-01

The classical mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 are activated upon stimulation of cells with a broad range of extracellular signals (including antigens) allowing cellular responses to occur. ERK3 is an atypical member of the MAPK family with highest homology to ERK1/2. Therefore, we evaluated the role of ERK3 in mature T cell response. Mouse resting T cells do not transcribe ERK3 but its expression is induced in both CD4+ and CD8+ T cells following T cell receptor (TCR)-induced T cell activation. This induction of ERK3 expression in T lymphocytes requires activation of the classical MAPK ERK1 and ERK2. Moreover, ERK3 protein is phosphorylated and associates with MK5 in activated primary T cells. We show that ERK3-deficient T cells have a decreased proliferation rate and are impaired in cytokine secretion following in vitro stimulation with low dose of anti-CD3 antibodies. Our findings identify the atypical MAPK ERK3 as a new and important regulator of TCR-induced T cell activation. PMID:24475167

Milk-induced eczema is associated with the expansion of T cells expressing cutaneous lymphocyte antigen.

PubMed Central

Abernathy-Carver, K J; Sampson, H A; Picker, L J; Leung, D Y

1995-01-01

The extravasation of T cells at sites of inflammation is critically dependent on the activity of homing receptors (HR) involved in endothelial cell recognition and binding. Two such HR (the cutaneous lymphocyte antigen [CLA] and L-selectin) have been shown to be selectively involved in T cell migration to skin and peripheral lymph nodes, respectively. This study was designed to assess the relationship between the organ specificity of an allergic reaction to food and the expression of HR on T cells activated in vitro by the relevant food allergen. Peripheral blood mononuclear cells were isolated from seven milk allergic children with a history of eczema when exposed to milk. All patients had a positive prick skin test and double-blind placebo-controlled food challenge to milk. 10 children with either allergic eosinophilic gastroenteritis or milk-induced enterocolitis and 8 nonatopic adults served as controls. Five-parameter flow cytometry using monoclonal antibodies was used for detection of the specific HR on freshly isolated T cells versus T cell blasts induced by a 6-d incubation with casein, as compared with Candida albicans. After in vitro stimulation with casein, but not C. albicans, patients with milk allergy and atopic dermatitis had a significantly greater percentage of CLA+ T cells (P < 0.01) than controls with milk-induced enterocolitis, allergic eosinophilic gastroenteritis, or nonatopic healthy controls. In contrast, the percentage of L-selectin-expressing T cells did not differ significantly between these groups. These data suggest that after casein stimulation allergic patients with milk-induced skin disease have an expanded population of CLA+ T cells, as compared with nonatopics or allergic patients without skin involvement. We postulate that heterogeneity in the regulation of HR expression on antigen-specific T cells may play a role in determining sites of involvement in tissue-directed allergic responses. Images PMID:7532192

Milk-induced eczema is associated with the expansion of T cells expressing cutaneous lymphocyte antigen.

PubMed

Abernathy-Carver, K J; Sampson, H A; Picker, L J; Leung, D Y

1995-02-01

The extravasation of T cells at sites of inflammation is critically dependent on the activity of homing receptors (HR) involved in endothelial cell recognition and binding. Two such HR (the cutaneous lymphocyte antigen [CLA] and L-selectin) have been shown to be selectively involved in T cell migration to skin and peripheral lymph nodes, respectively. This study was designed to assess the relationship between the organ specificity of an allergic reaction to food and the expression of HR on T cells activated in vitro by the relevant food allergen. Peripheral blood mononuclear cells were isolated from seven milk allergic children with a history of eczema when exposed to milk. All patients had a positive prick skin test and double-blind placebo-controlled food challenge to milk. 10 children with either allergic eosinophilic gastroenteritis or milk-induced enterocolitis and 8 nonatopic adults served as controls. Five-parameter flow cytometry using monoclonal antibodies was used for detection of the specific HR on freshly isolated T cells versus T cell blasts induced by a 6-d incubation with casein, as compared with Candida albicans. After in vitro stimulation with casein, but not C. albicans, patients with milk allergy and atopic dermatitis had a significantly greater percentage of CLA+ T cells (P < 0.01) than controls with milk-induced enterocolitis, allergic eosinophilic gastroenteritis, or nonatopic healthy controls. In contrast, the percentage of L-selectin-expressing T cells did not differ significantly between these groups. These data suggest that after casein stimulation allergic patients with milk-induced skin disease have an expanded population of CLA+ T cells, as compared with nonatopics or allergic patients without skin involvement. We postulate that heterogeneity in the regulation of HR expression on antigen-specific T cells may play a role in determining sites of involvement in tissue-directed allergic responses.

External stimulation strength controls actin response dynamics in Dictyostelium cells

NASA Astrophysics Data System (ADS)

Hsu, Hsin-Fang; Westendorf, Christian; Tarantola, Marco; Zykov, Vladimir; Bodenschatz, Eberhard; Beta, Carsten

2015-03-01

Self-sustained oscillation and the resonance frequency of the cytoskeletal actin polymerization/depolymerization have recently been observed in Dictyostelium, a model system for studying chemotaxis. Here we report that the resonance frequency is not constant but rather varies with the strength of external stimuli. To understand the underlying mechanism, we analyzed the polymerization and depolymerization time at different levels of external stimulation. We found that polymerization time is independent of external stimuli but the depolymerization time is prolonged as the stimulation increases. These observations can be successfully reproduced in the frame work of our time delayed differential equation model.

Estrogen biosynthesis in THP1 cells is regulated by promoter switching of the aromatase (CYP19) gene.

PubMed

Shozu, M; Zhao, Y; Simpson, E R

1997-12-01

The expression of aromatase, the enzyme responsible for estrogen biosynthesis, has been studied in THP-1 cells of human mononuclear leukemic origin, which exhibit high rates of aromatase activity. These cells have the capacity to differentiate in the presence of vitamin D into cells with osteoclast-like properties. Differentiated cells displayed higher rates of aromatase than undifferentiated cells, and, in both cases, activity was stimulated 10- to 20-fold by dexamethasone. Phorbol esters also increased aromatase activity, but the effect was the same in differentiated as in undifferentiated cells. In a similar fashion to adipose stromal cells, serum potentiated the response to dexamethasone but had no effect on phorbol ester-stimulated activity. By contrast to its action in adipose stromal cells, (Bu)2cAMP markedly inhibited aromatase activity of THP-1 cells, as did factors whose actions are mediated by cAMP, such as PTH and PTH-related peptide. This was true of control cells, as well as of dexamethasone- and phorbol ester-stimulated cells. Previously we have shown that type 1 cytokines as well as tumor necrosis factor-alpha stimulate aromatase activity of adipose stromal cells in the presence of dexamethasone. By contrast, interleukin-6, interleukin-11, and leukemia-inhibitory factor had no effect on aromatase activity of THP-1 cells, whereas tumor necrosis factor-alpha, oncostatin M, and platelet-derived growth factor were slightly inhibitory of aromatase activity. Exon-specific Southern analysis of rapid amplification of cDNA ends-amplified transcripts was employed to examine the distribution of the various 5'-termini of aromatase transcripts. In the control group, most of the clones contained transcripts specific for the proximal promoter II, whereas in dexamethasone-treated cells, most transcripts contained exon I.4. In the phorbol ester-treated cells, a broader spectrum of transcripts was present, with equal proportions of I.4, II, and I.3-containing clones. Additionally, one clone containing a new sequence, exon I.6, was found. This was shown to be located about 1 kb upstream of exon II. By contrast, all clones from cells treated with (Bu)2cAMP contained promoter II-specific sequences. In addition to these transcripts, two clones in the library from the dexamethasone-treated cells contained the sequence previously defined as the brain-specific sequence, 1f. In one of these, the 1f sequence was fused downstream of exon I.4, indicative that its expression likely employed promoter I.4. These results point to similarities and important differences between aromatase expression in THP-1 cells and other cells such as adipose stromal cells, indicative of unique regulatory pathways governing aromatase expression in these cells.

Effects of acupuncture on peripheral T lymphocyte subpopulation and amounts of cerebral catecholamines in mice.

PubMed

Okumura, M; Toriizuka, K; Iijima, K; Haruyama, K; Ishino, S; Cyong, J C

1999-01-01

The aim of this study was to investigate the effects of acupuncture on peripheral lymphocyte subpopulations and cerebral catecholamines. In order to examine the effects of acupuncture, two experiments were performed. Experiment 1: Eighteen female mice (strain; C57BL/6) at the age of 7 weeks were divided three groups, (a) sham operated (control; n=6), (b) ovariectomized (OVX; n=6), and (c) ovariectomized and stimulated by subcutaneous needles on acupuncture point, Shenshu (BL23) at the both sides of the back for 20 days (OVX+Acu; n=6). These animals were sacrificed at 20 days after needle insertion, and the splenic lymphoid cells were examined by two-color flow cytometry, using monoclonal antibodies (mAb) to the cell surface antigens, CD3, CD4, CD8a and NK1.1 (CD56). In the ovariectomized (OVX) group, the peripheral CD4/CD8 ratio was significantly increased and the ratio of natural killer (NK) cells (CD3-NK1.1+; CD3 negative, NK1.1 positive) to T lymphocytes was decreased compared to the sham control group. In the ovariectomized with needle insertion (OVX+Acu) group, the CD4/CD8 ratio was reduced, but the NK cells ratio was not changed compared to the OVX group. Experiment 2: To investigate the acute effects of subcutaneous needle insertion, male C57BL/6 mice (7 weeks old) were used (n=6, each group). The acupuncture points Shen-shu (BL23) on the backs of the male mice were also stimulated by subcutaneous needles for 3 and 7 days. As a result, the CD4/CD8 ratio was significantly decreased at day 3 and day 7, compared to the control group. On the other hand the NK cells ratio and activated T-cells were increased at day 7. The mitogenic activities in the splenic lymphocytes were also increased by acupuncture stimulation at day 3. Catecholamine contents in the hippocampus were measured by high performance liquid chromatography with the electro-chemical detector (ECD-HPLC) method. No significant change was observed in either dopamine contents or norepinephrine; however, dopamine metabolite, homovanillic acid (HVA) and DOPAC (3,4-dihydroxyphenylacetic acid) were increased at day 3. The study suggests that acupuncture has effects on peripheral lymphocyte subpopulations and may modulate mitogenic activity. In addition, acupuncture may stimulate dopamine turnover.

NK cells of the oldest seniors represent constant and resistant to stimulation high expression of cellular protective proteins SIRT1 and HSP70.

PubMed

Kaszubowska, Lucyna; Foerster, Jerzy; Kaczor, Jan Jacek; Schetz, Daria; Ślebioda, Tomasz Jerzy; Kmieć, Zbigniew

2018-01-01

Natural killer cells (NK cells) are cytotoxic lymphocytes of innate immunity that reveal some immunoregulatory properties, however, their role in the process of ageing is not completely understood. The study aimed to analyze the expression of proteins involved in cellular stress response: sirtuin 1 (SIRT1), heat shock protein 70 (HSP70) and manganese superoxide dismutase (SOD2) in human NK cells with reference to the process of ageing. Non-stimulated and stimulated with IL-2, LPS or PMA with ionomycin cells originated from peripheral blood samples of: seniors aged over 85 ('the oldest'; n  = 25; 88.5 ± 0.5 years, mean ± SEM), seniors aged under 85 ('the old'; n  = 30; 75.6 ± 0.9 years) and the young ( n  = 31; 20.9 ± 0.3 years). The relationships between the levels of expression of cellular protective proteins in the studied population were also analyzed. The concentrations of carbonyl groups and 8-isoprostanes, markers of oxidative stress, in both stimulated and non-stimulated cultured NK cells were measured to assess the level of the oxidative stress in the cells. The oldest seniors varied from the other age groups by significantly higher expression of SIRT1 and HSP70 both in non-stimulated and stimulated NK cells. These cells also appeared to be resistant to further stimulations with IL-2, LPS or PMA with ionomycin. Highly positive correlations between SIRT1 and intracellular HSP70 in both stimulated and non-stimulated NK cells were observed. SOD2 presented low expression in non-stimulated cells, whereas its sensitivity to stimulation increased with age of donors. High positive correlations between SOD2 and surface HSP70 were observed. We found that the markers of oxidative stress in NK cells did not change with ageing. The oldest seniors revealed well developed adaptive stress response in NK cells with increased, constant levels of SIRT1 and intracellular HSP70. They presented also very high positive correlations between expression of these cellular protective proteins both in stimulated and non-stimulated cells. These phenomena may contribute to the long lifespan of this group of elderly. Interestingly, in NK cells SOD2 revealed a distinct role in cellular stress response since it showed sensitivity to stimulation increasing with age of participants. These observations provide novel data concerning the role of NK cells in the process of ageing.

Differential monocyte responses to TLR ligands in children with autism spectrum disorders

PubMed Central

Enstrom, Amanda M; Onore, Charity E; Van de Water, Judy A; Ashwood, Paul

2010-01-01

Autism spectrum disorders (ASD) are characterized by impairment in social interactions, communication deficits, and restricted repetitive interests and behaviors. Recent evidence has suggested that impairments of innate immunity may play an important role in ASD. To test this hypothesis, we isolated peripheral blood monocytes from 17 children with ASD and 16 age-matched typically developing (TD) controls and stimulated these cell cultures in vitro with distinct toll-like receptors (TLR) ligands: TLR2 (lipoteichoic acid; LTA), TLR3 (poly I:C), TLR4 (lipopolysaccharide; LPS), TLR5 (flagellin) and TLR9 (CpG-B). Supernatants were harvested from the cell cultures and pro-inflammatory cytokine responses for IL-1β, IL-6, IL-8, TNFα, MCP-1, and GM-CSF were determined by multiplex Luminex analysis. After in vitro challenge with TLR ligands, differential cytokine responses were observed in monocyte cultures from children with ASD compared with TD control children. In particular, there was a marked increase in pro-inflammatory IL-1β, IL-6 and TNFα responses following TLR2, and IL-1β response following TLR4 stimulation in monocyte cultures from children with ASD (p<0.04). Conversely, following TLR9 stimulation there was a decrease in IL-1β, IL-6, GM-CSF and TNFα responses in monocyte cell cultures from children with ASD compared with controls (p<0.05). These data indicate that, monocyte cultures from children with ASD are more responsive to signaling via select TLRs. As monocytes are key regulators of the immune response, dysfunction in the response of these cells could result in long-term immune alterations in children with ASD that may lead to the development of adverse neuroimmune interactions and could play a role in the pathophysiology observed in ASD. PMID:19666104

Differential monocyte responses to TLR ligands in children with autism spectrum disorders.

PubMed

Enstrom, Amanda M; Onore, Charity E; Van de Water, Judy A; Ashwood, Paul

2010-01-01

Autism spectrum disorders (ASD) are characterized by impairment in social interactions, communication deficits, and restricted repetitive interests and behaviors. Recent evidence has suggested that impairments of innate immunity may play an important role in ASD. To test this hypothesis, we isolated peripheral blood monocytes from 17 children with ASD and 16 age-matched typically developing (TD) controls and stimulated these cell cultures in vitro with distinct toll-like receptors (TLR) ligands: TLR 2 (lipoteichoic acid; LTA), TLR 3 (poly I:C), TLR 4 (lipopolysaccharide; LPS), TLR 5 (flagellin), and TLR 9 (CpG-B). Supernatants were harvested from the cell cultures and pro-inflammatory cytokine responses for IL-1beta, IL-6, IL-8, TNFalpha, MCP-1, and GM-CSF were determined by multiplex Luminex analysis. After in vitro challenge with TLR ligands, differential cytokine responses were observed in monocyte cultures from children with ASD compared with TD control children. In particular, there was a marked increase in pro-inflammatory IL-1beta, IL-6, and TNFalpha responses following TLR 2, and IL-1beta response following TLR 4 stimulation in monocyte cultures from children with ASD (p<0.04). Conversely, following TLR 9 stimulation there was a decrease in IL-1beta, IL-6, GM-CSF, and TNFalpha responses in monocyte cell cultures from children with ASD compared with controls (p<0.05). These data indicate that, monocyte cultures from children with ASD are more responsive to signaling via select TLRs. As monocytes are key regulators of the immune response, dysfunction in the response of these cells could result in long-term immune alterations in children with ASD that may lead to the development of adverse neuroimmune interactions and could play a role in the pathophysiology observed in ASD.

A systems biology approach to the analysis of subset-specific responses to lipopolysaccharide in dendritic cells.

PubMed

Hancock, David G; Shklovskaya, Elena; Guy, Thomas V; Falsafi, Reza; Fjell, Chris D; Ritchie, William; Hancock, Robert E W; Fazekas de St Groth, Barbara

2014-01-01

Dendritic cells (DCs) are critical for regulating CD4 and CD8 T cell immunity, controlling Th1, Th2, and Th17 commitment, generating inducible Tregs, and mediating tolerance. It is believed that distinct DC subsets have evolved to control these different immune outcomes. However, how DC subsets mount different responses to inflammatory and/or tolerogenic signals in order to accomplish their divergent functions remains unclear. Lipopolysaccharide (LPS) provides an excellent model for investigating responses in closely related splenic DC subsets, as all subsets express the LPS receptor TLR4 and respond to LPS in vitro. However, previous studies of the LPS-induced DC transcriptome have been performed only on mixed DC populations. Moreover, comparisons of the in vivo response of two closely related DC subsets to LPS stimulation have not been reported in the literature to date. We compared the transcriptomes of murine splenic CD8 and CD11b DC subsets after in vivo LPS stimulation, using RNA-Seq and systems biology approaches. We identified subset-specific gene signatures, which included multiple functional immune mediators unique to each subset. To explain the observed subset-specific differences, we used a network analysis approach. While both DC subsets used a conserved set of transcription factors and major signalling pathways, the subsets showed differential regulation of sets of genes that 'fine-tune' the network Hubs expressed in common. We propose a model in which signalling through common pathway components is 'fine-tuned' by transcriptional control of subset-specific modulators, thus allowing for distinct functional outcomes in closely related DC subsets. We extend this analysis to comparable datasets from the literature and confirm that our model can account for cell subset-specific responses to LPS stimulation in multiple subpopulations in mouse and man.

Intracellular signaling required for CCL25-stimulated T cell adhesion mediated by the integrin alpha4beta1.

PubMed

Parmo-Cabañas, Marisa; García-Bernal, David; García-Verdugo, Rosa; Kremer, Leonor; Márquez, Gabriel; Teixidó, Joaquin

2007-08-01

The alpha4beta1 integrin is expressed on thymocytes and mediates cell attachment to its ligands CS-1/fibronectin (CS-1/FN) and VCAM-1 in the thymus. The chemokine CCL25 is highly expressed in the thymus, where it binds to its receptor CCR9 on thymocytes promoting migration and activation. We show here that alpha4beta1 and CCR9 are coexpressed mainly on double- and single-positive thymocytes and that CCL25 strongly stimulates CD4(+)CD8(+) and CD4(+)CD8(-) adhesion to CS-1/FN and VCAM-1. CCL25 rapidly activated the GTPases Rac and Rap1 on thymocytes, and this activation was required for stimulation of adhesion, as detected using the CCR9(+)/alpha4beta1(+) human T cell line Molt-4. To study the role on CCL25-stimulated adhesion of the Rac downstream effector Wiskott-Aldrich syndrome protein family verproline-homologous protein 2 (WAVE2) as well as of Rap1-GTP-interacting proteins, regulator of adhesion and cell polarization enriched in lymphoid tissues (RAPL) and Rap1-GTP-interacting adapter molecule (RIAM), we knocked down their expression and tested transfectant attachment to alpha4beta1 ligands. We found that WAVE2 and RAPL but not RIAM were required for efficient triggering by CCL25 of T cell adhesion to CS-1/FN and VCAM-1. Although Rac and Rap1 activation was required during early steps of T cell adhesion stimulated by CCL25, WAVE2 was needed for the development of actin-dependent T cell spreading subsequent to adhesion strengthening but not during initial alpha4beta1-ligand interactions. These results suggest that regulation by CCL25 of adhesion of thymocyte subpopulations mediated by alpha4beta1 could contribute to control their trafficking in the thymus during maturation, and identify Rac-WAVE2 and Rap1-RAPL as pathways whose activation is required in inside-out signaling, leading to stimulated adhesion.

Implication of matrix metalloproteinases 2 and 9 in ceramide 1-phosphate-stimulated macrophage migration.

PubMed

Ordoñez, Marta; Rivera, Io-Guané; Presa, Natalia; Gomez-Muñoz, Antonio

2016-08-01

Cell migration is a complex biological function involved in both physiologic and pathologic processes. Although this is a subject of intense investigation, the mechanisms by which cell migration is regulated are not completely understood. In this study we show that the bioactive sphingolipid ceramide 1-phosphate (C1P), which is involved in inflammatory responses, causes upregulation of metalloproteinases (MMP) -2 and -9 in J774A.1 macrophages. This effect was shown to be dependent on stimulation of phosphatidylinositol 3-kinase (PI3K) and extracellularly regulated kinases 1-2 (ERK1-2) as demonstrated by treating the cells with specific siRNA to knockdown the p85 regulatory subunit of PI3K, or ERK1-2. Inhibition of MMP-2 or MMP-9 pharmacologically or with specific siRNA to silence the genes encoding these MMPs abrogated C1P-stimulated macrophage migration. Also, C1P induced actin polymerization and potently increased phosphorylation of the focal adhesion protein paxillin, which are essential factors in the regulation of cell migration. As expected, blockade of paxillin activation with specific siRNA significantly reduced actin polymerization. In addition, inhibition of actin polymerization with cytochalasin D completely blocked C1P-induced MMP-2 and -9 expression as well as C1P-stimulated macrophage migration. It was also observed that pertussis toxin (Ptx) inhibited Akt, ERK1-2, and paxillin phosphorylation, and completely blocked cell migration. The latter findings support the notion that C1P-stimulated macrophage migration is a receptor mediated effect, and point to MMP-2 and -9 as possible therapeutic targets to control inflammation. Copyright © 2016 Elsevier Inc. All rights reserved.

Pancreatic Fibroblasts Stimulate the Motility of Pancreatic Cancer Cells through IGF1/IGF1R Signaling under Hypoxia.

PubMed

Hirakawa, Toshiki; Yashiro, Masakazu; Doi, Yosuke; Kinoshita, Haruhito; Morisaki, Tamami; Fukuoka, Tatsunari; Hasegawa, Tsuyoshi; Kimura, Kenjiro; Amano, Ryosuke; Hirakawa, Kosei

2016-01-01

Pancreatic ductal adenocarcinoma (PDAC) is characterized by its hypovascularity, with an extremely poor prognosis because of its highly invasive nature. PDAC proliferates with abundant stromal cells, suggesting that its invasive activity might be controlled by intercellular interactions between cancer cells and fibroblasts. Using four PDAC cell lines and two pancreas cancer-associated fibroblasts (CAFs), the expression of insulin-like growth factor-1 (IGF1) and IGF1 receptor (IGF1R) was evaluated by RT-PCR, FACScan, western blot, or ELISA. Correlation between IGF1R and the hypoxia marker carbonic anhydrase 9 (CA9) was examined by immunohistochemical staining of 120 pancreatic specimens. The effects of CAFs, IGF1, and IGF1R inhibitors on the motility of cancer cells were examined by wound-healing assay or invasion assay under normoxia (20% O2) and hypoxia (1% O2). IGF1R expression was significantly higher in RWP-1, MiaPaCa-2, and OCUP-AT cells than in Panc-1 cells. Hypoxia increased the expression level of IGF1R in RWP-1, MiaPaCa-2, and OCUP-AT cells. CA9 expression was correlated with IGF1R expression in pancreatic specimens. CAFs produced IGF1 under hypoxia, but PDAC cells did not. A conditioned medium from CAFs, which expressed αSMA, stimulated the migration and invasion ability of MiaPaCa-2, RWP-1, and OCUP-AT cells. The motility of all PDAC cells was greater under hypoxia than under normoxia. The motility-stimulating ability of CAFs was decreased by IGF1R inhibitors. These findings might suggest that pancreas CAFs stimulate the invasion activity of PDAC cells through paracrine IGF1/IGF1R signaling, especially under hypoxia. Therefore the targeting of IGF1R signaling might represent a promising therapeutic approach in IGF1R-dependent PDAC.

Electrophysiological and morphological features underlying neurotransmission efficacy at the splanchnic nerve-chromaffin cell synapse of bovine adrenal medulla.

PubMed

de Diego, Antonio M G

2010-02-01

The ability of adrenal chromaffin cells to fast-release catecholamines relies on their capacity to fire action potentials (APs). However, little attention has been paid to the requirements needed to evoke the controlled firing of APs. Few data are available in rodents and none on the bovine chromaffin cell, a model extensively used by researchers. The aim of this work was to clarify this issue. Short puffs of acetylcholine (ACh) were fast perifused to current-clamped chromaffin cells and produced the firing of single APs. Based on the currents generated by such ACh applications and previous literature, current waveforms that efficiently elicited APs at frequencies up to 20 Hz were generated. Complex waveforms were also generated by adding simple waveforms with different delays; these waveforms aimed at modeling the stimulation patterns that a chromaffin cell would conceivably undergo upon strong synaptic stimulation. Cholinergic innervation was assessed using the acetylcholinesterase staining technique on the supposition that the innervation pattern is a determinant of the kind of stimuli chromaffin cells can receive. It is concluded that 1) a reliable method to produce frequency-controlled APs by applying defined current injection waveforms is achieved; 2) the APs thus generated have essentially the same features as those spontaneously emitted by the cell and those elicited by fast-ACh perifusion; 3) the higher frequencies attainable peak at around 30 Hz; and 4) the bovine adrenal medulla shows abundant cholinergic innervation, and chromaffin cells show strong acetylcholinesterase staining, consistent with a tight cholinergic presynaptic control of firing frequency.

Two-Photon Holographic Stimulation of ReaChR

PubMed Central

Chaigneau, Emmanuelle; Ronzitti, Emiliano; Gajowa, Marta A.; Soler-Llavina, Gilberto J.; Tanese, Dimitrii; Brureau, Anthony Y. B.; Papagiakoumou, Eirini; Zeng, Hongkui; Emiliani, Valentina

2016-01-01

Optogenetics provides a unique approach to remotely manipulate brain activity with light. Reaching the degree of spatiotemporal control necessary to dissect the role of individual cells in neuronal networks, some of which reside deep in the brain, requires joint progress in opsin engineering and light sculpting methods. Here we investigate for the first time two-photon stimulation of the red-shifted opsin ReaChR. We use two-photon (2P) holographic illumination to control the activation of individually chosen neurons expressing ReaChR in acute brain slices. We demonstrated reliable action potential generation in ReaChR-expressing neurons and studied holographic 2P-evoked spiking performances depending on illumination power and pulse width using an amplified laser and a standard femtosecond Ti:Sapphire oscillator laser. These findings provide detailed knowledge of ReaChR's behavior under 2P illumination paving the way for achieving in depth remote control of multiple cells with high spatiotemporal resolution deep within scattering tissue. PMID:27803649

Stimulation of cell division in the rat by NaCl, KCl, MgCl2, and CaCl2, and inhibition of the sodium chloride effect on the glandular stomach by ascorbic acid and beta-carotene.

PubMed

Lugli, S M; Lutz, W K

1999-01-01

Three questions associated with the stimulation of cell division by chloride salts have been investigated: (i) whether cations other than sodium show a similar effect, (ii) whether vitamins can have a preventive activity, and (iii) whether subchronic treatment with sodium chloride in the diet is also effective. Male Fischer 344 rats were given solutions of the chloride salts of sodium, potassium, magnesium, and calcium by oral gavage. Water was used for control. After 4 h, a 24-h osmotic minipump containing 5-bromo-2'-deoxyuridine was implanted subcutaneously. The forestomach and glandular stomach, as well as liver and bladder were analyzed immunohistochemically 24 h later for the proportion of cells in S phase as an indicator of the rate of replicative DNA synthesis. For both the forestomach and the glandular stomach, potassium was as potent as sodium, and the divalent cations Mg and Ca were even more potent on a molar basis. Supplementation of the diet with ascorbic acid (2 g/kg food) or beta-carotene (12.5 mg/kg food) for 1 week before gavage of the sodium chloride solution resulted in an inhibition of the stimulation of cell division. A putative tumor-chemopreventive activity of the two vitamins might therefore not only rely on their antioxidative properties but may include effects on the cell cycle. A 4-week treatment with a sodium chloride supplement in the diet (2% and 4% supplement) resulted in a significant stimulation of cell division not only in both parts of the stomach and in the bladder (with the 4% supplement) but also in the liver (even with the 2% supplement). Sodium-chloride-stimulated cell turnover therefore is a sustained effect.

[Effect of moxa-burning heat stimulating Liangmen (ST 21) and Zusanli (ST 36) on proliferation and apoptosis signaling proteins in rats with stress-induced gastric ulcer].

PubMed

Peng, Li; Wang, Yadong; Chang, Xiaorong; Wu, Huangan; Liu, Mi; Wang, Hong; Chen, Jiaolong; Wang Chao; Quan, Renfu; Yang, Zongbao

2016-06-01

To observe the effect of moxa-burning heat stimulating acupoints of Liangmen (ST 21) and Zusanli (ST 36) on the proliferation and apoptosis signaling proteins in rats with stress-induced gastric ulcer. Forty rats were randomly divided into four groups: negative control (NC), ulcer control (UC), acupoints of stomach meridian (ASM), and acupoints control (AC). The acute gastric ulcer model was established by bound and water immersion. Rats in NC and UC groups didn't receive any moxa-burning heat stimulating treatment, while rats in ASM and AC groups were treated with buringmoxa heat stimulating the acupoints of Liangmen (ST 21) and Zusanli (ST 36) and their controlled points, respectively. Rats in all groups were sacrificed after 12 consecutive days treatment. The ulcer index was evaluated by using Guth's method. The expression of tumor necrosis factor-alpha (TNF-α), apoptotic protease activating facter-1 (Apaf-1), Caspase-3, p21 activated kinase 1 (PAK1), extracellular regulated protein kinases 2 (ERK2), phosphorylated ERK2 (pERK2), phosphoinositide 3-kinase (PI3K) and RAC-alpha serine/threonine-protein kinase (Akt) in gastric mucosa was detected by enzyme linked immunosorbent assay (ELISA). Compared with UC group, the ulcer index of ASM and AC groups decreased, and the injured gastric mucosa was improved, the expression of TNF-α, Apaf-1 and Caspase-3 in gastric mucosa was significantly reduced (P < 0.05), while the expression of PAK1, ERK2, pERK2, PI3K and Akt in gastric mucosa was significantly increased (P < 0.05). And ASM showed better effect than AC group (P < 0.05). Moxa-burning Heat stimulating of Liangmen (ST 21) and Zusanli (ST 36) could promote the recovery of gastric mucosal lesion probably by inhibiting cell apoptosis and promoting cell proliferation in stress-induced gastric ulcer.

Adrenaline Stimulates Glucagon Secretion by Tpc2-Dependent Ca2+ Mobilization From Acidic Stores in Pancreatic α-Cells.

PubMed

Hamilton, Alexander; Zhang, Quan; Salehi, Albert; Willems, Mara; Knudsen, Jakob G; Ringgaard, Anna K; Chapman, Caroline E; Gonzalez-Alvarez, Alejandro; Surdo, Nicoletta C; Zaccolo, Manuela; Basco, Davide; Johnson, Paul R V; Ramracheya, Reshma; Rutter, Guy A; Galione, Antony; Rorsman, Patrik; Tarasov, Andrei I

2018-06-01

Adrenaline is a powerful stimulus of glucagon secretion. It acts by activation of β-adrenergic receptors, but the downstream mechanisms have only been partially elucidated. Here, we have examined the effects of adrenaline in mouse and human α-cells by a combination of electrophysiology, imaging of Ca 2+ and PKA activity, and hormone release measurements. We found that stimulation of glucagon secretion correlated with a PKA- and EPAC2-dependent (inhibited by PKI and ESI-05, respectively) elevation of [Ca 2+ ] i in α-cells, which occurred without stimulation of electrical activity and persisted in the absence of extracellular Ca 2+ but was sensitive to ryanodine, bafilomycin, and thapsigargin. Adrenaline also increased [Ca 2+ ] i in α-cells in human islets. Genetic or pharmacological inhibition of the Tpc2 channel (that mediates Ca 2+ release from acidic intracellular stores) abolished the stimulatory effect of adrenaline on glucagon secretion and reduced the elevation of [Ca 2+ ] i Furthermore, in Tpc2-deficient islets, ryanodine exerted no additive inhibitory effect. These data suggest that β-adrenergic stimulation of glucagon secretion is controlled by a hierarchy of [Ca 2+ ] i signaling in the α-cell that is initiated by cAMP-induced Tpc2-dependent Ca 2+ release from the acidic stores and further amplified by Ca 2+ -induced Ca 2+ release from the sarco/endoplasmic reticulum. © 2018 by the American Diabetes Association.

Immunohistochemical localization of cannabinoid receptor 1 (CB1) in the submandibular gland of mice under normal conditions and when stimulated by isoproterenol or carbachol.

PubMed

Thoungseabyoun, Wipawee; Tachow, Apussara; Pakkarato, Sawetree; Rawangwong, Atsara; Krongyut, Suthankamon; Sakaew, Waraporn; Kondo, Hisatake; Hipkaeo, Wiphawi

2017-09-01

We wished to investigate the subcellular localization of CB1, a receptor for the endocannabinoids in mouse submandibular glands (SMGs) under normal conditions and when stimulated by adrenergic or cholinergic agonists. SMGs of both male and female adult mice were utilized for immunoblotting and immuno-light and -electron microscopic analyses. Isoproterenol and carbachol were used as adrenergic and cholinergic stimulants, respectively. SMGs were examined at 15, 30, 60 and 120min after intraperitoneal injection of these agents. Selective localization of intense immunoreactivity for CB1 in the granular convoluted ductal cells was confirmed by immunoblotting and the antigen absorption test. In SMGs of control male mice, CB1-immunoreactivity was evident on the basolateral plasma membranes, including the basal infoldings, but was absent on the apical membranes in the ductal cells. Localization and intensity of CB1-immunoreactivity were essentially the same in SMGs of female mice. The immunoreactivity was transiently localized in the apical plasmalemma of some acinar and granular ductal cells of male SMGs shortly after stimulation by isoproterenol, but not by carbachol. The present finding suggests that CB1 functions primarily in the basolateral membranes of the granular convoluted ductal cells of SMGs under normal conditions, and that the CB1 can function additionally in the apical membrane of acinar and granular ductal cells for modulation of the saliva secretory condition via adrenoceptors. Copyright © 2017 Elsevier Ltd. All rights reserved.

Application of low-frequency alternating current electric fields via interdigitated electrodes: effects on cellular viability, cytoplasmic calcium, and osteogenic differentiation of human adipose-derived stem cells.

PubMed

McCullen, Seth D; McQuilling, John P; Grossfeld, Robert M; Lubischer, Jane L; Clarke, Laura I; Loboa, Elizabeth G

2010-12-01

Electric stimulation is known to initiate signaling pathways and provides a technique to enhance osteogenic differentiation of stem and/or progenitor cells. There are a variety of in vitro stimulation devices to apply electric fields to such cells. Herein, we describe and highlight the use of interdigitated electrodes to characterize signaling pathways and the effect of electric fields on the proliferation and osteogenic differentiation of human adipose-derived stem cells (hASCs). The advantage of the interdigitated electrode configuration is that cells can be easily imaged during short-term (acute) stimulation, and this identical configuration can be utilized for long-term (chronic) studies. Acute exposure of hASCs to alternating current (AC) sinusoidal electric fields of 1 Hz induced a dose-dependent increase in cytoplasmic calcium in response to electric field magnitude, as observed by fluorescence microscopy. hASCs that were chronically exposed to AC electric field treatment of 1 V/cm (4 h/day for 14 days, cultured in the osteogenic differentiation medium containing dexamethasone, ascorbic acid, and β-glycerol phosphate) displayed a significant increase in mineral deposition relative to unstimulated controls. This is the first study to evaluate the effects of sinusoidal AC electric fields on hASCs and to demonstrate that acute and chronic electric field exposure can significantly increase intracellular calcium signaling and the deposition of accreted calcium under osteogenic stimulation, respectively.

The cell-mediated immune response to ectromelia virus infection. Secondary response in vitro: specificity, nature of effector and responder cells and requirements for induction of antigenic changes in stimulator cells.

PubMed

Pang, T; Blanden, R V

1976-06-01

An in vitro culture method was used to study secondary cell-mediated responses to ectromelia virus infection in mice. Infected, syngeneic spleen cells or peritoneal cells were efficient "stimulator" cells when cultured with "responder" cells obtained from mice infected with ectromelia 4-6 weeks previously. The kinetics of generation of cytotoxic cells in cultures were determined; a peak occurred on days 4-5. A separation procedure performed on the cytotoxic cells showed that activity was associated mainly with the Ig-negative subpopulation (T cell-rich) and that H-2 compatibility between cytotoxic cells and target cells was required. The secondary response was virus-specific, at the level of both induction and target cell lysis, at least so far as ectromelia and lymphocytic choriomeningitis (LCM) viruses are concerned. Seperation of responder cells prior to culture showed that a potent secondary response was generated with the Ig-negative (T cell-rich) subpopulation and only a weak response was observed when the responder cells were Ig-positive (rich in B cells). Infected stimulator cells did not appear to secrete significant amounts of soluble antigen into the medium over 4 days of culture. Thus, antigenic patterns effective in memory T cell stimulation may be largely associated with the surfaces of infected cells.Pretreatment of ectromelia virus with UV- or gamma-irradiation did not impair its ability to induce antigenic changes in stimulator cells. Stimulator cells treated with UV-or gamma-irradiated virus for 1 h and then immediately with pactamycin to inhibit further viral protein synthesis and replication were efficient stimulators, thus indicating that antigenic changes are induced very rapidly on the surface of stimulator cells after uptake of virus. These treatments are being used to further characterize the cellular requirements in the stimulator population.

β-Adrenergic receptor stimulation inhibits proarrhythmic alternans in postinfarction border zone cardiomyocytes: a computational analysis.

PubMed

Tomek, Jakub; Rodriguez, Blanca; Bub, Gil; Heijman, Jordi

2017-08-01

The border zone (BZ) of the viable myocardium adjacent to an infarct undergoes extensive autonomic and electrical remodeling and is prone to repolarization alternans-induced cardiac arrhythmias. BZ remodeling processes may promote or inhibit Ca 2+ and/or repolarization alternans and may differentially affect ventricular arrhythmogenesis. Here, we used a detailed computational model of the canine ventricular cardiomyocyte to study the determinants of alternans in the BZ and their regulation by β-adrenergic receptor (β-AR) stimulation. The BZ model developed Ca 2+ transient alternans at slower pacing cycle lengths than the control model, suggesting that the BZ may promote spatially heterogeneous alternans formation in an infarcted heart. β-AR stimulation abolished alternans. By evaluating all combinations of downstream β-AR stimulation targets, we identified both direct (via ryanodine receptor channels) and indirect [via sarcoplasmic reticulum (SR) Ca 2+ load] modulation of SR Ca 2+ release as critical determinants of Ca 2+ transient alternans. These findings were confirmed in a human ventricular cardiomyocyte model. Cell-to-cell coupling indirectly modulated the likelihood of alternans by affecting the action potential upstroke, reducing the trigger for SR Ca 2+ release in one-dimensional strand simulations. However, β-AR stimulation inhibited alternans in both single and multicellular simulations. Taken together, these data highlight a potential antiarrhythmic role of sympathetic hyperinnervation in the BZ by reducing the likelihood of alternans and provide new insights into the underlying mechanisms controlling Ca 2+ transient and repolarization alternans. NEW & NOTEWORTHY We integrated, for the first time, postmyocardial infarction electrical and autonomic remodeling in a detailed, validated computer model of β-adrenergic stimulation in ventricular cardiomyocytes. Here, we show that β-adrenergic stimulation inhibits alternans and provide novel insights into underlying mechanisms, adding to a recent controversy about pro-/antiarrhythmic effects of postmyocardial infarction hyperinnervation.Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/%CE%B2-ar-stimulation-and-alternans-in-border-zone-cardiomyocytes/. Copyright © 2017 the American Physiological Society.

Exogenous cytokines released by spleen and Peyer's patch cells removed from mice infected with Giardia muris.

PubMed

Djamiatun, K; Faubert, G M

1998-01-01

The role that T and B lymphocytes play in the clearance of Giardia muris in the mouse model is well known, but the cytokines produced by CD4+ T cells in response to Giardia antigenic stimulation are unknown. In this study, we have determined how Giardia trophozoite antigenic crude extract and T cell mitogens can trigger the production of cytokines by Peyer's patch and spleen cells removed from infected animals. When Giardia trophozoite proteins were used to challenge the cells in vitro, IL-4, IL-5 and IFN-gamma were not detected in the culture supernatant. When the cells were challenged with Con-A, all three cytokines were released in vitro. However, the level of each cytokine released by the spleen or Peyer's patch cells varied with the latent, acute and elimination phases of the infection. The high levels of IL-4 and IL-5 released by Peyer's patch cells confirm the importance of IgA in the control of the infection. However, we propose that the relative success of G. muris in completing its life cycle in a primary infection might be due, in part, to the stimulation of a Th2-type response (IL-4, IL-5). A stronger Th1 response (IFN-gamma) may lead to a better control of the primary infection.

Orphan nuclear receptor NR4A2 inhibits hepatic stellate cell proliferation through MAPK pathway in liver fibrosis.

PubMed

Chen, Pengguo; Li, Jie; Huo, Yan; Lu, Jin; Wan, Lili; Li, Bin; Gan, Run; Guo, Cheng

2015-01-01

Hepatic stellate cells (HSCs) play a crucial role in liver fibrosis, which is a pathological process characterized by extracellular matrix accumulation. NR4A2 is a nuclear receptor belonging to the NR4A subfamily and vital in regulating cell growth, metabolism, inflammation and other biological functions. However, its role in HSCs is unclear. We analyzed NR4A2 expression in fibrotic liver and stimulated HSCs compared with control group and studied the influence on cell proliferation, cell cycle, cell apoptosis and MAPK pathway after NR4A2 knockdown. NR4A2 expression was examined by real-time polymerase chain reaction, Western blotting, immunohistochemistry and immunofluorescence analyses. NR4A2 expression was significantly lower in fibrotic liver tissues and PDGF BB or TGF-β stimulated HSCs compared with control group. After NR4A2 knockdown α-smooth muscle actin and Col1 expression increased. In addition, NR4A2 silencing led to the promotion of cell proliferation, increase of cell percentage in S phase and reduced phosphorylation of ERK1/2, P38 and JNK in HSCs. These results indicate that NR4A2 can inhibit HSC proliferation through MAPK pathway and decrease extracellular matrix in liver fibrogenesis. NR4A2 may be a promising therapeutic target for liver fibrosis.

Differential expression of upstream stimulatory factor (USF) 2 variants in eutopic endometria from women with endometriosis: estradiol regulation.

PubMed

Castro, Jazmin; Araya, Germán; Inostroza, Pamela; Hidalgo, Paulina; González-Ramos, Reinaldo; Sovino, Hugo; Boric, M Angélica; Fuentes, Ariel; Johnson, M Cecilia

2015-10-09

Endometriosis, pro-inflammatory and invasive benign disease estrogen dependent, abnormally express in endometria the enzyme P450Arom, positively regulated by steroid factor-1 (SF-1). Our objective was to study the nuclear protein contents of upstream stimulating factor 2 (USF2a and USF2b), a positive regulator of SF-1, throughout the menstrual cycle in eutopic endometria from women with and without (control) endometriosis and the involvement of nuclear estrogen receptors (ER) and G-coupled protein estrogen receptor (GPER)-1. Upstream stimulating factor 2 protein contents were higher in mid (USF2b) and late (USF2a and USF2b) secretory phase in eutopic endometria from endometriosis than control (p < 0.05). In isolated control epithelial cells incubated with E2 and PGE2, to resemble the endometriosis condition, the data showed: (a) significant increase of USF2a and USF2b nuclear protein contents when treated with E2, PPT (specific agonist for ERα) or G1 (specific agonist for GPER1); (b) no increase in USF2 binding to SF-1 E-Box/DNA consensus sequence in E2-treated cells; (c) USF2 variants protein contents were not modified by PGE2; (d) SF-1 nuclear protein content was significantly higher than basal when treated with PGE2, E2 or G1, stimulation unaffected by ICI (nuclear ER antagonist); and (e) increased (p < 0.05) cytosolic protein contents of P450Arom when treated with PGE2, E2, PPT or G1 compared to basal, effect that was additive with E2 + PGE2 together. Nevertheless, in endometriosis cells, the high USF2, SF-1 and P450Arom protein contents in basal condition were unmodified. These data strongly suggest that USF2 variants and P450Arom are regulated by E2 through ERα and GPER1, whereas SF-1 through GPER1, visualized by the response of the cells obtained from control endometria, being unaffected the endogenously stimulated cells from endometriosis origin. The lack of E2 stimulation on USF2/SF-1 E-Box/DNA-sequence binding and the absence of PGE2 effect on USF2 variants opposite to the strong induction that they exert on SF1 and P450 proteins suggest different mechanisms and indirect regulations. The sustained USF2 variants protein expression during the secretory phase in eutopic endometria from women with endometriosis may participate in the pathophysiology of this disease strongly associated with infertility and its characteristic endometrial invasion to ectopic sites in the pelvic cavity.

Chip-based comparison of the osteogenesis of human bone marrow- and adipose tissue-derived mesenchymal stem cells under mechanical stimulation.

PubMed

Park, Sang-Hyug; Sim, Woo Young; Min, Byoung-Hyun; Yang, Sang Sik; Khademhosseini, Ali; Kaplan, David L

2012-01-01

Adipose tissue-derived stem cells (ASCs) are considered as an attractive stem cell source for tissue engineering and regenerative medicine. We compared human bone marrow-derived mesenchymal stem cells (hMSCs) and hASCs under dynamic hydraulic compression to evaluate and compare osteogenic abilities. A novel micro cell chip integrated with microvalves and microscale cell culture chambers separated from an air-pressure chamber was developed using microfabrication technology. The microscale chip enables the culture of two types of stem cells concurrently, where each is loaded into cell culture chambers and dynamic compressive stimulation is applied to the cells uniformly. Dynamic hydraulic compression (1 Hz, 1 psi) increased the production of osteogenic matrix components (bone sialoprotein, oateopontin, type I collagen) and integrin (CD11b and CD31) expression from both stem cell sources. Alkaline phosphatase and Alrizarin red staining were evident in the stimulated hMSCs, while the stimulated hASCs did not show significant increases in staining under the same stimulation conditions. Upon application of mechanical stimulus to the two types of stem cells, integrin (β1) and osteogenic gene markers were upregulated from both cell types. In conclusion, stimulated hMSCs and hASCs showed increased osteogenic gene expression compared to non-stimulated groups. The hMSCs were more sensitive to mechanical stimulation and more effective towards osteogenic differentiation than the hASCs under these modes of mechanical stimulation.

Chip-Based Comparison of the Osteogenesis of Human Bone Marrow- and Adipose Tissue-Derived Mesenchymal Stem Cells under Mechanical Stimulation

PubMed Central

Min, Byoung-Hyun; Yang, Sang Sik; Khademhosseini, Ali; Kaplan, David L.

2012-01-01

Adipose tissue-derived stem cells (ASCs) are considered as an attractive stem cell source for tissue engineering and regenerative medicine. We compared human bone marrow-derived mesenchymal stem cells (hMSCs) and hASCs under dynamic hydraulic compression to evaluate and compare osteogenic abilities. A novel micro cell chip integrated with microvalves and microscale cell culture chambers separated from an air-pressure chamber was developed using microfabrication technology. The microscale chip enables the culture of two types of stem cells concurrently, where each is loaded into cell culture chambers and dynamic compressive stimulation is applied to the cells uniformly. Dynamic hydraulic compression (1 Hz, 1 psi) increased the production of osteogenic matrix components (bone sialoprotein, oateopontin, type I collagen) and integrin (CD11b and CD31) expression from both stem cell sources. Alkaline phosphatase and Alrizarin red staining were evident in the stimulated hMSCs, while the stimulated hASCs did not show significant increases in staining under the same stimulation conditions. Upon application of mechanical stimulus to the two types of stem cells, integrin (β1) and osteogenic gene markers were upregulated from both cell types. In conclusion, stimulated hMSCs and hASCs showed increased osteogenic gene expression compared to non-stimulated groups. The hMSCs were more sensitive to mechanical stimulation and more effective towards osteogenic differentiation than the hASCs under these modes of mechanical stimulation. PMID:23029565

Lentivirus-mediated shRNA interference of ghrelin receptor blocks proliferation in the colorectal cancer cells.

PubMed

Liu, An; Huang, Chenggang; Xu, Jia; Cai, Xuehong

2016-09-01

Ghrelin, an orexigenic peptide, acts via the growth hormone secretagogue receptor (GHSR) to stimulate the release of growth hormone. Moreover, it has a range of biological actions, including the stimulation of food intake, modulation of insulin signaling and cardiovascular effects. Recently, it has been demonstrated that ghrelin has a proliferative and antiapoptotic effects in cancers, suggesting a potential role in promoting tumor growth. However, it remains unknown whether GHSR contributes to colorectal cancer proliferation. In this study, the therapeutic effect of lentivirus-mediated short hairpin RNA (shRNA) targeting ghrelin receptor 1a (GHSR1a) was analyzed in colorectal cancer cell line SW480 both in vitro and in vivo. Our study demonstrated that ghrelin and GHSR1a are significantly upregulated in cancerous colorectal tissue samples and cell lines. In vitro, human colorectal cancer cell line SW480 with downregulation of GHSR1a by shRNA showed significant inhibition of cell viability compared with blank control (BC) or scrambled control (SC) regardless of the application of exogenous ghrelin. Furthermore, GHSR1a silencing by target specific shRNA was shown capable of increasing PTEN, inhibiting AKT phosphorylation and promoting the release of p53 in SW480 cells. In addition, the effects of GHSR1a knockdown were further explored in vivo using colorectal tumor xenograft mouse model. The tumor weights were decreased markedly in GHSR1α knockdown SW480 mouse xenograft tumors compared with blank control or negative control tumors. Our results suggested that the expression of GHSR1a is significantly correlated with the growth of colorectal cancer cells, and the GHSR1a knockdown approach may be a potential therapy for the treatment of colorectal cancer. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Micro-particle transporting system using galvanotactically stimulated apo-symbiotic cells of Paramecium bursaria.

PubMed

Furukawa, Shunsuke; Karaki, Chiaki; Kawano, Tomonori

2009-01-01

It is well known that Paramecium species including green paramecia (Paramecium bursaria) migrate towards the anode when exposed to an electric field in a medium. This type of a cellular movement is known as galvanotaxis. Our previous study revealed that an electric stimulus given to P bursaria is converted to a galvanotactic cellular movement by involvement of T-type calcium channel on the plasma membrane [Aonuma et al. (2007), Z. Naturforsch. 62c, 93-102]. This phenomenon has attracted the attention of bioengineers in the fields of biorobotics or micro-robotics in order to develop electrically controllable micromachineries. Here, we demonstrate the galvanotactic controls of the cellular migration of P bursaria in capillary tubes (diameter, 1-2 mm; length, 30-240 mm). Since the Paramecium cells take up particles of various sizes, we attempted to use the electrically stimulated cells of P bursaria as the vehicle for transportation of micro-particles in the capillary system. By using apo-symbiotic cells of P bursaria obtained after forced removal of symbiotic algae, the uptake of the particles could be maximized and visualized. Then, electrically controlled transportations of particle-filled apo-symbiotic P bursaria cells were manifested. The particles transported by electrically controlled cells (varying in size from nm to /m levels) included re-introduced green algae, fluorescence-labeled polystyrene beads, magnetic microspheres, emerald green fluorescent protein (EmGFP)-labeled cells of E. coli, Indian ink, and crystals of zeolite (hydrated aluminosilicate minerals with a micro-porous structure) and some metal oxides. Since the above demonstrations were successful, we concluded that P bursaria has a potential to be employed as one of the micro-biorobotic devices used in BioMEMS (biological micro-electro-mechanical systems).

Tissue engineering bioreactor systems for applying physical and electrical stimulations to cells.

PubMed

Jin, GyuHyun; Yang, Gi-Hoon; Kim, GeunHyung

2015-05-01

Bioreactor systems in tissue engineering applications provide various types of stimulation to mimic the tissues in vitro and in vivo. Various bioreactors have been designed to induce high cellular activities, including initial cell attachment, cell growth, and differentiation. Although cell-stimulation processes exert mostly positive effects on cellular responses, in some cases such stimulation can also have a negative effect on cultured cells. In this review, we discuss various types of bioreactor and the positive and negative effects of stimulation (physical, chemical, and electrical) on various cultured cell types. © 2014 Wiley Periodicals, Inc.

Sigma Receptor 1 activation attenuates release of inflammatory cytokines MIP1γ, MIP2, MIP3α and IL12 (p40/p70) by retinal Müller glial cells

PubMed Central

Shanmugam, A.; Wang, J.; Markand, S.; Perry, R.L.; Tawfik, A.; Zorrilla, E.; Ganapathy, V.; Smith, S.B.

2015-01-01

The high affinity Sigma Receptor 1 (σR1) ligand (+)-pentazocine ((+)-PTZ) affords profound retinal neuroprotection in vitro and in vivo by a yet-unknown mechanism. A common feature of retinal disease is Müller cell reactive gliosis, which includes cytokine release. Here we investigated whether LPS stimulates cytokine release by primary mouse Müller cells and whether (+)-PTZ alters release. Using a highly sensitive inflammatory antibody array we observed significant release of macrophage inflammatory proteins (MIP1γ, MIP2, MIP3α) and interleukin-12 (IL12 (p40/p70)) in LPS-treated cells compared to controls, and a significant decrease in secretion upon (+)-PTZ treatment. Müller cells from σR1 knockout mice demonstrated increased MIP1γ, MIP2, MIP3α and IL12 (p40/p70) secretion when exposed to LPS compared to LPS-stimulated WT cells. We investigated whether cytokine secretion was accompanied by cytosolic-to-nuclear NFκB translocation and whether endothelial cell adhesion/migration was altered by released cytokines. Cells exposed to LPS demonstrated increased NFκB nuclear location, which was reduced significantly in (+)-PTZ-treated cells. Media conditioned by LPS-stimulated-Müller cells induced leukocyte-endothelial cell adhesion and endothelial cell migration, which was attenuated by (+)-PTZ treatment. The findings suggest that release of certain inflammatory cytokines by Müller cells can be attenuated by σR1 ligands providing insights into the retinal neuroprotective role of this receptor. PMID:25439327

Killer (FASL regulatory) B cells are present during latent TB and are induced by BCG stimulation in participants with and without latent tuberculosis.

PubMed

van Rensburg, Ilana C; Loxton, Andre G

2018-01-01

Regulatory B cells (Bregs) have been shown to be present during several disease states. The phenotype of the cells is not completely defined and the function of these cells differ between disease. The presence of FASL expressing (killer) B cells during latent and successfully treated TB disease have been shown but whether these cells are similar to regulatory B cells remain unclear. We assessed the receptor expression of FASL/IL5 (killer B cells), CD24/CD38 (regulatory B cells) on whole peripheral blood of participants with untreated active TB and healthy controls. We then isolated B cells from a second cohort of M.tb exposed (Quantiferon (QFN) positive) and unexposed (Quantiferon negative) HIV negative participants, and evaluated the frequency of killer B cells induced following stimulation with BCG and/or CD40 and IL5. Our data reveal no difference in the expression on CD24 and CD38 between participants with active TB and the controls. There was also no difference in the frequency of regulatory B cells measured in the peripheral blood mononuclear cells (PBMC) fraction between latent TB and uninfected controls. We did however notice that regulatory B cells (CD24hiCD38hi) population express the FASL receptor. The expression of killer B cell phenotype (CD178+IL5RA+) was significantly higher in controls compared to those with active TB disease (1,06% vs 0,455%). Furthermore, we found that BCG restimulation significantly induced the FASL/IL5RA B cells but this was only evident in the QFN positive group. Our data suggest that both regulatory and killer B cells are present during latent and active TB disease but that the frequency of these populations are increased during latent disease. We also show that the FASL+IL5RA+ B killer B cells are induced in latent TB infection following BCG restimulation but whether these cells are indicative of protection remains unclear. Copyright © 2017 Elsevier Ltd. All rights reserved.

Deciphering the role of a coleopteran steering muscle via free flight stimulation.

PubMed

Sato, Hirotaka; Vo Doan, Tat Thang; Kolev, Svetoslav; Huynh, Ngoc Anh; Zhang, Chao; Massey, Travis L; van Kleef, Joshua; Ikeda, Kazuo; Abbeel, Pieter; Maharbiz, Michel M

2015-03-16

Testing hypotheses of neuromuscular function during locomotion ideally requires the ability to record cellular responses and to stimulate the cells being investigated to observe downstream behaviors [1]. The inability to stimulate in free flight has been a long-standing hurdle for insect flight studies. The miniaturization of computation and communication technologies has delivered ultra-small, radio-enabled neuromuscular recorders and stimulators for untethered insects [2-8]. Published stimulation targets include the areas in brain potentially responsible for pattern generation in locomotion [5], the nerve chord for abdominal flexion [9], antennal muscles [2, 10], and the flight muscles (or their excitatory junctions) [7, 11-13]. However, neither fine nor graded control of turning has been demonstrated in free flight, and responses to the stimulation vary widely [2, 5, 7, 9]. Technological limitations have precluded hypotheses of function validation requiring exogenous stimulation during flight. We investigated the role of a muscle involved in wing articulation during flight in a coleopteran. We set out to identify muscles whose stimulation produced a graded turning in free flight, a feat that would enable fine steering control not previously demonstrated. We anticipated that gradation might arise either as a function of the phase of muscle firing relative to the wing stroke (as in the classic fly b1 muscle [14, 15] or the dorsal longitudinal and ventral muscles of moth [16]), or due to regulated tonic control, in which phase-independent summation of twitch responses produces varying amounts of force delivered to the wing linkages [15, 17, 18]. Copyright © 2015 Elsevier Ltd. All rights reserved.

YAP forms autocrine loops with the ERBB pathway to regulate ovarian cancer initiation and progression

PubMed Central

He, Chunbo; Lv, Xiangmin; Hua, Guohua; Lele, Subodh M; Remmenga, Steven; Dong, Jixin; Davis, John S; Wang, Cheng

2014-01-01

Mechanisms underlying ovarian cancer initiation and progression are unclear. Herein, we report that the Yes-associated protein (YAP), a major effector of the Hippo tumor suppressor pathway, interacts with ERBB signaling pathways to regulate the initiation and progression of ovarian cancer. Immunohistochemistry studies indicate that YAP expression is associated with poor clinical outcomes in patients. Overexpression or constitutive activation of YAP leads to transformation and tumorigenesis in human ovarian surface epithelial cells, and promotes growth of cancer cells in vivo and in vitro. YAP induces expression of EGF receptors (EGFR, ERBB3) and production of EGF-like ligands (HBEGF, NRG1 and NRG2). HBEGF or NRG1, in turn, activates YAP and stimulates cancer cell growth. Knockdown of ERBB3 or HBEGF eliminates YAP effects on cell growth and transformation, while knockdown of YAP abrogates NRG1- and HBEGF-stimulated cell proliferation. Collectively, our study demonstrates the existence of HBEGF&NRGs/ERBBs/YAP/HBEGF&NRGs autocrine loop that controls ovarian cell tumorigenesis and cancer progression. PMID:25798835

Lysosome trafficking is necessary for EGF-driven invasion and is regulated by p38 MAPK and Na+/H+ exchangers.

PubMed

Dykes, Samantha S; Steffan, Joshua J; Cardelli, James A

2017-10-04

Tumor invasion through a basement membrane is one of the earliest steps in metastasis, and growth factors, such as Epidermal Growth Factor (EGF) and Hepatocyte Growth Factor (HGF), stimulate this process in a majority of solid tumors. Basement membrane breakdown is one of the hallmarks of invasion; therefore, tumor cells secrete a variety of proteases to aid in this process, including lysosomal proteases. Previous studies demonstrated that peripheral lysosome distribution coincides with the release of lysosomal cathepsins. Immunofluorescence microscopy, western blot, and 2D and 3D cell culture techniques were performed to evaluate the effects of EGF on lysosome trafficking and cell motility and invasion. EGF-mediated lysosome trafficking, protease secretion, and invasion is regulated by the activity of p38 mitogen activated protein kinase (MAPK) and sodium hydrogen exchangers (NHEs). Interestingly, EGF stimulates anterograde lysosome trafficking through a different mechanism than previously reported for HGF, suggesting that there are redundant signaling pathways that control lysosome positioning and trafficking in tumor cells. These data suggest that EGF stimulation induces peripheral (anterograde) lysosome trafficking, which is critical for EGF-mediated invasion and protease release, through the activation of p38 MAPK and NHEs. Taken together, this report demonstrates that anterograde lysosome trafficking is necessary for EGF-mediated tumor invasion and begins to characterize the molecular mechanisms required for EGF-stimulated lysosome trafficking.

Cloning and expression of porcine Colony Stimulating Factor-1 (CSF-1) and Colony Stimulating Factor-1 Receptor (CSF-1R) and analysis of the species specificity of stimulation by CSF-1 and Interleukin 34

PubMed Central

Gow, Deborah J.; Garceau, Valerie; Kapetanovic, Ronan; Sester, David P.; Fici, Greg J.; Shelly, John A.; Wilson, Thomas L.; Hume, David A.

2012-01-01

Macrophage Colony Stimulating Factor (CSF-1) controls the survival, differentiation and proliferation of cells of the mononuclear phagocyte system. A second ligand for the CSF-1R, Interleukin 34 (IL-34), has been described, but its physiological role is not yet known. The domestic pig provides an alternative to traditional rodent models for evaluating potential therapeutic applications of CSF-1R agonists and antagonists. To enable such studies, we cloned and expressed active pig CSF-1. To provide a bioassay, pig CSF-1R was expressed in the factor-dependent Ba/F3 cell line. On this transfected cell line, recombinant porcine CSF-1 and human CSF-1 had identical activity. Mouse CSF-1 does not interact with the human CSF-1 receptor but was active on pig. By contrast, porcine CSF-1 was active on mouse, human, cat and dog cells. IL-34 was previously shown to be species-specific, with mouse and human proteins demonstrating limited cross-species activity. The pig CSF-1R was equally responsive to both mouse and human IL-34. Based upon the published crystal structures of CSF-1/CSF-1R and IL34/CSF-1R complexes, we discuss the molecular basis for the species specificity. PMID:22974529

A new bio-inspired stimulator to suppress hyper-synchronized neural firing in a cortical network.

PubMed

Amiri, Masoud; Amiri, Mahmood; Nazari, Soheila; Faez, Karim

2016-12-07

Hyper-synchronous neural oscillations are the character of several neurological diseases such as epilepsy. On the other hand, glial cells and particularly astrocytes can influence neural synchronization. Therefore, based on the recent researches, a new bio-inspired stimulator is proposed which basically is a dynamical model of the astrocyte biophysical model. The performance of the new stimulator is investigated on a large-scale, cortical network. Both excitatory and inhibitory synapses are also considered in the simulated spiking neural network. The simulation results show that the new stimulator has a good performance and is able to reduce recurrent abnormal excitability which in turn avoids the hyper-synchronous neural firing in the spiking neural network. In this way, the proposed stimulator has a demand controlled characteristic and is a good candidate for deep brain stimulation (DBS) technique to successfully suppress the neural hyper-synchronization. Copyright © 2016 Elsevier Ltd. All rights reserved.

Long-term in vivo provision of antigen-specific T cell immunity by programming hematopoietic stem cells

NASA Astrophysics Data System (ADS)

Yang, Lili; Baltimore, David

2005-03-01

A method to genetically program mouse hematopoietic stem cells to develop into functional CD8 or CD4 T cells of defined specificity in vivo is described. For this purpose, a bicistronic retroviral vector was engineered that efficiently delivers genes for both and chains of T cell receptor (TCR) to hematopoietic stem cells. When modified cell populations were used to reconstruct the hematopoietic lineages of recipient mice, significant percentages of antigen-specific CD8 or CD4 T cells were observed. These cells expressed normal surface markers and responded to peptide antigen stimulation by proliferation and cytokine production. Moreover, they could mature into memory cells after peptide stimulation. Using TCRs specific for a model tumor antigen, we found that the recipient mice were able to partially resist a challenge with tumor cells carrying the antigen. By combining cells modified with CD8- and CD4-specific TCRs, and boosting with dendritic cells pulsed with cognate peptides, complete suppression of tumor could be achieved and even tumors that had become established would regress and be eliminated after dendritic cell/peptide immunization. This methodology of "instructive immunotherapy" could be developed for controlling the growth of human tumors and attacking established pathogens.

Cell cycle in ascidian eggs and embryos.

PubMed

McDougall, Alex; Chenevert, Janet; Lee, Karen W; Hebras, Celine; Dumollard, Remi

2011-01-01

In ascidians the cell cycle machinery has been studied mainly in oocytes while ascidian embryos have been used to dissect the mechanism that controls asymmetric cell division (ACD). Here we overview the most specific and often exceptional points and events in cell cycle control in ascidian oocytes and early embryos. Mature stage IV eggs are arrested at metaphase I due to cytostatic factor (CSF). In vertebrates, unfertilized eggs are arrested at metaphase II by CSF. Meta II-CSF is mediated by the Mos/MEK/MAPK/Erp1 pathway, which inhibits the ubiquitin ligase APC/C(cdc20) preventing cyclin B destruction thus stabilizing MPF activity. CSF is inactivated by the fertilization Ca(2+) transient that stimulates the destruction of Erp1 thus releasing APC/C(cdc20) from inhibition. Although many of the components of CSF are conserved between the ascidian and the vertebrates, the lack of Erp1 in the ascidians (and indeed other invertebrates) is notable since the Mos/MAPK pathway nonetheless mediates Meta I-CSF. Moreover, since the fertilization Ca(2+) transient targets Erp1, it is not clear how the sperm-triggered Ca(2+) transient in ascidians (and again other invertebrates) stimulates cyclin B destruction in the absence of Erp1. Nonetheless, like mammalian eggs, sperm trigger a series of Ca(2+) oscillations that increases the rate of cyclin B destruction and the subsequent loss of MAPK activity leading to meiotic exit in ascidians. Positive feedback from MPF maintains the Ca(2+) oscillations in fertilized ascidian eggs ensuring the eventual loss of MPF stimulating the egg-to-embryo transition. Embryonic cell cycles in the ascidian are highly stereotyped where both the rate of cell division and the orientation of cell division planes are precisely controlled. Three successive rounds of ACD generate two small posterior germ cell precursors at the 64 cell stage. The centrosome-attracting body (CAB) is a macroscopic cortical structure visible by light microscopy that causes these three rounds of ACD. Entry into mitosis activates the CAB causing the whole mitotic spindle to rotate and migrate toward the cortical CAB leading to a highly ACD whereby one small cell is formed that inherits the CAB and approximately 40 maternal postplasmic/PEM RNAs including the germ cell marker vasa.

Induced pluripotent stem cells have similar immunogenic and more potent immunomodulatory properties compared with bone marrow-derived stromal cells in vitro

PubMed Central

Schnabel, Lauren V; Abratte, Christian M; Schimenti, John C; Felippe, M Julia Bevilaqua; Cassano, Jennifer M; Southard, Teresa L; Cross, Jessica A; Fortier, Lisa A

2015-01-01

Aim To evaluate the in vitro immunogenic and immunomodulatory properties of induced pluripotent stem cells (iPSCs) compared with bone marrow-derived mesenchymal stromal cells (MSCs). Materials & methods Mouse embryonic fibroblasts (MEFs) were isolated from C3HeB/FeJ and C57BL/6J mice, and reprogrammed to generate iPSCs. Mixed leukocyte reactions were performed using MHC-matched and -mismatched responder leukocytes and stimulator leukocytes, iPSCs or MSCs. To assess immunogenic potential, iPSCs and MSCs were used as stimulator cells for responder leukocytes. To assess immunomodulatory properties, iPSCs and MSCs were cultured in the presence of stimulator and responder leukocytes. MEFs were used as a control. Results iPSCs had similar immunogenic properties but more potent immunomodulatory effects than MSCs. Co-culture of MHC-mismatched leukocytes with MHC-matched iPSCs resulted in significantly less responder T-cell proliferation than observed for MHC-mismatched leukocytes alone and at more responder leukocyte concentrations than with MSCs. In addition, MHC-mismatched iPSCs significantly reduced responder T-cell proliferation when co-cultured with MHC-mismatched leukocytes, while MHC-mismatched MSCs did not. Conclusion These results provide important information when considering the use of iPSCs in place of MSCs in both regenerative and transplantation medicine. PMID:24773530

Combined effects of chemical priming and mechanical stimulation on mesenchymal stem cell differentiation on nanofiber scaffolds

PubMed Central

Subramony, Siddarth D.; Su, Amanda; Yeager, Keith; Lu, Helen H.

2014-01-01

Functional tissue engineering of connective tissues such as the anterior cruciate ligament (ACL) remains a significant clinical challenge, largely due to the need for mechanically competent scaffold systems for grafting, as well as a reliable cell source for tissue formation. We have designed an aligned, polylactide-co-glycolide (PLGA) nanofiber-based scaffold with physiologically relevant mechanical properties for ligament regeneration. The objective of this study is to identify optimal tissue engineering strategies for fibroblastic induction of human mesenchymal stem cells (hMSC), testing the hypothesis that basic fibroblast growth factor (bFGF) priming coupled with tensile loading will enhance hMSC-mediated ligament regeneration. It was observed that compared to the unloaded, as well as growth factor-primed but unloaded controls, bFGF stimulation followed by physiologically relevant tensile loading enhanced hMSC proliferation, collagen production and subsequent differentiation into ligament fibroblast-like cells, upregulating the expression of types I and III collagen, as well as tenasin-C and tenomodulin. The results of this study suggest that bFGF priming increases cell proliferation, while mechanical stimulation of the hMSCs on the aligned nanofiber scaffold promotes fibroblastic induction of these cells. In addition to demonstrating the potential of nanofiber scaffolds for hMSC-mediated functional ligament tissue engineering, this study yields new insights into the interactive effects of chemical and mechanical stimuli on stem cell differentiation. PMID:24267271

Glycolysis controls the induction of human regulatory T cells by modulating the expression of FOXP3 exon 2 splicing variants

PubMed Central

De Rosa, Veronica; Galgani, Mario; Porcellini, Antonio; Colamatteo, Alessandra; Santopaolo, Marianna; Zuchegna, Candida; Romano, Antonella; De Simone, Salvatore; Procaccini, Claudio; La Rocca, Claudia; Carrieri, Pietro Biagio; Maniscalco, Giorgia Teresa; Salvetti, Marco; Buscarinu, Maria Chiara; Franzese, Adriana; Mozzillo, Enza; La Cava, Antonio; Matarese, Giuseppe

2016-01-01

Human regulatory T cells (Treg cells) that develop from conventional T cells (Tconv cells) following suboptimal stimulation via the T cell antigen receptor (TCR) (induced Treg cells (iTreg cells)) express the transcription factor Foxp3, are suppressive, and display an active proliferative and metabolic state. Here we found that the induction and suppressive function of iTreg cells tightly depended on glycolysis, which controlled Foxp3 splicing variants containing exon 2 (Foxp3-E2) through the glycolytic enzyme enolase-1. The Foxp3-E2–related suppressive activity of iTreg cells was altered in human autoimmune diseases, including multiple sclerosis and type 1 diabetes, and was associated with impaired glycolysis and signaling via interleukin 2. This link between glycolysis and Foxp3-E2 variants via enolase-1 shows a previously unknown mechanism for controlling the induction and function of Treg cells in health and in autoimmunity. PMID:26414764

Inflammatory cells in rat skeletal muscle are elevated after electrically stimulated contractions.

PubMed

McLoughlin, Thomas J; Mylona, Eleni; Hornberger, Troy A; Esser, Karyn A; Pizza, Francis X

2003-03-01

We determined the effect of muscle contractions resulting from high-frequency electrical stimulation (HFES) on inflammatory cells in rat tibialis anterior (TA), plantaris (Pln), and soleus (Sol) muscles at 6, 24, and 72 h post-HFES. A minimum of four and a maximum of seven rats were analyzed at each time point. HFES, applied to the sciatic nerve, caused the Sol and Pln to contract concentrically and the TA to contract eccentrically. Neutrophils were higher (P < 0.05) at 6 and 24 h after HFES in the Sol, Pln, and TA muscles relative to control muscles. ED1(+) macrophages in the Pln were elevated at 6 and 24 h after HFES and were also elevated in the Sol and TA after HFES relative to controls. ED2(+) macrophages in the Sol and TA were elevated at 24 and 72 h after HFES, respectively, and were also elevated in the Pln after HFES relative to controls. In contrast to the TA muscles, the Pln and Sol muscles showed no gross histological abnormalities. Collectively, these data indicate that both eccentric and concentric contractions can increase inflammatory cells in muscle, regardless of whether overt histological signs of injury are apparent.

Compositos CNTs/bioceramico para a estimulacao eletrica ossea in situ

NASA Astrophysics Data System (ADS)

Mata, Diogo Miguel Rodrigues Marinho da

The present thesis aims to develop a biocompatible and electroconductor bone graft containing carbon nanotubes (CNTs) that allows the in situ regeneration of bone cells by applying pulsed external electrical stimuli. The CNTs were produced by chemical vapor deposition (CVD) by a semi-continuous method with a yield of 500 mg/day. The deposition parameters were optimised to obtain high pure CNTs 99.96% with controlled morphologies, fundamental requisites for the biomedical application under study. The chemical functionalisation of CNTs was also optimised to maximise their processability and biocompatibility. The CNTs were functionalised by the Diels-Alder cycloaddition of 1,3-butadiene. The biological behaviour of the functionalised CNTs was evaluated in vitro with the osteoblastic cells line MG63 and in vivo, by subcutaneous implantation in rats. The materials did not induce an expressed inflammatory response, but the functionalised CNTs showed a superior in vitro and in vivo biocompatibility than the non-functionalised ones. Composites of ceramic matrix, of bioglass (Glass) and hydroxyapatite (HA), reinforced with carbon nanotubes (CNT/Glass/HA) were processed by a wet approach. The incorporation of just 4.4 vol% of CNTs allowed the increase of 10 orders of magnitude of the electrical conductivity of the matrix. In vitro studies with MG63 cells show that the CNT/Glass/HA composites guarantee the adhesion and proliferation of bone cells, and stimulate their phenotype expression, namely the alkaline phosphate (ALP). The interactions between the composite materials and the culture medium (α-MEM), under an applied electrical external field, were studied by scanning vibrating electrode technique. An increase of the culture medium electrical conductivity and the electrical field confinement in the presence of the conductive samples submerged in the medium was demonstrated. The in vitro electrical stimulation of MG63 cells on the conductive composites promotes the increase of the cell metabolic activity and DNA content by 130% and 60%, relatively to the non-stimulated condition, after only 3 days of daily stimulation of 15 μA for 15 min. Moreover, the osteoblastic gene expression for Runx2, osteocalcin (OC) and ALP was enhanced by 80%, 50% and 25%, after 5 days of stimulation. Instead, for dielectric materials, the stimulus delivering was less efficient, giving an equal or lower cellular response than the non-stimulated condition. The proposed electroconductive bone grafts offer exciting possibilities in bone regeneration strategies by delivering in situ electrical stimulus to cells and consequent control of the new bone tissue formation rate. It is expected that conductive smart biomaterials might turn the selective bone electrotherapy of clinical relevance by decreasing the postoperative healing times.