Browsing by Subject "Cell interaction"

Now showing 1 - 6 of 6
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    Effects of carbon nanotubes on airway epithelial cells and model lipid bilayers : proteomic and biophysical studies
    (2014) Li, Pin; Blazer-Yost, Bonnie; Witzmann, F. A. (Frank A.); Randall, Stephen Karl, 1953-; Petrache, Horia; Wang, Xianzhong
    Carbon nanomaterials are widely produced and used in industry, medicine and scientific research. To examine the impact of exposure to nanoparticles on human health, the human airway epithelial cell line, Calu-3, was used to evaluate changes in the cellular proteome that could account for alterations in cellular function of airway epithelia after 24 h exposure to 10 μg/mL and 100 ng/mL of two common carbon nanoparticles, singleand multi-wall carbon nanotubes (SWCNT, MWCNT). After exposure to the nanoparticles, label-free quantitative mass spectrometry (LFQMS) was used to study differential protein expression. Ingenuity Pathway Analysis (IPA) was used to conduct a bioinformatics analysis of proteins identified by LFQMS. Interestingly, after exposure to a high concentration (10 μg/mL; 0.4 μg/cm2) of MWCNT or SWCNT, only 8 and 13 proteins, respectively, exhibited changes in abundance. In contrast, the abundance of hundreds of proteins was altered in response to a low concentration (100 ng/mL; 4 ng/cm2) of either CNT. Of the 281 and 282 proteins that were significantly altered in response to MWCNT or SWCNT, respectively, 231 proteins were the same. Bioinformatic analyses found that the proteins common to both kinds of nanotubes are associated with the cellular functions of cell death and survival, cell-to-cell signaling and interaction, cellular assembly and organization, cellular growth and proliferation, infectious disease, molecular transport and protein synthesis. The decrease in expression of the majority proteins suggests a general stress response to protect cells. The STRING database was used to analyze the various functional protein networks. Interestingly, some proteins like cadherin 1 (CDH1), signal transducer and activator of transcription 1 (STAT1), junction plakoglobin (JUP), and apoptosis-associated speck-like protein containing a CARD (PYCARD), appear in several functional categories and tend to be in the center of the networks. This central positioning suggests they may play important roles in multiple cellular functions and activities that are altered in response to carbon nanotube exposure. To examine the effect of nanotubes on the plasma membrane, we investigated the interaction of short purified MWCNT with model lipid membranes using a planar bilayer workstation. Bilayer lipid membranes were synthesized using neutral 1, 2-diphytanoylsn-glycero-3-phosphocholine (DPhPC) in 1 M KCl. The ion channel model protein, Gramicidin A (gA), was incorporated into the bilayers and used to measure the effect of MWCNT on ion transport. The opening and closing of ion channels, amplitude of current, and open probability and lifetime of ion channels were measured and analyzed by Clampfit. The presence of an intermediate concentration of MWCNT (2 μg/ml) could be related to a statistically significant decrease of the open probability and lifetime of gA channels. The proteomic studies revealed changes in response to CNT exposure. An analysis of the changes using multiple databases revealed alterations in pathways, which were consistent with the physiological changes that were observed in cultured cells exposed to very low concentrations of CNT. The physiological changes included the break down of the barrier function and the inhibition of the mucocillary clearance, both of which could increase the risk of CNT’s toxicity to human health. The biophysical studies indicate MWCNTs have an effect on single channel kinetics of Gramicidin A model cation channel. These changes are consistent with the inhibitory effect of nanoparticles on hormone stimulated transepithelial ion flux, but additional experiments will be necessary to substantiate this correlation.
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    Mechanisms of the downregulation of prostaglandin E₂-activated protein kinase A after chronic exposure to nerve growth factor or prostaglandin E₂
    (2013-10-07) Malty, Ramy Refaat Habashy; Vasko, Michael R.; Brustovetsky, Nickolay; Cummins, Theodore R.; Hudmon, Andy; Nicol, Grant D.
    Chronic inflammatory disorders are characterized by an increase in excitability of small diameter sensory neurons located in dorsal root ganglia (DRGs). This sensitization of neurons is a mechanism for chronic inflammatory pain and available therapies have poor efficacy and severe adverse effects when used chronically. Prostaglandin E₂ (PGE₂) is an inflammatory mediator that plays an important role in sensitization by activating G-protein coupled receptors (GPCRs) known as E-series prostaglandin receptors (EPs) coupled to the protein kinase A (PKA) pathway. EPs are known to downregulate upon prolonged exposure to PGE₂ or in chronic inflammation, however, sensitization persists and the mechanism for this is unknown. I hypothesized that persistence of PGE₂-induced hypersensitivity is associated with a switch in signaling caused by prolonged exposure to PGE₂ or the neurotrophin nerve growth factor (NGF), also a crucial inflammatory mediator. DRG cultures grown in the presence or absence of either PGE₂ or NGF were used to study whether re-exposure to the eicosanoid is able to cause sensitization and activate PKA. When cultures were grown in the presence of NGF, PGE₂-induced sensitization was not attenuated by inhibitors of PKA. Activation of PKA by PGE₂ was similar in DRG cultures grown in the presence or absence of NGF when phosphatase inhibitors were added to the lysis and assay buffers, but significantly less in cultures grown in the presence of NGF when phosphatase inhibitors were not added. In DRG cultures exposed to PGE₂ for 12 hours-5 days, sensitization after re-exposure to PGE₂ is maintained and resistant to PKA inhibition. Prolonged exposure to the eicosanoid caused complete loss of PKA activation after PGE₂ re-exposure. This desensitization was homologous, time dependent, reversible, and insurmountable by a higher concentration of PGE₂. Desensitization was attenuated by reduction of expression of G-protein receptor kinase 2 and was not mediated by PKA or protein kinase C. The presented work provides evidence for persistence of sensitization by PGE₂ as well as switch from the signaling pathway mediating this sensitization after long-term exposure to NFG or PGE₂.
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    Modeling and design optimization of a microfluidic chip for isolation of rare cells
    (2013-12) Gannavaram, Spandana; Zhu, Likun; Yu, Huidan (Whitney); Xie, Jian; Anwar, Sohel
    Cancer is still among those diseases that prominently contribute to the numerous deaths that are caused each year. But as technology and research is reaching new zeniths in the present times, cure or early detection of cancer is possible. The detection of rare cells can help understand the origin of many diseases. The current study deals with one such technology that is used for the capture or effective separation of these rare cells called Lab-on-a-chip microchip technology. The isolation and capture of rare cells is a problem uniquely suited to microfluidic devices, in which geometries on the cellular length scale can be engineered and a wide range of chemical functionalizations can be implemented. The performance of such devices is primarily affected by the chemical interaction between the cell and the capture surface and the mechanics of cell-surface collision and adhesion. This study focuses on the fundamental adhesion and transport mechanisms in rare cell-capture microdevices, and explores modern device design strategies in a transport context. The biorheology and engineering parameters of cell adhesion are defined; chip geometries are reviewed. Transport at the microscale, cell-wall interactions that result in cell motion across streamlines, is discussed. We have concentrated majorly on the fluid dynamics design of the chip. A simplified description of the device would be to say that the chip is at micro scale. There are posts arranged on the chip such that the arrangement will lead to a higher capture of rare cells. Blood consisting of rare cells will be passed through the chip and the posts will pose as an obstruction so that the interception and capture efficiency of the rare cells increases. The captured cells can be observed by fluorescence microscopy. As compared to previous studies of using solid microposts, we will be incorporating a new concept of cylindrical shell micropost. This type of micropost consists of a solid inner core and the annulus area is covered with a forest of silicon nanopillars. Utilization of such a design helps in increasing the interception and capture efficiency and reducing the hydrodynamic resistance between the cells and the posts. Computational analysis is done for different designs of the posts. Drag on the microposts due to fluid flow has a great significance on the capture efficiency of the chip. Also, the arrangement of the posts is important to contributing to the increase in the interception efficiency. The effects of these parameters on the efficiency in junction with other factors have been studied and quantified. The study is concluded by discussing design strategies with a focus on leveraging the underlying transport phenomena to maximize device performance.
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    Sphingosine 1-phosphate enhances excitability of sensory neurons through sphingosine 1-phosphate receptors 1 and/or 3
    (2014) Li, Chao; Vasko, Michael R.; Cummins, Theodore R.; Hudmon, Andrew; Nicol, Grant D.; Quilliam, Lawrence A.
    Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that has proven to be an important signaling molecule both as an extracellular primary messenger and as an intracellular second messenger. Extracellular S1P acts through a family of five S1P receptors, S1PR1-5, all of which are G protein-coupled receptors associated with different G proteins. Previous work from our laboratory shows that externally applied S1P increases the excitability of small-diameter sensory neurons by enhancing the action potential firing. The increased neuronal excitability is mediated primarily, but not exclusively, through S1PR1. This raises the question as to which other S1PRs mediate the enhanced excitability in sensory neurons. To address this question, the expression of different S1PR subtypes in small-diameter sensory neurons was examined by single-cell quantitative PCR. The results show that sensory neurons express the mRNAs for all five S1PRs, with S1PR1 mRNA level significantly greater than the other subtypes. To investigate the functional contribution of other S1PRs in augmenting excitability, sensory neurons were treated with a pool of three individual siRNAs targeted to S1PR1, R2 and R3. This treatment prevented S1P from augmenting excitability, indicating that S1PR1, R2 and/or R3 are essential in mediating S1P-induced sensitization. To study the role of S1PR2 in S1P-induced sensitization, JTE-013, a selective antagonist at S1PR2, was used. Surprisingly, JTE-013 by itself enhanced neuronal excitability. Alternatively, sensory neurons were pretreated with FTY720, which is an agonist at S1PR1/R3/R4/R5 and presumably downregulates these receptors. FTY720 pretreatment prevented S1P from increasing neuronal excitability, suggesting that S1PR2 does not mediate the S1P-induced sensitization. To test the hypothesis that S1PR1 and R3 mediate S1P-induced sensitization, sensory neurons were pretreated with specific antagonists for S1PR1 and R3, or with siRNAs targeted to S1PR1 and R3. Both treatments blocked the capacity of S1P to enhance neuronal excitability. Therefore my results demonstrate that the enhanced excitability produced by S1P is mediated by S1PR1 and/or S1PR3. Additionally, my results indicate that S1P/S1PR1 elevates neuronal excitability through the activation of mitogen-activated protein kinase kinase. The data from antagonism at S1PR1 to regulate neuronal excitability provides insight into the importance of S1P/S1PR1 axis in modulating pain signal transduction.
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    Tsg-6 : an inducible mediator of paracrine anti-inflammatory and myeloprotective effects of adipose stem cells
    (2014-01-29) Xie, Jie; March, Keith Leonard, 1963-; Petrache, Irina; Clauss, Matthias A.; Broxmeyer, Hal E.
    Tumor necrosis factor-induced protein 6 (TSG-6) has been shown to mitigate inflammation. Its presence in the secretome of adipose stem / stromal cells (ASC) and its role in activities of ASC have been overlooked. This thesis described for the first time the release of TSG-6 from ASC, and its modulation by endothelial cells. It also revealed that protection of endothelial barrier function was a novel mechanism underlying the anti-inflammatory activity of both ASC and TSG-6. Moreover, TSG-6 was found to inhibit mitogen-activated lymphocyte proliferation, extending the understanding of its pleiotropic effects on major cell populations involved in inflammation. Next, enzyme-linked immunosorbent assays (ELISA) were established to quantify secretion of TSG-6 from human and murine ASC. To study the importance of TSG-6 to specific activities of ASC, TSG-6 was knocked down in human ASC by siRNA. Murine ASC from TSG-6-/- mice were isolated and the down-regulation of TSG-6 was verified by ELISA. The subsequent attempt to determine the efficacy of ASC in ameliorating ischemic limb necrosis and the role of TSG-6, however, was hampered by the highly variable ischemic tissue necrosis in the BALB/c mouse strain. Afterwards in a mouse model of cigarette smoking (CS), in which inflammation also plays an important role, it was observed, for the first time, that 3-day CS exposure caused an acute functional exhaustion and cell cycle arrest of hematopoietic progenitor cells; and that 7-week CS exposure led to marked depletion of phenotypic bone marrow stem and progenitor cells (HSPC). Moreover, a dynamic crosstalk between human ASC and murine host inflammatory signals was described, and specifically TSG-6 was identified as a necessary and sufficient mediator accounting for the activity of the ASC secretome to ameliorate CS-induced myelotoxicity. These results implicate TSG-6 as a key mediator for activities of ASC in mitigation of inflammation and protection of HSPC from the myelotoxicity of cigarette smoke. They also prompt the notion that ASC and TSG-6 might potentially play therapeutic roles in other scenarios involving myelotoxicity.