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Sample records for adherent cell population

  1. Muscle-derived stem cells isolated as non-adherent population give rise to cardiac, skeletal muscle and neural lineages

    SciTech Connect

    Arsic, Nikola; Mamaeva, Daria; Lamb, Ned J.; Fernandez, Anne

    2008-04-01

    Stem cells with the ability to differentiate in specialized cell types can be extracted from a wide array of adult tissues including skeletal muscle. Here we have analyzed a population of cells isolated from skeletal muscle on the basis of their poor adherence on uncoated or collagen-coated dishes that show multi-lineage differentiation in vitro. When analysed under proliferative conditions, these cells express stem cell surface markers Sca-1 (65%) and Bcrp-1 (80%) but also MyoD (15%), Neuronal {beta} III-tubulin (25%), GFAP (30%) or Nkx2.5 (1%). Although capable of growing as non-attached spheres for months, when given an appropriate matrix, these cells adhere giving rise to skeletal muscle, neuronal and cardiac muscle cell lineages. A similar cell population could not be isolated from either bone marrow or cardiac tissue suggesting their specificity to skeletal muscle. When injected into damaged muscle, these non-adherent muscle-derived cells are retrieved expressing Pax7, in a sublaminar position characterizing satellite cells and participate in forming new myofibers. These data show that a non-adherent stem cell population can be specifically isolated and expanded from skeletal muscle and upon attachment to a matrix spontaneously differentiate into muscle, cardiac and neuronal lineages in vitro. Although competing with resident satellite cells, these cells are shown to significantly contribute to repair of injured muscle in vivo supporting that a similar muscle-derived non-adherent cell population from human muscle may be useful in treatment of neuromuscular disorders.

  2. Bayesian population modeling of drug dosing adherence.

    PubMed

    Fellows, Kelly; Stoneking, Colin J; Ramanathan, Murali

    2015-10-01

    Adherence is a frequent contributing factor to variations in drug concentrations and efficacy. The purpose of this work was to develop an integrated population model to describe variation in adherence, dose-timing deviations, overdosing and persistence to dosing regimens. The hybrid Markov chain-von Mises method for modeling adherence in individual subjects was extended to the population setting using a Bayesian approach. Four integrated population models for overall adherence, the two-state Markov chain transition parameters, dose-timing deviations, overdosing and persistence were formulated and critically compared. The Markov chain-Monte Carlo algorithm was used for identifying distribution parameters and for simulations. The model was challenged with medication event monitoring system data for 207 hypertension patients. The four Bayesian models demonstrated good mixing and convergence characteristics. The distributions of adherence, dose-timing deviations, overdosing and persistence were markedly non-normal and diverse. The models varied in complexity and the method used to incorporate inter-dependence with the preceding dose in the two-state Markov chain. The model that incorporated a cooperativity term for inter-dependence and a hyperbolic parameterization of the transition matrix probabilities was identified as the preferred model over the alternatives. The simulated probability densities from the model satisfactorily fit the observed probability distributions of adherence, dose-timing deviations, overdosing and persistence parameters in the sample patients. The model also adequately described the median and observed quartiles for these parameters. The Bayesian model for adherence provides a parsimonious, yet integrated, description of adherence in populations. It may find potential applications in clinical trial simulations and pharmacokinetic-pharmacodynamic modeling. PMID:26319548

  3. Physics of adherent cells

    NASA Astrophysics Data System (ADS)

    Schwarz, Ulrich S.; Safran, Samuel A.

    2013-07-01

    One of the most unique physical features of cell adhesion to external surfaces is the active generation of mechanical force at the cell-material interface. This includes pulling forces generated by contractile polymer bundles and networks, and pushing forces generated by the polymerization of polymer networks. These forces are transmitted to the substrate mainly by focal adhesions, which are large, yet highly dynamic adhesion clusters. Tissue cells use these forces to sense the physical properties of their environment and to communicate with each other. The effect of forces is intricately linked to the material properties of cells and their physical environment. Here a review is given of recent progress in our understanding of the role of forces in cell adhesion from the viewpoint of theoretical soft matter physics and in close relation to the relevant experiments.

  4. Characterization of a Distinct Population of Circulating Human Non-Adherent Endothelial Forming Cells and Their Recruitment via Intercellular Adhesion Molecule-3

    PubMed Central

    Thompson, Emma J.; Barrett, Jeffrey M.; Tooley, Katie; Sen, Shaundeep; Sun, Wai Yan; Grose, Randall; Nicholson, Ian; Levina, Vitalina; Cooke, Ira; Talbo, Gert; Lopez, Angel F.; Bonder, Claudine S.

    2012-01-01

    Circulating vascular progenitor cells contribute to the pathological vasculogenesis of cancer whilst on the other hand offer much promise in therapeutic revascularization in post-occlusion intervention in cardiovascular disease. However, their characterization has been hampered by the many variables to produce them as well as their described phenotypic and functional heterogeneity. Herein we have isolated, enriched for and then characterized a human umbilical cord blood derived CD133+ population of non-adherent endothelial forming cells (naEFCs) which expressed the hematopoietic progenitor cell markers (CD133, CD34, CD117, CD90 and CD38) together with mature endothelial cell markers (VEGFR2, CD144 and CD31). These cells also expressed low levels of CD45 but did not express the lymphoid markers (CD3, CD4, CD8) or myeloid markers (CD11b and CD14) which distinguishes them from ‘early’ endothelial progenitor cells (EPCs). Functional studies demonstrated that these naEFCs (i) bound Ulex europaeus lectin, (ii) demonstrated acetylated-low density lipoprotein uptake, (iii) increased vascular cell adhesion molecule (VCAM-1) surface expression in response to tumor necrosis factor and (iv) in co-culture with mature endothelial cells increased the number of tubes, tubule branching and loops in a 3-dimensional in vitro matrix. More importantly, naEFCs placed in vivo generated new lumen containing vasculature lined by CD144 expressing human endothelial cells (ECs). Extensive genomic and proteomic analyses of the naEFCs showed that intercellular adhesion molecule (ICAM)-3 is expressed on their cell surface but not on mature endothelial cells. Furthermore, functional analysis demonstrated that ICAM-3 mediated the rolling and adhesive events of the naEFCs under shear stress. We suggest that the distinct population of naEFCs identified and characterized here represents a new valuable therapeutic target to control aberrant vasculogenesis. PMID:23144795

  5. Micromolded Arrays for Separation of Adherent Cells

    PubMed Central

    Wang, Yuli; Phillips, Colleen; Xu, Wei; Pai, Jeng-Hao; Dhopeshwarkar, Rahul; Sims, Christopher E.; Allbritton, Nancy

    2010-01-01

    We present an efficient, yet inexpensive, approach for isolating viable single cells or colonies from a mixed population. This cell microarray platform possesses innovations in both the array manufacture and the manner of target cell release. Arrays of microwells with bases composed of detachable concave elements, termed microrafts, were fabricated by a dip-coating process using a polydimethylsiloxane mold as the template and the array substrate. This manufacturing approach enabled the use of materials other than photoresists to create the array elements. Thus microrafts possessing low autofluorescence could be fabricated for fluorescence-based identification of cells. Cells plated on the microarray settled and attached at the center of the wells due to the microrafts’ concavity. Individual microrafts were readily dislodged by the action of a needle inserted through the compliant polymer substrate. The hard polymer material (polystyrene or epoxy resin) of which the microrafts were composed protected the cells from damage by the needle. For cell analysis and isolation, cells of interest were identified using a standard inverted microscope and microrafts carrying target cells were dislodged with the needle. The released cells/microrafts could be efficiently collected, cultured and clonally expanded. During the separation and collection procedures, the cells remained adherent and provided a measure of protection during manipulation, thus providing an extremely high single-cell cloning rate (>95%). Generation of a transfected cell line based on expression of a fluorescent protein demonstrated an important application for performing on-chip cell separations. PMID:20838672

  6. Adherence of Mycoplasma hyopneumoniae to cell monolayers.

    PubMed

    Zielinski, G C; Young, T; Ross, R F; Rosenbusch, R F

    1990-03-01

    This work was an attempt to develop an in vitro adherence model for Mycoplasma hyopneumoniae, using monolayers of human and porcine lung fibroblasts and porcine kidney cells. Mycoplasma hyopneumoniae grown in Friis mycoplasma broth was radiolabeled with 35[S]-methionine, washed, concentrated, and inoculated on the monolayers. After 15 minutes of centrifugation to facilitate adherence, monolayers were washed 3 times, dissolved with 0.1N NaOH, and suspended in scintillation liquid, and the radioactivity was determined in a liquid scintillation counter. Adherence, measured as a percentage of counts added, varied according to the mycoplasma strain and the cell line used. Comparison of strains J, 144L, and 232 of M hyopneumoniae revealed 7.5 +/- 5.9, 31.9 +/- 13, and 9.6 +/- 5% adherence to porcine kidney cells, respectively. Slightly different, but proportionally the same relationships were obtained with swine or human fibroblasts. Adherence was decreased slightly by repeated washings of the mycoplasma-treated cell monolayers; however, a plateau was reached, indicating irreversibility of the adherence process. Pretreatment of cell monolayers with nonlabeled organisms substantially blocked adherence by labeled organisms. Dilution of labeled organisms resulted in an increased proportion adhering. Therefore, it appears that the adherence was a receptor-dependent event. Treatment of the mycoplasmas with trypsin prior to the inoculation of monolayers resulted in a marked reduction in adherence. Treatment of the mycoplasmas with hyperimmune swine serum against M hyopneumoniae or normal swine serum resulted in 80 to 90% reduction of adherence; however, no inhibition occurred when mycoplasmas were treated with purified IgG from the hyperimmune serum.

  7. Ferromagnetic Micropallets for Magnetic Capture of Single Adherent Cells

    PubMed Central

    Gunn, Nicholas M.; Chang, Ruth; Westerhof, Trisha; Li, Guann-Pyng; Bachman, Mark; Nelson, Edward L.

    2010-01-01

    We present a magnetic micropallet array and demonstration of its capacity to recover specific, individual adherent cells from large populations and deliver them for downstream single cell analysis. A ferromagnetic photopolymer was formulated, characterized, and used to fabricate magnetic micropallets, which are microscale pedestals that provide demarcated cell growth surfaces, with preservation of biophysical properties including photopatternability, biocompatibility, and optical clarity. Each micropallet holds a single adherent cell in culture and hundreds of thousands of micropallets compose a single micropallet array. Any micropallet in the array can be recovered on demand, carrying the adhered cell with it. We used this platform to selectively recover single cells, which were subsequently analyzed using single cell RT-qPCR. PMID:20968293

  8. Measurement of adherent cell mass and growth

    PubMed Central

    Park, Kidong; Millet, Larry J.; Kim, Namjung; Li, Huan; Jin, Xiaozhong; Popescu, Gabriel; Aluru, N. R.; Hsia, K. Jimmy; Bashir, Rashid

    2010-01-01

    The characterization of physical properties of cells such as their mass and stiffness has been of great interest and can have profound implications in cell biology, tissue engineering, cancer, and disease research. For example, the direct dependence of cell growth rate on cell mass for individual adherent human cells can elucidate the mechanisms underlying cell cycle progression. Here we develop an array of micro-electro-mechanical systems (MEMS) resonant mass sensors that can be used to directly measure the biophysical properties, mass, and growth rate of single adherent cells. Unlike conventional cantilever mass sensors, our sensors retain a uniform mass sensitivity over the cell attachment surface. By measuring the frequency shift of the mass sensors with growing (soft) cells and fixed (stiff) cells, and through analytical modeling, we derive the Young’s modulus of the unfixed cell and unravel the dependence of the cell mass measurement on cell stiffness. Finally, we grew individual cells on the mass sensors and measured their mass for 50+ hours. Our results demonstrate that adherent human colon epithelial cells have increased growth rates with a larger cell mass, and the average growth rate increases linearly with the cell mass, at 3.25%/hr. Our sensitive mass sensors with a position-independent mass sensitivity can be coupled with microscopy for simultaneous monitoring of cell growth and status, and provide an ideal method to study cell growth, cell cycle progression, differentiation, and apoptosis. PMID:21068372

  9. Allopurinol use in a New Zealand population: prevalence and adherence.

    PubMed

    Horsburgh, Simon; Norris, Pauline; Becket, Gordon; Arroll, Bruce; Crampton, Peter; Cumming, Jacqueline; Keown, Shirley; Herbison, Peter

    2014-07-01

    Allopurinol is effective for the control of gout and its long-term complications when taken consistently. There is evidence that adherence to allopurinol therapy varies across population groups. This may exacerbate differences in the burden of gout on population groups and needs to be accurately assessed. The aim of this study was to describe the prevalence of allopurinol use in a region of New Zealand using community pharmacy dispensing data and to examine the levels of suboptimal adherence in various population groups. Data from all community pharmacy dispensing databases in a New Zealand region were collected for a year covering 2005/2006 giving a near complete picture of dispensings to area residents. Prevalence of allopurinol use in the region by age, sex, ethnicity and socioeconomic position was calculated. Adherence was assessed using the medication possession ratio (MPR), with a MPR of 0.80 indicative of suboptimal adherence. Multiple logistic regression was used to explore variations in suboptimal adherence across population groups. A total of 953 people received allopurinol in the study year (prevalence 3%). Prevalence was higher in males (6%) than in females (1%) and Māori (5%) than non-Māori (3%). The overall MPR during the study was 0.88, with 161 (22%) of patients using allopurinol having suboptimal adherence. Non-Māori were 54% less likely to have suboptimal allopurinol adherence compared to Māori (95% CI 0.30-0.72, p = 0.001). These findings are consistent with those from other studies nationally and internationally and point to the important role for health professionals in improving patient adherence to an effective gout treatment.

  10. Characteristics and response of mouse bone marrow derived novel low adherent mesenchymal stem cells acquired by quantification of extracellular matrix

    PubMed Central

    Zheng, Ri-Cheng; Heo, Seong-Joo; Koak, Jai-Young; Lee, Joo-Hee; Park, Ji-Man

    2014-01-01

    PURPOSE The aim of present study was to identify characteristic and response of mouse bone marrow (BM) derived low-adherent bone marrow mesenchymal stem cells (BMMSCs) obtained by quantification of extracellular matrix (ECM). MATERIALS AND METHODS Non-adherent cells acquired by ECM coated dishes were termed low-adherent BMMSCs and these cells were analyzed by in vitro and in vivo methods, including colony forming unit fibroblast (CFU-f), bromodeoxyuridine (BrdU), multi-potential differentiation, flow cytometry and transplantation into nude mouse to measure the bone formation ability of these low-adherent BMMSCs. Titanium (Ti) discs with machined and anodized surfaces were prepared. Adherent and low-adherent BMMSCs were cultured on the Ti discs for testing their proliferation. RESULTS The amount of CFU-f cells was significantly higher when non-adherent cells were cultured on ECM coated dishes, which was made by 7 days culturing of adherent BMMSCs. Low-adherent BMMSCs had proliferation and differentiation potential as adherent BMMSCs in vitro. The mean amount bone formation of adherent and low-adherent BMMSCs was also investigated in vivo. There was higher cell proliferation appearance in adherent and low-adherent BMMSCs seeded on anodized Ti discs than machined Ti discs by time. CONCLUSION Low-adherent BMMSCs acquired by ECM from non-adherent cell populations maintained potential characteristic similar to those of the adherent BMMSCs and therefore could be used effectively as adherent BMMSCs in clinic. PMID:25352957

  11. Eikenella corrodens adherence to human buccal epithelial cells.

    PubMed Central

    Yamazaki, Y; Ebisu, S; Okada, H

    1981-01-01

    The mechanism of Eikenella corrodens adherence to human buccal epithelial cells in vitro was studied. Initial experiments to determine the optimal conditions for adherence of E. corrodens to buccal epithelial cells showed that adherence was dependent on time, temperature, bacterial concentration, and pH. Different strains of E. corrodens varied in their ability to adhere, and strain 1073 showed the greatest ability in adherence. Strain 1073 was selected for studies of adherence mechanisms. Trypsin treatment or heating (100 degrees C, 10 min) of the bacterial cells abolished their capacity to adhere to buccal epithelial cells. Treatment of buccal epithelial cells with trypsin also abolished adherence of E. corrodens 1073, whereas neuraminidase treatment of buccal epithelial cells enhanced the adherence. The adherence was inhibited by ethylenediaminetetraacetic acid and restored by adding Ca2+. The adherence was remarkably inhibited by sugars containing D-galactose and n-acetyl-D-galactosamine. Treatment of neuraminidase-treated epithelial cells with sodium metaperiodate or alpha- and beta-galactosidase did not decrease the adherence. These data suggest that adherence of E. corrodens 1073 to human buccal epithelial cells may require the interaction of lectin-like proteins on the bacterial surface with galactose-like receptors on the surface of epithelial cells. PMID:6260661

  12. Automated microinjection system for adherent cells

    NASA Astrophysics Data System (ADS)

    Youoku, Sachihiro; Suto, Yoshinori; Ando, Moritoshi; Ito, Akio

    2007-07-01

    We have developed an automated microinjection system that can handle more than 500 cells an hour. Microinjection injects foreign agents directly into cells using a micro-capillary. It can randomly introduce agents such as DNA, proteins and drugs into various types of cells. However, conventional methods require a skilled operator and suffer from low throughput. The new automated microinjection techniques we have developed consist of a Petri dish height measuring method and a capillary apex position measuring method. The dish surface height is measured by analyzing the images of cells that adhere to the dish surface. The contrast between the cell images is minimized when the focus plane of an object lens coincides with the dish surface. We have developed an optimized focus searching method with a height accuracy of +/-0.2 um. The capillary apex position detection method consists of three steps: rough, middle, and precise. These steps are employed sequentially to cover capillary displacements of up to +/-2 mm, and to ultimately accomplish an alignment accuracy of less than one micron. Experimental results using this system we developed show that it can introduce fluorescent material (Alexa488) into adherent cells, HEK293, with a success rate of 88.5%.

  13. Topography Influences Adherent Cell Regulation of Osteoclastogenesis.

    PubMed

    Nagasawa, M; Cooper, L F; Ogino, Y; Mendonca, D; Liang, R; Yang, S; Mendonca, G; Uoshima, K

    2016-03-01

    The importance of osteoclast-mediated bone resorption in the process of osseointegration has not been widely considered. In this study, cell culture was used to investigate the hypothesis that the function of implant-adherent bone marrow stromal cells (BMSCs) in osteoclastogenesis is influenced by surface topography. BMSCs isolated from femur and tibia of Sprague-Dawley rats were seeded onto 3 types of titanium surfaces (smooth, micro, and nano) and a control surface (tissue culture plastic) with or without osteogenic supplements. After 3 to 14 d, conditioned medium (CM) was collected. Subsequently, rat bone marrow-derived macrophages (BMMs) were cultured in media supplemented with soluble receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) as well as BMSC CM from each of the 4 surfaces. Gene expression levels of soluble RANKL, osteoprotegerin, tumor necrosis factor α, and M-CSF in cultured BMSCs at different time points were measured by real-time polymerase chain reaction. The number of differentiated osteoclastic cells was determined after tartrate-resistant acid phosphatase staining. Analysis of variance and t test were used for statistical analysis. The expression of prominent osteoclast-promoting factors tumor necrosis factor α and M-CSF was increased by BMSCs cultured on both micro- and nanoscale titanium topographies (P < 0.01). BMSC CM contained a heat-labile factor that increased BMMs osteoclastogenesis. CM from both micro- and nanoscale surface-adherent BMSCs increased the osteoclast number (P < 0.01). Difference in surface topography altered BMSC phenotype and influenced BMM osteoclastogenesis. Local signaling by implant-adherent cells at the implant-bone interface may indirectly control osteoclastogenesis and bone accrual around endosseous implants. PMID:26553885

  14. Active mechanics and geometry of adherent cells and cell colonies

    NASA Astrophysics Data System (ADS)

    Banerjee, Shiladitya

    2014-03-01

    Measurements of traction stresses exerted by adherent cells or cell colonies on elastic substrates have yielded new insight on how the mechanical and geometrical properties of the substrate regulate cellular force distribution, mechanical energy, spreading, morphology or stress ber architecture. We have developed a generic mechanical model of adherent cells as an active contractile gel mechanically coupled to an elastic substrate and to neighboring cells in a tissue. The contractile gel model accurately predicts the distribution of cellular and traction stresses as observed in single cell experiments, and captures the dependence of cell shape, traction stresses and stress ber polarization on the substrate's mechanical and geometrical properties. The model further predicts that the total strain energy of an adherent cell is solely regulated by its spread area, in agreement with recent experiments on micropatterned substrates with controlled geometry. When used to describe the behavior of colonies of adherent epithelial cells, the model demonstrates the crucial role of the mechanical cross-talk between intercellular and extracellular adhesion in regulating traction force distribution. Strong intercellular mechanical coupling organizes traction forces to the colony periphery, whereas weaker intercellular coupling leads to the build up of traction stresses at intercellular junctions. Furthermore, in agreement with experiments on large cohesive keratinocyte colonies, the model predicts a linear scaling of traction forces with the colony size. This scaling suggests the emergence of an effective surface tension as a scale-free material property of the adherent tissue, originating from actomyosin contractility.

  15. Late Adherent Human Bone Marrow Stromal Cells Form Bone and Restore the Hematopoietic Microenvironment In Vivo

    PubMed Central

    Vianna, Verônica Fernandes; Bonfim, Danielle Cabral; Cavalcanti, Amanda dos Santos; Fernandes, Marco Cury; Kahn, Suzana Assad; Casado, Priscila Ladeira; Lima, Inayá Correa; Murray, Samuel S.; Murray, Elsa J. Brochmann; Duarte, Maria Eugenia Leite

    2013-01-01

    Bone marrow stromal cells (BMSCs) are a valuable resource for skeletal regenerative medicine because of their osteogenic potential. In spite of the very general term “stem cell,” this population of cells is far from homogeneous, and different BMSCs clones have greatly different phenotypic properties and, therefore, potentially different therapeutic potential. Adherence to a culture flask surface is a primary defining characteristic of BMSCs. We hypothesized that based on the adherence time we could obtain an enriched population of cells with a greater therapeutic potential. We characterized two populations of bone marrow-derived cells, those that adhered by three days (R-cells) and those that did not adhere by three days but did by six days (L-cells). Clones derived from L-cells could be induced into adipogenic, chondrogenic, and osteogenic differentiation in vitro. L-cells appeared to have greater proliferative capacity, as manifested by larger colony diameter and clones with higher CD146 expression. Only clones from L-cells developed bone marrow stroma in vivo. We conclude that the use of late adherence of BMSCs is one parameter that can be used to enrich for cells that will constitute a superior final product for cell therapy in orthopedics. PMID:23710460

  16. Automated adherent human cell culture (mesenchymal stem cells).

    PubMed

    Thomas, Robert; Ratcliffe, Elizabeth

    2012-01-01

    Human cell culture processes developed at research laboratory scale need to be translated to large-scale production processes to achieve commercial application to a large market. To allow this transition of scale with consistent process performance and control of costs, it will be necessary to reduce manual processing and increase automation. There are a number of commercially available platforms that will reduce manual process intervention and improve process control for different culture formats. However, in many human cell-based applications, there is currently a need to remain close to the development format, usually adherent culture on cell culture plastic or matrix-coated wells or flasks due to deterioration of cell quality in other environments, such as suspension. This chapter presents an example method for adherent automated human stem cell culture using a specific automated flask handling platform, the CompacT SelecT.

  17. Adherence of Bilophila wadsworthia to cultured human embryonic intestinal cells.

    PubMed

    Gerardo, S H; Garcia, M M; Wexler, H M; Finegold, S M

    1998-02-01

    Adherence of Bilophila wadsworthia to the cultured human embryonic intestinal cell line, Intestine 407 (Int 407), varied among the strains tested from strongly adherent (76-100% cells positive for one or more adherent bacteria) to non- or weakly adherent (0-25% positive cells). Although negative staining revealed that infrequent cells of an adherent strain, WAL 9077, the adherent type-strain, WAL 7959, and a non-adherent strain, WAL 8448, expressed loosely associated fimbrial structures, a role for these structures in adhesion could not be confirmed with either scanning or thin-section electron micrography. Ruthenium red staining of thin-section preparations and subsequent electron microscopy failed to reveal an extensive extracellular polysaccharide layer. SDS-PAGE analysis of crude outer membrane fractions of WAL 9077 and WAL 8448 demonstrated clear differences in their major and minor outer membrane protein components. Thus, we postulate that the adherence of B. wadsworthia to Int 407 cells is mediated by an outer membrane or cell wall component. PMID:16887620

  18. Adherence to Mediterranean diet in a Spanish university population.

    PubMed

    García-Meseguer, María José; Burriel, Faustino Cervera; García, Cruz Vico; Serrano-Urrea, Ramón

    2014-07-01

    The aim of this work was to characterize food habits of Spanish University students and to assess the quality of their diet and some possible determinant factors according to Mediterranean food pattern among other indices. Two hundred eighty-four enrolled students during the academic year 2012-2013 participated in this survey. For each individual a questionnaire involving anthropometric measurements, types of housing, smoking habits and levels of physical activity were self-reported. Food consumption was gathered by two nonconsecutive 24 hour recalls including one weekend day. BMI within the normal range was showed by 72.5% of students and 75% of the sample reflected a sedentary lifestyle or low physical activity. The percentage of total energy from each macronutrient was approximately 17% proteins, 40% carbohydrates and 40% lipids. The ratio of polyunsaturated to monounsaturated fat only reached 0.32. Cholesterol consumption in men exceeded the intake in women by 70 mg/day but nutritional objectives were exceeded in both genders. The main source of protein had an animal origin from meat (38.1%), followed by cereals (19.4%) and dairy products (15.6%). The assessment of diet quality conducted by Healthy Eating Index (HEI) and Mediterranean Diet Score (MDS) revealed a low-intermediate score in both (51.2 ± 12.8 and 4.0 ± 1.5, respectively). The main deviations from Mediterranean pattern were a low intake of vegetables and fruit and a high consumption of meat and dairy products. According to HEI classification, 96.1% of subjects scored "poor" or "needs improvement" about the quality of their diet and only 5.3% of students achieved a high adherence to Mediterranean diet. It is necessary to foster changes toward a healthier diet pattern according to cultural context in this population for preventing cardiovascular diseases, type 2 diabetes and insulin resistance.

  19. Elasticity of adherent active cells on a compliant substrate

    NASA Astrophysics Data System (ADS)

    Banerjee, Shiladitya; Mertz, Aaron F.; Dufresne, Eric R.; Marchetti, M. Cristina

    2012-02-01

    We present a continuum mechanical model of rigidity sensing by livings cells adhering to a compliant substrate. The cell or cell colony is modeled as an elastic active gel, adapting recently developed continuum theories of active viscoelastic fluids. The coupling to the substrate enters as a boundary condition that relates the cell's deformation field to local stress gradients. In the presence of activity, the substrate induces spatially inhomogeneous contractile stresses and deformations, with a power law dependence of the total traction forces on cell or colony size. This is in agreement with recent experiments on keratinocyte colonies adhered to fibronectin coated surfaces. In the presence of acto-myosin activity, the substrate also enhances the cell polarization, breaking the cell's front-rear symmetry. Maximal polarization is observed when the substrate stiffness matches that of the cell, in agreement with experiments on stem cells.

  20. Microfluidic device capable of medium recirculation for non-adherent cell culture

    PubMed Central

    Dixon, Angela R.; Rajan, Shrinidhi; Kuo, Chuan-Hsien; Bersano, Tom; Wold, Rachel; Futai, Nobuyuki; Takayama, Shuichi; Mehta, Geeta

    2014-01-01

    We present a microfluidic device designed for maintenance and culture of non-adherent mammalian cells, which enables both recirculation and refreshing of medium, as well as easy harvesting of cells from the device. We demonstrate fabrication of a novel microfluidic device utilizing Braille perfusion for peristaltic fluid flow to enable switching between recirculation and refresh flow modes. Utilizing fluid flow simulations and the human promyelocytic leukemia cell line, HL-60, non-adherent cells, we demonstrate the utility of this RECIR-REFRESH device. With computer simulations, we profiled fluid flow and concentration gradients of autocrine factors and found that the geometry of the cell culture well plays a key role in cell entrapping and retaining autocrine and soluble factors. We subjected HL-60 cells, in the device, to a treatment regimen of 1.25% dimethylsulfoxide, every other day, to provoke differentiation and measured subsequent expression of CD11b on day 2 and day 4 and tumor necrosis factor-alpha (TNF-α) on day 4. Our findings display perfusion sensitive CD11b expression, but not TNF-α build-up, by day 4 of culture, with a 1:1 ratio of recirculation to refresh flow yielding the greatest increase in CD11b levels. RECIR-REFRESH facilitates programmable levels of cell differentiation in a HL-60 non-adherent cell population and can be expanded to other types of non-adherent cells such as hematopoietic stem cells. PMID:24753733

  1. Adherence of human basophils to cultured umbilical vein endothelial cells.

    PubMed Central

    Bochner, B S; Peachell, P T; Brown, K E; Schleimer, R P

    1988-01-01

    The mechanism by which circulating human basophils adhere to vascular endothelium and migrate to sites of allergic reactions is unknown. Agents have been identified which stimulate the adherence of purified basophils to cultured human umbilical vein vascular endothelial cells (HuVEC). Treatment of HuVEC with interleukin 1, tumor necrosis factor (TNF), bacterial endotoxin, and 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in time and dose-dependent increases of adhesiveness for basophils. Coincubation of basophils and HuVEC for 10 min with C5a, formyl-methionyl-leucyl-phenylalanine, the calcium ionophore A23187, platelet-activating factor, TNF, and TPA also resulted in significant dose-dependent increases in basophil adherence; this effect resulted from activation of the basophil. Adherence of basophils to HuVEC was time and temperature dependent, required divalent cations, and was unaffected by glucocorticoids. Monoclonal antibody 60.3, directed against the beta-subunit of the leukocyte adherence complex CD18, inhibited the binding of basophils to HuVEC. Adherence of basophils to vascular endothelium may be important in initiating basophil infiltrates in vivo. PMID:3130394

  2. Vaccine production: upstream processing with adherent or suspension cell lines.

    PubMed

    Genzel, Yvonne; Rödig, Jana; Rapp, Erdmann; Reichl, Udo

    2014-01-01

    The production of viral vaccines in cell culture can be accomplished with primary, diploid, or continuous (transformed) cell lines. Each cell line, each virus type, and each vaccine preparation require the specific design of upstream and downstream processing. Media have to be selected as well as production vessels, cultivation conditions, and modes of operation. Many viruses only replicate to high titers in adherently growing cells, but similar to processes established for recombinant protein production, an increasing number of suspension cell lines is being evaluated for future use. Here, we describe key issues to be considered for the establishment of large-scale virus production in bioreactors. As an example upstream processing of cell culture-derived influenza virus production is described in more detail for adherently growing and for suspension cells. In particular, use of serum-containing, serum-free, and chemically defined media as well as choice of cultivation vessel are considered. PMID:24297427

  3. Proliferative activity of vervet monkey bone marrow-derived adherent cells

    SciTech Connect

    Kramvis, A.; Garnett, H.M.

    1987-11-01

    Vervet monkey bone marrow-derived adherent cell population cultured in Fischer's medium supplemented with 12.5% fetal calf serum and 12.5% horse serum consists of two cell shapes: fusiform (type I) and polygonal (type II). Limiting-dilution cloning of the cells suggested that the two morphologically distinct cell types belong to the same cellular system even though they differ in their proliferative capabilities. The labeling index of type II cells, as measured by autoradiography, was found to be consistently lower than that of type I cells. It is probable that these two phenotypes represent different stages of differentiation, where progenitor type I gives rise to type II cells. The bone marrow-derived adherent cells were found to be cytokinetically at rest in vivo, using the thymidine suicide test, and relatively radioresistant with a D0 = 2.1 Gy and n = 2.36 at the time of explantation from the bone. Furthermore, in culture these cells are characterized by a relatively long cell cycle of 60 h, where the length of the S phase is 30 h, G2 is 12 h, M is 6 h, and G1 is 12 h. Thus, the vervet monkey bone marrow-derived adherent cells represent a cell population with a low turnover rate both in vivo and in vitro.

  4. Measuring Survival of Adherent Cells with the Colony-Forming Assay.

    PubMed

    Crowley, Lisa C; Christensen, Melinda E; Waterhouse, Nigel J

    2016-01-01

    Measuring cell death with colorimetric or fluorimetric dyes such as trypan blue and propidium iodide (PI) can provide an accurate measure of the number of dead cells in a population at a specific time; however, these assays cannot be used to distinguish cells that are dying or marked for future death. In many cases it is essential to measure the proliferative capacity of treated cells to provide an indirect measurement of cell death. This can be achieved using the colony-forming assay described here. This protocol specifically applies to measurement of HeLa cells but can be used for most adherent cell lines with limited motility. PMID:27480717

  5. Cell surface and transcriptional characterization of human adipose-derived adherent stromal (hADAS) cells.

    PubMed

    Katz, Adam J; Tholpady, Ashok; Tholpady, Sunil S; Shang, Hulan; Ogle, Roy C

    2005-03-01

    Adult human subcutaneous adipose tissue contains cells with intriguing multilineage developmental plasticity, much like marrow-derived mesenchymal stem cells. Putative stem or progenitor cells from fat have been given many different names in the literature, reflecting an early and evolving consensus regarding their phenotypic characterization. The study reported here used microarrays to evaluate over 170 genes relating to angiogenesis and extracellular matrix in undifferentiated, early-passage human adipose-derived adherent stromal (hADAS) cells isolated from three separate donors. The hADAS populations unanimously transcribed 66% of the screened genes, and 83% were transcribed by at least two of the three populations. The most highly transcribed genes relate to functional groupings such as cell adhesion, matrix proteins, growth factors and receptors, and proteases. The transcriptome of hADAS cells demonstrated by this work reveals many similarities to published profiles of bone marrow mesenchymal stem cells (MSCs). In addition, flow analysis of over 24 hADAS cell surface proteins (n = 7 donors) both confirms and expands on the existing literature and reveals strong intergroup correlation, despite an inconsistent nomenclature and the lack of standardized protocols for cell isolation and culture. Finally, based on flow analysis and reverse transcription polymerase chain reaction studies, our results suggest that hADAS cells do not express several proteins that are implicated as markers of "stemness" in other stem cell populations, including telomerase, CD133, and the membrane transporter ABCG2.

  6. ENHANCE—(Electronic Hydroxyurea Adherence): A Protocol to Increase Hydroxyurea Adherence in Patients with Sickle Cell Disease

    PubMed Central

    Chisolm, Deena J; O’Brien, Sarah H

    2016-01-01

    Background Hydroxyurea (HU) is the only disease-modifying medication for patients with sickle cell disease (SCD). HU can reduce SCD-related complications but only 35% to 50% of pediatric patients adhere to HU at the rates achieved in clinical trials and this limits its clinical effectiveness. Mobile Directly Observed Therapy (Mobile DOT) is a pilot-tested, electronic, multidimensional, HU adherence intervention that targets many components of the Health Behavior Model. Objective The aim of this study is to evaluate the impact of Mobile DOT on HU adherence in children with SCD. The objective of our study is to inform the development of future adherence interventions and pediatric SCD protocols. Methods This is a single-arm crossover study of pediatric patients with SCD. Participants self-record videos of their daily HU administrations and receive text message alerts to take HU, feedback on their HU adherence, and incentives when they achieve adherence goals during the 6-month Mobile DOT phase. Participants’ HU adherence during the Mobile DOT phase is compared with their baseline HU adherence (6 months prior to study entry) and to their HU adherence 6 months after completing the Mobile DOT phase. The primary outcome of this study is HU adherence measured by medication possession ratio. Results The trial is ongoing. Preliminary review of participant satisfaction results suggest that most participants can complete Mobile DOT in less than 5 minutes per day and are satisfied with the intervention. Conclusions If effective, the Mobile DOT strategy will increase HU adherence and this could improve patients’ clinical outcomes and reduce costs of care. PMID:27697749

  7. Optical painting and fluorescence activated sorting of single adherent cells labelled with photoswitchable Pdots

    PubMed Central

    Kuo, Chun-Ting; Thompson, Alison M.; Gallina, Maria Elena; Ye, Fangmao; Johnson, Eleanor S.; Sun, Wei; Zhao, Mengxia; Yu, Jiangbo; Wu, I-Che; Fujimoto, Bryant; DuFort, Christopher C.; Carlson, Markus A.; Hingorani, Sunil R.; Paguirigan, Amy L.; Radich, Jerald P.; Chiu, Daniel T.

    2016-01-01

    The efficient selection and isolation of individual cells of interest from a mixed population is desired in many biomedical and clinical applications. Here we show the concept of using photoswitchable semiconducting polymer dots (Pdots) as an optical ‘painting' tool, which enables the selection of certain adherent cells based on their fluorescence, and their spatial and morphological features, under a microscope. We first develop a Pdot that can switch between the bright (ON) and dark (OFF) states reversibly with a 150-fold contrast ratio on irradiation with ultraviolet or red light. With a focused 633-nm laser beam that acts as a ‘paintbrush' and the photoswitchable Pdots as the ‘paint', we select and ‘paint' individual Pdot-labelled adherent cells by turning on their fluorescence, then proceed to sort and recover the optically marked cells (with 90% recovery and near 100% purity), followed by genetic analysis. PMID:27118210

  8. Electroporation chip for adherent cells on photochemically modified polymer surfaces

    NASA Astrophysics Data System (ADS)

    Olbrich, Michael; Rebollar, Esther; Heitz, Johannes; Frischauf, Irene; Romanin, Christoph

    2008-01-01

    We present a polytetrafluoroethylene electroporation microchip with integrated electrodes for transfection of adherent biological cells. For fabrication, UV-surface modification was employed in combination with metal deposition. UV irradiation in reactive atmosphere resulted in introduction of polar chemical groups into the polytetrafluoroethylene surface for significant adhesion enhancement of both biological cells as well as metal electrodes thereon. Electroporation was demonstrated by transfection of human embryonic kidney cells with the enhanced green fluorescent protein. Transparent, working at low voltages, and easy to handle, this chip yields the potential to reduce the amount of sequential working steps necessary for transfection.

  9. Antiretroviral Drug Diversion Links Social Vulnerability to Poor Medication Adherence in Substance Abusing Populations

    PubMed Central

    Tsuyuki, Kiyomi; Surratt, Hilary L.

    2015-01-01

    Antiretroviral (ARV) medication diversion to the illicit market has been documented in South Florida, and linked to sub-optimal adherence in people living with HIV. ARV diversion reflects an unmet need for care in vulnerable populations that have difficulty engaging in consistent HIV care due to competing needs and co-morbidities. This study applies the Gelberg-Andersen Behavioral Model of Health Care Utilization for Vulnerable Populations to understand how social vulnerability is linked to ARV diversion and adherence. Cross-sectional data were collected from a targeted sample of vulnerable people living with HIV in South Florida between 2010 and 2012 (n=503). Structured interviews collected quantitative data on ARV diversion, access and utilization of care, and ARV adherence. Logistic regression was used to estimate the goodness-of-fit of additive models that test domain fit. Linear regression was used to estimate the effects of social vulnerability and ARV diversion on ARV adherence. The best fitting model to predict ARV diversion identifies having a low monthly income and unstable HIV care as salient enabling factors that promote ARV diversion. Importantly, health care need factors did not protect against ARV diversion, evidence that immediate competing needs are prioritized even in the face of poor health for this sample. We also find that ARV diversion provides a link between social vulnerability and sub-optimal ARV adherence, with ARV diversion and domains from the Behavioral Model explaining 25% of the variation in ARV adherence. Our analyses reveal great need to improve engagement in HIV care for vulnerable populations by strengthening enabling factors (e.g. patient-provider relationship) to improve retention in HIV care and ARV adherence for vulnerable populations. PMID:25893656

  10. Antiretroviral drug diversion links social vulnerability to poor medication adherence in substance abusing populations.

    PubMed

    Tsuyuki, Kiyomi; Surratt, Hilary L

    2015-05-01

    Antiretroviral (ARV) medication diversion to the illicit market has been documented in South Florida, and linked to sub-optimal adherence in people living with HIV. ARV diversion reflects an unmet need for care in vulnerable populations that have difficulty engaging in consistent HIV care due to competing needs and co-morbidities. This study applies the Gelberg-Andersen behavioral model of health care utilization for vulnerable populations to understand how social vulnerability is linked to ARV diversion and adherence. Cross-sectional data were collected from a targeted sample of vulnerable people living with HIV in South Florida between 2010 and 2012 (n = 503). Structured interviews collected quantitative data on ARV diversion, access and utilization of care, and ARV adherence. Logistic regression was used to estimate the goodness-of-fit of additive models that test domain fit. Linear regression was used to estimate the effects of social vulnerability and ARV diversion on ARV adherence. The best fitting model to predict ARV diversion identifies having a low monthly income and unstable HIV care as salient enabling factors that promote ARV diversion. Importantly, health care need factors did not protect against ARV diversion, evidence that immediate competing needs are prioritized even in the face of poor health for this sample. We also find that ARV diversion provides a link between social vulnerability and sub-optimal ARV adherence, with ARV diversion and domains from the Behavioral Model explaining 25 % of the variation in ARV adherence. Our analyses reveal great need to improve engagement in HIV care for vulnerable populations by strengthening enabling factors (e.g. patient-provider relationship) to improve retention in HIV care and ARV adherence for vulnerable populations.

  11. Dynamic mechanical measurement of the viscoelasticity of single adherent cells

    NASA Astrophysics Data System (ADS)

    Corbin, Elise A.; Adeniba, Olaoluwa O.; Ewoldt, Randy H.; Bashir, Rashid

    2016-02-01

    Many recent studies on the viscoelasticity of individual cells link mechanics with cellular function and health. Here, we introduce a measurement of the viscoelastic properties of individual human colon cancer cells (HT-29) using silicon pedestal microelectromechanical systems (MEMS) resonant sensors. We demonstrate that the viscoelastic properties of single adherent cells can be extracted by measuring a difference in vibrational amplitude of our resonant sensor platform. The magnitude of vibration of the pedestal sensor is measured using a laser Doppler vibrometer (LDV). A change in amplitude of the sensor, compared with the driving amplitude (amplitude ratio), is influenced by the mechanical properties of the adhered cells. The amplitude ratio of the fixed cells was greater than the live cells, with a p-value <0.0001. By combining the amplitude shift with the resonant frequency shift measure, we determined the elastic modulus and viscosity values of 100 Pa and 0.0031 Pa s, respectively. Our method using the change in amplitude of resonant MEMS devices can enable the determination of a refined solution space and could improve measuring the stiffness of cells.

  12. Factors that Influence Adherence to Antiretroviral Treatment in an Urban Population, Jakarta, Indonesia

    PubMed Central

    Weaver, Emma Rosamond Nony; Pane, Masdalina; Wandra, Toni; Windiyaningsih, Cicilia; Herlina; Samaan, Gina

    2014-01-01

    Introduction Although the number of people receiving antiretroviral therapy (ART) in Indonesia has increased in recent years, little is known about the specific characteristics affecting adherence in this population. Indonesia is different from most of its neighbors given that it is a geographically and culturally diverse country, with a large Muslim population. We aimed to identify the current rate of adherence and explore factors that influence ART adherence. Methods Data were collected from ART-prescribed outpatients on an HIV registry at a North Jakarta hospital in 2012. Socio-demographic and behavioral characteristics were explored as factors associated with adherence using logistics regression analyses. Chi squared test was used to compare the difference between proportions. Reasons for missing medication were analyzed descriptively. Results Two hundred and sixty-one patients participated, of whom 77% reported ART adherence in the last 3 months. The level of social support experienced was independently associated with adherence where some social support (p = 0.018) and good social support (p = 0.039) improved adherence compared to poor social support. Frequently cited reasons for not taking ART medication included forgetting to take medication (67%), busy with something else (63%) and asleep at medication time (60%). Discussion This study identified that an increase in the level of social support experienced by ART-prescribed patients was positively associated with adherence. Social support may minimize the impact of stigma among ART prescribed patients. Based on these findings, if social support is not available, alternative support through community-based organizations is recommended to maximize treatment success. PMID:25229671

  13. pH changes during in vitro adherence of Escherichia coli to HeLa cells.

    PubMed Central

    McCabe, K; Mann, M D; Bowie, M D

    1994-01-01

    Escherichia coli-induced acidic pH conditions were observed during the in vitro adherence of E. coli to HeLa cells. No pH changes occurred in the absence of adherence. This suggests that adherence affects the function or interaction of HeLa cells and E. coli. PMID:7927801

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

    PubMed

    Hoffmann, Else Kay

    2011-01-01

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

  15. Contractile Film Model for Polymorphism in Adherent Cells

    NASA Astrophysics Data System (ADS)

    Banerjee, Shiladitya; Giomi, Luca

    2013-03-01

    The optimal shapes attained by contractile cells on elastic substrates are determined by the crosstalk between intracellular forces and extracellular forces of adhesion. We model an adherent stationary cell as a contractile film bounded by an elastic cortex and connected to the substrate via elastic links. When the adhesion sites are continuously distributed, optimal cell shape is constrained by the adhesion geometry, with a spread area sensitively dependent on the substrate stiffness and contractile tension. For discrete adhesion sites, equilibrium cell shape is convex at weak contractility, while developing local concavities at intermediate values of contractility. Increasing contractility beyond a critical value, controlled by substrate stiffness, cell contour undergoes a discontinuous transition to a star-shaped configuration with cusps and protrusions, accompanied by a region of bistability and hysteresis.

  16. Strategies to improve adherence to medications for cardiovascular diseases in socioeconomically disadvantaged populations: a systematic review.

    PubMed

    Laba, Tracey-Lea; Bleasel, Jonathan; Brien, Jo-Anne; Cass, Alan; Howard, Kirsten; Peiris, David; Redfern, Julie; Salam, Abdul; Usherwood, Tim; Jan, Stephen

    2013-09-10

    Medication non-adherence poses a major barrier to reducing cardiovascular disease (CVD) burden globally, and is increasingly recognised as a socioeconomically determined problem. Strategies promoting CVD medication adherence appear of moderate effectiveness and cost-effectiveness. Potentially, 'one-size-fits-all' measures are ill-equipped to address heterogeneous adherence behaviour between social groups. This review aims to determine the effects of strategies to improve adherence to CVD-related medications in socioeconomically disadvantaged groups. Randomised/quasi-randomised controlled trials (1996-June 2012, English), testing strategies to increase adherence to CVD-related medications prescribed to adult patients who may experience health inequity (place of residence, occupation, education, or socioeconomic position) were reviewed. 772 abstracts were screened, 111 full-text articles retrieved, and 16 full-text articles reporting on 14 studies, involving 7739 patients (age range 41-66 years), were included. Methodological and clinical heterogeneity precluded quantitative data synthesis. Studies were thematically grouped by targeted outcomes; underlying interventions and policies were classified using Michie et al.'s Behaviour Change Wheel. Contrasting with patient or physician/practice strategies, those simultaneously directed at patients and physicians/practices resulted in statistically significant improvements in relative adherence (16-169%). Comparative cost and cost-effectiveness analyses from three studies did not find cost-saving or cost-effective strategies. Unlike much current evidence in general populations, promising evidence exists about what strategies improve adherence in disadvantaged groups. These strategies were generally complex: simultaneously targeting patients and physicians; addressing social, financial, and treatment-related adherence barriers; and supported by broader guidelines, regulatory and communication-based policies. Given their

  17. A fully automated system for adherent cells microinjection.

    PubMed

    Becattini, Gabriele; Mattos, Leonardo S; Caldwell, Darwin G

    2014-01-01

    This paper proposes an automated robotic system to perform cell microinjections to relieve human operators from this highly difficult and tedious manual procedure. The system, which uses commercial equipment currently found on most biomanipulation laboratories, consists of a multitask software framework combining computer vision and robotic control elements. The vision part features an injection pipette tracker and an automatic cell targeting system that is responsible for defining injection points within the contours of adherent cells in culture. The main challenge is the use of bright-field microscopy only, without the need for chemical markers normally employed to highlight the cells. Here, cells are identified and segmented using a threshold-based image processing technique working on defocused images. Fast and precise microinjection pipette positioning over the automatically defined targets is performed by a two-stage robotic system which achieves an average injection rate of 7.6 cells/min with a pipette positioning precision of 0.23 μm. The consistency of these microinjections and the performance of the visual targeting framework were experimentally evaluated using two cell lines (CHO-K1 and HEK) and over 500 cells. In these trials, the cells were automatically targeted and injected with a fluorescent marker, resulting in a correct cell detection rate of 87% and a successful marker delivery rate of 67.5%. These results demonstrate that the new system is capable of better performances than expert operators, highlighting its benefits and potential for large-scale application. PMID:24403406

  18. A fully automated system for adherent cells microinjection.

    PubMed

    Becattini, Gabriele; Mattos, Leonardo S; Caldwell, Darwin G

    2014-01-01

    This paper proposes an automated robotic system to perform cell microinjections to relieve human operators from this highly difficult and tedious manual procedure. The system, which uses commercial equipment currently found on most biomanipulation laboratories, consists of a multitask software framework combining computer vision and robotic control elements. The vision part features an injection pipette tracker and an automatic cell targeting system that is responsible for defining injection points within the contours of adherent cells in culture. The main challenge is the use of bright-field microscopy only, without the need for chemical markers normally employed to highlight the cells. Here, cells are identified and segmented using a threshold-based image processing technique working on defocused images. Fast and precise microinjection pipette positioning over the automatically defined targets is performed by a two-stage robotic system which achieves an average injection rate of 7.6 cells/min with a pipette positioning precision of 0.23 μm. The consistency of these microinjections and the performance of the visual targeting framework were experimentally evaluated using two cell lines (CHO-K1 and HEK) and over 500 cells. In these trials, the cells were automatically targeted and injected with a fluorescent marker, resulting in a correct cell detection rate of 87% and a successful marker delivery rate of 67.5%. These results demonstrate that the new system is capable of better performances than expert operators, highlighting its benefits and potential for large-scale application.

  19. Photosensitizer Adhered to Cell Culture Microplates Induces Phototoxicity in Carcinoma Cells

    PubMed Central

    Ziegler, Verena; Kiesslich, Tobias; Krammer, Barbara; Plaetzer, Kristjan

    2013-01-01

    In vitro experiments in plastic receptacles are the basis of characterization of new photosensitizers (PSs) for the photodynamic therapy. We recently reported that lipophilic PSs adhere to cell culture microplates in a kinetic-like manner (Engelhardt et al., 2011). In the current study, we examined the interaction and phototoxic effects of the microplate-adhered PS in cancer cells. Therefore, we preloaded microplates with hypericin, Foscan, PVP-hypericin, or aluminum (III) phthalocyanine tetrasulfonate chloride (AlPCS4) for 24 hours and measured the PS distribution after addition of A431 human carcinoma cells: following another 24 hours up to 68% of hypericin were detected in the cell fraction. The hydrophilic PVP-hypericin and AlPCS4 also diffused into the cells, but the quantities of PS adherence were considerably lower. Microplate-adhered Foscan appeared not to be redistributed. In contrast to the hydrophilic PSs, the cellular phototoxicity of microplate-adhered lipophilic PS was high, independent of whether the PS (i) was pre-loaded onto microplates or (ii) added simultaneously with the cells or (iii) one day after cell seeding. Based on these results, we suggest testing lipophilic PS dyes for their adherence to microplates. Furthermore, the ability of plastic materials to (reversibly) store PSs might represent a new approach for the PS delivery or the development of antimicrobial coatings. PMID:23509741

  20. Computational Tension Mapping of Adherent Cells Based on Actin Imaging

    PubMed Central

    Manifacier, Ian; Milan, Jean-Louis; Jeanneau, Charlotte; Chmilewsky, Fanny; Chabrand, Patrick; About, Imad

    2016-01-01

    Forces transiting through the cytoskeleton are known to play a role in adherent cell activity. Up to now few approaches haves been able to determine theses intracellular forces. We thus developed a computational mechanical model based on a reconstruction of the cytoskeleton of an adherent cell from fluorescence staining of the actin network and focal adhesions (FA). Our custom made algorithm converted the 2D image of an actin network into a map of contractile interactions inside a 2D node grid, each node representing a group of pixels. We assumed that actin filaments observed under fluorescence microscopy, appear brighter when thicker, we thus presumed that nodes corresponding to pixels with higher actin density were linked by stiffer interactions. This enabled us to create a system of heterogeneous interactions which represent the spatial organization of the contractile actin network. The contractility of this interaction system was then adapted to match the level of force the cell truly exerted on focal adhesions; forces on focal adhesions were estimated from their vinculin expressed size. This enabled the model to compute consistent mechanical forces transiting throughout the cell. After computation, we applied a graphical approach on the original actin image, which enabled us to calculate tension forces throughout the cell, or in a particular region or even in single stress fibers. It also enabled us to study different scenarios which may indicate the mechanical role of other cytoskeletal components such as microtubules. For instance, our results stated that the ratio between intra and extra cellular compression is inversely proportional to intracellular tension. PMID:26812601

  1. Adherence to cardiovascular medications in the South Asian population: A systematic review of current evidence and future directions

    PubMed Central

    Akeroyd, Julia M; Chan, Winston J; Kamal, Ayeesha K; Palaniappan, Latha; Virani, Salim S

    2015-01-01

    AIM: To review methods of assessing adherence and strategies to improve adherence to cardiovascular disease (CVD) medications, among South Asian CVD patients. METHODS: We conducted a systematic review of English language studies that examined CVD medication adherence in South Asian populations from 1966 to April 1, 2015 in SCOPUS and PubMed. Working in duplicate, we identified 61 studies. After exclusions, 26 studies were selected for full text review. Of these, 17 studies were included in the final review. We abstracted data on several factors including study design, study population, method of assessing adherence and adherence rate. RESULTS: These studies were conducted in India (n = 11), Pakistan (n = 3), Bangladesh (n = 1), Nepal (n = 1) and Sri Lanka (n = 1). Adherence rates ranged from 32%-95% across studies. Of the 17 total publications included, 10 focused on assessing adherence to CVD medications and 7 focused on assessing the impact of interventions on medication adherence. The validated Morisky Medication Adherence Scale (MMAS) was used as the primary method of assessing adherence in five studies. Three studies used validated questionnaires similar to the MMAS, and one study utilized Medication Event Monitoring System caps, with the remainder of the studies utilizing pill count and self-report measures. As expected, studies using non-validated self-report measures described higher rates of adherence than studies using validated scale measurements and pill count. The included intervention studies examined the use of polypill therapy, provider education and patient counseling to improve medication adherence. CONCLUSION: The overall medication adherence rates were low in the region, which suggest a growing need for future interventions to improve adherence. PMID:26730300

  2. Role of sulfated glycans in adherence of the microsporidian Encephalitozoon intestinalis to host cells in vitro.

    PubMed

    Hayman, J Russell; Southern, Timothy R; Nash, Theodore E

    2005-02-01

    Microsporidia are obligate intracellular opportunistic protists that infect a wide variety of animals, including humans, via environmentally resistant spores. Infection requires that spores be in close proximity to host cells so that the hollow polar tube can pierce the cell membrane and inject the spore contents into the cell cytoplasm. Like other eukaryotic microbes, microsporidia may use specific mechanisms for adherence in order to achieve target cell proximity and increase the likelihood of successful infection. Our data show that Encephalitozoon intestinalis exploits sulfated glycans such as the cell surface glycosaminoglycans (GAGs) in selection of and attachment to host cells. When exogenous sulfated glycans are used as inhibitors in spore adherence assays, E. intestinalis spore adherence is reduced by as much as 88%. However, there is no inhibition when nonsulfated glycans are used, suggesting that E. intestinalis spores utilize sulfated host cell glycans in adherence. These studies were confirmed by exposure of host cells to xylopyranoside, which limits host cell surface GAGs, and sodium chlorate, which decreases surface sulfation. Spore adherence studies with CHO mutant cell lines that are deficient in either surface GAGs or surface heparan sulfate also confirmed the necessity of sulfated glycans. Furthermore, when spore adherence is inhibited, host cell infection is reduced, indicating a direct association between spore adherence and infectivity. These data show that E. intestinalis specifically adheres to target cells by way of sulfated host cell surface GAGs and that this mechanism serves to enhance infectivity.

  3. Interventions to Increase Medication Adherence in African-American and Latino Populations: A Literature Review

    PubMed Central

    Juarez, Deborah Taira; Yeboah, Michelle; Castillo, Theresa P

    2014-01-01

    The objective of this systematic review was to investigate the effectiveness of interventions to improve medication adherence in ethnic minority populations. A literature search from January 2000 to August 2012 was conducted through PubMed/Medline, Web of Science, The Cochrane Library, and Google Scholar. Search terms used included: medication (MeSH), adherence, medication adherence (MeSH), compliance (MeSH), persistence, race, ethnicity, ethnic groups (MeSH), minority, African-American, Hispanic, Latino, Asian, Pacific Islander, and intervention. Studies which did not have ≥75% of the sample population comprised of individuals of any one ethnic background were excluded, unless the authors performed sub-group analyses by race/ethnicity. Of the 36 studies identified, 20 studies showed significant post-intervention differences. Sample population sizes ranged from 10 to 520, with a median of 126.5. The studies in this review were conducted with patients of mainly African-American and Latino descent. No studies were identified which focused on Asians, Pacific Islanders, or Native Americans. Interventions demonstrating mixed results included motivational interviewing, reminder devices, community health worker (CHW) delivered interventions, and pharmacist-delivered interventions. Directly observed therapy (DOT) was a successful intervention in two studies. Interventions which did not involve human contact with patients were ineffective. In this literature review, studies varied significantly in their methods and design as well as the populations studied. There was a lack of congruence among studies in the way adherence was measured and reported. No single intervention has been seen to be universally successful, particularly for patients from ethnic minority backgrounds. PMID:24470982

  4. Interventions to increase medication adherence in African-American and Latino populations: a literature review.

    PubMed

    Hu, Daniel; Juarez, Deborah Taira; Yeboah, Michelle; Castillo, Theresa P

    2014-01-01

    The objective of this systematic review was to investigate the effectiveness of interventions to improve medication adherence in ethnic minority populations. A literature search from January 2000 to August 2012 was conducted through PubMed/Medline, Web of Science, The Cochrane Library, and Google Scholar. Search terms used included: medication (MeSH), adherence, medication adherence (MeSH), compliance (MeSH), persistence, race, ethnicity, ethnic groups (MeSH), minority, African-American, Hispanic, Latino, Asian, Pacific Islander, and intervention. Studies which did not have ≥75% of the sample population comprised of individuals of any one ethnic background were excluded, unless the authors performed sub-group analyses by race/ethnicity. Of the 36 studies identified, 20 studies showed significant post-intervention differences. Sample population sizes ranged from 10 to 520, with a median of 126.5. The studies in this review were conducted with patients of mainly African-American and Latino descent. No studies were identified which focused on Asians, Pacific Islanders, or Native Americans. Interventions demonstrating mixed results included motivational interviewing, reminder devices, community health worker (CHW) delivered interventions, and pharmacist-delivered interventions. Directly observed therapy (DOT) was a successful intervention in two studies. Interventions which did not involve human contact with patients were ineffective. In this literature review, studies varied significantly in their methods and design as well as the populations studied. There was a lack of congruence among studies in the way adherence was measured and reported. No single intervention has been seen to be universally successful, particularly for patients from ethnic minority backgrounds.

  5. Evaluation of a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay (Keystone Sym)

    EPA Science Inventory

    Our goal is to establish an in vitro model system to evaluate chemical effects using a single stem cell culture technique that would improve throughput and provide quantitative markers of differentiation and cell number. To this end, we have used an adherent cell differentiation ...

  6. [Observation on the biological behavior of human umbilical cord blood adherent cells].

    PubMed

    Zhang, Xi; Wang, Pin; Chen, Xing-Hua; Liu, Lin; Peng, Xian-Gui; Kong, Pei-Yan; Liu, Hong; Zhang, Yi; Wang, Qing-Yu

    2005-02-01

    To study the possibility of separation and culture of human umbilical cord blood adherent cell (HUCBAC), the umbilical cord blood CD34(+) cells were cultured in Dexter system in order to evaluate and observe the biological behavior of adherent cells in vitro. The results showed that all cells were cultured with Dexter system. By day 9-14 (at a median of 11.2 days), adherent cell colonies formed and reached their maximum at 15-22 days (mean 19.6 days), by day 28, all adherent cells spread over the bottom of Petri dish. By means of light microscopy, these cells were found to differentiate into three kinds of cells in culture of 28 days: fibroblast-liked cell, macrophage liked cell and small-round cells. The ratio of these three kinds of cells was 56.8%, 38%, 5.5% respectively. Cytochemistry assay revealed that the positive rate reached 100% in NSE stain and PAS stain; the adherent cell by ALP stain were shown 35% positive, but in POX stain the result was negative. Immunohistochemistry stain revealed that the positive rate of cord adherent cells for CD106, CD29, CD44, CD45, CD50, Fn, Ln, collagen IV etc reached 96%, 93%, 98%, 68%, 72%, 92%, 74%, 83% respectively. It is concluded there are hematopoietic adherent precursors in cord blood CD34(+) cells and the HUCBAC shows some biological behavior of hematopoietic stromal cells.

  7. Heparin-mediated inhibition of Chlamydia psittaci adherence to HeLa cells.

    PubMed

    Gutiérrez-Martín, C B; Ojcius, D M; Hsia, R; Hellio, R; Bavoil, P M; Dautry-Varsat, A

    1997-01-01

    The adherence of human strains of Chlamydia trachomatis has been recently shown to be inhibitable by heparin and heparitinase, leading to the proposal that Chlamydia binding to host cells may be mediated by a glycosaminoglycan (GAG)-dependent mechanism. We here describe the adherence of the guinea-pig pathogen, Chlamydia psittaci GPIC, to HeLa cells, which was measured by cytofluorometry with chlamydiae whose DNA was fluorescently labelled. Adherence could be inhibited by heat or trypsin pretreatment of the bacteria, and binding was much faster at 37 degrees C (reaching a plateau within 1 h) than 4 degrees C. Little binding remained when host cells were pre-fixed with paraformaldehyde, suggesting that host cell receptor mobility may be required for effective adherence. Visualization by confocal microscopy confirmed that the bacteria were at or near the host cell surface during the entire time-course of these experiments. Adherence increased as a function of pH between pH 6 and pH 8.0-8.5. Both adherence and infection of HeLa cells could be inhibited with heparin when the adherence step was performed at 4 degrees C, but only infection was inhibited when the adherence step was performed at 37 degrees C, even though heparitinase could block adherence at either 4 degrees C or 37 degrees C. Even at 4 degrees C, heparin-mediated inhibition was significantly lower at pH 8 than pH 7.4, suggesting that GAG-independent mechanisms may play a role in the higher adherence observed at basic pH. These results therefore demonstrate that a GAG-dependent adherence step may be operative in C. psittaci, and raise the possibility that other adherence mechanisms may also contribute to binding by this chlamydial strain. Furthermore, they suggest that there may not be a strict correlation between C. psittaci adherence and the ability to cause productive infections.

  8. Surface modification of closed plastic bags for adherent cell cultivation

    NASA Astrophysics Data System (ADS)

    Lachmann, K.; Dohse, A.; Thomas, M.; Pohl, S.; Meyring, W.; Dittmar, K. E. J.; Lindenmeier, W.; Klages, C.-P.

    2011-07-01

    In modern medicine human mesenchymal stem cells are becoming increasingly important. However, a successful cultivation of this type of cells is only possible under very specific conditions. Of great importance, for instance, are the absence of contaminants such as foreign microbiological organisms, i.e., sterility, and the chemical functionalization of the ground on which the cells are grown. As cultivation of these cells makes high demands, a new procedure for cell cultivation has been developed in which closed plastic bags are used. For adherent cell growth chemical functional groups have to be introduced on the inner surface of the plastic bag. This can be achieved by a new, atmospheric-pressure plasma-based method presented in this paper. The method which was developed jointly by the Fraunhofer IST and the Helmholtz HZI can be implemented in automated equipment as is also shown in this contribution. Plasma process gases used include helium or helium-based gas mixtures (He + N2 + H2) and vapors of suitable film-forming agents or precursors such as APTMS, DACH, and TMOS in helium. The effect of plasma treatment is investigated by FTIR-ATR spectroscopy as well as surface tension determination based on contact angle measurements and XPS. Plasma treatment in nominally pure helium increases the surface tension of the polymer foil due to the presence of oxygen traces in the gas and oxygen diffusing through the gas-permeable foil, respectively, reacting with surface radical centers formed during contact with the discharge. Primary amino groups are obtained on the inner surface by treatment in mixtures with nitrogen and hydrogen albeit their amount is comparably small due to diffusion of oxygen through the gas-permeable bag, interfering with the plasma-amination process. Surface modifications introducing amino groups on the inner surface turned out to be most efficient in the promotion of cell growth.

  9. Adherence of Candida to cultured vascular endothelial cells: mechanisms of attachment and endothelial cell penetration.

    PubMed

    Rotrosen, D; Edwards, J E; Gibson, T R; Moore, J C; Cohen, A H; Green, I

    1985-12-01

    To elucidate the pathogenesis of hematogenous Candida infections, we developed an in vitro model of Candida adherence to and penetration of human endothelial cells. We enhanced or inhibited adherence in order to probe mechanisms of attachment. Adherence of Candida albicans showed a linear relation to Candida inoculum (range, 10(2)-10(5) cfu, r = .99, P less than .01) and exceeded that of less virulent Candida species and that of Saccharomyces cerevisiae (P less than .01). Candida immune serum blocked attachment (greater than 95% inhibition; P less than .001), however, this activity was abolished by immunoprecipitation of immune serum with C. albicans mannan (P less than .001) and was unaffected by immunoprecipitation with S. cerevisiae mannan or by adsorption with particulate chitin. Adherence was diminished by exposing C. albicans to heat (greater than 99% inhibition; P less than .01), UV light (98% inhibition; P less than .01), or sodium periodate (greater than 72% inhibition; P less than .01). An extract from heat-exposed C. albicans blocked adherence (greater than 51% inhibition; P less than .001). Transmission electron microscopy demonstrated that viable or killed Candida organisms were attached to endothelial cells, were enveloped by membrane processes from the endothelial cell surface, and were incorporated into the endothelial cells within phagosomes. Cytochalasin B blocked incorporation without blocking surface attachment. PMID:3905987

  10. Self-reported adherence to antiretroviral therapy in HIV+ population from Bata, Equatorial Guinea.

    PubMed

    Salmanton-García, Jon; Herrador, Zaida; Ruiz-Seco, Pilar; Nzang-Esono, Jesús; Bendomo, Veronica; Bashmakovic, Emma; Nseng-Nchama, Gloria; Benito, Agustín; Aparicio, Pilar

    2016-01-01

    The human immunodeficiency virus (HIV) and the acquired immune deficiency syndrome (AIDS) represent a serious public health problem in Equatorial Guinea, with a prevalence of 6.2% among adults. the high-activity antiretroviral treatment (HAART) coverage data is 10 points below the overall estimate for Sub-Saharan Africa, and only 61% patients continue with HAART 12 months after it started. This study aims to assess HAART adherence and related factors in Litoral Province of Equatorial Guinea. In this cross-sectional study, socio-demographic and clinical data were collected at Regional Hospital of Bata, during June-July 2014. Adherence to treatment was assessed by using the Spanish version of CEAT-VIH. Bivariate and linear regression analyses were employed to assess HAART adherence-related factors. We interviewed 50 men (35.5%) and 91 women (64.5%), with a mean age of 47.7 ± 8.9 and 36.2 ± 11.2, respectively (p < .001). Overall, 55% patients had low or insufficient adherence. CEAT-VIH score varied by ethnic group (p = .005). There was a positive correlation between CEAT-VIH score and current CD4 T-cells count (p = .013). The Cronbach's α value was 0.52. To our knowledge, this is the first study to assess HAART adherence in Equatorial Guinea. Internal reliability for CEAT-VIH was low, nonetheless the positive correlation between the CEAT-VIH score and the immunological status of patients add value to our findings. Our results serve as baseline for future research and will also assist stakeholders in planning and undertaking contextual and evidence-based policy initiatives.

  11. A validated measure of adherence to antibiotic prophylaxis in children with sickle cell disease

    PubMed Central

    Duncan, Natalie A; Kronenberger, William G; Hampton, Kisha C; Bloom, Ellen M; Rampersad, Angeli G; Roberson, Christopher P; Shapiro, Amy D

    2016-01-01

    Background Antibiotic prophylaxis is a mainstay in sickle cell disease management. However, adherence is estimated at only 66%. This study aimed to develop and validate a Sickle Cell Antibiotic Adherence Level Evaluation (SCAALE) to promote systematic and detailed adherence evaluation. Methods A 28-item questionnaire was created, covering seven adherence areas. General Adherence Ratings from the parent and one health care provider and medication possession ratios were obtained as validation measures. Results Internal consistency was very good to excellent for the total SCAALE (α=0.89) and four of the seven subscales. Correlations between SCAALE scores and validation measures were strong for the total SCAALE and five of the seven subscales. Conclusion The SCAALE provides a detailed, quantitative, multidimensional, and global measurement of adherence and can promote clinical care and research. PMID:27354768

  12. Interleukin-3 greatly expands non-adherent endothelial forming cells with pro-angiogenic properties.

    PubMed

    Moldenhauer, Lachlan M; Cockshell, Michaelia P; Frost, Lachlan; Parham, Kate A; Tvorogov, Denis; Tan, Lih Y; Ebert, Lisa M; Tooley, Katie; Worthley, Stephen; Lopez, Angel F; Bonder, Claudine S

    2015-05-01

    Circulating endothelial progenitor cells (EPCs) provide revascularisation for cardiovascular disease and the expansion of these cells opens up the possibility of their use as a cell therapy. Herein we show that interleukin-3 (IL3) strongly expands a population of human non-adherent endothelial forming cells (EXnaEFCs) with low immunogenicity as well as pro-angiogenic capabilities in vivo, making their therapeutic utilisation a realistic option. Non-adherent CD133(+) EFCs isolated from human umbilical cord blood and cultured under different conditions were maximally expanded by day 12 in the presence of IL3 at which time a 350-fold increase in cell number was obtained. Cell surface marker phenotyping confirmed expression of the hematopoietic progenitor cell markers CD133, CD117 and CD34, vascular cell markers VEGFR2 and CD31, dim expression of CD45 and absence of myeloid markers CD14 and CD11b. Functional experiments revealed that EXnaEFCs exhibited classical properties of endothelial cells (ECs), namely binding of Ulex europaeus lectin, up-take of acetylated-low density lipoprotein and contribution to EC tube formation in vitro. These EXnaEFCs demonstrated a pro-angiogenic phenotype within two independent in vivo rodent models. Firstly, a Matrigel plug assay showed increased vascularisation in mice. Secondly, a rat model of acute myocardial infarction demonstrated reduced heart damage as determined by lower levels of serum creatinine and a modest increase in heart functionality. Taken together, these studies show IL3 as a potent growth factor for human CD133(+) cell expansion with clear pro-angiogenic properties (in vitro and in vivo) and thus may provide clinical utility for humans in the future. PMID:25900163

  13. Isolation and manipulation of living adherent cells by micromolded magnetic rafts

    PubMed Central

    Gach, Philip C.; Wang, Yuli; Phillips, Colleen; Sims, Christopher E.; Allbritton, Nancy L.

    2011-01-01

    A new strategy for magnetically manipulating and isolating adherent cells with extremely high post-collection purity and viability is reported. Micromolded magnetic elements (termed microrafts) were fabricated in an array format and used as culture surfaces and carriers for living, adherent cells. A poly(styrene-co-acrylic acid) polymer containing well dispersed magnetic nanoparticles was developed for creating the microstructures by molding. Nanoparticles of γFe2O3 at concentrations up to 1% wt.∕wt. could be used to fabricate microrafts that were optically transparent, highly magnetic, biocompatible, and minimally fluorescent. To prevent cellular uptake of nanoparticles from the magnetic polymer, a poly(styrene-co-acrylic acid) layer lacking γFe2O3 nanoparticles was placed over the initial magnetic microraft layer to prevent cellular uptake of the γFe2O3 during culture. The microraft surface geometry and physical properties were altered by varying the polymer concentration or layering different polymers during fabrication. Cells plated on the magnetic microrafts were visualized using standard imaging techniques including brightfield, epifluorescence, and confocal microscopy. Magnetic microrafts possessing cells of interest were dislodged from the array and efficiently collected with an external magnet. To demonstrate the feasibility of cell isolation using the magnetic microrafts, a mixed population of wild-type cells and cells stably transfected with a fluorescent protein was plated onto an array. Microrafts possessing single, fluorescent cells were released from the array and magnetically collected. A post-sorting single-cell cloning rate of 92% and a purity of 100% were attained. PMID:22007266

  14. Localized electroporation effect on adherent cells in modified electric cell-substrate impedance sensing circuits

    NASA Astrophysics Data System (ADS)

    Kim, Yu Jin; Ram Song, Ka; Kim, Hee-Dae; Park, Bum Chul; Kim, Young Keun; Kang, Chi Jung

    2016-10-01

    Electroporation is a physical transfection method for introducing foreign genes or drugs into cells. It does not require toxic reagents or transfection vectors. However, its applications have been limited because of cell damage and nonspecific transport. Here, we present an effective method for selective and localized electroporation using atomic force microscopy. This electroporation method is applied to adherent cells on substrates, instead of conventionally used suspended cells, and offers relatively effective cell transfection. Moreover, this method enables localized transfection into targeted areas at the single-cell level.

  15. Inhibition of Pneumococcal Adherence to Human Nasopharyngeal Epithelial Cells by Anti-PsaA Antibodies

    PubMed Central

    Romero-Steiner, Sandra; Pilishvili, Tamar; Sampson, Jacquelyn S.; Johnson, Scott E.; Stinson, Annie; Carlone, George M.; Ades, Edwin W.

    2003-01-01

    The role of pneumococcal (Pnc) surface adhesin A (PsaA) in the adherence of Streptococcus pneumoniae (pneumococcus) to host cells is not well defined. We examined the effect of anti-PsaA antibodies in an inhibition of adherence assay using Detroit 562 nasopharyngeal human epithelial cells. Rabbit polyclonal (Pab) anti-recombinant PsaA (rPsaA) sera, a purified mouse monoclonal antibody (MAb) (MAb 6F62G8E12), and 22 healthy adult sera with known anti-PsaA IgG levels (obtained by enzyme-linked immunosorbent assay) were evaluated for their abilities to inhibit Pnc adherence to confluent monolayers (measured as percent reduction in CFU counts compared to those of uninhibited controls). Pnc adherence was dependent on capsular phenotype (no or low adherence for opaque strains). With an inoculum of 104 to 105 bacteria/well, the mean ± standard deviation count in controls was 163 ± 32 CFU/well for transparent strains. Low adherence was observed for a PsaA-minus mutant even at higher inoculum doses. Mean percent inhibitions of adherence with Pab and MAb were 54 and 50%, respectively. Adult sera showed inhibition in a dose-response fashion with a range of 98 to 8%, depending on the serum anti-PsaA antibody concentration. Absorption of Pab with rPsaA restored Pnc adherence to control levels. Absorption of sera with a PsaA-minus mutant did not result in a significant decrease (P >0.05) of inhibition of adherence activity. Additionally, nearly 100% of Pnc adherence was inhibited by lipidated rPsaA at 2.5 μg/ml. Our data support the argument that PsaA is an adhesin that mediates Pnc adherence to human nasopharyngeal cells. This functional assay may be useful in evaluating antibodies elicited in response to PsaA vaccination. PMID:12626450

  16. Adherence to treatment in a Western European paediatric population with haemophilia: reliability and validity of the VERITAS-Pro scale.

    PubMed

    Lock, J; Raat, H; Duncan, N; Shapiro, A; Beijlevelt, M; Peters, M; Tamminga, R Y J; Leebeek, F W G; Moll, H A; Cnossen, M H

    2014-09-01

    Treatment adherence in haemophilia is strongly associated with quality of life and the cost-benefit of treatment. Therefore, it is important to quantify and monitor it. This study aimed to validate a translation of the VERITAS-Pro cross-culturally and analyse treatment adherence in a Dutch population of paediatric haemophilia patients. Children aged 1-18 years with haemophilia were included from three Haemophilia Treatment Centres, on prophylactic clotting factor replacement therapy for more than 1 year. Parents and adolescents were analysed separately. The adherence scale for prophylactic therapy (VERITAS-Pro) was translated according to international guidelines. This instrument contains a total of six subscales ('Time', 'Dose', 'Plan', 'Remember', 'Skip' and 'Communicate') each with four items. Lower scores reflect higher adherence. Overall response rate was 85%, leading to a study population of 60 children. Mean age was 10 years (SD 4.1). Internal consistency reliability: Mean Cronbach's alphas were adequate (>0.70) for total score and the subscales 'Skip' and 'Communicate'. Item-own subscale correlations were stronger than most item-other subscale correlations. Convergent validity: Total scores were higher for non-adherent participants compared with adherent participants according to patient infusion logs (n = 48; P < 0.05). Test-retest correlations: Significant for all scales except 'Dose' (n = 58; P < 0.01). This study demonstrates applicability of VERITAS-Pro outside the United States, as total score and most subscales effectively quantified treatment adherence in a Dutch paediatric population on prophylactic therapy. Non-adherent respondents' total scores were significantly higher, demonstrating the ability of VERITAS-Pro to identify non-adherent individuals.

  17. Hepatitis B virus efficiently infects non-adherent hepatoma cells via human sodium taurocholate cotransporting polypeptide

    PubMed Central

    Okuyama-Dobashi, Kaori; Kasai, Hirotake; Tanaka, Tomohisa; Yamashita, Atsuya; Yasumoto, Jun; Chen, Wenjia; Okamoto, Toru; Maekawa, Shinya; Watashi, Koichi; Wakita, Takaji; Ryo, Akihide; Suzuki, Tetsuro; Matsuura, Yoshiharu; Enomoto, Nobuyuki; Moriishi, Kohji

    2015-01-01

    Sodium taurocholate cotransporting polypeptide (NTCP) has been reported as a functional receptor for hepatitis B virus (HBV) infection. However, HBV could not efficiently infect HepG2 cells expressing NTCP (NTCP-HepG2 cells) under adherent monolayer-cell conditions. In this study, NTCP was mainly detected in the basolateral membrane region, but not the apical site, of monolayer NTCP-HepG2 cells. We hypothesized that non-adherent cell conditions of infection would enhance HBV infectivity. Non-adherent NTCP-HepG2 cells were prepared by treatment with trypsin and EDTA, which did not degrade NTCP in the membrane fraction. HBV successfully infected NTCP-HepG2 cells at a viral dose 10 times lower in non-adherent phase than in adherent phase. Efficient infection of non-adherent NTCP-HepG2 cells with blood-borne or cell-culture-derived HBV was observed and was remarkably impaired in the presence of the myristoylated preS1 peptide. HBV could also efficiently infect HepaRG cells under non-adherent cell conditions. We screened several compounds using our culture system and identified proscillaridin A as a potent anti-HBV agent with an IC50 value of 7.2 nM. In conclusion, non-adherent host cell conditions of infection augmented HBV infectivity in an NTCP-dependent manner, thus providing a novel strategy to identify anti-HBV drugs and investigate the mechanism of HBV infection. PMID:26592202

  18. Hepatitis B virus efficiently infects non-adherent hepatoma cells via human sodium taurocholate cotransporting polypeptide.

    PubMed

    Okuyama-Dobashi, Kaori; Kasai, Hirotake; Tanaka, Tomohisa; Yamashita, Atsuya; Yasumoto, Jun; Chen, Wenjia; Okamoto, Toru; Maekawa, Shinya; Watashi, Koichi; Wakita, Takaji; Ryo, Akihide; Suzuki, Tetsuro; Matsuura, Yoshiharu; Enomoto, Nobuyuki; Moriishi, Kohji

    2015-01-01

    Sodium taurocholate cotransporting polypeptide (NTCP) has been reported as a functional receptor for hepatitis B virus (HBV) infection. However, HBV could not efficiently infect HepG2 cells expressing NTCP (NTCP-HepG2 cells) under adherent monolayer-cell conditions. In this study, NTCP was mainly detected in the basolateral membrane region, but not the apical site, of monolayer NTCP-HepG2 cells. We hypothesized that non-adherent cell conditions of infection would enhance HBV infectivity. Non-adherent NTCP-HepG2 cells were prepared by treatment with trypsin and EDTA, which did not degrade NTCP in the membrane fraction. HBV successfully infected NTCP-HepG2 cells at a viral dose 10 times lower in non-adherent phase than in adherent phase. Efficient infection of non-adherent NTCP-HepG2 cells with blood-borne or cell-culture-derived HBV was observed and was remarkably impaired in the presence of the myristoylated preS1 peptide. HBV could also efficiently infect HepaRG cells under non-adherent cell conditions. We screened several compounds using our culture system and identified proscillaridin A as a potent anti-HBV agent with an IC50 value of 7.2 nM. In conclusion, non-adherent host cell conditions of infection augmented HBV infectivity in an NTCP-dependent manner, thus providing a novel strategy to identify anti-HBV drugs and investigate the mechanism of HBV infection. PMID:26592202

  19. Adherence to HEp-2 cells and enteropathogenic potential of Aeromonas spp.

    PubMed

    Grey, P A; Kirov, S M

    1993-04-01

    Aeromonas strains (total = 60) of clinical, water and food origin were tested for adherence to HEp-2 cells. Environmental strains were selected (except for A. caviae) to include primarily those expressing other virulence-associated properties. Adhesion was markedly species-dependent (A. veronii biotype sobria, 15 of 26 [58%]. A caviae, 4 of 12 [33%] and A. hydrophila, 2 of 8 [11%]). A. veronii biotype sobria were adhesive, irrespective of source (62 and 54% for clinical and environmental strains, respectively). Adherent strains of this species were enterotoxin-positive and most (13 of 15) grew at 43 degrees C. A. caviae isolated from clinical specimens contained a higher proportion (75%) of adherent strains than environmental strains (13%). Virulent subsets of A. veronii biotype sobria and A. caviae are adherent to HEp-2 cells. The HEp-2 assay is a useful model for investigating mechanisms of adherence and enteropathogenicity of virulent Aeromonas species.

  20. Inhibitory effects of bovine lactoferrin on the adherence of enterotoxigenic Escherichia coli to host cells.

    PubMed

    Kawasaki, Y; Tazume, S; Shimizu, K; Matsuzawa, H; Dosako, S; Isoda, H; Tsukiji, M; Fujimura, R; Muranaka, Y; Isihida, H

    2000-02-01

    Adherence is an essential and prerequisite step for the colonization of mucosal surfaces by enterotoxigenic Escherichia coli (ETEC). We studied the effect of bovine lactoferrin (BLF) on the adherence of ETEC to human epithelial cells in vitro, and to intestinal mucosa of ICR germfree mice in vivo. In the in vitro study, BLF was found to inhibit the adherence of ETEC. This adhesion-inhibiting activity of BLF was found to lessen with decreasing BLF concentration, but the data obtained suggest a positive inhibitory effect of BLF against the adhesion of ETEC cells. In the in vivo study, the counts of adherent bacteria in various sections of the intestinal tract (duodenum, jejunoileum, and large intestine) were lower in the BLF group than in the control group, suggesting the possible action of BLF as an intestinal tract adherence-blocking agent with regards to ETEC.

  1. Immunoregulatory adherent cells in human tuberculosis: radiation-sensitive antigen-specific suppression by monocytes

    SciTech Connect

    Kleinhenz, M.E.; Ellner, J.J.

    1985-07-01

    In human tuberculosis, adherent mononuclear cells (AMC) selectively depress in vitro responses to the mycobacterial antigen tuberculin purified protein derivative (PPD). The phenotype of this antigen-specific adherent suppressor cell was characterized by examining the functional activity of adherent cells after selective depletion of sheep erythrocyte-rosetting T cells or OKM1-reactive monocytes. Adherent cell suppression was studied in the (/sup 3/H)thymidine-incorporation microculture assay by using T cells rigorously depleted of T cells with surface receptors for the Fc portion of IgG (T gamma cells) as antigen-responsive cells. PPD-induced (/sup 3/H)thymidine incorporation by these non gamma T cells was uniformly reduced (mean, 42% +/- 10% (SD)) when autologous AMC were added to non gamma T cells at a ratio of 1:2. Antigen-specific suppression by AMC was not altered by depletion of sheep erythrocyte-rosetting T cells or treatment with indomethacin. However, AMC treated with OKM1 and complement or gamma irradiation (1,500 rads) no longer suppressed tuberculin responses in vitro. These studies identify the antigen-specific adherent suppressor cell in tuberculosis as an OKM1-reactive, non-erythrocyte-rosetting monocyte. The radiosensitivity of this monocyte immunoregulatory function may facilitate its further definition.

  2. Cell fusion through a microslit between adhered cells and observation of their nuclear behavior.

    PubMed

    Wada, Ken-Ichi; Hosokawa, Kazuo; Kondo, Eitaro; Ito, Yoshihiro; Maeda, Mizuo

    2014-07-01

    This paper describes a novel cell fusion method which induces cell fusion between adhered cells through a microslit for preventing nuclear mixing. For this purpose, a microfluidic device which had ∼ 100 cell pairing structures (CPSs) making cell pairs through microslits with 2.1 ± 0.3 µm width was fabricated. After trapping NIH3T3 cells with hydrodynamic forces at the CPSs, the cells were fused through the microslit by the Sendai virus envelope method. With following timelapse observation, we discovered that the spread cells were much less susceptible to nuclear migration passing through the microslit compared with round cells, and that cytoplasmic fraction containing mitochondria was transferred through the microslit without nuclear mixing. These findings will provide an effective method for cell fusion without nuclear mixing, and will lead to an efficient method for reprograming and transdifferentiation of target cells toward regenerative medicine.

  3. Adherence of clinically isolated lactobacilli to human cervical cells in competition with Neisseria gonorrhoeae.

    PubMed

    Vielfort, Katarina; Sjölinder, Hong; Roos, Stefan; Jonsson, Hans; Aro, Helena

    2008-10-01

    Lactobacilli are normal inhabitants of our microbiota and are known to protect against pathogens. Neisseria gonorrhoeae is a human specific pathogenic bacterium that colonises the urogenital tract where it causes gonorrhoea. In this study we analysed early interactions between lactobacilli and gonococci and investigated how they compete for adherence to human epithelial cervical cells. We show that lactobacilli adhere at various levels and that the number of adherent bacteria does not correlate to the level of protection against gonococcal infection. Protection against gonococcal adhesion varied between Lactobacillus species. Lactobacillus crispatus, Lactobacillus gasseri and Lactobacillus reuteri were capable of reducing gonococcal adherence while Lactobacillus rhamnosus was not. Lactobacillus strains of vaginal origin had the best capacity to remain attached to the host cell during gonococcal adherence. Further, we show that gonococci and lactobacilli interact with each other with resultant lactobacilli incorporation into the gonococcal microcolony. Hence, gonococci bind to colonised lactobacilli and this complex frequently detaches from the epithelial cell surface, resulting in reduced bacterial colonisation. Also, purified gonococcal pili are capable of removing adherent lactobacilli from the cell surface. Taken together, we reveal novel data regarding gonococcal and lactobacilli competition for adherence that will benefit future gonococcal prevention and treatments.

  4. Adherence to Physical Activity Recommendations and Its Associated Factors: An Interregional Population-Based Study

    PubMed Central

    Alkerwi, Ala’a; Schuh, Barbara; Sauvageot, Nicolas; Zannad, Faiez; Olivier, Arnaud; Guillaume, Michèle; Albert, Adelin; Larsson, Charlotte A.

    2015-01-01

    Background Though the influence of physical activity in preventing cardiovascular diseases is well documented, only a few comparative studies have determined the degree of adherence to physical activity recommendations among populations and identified the demographic, socioeco-nomic, behavioural and health-related factors associated with good compliance. Design and methods Cross-sectional interregional NESCaV survey of 3133 subjects compared three populations, Luxembourg, Lorraine (France) and Wallonia (Belgium), by using the International Physical Activity Questionnaire. Age and gender prevalence rates of physical activity were standardized to the European population. Results The likelihood to meet the recommendations was higher in Luxembourg, after adjustment for age, gender, education, employment, weight status, morbidity score, health perception and level of importance attributed to the practice of physical activity (P<0.0001). The odds for meeting the recommendations were significantly higher among those with secondary than tertiary education. Compared to good self-health perception, subjects with poor or fair self-perceived health were less likely to meet the recommendations; this also applied to those attributing little or enough importance to physical activity compared with great importance. Conclusions Region, education, self-perceived health and perception of importance of physical activity were emerged as independent determinants of meeting the recommendations. Awareness of the positive health effects of physical activity might thus be crucial for motivating the people to become more active. Further research is needed to explore potential region-specific factors which might explain the difference in population behaviours with respect to physical activity. Significance for public health This manuscript describes the prevalence of physical activity level of adult population from three European regions, Luxembourg, Wallonia and Lorraine, based on the

  5. Adherence to Mediterranean diet and its relation with cardiovascular diseases in Turkish population

    PubMed Central

    Hoşcan, Yeşim; Yiğit, Fatma; Müderrisoğlu, Haldun

    2015-01-01

    Mediterranean diet (MD) is considered a model for healthy eating. However, prospective evidence in Turkey evaluating the relationship between MD and cardiovascular events is scarce. We surveyed the adherence of Alanya population to MD and its association with coronary heart diseases (CHD). The study population consisted of participants in Alanya, a region placed southern Turkey. Followed-up 900 participants (52 percent women) initially free of CHD during 5.1 years. The general dietary habits of study population were detected with a food frequency questionnaire. Data obtained from that questionnaire were tested with Mediterranean diet score in order to find out the relevance to Mediterranean diet. A MD score (scale 0-8) was computed reflecting high ratio of monounsaturated to saturated fat; high intake of legumes, cereals, vegetables, and fruits; low intakes of meat and it’s products, milk and dairy products. Scoring < 5 was defined as Low-MD consuming, while 5+ as High-MD consuming. We observed 25 incident cases of CHD. Consumption of High-MD was 21% in men and 19% in women. The risk for myocardial infarction, coronary bypass, coronary angioplasty, and any cardiovascular disease in men increased by 1.3 (P = 0.02), 1.4 (P = 0.03), 1.5 (P = 0.01), and 1.3 (P = 0.02), respectively, for each MD score decrease. In women, the risk for myocardial infarction and angioplasty increased by 1.3 (P = 0.02) and 1.5 (P = 0.01), respectively, for each MD score decrease. The risk for coronary bypass, and any cardiovascular disease in women, crude odds ratios ranged from 1.1 to 1.3 but were not statistically significant. The current rate of MD in Alanya is fairly low. There is an inverse association between adherence to MD and the incidence of fatal and non-fatal CHD in initially healthy adults. PMID:25932246

  6. Mortality benefits of population-wide adherence to national physical activity guidelines: a prospective cohort study.

    PubMed

    Long, Gráinne; Watkinson, Clare; Brage, Søren; Morris, Jerry; Tuxworth, Bill; Fentem, Peter; Griffin, Simon; Simmons, Rebecca; Wareham, Nicholas

    2015-01-01

    We quantified the mortality benefits and attributable fractions associated with engaging in physical activity across a range of levels, including those recommended by national guidelines. Data were from the Allied Dunbar National Fitness Survey, a population-based prospective cohort comprising 1,796 male and 2,122 female participants aged 16-96 years, randomly selected from 30 English constituencies in 1990. Participants were tagged for mortality at the Office for National Statistics. Cox multivariable regression quantified the association between self-reported achievement of activity guidelines--150 min of at least moderate activity per week, equivalent here to 30 or more 20-min episodes of at least moderate activity per month--and mortality adjusting for age, sex, smoking status, social class, geographical area, anxiety/depression and interview season. There were 1,175 deaths over a median (IQR) of 22.9 (3.9) years follow-up; a mortality rate of 15.2, 95% confidence interval (CI) 14.4-16.1 per 1,000 person years. Compared with being inactive (no 20-min bouts per month), meeting activity guidelines (30+ bouts) was associated with a 25% lower mortality rate, adjusting for measured confounders. If everyone adhered to recommended-, or even low-activity levels, a substantial proportion of premature mortality might be avoided (PAF, 95% CI 20.6, 6.9-32.3 and 8.9, 4.2-13.4%, respectively). Among a representative English population, adherence to activity guidelines was associated with significantly reduced mortality. Efforts to increase population-wide activity levels could produce large public health benefits and should remain a focus of health promotion efforts.

  7. Filamentous hemagglutinin has a major role in mediating adherence of Bordetella pertussis to human WiDr cells.

    PubMed Central

    Urisu, A; Cowell, J L; Manclark, C R

    1986-01-01

    [35S]methionine-labeled Bordetella pertussis adhered to monolayers of WiDr cells, an epitheliumlike cell line from a human intestinal carcinoma. Adherence was proportional to the density of the WiDr cells and to the concentration of B. pertussis in the assay. Adherence of virulent phase I strains Tohama phase I, 114, and BP338 was much greater than adherence of avirulent strains Tohama phase III and 423 phase IV. Mutants deficient in the production of the filamentous hemagglutinin (FHA) were hemagglutination negative and adhered to WiDr cells much less efficiently than the parent strains. Preincubation of B. pertussis cells with FHA increased their hemagglutination activity and adherence to WiDr cells. Goat antibody to FHA inhibited, in a dose-dependent manner, the adherence of strain Tohama I but not the adherence of FHA-deficient mutant Tohama 325. At similar protein concentrations, normal goat antibody, goat antibody to pertussis toxin, or the Fab fragments of goat antibody to serotype 2 fimbriae had no effect on adherence. Also, an FHA-positive strain without fimbriae showed high adherence, while a fimbriated FHA-deficient mutant adhered poorly. Our data indicate that FHA plays a major role in adherence of B. pertussis to human WiDr cells. Fimbriae do not appear to mediate attachment of B. pertussis to WiDr cells. PMID:2872165

  8. Inverting adherent cells for visualizing ECM interactions at the basal cell side.

    PubMed

    Gudzenko, Tetyana; Franz, Clemens M

    2013-05-01

    Interactions with the extracellular matrix (ECM) govern a wide range of cellular functions, including survival, migration and invasion. However, in adherent cells these interactions occur primarily on the basal cell side, making them inaccessible to high-resolution, surface-scanning imaging techniques such as atomic force microscopy (AFM) or scanning electron microscopy (SEM). Here we describe a fast and reliable method for inverting adherent cells, exposing the basal cell membrane for direct analysis by AFM or SEM in combination with fluorescence microscopy. Cells including their matrix adhesion sites remain intact during the inversion process and are transferred together with the complete array of basally associated ECM proteins. Molecular features of ECM proteins, such as the characteristic 67 nm collagen D-periodicity, are well preserved after inversion. To demonstrate the versatility of the method, we compared basal interactions of fibroblasts with fibrillar collagen I and fibronectin matrices. While fibroblasts remodel the fibronectin layer exclusively from above, they actively invade even thin collagen layers by contacting individual collagen nanofibrils both basally and apically through a network of cellular extensions. Cell-matrix entanglement coincides with enhanced cell spreading and flattening, indicating that nanoscale ECM interactions govern macroscopic changes in cell morphology. The presented cell inversion technique can thus provide novel insight into nanoscale cell-matrix interactions at the basal cell side.

  9. The HAART cell phone adherence trial (WelTel Kenya1): a randomized controlled trial protocol

    PubMed Central

    Lester, Richard T; Mills, Edward J; Kariri, Antony; Ritvo, Paul; Chung, Michael; Jack, William; Habyarimana, James; Karanja, Sarah; Barasa, Samson; Nguti, Rosemary; Estambale, Benson; Ngugi, Elizabeth; Ball, T Blake; Thabane, Lehana; Kimani, Joshua; Gelmon, Lawrence; Ackers, Marta; Plummer, Francis A

    2009-01-01

    Background The objectives are to compare the effectiveness of cell phone-supported SMS messaging to standard care on adherence, quality of life, retention, and mortality in a population receiving antiretroviral therapy (ART) in Nairobi, Kenya. Methods and Design A multi-site randomized controlled open-label trial. A central randomization centre provided opaque envelopes to allocate treatments. Patients initiating ART at three comprehensive care clinics in Kenya will be randomized to receive either a structured weekly SMS ('short message system' or text message) slogan (the intervention) or current standard of care support mechanisms alone (the control). Our hypothesis is that using a structured mobile phone protocol to keep in touch with patients will improve adherence to ART and other patient outcomes. Participants are evaluated at baseline, and then at six and twelve months after initiating ART. The care providers keep a weekly study log of all phone based communications with study participants. Primary outcomes are self-reported adherence to ART and suppression of HIV viral load at twelve months scheduled follow-up. Secondary outcomes are improvements in health, quality of life, social and economic factors, and retention on ART. Primary analysis is by 'intention-to-treat'. Sensitivity analysis will be used to assess per-protocol effects. Analysis of covariates will be undertaken to determine factors that contribute or deter from expected and determined outcomes. Discussion This study protocol tests whether a novel structured mobile phone intervention can positively contribute to ART management in a resource-limited setting. Trial Registration Trial Registration Number: NCT00830622 PMID:19772596

  10. Distinct mechanical behavior of HEK293 cells in adherent and suspended states.

    PubMed

    Haghparast, Seyed Mohammad Ali; Kihara, Takanori; Miyake, Jun

    2015-01-01

    The mechanical features of individual animal cells have been regarded as indicators of cell type and state. Previously, we investigated the surface mechanics of cancer and normal stromal cells in adherent and suspended states using atomic force microscopy. Cancer cells possessed specific mechanical and actin cytoskeleton features that were distinct from normal stromal cells in adherent and suspended states. In this paper, we report the unique mechanical and actin cytoskeletal features of human embryonic kidney HEK293 cells. Unlike normal stromal and cancer cells, the surface stiffness of adherent HEK293 cells was very low, but increased after cell detachment from the culture surface. Induced actin filament depolymerization revealed that the actin cytoskeleton was the underlying source of the stiffness in suspended HEK293 cells. The exclusive mechanical response of HEK293 cells to perturbation of the actin cytoskeleton resembled that of adherent cancer cells and suspended normal stromal cells. Thus, with respect to their special cell-surface mechanical features, HEK293 cells could be categorized into a new class distinct from normal stromal and cancer cells.

  11. Rethinking adherence.

    PubMed

    Steiner, John F

    2012-10-16

    In 2012, the Centers for Medicare & Medicaid Services (CMS) will introduce measures of adherence to oral hypoglycemic, antihypertensive, and cholesterol-lowering drugs into its Medicare Advantage quality program. To meet these quality goals, delivery systems will need to develop and disseminate strategies to improve adherence. The design of adherence interventions has too often been guided by the mistaken assumptions that adherence is a single behavior that can be predicted from readily available patient characteristics and that individual clinicians alone can improve adherence at the population level.Effective interventions require recognition that adherence is a set of interacting behaviors influenced by individual, social, and environmental forces; adherence interventions must be broadly based, rather than targeted to specific population subgroups; and counseling with a trusted clinician needs to be complemented by outreach interventions and removal of structural and organizational barriers. To achieve the adherence goals set by CMS, front-line clinicians, interdisciplinary teams, organizational leaders, and policymakers will need to coordinate efforts in ways that exemplify the underlying principles of health care reform.

  12. Establishment of an adherent cell feeder layer from human umbilical cord blood for support of long-term hematopoietic progenitor cell growth.

    PubMed Central

    Ye, Z Q; Burkholder, J K; Qiu, P; Schultz, J C; Shahidi, N T; Yang, N S

    1994-01-01

    Previous attempts to establish a stromal cell feeder layer from human umbilical cord blood (HUCB) have met with very limited success. It has been suggested that there is an insufficient number of stromal precursor cells in HUCB to form a hematopoietic-supporting feeder layer in primary cultures. The present study shows that HUCB does contain a significant accessory cell population that routinely develops into a confluent, adherent cell layer under defined primary culture conditions. HUCB-derived adherent layers were shown to support long-term hematopoietic activity for an average of 4 months. This was achieved by using a customized coverslip with a modified surface structure as the cell attachment substratum and using a specialized culture feeding regime. We have characterized the various cell types (including fibroblasts, macrophages, and endothelial cells) and extracellular matrix proteins (including fibronectin, collagen III, and laminin) that were present in abundance in the HUCB-derived adherent cell layer. In contrast, oil red O-staining fat cells were rarely detected. ELISA and bioassays showed that stem cell factor and interleukin 6 were produced by the HUCB stromal cell cultures, but interleukin 3 or granulocyte/macrophage colony-stimulating factor was not detected. Application of this hematopoietic culture system to transgenic and gene therapy studies of stem cells is discussed. Images PMID:7527553

  13. Missing signposts on the roadmap to quality: a call to improve medication adherence indicators in data collection for population research

    PubMed Central

    Granger, Bradi B.; Rusincovitch, Shelley A.; Avery, Suzanne; Batch, Bryan C.; Dunham, Ashley A.; Feinglos, Mark N.; Kelly, Katherine; Pierre-Louis, Marjorie; Spratt, Susan E.; Califf, Robert M.

    2013-01-01

    Purpose: Poor adherence to prescribed medicines is associated with increased rates of poor outcomes, including hospitalization, serious adverse events, and death, and is also associated with increased healthcare costs. However, current approaches to evaluation of medication adherence using real-world electronic health records (EHRs) or claims data may miss critical opportunities for data capture and fall short in modeling and representing the full complexity of the healthcare environment. We sought to explore a framework for understanding and improving data capture for medication adherence in a population-based intervention in four U.S. counties. Approach: We posited that application of a data model and a process matrix when designing data collection for medication adherence would improve identification of variables and data accessibility, and could support future research on medication-taking behaviors. We then constructed a use case in which data related to medication adherence would be leveraged to support improved healthcare quality, clinical outcomes, and efficiency of healthcare delivery in a population-based intervention for persons with diabetes. Because EHRs in use at participating sites were deemed incapable of supplying the needed data, we applied a taxonomic approach to identify and define variables of interest. We then applied a process matrix methodology, in which we identified key research goals and chose optimal data domains and their respective data elements, to instantiate the resulting data model. Conclusions: Combining a taxonomic approach with a process matrix methodology may afford significant benefits when designing data collection for clinical and population-based research in the arena of medication adherence. Such an approach can effectively depict complex real-world concepts and domains by “mapping” the relationships between disparate contributors to medication adherence and describing their relative contributions to the shared goals

  14. Adherence to human lung microvascular endothelial cells (HMVEC-L) of Plasmodium vivax isolates from Colombia

    PubMed Central

    2013-01-01

    Background For years Plasmodium vivax has been considered the cause of benign malaria. Nevertheless, it has been observed that this parasite can produce a severe disease comparable to Plasmodium falciparum. It has been suggested that some physiopathogenic processes might be shared by these two species, such as cytoadherence. Recently, it has been demonstrated that P. vivax-infected erythrocytes (Pv-iEs) have the capacity to adhere to endothelial cells, in which intercellular adhesion molecule-1 (ICAM-1) seems to be involved in this process. Methods Adherence capacity of 21 Colombian isolates, from patients with P. vivax mono-infection to a microvascular line of human lung endothelium (HMVEC-L) was assessed in static conditions and binding was evaluated at basal levels or in tumor necrosis factor (TNF) stimulated cells. The adherence specificity for the ICAM-1 receptor was determined through inhibition with an anti-CD54 monoclonal antibody. Results The majority of P. vivax isolates, 13 out of 21 (61.9%), adhered to the HMVEC-L cells, but P. vivax adherence was at least seven times lower when compared to the four P. falciparum isolates. Moreover, HMVEC-L stimulation with TNF led to an increase of 1.6-fold in P. vivax cytoadhesion, similar to P. falciparum isolates (1.8-fold) at comparable conditions. Also, blockage of ICAM-1 receptor with specific antibodies showed a significant 50% adherence reduction. Conclusions Plasmodium vivax isolates found in Colombia are also capable of adhering specifically in vitro to lung endothelial cells, via ICAM-1 cell receptor, both at basal state and after cell stimulation with TNF. Collectively, these findings reinforce the concept of cytoadherence for P. vivax, but here, to a different endothelial cell line and using geographical distinct isolates, thus contributing to understanding P. vivax biology. PMID:24080027

  15. Recent population adherence to and knowledge of United States federal nutrition guides, 1992-2013: a systematic review.

    PubMed

    Haack, Sarah A; Byker, Carmen J

    2014-10-01

    The Dietary Guidelines for Americans dictates the federal nutrition programs, policies, and recommendations of the United States. Corresponding nutrition guides have been established to help educate the public about the dietary intake patterns recommended in these guidelines as well as to ameliorate the US obesity epidemic and its health-related outcomes. The purpose of this systematic review was to summarize population adherence to and knowledge of these guiding US nutrition guides issued since 1992, including the Food Guide Pyramid, MyPyramid, and MyPlate. Of the 31 studies included in the review, 22 examined adherence, 6 examined knowledge, and 3 examined both adherence and knowledge. Across studies, adherence to nutrition guides was low, with participants consuming inadequate levels of fruit, vegetables, and dairy in particular. Knowledge of nutrition guides increased over time since publication and decreased with age of the participants. An association between knowledge of and adherence to nutrition guides was not found. Disparities in knowledge and adherence existed across demographic groups. Based on these findings, it is suggested that federal dietary guidance can be strengthened by increasing dissemination of nutrition guides to the public and tailoring promotional activities to specific demographic and socioeconomic groups. PMID:25209465

  16. Recent population adherence to and knowledge of United States federal nutrition guides, 1992-2013: a systematic review.

    PubMed

    Haack, Sarah A; Byker, Carmen J

    2014-10-01

    The Dietary Guidelines for Americans dictates the federal nutrition programs, policies, and recommendations of the United States. Corresponding nutrition guides have been established to help educate the public about the dietary intake patterns recommended in these guidelines as well as to ameliorate the US obesity epidemic and its health-related outcomes. The purpose of this systematic review was to summarize population adherence to and knowledge of these guiding US nutrition guides issued since 1992, including the Food Guide Pyramid, MyPyramid, and MyPlate. Of the 31 studies included in the review, 22 examined adherence, 6 examined knowledge, and 3 examined both adherence and knowledge. Across studies, adherence to nutrition guides was low, with participants consuming inadequate levels of fruit, vegetables, and dairy in particular. Knowledge of nutrition guides increased over time since publication and decreased with age of the participants. An association between knowledge of and adherence to nutrition guides was not found. Disparities in knowledge and adherence existed across demographic groups. Based on these findings, it is suggested that federal dietary guidance can be strengthened by increasing dissemination of nutrition guides to the public and tailoring promotional activities to specific demographic and socioeconomic groups.

  17. Relationship of cell surface morphology and composition of Streptococcus salivarius K+ to adherence and hydrophobicity.

    PubMed Central

    Weerkamp, A H; van der Mei, H C; Slot, J W

    1987-01-01

    The cell surfaces of a range of variants of Streptococcus salivarius HB, altered in cell wall antigen composition, were compared with those of the parent with respect to adherence, ability to adsorb to hexadecane, morphology, and exposure of lipoteichoic acid (LTA). Adherence to host surfaces was measured by using both saliva-coated hydroxyapatite beads and tissue-cultured HeLa cells, and interbacterial adherence was measured by using Veillonella alcalescens V1 cells. Progressive loss of the protease-sensitive fibril classes was generally associated with decreasing ability to adsorb to hexadecane. However, increased exposure of protein antigen C (AgC) increased the apparent hydrophobicity of the cell. This correlated with the finding that AgC was the most hydrophobic of the solubilized fibrillar cell wall antigens. Collectively, this demonstrates that adsorption to hydrophobic ligands is directly related to the density of the fibrillar layer on the cells and the properties and surface exposure of specific fibril classes. The involvement of hydrophobic interactions in AgC-associated attachment was suggested by its sensitivity to low levels of the hydrophobic bond-breaking agent tetramethyl urea, although the reduction was not to the level of adherence observed with strains lacking AgC. However, hydrophobicity was less essential to other adherence reactions. Circumstantial evidence, including immunoelectron microscopy, showing that LTA was virtually absent from the fibrillar layer, whole-cell enzyme-linked immunosorbent assay, suggesting that surface exposure of LTA related inversely to the density of the fibrillar layer, and agarose gel electrophoresis, showing that LTA was not specifically associated with protein fibrillar antigens, strongly suggested that LTA does not confer hydrophobic properties to these cells and is not involved in adherence reactions associated with the cell wall protein antigens. Images PMID:3804445

  18. Association between patients’ beliefs and oral antidiabetic medication adherence in a Chinese type 2 diabetic population

    PubMed Central

    Wu, Ping; Liu, Naifeng

    2016-01-01

    Purpose The objective of this study was to identify, using the theory of planned behavior (TPB), patients’ beliefs about taking oral antidiabetic drugs (OADs) as prescribed, and to measure the correlations between beliefs and medication adherence. Patients and methods We performed a cross-sectional study of type 2 diabetic patients using structured questionnaires in a Chinese tertiary hospital. A total of 130 patients were enrolled to be interviewed about TPB variables (behavioral, normative, and control beliefs) relevant to medication adherence. Medication adherence was assessed using the eight-item Morisky Medication Adherence Scale (MMAS-8). Spearman’s rank correlation was used to assess the association between TPB and MMAS-8. Logistic regression analysis was performed to examine the relationship between different variables and MMAS-8, with statistical significance determined at P<0.05. Results From 130 eligible Chinese patients with an average age of 60.6 years and a male proportion of 50.8%, a nonsignificant relationship between behavioral, normative, and the most facilitating control beliefs and OAD adherence was found in our study. Having the OADs on hand (P=0.037) was the only facilitating control belief associated with adherence behavior. Being away from home or eating out (P=0.000), not accepting the disease (P=0.000), ignorance of life-long drug adherence (P=0.038), being busy (P=0.001), or poor memory (P=0.008) were control belief barriers found to be correlated with poor adherence. TPB is the only important determinant influencing OAD adherence among all the factors (P=0.011). Conclusion The results indicate that the TPB model could be used to examine adherence to OADs. One facilitating control belief, and most of the barrier control beliefs of TPB were related to medication adherence among Chinese type 2 diabetes inpatients. It will be helpful to understand patients’ self-medication and provide methods to develop instruments for identifying

  19. Effect of respiratory syncytial virus (RSV) infection on the adherence of pathogenic bacteria to human epithelial cells

    SciTech Connect

    Faden, H.; Hong, J.J.; Ogra, P.L.

    1986-03-01

    The effect of RSV infection on the adherence of Streptococcus pneumoniae (SP), Haemophilus influenzae (HI) and Staphylococcus aureus (SA) to human epithelial cells was determined. RSV-infected Hep-2 cell cultures at different stages of expression of surface viral antigens and bacteria labeled with /sup 3/H-thymidine were employed to examine the kinetics of bacterial adherence to virus-infected cells. RSV infection did not alter the magnitude of adherence of HI or SA to HEp-2 cells. However, adherence of SP to HEp-2 cells was significantly (P < 0.01) enhanced by prior RSV infection. The degree of adherence was directly related to the amount of viral antigen expressed on the cell surface. The adherence was temperature dependent, with maximal adherence observed at 37/sup 0/C. Heat-inactivation of SP did not alter adherence characteristics. These data suggest that RSV infection increases adherence of SP to the surface of epithelial cells in vitro. Since attachment of bacteria to mucosal surfaces is the first step in many infections, it is suggested that viral infections of epithelial cells render them more susceptible to bacterial adherence. Thus, RSV infection in vivo may predispose children to SP infections, such as in otitis media, by increasing colonization with SP.

  20. Adherence to Mediterranean-style dietary pattern and risk of esophageal squamous cell carcinoma: a case-control study in Iran

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The benefit of adherence to a Mediterranean-style dietary pattern in relation to the risk of esophageal squamous cell carcinoma (ESCC) has not been investigated among non-Mediterranean high-risk populations. The objective of the present study was to examine the association of compliance with the Med...

  1. Self-Reported Prevalence of Gluten-Related Disorders and Adherence to Gluten-Free Diet in Colombian Adult Population.

    PubMed

    Cabrera-Chávez, Francisco; Granda-Restrepo, Diana María; Arámburo-Gálvez, Jesús Gilberto; Franco-Aguilar, Alejandro; Magaña-Ordorica, Dalia; Vergara-Jiménez, Marcela de Jesús; Ontiveros, Noé

    2016-01-01

    Background. Celiac disease seems to be rare in Colombians, but there are currently no data about the prevalence rates of symptomatic adverse reactions to gluten or adherence to gluten-free diet (GFD) in this population. Aim. to evaluate the self-reported prevalence rates of adverse reactions to gluten, adherence to GFD, and gluten-related disorders at population level in Colombia. Methods. A self-administered questionnaire-based cross-sectional study was conducted in a population from Northwest Colombia. Results. The estimated prevalence rates were (95% CI) 7.9% (6.5-9.6) and 5.3% (4.1-6.7) for adverse and recurrent adverse reactions to wheat/gluten, respectively, adherence to GFD 5.9% (4.7-7.4), wheat allergy 0.74% (0.3-1.4), and nonceliac gluten sensitivity 4.5% (3.5-5.8). There were no self-reported cases of celiac disease. Prevalence of self-reported physician-diagnosis of gluten-related disorders was 0.41% (0.17-0.96). Most respondents reported adherence to GFD without a physician-diagnosis of gluten-related disorders (97.2%). The proportion of gluten avoiders was 17.2% (15.2-19.5). Most of them did not report recurrent adverse reactions to wheat/gluten (87.0%). Conclusions. Nonceliac gluten sensitivity is rarely formally diagnosed in Colombia, but this population has the highest prevalence rate of adherence to GFD reported to date. Consequently, most respondents were avoiding wheat- and/or gluten-based products for reasons other than health-related symptoms.

  2. Self-Reported Prevalence of Gluten-Related Disorders and Adherence to Gluten-Free Diet in Colombian Adult Population

    PubMed Central

    Franco-Aguilar, Alejandro; Magaña-Ordorica, Dalia

    2016-01-01

    Background. Celiac disease seems to be rare in Colombians, but there are currently no data about the prevalence rates of symptomatic adverse reactions to gluten or adherence to gluten-free diet (GFD) in this population. Aim. to evaluate the self-reported prevalence rates of adverse reactions to gluten, adherence to GFD, and gluten-related disorders at population level in Colombia. Methods. A self-administered questionnaire-based cross-sectional study was conducted in a population from Northwest Colombia. Results. The estimated prevalence rates were (95% CI) 7.9% (6.5–9.6) and 5.3% (4.1–6.7) for adverse and recurrent adverse reactions to wheat/gluten, respectively, adherence to GFD 5.9% (4.7–7.4), wheat allergy 0.74% (0.3–1.4), and nonceliac gluten sensitivity 4.5% (3.5–5.8). There were no self-reported cases of celiac disease. Prevalence of self-reported physician-diagnosis of gluten-related disorders was 0.41% (0.17–0.96). Most respondents reported adherence to GFD without a physician-diagnosis of gluten-related disorders (97.2%). The proportion of gluten avoiders was 17.2% (15.2–19.5). Most of them did not report recurrent adverse reactions to wheat/gluten (87.0%). Conclusions. Nonceliac gluten sensitivity is rarely formally diagnosed in Colombia, but this population has the highest prevalence rate of adherence to GFD reported to date. Consequently, most respondents were avoiding wheat- and/or gluten-based products for reasons other than health-related symptoms. PMID:27648068

  3. Self-Reported Prevalence of Gluten-Related Disorders and Adherence to Gluten-Free Diet in Colombian Adult Population.

    PubMed

    Cabrera-Chávez, Francisco; Granda-Restrepo, Diana María; Arámburo-Gálvez, Jesús Gilberto; Franco-Aguilar, Alejandro; Magaña-Ordorica, Dalia; Vergara-Jiménez, Marcela de Jesús; Ontiveros, Noé

    2016-01-01

    Background. Celiac disease seems to be rare in Colombians, but there are currently no data about the prevalence rates of symptomatic adverse reactions to gluten or adherence to gluten-free diet (GFD) in this population. Aim. to evaluate the self-reported prevalence rates of adverse reactions to gluten, adherence to GFD, and gluten-related disorders at population level in Colombia. Methods. A self-administered questionnaire-based cross-sectional study was conducted in a population from Northwest Colombia. Results. The estimated prevalence rates were (95% CI) 7.9% (6.5-9.6) and 5.3% (4.1-6.7) for adverse and recurrent adverse reactions to wheat/gluten, respectively, adherence to GFD 5.9% (4.7-7.4), wheat allergy 0.74% (0.3-1.4), and nonceliac gluten sensitivity 4.5% (3.5-5.8). There were no self-reported cases of celiac disease. Prevalence of self-reported physician-diagnosis of gluten-related disorders was 0.41% (0.17-0.96). Most respondents reported adherence to GFD without a physician-diagnosis of gluten-related disorders (97.2%). The proportion of gluten avoiders was 17.2% (15.2-19.5). Most of them did not report recurrent adverse reactions to wheat/gluten (87.0%). Conclusions. Nonceliac gluten sensitivity is rarely formally diagnosed in Colombia, but this population has the highest prevalence rate of adherence to GFD reported to date. Consequently, most respondents were avoiding wheat- and/or gluten-based products for reasons other than health-related symptoms. PMID:27648068

  4. Self-Reported Prevalence of Gluten-Related Disorders and Adherence to Gluten-Free Diet in Colombian Adult Population

    PubMed Central

    Franco-Aguilar, Alejandro; Magaña-Ordorica, Dalia

    2016-01-01

    Background. Celiac disease seems to be rare in Colombians, but there are currently no data about the prevalence rates of symptomatic adverse reactions to gluten or adherence to gluten-free diet (GFD) in this population. Aim. to evaluate the self-reported prevalence rates of adverse reactions to gluten, adherence to GFD, and gluten-related disorders at population level in Colombia. Methods. A self-administered questionnaire-based cross-sectional study was conducted in a population from Northwest Colombia. Results. The estimated prevalence rates were (95% CI) 7.9% (6.5–9.6) and 5.3% (4.1–6.7) for adverse and recurrent adverse reactions to wheat/gluten, respectively, adherence to GFD 5.9% (4.7–7.4), wheat allergy 0.74% (0.3–1.4), and nonceliac gluten sensitivity 4.5% (3.5–5.8). There were no self-reported cases of celiac disease. Prevalence of self-reported physician-diagnosis of gluten-related disorders was 0.41% (0.17–0.96). Most respondents reported adherence to GFD without a physician-diagnosis of gluten-related disorders (97.2%). The proportion of gluten avoiders was 17.2% (15.2–19.5). Most of them did not report recurrent adverse reactions to wheat/gluten (87.0%). Conclusions. Nonceliac gluten sensitivity is rarely formally diagnosed in Colombia, but this population has the highest prevalence rate of adherence to GFD reported to date. Consequently, most respondents were avoiding wheat- and/or gluten-based products for reasons other than health-related symptoms.

  5. Apa is a trimeric autotransporter adhesin of Actinobacillus pleuropneumoniae responsible for autoagglutination and host cell adherence.

    PubMed

    Xiao, Longwen; Zhou, Liang; Sun, Changjiang; Feng, Xin; Du, ChongTao; Gao, Yu; Ji, Qun; Yang, Shuxin; Wang, Yu; Han, Wenyu; Langford, P R; Lei, Liancheng

    2012-10-01

    Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, and adherence to host cells is a key step in the pathogenic process. Although trimeric autotransporter adhesins (TAAs) were identified in many pathogenic bacteria in recent years, none in A. pleuropneumoniae have been characterized. In this study, we identified a TAA from A. pleuropneumoniae, Apa, and characterized the contribution of its amino acid residues to the adhesion process. Sequence analysis of the C-terminal amino acid residues of Apa revealed the presence of a putative translocator domain and six conserved HsfBD1-like or HsfBD2-like binding domains. Western blot analysis revealed that the 126 C-terminal amino acids of Apa could form trimeric molecules. By confocal laser scanning microscopy, one of these six domains (ApaBD3) was determined to mediate adherence to epithelial cells. Adherence assays and adherence inhibition assays using a recombinant E. coli- ApaBD3 strain which expressed ApaBD3 on the surface of E. coli confirmed that this domain was responsible for the adhesion activity. Moreover, cellular enzyme-linked immunosorbent assays demonstrated that ApaBD3 mediated high-level adherence to epithelial cell lines. Intriguingly, autoagglutination was observed with the E. coli- ApaBD3 strain, and this phenomenon was dependent upon the association of the expressed ApaBD3 with the C-terminal translocator domain.

  6. Contrasting effects of inflammatory stimuli on neutrophil and monocyte adherence to endothelial cells.

    PubMed

    Kamp, D W; Bauer, K D; Knap, A; Dunn, M M

    1989-08-01

    Leukocyte adherence to endothelial cells (EC) is an important early event in inflammatory responses, which are often characterized by a predominance of either neutrophils (PMN) or monocytes. However, there is little information concerning the molecular events important in leukocyte adherence to EC. Intracellular activation of protein kinase C and the calcium-second messenger system leads to the stimulation of a number of important functions in PMN and monocytes. We compared the effects of members of these pathways on human PMN and monocyte adherence to cultured bovine aortic EC. We observed that phorbol myristate acetate, phorbol, 12,13-dibutyrate, L-alpha-1-oleoyl-2-acetoyl-sn-3-glycerol, and ionomycin each induced significant dose-dependent increases in PMN adherence to EC monolayers. In contrast, similar concentrations of each of these agents induced significant decreases in EC adherence of monocytes enriched by countercurrent centrifugal elutriation. Separate experiments determined that the differences in PMN and monocyte adherence to EC were not related to differences in oxidant production because 1) phorbol myristate acetate and L-alpha-1-oleoyl-2-acetoyl-sn-3-glycerol caused similar marked increases in both PMN and monocyte superoxide anion and hydrogen peroxide production and 2) ionomycin, which had opposing effects on PMN and monocyte adherence, had no effect on PMN and monocyte superoxide anion or hydrogen peroxide release. We conclude that activators of protein kinase C and the Ca-second messenger pathway have opposite effects on PMN and monocyte adherence to EC and that these effects are mediated by O2 radical-independent mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Role of different classes of mammalian cell surface molecules in adherence of coagulase positive and coagulase negative staphylococci.

    PubMed

    Hafez, Mohamed M; Aboulwafa, Mohammad M; Yassien, Mahmoud A; Hassouna, Nadia A

    2008-10-01

    In the present study the role of different mammalian cell receptors in adherence of the coagulase positive pathogen, Staphylococcus aureus and some coagulase negative staphylococci, namely Staphylococcus epidermidis and Staphylococcus saprophyticus was investigated. Upon testing the adherence to Vero and Hep-2 cells, S. aureus isolates showed an adherence to both cell lines while S. epidermidis and S. saprophyticus isolates adhered to Vero cells only. According to the obtained results, both O-linked and N-linked mammalian cell surface glycoproteins are involved in the adherence of S. aureus isolates to Vero and Hep-2 cells, whereas only the O-linked ones serve as receptors for adherence of S. epidermidis and S. saprophyticus isolates to Vero cells. Of the O-linked glycoproteins, GAG-like receptors are involved in adherence of all tested isolates to Vero cells. The coagulase positive staphylococci preferred to adhere to the highly sulphated GAGs (Heparin and chondroitin sulphate B) while the coagulase negative isolates showed higher affinity to the less sulphated ones (Chondroitin sulphate A and C). Mucin like receptors appeared to be important for the adherence of all tested staphylococci. The role exhibited by fibronectin- and fibrinogen-like receptors was detected with S. aureus and S. epidermidis but not with S. saprophyticus isolates. While, collagen and gelatin were found to contribute to the adherence of S. aureus isolates only. Neither carbohydrate moieties of the glycoconjugates nor lipid molecules on the mammalian cell surface played a role in the adherence of the tested staphylococcal isolates. Taken together, the results revealed that both coagulase negative and coagulase positive staphylococcal tested isolates adhere to the same classes of mammalian cell surface receptors such as mucin-like, fibrinogen-like, fibronectin-like and GAG-like receptors. However, the tested isolates exhibited different degrees of affinities to such receptors.

  8. Use of a solid phase red blood cell adherence method for pretransfusion platelet compatibility testing.

    PubMed

    Rachel, J M; Summers, T C; Sinor, L T; Plapp, F V

    1988-07-01

    A solid phase red blood cell adherence method has been used for platelet antibody detection and crossmatching for refractory platelet recipients. Patient sera were first screened for HLA or platelet-specific antibodies, then crossmatched with potential apheresis platelet donors. The overall correlation of platelet crossmatch results with transfusion outcome was 97% in patients with no evidence of nonimmune platelet destruction. The solid phase red blood cell adherence method provided a feasible and effective alternative to HLA matching as a means of donor selection for refractory platelet recipients. The speed and simplicity of this method may allow most hospital laboratories to perform platelet antibody screening before routine platelet transfusions.

  9. Dynamic logistic regression model and population attributable fraction to investigate the association between adherence, missed visits and mortality: a study of HIV-infected adults surviving the first year of ART

    PubMed Central

    2013-01-01

    Background Adherence is one of the most important determinants of viral suppression and drug resistance in HIV-infected people receiving antiretroviral therapy (ART). Methods We examined the association between long-term mortality and poor adherence to ART in DART trial participants in Uganda and Zimbabwe randomly assigned to receive laboratory and clinical monitoring (LCM), or clinically driven monitoring (CDM). Since over 50% of all deaths in the DART trial occurred during the first year on ART, we focussed on participants continuing ART for 12 months to investigate the implications of longer-term adherence to treatment on mortality. Participants’ ART adherence was assessed by pill counts and structured questionnaires at 4-weekly clinic visits. We studied the effect of recent adherence history on the risk of death at the individual level (odds ratios from dynamic logistic regression model), and on mortality at the population level (population attributable fraction based on this model). Analyses were conducted separately for both randomization groups, adjusted for relevant confounding factors. Adherence behaviour was also confounded by a partial factorial randomization comparing structured treatment interruptions (STI) with continuous ART (CT). Results In the CDM arm a significant association was found between poor adherence to ART in the previous 3-9 months with increased mortality risk. In the LCM arm the association was not significant. The odds ratios for mortality in participants with poor adherence against those with optimal adherence was 1.30 (95% CI 0.78,2.10) in the LCM arm and 2.18 (1.47,3.22) in the CDM arm. The estimated proportions of deaths that could have been avoided with optimal adherence (population attributable fraction) in the LCM and CDM groups during the 5 years follow-up period were 16.0% (95% CI 0.7%,31.6%) and 33.1% (20.5%,44.8%), correspondingly. Conclusions Recurrent poor adherence determined even through simple measures is associated

  10. Adhesion and function of rat liver cells adherent to silk fibroin/collagen blend films.

    PubMed

    Cirillo, B; Morra, M; Catapano, G

    2004-01-01

    Collagen is often used in bioartificial livers as a biomimetic coating to promote liver cell adhesion and differentiation. Animal proteins are expensive and expose the host to risks of cross-species infection due to contamination with prions. Silk fibroin (SF) is a biocompatible protein produced by Bombyx mori silk worms and possibly an alternative to collagen. We prepared SF-collagen blend films with different SF content adherent to the bottom of standard tissue culture dishes, and characterized their surface morphology by SEM, their wettability and examined them for their capacity to support rat liver cell adhesion and metabolism. Cell metabolism was characterized by estimating the rate at which cells eliminated ammonia and synthesized urea for up to 48h of culture. SF-containing films were smooth, clear and more wettable than collagen. Cells readily adhered, formed junctions and small size aggregates on all films. As many cells adhered on SF as on collagen films. Cell adhesion to high collagen content blend films could not be reliably estimated because cells dwelt in the large cavities in the film. The effect of SF on cell metabolism differed with the investigated metabolic pathway. However, cells on SF-containing films eliminated ammonia and synthesized urea at rates generally comparable to, for urea synthesis at times higher than, that of cells on collagen. These results suggest that silk fibroin is a suitable substratum for liver cell attachment and culture, and a potential alternative to collagen as a biomimetic coating. PMID:14984185

  11. The evolution of pretransfusion testing: from agglutination to solid-phase red cell adherence tests.

    PubMed

    Plapp, F V; Sinor, L T; Rachel, J M

    1989-01-01

    Hospital transfusion services and blood centers still use manual hemagglutination tests for most of their serological procedures. Automation of hemagglutination reactions has proven to be difficult, primarily because hemagglutination lacks an objective endpoint which can be easily interpreted by inexpensive instruments. Alternatively, solid-phase red cell adherence assays for ABO cell and serum grouping, Rh typing, red cell and platelet antibody screening, red cell and platelet crossmatching, IgA deficiency screening, hepatitis B surface antigen, and HIV antibody screening have been developed. The performance of these assays compares favorably with current hemagglutination and enzyme immunoassay methods. All of these tests share a common objective endpoint of adherence or nonadherence of indicator red cells. This uniformity allows easy interpretation of results visually, spectrophotometrically, or by image analysis. The latter technique has the potential to revolutionize the reading and interpretation of all agglutination tests. Solid-phase red cell adherence tests in microplates are ideal for batch processing large numbers of specimens. However, adherence tests are not restricted to this format. Therefore, blood grouping dipsticks have been produced, which permit testing of individual blood samples even outside of the laboratory.

  12. Use of bovine primary mammary epithelial cells for the comparison of adherence and invasion ability of Staphylococcus aureus strains.

    PubMed

    Hensen, S M; Pavicić, M J; Lohuis, J A; Poutrel, B

    2000-03-01

    Adherence and invasion of epithelial cells are thought to play a role in the pathogenesis of Staphylococcus aureus mastitis. A cell culture model with primary mammary epithelial cells originating from the secretory tissue from the bovine udder was used to study adherence and invasion of S. aureus. The cells were characterized with antibodies against several cell markers that had been validated on histologic cryostat sections of bovine mammary tissue. All cells stained positively with the anticytokeratin antibodies, which are restricted to epithelial cells. The cell cultures contained a small number of alpha-smooth-muscle-actin positive cells (< 1%), probably myoepithelial cells. The use of bovine primary mammary epithelial cells and bovine S. aureus isolates, which were cultured in milk serum, results in a system similar to in vivo. Strain differences for adherence and invasion of S. aureus strains cultured in milk serum were studied. In addition, the correlation between adherence and invasion was evaluated. The number of adhered and invaded bacteria was strain dependent. The percentage of adherence after 5 min of incubation was correlated to the percentage of adherence after 3 h of incubation (r = 0.94; Pearson's correlation test). Fourteen of the 20 strains were able to invade epithelial cells. The percentage of invasion was correlated to the percentage of adherence after 5 min and to the percentage adherence after 3 h (r = 0.95 and 0.90, respectively; Pearson's correlation test). Results indicate that strain differences of adherence and invasion exist for S. aureus and that the invasion is a post adherence event.

  13. Characterization of antibody inhibiting adherence of Bordetella pertussis to human respiratory epithelial cells.

    PubMed Central

    Tuomanen, E I; Zapiain, L A; Galvan, P; Hewlett, E L

    1984-01-01

    We have recently established the topographic specificity of the adherence of Bordetella pertussis to human ciliated respiratory epithelial cells. For this study, we employed the same quantitative, immunofluorescent adherence assay to test the possibility that sera of patients recovering from naturally acquired whooping cough or immunized with pertussis vaccine may contain activity capable of interfering with this specific adherence. Evaluation of paired sera from six children with culture-proven pertussis demonstrated that antiadherence activity appeared in serum during convalescence from disease. Nine children immunized with diptheria-pertussin-tetanus vaccine also showed activity against adherence, although it was significantly less than in those with clinical disease. Naturally acquired serum antiadherence activity was identified in both immunoglobulin G (IgG) and IgA antibody classes, whereas, as expected, only IgG antibody was present in children receiving the parenteral vaccine. The findings suggest that natural infection or vaccination are associated with the acquisition of serum activity inhibiting the adherence of B. pertussis to ciliated cells. Immunization may fail to elicit IgA antiadherence activity. PMID:6092416

  14. Endometrial regenerative cells: a novel stem cell population.

    PubMed

    Meng, Xiaolong; Ichim, Thomas E; Zhong, Jie; Rogers, Andrea; Yin, Zhenglian; Jackson, James; Wang, Hao; Ge, Wei; Bogin, Vladimir; Chan, Kyle W; Thébaud, Bernard; Riordan, Neil H

    2007-01-01

    Angiogenesis is a critical component of the proliferative endometrial phase of the menstrual cycle. Thus, we hypothesized that a stem cell-like population exist and can be isolated from menstrual blood. Mononuclear cells collected from the menstrual blood contained a subpopulation of adherent cells which could be maintained in tissue culture for >68 doublings and retained expression of the markers CD9, CD29, CD41a, CD44, CD59, CD73, CD90 and CD105, without karyotypic abnormalities. Proliferative rate of the cells was significantly higher than control umbilical cord derived mesenchymal stem cells, with doubling occurring every 19.4 hours. These cells, which we termed "Endometrial Regenerative Cells" (ERC) were capable of differentiating into 9 lineages: cardiomyocytic, respiratory epithelial, neurocytic, myocytic, endothelial, pancreatic, hepatic, adipocytic, and osteogenic. Additionally, ERC produced MMP3, MMP10, GM-CSF, angiopoietin-2 and PDGF-BB at 10-100,000 fold higher levels than two control cord blood derived mesenchymal stem cell lines. Given the ease of extraction and pluripotency of this cell population, we propose ERC as a novel alternative to current stem cells sources. PMID:18005405

  15. Cell prestress. I. Stiffness and prestress are closely associated in adherent contractile cells

    NASA Technical Reports Server (NTRS)

    Wang, Ning; Tolic-Norrelykke, Iva Marija; Chen, Jianxin; Mijailovich, Srboljub M.; Butler, James P.; Fredberg, Jeffrey J.; Stamenovic, Dimitrije; Ingber, D. E. (Principal Investigator)

    2002-01-01

    The tensegrity hypothesis holds that the cytoskeleton is a structure whose shape is stabilized predominantly by the tensile stresses borne by filamentous structures. Accordingly, cell stiffness must increase in proportion with the level of the tensile stress, which is called the prestress. Here we have tested that prediction in adherent human airway smooth muscle (HASM) cells. Traction microscopy was used to measure the distribution of contractile stresses arising at the interface between each cell and its substrate; this distribution is called the traction field. Because the traction field must be balanced by tensile stresses within the cell body, the prestress could be computed. Cell stiffness (G) was measured by oscillatory magnetic twisting cytometry. As the contractile state of the cell was modulated with graded concentrations of relaxing or contracting agonists (isoproterenol or histamine, respectively), the mean prestress ((t)) ranged from 350 to 1,900 Pa. Over that range, cell stiffness increased linearly with the prestress: G (Pa) = 0.18(t) + 92. While this association does not necessarily preclude other interpretations, it is the hallmark of systems that secure shape stability mainly through the prestress. Regardless of mechanism, these data establish a strong association between stiffness of HASM cells and the level of tensile stress within the cytoskeleton.

  16. Screening ToxCast™ Phase I Chemicals in a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) Assay

    EPA Science Inventory

    An Adherent Cell Differentiation and Cytotoxicity (ACDC) in vitro assay with mouse embryonic stem cells was used to screen the ToxCast Phase I chemical library for effects on cellular differentiation and cell number. The U.S. Environmental Protection Agency (EPA) established the ...

  17. Soluble fibrin inhibits monocyte adherence and cytotoxicity against tumor cells: implications for cancer metastasis

    PubMed Central

    Biggerstaff, John P; Weidow, Brandy; Vidosh, Jacqueline; Dexheimer, Judith; Patel, Shonak; Patel, Pretesh

    2006-01-01

    Background Soluble fibrin (sFn) is a marker for disseminated intravascular coagulation and may have prognostic significance, especially in metastasis. However, a role for sFn in the etiology of metastatic cancer growth has not been extensively studied. We have reported that sFn cross-linked platelet binding to tumor cells via the major platelet fibrin receptor αIIbβ3, and tumor cell CD54 (ICAM-1), which is the receptor for two of the leukocyte β2 integrins (αLβ2 and aMβ2). We hypothesized that sFn may also affect leukocyte adherence, recognition, and killing of tumor cells. Furthermore, in a rat experimental metastasis model sFn pre-treatment of tumor cells enhanced metastasis by over 60% compared to untreated cells. Other studies have shown that fibrin(ogen) binds to the monocyte integrin αMβ2. This study therefore sought to investigate the effect of sFn on β2 integrin mediated monocyte adherence and killing of tumor cells. Methods The role of sFn in monocyte adherence and cytotoxicity against tumor cells was initially studied using static microplate adherence and cytotoxicity assays, and under physiologically relevant flow conditions in a microscope perfusion incubator system. Blocking studies were performed using monoclonal antibodies specific for β2 integrins and CD54, and specific peptides which inhibit sFn binding to these receptors. Results Enhancement of monocyte/tumor cell adherence was observed when only one cell type was bound to sFn, but profound inhibition was observed when sFn was bound to both monocytes and tumor cells. This effect was also reflected in the pattern of monocyte cytotoxicity. Studies using monoclonal blocking antibodies and specific blocking peptides (which did not affect normal coagulation) showed that the predominant mechanism of fibrin inhibition is via its binding to αMβ2 on monocytes, and to CD54 on both leukocytes and tumor cells. Conclusion sFn inhibits monocyte adherence and cytotoxicity of tumor cells by blocking

  18. Triggering Death of Adherent Cells with Ultraviolet Radiation.

    PubMed

    Crowley, Lisa C; Waterhouse, Nigel J

    2016-01-01

    Ultraviolet (UV) radiation is a convenient stimulus for triggering cell death that is available in most laboratories. We use a Stratalinker UV cross-linker because it is a safe, cheap, reliable, consistent, and easily controlled source of UV irradiation. This protocol describes using a Stratalinker to trigger UV-induced death of HeLa cells. PMID:27371593

  19. Campylobacter jejuni: components for adherence to and invasion of eukaryotic cells.

    PubMed

    Lugert, Raimond; Gross, Uwe; Zautner, Andreas E

    2015-01-01

    Campylobacter (C.) jejuni is the most important reported cause for bacterial diarrhoea. The infection can be accompanied by fever and abdominal cramps and in rare cases the Guillain-Barré syndrome or reactive arthritis can develop as a post-infection complication. Several biological properties of Cjejuni, e. g. motility and chemotaxis, contribute to the biological fitness of the pathogen. For this, deficiencies in the function of these features clearly reduce the pathogenicity of C. jejuni without being a virulence factor per se. Opposing to this, there are two essential requirements to determine the virulence of C. jejuni which represent the adherence to, and the invasion of host cells. Thereby, adherence, as a virulence factor, is mediated by many different bacterial-derived components like proteins but also by several oligo- and polysaccharide structures that are linked to surface proteins but also to the flagella. In addition, several invasion-relevant features of C. jejuni have been detected so far. Whereas some of them are described functionally to modulate cytoskeleton arrangement of the host cell, others are only described as invasion relevant. Indeed, investigations with respect to the pathogenic potential of some adherence- and invasion-relevant components did not give identical results indicating that their relevance might depend on the interplay of the respective C. jejuni strains used in these studies with the corresponding host cells. This review summarizes the C. jejuni components for adherence to and invasion of host cells together with their particular mode of action if known.

  20. Patient–provider communication, self-reported medication adherence, and race in a postmyocardial infarction population

    PubMed Central

    Zullig, Leah L; Shaw, Ryan J; Shah, Bimal R; Peterson, Eric D; Lindquist, Jennifer H; Crowley, Matthew J; Grambow, Steven C; Bosworth, Hayden B

    2015-01-01

    Objectives Our objectives were to: 1) describe patient-reported communication with their provider and explore differences in perceptions of racially diverse adherent versus nonadherent patients; and 2) examine whether the association between unanswered questions and patient-reported medication nonadherence varied as a function of patients’ race. Methods We conducted a cross-sectional analysis of baseline in-person survey data from a trial designed to improve postmyocardial infarction management of cardiovascular disease risk factors. Results Overall, 298 patients (74%) reported never leaving their doctor’s office with unanswered questions. Among those who were adherent and nonadherent with their medications, 183 (79%) and 115 (67%) patients, respectively, never left their doctor’s office with unanswered questions. In multivariable logistic regression, although the simple effects of the interaction term were different for patients of nonminority race (odds ratio [OR]: 2.16; 95% confidence interval [CI]: 1.19–3.92) and those of minority race (OR: 1.19; 95% CI: 0.54–2.66), the overall interaction effect was not statistically significant (P=0.24). Conclusion The quality of patient–provider communication is critical for cardiovascular disease medication adherence. In this study, however, having unanswered questions did not impact medication adherence differently as a function of patients’ race. Nevertheless, there were racial differences in medication adherence that may need to be addressed to ensure optimal adherence and health outcomes. Effort should be made to provide training opportunities for both patients and their providers to ensure strong communication skills and to address potential differences in medication adherence in patients of diverse backgrounds. PMID:25737633

  1. IL-2 induces T cell adherence to extracellular matrix: inhibition of adherence and migration by IL-2 peptides generated by leukocyte elastase.

    PubMed

    Ariel, A; Yavin, E J; Hershkoviz, R; Avron, A; Franitza, S; Hardan, I; Cahalon, L; Fridkin, M; Lider, O

    1998-09-01

    Migration of inflammatory cells requires cell adhesion and their subsequent detachment from the extracellular matrix (ECM). Leukocyte activation and migration must be terminated to stop inflammation. Here, we report that IL-2 enhances human T cell adherence to laminin, collagen type IV, and fibronectin (FN). In contrast, neutrophil elastase, an enzyme activated during inflammation, degrades IL-2 to yield IL-2 fractions that inhibit IL-2-induced T cell adhesion to FN. The amino acid composition of two of these IL-2 fractions, which appear to block T cell adherence to FN, were analyzed, and three peptides were consequently synthesized. The three peptides IVL, RMLT, and EFLNRWIT, but not the corresponding inversely synthesized peptides, inhibited T cell adhesion to FN induced by a variety of activators: IL-2, IL-7, macrophage inflammatory protein (MIP)-1beta, and PMA, as well as anti-CD3 and anti-beta1 integrin-activating mAb. Moreover, these IL-2 peptides inhibited T cell chemotaxis via FN-coated membranes induced by IL-2 and MIP-1beta. Inhibition of T cell adherence and migration apparently involves abrogation of the rearrangement of the T cell actin cytoskeleton. Thus, the migrating immune cells, the cytokines, and the ECM can create a functional relationship in which both inflammation-inducing signals and inhibitory molecules of immune responses can coexist; the enzymatic products of IL-2 may serve as natural feedback inhibitors of inflammation. PMID:9725245

  2. Adherence of Candida albicans to a cell surface polysaccharide receptor on Streptococcus gordonii.

    PubMed Central

    Holmes, A R; Gopal, P K; Jenkinson, H F

    1995-01-01

    Candida albicans ATCC 10261 and CA2 bound to cells of the oral bacteria Streptococcus gordonii, Streptococcus oralis, and Streptococcus sanguis when these bacteria were immobilized onto microtiter plate wells, but they did not bind to cells of Streptococcus mutans or Streptococcus salivarius. Cell wall polysaccharide was extracted with alkali from S. gordonii NCTC 7869, the streptococcal species to which C. albicans bound with highest affinity, and was effective in blocking the coaggregation of C. albicans and S. gordonii cells in the fluid phase. When fixed to microtiter plate wells, the S. gordonii polysaccharide was bound by all strains of C. albicans tested. The polysaccharide contained Rha, Glc, GalNAc, GlcNAc, and Gal and was related compositionally to previously characterized cell wall polysaccharides from strains of S. oralis and S. sanguis. The adherence of yeast cells to the immobilized polysaccharide was not inhibitable by a number of saccharides. Antiserum raised to the S. gordonii NCTC 7869 polysaccharide blocked adherence of C. albicans ATCC 10261 to the polysaccharide. The results identify a complex cell wall polysaccharide of S. gordonii as the coaggregation receptor for C. albicans. Adherent interactions of yeast cells with streptococci and other bacteria may be important for colonization of both hard and soft oral surfaces by C. albicans. PMID:7729891

  3. The functions of the variable lipoprotein family of Mycoplasma hyorhinis in adherence to host cells.

    PubMed

    Xiong, Qiyan; Wang, Jia; Ji, Yan; Ni, Bo; Zhang, Bixiong; Ma, Qinghong; Wei, Yanna; Xiao, Shaobo; Feng, Zhixin; Liu, Maojun; Shao, Guoqing

    2016-04-15

    Mycoplasma hyorhinis (M. hyorhinis) is a swine pathogen that is associated with various human cancers and contamination in cell cultures. However, no studies on the adhesion molecules of this pathogen have yet been reported. The variable lipoprotein (Vlp) family is an important surface component of M. hyorhinis. Herein, we performed several experiments to identify the function of the Vlp family in adherence to host cells. Seven recombinant Vlp (rVlp) proteins were expressed in Escherichia coli and purified by affinity chromatography. The potential role of rVlp adherence to pig kidney (PK-15) and swine tracheal epithelial (STEC) cells was then studied by indirect immunofluorescence assay and microtiter plate adherence assay. Adhesion of M. hyorhinis to PK-15 and STEC cells was specifically inhibited by the addition of a cocktail of rVlp proteins. The rVlp protein mixture was shown to bind to both PK-15 and STEC cells. The binding increased in a dose-dependent manner and could be blocked by antisera against the rVlp proteins. Most of the rVlp proteins could bind individually to both PK-15 and STEC cells except for rVlpD and rVlpF, which bound only to STEC cells. Because Vlp members vary in size among different strains and generations, they may vary in their cytoadhesion capabilities in various strains. In summary, the present results indicate that the Vlp family functions as adhesins of M. hyorhinis. PMID:27016761

  4. Comparative study of the radiobiological effects induced on adherent vs suspended cells by BNCT, neutrons and gamma rays treatments.

    PubMed

    Cansolino, L; Clerici, A M; Zonta, C; Dionigi, P; Mazzini, G; Di Liberto, R; Altieri, S; Ballarini, F; Bortolussi, S; Carante, M P; Ferrari, M; González, S J; Postuma, I; Protti, N; Santa Cruz, G A; Ferrari, C

    2015-12-01

    The present work is part of a preclinical in vitro study to assess the efficacy of BNCT applied to liver or lung coloncarcinoma metastases and to limb osteosarcoma. Adherent growing cell lines can be irradiated as adherent to the culture flasks or as cell suspensions, differences in radio-sensitivity of the two modalities of radiation exposure have been investigated. Dose related cell survival and cell cycle perturbation results evidenced that the radiosensitivity of adherent cells is higher than that of the suspended ones. PMID:26256647

  5. Comparative study of the radiobiological effects induced on adherent vs suspended cells by BNCT, neutrons and gamma rays treatments.

    PubMed

    Cansolino, L; Clerici, A M; Zonta, C; Dionigi, P; Mazzini, G; Di Liberto, R; Altieri, S; Ballarini, F; Bortolussi, S; Carante, M P; Ferrari, M; González, S J; Postuma, I; Protti, N; Santa Cruz, G A; Ferrari, C

    2015-12-01

    The present work is part of a preclinical in vitro study to assess the efficacy of BNCT applied to liver or lung coloncarcinoma metastases and to limb osteosarcoma. Adherent growing cell lines can be irradiated as adherent to the culture flasks or as cell suspensions, differences in radio-sensitivity of the two modalities of radiation exposure have been investigated. Dose related cell survival and cell cycle perturbation results evidenced that the radiosensitivity of adherent cells is higher than that of the suspended ones.

  6. Differences in Haemophilus parasuis adherence to and invasion of AOC-45 porcine aorta endothelial cells

    PubMed Central

    2013-01-01

    Background The pathogenesis of Haemophilus parasuis depends on the bacterium’s ability to interact with endothelial cells and invade adjacent tissues. In this study, we investigated the abilities of eight H. parasuis reference strains belonging to serovars 1, 2, 4, 5, 7, 9, 10 and 13 to adhere to and invade porcine aortic endothelial cells (AOC-45 cell line). Results The strains belonging to serovars 1, 2 and 5 were able to attach at high rates between 60 and 240 min of incubation, and serovars 4, 7 and 13 had moderate attachment rates; however, the strains belonging to serovars 9 and 10 had low adherence at all time points. Strong adherence was observed by scanning electron microscopy for the strains of serovars 5 and 4, which had high and moderate numbers, respectively, of H. parasuis cells attached to AOC-45 cells after 240 min of incubation. The highest invasiveness was reached at 180 min by the serovar 4 strain, followed by the serovar 5 strain at 240 min. The invasion results differed substantially depending on the strain. Conclusion The reference strains of H. parasuis serovars 1, 2, 4 and 5 exhibited high adhesion and invasion levels to AOC-45 porcine aorta endothelial cells, and these findings could aid to better explain the pathogenesis of the disease caused by these serovars. PMID:24119995

  7. Pseudomonas cepacia adherence to respiratory epithelial cells is enhanced by Pseudomonas aeruginosa

    SciTech Connect

    Saiman, L.; Cacalano, G.; Prince, A. )

    1990-08-01

    Pseudomonas aeruginosa and Pseudomonas cepacia are both opportunistic pathogens of patients with cystic fibrosis. The binding characteristics of these two species were compared to determine if they use similar mechanisms to adhere to respiratory epithelial cells. P. cepacia 249 was shown to be piliated, but there was no detectable homology between P. aeruginosa pilin gene probes and P. cepacia genomic DNA. P. cepacia and P. aeruginosa did not appear to compete for epithelial receptors. In the presence of purified P. aeruginosa pili, the adherence of 35S-labeled strain 249 to respiratory epithelial monolayers was unaffected, while that of P. aeruginosa PAO1 was decreased by 55%. The binding of P. cepacia 249 and 715j was increased by 2.4-fold and 1.5-fold, respectively, in the presence of an equal inoculum of PAO1. Interbacterial agglutination contributed to the increased adherence of P. cepacia, as the binding of 249 was increased twofold in the presence of irradiated PAO1. PAO1 exoproducts had a marked effect in enhancing the ability of the P. cepacia strains to adhere to the epithelial monolayers. A PAO1 supernatant increased the binding of 249 by eightfold and that of 715j by fourfold. Thus, there appears to be a synergistic relationship between P. aeruginosa and P. cepacia in which PAO1 exoproducts modify the epithelial cell surface, exposing receptors and facilitating increased P. cepacia attachment.

  8. A dual fluorescence flow cytometric analysis of bacterial adherence to mammalian host cells

    PubMed Central

    Hara-Kaonga, Bochiwe; Pistole, Thomas G.

    2009-01-01

    Flow cytometry has provided a powerful tool for analyzing bacteria-host cell associations. Established approaches have used bacteria, labeled either directly with fluorochromes or indirectly with fluorescently conjugated antibodies, to detect these associations. Although useful, these techniques are unable consistently to include all host cells in the analysis while excluding free, aggregated bacteria. This study describes a new flow cytometry method of assessing bacterial adherence to host cells based on direct fluorescent labeling of both bacteria and host cells. Eukaryotic host cells were labeled with PKH-26, a red fluorescent dye, and bacteria were labeled with fluorescein isothiocyanate, a green fluorescent dye. The red host cells were gated and the mean green fluorescence intensity (MFI) of these red cells was determined. We used MFI values obtained from control samples (unlabeled and labeled host cells with unlabeled bacteria) to eliminate contributions due to autofluorescence. The final MFI values represent fluorescence of host cells resulting from the adherent bacteria. Because all red fluorescent cells are analyzed, this method includes all the eukaryotic cells for analysis but excludes all free or aggregated bacteria that are not bound to target cells. PMID:17222473

  9. A dual fluorescence flow cytometric analysis of bacterial adherence to mammalian host cells.

    PubMed

    Hara-Kaonga, Bochiwe; Pistole, Thomas G

    2007-04-01

    Flow cytometry has provided a powerful tool for analyzing bacteria-host cell associations. Established approaches have used bacteria, labeled either directly with fluorochromes or indirectly with fluorescently conjugated antibodies, to detect these associations. Although useful, these techniques are consistently unable to include all host cells in the analysis while excluding free, aggregated bacteria. This study describes a new flow cytometry method of assessing bacterial adherence to host cells based on direct fluorescent labeling of both bacteria and host cells. Eukaryotic host cells were labeled with PKH-26, a red fluorescent dye, and bacteria were labeled with fluorescein isothiocyanate, a green fluorescent dye. The red host cells were gated and the mean green fluorescence intensity (MFI) of these red cells was determined. We used MFI values obtained from control samples (unlabeled and labeled host cells with unlabeled bacteria) to eliminate contributions due to autofluorescence. The final MFI values represent fluorescence of host cells resulting from the adherent bacteria. Because all red fluorescent cells are analyzed, this method includes all the eukaryotic cells for analysis but excludes all free or aggregated bacteria that are not bound to target cells.

  10. Manipulation of a quasi-natural cell block for high-efficiency transplantation of adherent somatic cells.

    PubMed

    Chung, H J; Hassan, M M; Park, J O; Kim, H J; Hong, S T

    2015-05-01

    Recent advances have raised hope that transplantation of adherent somatic cells could provide dramatic new therapies for various diseases. However, current methods for transplanting adherent somatic cells are not efficient enough for therapeutic applications. Here, we report the development of a novel method to generate quasi-natural cell blocks for high-efficiency transplantation of adherent somatic cells. The blocks were created by providing a unique environment in which cultured cells generated their own extracellular matrix. Initially, stromal cells isolated from mice were expanded in vitro in liquid cell culture medium followed by transferring the cells into a hydrogel shell. After incubation for 1 day with mechanical agitation, the encapsulated cell mass was perforated with a thin needle and then incubated for an additional 6 days to form a quasi-natural cell block. Allograft transplantation of the cell block into C57BL/6 mice resulted in perfect adaptation of the allograft and complete integration into the tissue of the recipient. This method could be widely applied for repairing damaged cells or tissues, stem cell transplantation, ex vivo gene therapy, or plastic surgery.

  11. Establishment and characterization of a novel, spontaneously immortalized retinoblastoma cell line with adherent growth.

    PubMed

    Kim, Jeong Hun; Kim, Jin Hyoung; Yu, Young Suk; Kim, Dong Hun; Kim, Chong Jai; Kim, Kyu-Won

    2007-09-01

    Retinoblastoma is the most common intraocular cancer of childhood, however, only a few cultured retinoblastoma cell lines are available to date. In the present study, we established a new human retinoblastoma cell line with adherent growth, named SNUOT-Rb1. The SNUOT-Rb1 cell line was established from an eye with retinoblastoma, which was enucleated from a 3-year-old Korean child. SNUOT-Rb1 has morphological and biochemical characteristics common to previous human retinoblastoma cell line, Y79: morphological features of fibroblast- or ganglion-like cells, and biochemical features of expression of glial fibrillary acidic protein and neuron-specific enolase. However, compared to Y79, SNUOT-Rb1 has a unique characteristic of growing in adherence, and the doubling time of SNUOT-Rb1 is shorter than Y79 in adherent or floating growth. In analysis of the tumorigenic potential of SNUOT-Rb1 in nude mice, orthotopic implantation of SNUOT-Rb1 mimics the pattern of local growth of retinoblastoma. In comparative genomic hybridization analysis, we found that SNUOT-Rb1 has significant chromosomal imbalances on chromosome 3, 9, 10, 11, 14, 16, 17, and 22. Therefore, SNUOT-Rb1 could be useful in studying the biological and genetic characteristics of retinoblastoma for insights into the heredity and genetics of childhood cancer. PMID:17671685

  12. Impact Mediated Loading Cytoplasmic Loading of Macromolecules into Adherent Cells

    NASA Technical Reports Server (NTRS)

    Clarke, Mark S. F.; Feeback, Daniel L.; Vanderburg, Charles R.

    2003-01-01

    The advent of modern molecular biology, including the development of gene array technologies, has resulted in an explosion of information concerning the specific genes activated during normal cellular development, as well as those associated with a variety of pathological conditions. These techniques have served as a highly efficient, broacI.-based screening approach for those specific genes involved. in regulating normal cellular physiology and identifying candidate genes directly associated with the etiology of specific disease states. However, this approach provides information at the transcriptional' level only and does not necessarily indicate . that the gene in question is in fact translated ito a protein, or whether or not post-translational modification of the protein occurs. The critical importance of post-translational modification (i.e. phosphorylation, glycosylation, sialyation, etc.) to protein function has been recognized with regard to a number of proteins involved in a variety of important disease states. For example, altered glycosylation of beta-amyloid precursor protein results in an increase in the amount of beta-amyloid peptide generated and hence secreted as insoluble extracellular amyloid deposits (Georgopoulou, McLaughlin et al. 2001; Walter, Fluhrer et al. 2001), a pathological hal1nark of Alzheimer's disease. Abnormal phosphorylaion of synapsin I has been linked to alterations in synaptic vesicle trafficking leading to defective neurotransmission in Huntington's disease (Lievens, Woodman et al. 2002). Altered phosphorylation of the TAU protein involved in microtubule function has been linked to a number of neurodegenative diseases such as Alzheimer's disease (Billingsley and Kincaid 1997; Sanchez, Alvarez-Tllada et a1. 2001). Aberrant siaIyation of cell/I surface antigens has been detected in a number of different tumor cell types and has been linked to the acquisition of a neoplastic phenotype (Sell 1990), while improper' sia1yation of

  13. A Longitudinal Study of Adherence to the Mediterranean Dietary Pattern and Metabolic Syndrome in a Non-Mediterranean Population

    PubMed Central

    Mirmiran, Parvin; Moslehi, Nazanin; Mahmoudof, Hessameddin; Sadeghi, Mahbubeh; Azizi, Fereidoun

    2015-01-01

    Background: Adherence to the Mediterranean diet (MedDiet) has been proposed to reduce the risk of metabolic syndrome (MetS), but the association of the diet on MetS in non-Mediterranean populations remains unclear. Objectives: The aim of this study was to investigate whether adherence to the MedDiet is related to the incident MetS and its components during a 3-year follow-up among Iranian adults. Patients and Methods: Longitudinal associations between the MedDiet and MetS components were investigated on 2241 adults, aged 18 - 74 years, without type 2 diabetes selected from participants of the Tehran lipid and glucose study (TLGS). The association between the MedDiet and MetS incidence was also examined in 1661 participants, free of Mets at baseline. Adherence to the MedDiet was assessed using the traditional Mediterranean diet score (MDS) and a recently posteriori adaptation of the MDS of Sofi et al. (Sofi-MDS). MetS was defined according to the recent interim consensus. Results: After adjusting for potential confounders, neither higher MDS nor higher Sofi-MDS were significantly associated with the mean values of MetS components and the risk of metabolic abnormalities incidence after 3 years follow-up. During the 3 years of follow-up, 246 developed MetS were identified. In multivariable model, the adjusted odds ratio (OR) of developing MetS did not differ significantly in participants with the highest tertile of the MDS (OR (95% CI): 0.88 (0.62 - 1.23)) or sofi-MDS (OR (95% CI):1.12 (0.77 - 1.62)) compared to those in the lowest tertiles. Conclusions: Adherence to the MedDiet, according to MDS and Sofi-MDS, may not predict MetS components and MetS incidence after 3 years of follow-up in Iranian adult populations. PMID:26425127

  14. Estimating cell populations

    NASA Technical Reports Server (NTRS)

    White, B. S.; Castleman, K. R.

    1981-01-01

    An important step in the diagnosis of a cervical cytology specimen is estimating the proportions of the various cell types present. This is usually done with a cell classifier, the error rates of which can be expressed as a confusion matrix. We show how to use the confusion matrix to obtain an unbiased estimate of the desired proportions. We show that the mean square error of this estimate depends on a 'befuddlement matrix' derived from the confusion matrix, and how this, in turn, leads to a figure of merit for cell classifiers. Finally, we work out the two-class problem in detail and present examples to illustrate the theory.

  15. Heparin modulates intracellular cyclic AMP in human trabecular bone cells and adherent rheumatoid synovial cells.

    PubMed Central

    Crisp, A J; Roelke, M S; Goldring, S R; Krane, S M

    1984-01-01

    Cells were cultured from explants of human trabecular bone excised from eight patients and incubated usually for 20 minutes with bovine parathyroid hormone, salmon calcitonin, prostaglandin E2, or heparin. The intracellular content of cyclic AMP was measured by radioimmunoassay and was significantly increased by parathyroid hormone in four, by calcitonin in two, by prostaglandin E2 in eight, and by heparin in seven out of eight cultures. In the two cultures containing calcitonin-responsive cells heparin inhibited the cyclic AMP response induced by calcitonin. Heparin did not affect the cyclic AMP response to parathyroid hormone or prostaglandin E2. Heparin also increased the cyclic AMP content of cultured adherent rheumatoid synovial cells. It is proposed that, in certain situations of focal pathological bone resorption, although concentrations of circulating hormones may be normal, the local release of products such as heparin may modify the effect of hormones which regulate connective tissue homoeostasis. local changes in hormone responses could contribute to the enhanced bone resorption associated with inflammatory processes such as rheumatoid arthritis. Images PMID:6089675

  16. Increased neutrophil adherence and adhesion molecule mRNA expression in endothelial cells during selenium deficiency.

    PubMed

    Maddox, J F; Aherne, K M; Reddy, C C; Sordillo, L M

    1999-05-01

    Leukocyte aggregation and activation on endothelial cells (EC) are important preliminary events in leukocyte migration into tissue and subsequent inflammation. Thus, an increase in leukocyte adherence has the potential to affect inflammatory disease outcome. Selenium (Se) is an integral part of the antioxidant enzyme glutathione peroxidase (GSH-Px) and plays an important role in the maintenance of the redox state of a cell. Se supplementation in the bovine has been shown to improve the outcome of acute mastitis caused by coliform bacteria, in part by enhancing the speed of neutrophil migration into the affected mammary gland. However, the mechanisms by which Se modulates neutrophil migration have not been elucidated. Therefore, an in vitro model of Se deficiency in primary bovine mammary artery EC was used to examine the impact of Se status on the adhesive properties of EC. The effect of Se on functional activities was examined by measuring neutrophil adherence to Se-deficient and Se-supplemented EC. Se-deficient EC showed significantly enhanced neutrophil adherence when stimulated with tumor necrosis factor alpha (TNF-alpha) for 4 or 24 h, interleukin-1 for 12 h, or H2O2 for 20 min (P < 0.05). To determine the mechanisms underlying these changes in neutrophil adherence, the expression of EC adhesion molecules, ICAM-1, E-selectin, and P-selectin were examined at the molecular level by a competitive reverse transcription-polymerase chain reaction. Results revealed higher mRNA expression for E-selectin and ICAM-1 in Se-deficient EC stimulated with TNF-alpha for 3 and 6 h, and greater expression of P-selectin mRNA in Se-supplemented EC with 3-h TNF-alpha stimulation. These studies provide new information to establish the role of Se nutrition in the initiation of leukocyte adherence to endothelium. PMID:10331495

  17. Neurosphere and adherent culture conditions are equivalent for malignant glioma stem cell lines.

    PubMed

    Rahman, Maryam; Reyner, Karina; Deleyrolle, Loic; Millette, Sebastien; Azari, Hassan; Day, Bryan W; Stringer, Brett W; Boyd, Andrew W; Johns, Terrance G; Blot, Vincent; Duggal, Rohit; Reynolds, Brent A

    2015-03-01

    Certain limitations of the neurosphere assay (NSA) have resulted in a search for alternative culture techniques for brain tumor-initiating cells (TICs). Recently, reports have described growing glioblastoma (GBM) TICs as a monolayer using laminin. We performed a side-by-side analysis of the NSA and laminin (adherent) culture conditions to compare the growth and expansion of GBM TICs. GBM cells were grown using the NSA and adherent culture conditions. Comparisons were made using growth in culture, apoptosis assays, protein expression, limiting dilution clonal frequency assay, genetic affymetrix analysis, and tumorigenicity in vivo. In vitro expansion curves for the NSA and adherent culture conditions were virtually identical (P=0.24) and the clonogenic frequencies (5.2% for NSA vs. 5.0% for laminin, P=0.9) were similar as well. Likewise, markers of differentiation (glial fibrillary acidic protein and beta tubulin III) and proliferation (Ki67 and MCM2) revealed no statistical difference between the sphere and attachment methods. Several different methods were used to determine the numbers of dead or dying cells (trypan blue, DiIC, caspase-3, and annexin V) with none of the assays noting a meaningful variance between the two methods. In addition, genetic expression analysis with microarrays revealed no significant differences between the two groups. Finally, glioma cells derived from both methods of expansion formed large invasive tumors exhibiting GBM features when implanted in immune-compromised animals. A detailed functional, protein and genetic characterization of human GBM cells cultured in serum-free defined conditions demonstrated no statistically meaningful differences when grown using sphere (NSA) or adherent conditions. Hence, both methods are functionally equivalent and remain suitable options for expanding primary high-grade gliomas in tissue culture.

  18. Urease prevents adherence of Helicobacter pylori to Kato III gastric epithelial cells.

    PubMed

    Makristathis, A; Rokita, E; Pasching, E; Apfalter, P; Willinger, B; Rotter, M L; Hirschl, A M

    2001-08-15

    The role of urease in Helicobacter pylori adherence to and internalization by Kato III cells was investigated. Kato III cells were incubated with wild-type strains (N6 or P1), with isogenic mutants lacking urease (N6ureB::TnKm or P1ureA::TnMax5) or producing the inactive apoprotein (N6ureG::TnKm), and with urease-positive clones recovered after complementation of N6ureB::TnKm with ureAB. Bacteria were stained with the green fluorescent dye PKH2, and the bacteria load of cells was analyzed by flow cytometry. With mutants lacking urease, the bacteria load was considerably increased, in comparison with the corresponding parental strains (P<.001). With clone K2(3), producing larger amounts of urease than N6, a significant reduction of bacteria load was observed, in comparison with the wild type (P<.001). N6ureG::TnKm showed adherence characteristics similar to those of N6. The role of urease in internalization was not clear. Thus, urease significantly inhibits H. pylori adherence to Kato III cells by a mechanism largely independent of enzymatic activity. PMID:11471101

  19. Temperature-induced labelling of Fluo-3 AM selectively yields brighter nucleus in adherent cells

    SciTech Connect

    Meng, Guixian; Pan, Leiting; Li, Cunbo; Hu, Fen; Shi, Xuechen; Lee, Imshik; Drevenšek-Olenik, Irena; Zhang, Xinzheng; Xu, Jingjun

    2014-01-17

    Highlights: •We detailedly examine temperature effects of Fluo-3 AM labelling in adherent cells. •4 °C Loading and 20 °C de-esterification of Fluo-3 AM yields brighter nuclei. •Brighter nuclei labelling by Fluo-3 AM also depends on cell adhesion quality. •A qualitative model of the brighter nucleus is proposed. -- Abstract: Fluo-3 is widely used to study cell calcium. Two traditional approaches: (1) direct injection and (2) Fluo-3 acetoxymethyl ester (AM) loading, often bring conflicting results in cytoplasmic calcium ([Ca{sup 2+}]{sub c}) and nuclear calcium ([Ca{sup 2+}]{sub n}) imaging. AM loading usually yields a darker nucleus than in cytoplasm, while direct injection always induces a brighter nucleus which is more responsive to [Ca{sup 2+}]{sub n} detection. In this work, we detailedly investigated the effects of loading and de-esterification temperatures on the fluorescence intensity of Fluo-3 in response to [Ca{sup 2+}]{sub n} and [Ca{sup 2+}]{sub c} in adherent cells, including osteoblast, HeLa and BV2 cells. Interestingly, it showed that fluorescence intensity of nucleus in osteoblast cells was about two times larger than that of cytoplasm when cells were loaded with Fluo-3 AM at 4 °C and allowed a subsequent step for de-esterification at 20 °C. Brighter nuclei were also acquired in HeLa and BV2 cells using the same experimental condition. Furthermore, loading time and adhesion quality of cells had effect on fluorescence intensity. Taken together, cold loading and room temperature de-esterification treatment of Fluo-3 AM selectively yielded brighter nucleus in adherent cells.

  20. Adherence of bacteria, yeast, blood cells, and latex spheres to large-porosity membrane filters.

    PubMed Central

    Zierdt, C H

    1979-01-01

    Strong adherence of bacteria, yeast, erythrocytes, leukocytes, platelets, spores, and polystyrene spheres to membrane filter materials was noted during filtration through membranes with pore size diameters much larger than the particles themselves. Quantitative recovery on the membrane filters of these particles from low-concentration suspensions was achieved during gravity- or vacuum-assisted filtration through membranes with pore diameters as much as 30 times that of the filtered particles. Mechanical sieving was not responsible. The phenomenon was judged to be electrostatic. It could be partially blocked by pretreating the filter with a nonionic surfactant (Tween 20), and elution of adherent particles was achieved with 0.05% Tween 20. Gram-positive cocci were removed from suspension more efficiently than gram-negative rods. The commonly used cellulose membranes adsorbed more bacteria, blood cells, and other particles than did polycarbonate filters. Of lesser adsorptive capacity were vinyl acetate, nylon, acrylic, and Teflon membranes. Backwashing with saline, serum, 6% NaCl, dextran solutions, or phosphate buffers of varying molality and pH removed only a fraction of adherent particles. Tween 20 (0.05%) eluted up to 45% of adherent particles in a single back-filtration. Selected filters quantitatively removed the particles tested, which then could be washed and subjected to reagents for a variety of purposes. It is important to anticipate the removal of particles during membrane filtration, since it is not a simple mechanical event. Images PMID:393171

  1. Upon impact: the fate of adhering Pseudomonas fluorescens cells during nanofiltration.

    PubMed

    Habimana, Olivier; Semião, Andrea J C; Casey, Eoin

    2014-08-19

    Nanofiltration (NF) is a high-pressure membrane filtration process increasingly applied in drinking water treatment and water reuse processes. NF typically rejects divalent salts, organic matter, and micropollutants. However, the efficiency of NF is adversely affected by membrane biofouling, during which microorganisms adhere to the membrane and proliferate to create a biofilm. Here we show that adhered Pseudomonas fluorescens cells under high permeate flux conditions are met with high fluid shear and convective fluxes at the membrane-liquid interface, resulting in their structural damage and collapse. These results were confirmed by fluorescent staining, flow cytometry, and scanning electron microscopy. This present study offers a "first-glimpse" of cell damage and death during the initial phases of bacterial adhesion to NF membranes and raises a key question about the role of this observed phenomena during early-stage biofilm formation under permeate flux and cross-flow conditions. PMID:25072514

  2. Non-invasive and non-destructive measurements of confluence in cultured adherent cell lines.

    PubMed

    Busschots, Steven; O'Toole, Sharon; O'Leary, John J; Stordal, Britta

    2015-01-01

    Many protocols used for measuring the growth of adherent monolayer cells in vitro are invasive, destructive and do not allow for the continued, undisturbed growth of cells within flasks. Protocols often use indirect methods for measuring proliferation. Microscopy techniques can analyse cell proliferation in a non-invasive or non-destructive manner but often use expensive equipment and software algorithms. In this method images of cells within flasks are captured by photographing under a standard inverted phase contract light microscope using a digital camera with a camera lens adaptor. Images are analysed for confluence using ImageJ freeware resulting in a measure of confluence known as an Area Fraction (AF) output. An example of the AF method in use on OVCAR8 and UPN251 cell lines is included. •Measurements of confluence from growing adherent cell lines in cell culture flasks is obtained in a non-invasive, non-destructive, label-free manner.•The technique is quick, affordable and eliminates sample manipulation.•The technique provides an objective, consistent measure of when cells reach confluence and is highly correlated to manual counting with a haemocytometer. The average correlation co-efficient from a Spearman correlation (n = 3) was 0.99 ± 0.008 for OVCAR8 (p = 0.01) and 0.99 ± 0.01 for UPN251 (p = 0.01) cell lines.

  3. Non-invasive and non-destructive measurements of confluence in cultured adherent cell lines

    PubMed Central

    Busschots, Steven; O’Toole, Sharon; O’Leary, John J.; Stordal, Britta

    2014-01-01

    Many protocols used for measuring the growth of adherent monolayer cells in vitro are invasive, destructive and do not allow for the continued, undisturbed growth of cells within flasks. Protocols often use indirect methods for measuring proliferation. Microscopy techniques can analyse cell proliferation in a non-invasive or non-destructive manner but often use expensive equipment and software algorithms. In this method images of cells within flasks are captured by photographing under a standard inverted phase contract light microscope using a digital camera with a camera lens adaptor. Images are analysed for confluence using ImageJ freeware resulting in a measure of confluence known as an Area Fraction (AF) output. An example of the AF method in use on OVCAR8 and UPN251 cell lines is included. • Measurements of confluence from growing adherent cell lines in cell culture flasks is obtained in a non-invasive, non-destructive, label-free manner. • The technique is quick, affordable and eliminates sample manipulation. • The technique provides an objective, consistent measure of when cells reach confluence and is highly correlated to manual counting with a haemocytometer. The average correlation co-efficient from a Spearman correlation (n = 3) was 0.99 ± 0.008 for OVCAR8 (p = 0.01) and 0.99 ± 0.01 for UPN251 (p = 0.01) cell lines. PMID:26150966

  4. Morphological investigations of cells that adhered to the irregular patterned polydimethylsiloxane (PDMS) surface without reagents.

    PubMed

    Chung, Sung Hee; Min, Junhong

    2009-07-01

    Polydimethylsiloxane (PDMS) surface consisting irregular pattern was investigated to develop cell-based biochip using PDMS. PDMS surface was modified with nano- and micro-combined patterns using surface deformation technology. Hydrophobicity of nano-patterned PDMS surface was sustained. Nevertheless it has irregular patterns consisting of micro- and nano-patterns. According to atomic force microscopy (AFM), scanning electron microscopy (SEM) and confocal microscopy results by immunostaining method, human mammary epithelial cells (HMEC) adhered well on irregularly patterned surface without any reagents such as gelatin and collagen, compared to commercial culture dish. It implies PDMS material can be utilized as template for cell-based biochip without any reagents. PMID:19427124

  5. Is there an association between immunosuppressant therapy medication adherence and depression, quality of life, and personality traits in the kidney and liver transplant population?

    PubMed Central

    Gorevski, Elizabeth; Succop, Paul; Sachdeva, Jyoti; Cavanaugh, Teresa M; Volek, Paul; Heaton, Pamela; Chisholm-Burns, Marie; Martin-Boone, Jill E

    2013-01-01

    Objectives To measure the association of transplant patients’ personality, depression, and quality of life with medication adherence in kidney and liver transplant recipients. Methods A cross-sectional study of liver and kidney transplant recipients greater than 1 year post-transplant was conducted. Patients’ adherence with medications was assessed using the Immunosuppressive Therapy Adherence Scale. Personality and depression were assessed using the NEO Five-Factor Inventory Scale and Patient Health Questionnaire 9, respectively. Quality of life was assessed using the Short Form-36, and functional status was determined using the Karnofsky Performance Status Scale. Results A total of 86 kidney and 50 liver transplant patients completed the surveys. Logistic regression analysis demonstrated an association between depression and adherence with immunosuppressive medications in kidney transplant recipients. Kidney transplant patients who exhibited “low openness” scores were 91% more likely to be nonadherent. Kidney transplant patients’ physical functional status was strongly associated with nonadherence, and for each point increase in functionality the patients’ adherence increased by 4%. In the liver sample, age was associated with adherence. For every year increase in age, adherence increased by 7%. Conclusion The presence of low openness as a personality trait, poor physical functional status, and depression were associated with adherence in the kidney transplant population. In the liver transplant population, younger age was associated with nonadherence. PMID:23620661

  6. Trichomonas vaginalis surface proteinase activity is necessary for parasite adherence to epithelial cells.

    PubMed Central

    Arroyo, R; Alderete, J F

    1989-01-01

    The role of cysteine proteinases in adherence of Trichomonas vaginalis NYH 286 to HeLa and human vaginal epithelial cells was evaluated. Only pretreatment of trichomonads, but not epithelial cells, with N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), an inhibitor of trichomonad cysteine proteinases, greatly diminished the ability of T. vaginalis to recognize and bind to epithelial cells. Leupeptin and L-1-tosylamide-2-phenylethyl chloromethyl ketone, other cysteine proteinase inhibitors, also decreased T. vaginalis cytadherence. Parasites incubated with TLCK and washed extensively still did not adhere to cells at levels equal to those seen for control trichomonads treated with phosphate-buffered saline or culture medium alone. Exposure of TLCK-treated organisms with other cysteine proteinases restored cytadherence levels, indicating that proteinase action on the parasite surface is prerequisite for host cell attachment. Concentrations of TLCK which inhibited cytadherence did not alter the metabolism of T. vaginalis, as determined by metabolic labeling of trichomonad proteins; the protein patterns of T. vaginalis in the presence and absence of TLCK were identical. Kinetics of TLCK-mediated inhibition of cytadherence of other T. vaginalis isolates with different levels of epithelial-cell parasitism were similar to the concentration-dependent inhibition seen for isolate NYH 286. Incubation of TLCK-treated, washed organisms in growth medium resulted in regeneration of adherence. Finally, treatment of T. vaginalis organisms with proteinase inhibitors for abrogation of cytadherence effectively rendered the trichomonads unable to kill host cells, which is consistent with the contact-dependent nature of host cytotoxicity. These data show for the first time the involvement of T. vaginalis cysteine proteinases in parasite attachment to human epithelial cells. These results have implications for future pharmacologic intervention at a key step in infection. PMID:2789190

  7. Transglutaminase 2 is essential for adherence of Porphyromonas gingivalis to host cells

    PubMed Central

    Boisvert, Heike; Lorand, Laszlo; Duncan, Margaret J.

    2014-01-01

    Porphyromonas gingivalis is the major causative agent of periodontitis, and it may also be involved in the development of systemic diseases (atherosclerosis, rheumatoid arthritis). P. gingivalis is found on and within oral and gingival epithelial cells following binding to surface components of host cells, which serve as receptors for the bacterium. Evidence is presented in this study that shows that transglutaminase 2 (TG2) plays a critical role in the adherence of P. gingivalis to host cells. Studies of confocal microscopy indicate colocalization of P. gingivalis with TG2 on the surface of HEp-2 epithelial cells, with clusters of TG2 seen at bacterial attachment sites. By silencing the expression of TG2 with siRNA in HEp-2 cells, P. gingivalis association was greatly diminished. The bacterium does not bind well to a mouse fibroblast cell line that produces low amounts of surface TG2, but binding can be restored by introduction of TG2 expressed on a plasmid. TG2 can form very tight complexes with fibronectin (FN), and the complementary binding sites of the two proteins are known. A synthetic peptide that mimics the main FN-binding sequence of TG2 blocks the formation of TG2–FN complexes and is highly effective in inhibiting adherence of P. gingivalis to host cells. These findings provide evidence of a role for cell-surface TG2 in bacterial attachment and subsequent internalization. PMID:24706840

  8. Towards high-throughput automated targeted femtosecond laser-based transfection of adherent cells

    NASA Astrophysics Data System (ADS)

    Antkowiak, Maciej; Torres-Mapa, Maria Leilani; Gunn-Moore, Frank; Dholakia, Kishan

    2011-03-01

    Femtosecond laser induced cell membrane poration has proven to be an attractive alternative to the classical methods of drug and gene delivery. It is a selective, sterile, non-contact technique that offers a highly localized operation, low toxicity and consistent performance. However, its broader application still requires the development of robust, high-throughput and user-friendly systems. We present a system capable of unassisted enhanced targeted optoinjection and phototransfection of adherent mammalian cells with a femtosecond laser. We demonstrate the advantages of a dynamic diffractive optical element, namely a spatial light modulator (SLM) for precise three dimensional positioning of the beam. It enables the implementation of a "point-and-shoot" system in which using the software interface a user simply points at the cell and a predefined sequence of precisely positioned doses can be applied. We show that irradiation in three axial positions alleviates the problem of exact beam positioning on the cell membrane and doubles the number of viably optoinjected cells when compared with a single dose. The presented system enables untargeted raster scan irradiation which provides transfection of adherent cells at the throughput of 1 cell per second.

  9. Bio-chemo-mechanical models for nuclear deformation in adherent eukaryotic cells.

    PubMed

    Nava, Michele M; Raimondi, Manuela T; Pietrabissa, Riccardo

    2014-10-01

    Adherent eukaryotic cells are subjected to a broad variety of extracellular and intracellular stimuli regulating their behaviour. These stimuli can be either purely chemical, for example soluble factors binding to the cell membrane, or mechano-chemical, for example integrin-based adhesion complexes stretching the cell cytoskeleton. Here, we focus on mechano-chemical stimuli such as extracellular forces (interstitial flow, pressurization) and intracellular forces (due to cell adhesion), which may combine generating stress-strain states in the cytoskeleton. These states are transferred to the nucleus to influence the transcription of specific genes, likely by changing the chromatin organization and by altering the permeability of the nuclear membrane. While there exists increasing experimental evidence of the mechanosensing role of the cell nucleus, both the underlying molecular mechanisms involved, and the nuclear structural behaviour in response to forces, are still poorly understood. Here, we review the existing literature on computational models developed to investigate the chemo-mechanical behaviour of adherent eukaryotic cells. We analyse two main classes of models of single-cell mechanics, based either on the discrete or on the continuum approaches. We focus on the bio-chemo-mechanical model and modelling techniques accounting for the nuclear body. The modelling techniques are discussed highlighting their ability in predicting cytoskeletal contractility states and nuclear stress-strain states.

  10. Effect of enteroviruses on adherence to and invasion of HEp-2 cells by Campylobacter isolates.

    PubMed Central

    Konkel, M E; Joens, L A

    1990-01-01

    Coinfection of HEp-2 epithelial cells with coxsackievirus B3, echovirus 7, poliovirus (LSc type 1), porcine enterovirus, and Campylobacter isolates was performed to determine if a synergistic effect could be obtained. The invasiveness of Campylobacter jejuni ATCC 33560 was significantly increased for HEp-2 cells preinfected with echovirus 7, coxsackievirus B3, and UV-inactivated (noninfectious) coxsackievirus B3 particles. Additionally, the invasiveness of C. jejuni M96, a clinical isolate, was significantly increased for HEp-2 cells preinfected with coxsackievirus B3. Poliovirus and porcine enterovirus had no effect on C. jejuni ATCC 33560 adherence and invasiveness. Furthermore, poliovirus had no effect on the ability of C. jejuni M96 to adhere to and invade HEp-2 cells. Campylobacter hyointestinalis and Campylobacter mucosalis, two noninvasive isolates, did not invade virus-infected HEp-2 cells. The increase in the invasiveness of C. jejuni appeared to be the result of specific interactions between the virus and the HEp-2 cell membrane. The data suggest that the invasiveness of Campylobacter spp. is dependent upon the inherent properties of the organism. Virus-induced cell alterations can potentiate the invasiveness of virulent Campylobacter spp. but are not sufficient to allow internalization of noninvasive bacteria. PMID:2156779

  11. Pathogenic hantaviruses direct the adherence of quiescent platelets to infected endothelial cells.

    PubMed

    Gavrilovskaya, Irina N; Gorbunova, Elena E; Mackow, Erich R

    2010-05-01

    Hantavirus infections are noted for their ability to infect endothelial cells, cause acute thrombocytopenia, and trigger 2 vascular-permeability-based diseases. However, hantavirus infections are not lytic, and the mechanisms by which hantaviruses cause capillary permeability and thrombocytopenia are only partially understood. The role of beta(3) integrins in hemostasis and the inactivation of beta(3) integrin receptors by pathogenic hantaviruses suggest the involvement of hantaviruses in altered platelet and endothelial cell functions that regulate permeability. Here, we determined that pathogenic hantaviruses bind to quiescent platelets via a beta(3) integrin-dependent mechanism. This suggests that platelets may contribute to hantavirus dissemination within infected patients and provides a means by which hantavirus binding to beta(3) integrin receptors prevents platelet activation. The ability of hantaviruses to bind platelets further suggested that cell-associated hantaviruses might recruit platelets to the endothelial cell surface. Our findings indicate that Andes virus (ANDV)- or Hantaan virus (HTNV)-infected endothelial cells specifically direct the adherence of calcein-labeled platelets. In contrast, cells comparably infected with nonpathogenic Tula virus (TULV) failed to recruit platelets to the endothelial cell surface. Platelet adherence was dependent on endothelial cell beta(3) integrins and neutralized by the addition of the anti-beta(3) Fab fragment, c7E3, or specific ANDV- or HTNV-neutralizing antibodies. These findings indicate that pathogenic hantaviruses displayed on the surface of infected endothelial cells bind platelets and that a platelet layer covers the surface of infected endothelial cells. This fundamentally changes the appearance of endothelial cells and has the potential to alter cellular immune responses, platelet activation, and endothelial cell functions that affect vascular permeability. Hantavirus-directed platelet quiescence and

  12. Moraxella catarrhalis Expresses a Cardiolipin Synthase That Impacts Adherence to Human Epithelial Cells

    PubMed Central

    Buskirk, Sean W.

    2014-01-01

    The major phospholipid constituents of Moraxella catarrhalis membranes are phosphatidylglycerol, phosphatidylethanolamine, and cardiolipin (CL). However, very little is known regarding the synthesis and function of these phospholipids in M. catarrhalis. In this study, we discovered that M. catarrhalis expresses a cardiolipin synthase (CLS), termed MclS, that is responsible for the synthesis of CL within the bacterium. The nucleotide sequence of mclS is highly conserved among M. catarrhalis isolates and is predicted to encode a protein with significant amino acid similarity to the recently characterized YmdC/ClsC protein of Escherichia coli. Isogenic mclS mutant strains were generated in M. catarrhalis isolates O35E, O12E, and McGHS1 and contained no observable levels of CL. Site-directed mutagenesis of a highly conserved HKD motif of MclS also resulted in a CL-deficient strain. Moraxella catarrhalis, which depends on adherence to epithelial cells for colonization of the human host, displays significantly reduced levels of adherence to HEp-2 and A549 cell lines in the mclS mutant strains compared to wild-type bacteria. The reduction in adherence appears to be attributed to the absence of CL. These findings mark the first instance in which a CLS has been related to a virulence-associated trait. PMID:24142255

  13. Subinhibitory concentrations of triclosan promote Streptococcus mutans biofilm formation and adherence to oral epithelial cells.

    PubMed

    Bedran, Telma Blanca Lombardo; Grignon, Louis; Spolidorio, Denise Palomari; Grenier, Daniel

    2014-01-01

    Triclosan is a general membrane-active agent with a broad-spectrum antimicrobial activity that is commonly used in oral care products. In this study, we investigated the effect of sub-minimum inhibitory concentrations (MICs) of triclosan on the capacity of the cariogenic bacterium Streptococcus mutans to form biofilm and adhere to oral epithelial cells. As quantified by crystal violet staining, biofilm formation by two reference strains of S. mutans was dose-dependently promoted, in the range of 2.2- to 6.2-fold, by 1/2 and 1/4 MIC of triclosan. Observations by scanning electron microscopy revealed the presence of a dense biofilm attached to the polystyrene surface. Growth of S. mutans in the presence of triclosan at sub-MICs also increased its capacity to adhere to a monolayer of gingival epithelial cells. The expression of several genes involved in adherence and biofilm formation in S. mutans was investigated by quantitative RT-PCR. It was found that sub-MICs of triclosan significantly increased the expression of comD, gtfC, and luxS, and to a lesser extent of gtfB and atlA genes. These findings stress the importance of maintaining effective bactericidal concentrations of therapeutic triclosan since sub-MICs may promote colonization of the oral cavity by S. mutans.

  14. Toxicity Minimized Cryoprotectant Addition and Removal Procedures for Adherent Endothelial Cells

    PubMed Central

    Davidson, Allyson Fry; Glasscock, Cameron; McClanahan, Danielle R.; Benson, James D.; Higgins, Adam Z.

    2015-01-01

    Ice-free cryopreservation, known as vitrification, is an appealing approach for banking of adherent cells and tissues because it prevents dissociation and morphological damage that may result from ice crystal formation. However, current vitrification methods are often limited by the cytotoxicity of the concentrated cryoprotective agent (CPA) solutions that are required to suppress ice formation. Recently, we described a mathematical strategy for identifying minimally toxic CPA equilibration procedures based on the minimization of a toxicity cost function. Here we provide direct experimental support for the feasibility of these methods when applied to adherent endothelial cells. We first developed a concentration- and temperature-dependent toxicity cost function by exposing the cells to a range of glycerol concentrations at 21°C and 37°C, and fitting the resulting viability data to a first order cell death model. This cost function was then numerically minimized in our state constrained optimization routine to determine addition and removal procedures for 17 molal (mol/kg water) glycerol solutions. Using these predicted optimal procedures, we obtained 81% recovery after exposure to vitrification solutions, as well as successful vitrification with the relatively slow cooling and warming rates of 50°C/min and 130°C/min. In comparison, conventional multistep CPA equilibration procedures resulted in much lower cell yields of about 10%. Our results demonstrate the potential for rational design of minimally toxic vitrification procedures and pave the way for extension of our optimization approach to other adherent cell types as well as more complex systems such as tissues and organs. PMID:26605546

  15. Applications of electroporation of adherent cells in situ, on a partly conductive slide.

    PubMed

    Raptis, L H; Brownell, H L; Liu, S K; Firth, K L; MacKenzie, L W; Stiles, C D; Alberta, J A

    1995-10-01

    Nontraumatic, simple, and reproducible procedures for the introduction of nonpermeant molecules into adherent mammalian cells by in situ electroporation are described. Cells are grown on a glass slide, half of which is coated with electrically conductive, optically transparent, indium-tin oxide. An electric pulse is applied in the presence of the molecules to be introduced, and their effect on the cellular phenotype can be observed. The cells growing on the nonconductive side of the slide do not receive any pulse and serve as controls. Careful adjustment of electric field strength can achieve the introduction of the molecules into essentially 100% of the cells, and this treatment causes no detectable disruption to cellular metabolism. This is applied in the presence of the fluorescent dye, Lucifer yellow, causing its penetration into the cells growing on the conductive half of the slide. The migration of the dye to the nonelectroporated cells growing on the nonconductive area is microscopically observed under fluorescence illumination. PMID:8556428

  16. Fibrin clots keep non-adhering living cells in place on glass for perfusion or fixation.

    PubMed

    Forer, Arthur; Pickett-Heaps, Jeremy

    2005-09-01

    We describe a method to hold living cells in place that ordinarily do not adhere to glass coverslips. The method, developed for insect spermatocytes but with application to other cell types, consists of embedding cells in a fibrin clot that forms after the enzyme thrombin cleaves the blood protein fibrinogen. The method permits continuous observation of living cells as they are treated with and recover from drug or other treatments: when held in the clot the living cells remain in place and keep their shapes when perfused with drugs that ordinarily cause drastic shape changes, and they remain in place and keep their shapes through lysis/fixation procedures. We describe how to place live cells in a fibrin clot and how subsequently to perfuse them. PMID:16095930

  17. Reduction of Adherence of E. coli O157:H7 to HEp-2 Cells and to Bovine Large Intestinal Mucosal Explants by Colicinogenic E. coli

    PubMed Central

    Etcheverría, A. I.; Arroyo, G. H.; Alzola, R.; Parma, A. E.

    2011-01-01

    Enterohemorrhagic E. coli strains (EHEC) had emerged as foodborne pathogens and cause in human diarrhea and hemolytic-uremic syndrome. Because of the widespread distribution of EHEC serotypes and O157 and non-O157 in cattle population, its control will require interventions at the farm level such as the administration of probiotics that produce inhibitory metabolites. E. coli O157:H7 shows tissue tropisms for the gastrointestinal tract (GIT) of cattle. The aim of this study was to test the ability of a colicinogenic E. coli (isolated from bovine) to reduce the adherence of E. coli O157:H7 to HEp-2 cells and to GIT of cattle. We inoculated HEp-2 cells and bovine colon explants with both kinds of strains. Colicinogenic E. coli was able to reduce the adherence of E. coli O157:H7 to HEp-2 cells and to bovine tissues. PMID:23724308

  18. Adherence of Staphylococcus epidermidis to human endothelial cells is associated with a polysaccharidic component of its extracellular mucous layer.

    PubMed

    Krevvata, Maria I; Spiliopoulou, Anastasia; Anastassiou, Evangelos D; Karamanos, Nikos; Kolonitsiou, Fevronia

    2011-06-01

    Bacterial adherence to eukaryotic cells is highly contributing to microbial pathogenesis. Bacterial adhesins, macromolecules, and glycosaminoglycan chains of the endothelial cell surface have been implicated in staphylococcal attachment. Our research group has isolated an antigenic polysaccharidic component of Staphylococcus epidermidis extracellular layer, known as 20-kDa PS (PS), and showed that antibodies against this polysaccharide protect from infections. Therefore, the role of PS in S. epidermidis adherence to endothelial cells was studied. For this purpose we examined the impact of PS on the ability of two S. epidermidis strains (a PS-producing and a non-PS-producing strain) to adhere to human endothelial cells in the presence or absence of specific antibodies to this polysaccharide. Hence, it is established that exogenous chondroitin sulfate (CS) decreases, in part, the S. epidermidis' attachment to endothelial cells and the antagonistic binding effect of CS and PS was also studied. The results obtained demonstrate that PS facilitates the adherence of S. epidermidis to both strains. CS abolished the PS-induced adherence in PS-producing strain and partially in the non-PS-producing one. Conclusively, the adherence of S. epidermidis to human endothelial cells is associated with its extracellular PS component and it is suggested that the bacterial binding via glycosaminoglycan chains is an important mechanism underlining the PS-induced binding to endothelial cells.

  19. Participation of Integrin α5β1 in the Fibronectin-Mediated Adherence of Enteroaggregative Escherichia coli to Intestinal Cells

    PubMed Central

    Izquierdo, Mariana; Nataro, James P.; Ruiz-Perez, Fernando; Farfan, Mauricio J.

    2014-01-01

    Adherence to the intestinal epithelia is a key feature in enteroaggregative Escherichia coli (EAEC) infection. The aggregative adherence fimbriae (AAFs) are involved in EAEC interaction with receptors at the surface of intestinal cells. We and others have demonstrated that fibronectin is a receptor for AAF/II fimbriae. Considering that the major cellular receptor of fibronectin is integrin α5β1, in this study we evaluated the participation of this receptor in the fibronectin-mediated adherence of EAEC strain 042 to intestinal cells. We found that EAEC strain 042 has the ability to bind directly and indirectly to integrin α5β1; direct binding was not mediated by AAF/II fimbriae and indirect binding was mediated by AAF/II and fibronectin. Coimmunoprecipitation assays confirmed the formation of the complex AafA/fibronectin/integrin α5β1. To evaluate EAEC adherence to intestinal cells, T84 cells were incubated with fibronectin and an antibody that blocks the interaction region of integrin α5β1 to fibronectin, the RGD site. Under these conditions, we found the number of adherent bacteria to epithelial cells significantly reduced. Additionally, fibronectin-mediated adherence of EAEC strain 042 was abolished in HEp-2 cells transfected with integrin α5 shRNA. Altogether, our data support the involvement of integrin α5β1 in the fibronectin-mediated EAEC binding to intestinal cells. PMID:25177698

  20. Participation of integrin α5β1 in the fibronectin-mediated adherence of enteroaggregative Escherichia coli to intestinal cells.

    PubMed

    Izquierdo, Mariana; Alvestegui, Alejandra; Nataro, James P; Ruiz-Perez, Fernando; Farfan, Mauricio J

    2014-01-01

    Adherence to the intestinal epithelia is a key feature in enteroaggregative Escherichia coli (EAEC) infection. The aggregative adherence fimbriae (AAFs) are involved in EAEC interaction with receptors at the surface of intestinal cells. We and others have demonstrated that fibronectin is a receptor for AAF/II fimbriae. Considering that the major cellular receptor of fibronectin is integrin α5β1, in this study we evaluated the participation of this receptor in the fibronectin-mediated adherence of EAEC strain 042 to intestinal cells. We found that EAEC strain 042 has the ability to bind directly and indirectly to integrin α5β1; direct binding was not mediated by AAF/II fimbriae and indirect binding was mediated by AAF/II and fibronectin. Coimmunoprecipitation assays confirmed the formation of the complex AafA/fibronectin/integrin α5β1. To evaluate EAEC adherence to intestinal cells, T84 cells were incubated with fibronectin and an antibody that blocks the interaction region of integrin α5β1 to fibronectin, the RGD site. Under these conditions, we found the number of adherent bacteria to epithelial cells significantly reduced. Additionally, fibronectin-mediated adherence of EAEC strain 042 was abolished in HEp-2 cells transfected with integrin α5 shRNA. Altogether, our data support the involvement of integrin α5β1 in the fibronectin-mediated EAEC binding to intestinal cells.

  1. Participation of integrin α5β1 in the fibronectin-mediated adherence of enteroaggregative Escherichia coli to intestinal cells.

    PubMed

    Izquierdo, Mariana; Alvestegui, Alejandra; Nataro, James P; Ruiz-Perez, Fernando; Farfan, Mauricio J

    2014-01-01

    Adherence to the intestinal epithelia is a key feature in enteroaggregative Escherichia coli (EAEC) infection. The aggregative adherence fimbriae (AAFs) are involved in EAEC interaction with receptors at the surface of intestinal cells. We and others have demonstrated that fibronectin is a receptor for AAF/II fimbriae. Considering that the major cellular receptor of fibronectin is integrin α5β1, in this study we evaluated the participation of this receptor in the fibronectin-mediated adherence of EAEC strain 042 to intestinal cells. We found that EAEC strain 042 has the ability to bind directly and indirectly to integrin α5β1; direct binding was not mediated by AAF/II fimbriae and indirect binding was mediated by AAF/II and fibronectin. Coimmunoprecipitation assays confirmed the formation of the complex AafA/fibronectin/integrin α5β1. To evaluate EAEC adherence to intestinal cells, T84 cells were incubated with fibronectin and an antibody that blocks the interaction region of integrin α5β1 to fibronectin, the RGD site. Under these conditions, we found the number of adherent bacteria to epithelial cells significantly reduced. Additionally, fibronectin-mediated adherence of EAEC strain 042 was abolished in HEp-2 cells transfected with integrin α5 shRNA. Altogether, our data support the involvement of integrin α5β1 in the fibronectin-mediated EAEC binding to intestinal cells. PMID:25177698

  2. Medication Adherence Patterns after Hospitalization for Coronary Heart Disease. A Population-Based Study Using Electronic Records and Group-Based Trajectory Models

    PubMed Central

    Librero, Julián; Sanfélix-Gimeno, Gabriel; Peiró, Salvador

    2016-01-01

    Objective To identify adherence patterns over time and their predictors for evidence-based medications used after hospitalization for coronary heart disease (CHD). Patients and Methods We built a population-based retrospective cohort of all patients discharged after hospitalization for CHD from public hospitals in the Valencia region (Spain) during 2008 (n = 7462). From this initial cohort, we created 4 subcohorts with at least one prescription (filled or not) from each therapeutic group (antiplatelet, beta-blockers, ACEI/ARB, statins) within the first 3 months after discharge. Monthly adherence was defined as having ≥24 days covered out of 30, leading to a repeated binary outcome measure. We assessed the membership to trajectory groups of adherence using group-based trajectory models. We also analyzed predictors of the different adherence patterns using multinomial logistic regression. Results We identified a maximum of 5 different adherence patterns: 1) Nearly-always adherent patients; 2) An early gap in adherence with a later recovery; 3) Brief gaps in medication use or occasional users; 4) A slow decline in adherence; and 5) A fast decline. These patterns represented variable proportions of patients, the descending trajectories being more frequent for the beta-blocker and ACEI/ARB cohorts (16% and 17%, respectively) than the antiplatelet and statin cohorts (10% and 8%, respectively). Predictors of poor or intermediate adherence patterns were having a main diagnosis of unstable angina or other forms of CHD vs. AMI in the index hospitalization, being born outside Spain, requiring copayment or being older. Conclusion Distinct adherence patterns over time and their predictors were identified. This may be a useful approach for targeting improvement interventions in patients with poor adherence patterns. PMID:27551748

  3. Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay-Book Chapter*

    EPA Science Inventory

    There are thousands of environmental chemicals for which there is limited toxicological information, motivating the development and application of in vitro systems to profile the biological effects of xenobiotic exposure and predict their potential developmental hazard. An adhere...

  4. Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) Assay: Book Chapter

    EPA Science Inventory

    There are thousands of environmental chemicals for which there is limited toxicological information, motivating the development and application of in vitro systems to profile the biological effects of xenobiotic exposure and predict their potential developmental hazard. An adher...

  5. A review of the role of food insecurity in adherence to care and treatment among adult and pediatric populations living with HIV and AIDS

    PubMed Central

    Young, Sera; Wheeler, Amanda; McCoy, Sandi; Weiser, Sheri D.

    2013-01-01

    Adherence to antiretroviral therapy (ART) is critical for reducing HIV/AIDS morbidity and mortality. Food insecurity (FI) is emerging as an important barrier to adherence to care and treatment recommendations for people living with HIV (PLHIV), but this relationship has not been comprehensively examined. Therefore, we reviewed the literature to explore how FI may impact ART adherence, retention in medical care, and adherence to health care recommendations among PLHIV. We found data to support FI as a critical barrier to adherence to ART and to other health care recommendations among HIV-infected adults, HIV-infected pregnant women and their HIV-exposed infants, and child and adolescent populations of PLHIV. Associations between FI and ART non-adherence were seen in qualitative and quantitative studies. We identified a number of mechanisms to explain how food insecurity and ART non-adherence may be causally linked, including the exacerbation of hunger or ART side effects in the absence of adequate food and competing resource demands. Interventions that address FI may improve adherence to care and treatment recommendations for PLHIV. PMID:23842717

  6. Study protocol for a randomized controlled trial to assess the feasibility of an open label intervention to improve hydroxyurea adherence in youth with sickle cell disease

    PubMed Central

    Smaldone, Arlene; Findley, Sally; Bakken, Suzanne; Matiz, L. Adriana; Rosenthal, Susan L.; Jia, Haomiao; Matos, Sergio; Manwani, Deepa; Green, Nancy S.

    2016-01-01

    Background Community health workers (CHW) are increasingly recognized as a strategy to improve health outcomes for the underserved with chronic diseases but has not been formally explored in adolescents with sickle cell disease (SCD). SCD primarily affects African American, Hispanic and other traditionally underserved populations. Hydroxyurea (HU), an oral, once-daily medication, is the only approved therapeutic drug for sickle cell disease and markedly reduces symptoms, morbidity and mortality and improves quality of life largely by increasing hemoglobin F blood levels. This paper presents the rationale, study design and protocol for an open label randomized controlled trial to improve parent-youth partnerships in self-management and medication adherence to HU in adolescents with SCD. Methods/Design A CHW intervention augmented by text messaging was designed for adolescents with SCD ages 10–18 years and their parents to improve daily HU adherence. Thirty adolescent parent dyads will be randomized with 2:1 intervention group allocation. Intervention dyads will establish a relationship with a culturally aligned CHW to identify barriers to HU use, identify cues to build a habit, and develop a dyad partnership to improve daily HU adherence and achieve their individualized “personal best” hemoglobin F target. Intervention feasibility, acceptability and efficacy will be assessed via a 2-site trial. Outcomes of interest are HU adherence, dyad self-management communication, quality of life, and resource use. Discussion Despite known benefits, poor HU adherence is common. If feasible and acceptable, the proposed intervention may improve health of underserved adolescents with SCD by enhancing long-term HU adherence. PMID:27327779

  7. Enterotoxigenic Escherichia coli TibA Glycoprotein Adheres to Human Intestine Epithelial Cells

    PubMed Central

    Lindenthal, Christoph; Elsinghorst, Eric A.

    2001-01-01

    Enterotoxigenic Escherichia coli (ETEC) is capable of invading epithelial cell lines derived from the human ileum and colon. Two separate invasion loci (tia and tib) that direct noninvasive E. coli strains to adhere to and invade cultured human intestine epithelial cells have previously been isolated from the classical ETEC strain H10407. The tib locus directs the synthesis of TibA, a 104-kDa outer membrane glycoprotein. Synthesis of TibA is directly correlated with the adherence and invasion phenotypes of the tib locus, suggesting that this protein is an adhesin and invasin. Here we report the purification of TibA and characterization of its biological activity. TibA was purified by continuous-elution preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified TibA was biotin labeled and then shown to bind to HCT8 human ileocecal epithelial cells in a specific and saturable manner. Unlabeled TibA competed with biotin-labeled TibA, suggesting the presence of a specific TibA receptor in HCT8 cells. These results show that TibA acts as an adhesin. Polyclonal anti-TibA antiserum inhibited invasion of ETEC strain H10407 and of recombinant E. coli bearing tib locus clones, suggesting that TibA also acts as an invasin. The ability of TibA to direct epithelial cell adhesion suggests a role for this protein in ETEC pathogenesis. PMID:11119488

  8. Enterotoxigenic Escherichia coli TibA glycoprotein adheres to human intestine epithelial cells.

    PubMed

    Lindenthal, C; Elsinghorst, E A

    2001-01-01

    Enterotoxigenic Escherichia coli (ETEC) is capable of invading epithelial cell lines derived from the human ileum and colon. Two separate invasion loci (tia and tib) that direct noninvasive E. coli strains to adhere to and invade cultured human intestine epithelial cells have previously been isolated from the classical ETEC strain H10407. The tib locus directs the synthesis of TibA, a 104-kDa outer membrane glycoprotein. Synthesis of TibA is directly correlated with the adherence and invasion phenotypes of the tib locus, suggesting that this protein is an adhesin and invasin. Here we report the purification of TibA and characterization of its biological activity. TibA was purified by continuous-elution preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified TibA was biotin labeled and then shown to bind to HCT8 human ileocecal epithelial cells in a specific and saturable manner. Unlabeled TibA competed with biotin-labeled TibA, suggesting the presence of a specific TibA receptor in HCT8 cells. These results show that TibA acts as an adhesin. Polyclonal anti-TibA antiserum inhibited invasion of ETEC strain H10407 and of recombinant E. coli bearing tib locus clones, suggesting that TibA also acts as an invasin. The ability of TibA to direct epithelial cell adhesion suggests a role for this protein in ETEC pathogenesis. PMID:11119488

  9. Visualization of adherent cell monolayers by cryo-electron microscopy: A snapshot of endothelial adherens junctions.

    PubMed

    Le Bihan, Olivier; Decossas, Marion; Gontier, Etienne; Gerbod-Giannone, Marie-Christine; Lambert, Olivier

    2015-12-01

    Cryo-electron microscopy (cryo-EM) allows the visualization of the cell architecture in its native state. We developed a robust solution to adapt cryo-electron microscopy of vitreous sections (CEMOVIS) to a monolayer of adherent cells using a functionalized polyacrylamide hydrogel growing substrate. We applied this method to reconstitute an endothelial cell monolayer to visualize the morphology of adherens junctions (AJs) which regulate permeability and integrity of the vascular barrier. The fine morphology and ultrastructure of AJs from cultured primary human coronary artery endothelial cells (HCAECs) were analyzed in their native state by using CEMOVIS. Doxycycline and sphingosine-1-phosphate (S1P) are known as efficient regulators of endothelial permeability. Doxycycline and S1P treatments both led to a drastic morphological switch from very uneven to standardized 14-17 nm wide AJs over several microns indicative of a better membrane tethering. Repetitive structures were occasionally noticed within the AJ cleft reflecting a local improved structural organization of VE-cadherin molecules. The ultrastructural stabilization of AJs observed upon treatment likely indicates a better adhesion and thus provides structural clues on the mechanism by which these treatments improve the endothelial barrier function. This method was also successfully extended to a thick epithelial barrier model. We expect our strategy to extend the reliable application of CEMOVIS to virtually any adherent cultured cell systems.

  10. Cell Phone-Based and Adherence Device Technologies for HIV Care and Treatment in Resource-Limited Settings: Recent Advances.

    PubMed

    Campbell, Jeffrey I; Haberer, Jessica E

    2015-12-01

    Numerous cell phone-based and adherence monitoring technologies have been developed to address barriers to effective HIV prevention, testing, and treatment. Because most people living with HIV and AIDS reside in resource-limited settings (RLS), it is important to understand the development and use of these technologies in RLS. Recent research on cell phone-based technologies has focused on HIV education, linkage to and retention in care, disease tracking, and antiretroviral therapy adherence reminders. Advances in adherence devices have focused on real-time adherence monitors, which have been used for both antiretroviral therapy and pre-exposure prophylaxis. Real-time monitoring has recently been combined with cell phone-based technologies to create real-time adherence interventions using short message service (SMS). New developments in adherence technologies are exploring ingestion monitoring and metabolite detection to confirm adherence. This article provides an overview of recent advances in these two families of technologies and includes research on their acceptability and cost-effectiveness when available. It additionally outlines key challenges and needed research as use of these technologies continues to expand and evolve.

  11. Characterization and classification of adherent cells in monolayer culture using automated tracking and evolutionary algorithms.

    PubMed

    Zhang, Zhen; Bedder, Matthew; Smith, Stephen L; Walker, Dawn; Shabir, Saqib; Southgate, Jennifer

    2016-08-01

    This paper presents a novel method for tracking and characterizing adherent cells in monolayer culture. A system of cell tracking employing computer vision techniques was applied to time-lapse videos of replicate normal human uro-epithelial cell cultures exposed to different concentrations of adenosine triphosphate (ATP) and a selective purinergic P2X antagonist (PPADS), acquired over a 24h period. Subsequent analysis following feature extraction demonstrated the ability of the technique to successfully separate the modulated classes of cell using evolutionary algorithms. Specifically, a Cartesian Genetic Program (CGP) network was evolved that identified average migration speed, in-contact angular velocity, cohesivity and average cell clump size as the principal features contributing to the separation. Our approach not only provides non-biased and parsimonious insight into modulated class behaviours, but can be extracted as mathematical formulae for the parameterization of computational models. PMID:27267455

  12. Modification of Solid Phase Red Cell Adherence Assay for the Detection of Platelet Antibodies in Patients With Thrombocytopenia

    PubMed Central

    Vongchan, Preeyanat; Nawarawong, Weerasak; Linhardt, Robert J.

    2009-01-01

    Platelet refractoriness is caused by HLA antibodies and platelet-specific antibodies. Current methods used to detect antiplatelet antibodies have limitations. Solid phase red cell adherence (SPRCA) lacks sensitivity and requires a second assay using chloroquine-treated intact platelets to specify the response due to anti-HLA. We modified SPRCA by using 2 types of antihuman platelet antibodies with different specificities toward platelet lysate and tested samples from 361 patients (69 with unexplained thrombocytopenia and 292 with poor response to platelet transfusions not explicable by alloimmunization or the clinical situation) and 50 from healthy volunteers. Our method compared favorably with platelet suspension direct immunofluorescence. All samples from healthy volunteers were negative; of the samples from the patient population, 240 were positive (147 samples had only antiplatelet and 3 samples had only anti-HLA antibodies). This modified technique had a sensitivity of 98% and a specificity of 91%. PMID:18701420

  13. Modification of solid phase red cell adherence assay for the detection of platelet antibodies in patients with thrombocytopenia.

    PubMed

    Vongchan, Preeyanat; Nawarawong, Weerasak; Linhardt, Robert J

    2008-09-01

    Platelet refractoriness is caused by HLA antibodies and platelet-specific antibodies. Current methods used to detect antiplatelet antibodies have limitations. Solid phase red cell adherence (SPRCA) lacks sensitivity and requires a second assay using chloroquine-treated intact platelets to specify the response due to anti-HLA. We modified SPRCA by using 2 types of antihuman platelet antibodies with different specificities toward platelet lysate and tested samples from 361 patients (69 with unexplained thrombocytopenia and 292 with poor response to platelet transfusions not explicable by alloimmunization or the clinical situation) and 50 from healthy volunteers. Our method compared favorably with platelet suspension direct immunofluorescence. All samples from healthy volunteers were negative; of the samples from the patient population, 240 were positive (147 samples had only antiplatelet and 3 samples had only anti-HLA antibodies). This modified technique had a sensitivity of 98% and a specificity of 91%.

  14. Adherence to hydroxyurea medication by children with sickle cell disease (SCD) using an electronic device: a feasibility study.

    PubMed

    Inoue, Susumu; Kodjebacheva, Gergana; Scherrer, Tammy; Rice, Gary; Grigorian, Matthew; Blankenship, Jeremy; Onwuzurike, Nkechi

    2016-08-01

    Adherence to hydroxyurea (HU) is a significant modifying factor in sickle cell vaso-occlusive pain. We conducted a study using an electronic medication container-monitor-reminder device (GlowCap™) to track adherence and determine whether use of this device affected rates of HU adherence. Subjects were regular attendees to our clinic. They were given a 37-item questionnaire and were asked to use a GlowCap containing HU. When the device cap is opened, it makes a remote "medication taken" record. The device also provides usage reminder in the form of lights and alarm sounds if the cap opening is delayed. Nineteen subjects participated in the survey, and 17 in the intervention phase. Of the 17, 12 had reliable adherence data. Seventeen caregivers of patients and two patients completed the survey. Two most common barriers to adherence identified were lack of reminders and absence of medicine home delivery. The intervention component of this study, which used both the electronic (GlowCap) method and medication possession ratio showed that the median adherence rate for the 12 patients evaluated was 85 %. The GlowCap device accurately kept a record of adherence rates. This device may be an effective tool for increasing HU medication adherence. PMID:27225236

  15. The Acinetobacter baumannii biofilm-associated protein plays a role in adherence to human epithelial cells.

    PubMed

    Brossard, Kari A; Campagnari, Anthony A

    2012-01-01

    Acinetobacter baumannii is a significant source of nosocomial infections worldwide. This bacterium has the ability to survive and persist on multiple abiotic surfaces in health care facilities, and once a focus has been established, this opportunistic pathogen is difficult to eradicate. This paper demonstrates that the A. baumannii biofilm-associated protein (Bap) is necessary for mature biofilm formation on medically relevant surfaces, including polypropylene, polystyrene, and titanium. Scanning electron microscopy analyses of biofilms show that Bap is required for three-dimensional tower structure and water channel formation. In conjunction with persistence on abiotic surfaces, adherence to eukaryotic cells is an important step in bacterial colonization resulting in infection of the host. We have described Bap as the surface structure involved in adherence of A. baumannii to both normal human bronchial epithelial cells and normal human neonatal keratinocytes. However, Bap is not involved in internalization of the bacterium in these two cell lines. Furthermore, this study shows that the presence of Bap increases the bacterial cell surface hydrophobicity. The results of this study are pertinent, as the data lead to a better understanding of the role of Bap in biofilm formation on medical surfaces and in colonization of the host.

  16. Adhere to Marxist viewpoint and ways, compile "A dictionary of population science" (first edition) well.

    PubMed

    Xu, D

    1983-01-01

    Chinese scholars are now compiling the first Chinese population dictionary, which shows the advance being made in the social sciences in China today. This dictionary represents a very important effort because China's population problem remains serious. The population now approaches 1 billion; no other country has a population so large. In the first 5 year plan, China achieved increasing rates of economic development but did not then realize that population control was essential for economic growth. Population growth became out-of-control during the Cultural Revolution due to sabotage from counter revolutionary cliques. Family planning was introduced again in 1971, thanks to the efforts of Zhou Enlai. The contract responsibility system, which has increased grain production by allowing peasants to keep whatever grain remains after their government quota is met, has increased grain production but unfortuantely also provides an incentive for having a big family of farm workers. Education and publicity provide the key to coping with this problem. Socialist production requires planning, including population planning. Population control should not be equated with Malthusianism or NeoMalthusianism, which characterize capitalist systems. China wants socialism. Compiling a disctionary will help to provide the theoretical basis for the practical problem of controlling population growth. Finally, compiling a dictionary requires experience in subjects and in writing and editing. Since the right people must accomplish the work, a team of experts remains the best choice for this task. PMID:12313985

  17. Differences in diet pattern adherence by nativity and duration of US residence in the Mexican-American population.

    PubMed

    Sofianou, Anastasia; Fung, Teresa T; Tucker, Katherine L

    2011-10-01

    Literature on the diet of the Mexican-American population suggests that food consumption differs by nativity and duration of residence in the United States. However, no studies have used dietary pattern analysis to investigate these differences. To create dietary patterns, we used principal components analysis on food frequency questionnaire data for all adult respondents of the National Health and Nutrition Examination Survey years 2003-2006. Four dietary patterns were identified: Western, Healthy, Tomato/Tortilla, and Coffee/Sugar. Least squares means regression was used to test differences in adjusted mean dietary adherence scores among Mexico-born Mexican-American adults residing in the United States for <15 years (n=302), Mexico-born Mexican-American adults residing in the United States for ≥15 years (n=234), US-born Mexican-American adults (n=509), and US-born non-Hispanic whites (n=2,530) aged 18 to 69 years. Mean score for each diet pattern did not differ significantly by duration of US residence category in the Mexico-born Mexican-American population. However, in comparison to all Mexico-born Mexican Americans, US-born Mexican Americans had significantly lower score for the Tomato/Tortilla pattern, and significantly higher score for the Western pattern. Scores for the Healthy pattern were relatively low in all Mexican-American subgroups, indicating low adherence to the Healthy diet. However, Healthy diet scores of Mexico-born Mexican Americans among those aged ≤41 years appeared more similar to those of US-born non-Hispanic whites than US-born Mexican Americans. Education and policy action promoting healthy food access in Hispanic neighborhoods could help limit consumption of Western and Coffee/Sugar diet patterns and promote healthier choices in the Mexican-American population. PMID:21963024

  18. Differences in diet pattern adherence by nativity and duration of US residence in the Mexican-American population.

    PubMed

    Sofianou, Anastasia; Fung, Teresa T; Tucker, Katherine L

    2011-10-01

    Literature on the diet of the Mexican-American population suggests that food consumption differs by nativity and duration of residence in the United States. However, no studies have used dietary pattern analysis to investigate these differences. To create dietary patterns, we used principal components analysis on food frequency questionnaire data for all adult respondents of the National Health and Nutrition Examination Survey years 2003-2006. Four dietary patterns were identified: Western, Healthy, Tomato/Tortilla, and Coffee/Sugar. Least squares means regression was used to test differences in adjusted mean dietary adherence scores among Mexico-born Mexican-American adults residing in the United States for <15 years (n=302), Mexico-born Mexican-American adults residing in the United States for ≥15 years (n=234), US-born Mexican-American adults (n=509), and US-born non-Hispanic whites (n=2,530) aged 18 to 69 years. Mean score for each diet pattern did not differ significantly by duration of US residence category in the Mexico-born Mexican-American population. However, in comparison to all Mexico-born Mexican Americans, US-born Mexican Americans had significantly lower score for the Tomato/Tortilla pattern, and significantly higher score for the Western pattern. Scores for the Healthy pattern were relatively low in all Mexican-American subgroups, indicating low adherence to the Healthy diet. However, Healthy diet scores of Mexico-born Mexican Americans among those aged ≤41 years appeared more similar to those of US-born non-Hispanic whites than US-born Mexican Americans. Education and policy action promoting healthy food access in Hispanic neighborhoods could help limit consumption of Western and Coffee/Sugar diet patterns and promote healthier choices in the Mexican-American population.

  19. Phenotypic analysis of nylon-wool-adherent suppressor cells that inhibit the effector process of tumour cell lysis by lymphokine-activated killer cells in patients with advanced gastric carcinoma.

    PubMed

    Koyama, S; Fukao, K

    1994-01-01

    The causes of down-regulation of cytotoxic immune responses in cancer patients have not been fully evaluated. We previously demonstrated that T-cell-growth-factor-activated peripheral blood lymphocytes (PBL) with the surface phenotype CD8+ CD11b-, from patients with widespread metastasis of gastric carcinoma, inhibited the effector process of lymphokine-activated-killer(LAK)-cell-mediated cytolysis. In this study, we examined suppressor cell activity in freshly prepared PBL from 18 patients with advanced gastric carcinoma, and 10 normal healthy individuals. The suppressor cell activity was assayed by recording whether or not PBL inhibited directly the effector process of LAK cell cytotoxicity. Most of the PBL suspensions from cancer patients showed that they contained a population of cells that can directly inhibit the effector phase of tumor cell lysis of the cytotoxic cells. To analyze further the PBL responsible for the suppression, the cells were passed over a nylon-wool column. Nylon-wool-adherent cells significantly augmented the suppression, while the cells passing through abrogated the suppressive effect. Most nylon-wool-adherent cells from 10 normal healthy controls did not inhibit the cytotoxic reaction. To determine further the suppressor-effector population in nylon-wool-adherent cells, negative-selection studies using CD8-, CD4- or CD11b-coated magnetic beads, and positive-selection studies using CD8- or CD4-coated magnetic beads were performed. Finally the results suggest that the suppressor-effector cells comprise at least two different surface phenotypes: CD8+ T and CD8-CD11b+ cells. The possible role of CD4+ T cells and HLA-DR+ LeuM3+ macrophages as suppressor cells was ruled out in nylon-wool-adherent cells. CD8+ T and possibly CD8-CD11b+ cells apparently suppressed the efferent limb of the antitumor immunity. The selective immune suppression mediated by these cells may partly be concerned with escape mechanisms of gastric carcinoma from the host

  20. Fucoidans Disrupt Adherence of Helicobacter pylori to AGS Cells In Vitro.

    PubMed

    Chua, Eng-Guan; Verbrugghe, Phebe; Perkins, Timothy T; Tay, Chin-Yen

    2015-01-01

    Fucoidans are complex sulphated polysaccharides derived from abundant and edible marine algae. Helicobacter pylori is a stomach pathogen that persists in the hostile milieu of the human stomach unless treated with antibiotics. This study aims to provide preliminary data to determine, in vitro, if fucoidans can inhibit the growth of H. pylori and its ability to adhere to gastric epithelial cells (AGS). We analysed the activity of three different fucoidan preparations (Fucus A, Fucus B, and Undaria extracts). Bacterial growth was not arrested or inhibited by the fucoidan preparations supplemented into culture media. All fucoidans, when supplemented into tissue culture media at 1000 µg mL(-1), were toxic to AGS cells and reduced the viable cell count significantly. Fucoidan preparations at 100 µg mL(-1) were shown to significantly reduce the number of adherent H. pylori. These in vitro findings provide the basis for further studies on the clinical use of sulphated polysaccharides as complementary therapeutic agents. PMID:26604968

  1. Extending metabolome coverage for untargeted metabolite profiling of adherent cultured hepatic cells.

    PubMed

    García-Cañaveras, Juan Carlos; López, Silvia; Castell, José Vicente; Donato, M Teresa; Lahoz, Agustín

    2016-02-01

    MS-based metabolite profiling of adherent mammalian cells comprises several challenging steps such as metabolism quenching, cell detachment, cell disruption, metabolome extraction, and metabolite measurement. In LC-MS, the final metabolome coverage is strongly determined by the separation technique and the MS conditions used. Human liver-derived cell line HepG2 was chosen as adherent mammalian cell model to evaluate the performance of several commonly used procedures in both sample processing and LC-MS analysis. In a first phase, metabolite extraction and sample analysis were optimized in a combined manner. To this end, the extraction abilities of five different solvents (or combinations) were assessed by comparing the number and the levels of the metabolites comprised in each extract. Three different chromatographic methods were selected for metabolites separation. A HILIC-based method which was set to specifically separate polar metabolites and two RP-based methods focused on lipidome and wide-ranging metabolite detection, respectively. With regard to metabolite measurement, a Q-ToF instrument operating in both ESI (+) and ESI (-) was used for unbiased extract analysis. Once metabolite extraction and analysis conditions were set up, the influence of cell harvesting on metabolome coverage was also evaluated. Therefore, different protocols for cell detachment (trypsinization or scraping) and metabolism quenching were compared. This study confirmed the inconvenience of trypsinization as a harvesting technique, and the importance of using complementary extraction solvents to extend metabolome coverage, minimizing interferences and maximizing detection, thanks to the use of dedicated analytical conditions through the combination of HILIC and RP separations. The proposed workflow allowed the detection of over 300 identified metabolites from highly polar compounds to a wide range of lipids.

  2. Acinetobacter baumannii and A. pittii clinical isolates lack adherence and cytotoxicity to lung epithelial cells in vitro.

    PubMed

    Lázaro-Díez, María; Navascués-Lejarza, Teresa; Remuzgo-Martínez, Sara; Navas, Jesús; Icardo, José Manuel; Acosta, Felix; Martínez-Martínez, Luis; Ramos-Vivas, José

    2016-09-01

    The molecular and genetic basis of Acinetobacter baumannii and Acinetobacter pittii virulence remains poorly understood, and there is still lack of knowledge in host cell response to these bacteria. In this study, we have used eleven clinical Acinetobacter strains (A. baumannii n = 5; A. pittii n = 6) to unravel bacterial adherence, invasion and cytotoxicity to human lung epithelial cells. Our results showed that adherence to epithelial cells by Acinetobacter strains is scarce and cellular invasion was not truly detected. In addition, all Acinetobacter strains failed to induce any cytotoxic effect on A549 cells.

  3. In vitro adherence patterns of Shigella serogroups to bovine recto-anal junction squamous epithelial (RSE) cells are similar to those of Escherichia coli O157

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aim of this study was to determine whether Shigella species, which are human gastrointestinal pathogens, can adhere to cattle recto-anal junction squamous epithelial (RSE) cells using a recently standardized adherence assay, and to compare their adherence patterns to that of Escherichia coli O15...

  4. The role of alginate in Pseudomonas aeruginosa EPS adherence, viscoelastic properties and cell attachment.

    PubMed

    Orgad, Oded; Oren, Yoram; Walker, Sharon L; Herzberg, Moshe

    2011-08-01

    Among various functions, extracellular polymeric substances (EPS) provide microbial biofilms with mechanical stability and affect initial cell attachment, the first stage in the biofilm formation process. The role of alginate, an abundant polysaccharide in Pseudomonas aeruginosa biofilms, in the viscoelastic properties and adhesion kinetics of EPS was analyzed using a quartz crystal microbalance with dissipation (QCM-D) monitoring technology. EPS was extracted from two P. aeruginosa biofilms, a wild type strain, PAO1, and a mucoid strain, PAOmucA22 that over-expresses alginate production. The higher alginate content in the EPS originating from the mucoid biofilms was clearly shown to increase both the rate and the extent of attachment of the EPS, as well as the layer's thickness. Also, the presence of calcium and elevated ionic strength increased the thickness of the EPS layer. Dynamic light scattering (DLS) showed that the presence of calcium and elevated ionic strength induced intermolecular attractive interactions in the mucoid EPS molecules. For the wild type EPS, in the presence of calcium, an elevated shift in the distribution of the diffusion coefficients was observed with DLS due to a more compacted conformation of the EPS molecules. Moreover, the alginate over-expression effect on EPS adherence was compared to the effect of alginate over-expression on P. aeruginosa cell attachment. In a parallel plate flow cell, under similar hydraulic and aquatic conditions as those applied for the EPS adsorption tests in the QCM-D flow cell, reduced adherence of the mucoid strain was clearly observed compared to the wild type isogenic bacteria. The results suggest that alginate contributes to steric hindrance and shielding of cell surface features and adhesins that are known to promote cell attachment.

  5. Lactobacilli Interfere with Streptococcus pyogenes Hemolytic Activity and Adherence to Host Epithelial Cells.

    PubMed

    Saroj, Sunil D; Maudsdotter, Lisa; Tavares, Raquel; Jonsson, Ann-Beth

    2016-01-01

    Streptococcus pyogenes [Group A streptococcus (GAS)], a frequent colonizer of the respiratory tract mucosal surface, causes a variety of human diseases, ranging from pharyngitis to the life-threatening streptococcal toxic shock-like syndrome. Lactobacilli have been demonstrated to colonize the respiratory tract. In this study, we investigated the interference of lactobacilli with the virulence phenotypes of GAS. The Lactobacillus strains L. rhamnosus Kx151A1 and L. reuteri PTA-5289, but not L. salivarius LMG9477, inhibited the hemolytic activity of S. pyogenes S165. The inhibition of hemolytic activity was attributed to a decrease in the production of streptolysin S (SLS). Conditioned medium (CM) from the growth of L. rhamnosus Kx151A1 and L. reuteri PTA-5289 was sufficient to down-regulate the expression of the sag operon, encoding SLS. The Lactobacillus strains L. rhamnosus Kx151A1, L. reuteri PTA-5289, and L. salivarius LMG9477 inhibited the initial adherence of GAS to host epithelial cells. Intriguingly, competition with a combination of Lactobacillus species reduced GAS adherence to host cells most efficiently. The data suggest that an effector molecule released from certain Lactobacillus strains attenuates the production of SLS at the transcriptional level and that combinations of Lactobacillus strains may protect the pharyngeal mucosa more efficiently from the initial colonization of GAS. The effector molecules released from Lactobacillus strains affecting the virulence phenotypes of pathogens hold potential in the development of a new generation of therapeutics. PMID:27524981

  6. An optical pressure chamber designed for high numerical aperture studies on adherent living cells.

    PubMed

    Pagliaro, L; Reitz, F; Wang, J

    1995-06-01

    We have developed an optical pressure chamber designed for use with high numerical aperture oil immersion microscope objectives at working pressures up to 1,000 psi (67 atm abs). The chamber is optimized for studies of living, adherent, cultured mammalian cells using high resolution epifluorescence and phase contrast microscopy, and biophysical techniques such as fluorescence redistribution after photobleaching and optical trapping. The primary optical window assembly of the chamber can be removed and placed into a standard 35-mm tissue culture dish, allowing for culture, microinjection, and micromanipulation of adherent cells before they are loaded into the chamber. The chamber is designed to fit into a commercially available stage heater for temperature control, and we used a computer-controlled high pressure liquid chromatography pump for pressure control. A graphic software interface allows the user to program "dive" profiles and to link temperature and pressure data with digital image files of specimens under study. A minor modification of the present design will allow perfusion at high pressure. PMID:7633279

  7. Lactobacilli Interfere with Streptococcus pyogenes Hemolytic Activity and Adherence to Host Epithelial Cells.

    PubMed

    Saroj, Sunil D; Maudsdotter, Lisa; Tavares, Raquel; Jonsson, Ann-Beth

    2016-01-01

    Streptococcus pyogenes [Group A streptococcus (GAS)], a frequent colonizer of the respiratory tract mucosal surface, causes a variety of human diseases, ranging from pharyngitis to the life-threatening streptococcal toxic shock-like syndrome. Lactobacilli have been demonstrated to colonize the respiratory tract. In this study, we investigated the interference of lactobacilli with the virulence phenotypes of GAS. The Lactobacillus strains L. rhamnosus Kx151A1 and L. reuteri PTA-5289, but not L. salivarius LMG9477, inhibited the hemolytic activity of S. pyogenes S165. The inhibition of hemolytic activity was attributed to a decrease in the production of streptolysin S (SLS). Conditioned medium (CM) from the growth of L. rhamnosus Kx151A1 and L. reuteri PTA-5289 was sufficient to down-regulate the expression of the sag operon, encoding SLS. The Lactobacillus strains L. rhamnosus Kx151A1, L. reuteri PTA-5289, and L. salivarius LMG9477 inhibited the initial adherence of GAS to host epithelial cells. Intriguingly, competition with a combination of Lactobacillus species reduced GAS adherence to host cells most efficiently. The data suggest that an effector molecule released from certain Lactobacillus strains attenuates the production of SLS at the transcriptional level and that combinations of Lactobacillus strains may protect the pharyngeal mucosa more efficiently from the initial colonization of GAS. The effector molecules released from Lactobacillus strains affecting the virulence phenotypes of pathogens hold potential in the development of a new generation of therapeutics.

  8. Lactobacilli Interfere with Streptococcus pyogenes Hemolytic Activity and Adherence to Host Epithelial Cells

    PubMed Central

    Saroj, Sunil D.; Maudsdotter, Lisa; Tavares, Raquel; Jonsson, Ann-Beth

    2016-01-01

    Streptococcus pyogenes [Group A streptococcus (GAS)], a frequent colonizer of the respiratory tract mucosal surface, causes a variety of human diseases, ranging from pharyngitis to the life-threatening streptococcal toxic shock-like syndrome. Lactobacilli have been demonstrated to colonize the respiratory tract. In this study, we investigated the interference of lactobacilli with the virulence phenotypes of GAS. The Lactobacillus strains L. rhamnosus Kx151A1 and L. reuteri PTA-5289, but not L. salivarius LMG9477, inhibited the hemolytic activity of S. pyogenes S165. The inhibition of hemolytic activity was attributed to a decrease in the production of streptolysin S (SLS). Conditioned medium (CM) from the growth of L. rhamnosus Kx151A1 and L. reuteri PTA-5289 was sufficient to down-regulate the expression of the sag operon, encoding SLS. The Lactobacillus strains L. rhamnosus Kx151A1, L. reuteri PTA-5289, and L. salivarius LMG9477 inhibited the initial adherence of GAS to host epithelial cells. Intriguingly, competition with a combination of Lactobacillus species reduced GAS adherence to host cells most efficiently. The data suggest that an effector molecule released from certain Lactobacillus strains attenuates the production of SLS at the transcriptional level and that combinations of Lactobacillus strains may protect the pharyngeal mucosa more efficiently from the initial colonization of GAS. The effector molecules released from Lactobacillus strains affecting the virulence phenotypes of pathogens hold potential in the development of a new generation of therapeutics. PMID:27524981

  9. Bead transfection: rapid and efficient gene transfer into marrow stromal and other adherent mammalian cells.

    PubMed

    Matthews, K E; Mills, G B; Horsfall, W; Hack, N; Skorecki, K; Keating, A

    1993-05-01

    We report a simple, rapid, efficient and cost-effective method of gene transfer into bone marrow stromal and other adherent mammalian cells. Our approach involves brief incubation of cells with glass beads in a solution containing the DNA to be transferred. We optimized the technique using COS cells (SV40 transformed kidney cell line from African green monkey) and a transient expression assay for chloramphenicol acetyl transferase (CAT). Factors affecting gene transfer include size and condition of the beads and DNA concentration, but not DNA conformation. Gene transfer efficiency, assessed in a transient expression assay for beta-galactosidase activity, was 5 and 3% in nontransformed human bone marrow stromal cells and COS cells, respectively. Long-term stable expression with the selectable marker, neomycin phosphotransferase, was demonstrated in clonogenic COS cells at a frequency of 27%. Southern analysis of resistant clones revealed the transferred DNA to be integrated in low copy number at one or two sites in the host cell genome. Comparison with electroporation and DEAE-dextran indicates that bead transfection is more efficient than the latter and less costly than either of these methods. In view of its simplicity and because the use of retroviral sequences can be avoided, bead transfection may be an attractive means of gene insertion for gene therapy.

  10. Viability of adhered bacterial cells: tracking MinD protein oscillations

    NASA Astrophysics Data System (ADS)

    Barrett, Matt; Colville, Keegan; Schultz-Nielsen, Chris; Jericho, Manfred; Dutcher, John

    2010-03-01

    To study bacterial cells using atomic force microscopy, it is necessary to immobilize the cells on a substrate. Because bacterial cells and common substrates such as glass and mica have a net negative charge, positively charged polymers such as poly-L-lysine (PLL) and polyethyleneimine (PEI) are commonly used as adhesion layers. However, the use of adhesion polymers could stress the cell and even render it inviable. Viable E. coli cells use oscillations of Min proteins along the axis of the rod-shaped cells to ensure accurate cell division. By tagging MinD proteins with GFP, oscillations can be observed using fluorescence microscopy. For a healthy cell in an ideal environment, the oscillation period is measured to be ˜40 s. Prior experiments have shown that PLL increases the oscillation period significantly (up to 80%). In the present study, we have used epifluorescence and total internal reflection fluorescence (TIRF) to track MinD protein oscillations in E. coli bacteria adhered to a variety of positively charged polymers on mica as a function of polymer surface coverage.

  11. Stepwise, non-adherent differentiation of human pluripotent stem cells to generate basal forebrain cholinergic neurons via hedgehog signaling.

    PubMed

    Crompton, Lucy A; Byrne, Meg L; Taylor, Hannah; Kerrigan, Talitha L; Bru-Mercier, Gilles; Badger, Jennifer L; Barbuti, Peter A; Jo, Jihoon; Tyler, Sue J; Allen, Shelley J; Kunath, Tilo; Cho, Kwangwook; Caldwell, Maeve A

    2013-11-01

    Basal forebrain cholinergic neurons (bfCNs) which provide innervation to the hippocampus and cortex, are required for memory and learning, and are primarily affected in Alzheimer's Disease (AD), resulting in related cognitive decline. Therefore generation of a source of bfCNs from human pluripotent stem cells (hPSCs) is crucial for in vitro disease modeling and development of novel AD therapies. In addition, for the advancement of regenerative approaches there is a requirement for an accurate developmental model to study the neurogenesis and survival of this population. Here we demonstrate the efficient production of bfCNs, using a novel embryoid body (EB) based non-adherent differentiation (NAdD) protocol. We establish a specific basal forebrain neural stem cell (NSC) phenotype via expression of the basal forebrain transcription factors NKX2.1 and LHX8, as well as the general forebrain marker FOXG1. We present evidence that this lineage is achieved via recapitulation of embryonic events, with induction of intrinsic hedgehog signaling, through the use of a 3D non-adherent differentiation system. This is the first example of hPSC-derived basal forebrain-like NSCs, which are scalable via self-renewal in prolonged culture. Furthermore upon terminal differentiation these basal forebrain-like NSCs generate high numbers of cholinergic neurons expressing the specific markers ChAT, VACht and ISL1. These hPSC-derived bfCNs possess characteristics that are crucial in a model to study AD related cholinergic neuronal loss in the basal forebrain. Examples are expression of the therapeutic target p75(NTR), the release of acetylcholine, and demonstration of a mature, and functional electrophysiological profile. In conclusion, this work provides a renewable source of human functional bfCNs applicable for studying AD specifically in the cholinergic system, and also provides a model of the key embryonic events in human bfCN development. PMID:24013066

  12. Effect of Iron on Adherence and Cytotoxicity of Entamoeba histolytica to CHO Cell Monolayers

    PubMed Central

    Lee, Jongweon; Park, Soon-Jung

    2008-01-01

    Iron is an essential element for almost all living organisms. The possible role of iron for growth, adherence and cytotoxicity of Entamoeba histolytica was evaluated in this study. The absence of iron from TYI-S-33 medium stopped amebic growth in vitro. However, iron concentrations in the culture media of 21.4-285.6 µM did not affect the growth of the amebae. Although growth was not retarded at these concentrations, the adhesive abilities of E. histolytica and their cytotoxicities to CHO cell monolayer were correlated with iron concentration. Amebic adhesion to CHO cell monolayers was significantly reduced by low-iron (24.6 ± 2.1%) compared with 62.7 ± 2.8 and 63.1 ± 1.4% of amebae grown in a normal-iron and high-iron media, respectively. E. histolytica cultured in the normal- and high-iron media destroyed 69.1 ± 4.3% and 72.6 ± 5.7% of cultured CHO cell monolayers, but amebae grown in the low-iron medium showed a significantly reduced level of cytotoxicity to CHO cells (2.8 ± 0.2%). Addition of divalent cations other than iron to amebic trophozoites grown in the low-iron medium failed to restore levels of the cytotoxicity. However, when E. histolytica grown in low-iron medium were transferred to normal-iron medium, the amebae showed completely restored cytotoxicity within 7 days. The result suggests that iron is an important factor in the adherence and cytotoxicity of E. histolytica to CHO cell monolayer. PMID:18344676

  13. Silencing the ap65 gene reduces adherence to vaginal epithelial cells by Trichomonas vaginalis.

    PubMed

    Mundodi, V; Kucknoor, A S; Klumpp, D J; Chang, T-H; Alderete, J F

    2004-08-01

    Host parasitism by Trichomonas vaginalis is complex and in part mediated by adherence to vaginal epithelial cells (VECs). Four trichomonad surface proteins bind VECs as adhesins, and AP65 is a major adhesin with sequence identity to an enzyme of the hydrogenosome organelle that is involved in energy generation. In order to perform genetic analysis and assess the role of AP65 in T. vaginalis adherence, we silenced expression of ap65 using antisense RNA. The gene for ap65 was inserted into the vector pBS-neo in sense and antisense orientations to generate plasmids pBS-neoS (S) and pBS-neoAS (AS), respectively. Trichomonads were then transfected with S and AS plasmids for selection of stable transfectants using Geneticin, and the presence of plasmid in transfectants was confirmed by polymerase chain reaction of the neo gene. Reverse transcription polymerase chain reaction and Northern blot analysis showed decreased amounts of ap65 transcript in AS transfected parasites. Growth kinetics of the antisense-transfected and wild type organisms were similar, suggesting that silencing AP65 did not affect overall energy generation for growth. Immunoblot analysis using monoclonal antibody (mAb) to AP65 of AS transfectants showed decreased amounts of AP65 when compared to wild type or S transfectants. Not unexpectedly, this corresponded to decreased amounts of AP65 bound to VECs in a functional ligand assay. Reduction in parasite surface expression of AP65 was related to lower levels of adherence to VECs by AS-transfectants compared to control organisms. Antisense silencing of ap65 was not alleviated by growth of trichomonads in high iron, which up-regulates transcription of ap65. Our work reaffirms the role for AP65 as an adhesin, and in addition, we demonstrate antisense RNA gene silencing in T. vaginalis to study the contribution of specific genes in pathogenesis. PMID:15306014

  14. Silencing the ap65 gene reduces adherence to vaginal epithelial cells by Trichomonas vaginalis

    PubMed Central

    Mundodi, V.; Kucknoor, A. S.; Klumpp, D. J.; Chang, T.-H.; Alderete, J. F.

    2007-01-01

    Summary Host parasitism by Trichomonas vaginalis is complex and in part mediated by adherence to vaginal epithelial cells (VECs). Four trichomonad surface proteins bind VECs as adhesins, and AP65 is a major adhesin with sequence identity to an enzyme of the hydrogenosome organelle that is involved in energy generation. In order to perform genetic analysis and assess the role of AP65 in T. vaginalis adherence, we silenced expression of ap65 using antisense RNA. The gene for ap65 was inserted into the vector pBS-neo in sense and antisense orientations to generate plasmids pBS-neoS (S) and pBS-neoAS (AS), respectively. Trichomonads were then transfected with S and AS plasmids for selection of stable transfectants using Geneticin, and the presence of plasmid in transfectants was confirmed by polymerase chain reaction of the neo gene. Reverse transcription polymerase chain reaction and Northern blot analysis showed decreased amounts of ap65 transcript in AS transfected parasites. Growth kinetics of the antisense-transfected and wild type organisms were similar, suggesting that silencing AP65 did not affect overall energy generation for growth. Immunoblot analysis using monoclonal antibody (mAb) to AP65 of AS transfectants showed decreased amounts of AP65 when compared to wild type or S transfectants. Not unexpectedly, this corresponded to decreased amounts of AP65 bound to VECs in a functional ligand assay. Reduction in parasite surface expression of AP65 was related to lower levels of adherence to VECs by AS-transfectants compared to control organisms. Antisense silencing of ap65 was not alleviated by growth of trichomonads in high iron, which up-regulates transcription of ap65. Our work reaffirms the role for AP65 as an adhesin, and in addition, we demonstrate antisense RNA gene silencing in T. vaginalis to study the contribution of specific genes in pathogenesis. PMID:15306014

  15. Flow-induced detachment of red blood cells adhering to surfaces by specific antigen-antibody bonds.

    PubMed Central

    Xia, Z; Goldsmith, H L; van de Ven, T G

    1994-01-01

    Fixed spherical swollen human red blood cells of blood type B adhering on a glass surface through antigen-antibody bonds to monoclonal mouse antihuman IgM, adsorbed or covalently linked on the surface, were detached by known hydrodynamic forces created in an impinging jet. The dynamic process of detachment of the specifically bound cells was recorded and analyzed. The fraction of adherent cells remaining on the surface decreased with increasing hydrodynamic force. For an IgM coverage of 0.26%, a tangential force on the order of 100 pN was able to detach almost all of the cells from the surface within 20 min. After a given time of exposure to hydrodynamic force, the fraction of adherent cells remaining increased with time, reflecting an increase in adhesion strength. The characteristic time for effective aging was approximately 4 h. Results from experiments in which the adsorbed antibody molecules were immobilized through covalent coupling and from evanescent wave light scattering of adherent cells, imply that deformation of red cells at the contact area was the principal cause for aging, rather than local clustering of the antibody through surface diffusion. Experiments with latex beads specifically bound to red blood cells suggest that, instead of breaking the antigen-antibody bonds, antigen molecules were extracted from the cell membrane during detachment. Images FIGURE 6 FIGURE 7 PMID:8038393

  16. NLRP3 protects alveolar barrier integrity by an inflammasome-independent increase of epithelial cell adherence

    PubMed Central

    Kostadinova, Elena; Chaput, Catherine; Gutbier, Birgitt; Lippmann, Juliane; Sander, Leif E.; Mitchell, Timothy J.; Suttorp, Norbert; Witzenrath, Martin; Opitz, Bastian

    2016-01-01

    Bacterial pneumonia is a major cause of acute lung injury and acute respiratory distress syndrome, characterized by alveolar barrier disruption. NLRP3 is best known for its ability to form inflammasomes and to regulate IL-1β and IL-18 production in myeloid cells. Here we show that NLRP3 protects the integrity of the alveolar barrier in a mouse model of Streptococcus pneumoniae-induced pneumonia, and ex vivo upon treatment of isolated perfused and ventilated lungs with the purified bacterial toxin, pneumolysin. We reveal that the preserving effect of NLRP3 on the lung barrier is independent of inflammasomes, IL-1β and IL-18. NLRP3 improves the integrity of alveolar epithelial cell monolayers by enhancing cellular adherence. Collectively, our study uncovers a novel function of NLRP3 by demonstrating that it protects epithelial barrier function independently of inflammasomes. PMID:27476670

  17. Mechanical Restrictions on Biological Responses by Adherent Cells within Collagen Gels

    PubMed Central

    Simon, D.D.; Horgan, C.O.; Humphrey, J.D.

    2012-01-01

    Cell-seeded collagen and fibrin gels represent excellent assays for studying interactions between adherent interstitial cells and the three-dimensional extracellular matrix in which they reside. Over one hundred papers have employed the free-floating collagen gel assay alone since its introduction in 1979 and much has been learned about mechanobiological responses of diverse types of cells. Yet, given that mechanobiology is the study of biological responses by cells to mechanical stimuli that must respect the basic laws of mechanics, we must quantify better the mechanical conditions that are imposed on or arise in cell-seeded gels. In this paper, we suggest that cell responses and associated changes in matrix organization within the classical free-floating gel assay are highly restricted by the mechanics. In particular, many salient but heretofore unexplained or misinterpreted observations in free-floating gels can be understood in terms of apparent cell-mediated residual stress fields that satisfy quasi-static equilibria and continuity of tractions. There is a continuing need, therefore, to bring together the allied fields of mechanobiology and biomechanics as we continue to elucidate cellular function within both native connective tissues and tissue equivalents that are used in basic scientific investigations or regenerative medicine. PMID:23022259

  18. Investigation on cytoskeleton dynamics for non-adherent cells under point-like stimuli

    NASA Astrophysics Data System (ADS)

    Miccio, Lisa; Memmolo, Pasquale; Merola, Francesco; Mugnano, Martina; Fusco, Sabato; Paciello, Antonio; Ferraro, Pietro; Netti, Paolo A.

    2015-05-01

    In the present paper, Holographic Optical Tweezers (HOT) is employed to trap and manage functionalized micrometric latex beads with the aim at probing cellular forces in no-adherent state. For the first time at best of our knowledge, a suspended cell, subjected to mechanical stress, structures its cytoskeleton when anchored to point-like bonds. We exploit the HOT arrangement to induce mechanical deformation in suspended NIH 3T3 fibroblast. Our investigation is devoted to understand the inner cell mechanism when it is mechanically stressed by point-like stimulus without the substrate influence. In our experiment, cell adhesion is prevented and the stimulus is applied through latex beads trapped by HOT and positioned externally to the cell membrane. Our aims are devoted to analyze cell response during the transition from an homogeneous and isotropic structure (as it's in suspension) to a mechanically stressed state. To analyze the cell material interaction we combine the HOT arrangement with two imaging systems: a Digital Holography (DH) setup in microscope configuration that is an investigation method useful for quantitative, label-free and full-field analysis of low contrast object and a fluorescence modulus. HOT are exploited to induce cellular response to specific stimuli while DH allows to measure such responses in no-invasive way. Finally, fluorescence imaging is added to discriminate the inner cell structures.

  19. Laser-generated Micro-bubbles for Molecular Delivery to Adherent Cells

    NASA Astrophysics Data System (ADS)

    Genc, Suzanne Lee

    We examine the use of optical breakdown in aqueous media as a means to deliver molecules into live adherent cell cultures. This process, called optoinjection (OI), is affected both by the media composition and the cellular exposure to hydrodynamic stresses associated with the cavitation bubble formed by the optical breakdown process. Here we explore the possibility of performing OI using laser microbeams focused at low numerical aperture to provide conditions where OI can be performed at high-throughput. We first investigate the effect of media composition on plasma and cavitation bubble formation. We make the discovery that irradiation of minimal essential media, supports the formation of low-density plasmas (LDP) resulting in the generation of small (2--20 mum radius) cavitation bubbles. This provides gentle specific hydrodynamic perturbations to single or small groups of cells. The addition of supplemental fetal bovine serum to the medium prevents the formation LDPs and the resulting avalanche ionization generates larger (> 100 mum radius) bubbles and more violent hydrodynamic effects. Second, using high-speed photography we provide the first visualization of LDP-generated cavitation bubbles at precise offset locations relative to a boundary on which a cell monolayer can be cultured. These images depict the cellular exposure to different hydrodynamic conditions depending on the normalized offset distance (gamma = s/Rmax) and show how it affects the cellular exposure to shear stresses upon bubble expansion and different distributions of bubble energy upon collapse. Lastly, we examine the effects of pulse energy, parameters, and single vs. multiple laser exposures on the ability to deliver 3-5 kDa dextrans into adherent cells using both small (< 20 mum) and large (100mu m) radius bubbles. For single exposures, we identify several conditions under which OI can be optimized: (a) conditions where cell viability is maximized (˜90%) but optoinjection of viable cells

  20. Refill Adherence in Relation to Substitution and the Use of Multiple Medications: A Nationwide Population Based Study on New ACE-Inhibitor Users

    PubMed Central

    Jönsson, Anna K.; Lesén, Eva; Mårdby, Ann-Charlotte; Sundell, Karolina Andersson

    2016-01-01

    Objective Generic substitution has contributed to economic savings but switching products may affect patient adherence, particularly among those using multiple medications. The aim was to analyse if use of multiple medications influenced the association between switching products and refill adherence to angiotensin-converting-enzyme (ACE) inhibitors in Sweden. Study Design and Setting New users of ACE-inhibitors, starting between 1 July 2006 and 30 June 2007, were identified in the Swedish Prescribed Drug Register. Refill adherence was assessed using the continuous measure of medication acquisition (CMA) and analysed with linear regression and analysis of covariance. Results The study population included 42735 individuals whereof 51.2% were exposed to switching ACE-inhibitor and 39.6% used multiple medications. Refill adherence was higher among those exposed to switching products than those not, but did not vary depending on the use of multiple medications or among those not. Refill adherence varied with age, educational level, household income, country of birth, previous hospitalisation and previous cardiovascular diagnosis. Conclusion The results indicate a positive association between refill adherence and switching products, mainly due to generic substitution, among new users of ACE-inhibitors in Sweden. This association was independent of use of multiple medications. PMID:27192203

  1. Enhanced adherence of mouse fibroblast and vascular cells to plasma modified polyethylene.

    PubMed

    Reznickova, Alena; Novotna, Zdenka; Kolska, Zdenka; Kasalkova, Nikola Slepickova; Rimpelova, Silvie; Svorcik, Vaclav

    2015-01-01

    Since the last decade, tissue engineering has shown a sensational promise in providing more viable alternatives to surgical procedures for harvested tissues, implants and prostheses. Biomedical polymers, such as low-density polyethylene (LDPE), high-density polyethylene (HDPE) and ultra-high molecular weight polyethylene (UHMWPE), were activated by Ar plasma discharge. Degradation of polymer chains was examined by determination of the thickness of ablated layer. The amount of an ablated polymer layer was measured by gravimetry. Contact angle, measured by goniometry, was studied as a function of plasma exposure and post-exposure aging times. Chemical structure of modified polymers was characterized by angle resolved X-ray photoelectron spectroscopy. Surface chemistry and polarity of the samples were investigated by electrokinetic analysis. Changes in surface morphology were followed using atomic force microscopy. Cytocompatibility of plasma activated polyethylene foils was studied using two distinct model cell lines; VSMCs (vascular smooth muscle cells) as a model for vascular graft testing and connective tissue cells L929 (mouse fibroblasts) approved for standardized material cytotoxicity testing. Specifically, the cell number, morphology, and metabolic activity of the adhered and proliferated cells on the polyethylene matrices were studied in vitro. It was found that the plasma treatment caused ablation of the polymers, resulting in dramatic changes in their surface morphology and roughness. ARXPS and electrokinetic measurements revealed oxidation of the polymer surface. It was found that plasma activation has a positive effect on the adhesion and proliferation of VSMCs and L929 cells. PMID:25953566

  2. On-chip integrated lensless microscopy module for optical monitoring of adherent growing mammalian cells.

    PubMed

    Li, Wei; Knoll, Thorsten; Thielecke, Hagen

    2010-01-01

    Lab-on-a-chip systems are increasingly applied in cell-based assays for toxicology and drug testing. In this paper, an on-chip integrated lensless microscopy module using a direct projection method for optical monitoring of the shadow images of adherent growing mammalian cells is presented. The biological cells are conserved and interfaced by a microfabricated cavity chip with a 1 microm thick silicon nitride (Si(3)N(4)) substrate onto the surface of a 5 megapixel CMOS image sensor with 2.2 microm pixel size. The optical resolution of the assembly is estimated by the contact/proximate printing theory from optical lithography. Further characterization is made by imaging microbeads in chips with the Si(3)N(4)-membrane as well as in cavity chips with membranes made from dry film resist (DFR, thickness 20, 40 and 60 microm). The module represents a 3 × optical microscope for cell morphology imaging. The function is demonstrated by the growth monitoring of L929 cells cultured in cavity chips with Si(3)N(4) substrate for 2 days and by checking the colorimetric staining of cells with a compromised membrane. PMID:21096993

  3. Enhanced adherence of mouse fibroblast and vascular cells to plasma modified polyethylene.

    PubMed

    Reznickova, Alena; Novotna, Zdenka; Kolska, Zdenka; Kasalkova, Nikola Slepickova; Rimpelova, Silvie; Svorcik, Vaclav

    2015-01-01

    Since the last decade, tissue engineering has shown a sensational promise in providing more viable alternatives to surgical procedures for harvested tissues, implants and prostheses. Biomedical polymers, such as low-density polyethylene (LDPE), high-density polyethylene (HDPE) and ultra-high molecular weight polyethylene (UHMWPE), were activated by Ar plasma discharge. Degradation of polymer chains was examined by determination of the thickness of ablated layer. The amount of an ablated polymer layer was measured by gravimetry. Contact angle, measured by goniometry, was studied as a function of plasma exposure and post-exposure aging times. Chemical structure of modified polymers was characterized by angle resolved X-ray photoelectron spectroscopy. Surface chemistry and polarity of the samples were investigated by electrokinetic analysis. Changes in surface morphology were followed using atomic force microscopy. Cytocompatibility of plasma activated polyethylene foils was studied using two distinct model cell lines; VSMCs (vascular smooth muscle cells) as a model for vascular graft testing and connective tissue cells L929 (mouse fibroblasts) approved for standardized material cytotoxicity testing. Specifically, the cell number, morphology, and metabolic activity of the adhered and proliferated cells on the polyethylene matrices were studied in vitro. It was found that the plasma treatment caused ablation of the polymers, resulting in dramatic changes in their surface morphology and roughness. ARXPS and electrokinetic measurements revealed oxidation of the polymer surface. It was found that plasma activation has a positive effect on the adhesion and proliferation of VSMCs and L929 cells.

  4. Review: An urgent need for research on factors impacting adherence to and retention in care among HIV-positive youth and adolescents from key populations

    PubMed Central

    Lall, Priya; Lim, Sin How; Khairuddin, Norliana; Kamarulzaman, Adeeba

    2015-01-01

    Introduction The 50% increase in HIV-related deaths in youth and adolescents (aged 10–24) from 2005 to 2012 highlights the need to improve HIV treatment and care in this population, including treatment adherence and retention. Youth and adolescents from key populations or young key populations (YKP) in particular are highly stigmatized and may face additional barrier(s) in adhering to HIV treatment and services. We reviewed the current knowledge on treatment adherence and retention in HIV care among YKP to identify gaps in the literature and suggest future directions to improve HIV care for YKP. Methods We conducted a comprehensive literature search for YKP and their adherence to antiretroviral therapy (ART) and retention in HIV care on PsycInfo (Ovid), PubMed and Google Scholar using combinations of the keywords HIV/AIDS, ART, adolescents, young adults, adherence (or compliance), retention, men who have sex with men, transgender, injection drug users, people who inject drugs and prisoners. We included empirical studies on key populations defined by WHO; included the terms youth and adolescents and/or aged between 10 and 24; examined adherence to or retention in HIV care; and published in English-language journals. All articles were coded using NVivo. Results and discussion The systematic search yielded 10 articles on YKP and 16 articles on behaviourally infected youth and adolescents from 1999 to 2014. We found no studies reporting on youth and adolescents identified as sex workers, transgender people and prisoners. From existing literature, adherence to ART was reported to be influenced by age, access to healthcare, the burden of multiple vulnerabilities, policy involving risk behaviours and mental health. A combination of two or more of these factors negatively impacted adherence to ART among YKP. Collectively, these studies demonstrated that future programmes need to be tailored specifically to YKP to ensure adherence. Conclusions There is an urgent need for

  5. Soluble fibrin augments platelet/tumor cell adherence in vitro and in vivo, and enhances experimental metastasis.

    PubMed

    Biggerstaff, J P; Seth, N; Amirkhosravi, A; Amaya, M; Fogarty, S; Meyer, T V; Siddiqui, F; Francis, J L

    1999-01-01

    There is considerable evidence for a relationship between hemostasis and malignancy. Since platelet adhesion to tumor cells has been implicated in the metastatic process and plasma levels of fibrinogen (Fg) and soluble fibrin (sFn) monomer are increased in cancer, we hypothesized that these molecules might enhance tumor-platelet interaction. We therefore studied binding of sFn monomer to tumor cells in a static microplate adhesion assay and determined the effect of pre-treating tumor cells with sFn on tumor cell-induced thrombocytopenia and experimental metastasis. Soluble fibrin (produced by adding thrombin to FXIII- and plasminogen-free Fg in the presence of Gly-Pro-Arg-Pro-amide (GPRP-NH2) significantly increased platelet adherence to tumor cells. This effect was primarily mediated by the integrins alphaIIb beta3 on the platelet and CD 54 (ICAM-1) on the tumor cells. Platelets adhered to untreated A375 cells (28 +/- 8 platelets/tumor cell) and this was not significantly affected by pre-treatment of the tumor cells with fibrinogen or GPRP-NH2. Although thrombin treatment increased adherence, pre-incubation of the tumor cells with sFn resulted in a further increase in platelet binding to tumor cells. In contrast to untreated tumor cells, intravenous injection of sFn-treated A 375 cells reduced the platelet count in anticoagulated mice, supporting the in vitro finding that sFn enhanced tumor cell-platelet adherence. In a more aggressive model of experimental metastasis, treating tumor cells with sFn enhanced lung seeding by 65% compared to untreated cells. Extrapolation of our data to the clinical situation suggests that coagulation activation, and subsequent increase in circulating Fn monomer, may enhance platelet adhesion to circulating tumor cells and thereby facilitate metastatic spread.

  6. Soluble fibrin augments platelet/tumor cell adherence in vitro and in vivo, and enhances experimental metastasis.

    PubMed

    Biggerstaff, J P; Seth, N; Amirkhosravi, A; Amaya, M; Fogarty, S; Meyer, T V; Siddiqui, F; Francis, J L

    1999-01-01

    There is considerable evidence for a relationship between hemostasis and malignancy. Since platelet adhesion to tumor cells has been implicated in the metastatic process and plasma levels of fibrinogen (Fg) and soluble fibrin (sFn) monomer are increased in cancer, we hypothesized that these molecules might enhance tumor-platelet interaction. We therefore studied binding of sFn monomer to tumor cells in a static microplate adhesion assay and determined the effect of pre-treating tumor cells with sFn on tumor cell-induced thrombocytopenia and experimental metastasis. Soluble fibrin (produced by adding thrombin to FXIII- and plasminogen-free Fg in the presence of Gly-Pro-Arg-Pro-amide (GPRP-NH2) significantly increased platelet adherence to tumor cells. This effect was primarily mediated by the integrins alphaIIb beta3 on the platelet and CD 54 (ICAM-1) on the tumor cells. Platelets adhered to untreated A375 cells (28 +/- 8 platelets/tumor cell) and this was not significantly affected by pre-treatment of the tumor cells with fibrinogen or GPRP-NH2. Although thrombin treatment increased adherence, pre-incubation of the tumor cells with sFn resulted in a further increase in platelet binding to tumor cells. In contrast to untreated tumor cells, intravenous injection of sFn-treated A 375 cells reduced the platelet count in anticoagulated mice, supporting the in vitro finding that sFn enhanced tumor cell-platelet adherence. In a more aggressive model of experimental metastasis, treating tumor cells with sFn enhanced lung seeding by 65% compared to untreated cells. Extrapolation of our data to the clinical situation suggests that coagulation activation, and subsequent increase in circulating Fn monomer, may enhance platelet adhesion to circulating tumor cells and thereby facilitate metastatic spread. PMID:10919717

  7. Adherent-phagocytic cells influence suppressed concanavalin-A induced proliferation of spleen lymphoid cells in copper deficient rats

    SciTech Connect

    Kramer, T.R.; Briske-Anderson, M.; Johnson, W.T.

    1986-03-01

    Weanling male Lewis rats (N = 10/group) were fed ad-libitum for 42 days diets based on AIN standards containing 21% casein, 5% safflower oil, and deficient (0.6 ..mu..g/g) or adequate (5.6 ..mu..g/g) levels of cu. Cu-deficient rats showed typical biochemical and hematological changes. Immunological changes exhibited by Cu-deficient rats were influenced by the presence of splenic adherent-phagocytic cells (macrophage-like), but not by cytochrome-c oxidase activity of spleen lymphoid cells (SLC). Decreased proliferation was exhibited by concanavalin-A (Con-A) stimulated SLC of Cu-deficient rats. Following removal of plastic-adherent phagocytic cells from the SLC suspensions, equivalent proliferation was exhibited by Con-A stimulated nonadherent-SLC of Cu-deficient and Cu-adequate rats. Decreased cytochrome-c oxidase activity was exhibited by both unstimulated SLC and nonadherent-SLC of Cu-deficient rats, but decreased proliferation was exhibited only in Con-A stimulated SLC of Cu-deficient rats. These findings indicate that nonadherent splenic T-lymphocytes of Cu-deficient rats are not impaired in their ability to proliferate, and that cytochrome-c oxidase activity in unstimulated lymphoid cells of Cu-deficient rats is apparently not related to levels of proliferation by the Con-A stimulated cells.

  8. Isolation of an Escherichia coil strain mutant unable to form biofilm on polystyrene and to adhere to human pneumocyte cells: involvement of tryptophanase.

    PubMed

    Di Martino, P; Merieau, A; Phillips, R; Orange, N; Hulen, C

    2002-02-01

    Escherichia coli adherence to biotic and abiotic surfaces constitutes the first step of infection by promoting colonization and biofilm formation. The aim of this study was to gain a better understanding of the relationship between E. coli adherence to different biotic surfaces and biofilm formation on abiotic surfaces. We isolated mutants defective in A549 pneumocyte cells adherence, fibronectin adherence, and biofilm formation by random transposition mutagenesis and sequential passages over A549 cell monolayers. Among the 97 mutants tested, 80 were decreased in biofilm formation, 8 were decreased in A549 cells adherence, 7 were decreased in their adherence to fibronectin, and 17 had no perturbations in either of the three phenotypes. We observed a correlation between adherence to fibronectin or A549 cells and biofilm formation, indicating that biotic adhesive factors are involved in biofilm formation by E. coli. Molecular analysis of the mutants revealed that a transposon insertion in the tnaA gene encoding for tryptophanase was associated with a decrease in both A549 cells adherence and biofilm formation by E. coli. The complementation of the tnaA mutant with plasmid-located wild-type tnaA restored the tryptophanase activity, epithelial cells adherence, and biofilm formation on polystyrene. The possible mechanism of tryptophanase involvement in E. coli adherence and biofilm formation is discussed.

  9. Akkermansia muciniphila Adheres to Enterocytes and Strengthens the Integrity of the Epithelial Cell Layer

    PubMed Central

    Reunanen, Justus; Kainulainen, Veera; Huuskonen, Laura; Ottman, Noora; Belzer, Clara; Huhtinen, Heikki; de Vos, Willem M.

    2015-01-01

    Akkermansia muciniphila is a Gram-negative mucin-degrading bacterium that resides in the gastrointestinal tracts of humans and animals. A. muciniphila has been linked with intestinal health and improved metabolic status in obese and type 2 diabetic subjects. Specifically, A. muciniphila has been shown to reduce high-fat-diet-induced endotoxemia, which develops as a result of an impaired gut barrier. Despite the accumulating evidence of the health-promoting effects of A. muciniphila, the mechanisms of interaction of the bacterium with the host have received little attention. In this study, we used several in vitro models to investigate the adhesion of A. muciniphila to the intestinal epithelium and its interaction with the host mucosa. We found that A. muciniphila adheres strongly to the Caco-2 and HT-29 human colonic cell lines but not to human colonic mucus. In addition, A. muciniphila showed binding to the extracellular matrix protein laminin but not to collagen I or IV, fibronectin, or fetuin. Importantly, A. muciniphila improved enterocyte monolayer integrity, as shown by a significant increase in the transepithelial electrical resistance (TER) of cocultures of Caco-2 cells with the bacterium. Further, A. muciniphila induced interleukin 8 (IL-8) production by enterocytes at cell concentrations 100-fold higher than those for Escherichia coli, suggesting a very low level of proinflammatory activity in the epithelium. In conclusion, our results demonstrate that A. muciniphila adheres to the intestinal epithelium and strengthens enterocyte monolayer integrity in vitro, suggesting an ability to fortify an impaired gut barrier. These results support earlier associative in vivo studies and provide insights into the interaction of A. muciniphila with the host. PMID:25795669

  10. Numerical fluid-dynamic optimization of microchannel-provided porous scaffolds for the co-culture of adherent and non-adherent cells.

    PubMed

    Cantini, Marco; Fiore, Gianfranco B; Redaelli, Alberto; Soncini, Monica

    2009-03-01

    Computational fluid dynamic (CFD) techniques were used to optimize the microenvironment inside scaffolds for hematopoietic stem cell (HSC) culture in a perfusion bioreactor. These matrices are meant to be seeded with adherent bone marrow stromal cells and then co-cultivated with HSCs; the scaffold micro-architecture and the fluid-dynamic conditions have to be optimized to avoid non-adherent stem cells being dragged away while ensuring adequate nutrient supply. The insertion of longitudinal microchannels was tested as a tool to improve perfusion in a homogeneous porous scaffold. Models of microchannel-provided scaffolds, characterized by different values of geometric parameters concerning pores and channels, were built, and numerical fluid-dynamic and oxygen-transfer analyses were carried out. The results of the computations indicated that the microchannels created preferential paths for culture medium flow, causing low shear stresses and drag forces within the pores; meanwhile, they improved oxygen delivery by forcing its penetration into the scaffold bulk. In particular, an 85% porous, 3-mm-thick scaffold with 175-microm-diameter pores was considered; at a constant average drag force guaranteeing stem cell suspension inside this porous bulk, the addition of approximately 260-microm-diameter, 700-microm-spaced channels resulted in 34% higher oxygen partial pressure at the exit (approximately 135 vs 101 mmHg), maintaining a wall shear stress median value of approximately 0.14 mPa. The present work demonstrates the capacity of microchannel-provided scaffolds to ensure suitable conditions for HSC culture and shows that CFD methods are a valuable tool to retrieve significant clues for the design of the culture environment.

  11. Streptococcus pneumoniae ClpL Modulates Adherence to A549 Human Lung Cells through Rap1/Rac1 Activation

    PubMed Central

    Nguyen, Cuong Thach; Le, Nhat-Tu; Tran, Thao Dang-Hien; Kim, Eun-Hye; Park, Sang-Sang; Luong, Truc Thanh; Chung, Kyung-Tae; Pyo, Suhkneung

    2014-01-01

    Caseinolytic protease L (ClpL) is a member of the HSP100/Clp chaperone family, which is found mainly in Gram-positive bacteria. ClpL is highly expressed during infection for refolding of stress-induced denatured proteins, some of which are important for adherence. However, the role of ClpL in modulating pneumococcal virulence is poorly understood. Here, we show that ClpL impairs pneumococcal adherence to A549 lung cells by inducing and activating Rap1 and Rac1, thus increasing phosphorylation of cofilin (inactive form). Moreover, infection with a clpL mutant (ΔclpL) causes a greater degree of filopodium formation than D39 wild-type (WT) infection. Inhibition of Rap1 and Rac1 impairs filopodium formation and pneumococcal adherence. Therefore, ClpL can reduce pneumococcal adherence to A549 cells, likely via modulation of Rap1- and Rac1-mediated filopodium formation. These results demonstrate a potential role for ClpL in pneumococcal resistance to host cell adherence during infection. This study provides insight into further understanding the interactions between hosts and pathogens. PMID:24980975

  12. Adherence to lifestyle modifications after a cardiac rehabilitation program and endothelial progenitor cells. A six-month follow-up study.

    PubMed

    Cesari, F; Marcucci, R; Gori, A M; Burgisser, C; Francini, S; Roberts, A T; Sofi, F; Gensini, G F; Abbate, R; Fattirolli, F

    2014-07-01

    An increase of endothelial progenitor cells (EPCs) among acute myocardial infarction (AMI) patients participating in a cardiac rehabilitation (CR) program has been reported, but no data on the impact of adherence to lifestyle recommendations provided during a CR program on EPCs are available. It was our aim to investigate the effect of adherence to lifestyle recommendations on EPCs, inflammatory and functional parameters after six months of a CR program in AMI patients. In 110 AMI patients (90 male/20 female; mean age 57.9 ± 9.4 years) EPCs, high sensitivity C-reactive protein (hsCRP), N-terminal pro-brain natriuretic peptide (NT-ProBNP) levels, and cardiopulmonary testings were determined at the end of the CR (T1) and at a six-month follow-up (T2). At T2 we administered a questionnaire assessing dietary habits and physical activity. At T2, we observed a decrease of EPCs (p<0.05), of hsCRP (p=0.009) and of NT-ProBNP (p<0.0001). Patient population was divided into three categories by Healthy Lifestyle (HL) score (none/low, moderate and high adherence to lifestyle recommendations). We observed a significant association between adherence to lifestyle recommendations, increase in EPCs and exercise capacity between T1 and T2 (Δ EPCs p for trend <0.05; ΔWatt max p for trend=0.004). In a multivariate logistic regression analyses, being in the highest tertile of HL score affected the likelihood of an increase of EPC levels at T2 [OR (95% confidence interval): 3.36 (1.0-10.72) p=0.04]. In conclusion, adherence to lifestyle recommendations provided during a CR program positively influences EPC levels and exercise capacity.

  13. Cyclic Amphipathic Peptide-DNA Complexes Mediate High-Efficiency Transfection of Adherent Mammalian Cells

    NASA Astrophysics Data System (ADS)

    Legendre, Jean-Yves; Szoka, Francis C., Jr.

    1993-02-01

    A DNA transfection protocol has been developed that makes use of the cyclic cationic amphipathic peptide gramicidin S and dioleoyl phosphatidylethanolamine. The DNA complex is formed by mixing gramicidin S with DNA at a 1:1 charge ratio and then adding phosphatidylethanolamine at a lipid/peptide molar ratio of 5:1. The complex mediates rapid association of DNA with cells and leads to transient expression levels of β-galactosidase ranging from 1 to 30% of the transfected cells, with long-term expression being about an order of magnitude lower. The respective roles of peptide and phospholipid are not yet resolved but optimal transfection requires both the cyclic peptide and the hexagonal phase-competent phospholipid PtdEtn. Transfection in CV-1 cells is not affected by lysomotrophic agents, which suggests that DNA entry into the cell is via the plasma membrane. This technique that is simple, economical, and reproducible mediates transfection levels up to 20-fold higher than cationic liposomes in adherent mammalian cells.

  14. A novel multi-coaxial hollow fiber bioreactor for adherent cell types. Part 1: hydrodynamic studies.

    PubMed

    Wolfe, Stephen P; Hsu, Edward; Reid, Lola M; Macdonald, Jeffrey M

    2002-01-01

    A novel multi-coaxial bioreactor for three-dimensional cultures of adherent cell types, such as liver, is described. It is composed of four tubes of increasing diameter placed one inside the other, creating four spatially isolated compartments. Liver acinar structure and physiological parameters are mimicked by sandwiching cells in the space between the two innermost semi-permeable tubes, or hollows fibers, and creating a radial flow of media from an outer compartment (ECC), through the cell mass compartment, and to an inner compartment (ICC). The outermost compartment is created by gas-permeable tubing, and the housing is used to oxygenate the perfusion media to periportal levels in the ECC. Experiments were performed using distilled water to correlate the radial flow rate (Q(r)) with (1) the pressure drop (DeltaP) between the media compartments that sandwich the cell compartment and (2) the pressure in the cell compartment (P(c)). These results were compared with the theoretical profile calculated based on the hydraulic permeability of the two innermost fibers. Phase-contrast velocity-encoded magnetic resonance imaging was used to visualize directly the axial velocities inside the bioreactor and confirm the assumptions of laminar flow and zero axial velocity at the boundaries of each compartment in the bioreactor. Axial flow rates were calculated from the magnetic resonance imaging results and were similar to the measured axial flow rates for the previously described experiments.

  15. Surface glycosaminoglycans mediate adherence between HeLa cells and Lactobacillus salivarius Lv72

    PubMed Central

    2013-01-01

    Background The adhesion of lactobacilli to the vaginal surface is of paramount importance to develop their probiotic functions. For this reason, the role of HeLa cell surface proteoglycans in the attachment of Lactobacillus salivarius Lv72, a mutualistic strain of vaginal origin, was investigated. Results Incubation of cultures with a variety of glycosaminoglycans (chondroitin sulfate A and C, heparin and heparan sulfate) resulted in marked binding interference. However, no single glycosaminoglycan was able to completely abolish cell binding, the sum of all having an additive effect that suggests cooperation between them and recognition of specific adhesins on the bacterial surface. In contrast, chondroitin sulfate B enhanced cell to cell attachment, showing the relevance of the stereochemistry of the uronic acid and the sulfation pattern on binding. Elimination of the HeLa surface glycosaminoglycans with lyases also resulted in severe adherence impairment. Advantage was taken of the Lactobacillus-glycosaminoglycans interaction to identify an adhesin from the bacterial surface. This protein, identify as a soluble binding protein of an ABC transporter system (OppA) by MALDI-TOF/(MS), was overproduced in Escherichia coli, purified and shown to interfere with L. salivarius Lv72 adhesion to HeLa cells. Conclusions These data suggest that glycosaminoglycans play a fundamental role in attachment of mutualistic bacteria to the epithelium that lines the cavities where the normal microbiota thrives, OppA being a bacterial adhesin involved in the process. PMID:24044741

  16. Raman micro-spectroscopy study of living SH-SY5Y cells adhering on different substrates.

    PubMed

    Caponi, S; Mattana, S; Ricci, M; Sagini, K; Urbanelli, L; Sassi, P; Morresi, A; Emiliani, C; Dalla Serra, M; Iannotta, S; Musio, C; Fioretto, D

    2016-01-01

    In this paper we test the ability of Raman micro-spectroscopy and Raman mapping to investigate the status of cells grown in adhesion on different substrates. The spectra of immortalized SH-SY5Y cells, grown on silicon and on metallic substrates are compared with those obtained for the same type of cells adhering on organic polyaniline (PANI), a memristive substrate chosen to achieve a living bio-hybrid system. Raman spectra give information on the status of the single cell, its local biochemical composition, and on the modifications induced by the substrate interaction. The good agreement between Raman spectra collected from cells adhering on different substrates confirms that the PANI, besides allowing the cell growth, doesn't strongly affect the general biochemical properties of the cell. The investigation of the cellular state in a label free condition is challenging and the obtained results confirm the Raman ability to achieve this information. PMID:26256426

  17. Incidence and Predictors of Multiple Fractures Despite High Adherence to Oral Bisphosphonates: A Binational Population-Based Cohort Study.

    PubMed

    Hawley, Samuel; Javaid, M Kassim; Rubin, Katrine H; Judge, Andrew; Arden, Nigel K; Vestergaard, Peter; Eastell, Richard; Diez-Perez, Adolfo; Cooper, Cyrus; Abrahamsen, Bo; Prieto-Alhambra, Daniel

    2016-01-01

    Oral bisphosphonates (BPs) are highly effective in preventing fractures and are recommended first-line therapies for patients with osteoporosis. We identified the incidence and predictors of oral BP treatment failure, defined as the incidence of two or more fractures while on treatment (≥2 FWOT) among users with high adherence. Fractures were considered from 6 months after treatment initiation and up to 6 months after discontinuation. Data from computerized records and pharmacy invoices were obtained from Sistema d'Informació per al Desenvolupament de l'Investigació en Atenció Primària (SIDIAP; Catalonia, Spain) and Danish Health Registries (Denmark) for all incident users of oral BPs in 2006-2007 and 2000-2001, respectively. Fine and Gray survival models using backward-stepwise selection (p-entry 0.049; p- exit 0.10) and accounting for the competing risk of therapy cessation were used to identify predictors of ≥2 FWOT among patients having persisted with treatment ≥6 months with overall medication possession ratio (MPR) ≥80%. Incidence of ≥2 FWOT was 2.4 (95% confidence interval [CI], 1.8 to 3.2) and 1.7 (95% CI, 1.2 to 2.2) per 1000 patient-years (PYs) within Catalonia and Denmark, respectively. Older age was predictive of ≥2 FWOT in both Catalonian and Danish cohorts: subhazard ratio (SHR) = 2.28 (95% CI, 1.11 to 4.68) and SHR = 2.61 (95% CI, 0.98 to 6.95), respectively, for 65 to <80 years; and SHR = 3.19 (95% CI, 1.33 to 7.69) and SHR = 4.88 (95% CI, 1.74 to 13.7), respectively, for ≥80 years. Further significant predictors of ≥2 FWOT identified within only one cohort were dementia, SHR = 4.46 (95% CI, 1.02 to 19.4) (SIDIAP); and history of recent or older fracture, SHR = 3.40 (95% CI, 1.50 to 7.68) and SHR = 2.08 (95% CI: 1.04-4.15), respectively (Denmark). Even among highly adherent users of oral BP therapy, a minority sustain multiple fractures while on treatment. Older age was predictive of increased risk

  18. A computational model of the response of adherent cells to stretch and changes in substrate stiffness

    PubMed Central

    Lutchen, Kenneth R.; Suki, Béla

    2014-01-01

    Cells in the body exist in a dynamic mechanical environment where they are subject to mechanical stretch as well as changes in composition and stiffness of the underlying extracellular matrix (ECM). However, the underlying mechanisms by which cells sense and adapt to their dynamic mechanical environment, in particular to stretch, are not well understood. In this study, we hypothesized that emergent phenomena at the level of the actin network arising from active structural rearrangements driven by nonmuscle myosin II molecular motors play a major role in the cellular response to both stretch and changes in ECM stiffness. To test this hypothesis, we introduce a simple network model of actin-myosin interactions that links active self-organization of the actin network to the stiffness of the network and the traction forces generated by the network. We demonstrate that such a network replicates not only the effect of changes in substrate stiffness on cellular traction and stiffness and the dependence of rate of force development by a cell on the stiffness of its substrate, but also explains the physical response of adherent cells to transient and cyclic stretch. Our results provide strong indication that network phenomena governed by the active reorganization of the actin-myosin structure plays an important role in cellular mechanosensing and response to both changes in ECM stiffness and externally applied mechanical stretch. PMID:24408996

  19. CsrRS and Environmental pH Regulate Group B Streptococcus Adherence to Human Epithelial Cells and Extracellular Matrix

    PubMed Central

    Park, Su Eun; Jiang, Shengmei

    2012-01-01

    Streptococcus agalactiae (group B Streptococcus or GBS) is a common colonizer of the gastrointestinal and genital tracts and an important cause of invasive infections in newborn infants and in adults with predisposing chronic conditions or advanced age. Attachment to epithelial surfaces at mucosal sites is a critical step in the successful colonization of a human host, and regulation of this process is likely to play an important role in both commensalism and dissemination to cause invasive disease. We found that inactivation of the CsrRS (or CovRS) two-component system increased GBS adherence to epithelial cells derived from human vaginal, cervical, and respiratory epithelium, as well as increasing adherence to extracellular matrix proteins and increasing biofilm formation on polystyrene. Neutral (as opposed to acidic) pH enhanced GBS binding to vaginal epithelial cells and to fibrinogen and fibronectin, effects that were partially dependent on CsrRS. The regulatory effects of CsrRS and environmental pH on bacterial adherence correlated with their effects on the expression of multiple surface adhesins, as assessed by quantitative reverse transcription-PCR. We conclude that GBS adherence to epithelial and abiotic surfaces is regulated by the CsrRS two-component system and by environmental pH through their regulatory effects on the expression of bacterial surface adhesins. Dynamic regulation of GBS adherence enhances the organism's adaptability to survival in multiple niches in the human host. PMID:22949550

  20. Trichomonas vaginalis adherence mediates differential gene expression in human vaginal epithelial cells

    PubMed Central

    Kucknoor, Ashwini; Mundodi, Vasanthakrishna; Alderete, John F.

    2007-01-01

    Summary Trichomonas vaginalis, an ancient protist, colonizes the vaginal mucosa causing trichomonosis, a vaginitis that sometimes leads to severe health complications. Preparatory to colonization of the vagina is the adhesion to vaginal epithelial cells (VECs) by trichomonads. We hypothesized that VECs alter the gene expression to form a complex signalling cascade in response to trichomonal adherence. In order to identify the genes that are upregulated, we constructed a subtraction cDNA library after contact with parasites that is enriched for differentially expressed genes from the immortalized MS-74 VECs. Sixty cDNA clones were sequenced and to our knowledge for the first time, differentially regulated genes were identified in response to early trichomonal infection. The identified genes were found to encode functional proteins with specific functions associated with cell structure maintenance and extracellular matrix components, proinflammatory molecules and apoptosis. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) confirmed expression of selected genes. Further, cyclooxygenase 2 (COX-2) protein expression was analysed using Western blot and immunofluorescence assays. Data suggest that p38 mitogen-activated protein (MAP) kinase and tyrosine kinases play a role in COX-2 induction. Finally, T. vaginalis and Tritrichomonas foetus but not Pentatrichomonas hominis induce expression of COX-2. This is a first attempt at elucidating the basis of interaction of trichomonads with host cells and the corresponding host responses triggered by the parasites. PMID:15888089

  1. Assessing the adherence to and the therapeutic effectiveness of hypolipidemic agents in a population of patients in Brazil: a retrospective cohort study

    PubMed Central

    Cunico, Cássia; Picheth, Geraldo; Correr, Cassyano J.; Scartezini, Marileia

    2013-01-01

    Objective to evaluate the relation between patient adherence and therapeutic effectiveness of hypolipidemic agents in clinical practice. Methods A retrospective cohort study of 417 patients using hypolipidemic drugs (simvastatin, atorvastatin) between 2003 and 2010 was performed. The population studied consists of patients assisted by the Public Health Service in the far-west region of the State of Santa Catarina, Brazil. The Medication Possession Ratio obtained from pharmacy refill data was used to measure patient adherence. Therapeutic effectiveness was evaluated based on the difference obtained in the serum levels of total cholesterol, LDL-cholesterol, HDL-cholesterol and triglycerides, before and after taking the drug, in an average time of 8.3 months. Results Following the treatment with hypolipidemic agents, it has been observed a reduction of 14.3% for total cholesterol, 19.6% for LDL-cholesterol, and 14.4% for triglycerides. HDL-cholesterol increased by an 8.0% average. The major changes in lipid profile were promoted by atorvastatin 20 mg daily. The medication adherence rate decreased over the monitoring period. Adherence rates below 60% were associated with therapeutic failure, while rates equal to 80% or higher were associated with the best response to the lipid-lowering drugs. Conclusion Adherence to hypolipidemic agents is higher at the beginning of the treatment, but it decreases over time, affecting the achievement of therapeutic goals. PMID:25035713

  2. Porcine intestinal epithelial cell lines as a new in vitro model for studying adherence and pathogenesis of enterotoxigenic Escherichia coli.

    PubMed

    Koh, Seung Y; George, Sajan; Brözel, Volker; Moxley, Rodney; Francis, David; Kaushik, Radhey S

    2008-07-27

    Enterotoxigenic Escherichia coli (ETEC) infections result in large economic losses in the swine industry worldwide. The organism causes diarrhea by adhering to and colonizing enterocytes in the small intestines. While much progress has been made in understanding the pathogenesis of ETEC, no homologous intestinal epithelial cultures suitable for studying porcine ETEC pathogenesis have been described prior to this report. In the current study, we investigated the adherence of various porcine ETEC strains to two porcine (IPEC-1 and IPEC-J2) and one human (INT-407) small intestinal epithelial cell lines. Each cell line was assessed for its ability to support the adherence of E. coli expressing fimbrial adhesins K88ab, K88ac, K88ad, K99, F41, 987P, and F18. Wild-type ETEC expressing K88ab, K88ac, and K88ad efficiently bound to both IPEC-1 and IPEC-J2 cells. An ETEC strain expressing both K99 and F41 bound heavily to both porcine cell lines but an E. coli strain expressing only K99 bound very poorly to these cells. E. coli expressing F18 adhesin strongly bound to IPEC-1 cells but did not adhere to IPEC-J2 cells. The E. coli strains G58-1 and 711 which express no fimbrial adhesins and those that express 987P fimbriae failed to bind to either porcine cell line. Only strains B41 and K12:K99 bound in abundance to INT-407 cells. The binding of porcine ETEC to IPEC-J2, IPEC-1 and INT-407 with varying affinities, together with lack of binding of 987P ETEC and non-fimbriated E. coli strains, suggests strain-specific E. coli binding to these cell lines. These findings suggest the potential usefulness of porcine intestinal cell lines for studying ETEC pathogenesis.

  3. The role of Listeria monocytogenes cell wall surface anchor protein LapB in virulence, adherence, and intracellular replication

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lmof2365_2117 is a Listeria monocytogenes putative cell wall surface anchor protein with a conserved domain found in collagen binding proteins. We constructed a deletion mutation in lmof2365_2117 in serotype 4b strain F2365, evaluated its virulence, and determined its ability to adhere and invade co...

  4. Non-invasive, label-free cell counting and quantitative analysis of adherent cells using digital holography.

    PubMed

    Mölder, A; Sebesta, M; Gustafsson, M; Gisselson, L; Wingren, A Gjörloff; Alm, K

    2008-11-01

    Manual cell counting is time consuming and requires a high degree of skill on behalf of the person performing the count. Here we use a technique that utilizes digital holography, allowing label-free and completely non-invasive cell counting directly in cell culture vessels with adherent viable cells. The images produced can provide both quantitative and qualitative phase information from a single hologram. The recently constructed microscope Holomonitor (Phase Holographic Imaging AB, Lund, Sweden) combines the commonly used phase contrast microscope with digital holography, the latter giving us the possibility of achieving quantitative information on cellular shape, area, confluence and optical thickness. This project aimed at determining the accuracy and repeatability of cell counting measurements using digital holography compared to the conventional manual cell counting method using a haemocytometer. The collected data were also used to determine cell size and cellular optical thickness. The results show that digital holography can be used for non-invasive automatic cell counting as precisely as conventional manual cell counting. PMID:19017223

  5. Selective adherence of non-typeable Haemophilus influenzae (NTHi) to mucus or epithelial cells in the chinchilla eustachian tube and middle ear.

    PubMed

    Miyamoto, N; Bakaletz, L O

    1996-11-01

    Frozen sections of chinchilla Eustachian tube (ET) and middle ear mucosa were incubated with either FITC-labeled non-typeable Haemophilus influenzae (NTHi) or Bordetella pertussis. The number of bacteria adherent to "roof" vs "floor" regions was compared for each of three anatomic portions of the ET and for middle ear epithelium noting whether bacteria adhered to mucus or to epithelial cells. NTHi strains adhered significantly greater to mucus in the ET lumen whereas B. pertussis preferentially adhered to epithelial cells lining the ET (P < or = 0.05). A non-fimbriated isogenic mutant of NTHi adhered significantly less to mucus than the parental isolate at all sites of the ET floor (P < or = 0.05). Isolated fimbrin protein adhered to ET mucus and blocked adherence of whole organisms. Treatment with the mucolytic agent N-acetyl-L-cysteine resulted in significantly reduced adherence of NTHi to mucus (P < or = 0.001) and eliminated the ability to detect binding of isolated fimbrin protein. N-acetyl-L-cysteine treatment did not affect adherence of either B. pertussis or NTHi to epithelial cells. These data indicated that NTHi may mediate ascension of the ET from the nasopharynx primarily via adherence to and growth in mucus overlying the floor region of the tubal lumen. The OMP P5-homologous fimbriae were shown to contribute to this binding.

  6. T lymphocytes adhere to airway smooth muscle cells via integrins and CD44 and induce smooth muscle cell DNA synthesis

    PubMed Central

    1994-01-01

    Asthma is a disease of airway inflammation and hyperreactivity that is associated with a lymphocytic infiltrate in the bronchial submucosa. The interactions between infiltrating T lymphocytes with cellular and extracellular matrix components of the airway and the consequences of these interactions have not been defined. We demonstrate the constitutive expression of CD44 on human airway smooth muscle (ASM) cells in culture as well as in human bronchial tissue transplanted into severe combined immunodeficient mice. In contrast, basal levels of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expression are minimal but are induced on ASM by inflammatory mediators such as tumor necrosis factor alpha (TNF-alpha). Activated, but not resting T cells, adhere to cultured ASM; stimulation of the ASM with TNF-alpha enhanced this adhesion. Adhesion was partially blocked by monoclonal antibodies (mAb) specific for lymphocyte function-associated antigen 1 (LFA-1) and very late antigen 4 (VLA-4) on T cells and ICAM-1 and VCAM-1 on ASM cells. The observed integrin-independent adhesion was mediated by CD44/hyaluronate interactions as it was inhibited by anti-CD44 mAb 5F12 and by hyaluronidase. Furthermore, the adhesion of activated T lymphocytes induced DNA synthesis in growth-arrested ASM cells. Thus, the interaction between T cells and ASM may provide insight into the mechanisms that induce bronchial inflammation and possibly ASM cell hyperplasia seen in asthma. PMID:7520473

  7. Fimbria-mediated adherence of Candida albicans to glycosphingolipid receptors on human buccal epithelial cells.

    PubMed Central

    Yu, L; Lee, K K; Sheth, H B; Lane-Bell, P; Srivastava, G; Hindsgaul, O; Paranchych, W; Hodges, R S; Irvin, R T

    1994-01-01

    Candida albicans is an opportunist fungal pathogen that has the ability to adhere to host cell surface receptors via a number of adhesins. Yu et al. (L. Yu, K. K. Lee, K. Ens, P. C. Doig, M. R. Carpenter, W. Staddon, R. S. Hodges, W. Paranchych, and R. T. Irvin, Infect. Immun. 62:2834-2842, 1994) described the purification and initial characterization of a fimbrial adhesin from C. albicans. In this paper, we show that C. albicans fimbriae also bind to asialo-GM1 [gangliotetraosylceramide: beta Gal(1-3)beta GalNAc(1-4) beta Gal(1-4)beta Glc(1-1)Cer] immobilized on microtiter plates in a saturable and concentration-dependent manner. C. albicans fimbrial binding to exfoliated human buccal epithelial cells (BECs) was inhibited by asialo-GM1 in in vitro binding assays. The fimbriae interact with the glycosphingolipid receptors via the carbohydrate portion of the receptors, since fimbriae were observed to bind to synthetic beta GalNAc(1-4)beta Gal-protein conjugates and the disaccharide was able to inhibit binding of fimbriae to BECs in in vitro binding assays. We conclude from these results that the C. albicans yeast form expresses a fimbrial adhesin that binds to glycosphingolipids displayed on the surface of human BECs. Images PMID:8005674

  8. Protein phosphatase 2A activity is required for functional adherent junctions in endothelial cells.

    PubMed

    Kása, Anita; Czikora, István; Verin, Alexander D; Gergely, Pál; Csortos, Csilla

    2013-09-01

    Reversible Ser/Thr phosphorylation of cytoskeletal and adherent junction (AJ) proteins has a critical role in the regulation of endothelial cell (EC) barrier function. We have demonstrated earlier that protein phosphatase 2A (PP2A) activity is important in EC barrier integrity. In the present work, macro- and microvascular EC were examined and we provided further evidence on the significance of PP2A in the maintenance of EC cytoskeleton and barrier function with special focus on the Bα (regulatory) subunit of PP2A. Immunofluorescent staining revealed that the inhibition of PP2A results in changes in the organization of EC cytoskeleton as microtubule dissolution and actin re-arrangement were detected. Depletion of Bα regulatory subunit of PP2A had similar effect on the cytoskeleton structure of the cells. Furthermore, transendothelial electric resistance measurements demonstrated significantly slower barrier recovery of Bα depleted EC after thrombin treatment. AJ proteins, VE-cadherin and β-catenin, were detected along with Bα in pull-down assay. Also, the inhibition of PP2A (by okadaic acid or fostriecin) or depletion of Bα caused β-catenin translocation from the membrane to the cytoplasm in parallel with its phosphorylation on Ser552. In conclusion, our data suggest that the A/Bα/C holoenzyme form of PP2A is essential in EC barrier integrity both in micro- and macrovascular EC. PMID:23721711

  9. Self-Reported Prevalence of Symptomatic Adverse Reactions to Gluten and Adherence to Gluten-Free Diet in an Adult Mexican Population.

    PubMed

    Ontiveros, Noe; López-Gallardo, Jesús A; Vergara-Jiménez, Marcela J; Cabrera-Chávez, Francisco

    2015-07-21

    The prevalence of symptomatic adverse reactions to gluten and adherence to gluten-free diet in Latin American countries is unknown. These measurements are strongly linked to gluten-related disorders. This work aimed to estimate the prevalence of adverse reactions to oral gluten and the adherence to gluten-free diet in the adult Mexican population. To reach this aim, a self-administered questionnaire was designed and tested for clarity/comprehension and reproducibility. Then, a self-administered questionnaire-based cross-sectional study was conducted in the Mexican population. The estimated prevalence rates were (95% CI): 11.9% (9.9-13.5) and 7.8 (6.4-9.4) for adverse and recurrent adverse reactions to gluten respectively; adherence to gluten-free diet 3.7% (2.7-4.8), wheat allergy 0.72% (0.38-1.37); celiac disease 0.08% (0.01-0.45), and NCGS 0.97% (0.55-1.68). Estimated pooled prevalence of self-reported physician-diagnosis of gluten-related disorders was 0.88% (0.49-1.5), and 93.3% respondents reported adherence to gluten-free diet without a physician-diagnosis of gluten-related disorders. Symptom comparisons between those who reported recurrent adverse reactions to gluten and other foods showed statistically significant differences for bloating, constipation, and tiredness (p < 0.05). Gluten-related disorders may be underdiagnosed in the Mexican population and most people adhering to a gluten-free diet are doing it without proper diagnostic work-up of these disorders, and probably without medical/dietician advice.

  10. Self-Reported Prevalence of Symptomatic Adverse Reactions to Gluten and Adherence to Gluten-Free Diet in an Adult Mexican Population

    PubMed Central

    Ontiveros, Noe; López-Gallardo, Jesús A.; Vergara-Jiménez, Marcela J.; Cabrera-Chávez, Francisco

    2015-01-01

    The prevalence of symptomatic adverse reactions to gluten and adherence to gluten-free diet in Latin American countries is unknown. These measurements are strongly linked to gluten-related disorders. This work aimed to estimate the prevalence of adverse reactions to oral gluten and the adherence to gluten-free diet in the adult Mexican population. To reach this aim, a self-administered questionnaire was designed and tested for clarity/comprehension and reproducibility. Then, a self-administered questionnaire-based cross-sectional study was conducted in the Mexican population. The estimated prevalence rates were (95% CI): 11.9% (9.9–13.5) and 7.8 (6.4–9.4) for adverse and recurrent adverse reactions to gluten respectively; adherence to gluten-free diet 3.7% (2.7–4.8), wheat allergy 0.72% (0.38–1.37); celiac disease 0.08% (0.01–0.45), and NCGS 0.97% (0.55–1.68). Estimated pooled prevalence of self-reported physician-diagnosis of gluten-related disorders was 0.88% (0.49–1.5), and 93.3% respondents reported adherence to gluten-free diet without a physician-diagnosis of gluten-related disorders. Symptom comparisons between those who reported recurrent adverse reactions to gluten and other foods showed statistically significant differences for bloating, constipation, and tiredness (p < 0.05). Gluten-related disorders may be underdiagnosed in the Mexican population and most people adhering to a gluten-free diet are doing it without proper diagnostic work-up of these disorders, and probably without medical/dietician advice. PMID:26197336

  11. Self-Reported Prevalence of Symptomatic Adverse Reactions to Gluten and Adherence to Gluten-Free Diet in an Adult Mexican Population.

    PubMed

    Ontiveros, Noe; López-Gallardo, Jesús A; Vergara-Jiménez, Marcela J; Cabrera-Chávez, Francisco

    2015-07-01

    The prevalence of symptomatic adverse reactions to gluten and adherence to gluten-free diet in Latin American countries is unknown. These measurements are strongly linked to gluten-related disorders. This work aimed to estimate the prevalence of adverse reactions to oral gluten and the adherence to gluten-free diet in the adult Mexican population. To reach this aim, a self-administered questionnaire was designed and tested for clarity/comprehension and reproducibility. Then, a self-administered questionnaire-based cross-sectional study was conducted in the Mexican population. The estimated prevalence rates were (95% CI): 11.9% (9.9-13.5) and 7.8 (6.4-9.4) for adverse and recurrent adverse reactions to gluten respectively; adherence to gluten-free diet 3.7% (2.7-4.8), wheat allergy 0.72% (0.38-1.37); celiac disease 0.08% (0.01-0.45), and NCGS 0.97% (0.55-1.68). Estimated pooled prevalence of self-reported physician-diagnosis of gluten-related disorders was 0.88% (0.49-1.5), and 93.3% respondents reported adherence to gluten-free diet without a physician-diagnosis of gluten-related disorders. Symptom comparisons between those who reported recurrent adverse reactions to gluten and other foods showed statistically significant differences for bloating, constipation, and tiredness (p < 0.05). Gluten-related disorders may be underdiagnosed in the Mexican population and most people adhering to a gluten-free diet are doing it without proper diagnostic work-up of these disorders, and probably without medical/dietician advice. PMID:26197336

  12. Adherence to a Mediterranean diet in Morocco and its correlates: cross-sectional analysis of a sample of the adult Moroccan population

    PubMed Central

    2012-01-01

    Background Dietary habits in Morocco are changing and the causes are not well understood. This study aimed to analyse socio-demographic factors associated with adherence to the Mediterranean diet (MeDi) in a national random sample of the adult Moroccan population. Methods The data collected in this cross-sectional survey included socio-demographic factors and a food frequency questionnaire. MeDi adherence was assessed in 2214 individuals with complete dietary data. MeDi adherence was measured according to a simplified MeDi score based on the weekly frequency of intake of eight food groups (vegetables, legumes, fruits, cereal or potatoes, fish, red meat, dairy products and olive oil) with the use of the sex specific medians of the sample as cut-offs. A value of 0 or 1 was assigned to consumption of each component according to its presumed detrimental or beneficial effect on health. Logistic regression was used to estimate the association between MeDi adherence (low score 1-4 vs. high 5-8) and other factors. Results Mean age of the sample was 41.4 (standard deviation 15.3) years, 45.4% were men and 29.9% had a low MeDi adherence. Married subjects (adjusted odds ratio ORa=0.68, 95% CI 0.55-0.84) were less likely to have a low MeDi adherence compared to single, divorced or widowed persons. Persons from rural areas (ORa=1.46, 95% CI: 1.02-2.08), were more often low MeDi adherents compared to those from urban areas. Obese persons (ORa=1.56, 95% CI: 1.16-2.11) were more prone to low MeDi adherence than normal weight individuals. Conclusion MeDi is far from being a universal pattern in the Moroccan population. Intervention strategies should be implemented in target groups to maintain the traditional MeDi pattern considered as the original diet in Morocco. PMID:22578133

  13. Evaluation of a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay (SOT)

    EPA Science Inventory

    The Embryonic Stem Cell Test (EST) has been used to evaluate the effects of xenobiotics using three endpoints, stem cell differentiation, stem cell viability and 3T3-cell viability. Our research goal is to establish amodel system that would evaluate chemical effects using a singl...

  14. Comparative analysis of non-adherence to medication treatment for systemic arterial hypertension in urban and rural populations 1

    PubMed Central

    Magnabosco, Patricia; Teraoka, Eliana Cavalari; de Oliveira, Edward Meirelles; Felipe, Elisangela Aparecida; Freitas, Dayana; Marchi-Alves, Leila Maria

    2015-01-01

    OBJECTIVE: to evaluate the indexes and the main factors associated with non-adherence to medication treatment for systemic arterial hypertension between urban and rural areas. METHOD: analytical study based on an epidemiological survey with a sample of 247 hypertensive residents of rural and urban areas, with application of a socio-demographic and economic questionnaire, and treatment adherence assessment. The Pearson's Chi-square test was used and the odds ratio (OD) was calculated to analyze the factors related to non-adherence. RESULTS: the prevalence of non-adherence was 61.9% and it was higher in urban areas (63.4%). Factors significantly associated with non-adherence were: male gender (OR=1.95; 95% CI 1.08-3.50), age 20-59 years old (OR=2.51; 95% CI 1.44-4.39), low economic status (OR=1.95; 95% CI 1.09-3.47), alcohol consumption (OR=5.92, 95% CI 1.73-20.21), short time of hypertension diagnosis (OR=3.07; 95% CI 1.35-6.96) and not attending the health service for routine consultations (OR=2.45; 1.35-4.42). CONCLUSION: the socio-demographic/economic characteristics, lifestyle habits and how to relate to health services were the factors that presented association with non-adherence regardless of the place of residence. PMID:25806627

  15. Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay

    EPA Science Inventory

    The Embryonic Stem Cell Test (EST) is an assay which evaluates xenobiotic-induced effects using three endpoints: mouse embryonic stem cell (mESC) differentiation, mESC viability, and 3T3-cell viability. Our research goal was to develop an improved high-throughput assay by establi...

  16. Correlative VIS-fluorescence and soft X-ray cryo-microscopy/tomography of adherent cells.

    PubMed

    Hagen, Christoph; Guttmann, Peter; Klupp, Barbara; Werner, Stephan; Rehbein, Stefan; Mettenleiter, Thomas C; Schneider, Gerd; Grünewald, Kay

    2012-02-01

    Soft X-ray cryo-microscopy/tomography of vitreous samples is becoming a valuable tool in structural cell biology. Within the 'water-window' wavelength region (2.34-4.37nm), it provides absorption contrast images with high signal to noise ratio and resolution of a few tens of nanometer. Soft X-rays with wavelengths close to the K-absorption edge of oxygen penetrate biological samples with thicknesses in the micrometer range. Here, we report on the application of a recently established extension of the transmission soft X-ray cryo-microscope (HZB TXM) at the beamline U41-XM of the BESSY II electron storage ring by an in-column epi-fluorescence and reflected light cryo-microscope. We demonstrate the new capability for correlative fluorescence and soft X-ray cryo-microscopy/tomography of this instrument along a typical life science experimental approach - the correlation of a fluorophore-tagged protein (pUL34-GFP of pseudorabies virus, PrV, the nuclear membrane-anchored component of the nuclear egress complex of the Herpesviridae which interacts with viral pUL31) in PrV pUL34-GFP/pUL31 coexpressing mammalian cells, with virus-induced vesicular structures in the nucleus, expanding the nucleoplasmic reticulum. Taken together, our results demonstrate new possibilities to study the role of specific proteins in substructures of adherent cells, especially of the nucleus in toto, accessible to electron microscopy in thinned samples only. PMID:22210307

  17. Correlative VIS-fluorescence and soft X-ray cryo-microscopy/tomography of adherent cells

    PubMed Central

    Hagen, Christoph; Guttmann, Peter; Klupp, Barbara; Werner, Stephan; Rehbein, Stefan; Mettenleiter, Thomas C.; Schneider, Gerd; Grünewald, Kay

    2012-01-01

    Soft X-ray cryo-microscopy/tomography of vitreous samples is becoming a valuable tool in structural cell biology. Within the ‘water-window’ wavelength region (2.34–4.37 nm), it provides absorption contrast images with high signal to noise ratio and resolution of a few tens of nanometer. Soft X-rays with wavelengths close to the K-absorption edge of oxygen penetrate biological samples with thicknesses in the micrometer range. Here, we report on the application of a recently established extension of the transmission soft X-ray cryo-microscope (HZB TXM) at the beamline U41-XM of the BESSY II electron storage ring by an in-column epi-fluorescence and reflected light cryo-microscope. We demonstrate the new capability for correlative fluorescence and soft X-ray cryo-microscopy/tomography of this instrument along a typical life science experimental approach – the correlation of a fluorophore-tagged protein (pUL34-GFP of pseudorabies virus, PrV, the nuclear membrane-anchored component of the nuclear egress complex of the Herpesviridae which interacts with viral pUL31) in PrV pUL34-GFP/pUL31 coexpressing mammalian cells, with virus-induced vesicular structures in the nucleus, expanding the nucleoplasmic reticulum. Taken together, our results demonstrate new possibilities to study the role of specific proteins in substructures of adherent cells, especially of the nucleus in toto, accessible to electron microscopy in thinned samples only. PMID:22210307

  18. Comparative adherence of Candida albicans and Candida dubliniensis to human buccal epithelial cells and extracellular matrix proteins.

    PubMed

    Jordan, Rachael P C; Williams, David W; Moran, Gary P; Coleman, David C; Sullivan, Derek J

    2014-04-01

    Candida albicans and Candida dubliniensis are very closely related pathogenic yeast species. Despite their close relationship, C. albicans is a far more successful colonizer and pathogen of humans. The purpose of this study was to determine if the disparity in the virulence of the two species is attributed to differences in their ability to adhere to human buccal epithelial cells (BECs) and/or extracellular matrix proteins. When grown overnight at 30°C in yeast extract peptone dextrose, genotype 1 C. dubliniensis isolates were found to be significantly more adherent to human BECs than C. albicans or C. dubliniensis genotypes 2-4 (P < 0.001). However, when the yeast cells were grown at 37°C, no significant difference between the adhesion of C. dubliniensis genotype 1 and C. albicans to human BECs was observed, and C. dubliniensis genotype 1 and C. albicans adhered to BECs in significantly greater numbers than the other C. dubliniensis genotypes (P < 0.001). Using surface plasmon resonance analysis, C. dubliniensis isolates were found to adhere in significantly greater numbers than C. albicans to type I and IV collagen, fibronectin, laminin, vitronectin, and proline-rich peptides. These data suggest that C. albicans is not more adherent to epithelial cells or matrix proteins than C. dubliniensis and therefore other factors must contribute to the greater levels of virulence exhibited by C. albicans.

  19. Evaluation of a solid phase red cell adherence technique for platelet antibody screening.

    PubMed

    Lown, J A; Ivey, J G

    1991-09-01

    Solid-phase red-cell adherence (SPRCA) techniques in platelet serology are used mainly for crossmatching. A SPRCA method for general diagnostic application was evaluated in parallel with the platelet suspension immunofluorescence test (PIFT). Of 149 patient sera sent for investigation of thrombocytopaenia, 76 were negative and 59 positive when studied by both methods, eight positive by PIFT only and six positive by SPRCA only. The reactivity observed for 24 sera containing HLA antibodies tested with chloroquine-treated and untreated platelets was similar for both methods. All of 14 sera containing quinine-associated antibodies reacted strongly to both techniques in the presence of added quinine. In comparison, however, whereas all sera were nonreactive to SPRCA in the absence of added quinine, and with PIFT, seven of the sera reacted weakly. Titration studies with three examples of anti-PlA1 and five sera containing HLA antibodies generally showed a one doubling dilution lower titre with the SPRCA procedure. End-point interpretation, however, was more readily achieved with the SPRCA method. The SPRCA technique displays similar sensitivity and specificity to the PIFT and is recommended for use by routine hospital laboratories to screen platelet antibodies.

  20. Fibrin monomer increases platelet adherence to tumor cells in a flowing system: a possible role in metastasis?

    PubMed

    Biggerstaff, J P; Seth, N B; Meyer, T V; Amirkhosravi, A; Francis, J L

    1998-12-15

    Considerable evidence exists linking hemostasis and malignancy. Platelet adhesion to tumor cells has been implicated in the metastatic process. Plasma fibrinogen (Fg) and fibrin (Fn) monomer, increased in cancer, may play a role in tumor biology. Binding of Fn monomer to tumor cells and its effect on platelet-tumor cell adhesion in a flowing system were studied. Fn monomer was produced by adding thrombin (1 micro/mL) to FXIII- and plasminogen-free Fg in the presence of Gly-Pro-Arg-Pro (GPRP) amide. Fn monomer binding to live A375 cells was visualized by confocal laser scanning microscopy (CLSM). Adherent cells were perfused for 1h with Fn monomer, washed and stained in situ with anti-human Fn (American Biogenetic Sciences, Inc.) followed by goat anti-mouse IgG(FITC). Platelet adherence to Fn monomer treated A375 cells was performed under flow conditions by passing platelets (5x10(4)/microl 0.25 mL/min; labeled with the carbocyanine dye DiI) over the tumor cells for 30 min. CLSM images were obtained after washing. There was considerable binding of Fn monomer, but not Fg alone. Platelets adhered relatively weakly to untreated A375 cells and this was not significantly affected by pre-treatment of the tumor cells with fibrinogen or thrombin. However, pre-treatment with Fn monomer resulted in extensive platelet binding to tumor cells, suggesting that coagulation activation and the subsequent increase in circulating Fn monomer may enhance platelet adhesion to circulating tumor cells and thereby facilitate metastatic spread.

  1. Fibrin monomer increases platelet adherence to tumor cells in a flowing system: a possible role in metastasis?

    PubMed

    Biggerstaff, J P; Seth, N B; Meyer, T V; Amirkhosravi, A; Francis, J L

    1998-12-15

    Considerable evidence exists linking hemostasis and malignancy. Platelet adhesion to tumor cells has been implicated in the metastatic process. Plasma fibrinogen (Fg) and fibrin (Fn) monomer, increased in cancer, may play a role in tumor biology. Binding of Fn monomer to tumor cells and its effect on platelet-tumor cell adhesion in a flowing system were studied. Fn monomer was produced by adding thrombin (1 micro/mL) to FXIII- and plasminogen-free Fg in the presence of Gly-Pro-Arg-Pro (GPRP) amide. Fn monomer binding to live A375 cells was visualized by confocal laser scanning microscopy (CLSM). Adherent cells were perfused for 1h with Fn monomer, washed and stained in situ with anti-human Fn (American Biogenetic Sciences, Inc.) followed by goat anti-mouse IgG(FITC). Platelet adherence to Fn monomer treated A375 cells was performed under flow conditions by passing platelets (5x10(4)/microl 0.25 mL/min; labeled with the carbocyanine dye DiI) over the tumor cells for 30 min. CLSM images were obtained after washing. There was considerable binding of Fn monomer, but not Fg alone. Platelets adhered relatively weakly to untreated A375 cells and this was not significantly affected by pre-treatment of the tumor cells with fibrinogen or thrombin. However, pre-treatment with Fn monomer resulted in extensive platelet binding to tumor cells, suggesting that coagulation activation and the subsequent increase in circulating Fn monomer may enhance platelet adhesion to circulating tumor cells and thereby facilitate metastatic spread. PMID:9886911

  2. An adherent cell perifusion technique to study the overall and sequential response of rat alveolar macrophages to toxic substances.

    PubMed Central

    Forget, G; Lacroix, M J; Cadieux, A; Calvert, R; Grose, J H; Sirois, P

    1983-01-01

    Essentially pure (97%) alveolar macrophages were isolated by bronchoalveolar lavage of rats with warm (37 degrees C) PBS solution. These cells were allowed to adhere to the inside walls of open-ended glass cylinders which were closed off at each end by three-way stopcocks. The adhering cells were perifused with RPMI-1640 medium supplemented with 5% fetal bovine serum for 18 hr at the rate of 1 mL/hr, and the effluent medium was collected automatically in 2-mL aliquots. Cell recoveries and viabilities did not differ from those found for Petri cultures treated similarly, indicating that the perifusion method under study offered an adequate milieu for short-term primary cultures. The alveolar macrophages in culture were subjected to the presence of particulate (chrysotile asbestos) and soluble (phorbol myristate) toxicants, and their response was monitored in the effluent medium by measuring the release of prostaglandins (PGE) by radioimmunoassay. A significant increase in the sequential release of PGE was observed in the presence of asbestos (100 micrograms/mL) or phorbol myristate (200 ng/mL). Treatment of the cells with indomethacin (20 microM) completely abolished the release of PGE stimulated with phorbol myristate. A cumulative response to the toxicants was also observed when cells were harvested manually from the chambers: asbestos caused a 2-fold increase in cell mortality relative to control, while phorbol myristate brought about a 3-fold increase in the number of dead cells. This effect was not prevented by the presence of indomethacin. Cell aggregation was also observed when cells were perifused in the presence of phorbol myristate, whether indomethacin was present or absent. Our results indicate that the cell perifusion system combines the advantages of conventional adherent cell cultures (viability, aggregation) with those of perifusion techniques (sequential metabolism studies). Images FIGURE 1. FIGURE 2. FIGURE 3. FIGURE 6. PMID:6641651

  3. Evidence for Technology Interventions to Promote ART Adherence in Adult Populations: a Review of the Literature 2012-2015.

    PubMed

    Amico, K Rivet

    2015-12-01

    Leveraging technology to enhance antiretroviral therapy (ART) adherence, and factors associated with it, has tremendous appeal as a low-cost, generalizable strategy to offer high-quality adherence support given an increasingly limited workforce. As the numbers of individuals living with HIV entering care and initiating ART are expected to increase substantially worldwide in the next decade, capacity to support ART adherence is needed and use of computers, internet, and mobile phones has the potential to offer those efficiencies-provided they are effective. This review summarizes recent advances in the evidence base for technology-driven, technology-delivered, or technology-enhanced ART adherence intervention approaches. A PubMed search limited to January 2013 through July 2015 identified 13 qualifying studies evaluating computer-delivered interventions, internet approaches, mobile phone technologies, and electronic dose monitoring with triggered messaging and data-informed counseling. Considerable support for each area has emerged, with the majority of studies reviewed demonstrating significant effects on ART adherence and clinical outcomes. Gaps are identified and recommendations offered.

  4. Identification of Cell Surface-Exposed Proteins Involved in the Fimbria-Mediated Adherence of Enteroaggregative Escherichia coli to Intestinal Cells

    PubMed Central

    Izquierdo, Mariana; Navarro-Garcia, Fernando; Nava-Acosta, Raul; Nataro, James P.; Ruiz-Perez, Fernando

    2014-01-01

    Fimbria-mediated adherence to the intestinal epithelia is a key step in enteroaggregative Escherichia coli (EAEC) pathogenesis. To date, four fimbriae have been described for EAEC; aggregative adherence fimbria II (AAF/II) is the most important adherence factor for EAEC prototype strain 042. Previously, we described results showing that extracellular matrix (ECM) components might be involved in the recognition of AAF/II fimbriae by intestinal cells. In this study, we sought to identify novel potential receptors on intestinal epithelial cells recognized by the AAF/II fimbriae. Purified AafA-dsc protein, the major subunit of AAF/II fimbriae, was incubated with a monolayer of T84 cells, cross-linked to the surface-exposed T84 cell proteins, and immunoprecipitated by using anti-AafA antibodies. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of cellular proteins bound to AafA-dsc protein identified laminin (previously recognized as a potential receptor for AAF/II) and cytokeratin 8 (CK8). Involvement of the major subunit of AAF/II fimbriae (AafA protein) in the binding to recombinant CK8 was confirmed by adherence assays with purified AAF/II fimbriae, AafA-dsc protein, and strain 042. Moreover, HEp-2 cells transfected with CK8 small interfering RNA (siRNA) showed reduced 042 adherence compared with cells transfected with scrambled siRNA as a control. Adherence of 042 to HEp-2 cells preincubated with antibodies against ECM proteins or CK8 was substantially reduced. Altogether, our results supported the idea of a role of CK8 as a potential receptor for EAEC. PMID:24516112

  5. Identification of cell surface-exposed proteins involved in the fimbria-mediated adherence of enteroaggregative Escherichia coli to intestinal cells.

    PubMed

    Izquierdo, Mariana; Navarro-Garcia, Fernando; Nava-Acosta, Raul; Nataro, James P; Ruiz-Perez, Fernando; Farfan, Mauricio J

    2014-04-01

    Fimbria-mediated adherence to the intestinal epithelia is a key step in enteroaggregative Escherichia coli (EAEC) pathogenesis. To date, four fimbriae have been described for EAEC; aggregative adherence fimbria II (AAF/II) is the most important adherence factor for EAEC prototype strain 042. Previously, we described results showing that extracellular matrix (ECM) components might be involved in the recognition of AAF/II fimbriae by intestinal cells. In this study, we sought to identify novel potential receptors on intestinal epithelial cells recognized by the AAF/II fimbriae. Purified AafA-dsc protein, the major subunit of AAF/II fimbriae, was incubated with a monolayer of T84 cells, cross-linked to the surface-exposed T84 cell proteins, and immunoprecipitated by using anti-AafA antibodies. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of cellular proteins bound to AafA-dsc protein identified laminin (previously recognized as a potential receptor for AAF/II) and cytokeratin 8 (CK8). Involvement of the major subunit of AAF/II fimbriae (AafA protein) in the binding to recombinant CK8 was confirmed by adherence assays with purified AAF/II fimbriae, AafA-dsc protein, and strain 042. Moreover, HEp-2 cells transfected with CK8 small interfering RNA (siRNA) showed reduced 042 adherence compared with cells transfected with scrambled siRNA as a control. Adherence of 042 to HEp-2 cells preincubated with antibodies against ECM proteins or CK8 was substantially reduced. Altogether, our results supported the idea of a role of CK8 as a potential receptor for EAEC.

  6. Identification of cell surface-exposed proteins involved in the fimbria-mediated adherence of enteroaggregative Escherichia coli to intestinal cells.

    PubMed

    Izquierdo, Mariana; Navarro-Garcia, Fernando; Nava-Acosta, Raul; Nataro, James P; Ruiz-Perez, Fernando; Farfan, Mauricio J

    2014-04-01

    Fimbria-mediated adherence to the intestinal epithelia is a key step in enteroaggregative Escherichia coli (EAEC) pathogenesis. To date, four fimbriae have been described for EAEC; aggregative adherence fimbria II (AAF/II) is the most important adherence factor for EAEC prototype strain 042. Previously, we described results showing that extracellular matrix (ECM) components might be involved in the recognition of AAF/II fimbriae by intestinal cells. In this study, we sought to identify novel potential receptors on intestinal epithelial cells recognized by the AAF/II fimbriae. Purified AafA-dsc protein, the major subunit of AAF/II fimbriae, was incubated with a monolayer of T84 cells, cross-linked to the surface-exposed T84 cell proteins, and immunoprecipitated by using anti-AafA antibodies. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of cellular proteins bound to AafA-dsc protein identified laminin (previously recognized as a potential receptor for AAF/II) and cytokeratin 8 (CK8). Involvement of the major subunit of AAF/II fimbriae (AafA protein) in the binding to recombinant CK8 was confirmed by adherence assays with purified AAF/II fimbriae, AafA-dsc protein, and strain 042. Moreover, HEp-2 cells transfected with CK8 small interfering RNA (siRNA) showed reduced 042 adherence compared with cells transfected with scrambled siRNA as a control. Adherence of 042 to HEp-2 cells preincubated with antibodies against ECM proteins or CK8 was substantially reduced. Altogether, our results supported the idea of a role of CK8 as a potential receptor for EAEC. PMID:24516112

  7. The adherence of endothelial cells to Dacron induces the expression of the intercellular adhesion molecule (ICAM-1).

    PubMed Central

    Margiotta, M S; Robertson, F S; Greco, R S

    1992-01-01

    The intercellular adhesion molecule (ICAM-1) is a glycoprotein expressed by endothelial cells activated by cytokines. The lymphocyte-function-associated antigen (LFA-1) is an integrin expressed by activated white blood cells. Together, this receptor-ligand pair is responsible, in part, for the localization of neutrophils at sites of inflammation. Using an in vitro model, the authors studied the binding of antibodies against ICAM-1 by human saphenous vein endothelial cells (HSVEC) adherent to Dacron and control cultureware. After adherence to Dacron pretreated with fibronectin, 24% more HSVEC-bound antibody against ICAM-1 compared with HSVEC on controls. In contrast, 90% more HSVEC adherent to Dacron incubated with whole blood bound anti-ICAM-1 antibodies. These cells bound 17.7-fold greater amounts of antibody compared with HSVEC on controls. Pretreating Dacron with plasma resulted in no increase in antibody binding compared with control. Our studies suggest that the cellular components of blood in contact with Dacron create a microenvironment that activates HSVEC and enhances ICAM-1 expression. Induction of this adhesion molecule may play a pivotal role in the migration and localization of leukocytes at the site of the vascular prosthesis. PMID:1359845

  8. Accessory cells with a veiled morphology and movement pattern generated from monocytes after avoidance of plastic adherence and of NADPH oxidase activation. A comparison with GM-CSF/IL-4-induced monocyte-derived dendritic cells.

    PubMed

    Ruwhof, Cindy; Canning, Martha O; Grotenhuis, Kristel; de Wit, Harm J; Florencia, Zenovia Z; de Haan-Meulman, Meeny; Drexhage, Hemmo A

    2002-07-01

    Veiled cells (VC) present in afferent lymph transport antigen from the periphery to the draining lymph nodes. Although VC in lymph form a heterogeneous population, some of the cells clearly belong on morphological grounds to the Langerhans cell (LC)/ dendritic cell (DC) series. Here we show that culturing monocytes for 24 hrs while avoiding plastic adherence (polypropylene tubes) and avoiding the activation of NADPH oxidase (blocking agents) results in the generation of a population of veiled accessory cells. The generated VC were actively moving cells like lymph-borne VC in vivo. The monocyte (mo)-derived VC population existed of CD14(dim/-) and CD14(brighT) cells. Of these the CD14(dim/-) VC were as good in stimulating allogeneic T cell proliferation as immature DC (iDC) obtained after one week of adherent culture of monocytes in granulocyte-macrophage-colony stimulating factor (GM-CSF)/interleukin (IL)-4. This underscores the accessory cell function of the mo-derived CD14(dim/-) VC. Although the CD14(dim/-)VC had a modest expression of the DC-specific marker CD83 and were positive for S100, expression of the DC-specific markers CD1a, Langerin, DC-SIGN, and DC-LAMP were absent. This indicates that the here generated CD14(dim/-) VC can not be considered as classical LC/DC. It was also impossible to turn the CD14(dim/-) mo-derived VC population into typical DC by culture for one week in GM-CSF/IL-4 or LPS. In fact the cells died tinder such circumstances, gaining some macrophage characteristics before dying. The IL-12 production from mo-derived CD14(dim/-) VC was lower, whereas the production of IL-10 was higher as compared to iDC. Consequently the T cells that were stimulated by these mo-derived VC produced less IFN-gamma as compared with T cells stimulated by iDC. Our data indicate that it is possible to rapidly generate a population of CD14(dim/-) veiled accessory cells from monocytes. The marker pattern and cytokine production of these VC indicate that this

  9. Cell receptor and surface ligand density effects on dynamic states of adhering circulating tumor cells.

    PubMed

    Zheng, Xiangjun; Cheung, Luthur Siu-Lun; Schroeder, Joyce A; Jiang, Linan; Zohar, Yitshak

    2011-10-21

    Dynamic states of cancer cells moving under shear flow in an antibody-functionalized microchannel are investigated experimentally and theoretically. The cell motion is analyzed with the aid of a simplified physical model featuring a receptor-coated rigid sphere moving above a solid surface with immobilized ligands. The motion of the sphere is described by the Langevin equation accounting for the hydrodynamic loadings, gravitational force, receptor-ligand bindings, and thermal fluctuations; the receptor-ligand bonds are modeled as linear springs. Depending on the applied shear flow rate, three dynamic states of cell motion have been identified: (i) free motion, (ii) rolling adhesion, and (iii) firm adhesion. Of particular interest is the fraction of captured circulating tumor cells, defined as the capture ratio, via specific receptor-ligand bonds. The cell capture ratio decreases with increasing shear flow rate with a characteristic rate. Based on both experimental and theoretical results, the characteristic flow rate increases monotonically with increasing either cell-receptor or surface-ligand density within certain ranges. Utilizing it as a scaling parameter, flow-rate dependent capture ratios for various cell-surface combinations collapse onto a single curve described by an exponential formula.

  10. Effect of selective serotonin reuptake inhibitors use on endocrine therapy adherence and breast cancer mortality: a population-based study.

    PubMed

    Valachis, Antonis; Garmo, Hans; Weinman, John; Fredriksson, Irma; Ahlgren, Johan; Sund, Malin; Holmberg, Lars

    2016-09-01

    The purpose of the study was to investigate whether the concomitant use of selective serotonin reuptake inhibitors (SSRI) with tamoxifen influences the risk of death due to breast cancer, and we also investigated the association between SSRI use and adherence to oral endocrine therapy (ET). We analyzed data from BCBaSe Sweden, which is a database created by the data linkage of Registries from three different regions of Sweden. To investigate the association between ET adherence and SSRI use, we included all women who were diagnosed with non-distant metastatic ER-positive invasive breast cancer from July 2007 to July 2011 and had at least one dispensed prescription of oral tamoxifen or aromatase inhibitor. To investigate the role of concurrent administration of SSRI and tamoxifen on breast cancer prognosis, we performed a nested case-control study. In the adherence cohort, 9104 women were included in the analyses. Women who received SSRI, either before or after breast cancer diagnosis, were at higher risk for low adherence to ET. However, when the overlapping period between SSRI use and ET was >50 %, no excess risk for low adherence was observed. Non-adherence (<80 %) to ET was significantly associated with worse breast cancer survival (OR 4.07; 95 % CI 3.27-5.06). In the case-control study, 445 cases and 11125 controls were included. The concomitant administration of SSRI and tamoxifen did not influence breast cancer survival, neither in short-term (OR 1.41; 95 % CI 0.74-2.68) nor in long-term SSRI users (OR 0.85; 95 % CI 0.35-2.08). Concomitant SSRI and tamoxifen use does not seem to increase risk for death due to breast cancer. Given the positive association between continuing antidepressive pharmacotherapy for a longer period of time and adherence to ET, it is essential to capture and treat depression in breast cancer patients to secure adherence to ET. PMID:27492739

  11. Solid Phase Red Cell Adherence Assay: a tubeless method for pretransfusion testing and other applications in transfusion science.

    PubMed

    Ching, Eric

    2012-06-01

    Solid Phase Red Cell Adherence Assay (SPRCA) is one of the two tubeless methods developed to improve sensitivity and specificity in blood group serology. The SPRCA (solid phase) and the column agglutination (gel) technology have gained wide acceptance following successful adaptation to fully automated platforms, The purpose of this paper is to discuss the development, principle, procedures as well as laboratory and clinical applications of the SPRCA in transfusion medicine.

  12. Predictors of adherence to screening guidelines for chronic diseases of lifestyle, cancers, and HIV in a health-insured population in South Africa

    PubMed Central

    Adonis, Leegale; Basu, Debashis; Luiz, John

    2014-01-01

    Background Adherence to screening guidelines has been widely accepted to reduce morbidity, mortality, and cost outcomes. The aim of this study was to identify predictors of adherence to screening guidelines for chronic diseases of lifestyle (CDL), cancers, and HIV in a health-insured population in South Africa, some of whom voluntarily opt into a wellness program that incentivizes screening. Method A cross-sectional study for the period 2007–2011 was conducted using a random sample of 170,471 health insurance members from a single insurer. Adherence to screening guidelines was calculated from medical claims data. Results Adherence to screening guidelines ranged from 1.1% for colorectal cancer to 40.9% for cholesterol screening. Members of the wellness program were up to three times more likely to screen for diseases (odds ratio [OR]=3.2 for HIV screening, confidence interval [CI]=2.75–3.73). Plan type (full comprehensive plan) was most strongly associated with cholesterol screening (OR=3.53, CI=3.27–3.80), and most negatively associated (hospital-only core plan) with cervical cancer screening (OR= 0.44, CI=0.28–0.70). Gender was a negative predictor for glucose screening (OR=0.88, CI=0.82–0.96). Provincial residence was most strongly associated with cervical cancer screening (OR=1.89, CI=0.65–5.54). Conclusion Adherence to screening recommendations was <50%. Plan type, gender, provincial residence, and belonging to an incentivized wellness program were associated with disproportionate utilization of screening services, even with equal payment access. PMID:24647130

  13. Suppression of unprimed T and B cells in antibody responses by irradiation-resistant and plastic-adherent suppressor cells in Toxoplasma gondii-infected mice.

    PubMed Central

    Suzuki, Y; Kobayashi, A

    1983-01-01

    In the acute phase of Toxoplasma infection, the function of both helper T and B cells was suppressed in primary antibody responses to dinitrophenol (DNP)-conjugated protein antigens. During the course of infection, the suppressive effect on T cells seems to continue longer than that on B cells, since suppression in responses to sheep erythrocytes, a T-dependent antigen, persisted longer than those to DNP-Ficoll, a T-independent antigen. Plastic-adherent cells from the spleens of Toxoplasma-infected and X-irradiated (400 rads) mice had strong suppressor activity in primary anti-sheep erythrocyte antibody responses of normal mouse spleen cells in vitro. These data suggest that the activation of irradiation-resistant and plastic-adherent suppressor cells causes the suppression of both T and B cells in Toxoplasma-infected mice. PMID:6219954

  14. Contribution of Efa1/LifA to the adherence of enteropathogenic Escherichia coli to epithelial cells.

    PubMed

    Badea, Luminita; Doughty, Stephen; Nicholls, Larissa; Sloan, Joan; Robins-Browne, Roy M; Hartland, Elizabeth L

    2003-05-01

    Enteropathogenic E. coli(EPEC) is an important diarrhoeal pathogen that induces characteristic lesions on the host intestine termed attaching and effacing (A/E) lesions. In this study we have examined the contribution of a large gene, efa1, which is present in all A/E pathogens, to the adherence phenotype of EPEC. An efa- derivative of EPEC JPN15 was constructed and this mutant was significantly less adherent to epithelial cells than the parent strain. The JPN15 efa- derivative was FAS-positive, produced EspA filaments and showed comparable levels of EspA secretion to JPN15. In addition, polyclonal antibodies raised to Efa1 partially inhibited the adherence of JPN15 to cultured epithelial cells. In further work, we showed that human and rabbit hosts infected with an A/E pathogen produced antibodies to Efa1 and we observed that the truncated form of efa1 present in EHEC O157:H7 was specific to that serotype. Generally efa1 was present in its entirety in the genomes of other A/E pathogens. Overall our data suggest that Efa1 has host cell binding activity, at least in tissue culture, and that it is produced during infection. These findings suggest that Efa1 may play a direct role in the pathogenesis of infections caused by A/E pathogens.

  15. OmpD but not OmpC is involved in adherence of Salmonella enterica serovar typhimurium to human cells.

    PubMed

    Hara-Kaonga, Bochiwe; Pistole, Thomas G

    2004-09-01

    Conflicting reports exist regarding the role of porins OmpC and OmpD in infections due to Salmonella enterica serovar Typhimurium. This study investigated the role of these porins in bacterial adherence to human macrophages and intestinal epithelial cells. ompC and ompD mutant strains were created by transposon mutagenesis using P22-mediated transduction of Tn10 and Tn5 insertions, respectively, into wild-type strain 14028. Fluorescein-labeled wild-type and mutant bacteria were incubated with host cells at various bacteria to cell ratios for 1 h at 37 degrees C and analyzed by flow cytometry. The mean fluorescence intensity of cells with associated wild-type and mutant bacteria was used to estimate the number of bacteria bound per host cell. Adherence was also measured by fluorescence microscopy. Neither assay showed a significant difference in binding of the ompC mutant and wild-type strains to the human cells. In contrast, the ompD mutant exhibited lowered binding to both cell types. Our findings suggest that OmpD but not OmpC is involved in the recognition of Salmonella serovar Typhimurium by human macrophages and intestinal epithelial cells.

  16. Methanocaldococcus villosus sp. nov., a heavily flagellated archaeon that adheres to surfaces and forms cell-cell contacts.

    PubMed

    Bellack, Annett; Huber, Harald; Rachel, Reinhard; Wanner, Gerhard; Wirth, Reinhard

    2011-06-01

    A novel chemolithoautotrophic, hyperthermophilic methanogen was isolated from a submarine hydrothermal system at the Kolbeinsey Ridge, north of Iceland. Based on its 16S rRNA gene sequence, the strain belongs to the order Methanococcales within the genus Methanocaldococcus, with approximately 95 % sequence similarity to Methanocaldococcus jannaschii as its closest relative. Cells of the novel organism stained Gram-negative and appeared as regular to irregular cocci possessing more than 50 polar flagella. These cell appendages mediated not only motility but also adherence to abiotic surfaces and the formation of cell-cell contacts. The new isolate grew at 55-90 °C, with optimum growth at 80 °C. The optimum NaCl concentration for growth was 2.5 % (w/v), and the optimal pH was 6.5. The cells gained their energy exclusively by reduction of CO(2) with H(2). Selenate, tungstate and yeast extract stimulated growth significantly. The genome size was determined to be in the range 1.8-2.0 kb, and the G+C content of the genomic DNA was 30 mol%. Despite being physiologically nearly identical to the other members of the genus Methanocaldococcus, analysis of whole-cell proteins revealed significant differences. Based on the results from phylogenetic, morphological and protein analyses, we conclude that the novel strain represents a novel species of the genus Methanocaldococcus, for which the name Methanocaldococcus villosus sp. nov. is proposed (type strain KIN24-T80(T)  = DSM 22612(T)  = JCM 16315(T)). PMID:20622057

  17. Escherichia coli isolated from a Crohn's disease patient adheres, invades, and induces inflammatory responses in polarized intestinal epithelial cells.

    PubMed

    Eaves-Pyles, Tonyia; Allen, Christopher A; Taormina, Joanna; Swidsinski, Alexander; Tutt, Christopher B; Jezek, G Eric; Islas-Islas, Martha; Torres, Alfredo G

    2008-07-01

    Inflammatory diseases of the intestinal tract are a major health concern both in the United States and around the world. Evidence now suggests that a new category of Escherichia coli, designated Adherent Invasive E. coli (AIEC) is highly prevalent in Crohn's Disease (CD) patients. AIEC strains have been shown to colonize and adhere to intestinal epithelial cells (IEC). However, the role AIEC strains play in the induction of an inflammatory response is not known. Therefore, we examined several E. coli strains (designated LF82, O83:H1, 6604 and 6655) that were isolated from CD patients for their ability to induce inflammation in two IEC, Caco-2BBe and T-84 cells. Results showed that each strain had varying abilities to adhere to and invade IEC as well as induced cytokine secretion from polarized IEC. However, E. coli O83:H1 displayed the best characteristics of AIEC strains as compared to the prototype AIEC strain LF82, inducing cytokine secretion from IEC and promoting immune cell migration through IEC. Upon further analysis, E. coli O83:H1 did not harbor virulence genes present in known pathogenic intestinal organisms. Further characterization of E. coli O83:H1 virulence determinants showed that a non-flagellated O83:H1 strain significantly decreased the organism's ability to adhere to and invade both IEC and elicit IEC cytokine secretion compared to the wild type and complemented strains. These findings demonstrate that E. coli O83:H1 possesses the characteristics of the AIEC LF82 strain that may contribute to the low-grade, chronic inflammation observed in Crohn's disease. PMID:17900983

  18. Stationary nanoliter droplet array with a substrate of choice for single adherent/nonadherent cell incubation and analysis

    PubMed Central

    Shemesh, Jonathan; Ben Arye, Tom; Avesar, Jonathan; Kang, Joo H.; Fine, Amir; Super, Michael; Meller, Amit; Ingber, Donald E.; Levenberg, Shulamit

    2014-01-01

    Microfluidic water-in-oil droplets that serve as separate, chemically isolated compartments can be applied for single-cell analysis; however, to investigate encapsulated cells effectively over prolonged time periods, an array of droplets must remain stationary on a versatile substrate for optimal cell compatibility. We present here a platform of unique geometry and substrate versatility that generates a stationary nanodroplet array by using wells branching off a main microfluidic channel. These droplets are confined by multiple sides of a nanowell and are in direct contact with a biocompatible substrate of choice. The device is operated by a unique and reversed loading procedure that eliminates the need for fine pressure control or external tubing. Fluorocarbon oil isolates the droplets and provides soluble oxygen for the cells. By using this approach, the metabolic activity of single adherent cells was monitored continuously over time, and the concentration of viable pathogens in blood-derived samples was determined directly by measuring the number of colony-formed droplets. The method is simple to operate, requires a few microliters of reagent volume, is portable, is reusable, and allows for cell retrieval. This technology may be particularly useful for multiplexed assays for which prolonged and simultaneous visual inspection of many isolated single adherent or nonadherent cells is required. PMID:25053808

  19. Genome Wide assessment of Early Osseointegration in Implant-Adherent Cells

    NASA Astrophysics Data System (ADS)

    Thalji, Ghadeer N.

    Objectives: To determine the molecular processes involved in osseointegration. Materials and methods: A structured literature review concerning in vitro and in vivo molecular assessment of osseointegration was performed. A rat and a human model were then used to identify the early molecular processes involved in osseointegration associated with a micro roughened and nanosurface superimposed featured implants. In the rat model, 32 titanium implants with surface topographies exhibiting a micro roughened (AT-II) and nanosurface superimposed featured implants (AT-I) were placed in the tibiae of 8 rats and subsequently harvested at 2 and 4 days after placement. Whereas in the human model, four titanium mini-implants with either a moderately roughened surface (TiOblast) or super-imposed nanoscale topography (Osseospeed) were placed in edentulous sites of eleven systemically healthy subjects and subsequently removed after 3 and 7 days. Total RNA was isolated from cells adherent to retrieved implants. A whole genome microarray using the Affymetrix 1.1 ST Array platform was used to describe the gene expression profiles that were differentially regulated by the implant surfaces. Results: The literature review provided evidence that particular topographic cues can be specifically integrated among the many extracellular signals received by the cell in its signal transduction network. In the rat model, functionally relevant categories related to ossification, skeletal system development, osteoblast differentiation, bone development and biomineral tissue development were upregulated and more prominent at AT-I compared to AT-II. In the human model, there were no significant differences when comparing the two-implant surfaces at each time point. However, the microarray identified several genes that were differentially regulated at day 7 vs. day 3 for both implant surfaces. Functionally relevant categories related to the extracellular matrix, collagen fibril organization and

  20. Method of making a membrane having hydrophilic and hydrophobic surfaces for adhering cells or antibodies by using atomic oxygen or hydroxyl radicals

    NASA Technical Reports Server (NTRS)

    Koontz, Steven L. (Inventor); Spaulding, Glenn F. (Inventor)

    1994-01-01

    A portion of an organic polymer article such as a membrane is made hydrophilic by exposing a hydrophobic surface of the article to a depth of about 50 to about 5000 angstroms to atomic oxygen or hydroxyl radicals at a temperature below 100C., preferably below 40 C, to form a hydrophilic uniform surface layer of hydrophilic hydroxyl groups. The atomic oxygen and hydroxyl radicals are generated by a flowing afterglow microwave discharge, and the surface is outside of a plasma produced by the discharge. A membrane having both hydrophilic and hydrophobic surfaces can be used in an immunoassay by adhering antibodies to the hydrophobic surface. In another embodiment, the membrane is used in cell culturing where cells adhere to the hydrophilic surface. Prior to adhering cells, the hydrophilic surface may be grafted with a compatibilizing compound. A plurality of hydrophilic regions bounded by adjacent hydrophobic regions can be produced such that a maximum of one cell per each hydrophilic region adheres.

  1. Comparisons of mouse mesenchymal stem cells in primary adherent culture of compact bone fragments and whole bone marrow.

    PubMed

    Cai, Yiting; Liu, Tianshu; Fang, Fang; Xiong, Chengliang; Shen, Shiliang

    2015-01-01

    The purification of mouse bone marrow mesenchymal stem cells (BMSCs) by using the standard method of whole bone marrow adherence to plastic still remains ineffective. An increasing number of studies have indicated compact bone as an alternative source of BMSCs. We isolated BMSCs from cultured compact bone fragments and investigated the proliferative capacity, surface immunophenotypes, and osteogenic and adipogenic differentiations of the cells after the first trypsinization. The fragment culture was based on the fact that BMSCs were assembled in compact bones. Thus, the procedure included flushing bone marrow out of bone cavity and culturing the fragments without any collagenase digestion. The cell yield from cultured fragments was slightly less than that from cultured bone marrow using the same bone quantity. However, the trypsinized cells from cultured fragments exhibited significantly higher proliferation and were accompanied with more CD90 and CD44 expressions and less CD45 expression. The osteogenic and adipogenic differentiation capacity of cells from cultured fragments were better than those of cells from bone marrow. The directly adherent culture of compact bone is suitable for mouse BMSC isolation, and more BMSCs with potentially improved proliferation capacity can be obtained in the primary culture.

  2. Automated method for the rapid and precise estimation of adherent cell culture characteristics from phase contrast microscopy images.

    PubMed

    Jaccard, Nicolas; Griffin, Lewis D; Keser, Ana; Macown, Rhys J; Super, Alexandre; Veraitch, Farlan S; Szita, Nicolas

    2014-03-01

    The quantitative determination of key adherent cell culture characteristics such as confluency, morphology, and cell density is necessary for the evaluation of experimental outcomes and to provide a suitable basis for the establishment of robust cell culture protocols. Automated processing of images acquired using phase contrast microscopy (PCM), an imaging modality widely used for the visual inspection of adherent cell cultures, could enable the non-invasive determination of these characteristics. We present an image-processing approach that accurately detects cellular objects in PCM images through a combination of local contrast thresholding and post hoc correction of halo artifacts. The method was thoroughly validated using a variety of cell lines, microscope models and imaging conditions, demonstrating consistently high segmentation performance in all cases and very short processing times (<1 s per 1,208 × 960 pixels image). Based on the high segmentation performance, it was possible to precisely determine culture confluency, cell density, and the morphology of cellular objects, demonstrating the wide applicability of our algorithm for typical microscopy image processing pipelines. Furthermore, PCM image segmentation was used to facilitate the interpretation and analysis of fluorescence microscopy data, enabling the determination of temporal and spatial expression patterns of a fluorescent reporter. We created a software toolbox (PHANTAST) that bundles all the algorithms and provides an easy to use graphical user interface. Source-code for MATLAB and ImageJ is freely available under a permissive open-source license. PMID:24037521

  3. Automated method for the rapid and precise estimation of adherent cell culture characteristics from phase contrast microscopy images.

    PubMed

    Jaccard, Nicolas; Griffin, Lewis D; Keser, Ana; Macown, Rhys J; Super, Alexandre; Veraitch, Farlan S; Szita, Nicolas

    2014-03-01

    The quantitative determination of key adherent cell culture characteristics such as confluency, morphology, and cell density is necessary for the evaluation of experimental outcomes and to provide a suitable basis for the establishment of robust cell culture protocols. Automated processing of images acquired using phase contrast microscopy (PCM), an imaging modality widely used for the visual inspection of adherent cell cultures, could enable the non-invasive determination of these characteristics. We present an image-processing approach that accurately detects cellular objects in PCM images through a combination of local contrast thresholding and post hoc correction of halo artifacts. The method was thoroughly validated using a variety of cell lines, microscope models and imaging conditions, demonstrating consistently high segmentation performance in all cases and very short processing times (<1 s per 1,208 × 960 pixels image). Based on the high segmentation performance, it was possible to precisely determine culture confluency, cell density, and the morphology of cellular objects, demonstrating the wide applicability of our algorithm for typical microscopy image processing pipelines. Furthermore, PCM image segmentation was used to facilitate the interpretation and analysis of fluorescence microscopy data, enabling the determination of temporal and spatial expression patterns of a fluorescent reporter. We created a software toolbox (PHANTAST) that bundles all the algorithms and provides an easy to use graphical user interface. Source-code for MATLAB and ImageJ is freely available under a permissive open-source license.

  4. Assessment of mechanical properties of adherent living cells by bead micromanipulation: comparison of magnetic twisting cytometry vs optical tweezers.

    PubMed

    Laurent, Valérie M; Hénon, Sylvie; Planus, Emmanuelle; Fodil, Redouane; Balland, Martial; Isabey, Daniel; Gallet, François

    2002-08-01

    We compare the measurements of viscoelastic properties of adherent alveolar epithelial cells by two micromanipulation techniques: (i) magnetic twisting cytometry and (ii) optical tweezers, using microbeads of same size and similarly attached to F-actin. The values of equivalent Young modulus E, derived from linear viscoelasticity theory, become consistent when the degree of bead immersion in the cell is taken into account. E-values are smaller in (i) than in (ii): approximately 34-58 Pa vs approximately 29-258 Pa, probably because higher stress in (i) reinforces nonlinearity and cellular plasticity. Otherwise, similar relaxation time constants, around 2 s, suggest similar dissipative mechanisms.

  5. Varenicline for smoking cessation: a narrative review of efficacy, adverse effects, use in at-risk populations, and adherence

    PubMed Central

    Burke, Michael V; Hays, J Taylor; Ebbert, Jon O

    2016-01-01

    Treating tobacco dependence is the most effective way to reduce tobacco-related death and disability. Counseling and pharmacotherapy have been shown to increase tobacco abstinence rates among smokers. Varenicline is the most effective monotherapy treatment for tobacco dependence; however, it is prescribed less often than indicated, and adherence is less than optimal. We conducted a literature review of the development, efficacy, safety, contraindications, and adverse effects of varenicline; including reviewing data regarding combination therapy, extended duration, and patient adherence. Varenicline was developed to work specifically on the factors that underlie nicotine addiction. Phase II and Phase III trials established dosing, safety profiles, and efficacy. Postmarketing research raised concerns about neuropsychiatric and cardiac effects, resulting in warning labels being added and modified to encourage discussions with patients weighing the risks and benefits. While more research is needed, evidence is strong that varenicline is safe and effective in treating tobacco dependence among people who are at higher risk for neuropsychiatric symptoms and cardiovascular disease. The effectiveness of varenicline can be improved by taking it in combination with other medications, enhancing patient adherence and extending the duration of treatment. PMID:27099479

  6. Adherence to oral anticoagulants in patients with atrial fibrillation—a population-based retrospective cohort study linking health information systems in the Valencia region, Spain: a study protocol

    PubMed Central

    Sanfélix-Gimeno, G; Rodríguez-Bernal, C L; Hurtado, I; Baixáuli-Pérez, C; Librero, J; Peiró, S

    2015-01-01

    Introduction Adherence to oral anticoagulation (OAC) treatment, vitamin K antagonists or new oral anticoagulants, is an essential element for effectiveness. Information on adherence to OAC in atrial fibrillation (AF) and the impact of adherence on clinical outcomes using real-world data barely exists. We aim to describe the patterns of adherence to OAC over time in patients with AF, estimate the associated factors and their impact on clinical events, and assess the same issues with conventional measures of primary and secondary adherence—proportion of days covered (PDC) and persistence—in routine clinical practice. Methods and analysis This is a population-based retrospective cohort study including all patients with AF treated with OAC from 2010 to date in Valencia, Spain; data will be obtained from diverse electronic records of the Valencia Health Agency. Primary outcome measure: adherence trajectories. Secondary outcomes: (1) primary non-adherence; (2) secondary adherence: (a) PDC, (b) persistence. Clinical outcomes: hospitalisation for haemorrhagic or thromboembolic events and death during follow-up. Analysis: (1) description of baseline characteristics, adherence patterns (trajectory models or latent class growth analysis models) and conventional adherence measures; (2) logistic or Cox multivariate regression models, to assess the associations between adherence measures and the covariates, and logistic multinomial regression models, to identify characteristics associated with each trajectory; (3) Cox proportional hazard models, to assess the relationship between adherence and clinical outcomes, with propensity score adjustment applied to further control for potential confounders; (4) to estimate the importance of different healthcare levels in the variations of adherence, logistic or Cox multilevel regression models. Ethics and dissemination This study has been approved by the corresponding Clinical Research Ethics Committee. We plan to disseminate the

  7. FOXA1 expression affects the proliferation activity of luminal breast cancer stem cell populations.

    PubMed

    Tachi, Kana; Shiraishi, Akira; Bando, Hiroko; Yamashita, Toshiharu; Tsuboi, Ikki; Kato, Toshiki; Hara, Hisato; Ohneda, Osamu

    2016-03-01

    The expression of estrogen receptor is the key in most breast cancers (BC) and binding of estrogen receptor to the genome correlates to Forkhead protein (FOXA1) expression. We herein assessed the correlation between the cancer stem cell (CSC) population and FOXA1 expression in luminal BC. We established luminal BC cells derived from metastatic pleural effusion and analyzed the potency of CSC and related factors with established luminal BC cell lines. We also confirmed that mammosphere cultures have an increased aldehyde dehydrogenase-positive population, which is one of the CSC markers, compared with adherent culture cells. Using a quantitative PCR analysis, we found that mammosphere forming cells showed a higher expression of FOXA1 and stemness-related genes compared with adherent culture cells. Furthermore, the growth activity and colony-forming activity of 4-hydroxytamoxifen-treated BC cells were inhibited in a mammosphere assay. Interestingly, 4-hydroxytamoxifen-resistant cells had significantly increased FOXA1 gene expression levels. Finally, we established short hairpin RNA of FOXA1 (shFOXA1) MCF-7 cells and investigated the relationship between self-renewal potential and FOXA1 expression. As a result, we found no significant difference in the number of mammospheres but decreased colony formation in shFOXA1 MCF-7 cells compared with control. These results suggest that the expression of FOXA1 appears to be involved in the proliferation of immature BC cells rather than the induction of stemness-related genes and self-renewal potency of CSCs.

  8. Catechin-based procyanidins from Peumus boldus Mol. aqueous extract inhibit Helicobacter pylori urease and adherence to adenocarcinoma gastric cells.

    PubMed

    Pastene, Edgar; Parada, Víctor; Avello, Marcia; Ruiz, Antonieta; García, Apolinaria

    2014-11-01

    In this work, the anti-Helicobacter pylori effect of an aqueous extract from dried leaves of Peumus boldus Mol. (Monimiaceae) was evaluated. This extract displayed high inhibitory activity against H. pylori urease. Therefore, in order to clarify the type of substances responsible for such effect, a bioassay-guided fractionation strategy was carried out. The active compounds in the fractions were characterized through different chromatographic methods (RP-HPLC; HILIC-HPLC). The fraction named F5 (mDP = 7.8) from aqueous extract was the most active against H. pylori urease with an IC50  = 15.9 µg gallic acid equivalents (GAE)/mL. HPLC analysis evidenced that F5 was composed mainly by catechin-derived proanthocyanidins (LC-MS and phloroglucinolysis). The anti-adherent effect of boldo was assessed by co-culture of H. pylori and AGS cells. Both the aqueous extract and F5 showed an anti-adherent effect in a concentration-dependent manner. An 89.3% of inhibition was reached at 2.0 mg GAE/mL of boldo extract. In conjunction, our results suggest that boldo extract has a potent anti-urease activity and anti-adherent effect against H. pylori, properties directly linked with the presence of catechin-derived proanthocyanidins. PMID:24853276

  9. Catechin-based procyanidins from Peumus boldus Mol. aqueous extract inhibit Helicobacter pylori urease and adherence to adenocarcinoma gastric cells.

    PubMed

    Pastene, Edgar; Parada, Víctor; Avello, Marcia; Ruiz, Antonieta; García, Apolinaria

    2014-11-01

    In this work, the anti-Helicobacter pylori effect of an aqueous extract from dried leaves of Peumus boldus Mol. (Monimiaceae) was evaluated. This extract displayed high inhibitory activity against H. pylori urease. Therefore, in order to clarify the type of substances responsible for such effect, a bioassay-guided fractionation strategy was carried out. The active compounds in the fractions were characterized through different chromatographic methods (RP-HPLC; HILIC-HPLC). The fraction named F5 (mDP = 7.8) from aqueous extract was the most active against H. pylori urease with an IC50  = 15.9 µg gallic acid equivalents (GAE)/mL. HPLC analysis evidenced that F5 was composed mainly by catechin-derived proanthocyanidins (LC-MS and phloroglucinolysis). The anti-adherent effect of boldo was assessed by co-culture of H. pylori and AGS cells. Both the aqueous extract and F5 showed an anti-adherent effect in a concentration-dependent manner. An 89.3% of inhibition was reached at 2.0 mg GAE/mL of boldo extract. In conjunction, our results suggest that boldo extract has a potent anti-urease activity and anti-adherent effect against H. pylori, properties directly linked with the presence of catechin-derived proanthocyanidins.

  10. Desmosomal molecules in and out of adhering junctions: normal and diseased States of epidermal, cardiac and mesenchymally derived cells.

    PubMed

    Pieperhoff, Sebastian; Barth, Mareike; Rickelt, Steffen; Franke, Werner W

    2010-01-01

    Current cell biology textbooks mention only two kinds of cell-to-cell adhering junctions coated with the cytoplasmic plaques: the desmosomes (maculae adhaerentes), anchoring intermediate-sized filaments (IFs), and the actin microfilament-anchoring adherens junctions (AJs), including both punctate (puncta adhaerentia) and elongate (fasciae adhaerentes) structures. In addition, however, a series of other junction types has been identified and characterized which contain desmosomal molecules but do not fit the definition of desmosomes. Of these special cell-cell junctions containing desmosomal glycoproteins or proteins we review the composite junctions (areae compositae) connecting the cardiomyocytes of mature mammalian hearts and their importance in relation to human arrhythmogenic cardiomyopathies. We also emphasize the various plakophilin-2-positive plaques in AJs (coniunctiones adhaerentes) connecting proliferatively active mesenchymally-derived cells, including interstitial cells of the heart and several soft tissue tumor cell types. Moreover, desmoplakin has also been recognized as a constituent of the plaques of the complexus adhaerentes connecting certain lymphatic endothelial cells. Finally, we emphasize the occurrence of the desmosomal transmembrane glycoprotein, desmoglein Dsg2, out of the context of any junction as dispersed cell surface molecules in certain types of melanoma cells and melanocytes. This broadening of our knowledge on the diversity of AJ structures indicates that it may still be too premature to close the textbook chapters on cell-cell junctions. PMID:20671973

  11. Impact of release dynamics of laser-irradiated polymer micropallets on the viability of selected adherent cells

    PubMed Central

    Ma, Huan; Mismar, Wael; Wang, Yuli; Small, Donald W.; Ras, Mat; Allbritton, Nancy L.; Sims, Christopher E.; Venugopalan, Vasan

    2012-01-01

    We use time-resolved interferometry, fluorescence assays and computational fluid dynamics (CFD) simulations to examine the viability of confluent adherent cell monolayers to selection via laser microbeam release of photoresist polymer micropallets. We demonstrate the importance of laser microbeam pulse energy and focal volume position relative to the glass–pallet interface in governing the threshold energies for pallet release as well as the pallet release dynamics. Measurements using time-resolved interferometry show that increases in laser pulse energy result in increasing pallet release velocities that can approach 10 m s−1 through aqueous media. CFD simulations reveal that the pallet motion results in cellular exposure to transient hydrodynamic shear stress amplitudes that can exceed 100 kPa on microsecond timescales, and which produces reduced cell viability. Moreover, CFD simulation results show that the maximum shear stress on the pallet surface varies spatially, with the largest shear stresses occurring on the pallet periphery. Cell viability of confluent cell monolayers on the pallet surface confirms that the use of larger pulse energies results in increased rates of necrosis for those cells situated away from the pallet centre, while cells situated at the pallet centre remain viable. Nevertheless, experiments that examine the viability of these cell monolayers following pallet release show that proper choices for laser microbeam pulse energy and focal volume position lead to the routine achievement of cell viability in excess of 90 per cent. These laser microbeam parameters result in maximum pallet release velocities below 6 m s−1 and cellular exposure of transient hydrodynamic shear stresses below 20 kPa. Collectively, these results provide a mechanistic understanding that relates pallet release dynamics and associated transient shear stresses with subsequent cellular viability. This provides a quantitative, mechanistic basis for determining

  12. Decreased Adherence of Enterohemorrhagic Escherichia coli to HEp-2 Cells in the Presence of Antibodies That Recognize the C-Terminal Region of Intimin

    PubMed Central

    Gansheroff, Lisa J.; Wachtel, Marian R.; O'Brien, Alison D.

    1999-01-01

    Antiserum raised against intimin from enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain 86-24 has been shown previously by our laboratory to inhibit adherence of this strain to HEp-2 cells. In the present study, we sought to identify the region(s) of intimin important for the effect of anti-intimin antisera on EHEC adherence and to determine whether antisera raised against intimin from an O157:H7 strain could reduce adherence of other strains. Compared to preimmune serum controls, polyclonal sera raised against the histidine-tagged intimin protein RIHisEae (intiminO157) or against His-tagged C-terminal fragments of intimin from strain 86-24 reduced adherence of this strain. Furthermore, an antibody fraction purified from the anti-RIHisEae serum that contained antibodies to the C-terminal third of intimin, the putative receptor-binding domain, also reduced adherence of strain 86-24, but a purified fraction containing antibodies to the N-terminal two-thirds of intimin did not inhibit adherence. The polyclonal anti-intiminO157 serum raised against RIHisEae inhibited, to different degrees, the adherence of another O157:H7 strain, an EHEC O55:H7 strain, one of two independent EHEC O111:NM isolates tested, and one of two EHEC O26:H11 strains tested. Adherence of the other O26:H11 and O111:NM strains and an EPEC O127:H6 strain was not reduced. Finally, immunoblot analysis indicated a correlation between the antigenic divergence in the C-terminal third of intimins from different strains and the capacity of anti-intiminO157 antiserum to reduce adherence of heterologous strains. Taken together, these data suggest that intiminO157 could be used as an immunogen to elicit adherence-blocking antibodies against O157:H7 strains and closely-related EHEC. PMID:10569757

  13. Acute myelogenous leukemia cells with the MLL-ELL translocation convert morphologically and functionally into adherent myofibroblasts

    SciTech Connect

    Tashiro, Haruko; Mizutani-Noguchi, Mitsuho; Shirasaki, Ryosuke

    2010-01-01

    Bone marrow-myofibroblasts, a major component of bone marrow-stroma, are reported to originate from hematopoietic stem cells. We show in this paper that non-adherent leukemia blasts can change into myofibroblasts. When myeloblasts from two cases of acute myelogenous leukemia with a fusion product comprising mixed lineage leukemia and RNA polymerase II elongation factor, were cultured long term, their morphology changed to that of myofibroblasts with similar molecular characteristics to the parental myeloblasts. The original leukemia blasts, when cultured on the leukemia blast-derived myofibroblasts, grew extensively. Leukemia blasts can create their own microenvironment for proliferation.

  14. Asthma prevalence, cost, and adherence with expert guidelines on the utilization of health care services and costs in a state Medicaid population.

    PubMed Central

    Piecoro, L T; Potoski, M; Talbert, J C; Doherty, D E

    2001-01-01

    OBJECTIVE: To provide a descriptive analysis of asthma prevalence and costs in a Medicaid population and gauge the degree of adherence with expert guidelines for asthma medication management from the National Asthma Education and Prevention Program. DATA SOURCES: Kentucky Medicaid administrative data for 1996. STUDY DESIGN: A cross-sectional retrospective analysis was used to determine adherence with asthma medication guidelines and utilization of asthma-related health care services and costs. Multivariate logistic regression was used to determine the relationship between nonadherence with the guidelines and utilization of health care services. PRINCIPAL FINDINGS: Of the 530,000 Medicaid recipients, 24,365 (4.6 percent) were identified as having asthma. Average annual asthma-related costs ($616) accounted for less than 20 percent of total health care costs ($3,645). Nonadherence to the guidelines was prevalent. Less than 40 percent of the patients received a prescription for a rescue medication, and fewer than 10 percent of the patients who received daily inhaled short-acting beta-2 agonists were regular users of inhaled steroids. Nonadherence to the guidelines was associated with an increased risk of an asthma-related hospitalization (odds ratio = 1.5, p < .05). CONCLUSIONS: Guideline nonadherence was widespread and associated with an increase in exacerbations of asthma that resulted in hospitalizations. Asthma prevalence and utilization of health care services in a Medicaid population were similar to previous estimates reported nationally and in health maintenance organizations. PMID:11409817

  15. Tracking in real time the crawling dynamics of adherent living cells with a high resolution surface plasmon microscope

    NASA Astrophysics Data System (ADS)

    Streppa, L.; Berguiga, L.; Boyer Provera, E.; Ratti, F.; Goillot, E.; Martinez Torres, C.; Schaeffer, L.; Elezgaray, Juan; Arneodo, A.; Argoul, F.

    2016-03-01

    We introduce a high resolution scanning surface plasmon microscope for long term imaging of living adherent mouse myoblast cells. The coupling of a high numerical aperture objective lens with a fibered heterodyne interferometer provides both enhanced sensitivity and long term stability. This microscope takes advantage of the plasmon resonance excitation and the amplification of the electromagnetic field in near-field distance to the gold coated coverslip. This plasmon enhanced evanescent wave microscopy is particularly attractive for the study of cell adhesion and motility since it can be operated without staining of the biological sample. We show that this microscope allows very long-term imaging of living samples, and that it can capture and follow the temporal deformation of C2C12 myoblast cell protusions (lamellipodia), during their migration on a at surface.

  16. Phenotype heterogeneity in cancer cell populations

    NASA Astrophysics Data System (ADS)

    Almeida, Luis; Chisholm, Rebecca; Clairambault, Jean; Escargueil, Alexandre; Lorenzi, Tommaso; Lorz, Alexander; Trélat, Emmanuel

    2016-06-01

    Phenotype heterogeneity in cancer cell populations, be it of genetic, epigenetic or stochastic origin, has been identified as a main source of resistance to drug treatments and a major source of therapeutic failures in cancers. The molecular mechanisms of drug resistance are partly understood at the single cell level (e.g., overexpression of ABC transporters or of detoxication enzymes), but poorly predictable in tumours, where they are hypothesised to rely on heterogeneity at the cell population scale, which is thus the right level to describe cancer growth and optimise its control by therapeutic strategies in the clinic. We review a few results from the biological literature on the subject, and from mathematical models that have been published to predict and control evolution towards drug resistance in cancer cell populations. We propose, based on the latter, optimisation strategies of combined treatments to limit emergence of drug resistance to cytotoxic drugs in cancer cell populations, in the monoclonal situation, which limited as it is still retains consistent features of cell population heterogeneity. The polyclonal situation, that may be understood as "bet hedging" of the tumour, thus protecting itself from different sources of drug insults, may lie beyond such strategies and will need further developments. In the monoclonal situation, we have designed an optimised therapeutic strategy relying on a scheduled combination of cytotoxic and cytostatic treatments that can be adapted to different situations of cancer treatments. Finally, we review arguments for biological theoretical frameworks proposed at different time and development scales, the so-called atavistic model (diachronic view relying on Darwinian genotype selection in the coursof billions of years) and the Waddington-like epigenetic landscape endowed with evolutionary quasi-potential (synchronic view relying on Lamarckian phenotype instruction of a given genome by reversible mechanisms), to

  17. Automated Method for the Rapid and Precise Estimation of Adherent Cell Culture Characteristics from Phase Contrast Microscopy Images

    PubMed Central

    Jaccard, Nicolas; Griffin, Lewis D; Keser, Ana; Macown, Rhys J; Super, Alexandre; Veraitch, Farlan S; Szita, Nicolas

    2014-01-01

    The quantitative determination of key adherent cell culture characteristics such as confluency, morphology, and cell density is necessary for the evaluation of experimental outcomes and to provide a suitable basis for the establishment of robust cell culture protocols. Automated processing of images acquired using phase contrast microscopy (PCM), an imaging modality widely used for the visual inspection of adherent cell cultures, could enable the non-invasive determination of these characteristics. We present an image-processing approach that accurately detects cellular objects in PCM images through a combination of local contrast thresholding and post hoc correction of halo artifacts. The method was thoroughly validated using a variety of cell lines, microscope models and imaging conditions, demonstrating consistently high segmentation performance in all cases and very short processing times (<1 s per 1,208 × 960 pixels image). Based on the high segmentation performance, it was possible to precisely determine culture confluency, cell density, and the morphology of cellular objects, demonstrating the wide applicability of our algorithm for typical microscopy image processing pipelines. Furthermore, PCM image segmentation was used to facilitate the interpretation and analysis of fluorescence microscopy data, enabling the determination of temporal and spatial expression patterns of a fluorescent reporter. We created a software toolbox (PHANTAST) that bundles all the algorithms and provides an easy to use graphical user interface. Source-code for MATLAB and ImageJ is freely available under a permissive open-source license. Biotechnol. Bioeng. 2014;111: 504–517. © 2013 Wiley Periodicals, Inc. PMID:24037521

  18. Risk factors for AIDS-defining illnesses among a population of poorly adherent people living with HIV/AIDS in Atlanta, Georgia.

    PubMed

    Chow, Jeremy Y; Alsan, Marcella; Armstrong, Wendy; del Rio, Carlos; Marconi, Vincent C

    2015-01-01

    In order to achieve the programmatic goals established in the National HIV/AIDS Strategy, virologic suppression remains the most important outcome within the HIV care continuum for individuals receiving antiretroviral therapy (ART). Therefore, clinicians have dedicated substantial resources to improve adherence and clinic retention for individuals on ART; however, these efforts should be focused first on those most at risk of morbidity and mortality related to AIDS. Our study aimed to characterize the factors that are associated with AIDS-defining illnesses (ADIs) amongst people living with HIV (PLHIV) who are poorly adherent or retained in care in order to identify those at highest risk of poor clinical outcomes. We recruited 99 adult PLHIV with a history of poor adherence to ART, poor clinic attendance, or unsuppressed viral load (VL) from the Infectious Disease Program (IDP) of the Grady Health System in Atlanta, Georgia between January and May 2011 to participate in a survey investigating the acceptability of a financial incentive for improving adherence. Clinical outcomes including the number of ADI episodes in the last five years, VLs, and CD4 counts were abstracted from medical records. Associations between survey items and number of ADIs were performed using chi-square analysis. In our study, 36.4% of participants had ≥1 ADI in the last five years. The most common ADIs were Pneumocystis jirovecii pneumonia, recurrent bacterial pneumonia, and esophageal candidiasis. Age <42.5 years (OR 2.52, 95% CI = 1.08-5.86), male gender (OR 3.51, 95% CI = 1.08-11.34), CD4 nadir <200 cells/µL (OR 11.92, 95% CI = 1.51-94.15), unemployment (OR 3.54, 95% CI = 1.20-10.40), and travel time to clinic <30 minutes (OR 2.80, 95% CI = 1.20-6.52) were all significantly associated with a history of ≥1 ADI in the last five years. Awareness of factors associated with ADIs may help clinicians identify which poorly adherent PLHIV are at highest risk of HIV-related morbidity.

  19. Cardiac side population cells and Sca-1-positive cells.

    PubMed

    Nagai, Toshio; Matsuura, Katsuhisa; Komuro, Issei

    2013-01-01

    Since the resident cardiac stem/progenitor cells were discovered, their ability to maintain the architecture and functional integrity of adult heart has been broadly explored. The methods for isolation and purification of the cardiac stem cells are crucial for the precise analysis of their developmental origin and intrinsic potential as tissue stem cells. Stem cell antigen-1 (Sca-1) is one of the useful cell surface markers to purify the cardiac progenitor cells. Another purification strategy is based on the high efflux ability of the dye, which is a common feature of tissue stem cells. These dye-extruding cells have been called side population cells because they locate in the side of dye-retaining cells after fluorescent cell sorting. In this chapter, we describe the methodology for the isolation of cardiac SP cells and Sca-1 positive cells.

  20. Receptor-like glycocompounds in human milk that inhibit classical and El Tor Vibrio cholerae cell adherence (hemagglutination).

    PubMed Central

    Holmgren, J; Svennerholm, A M; Lindblad, M

    1983-01-01

    The two biotypes of Vibrio cholerae were found to have cell-associated hemagglutinins which differ with regard to binding to different species of erythrocytes and inhibition by monosaccharides. A total of 12 classical V. cholerae strains (Inaba or Ogawa) strongly agglutinated human erythrocytes in a reaction specifically inhibited by L-fucose, whereas 12 El Tor strains preferably agglutinated chicken erythrocytes, a reaction reversed by D-mannose or by higher concentrations of D-fructose, D-glucose, alpha-methyl-D-mannoside, or sucrose. Milk from Swedish women inhibited both of these adherence reactions, and the predominating inhibitory activity for each reaction resisted boiling, was destroyed by periodate treatment, and bound a concanavalin A-Sepharose column, suggesting a carbohydrate structure. Further characterization indicated that the inhibitory activity for classical V. cholerae hemagglutination was distributed about equally on glycoprotein and free oligosaccharide, but was not present on glycolipid. The El Tor inhibiting activity, on the other hand, was almost exclusively of a high-molecular-weight glycoprotein nature. These results support our previous suggestion (Holmgren et al., Infect. Immun. 33:136-141, 1981) that human milk may contain receptor-like glycocompounds which can prevent bacterial adherence by competition with receptors on target cells. PMID:6295953

  1. Dual Pili Post-translational Modifications Synergize to Mediate Meningococcal Adherence to Platelet Activating Factor Receptor on Human Airway Cells

    PubMed Central

    Schulz, Benjamin L.; Power, Peter M.; Swords, W. Edward; Weiser, Jeffery N.; Apicella, Michael A.; Edwards, Jennifer L.; Jennings, Michael P.

    2013-01-01

    Pili of pathogenic Neisseria are major virulence factors associated with adhesion, twitching motility, auto-aggregation, and DNA transformation. Pili of N. meningitidis are subject to several different post-translational modifications. Among these pilin modifications, the presence of phosphorylcholine (ChoP) and a glycan on the pilin protein are phase-variable (subject to high frequency, reversible on/off switching of expression). In this study we report the location of two ChoP modifications on the C-terminus of N. meningitidis pilin. We show that the surface accessibility of ChoP on pili is affected by phase variable changes to the structure of the pilin-linked glycan. We identify for the first time that the platelet activating factor receptor (PAFr) is a key, early event receptor for meningococcal adherence to human bronchial epithelial cells and tissue, and that synergy between the pilin-linked glycan and ChoP post-translational modifications is required for pili to optimally engage PAFr to mediate adherence to human airway cells. PMID:23696740

  2. Spatially and temporally controlled gene transfer by electroporation into adherent cells on plasmid DNA-loaded electrodes.

    PubMed

    Yamauchi, Fumio; Kato, Koichi; Iwata, Hiroo

    2004-01-01

    Functional characterization of human genes is one of the most challenging tasks in current genomics. Owing to a large number of newly discovered genes, high-throughput methodologies are greatly needed to express in parallel each gene in living cells. To develop a method that allows efficient transfection of plasmids into adherent cells in spatial- and temporal-specific manners, we studied electric pulse-triggered gene transfer using a plasmid-loaded electrode. A plasmid was loaded on a gold electrode surface having an adsorbed layer of poly(ethyleneimine), and cells were then plated directly onto this modified surface. The plasmid was detached from the electrode by applying a short electric pulse and introduced into the cells cultured on the electrode, resulting in efficient gene expression, even in primary cultured cells. The location of transfected cells could be restricted within a small area on a micropatterned electrode, showing the versatility of the method for spatially controlled transfection. Plasmid transfection could also be performed in a temporally controlled manner without a marked loss of the efficiency when an electric pulse was applied within 3 days after cell plating. The method described here will provide an efficient means to transfer multiple genes, in parallel, into cultured mammalian cells for high-throughput reverse genetics research. PMID:15613595

  3. Contributions of O Island 48 to Adherence of Enterohemorrhagic Escherichia coli O157:H7 to Epithelial Cells In Vitro and in Ligated Pig Ileal Loops▿

    PubMed Central

    Yin, Xianhua; Wheatcroft, Roger; Chambers, James R.; Liu, Bianfang; Zhu, Jing; Gyles, Carlton L.

    2009-01-01

    O island 48 (OI-48) of Escherichia coli consists of three functional gene clusters that encode urease, tellurite resistance (Ter), and putative adhesins Iha and AIDA-1. The functions of these clusters in enterohemorrhagic E. coli (EHEC) O157:H7 infection are unknown. Deletion mutants for these three regions were constructed and evaluated for their ability to adhere to epithelial cells in vitro and in ligated pig ileal loops. Deletion of the Ter gene cluster reduced the ability of the organism to adhere to and form large clusters on IPEC-J2 and HEp-2 cells. Complementation of the mutation by introducing the wild-type ter genes restored adherence and large-cluster formation. Tests in ligated pig ileal loops showed a decrease in colonization by the Ter-negative mutant, but the difference was not significant compared to colonization by the wild type (26.4% ± 21.2% versus 40.1% ± 19.1%; P = 0.168). The OI-48 aidA gene deletion had no effect on adherence in vitro or in vivo. Deletion of the iha and ureC genes had no effect on adherence in vitro but significantly reduced the colonization of EHEC O157:H7 in the ligated pig intestine. These data suggest that Ter, Iha, and urease may contribute to EHEC O157:H7 pathogenesis by promoting adherence of the pathogen to the host intestinal epithelium. PMID:19633120

  4. Heat-labile enterotoxin-induced activation of NF-κB and MAPK pathways in intestinal epithelial cells impacts enterotoxigenic Escherichia coli (ETEC) adherence.

    PubMed

    Wang, Xiaogang; Gao, Xiaofei; Hardwidge, Philip R

    2012-08-01

    Enterotoxigenic Escherichia coli (ETEC) causes human morbidity and mortality in developing nations and is an emerging threat to food safety in developed nations. The ETEC heat-labile enterotoxin (LT) not only causes diarrheal disease by deregulating host adenylate cyclase, but also enhances ETEC adherence to intestinal epithelial cells. The mechanism governing this LT pro-adherence phenotype is unclear. Here we investigated intestinal epithelial cell signal transduction pathways activated by ETEC and quantified the relative importance of these host pathways to LT-induced ETEC adherence. We show that ETEC activates both NF-κB and mitogen-activated protein kinase signalling pathways through mechanisms that are primarily dependent upon LT. LT-induced NF-κB activation depends upon the cAMP-dependent activation of the Ras-like GTPase Rap1 but is independent of protein kinase A (PKA). By using inhibitors of these pathways, we demonstrate that inhibiting the p38 mitogen-activated protein kinase prevents LT from increasing ETEC adherence. By contrast, the LT pro-adherence phenotype appears unrelated to both LT-induced Rap1 activity and to subsequent NF-κB activation. We speculate that LT may alter host signal transduction to induce the presentation of ligands for ETEC adhesins in such a way that promotes ETEC adherence. Our findings provide insight into previously unexplored functions of LT and their relative importance to ETEC virulence. PMID:22452361

  5. Differential Flo8p-dependent regulation of FLO1 and FLO11 for cell-cell and cell-substrate adherence of S. cerevisiae S288c.

    PubMed

    Fichtner, Lars; Schulze, Florian; Braus, Gerhard H

    2007-12-01

    Cell-cell and cell-surface adherence represents initial steps in forming multicellular aggregates or in establishing cell-surface interactions. The commonly used Saccharomyces cerevisiae laboratory strain S288c carries a flo8 mutation, and is only able to express the flocculin-encoding genes FLO1 and FLO11, when FLO8 is restored. We show here that the two flocculin genes exhibit differences in regulation to execute distinct functions under various environmental conditions. In contrast to the laboratory strain Sigma1278b, haploids of the S288c genetic background require FLO1 for cell-cell and cell-substrate adhesion, whereas FLO11 is required for pseudohyphae formation of diploids. In contrast to FLO11, FLO1 repression requires the Sin4p mediator tail component, but is independent of the repressor Sfl1p. FLO1 regulation also differs from FLO11, because it requires neither the KSS1 MAP kinase cascade nor the pathways which lead to the transcription factors Gcn4p or Msn1p. The protein kinase A pathway and the transcription factors Flo8p and Mss11p are the major regulators for FLO1 expression. Therefore, S. cerevisiae is prepared to simultaneously express two genes of its otherwise silenced FLO reservoir resulting in an appropriate cellular surface for different environments. PMID:18001350

  6. Coculture with endothelial cells reduces the population of cycling LeX neural precursors but increases that of quiescent cells with a side population phenotype

    SciTech Connect

    Mathieu, Celine . E-mail: marc-andre.mouthon@cea.fr

    2006-04-01

    Neural stem cell proliferation and differentiation are regulated by external cues from their microenvironment. As endothelial cells are closely associated with neural stem cell in brain germinal zones, we investigated whether endothelial cells may interfere with neurogenesis. Neural precursor cells (NPC) from telencephalon of EGFP mouse embryos were cocultured in direct contact with endothelial cells. Endothelial cells did not modify the overall proliferation and apoptosis of neural cells, albeit they transiently delayed spontaneous apoptosis. These effects appeared to be specific to endothelial cells since a decrease in proliferation and a raise in apoptosis were observed in cocultures with fibroblasts. Endothelial cells stimulated the differentiation of NPC into astrocytes and into neurons, whereas they reduced differentiation into oligodendrocytes in comparison to adherent cultures on polyornithine. Determination of NPC clonogenicity and quantification of LeX expression, a marker for NPC, showed that endothelial cells decreased the number of cycling NPC. On the other hand, the presence of endothelial cells increased the number of neural cells having 'side population' phenotype, another marker reported on NPC, which we have shown to contain quiescent cells. Thus, we show that endothelial cells may regulate neurogenesis by acting at different level of NPC differentiation, proliferation and quiescence.

  7. The effect of nedocromil sodium on human airway epithelial cell-induced eosinophil chemotaxis and adherence to human endothelial cell in vitro.

    PubMed

    Abdelaziz, M M; Devalia, J L; Khair, O A; Rusznak, C; Calderon, M; Sapsford, R J; Bayram, H; Davies, R J

    1997-04-01

    Although some studies have shown that long-term treatment of asthmatics with nedocromil sodium can reduce airway hyperresponsiveness and improve symptoms and lung function, the mechanisms underlying its effects are not well understood. We have investigated the effect of nedocromil sodium on eosinophil chemotaxis, eosinophil adherence to human endothelial cells and release of soluble intercellular adhesion molecule-1 (sICAM-1) from endothelial cells, induced by conditioned medium collected from cultured human bronchial epithelial cells. Conditioned medium significantly increased eosinophil chemotaxis from a baseline median value of 2.1 (range 1.9-4.5) cells-high power field(-1) (HPF) to 10.5 (range 7.8-12.3) cells-HPF(-1) (p<0.05). Similarly, conditioned medium significantly increased eosinophil adherence to endothelial cells from a baseline value of 9 (range 8-12)% to 23 (range 21-30)% (p<0.05). Nedocromil sodium, at 10(-5) M concentration, significantly attenuated the eosinophil chemotaxis and adherence induced by conditioned medium. Conditioned medium also significantly increased the release of sICAM-1 from endothelial cells, from a baseline value of 11.5 (range 8.1-15.4) pg x microg(-1) protein to 67.6 (range 55.6-73.5) pg x microg(-1) protein (p<0.05). This was significantly attenuated by anti-tumour necrosis factor-alpha (TNF-alpha), anti-interleukin-1beta (IL-1beta) and 10(-5) M nedocromil sodium. These findings suggest that human bronchial epithelial cell-derived mediators may potentiate eosinophil activity, and that this can be modulated by nedocromil sodium, suggesting a possible mechanism underlying its anti-inflammatory effect.

  8. Nanoscopic vibrations of bacteria with different cell-wall properties adhering to surfaces under flow and static conditions.

    PubMed

    Song, Lei; Sjollema, Jelmer; Sharma, Prashant K; Kaper, Hans J; van der Mei, Henny C; Busscher, Henk J

    2014-08-26

    Bacteria adhering to surfaces demonstrate random, nanoscopic vibrations around their equilibrium positions. This paper compares vibrational amplitudes of bacteria adhering to glass. Spring constants of the bond are derived from vibrational amplitudes and related to the electrophoretic softness of the cell surfaces and dissipation shifts measured upon bacterial adhesion in a quartz-crystal-microbalance (QCM-D). Experiments were conducted with six bacterial strains with pairwise differences in cell surface characteristics. Vibrational amplitudes were highest in low ionic strength suspensions. Under fluid flow, vibrational amplitudes were lower in the direction of flow than perpendicular to it because stretching of cell surface polymers in the direction of flow causes stiffening of the polyelectrolyte network surrounding a bacterium. Under static conditions (0.57 mM), vibrational amplitudes of fibrillated Streptococcus salivarius HB7 (145 nm) were higher than that of a bald mutant HB-C12 (76 nm). Amplitudes of moderately extracellular polymeric substance (EPS) producing Staphylococcus epidermidis ATCC35983 (47 nm) were more than twice the amplitudes of strongly EPS producing S. epidermidis ATCC35984 (21 nm). No differences were found between Staphylococcus aureus strains differing in membrane cross-linking. High vibrational amplitudes corresponded with low dissipation shifts in QCM-D. In streptococci, the polyelectrolyte network surrounding a bacterium is formed by fibrillar surface appendages and spring constants derived from vibrational amplitudes decreased with increasing fibrillar density. In staphylococci, EPS constitutes the main network component, and larger amounts of EPS yielded higher spring constants. Spring constants increased with increasing ionic strength and strains with smaller electrophoretically derived bacterial cell surface softnesses possessed the highest spring constants. PMID:25025495

  9. Single cell dual adherent-suspension co-culture micro-environment for studying tumor-stromal interactions with functionally selected cancer stem-like cells.

    PubMed

    Chen, Yu-Chih; Zhang, Zhixiong; Fouladdel, Shamileh; Deol, Yadwinder; Ingram, Patrick N; McDermott, Sean P; Azizi, Ebrahim; Wicha, Max S; Yoon, Euisik

    2016-08-01

    Considerable evidence suggests that cancer stem-like cells (CSCs) are critical in tumor pathogenesis, but their rarity and transience has led to much controversy about their exact nature. Although CSCs can be functionally identified using dish-based tumorsphere assays, it is difficult to handle and monitor single cells in dish-based approaches; single cell-based microfluidic approaches offer better control and reliable single cell derived sphere formation. However, like normal stem cells, CSCs are heavily regulated by their microenvironment, requiring tumor-stromal interactions for tumorigenic and proliferative behaviors. To enable single cell derived tumorsphere formation within a stromal microenvironment, we present a dual adherent/suspension co-culture device, which combines a suspension environment for single-cell tumorsphere assays and an adherent environment for co-culturing stromal cells in close proximity by selectively patterning polyHEMA in indented microwells. By minimizing dead volume and improving cell capture efficiency, the presented platform allows for the use of small numbers of cells (<100 cells). As a proof of concept, we co-cultured single T47D (breast cancer) cells and primary cancer associated fibroblasts (CAF) on-chip for 14 days to monitor sphere formation and growth. Compared to mono-culture, co-cultured T47D have higher tumorigenic potential (sphere formation rate) and proliferation rates (larger sphere size). Furthermore, 96-multiplexed single-cell transcriptome analyses were performed to compare the gene expression of co-cultured and mono-cultured T47D cells. Phenotypic changes observed in co-culture correlated with expression changes in genes associated with proliferation, apoptotic suppression, tumorigenicity and even epithelial-to-mesechymal transition. Combining the presented platform with single cell transcriptome analysis, we successfully identified functional CSCs and investigated the phenotypic and transcriptome effects induced

  10. Interval scanning photomicrography of microbial cell populations.

    NASA Technical Reports Server (NTRS)

    Casida, L. E., Jr.

    1972-01-01

    A single reproducible area of the preparation in a fixed focal plane is photographically scanned at intervals during incubation. The procedure can be used for evaluating the aerobic or anaerobic growth of many microbial cells simultaneously within a population. In addition, the microscope is not restricted to the viewing of any one microculture preparation, since the slide cultures are incubated separately from the microscope.

  11. Identification of a population of cells with hematopoietic stem cell properties in mouse aorta-gonad-mesonephros cultures

    SciTech Connect

    Nobuhisa, Ikuo; Ohtsu, Naoki; Okada, Seiji; Nakagata, Naomi; Taga, Tetsuya . E-mail: taga@kaiju.medic.kumamoto-u.ac.jp

    2007-03-10

    The aorta-gonad-mesonephros (AGM) region is a primary source of definitive hematopoietic cells in the midgestation mouse embryo. In cultures of dispersed AGM regions, adherent cells containing endothelial cells are observed first, and then non-adherent hematopoietic cells are produced. Here we report on the characterization of hematopoietic cells that emerge in the AGM culture. Based on the expression profiles of CD45 and c-Kit, we defined three cell populations: CD45{sup low} c-Kit{sup +} cells that had the ability to form hematopoietic cell colonies in methylcellulose media and in co-cultures with stromal cells; CD45{sup low} c-Kit{sup -} cells that showed a granulocyte morphology; CD45{sup high} c-Kit{sup low/-} that exhibited a macrophage morphology. In co-cultures of OP9 stromal cells and freshly prepared AGM cultures, CD45{sup low} c-Kit{sup +} cells from the AGM culture had the abilities to reproduce CD45{sup low} c-Kit{sup +} cells and differentiate into CD45{sup low} c-Kit{sup -} and CD45{sup high} c-Kit{sup low/-} cells, whereas CD45{sup low} c-Kit{sup -} and CD45{sup high} c-Kit{sup low/-} did not produce CD45{sup low} c-Kit{sup +} cells. Furthermore, CD45{sup low} c-Kit{sup +} cells displayed a long-term repopulating activity in adult hematopoietic tissue when transplanted into the liver of irradiated newborn mice. These results indicate that CD45{sup low} c-Kit{sup +} cells from the AGM culture have the potential to reconstitute multi-lineage hematopoietic cells.

  12. Dynamic expression of the Robo ligand Slit2 in bone marrow cell populations.

    PubMed

    Smith-Berdan, Stephanie; Schepers, Koen; Ly, Alan; Passegué, Emmanuelle; Forsberg, E Camilla

    2012-02-15

    The bone marrow (BM) niche is essential for lifelong hematopoietic stem cell (HSC) maintenance, proliferation and differentiation. Several BM cell types, including osteoblast lineage cells (OBC), mesenchymal stem cells (MSC) and endothelial cells (EC) have been implicated in supporting HSC location and function, but the relative importance of these cell types and their secreted ligands remain controversial. We recently found that the cell surface receptors Robo4 and CXCR4 cooperate to localize HSC to BM niches. We hypothesized that Slit2, a putative ligand for Robo4, cooperates with the CXCR4 ligand SDF1 to direct HSC to specific BM niche sites. Here, we have isolated OBC, MSC and EC by flow cytometry and determined their frequency within the bone marrow and the relative mRNA levels of Slit2, SDF1 and Robo4. We found that expression of Slit2 and SDF1 were dynamically regulated in MSC and OBC-like populations following radiation, while Robo4 expression was restricted to EC. Radiation also significantly affected the cellularity and frequency of both the non-adherent and adherent cells within the BM stroma. These data support a physiological role for Slit2 in regulating the dynamic function of Robo-expressing cells within BM niches at steady state and following radiation.

  13. Characterization and Comparison of Intercellular Adherent Junctions Expressed by Human Corneal Endothelial Cells in Vivo and in Vitro

    PubMed Central

    Ying-Ting, Zhu; Hayashida, Yasutaka; Kheirkhah, Ahmad; He, Hua; Sue-Yue, Chen; Tseng, Scheffer C. G.

    2008-01-01

    Purpose Human corneal endothelial cell (HCEC) proliferation is controlled by their cell junctions, of which the mechanism remains unknown. We sought to characterize adherent junction components of in vivo HCECs, and compare their gene expression and their proliferative potential to those of in vitro counterparts. Methods Stripped human Descemet’s membranes were digested with collagenase A, and the resultant HCEC aggregates were cultured for 7, 14, and 21 days in supplemented hormonal epithelial medium (SHEM). Growth of HCEC monolayers was monitored by BrdU labeling performed 24 h before termination. Both in vivo and in vitro HCECs were subjected to immunostaining to FITC-phalloidin and antibodies to different junction components and BrdU. Their mRNA expressions were determined by RT-PCR. Results In vivo HCECs expressed transcripts of N-, VE-, E-, and P-cadherins, α-, β-, γ-, and p120-catenins, and p190. In vitro HCEC counterparts also expressed all these mRNAs except P-cadherin. In vivo HCECs displayed continuous circular F-actin, N-cadherin, β- and p120-catenins, and p190, discontinuous circular VE-cadherin bands at/close to cell junctions, and E-cadherin in the cytoplasm. Such an in vivo pattern was gradually achieved by in vitro HCECs at day 21 and was correlated with a progressive decline of BrdU labeling. Conclusions Both in vivo and in vitro HCECs displayed distinct protein cytolocalization of N-, VE-, and E-cadherins, β- and p120-catenins, and p190. Progressive maturation of adherent junctions was associated with a decline of the proliferative potential. This information allows us to devise new strategies to engineer in vitro HCECs by targeting these components. PMID:18502989

  14. Inhibition of mitogenesis induced by phytohemagglutinin and Lens culinaris lectin in adherent-cell supernatants treated with protein extract of Mycobacterium tuberculosis.

    PubMed Central

    Parra, C; Montaño, L F; Huesca, M; Rayón, I; Willms, K; Goodsaid, F

    1986-01-01

    Specific stimulation of T cells by phytohemagglutinin and Lens culinaris lectin was inhibited by a soluble factor(s) secreted by normal adherent cells stimulated with culture filtrate protein extract (CFPE) derived from bacterial cultures of Mycobacterium tuberculosis H37Ra (avirulent) and H37Rv (virulent). The induction of the inhibitory factor was blocked by the presence of hyperimmune antisera to H37Rv or H37Ra CFPE. The inhibitory factor did not seem to be a CFPE reprocessed by the adherent cells. Inhibitory activity was maximal in supernatants of adherent-cell cultures incubated for 48 h; the inhibitory factor was heat labile, and its production was dependent on the concentration of M. tuberculosis CFPE. A mouse monocyte-macrophage cell line, ATCC J774A.1, produced an identical inhibitory factor, thus excluding a non-macrophage-contaminating adherent cell as the source of the factor. This inhibitory factor also interfered with the recognition of phytohemagglutinin and Lens culinaris lectin by T cells. PMID:3082760

  15. Open-channel microfluidic membrane device for long-term FT-IR spectromicroscopy of live adherent cells.

    PubMed

    Loutherback, Kevin; Chen, Liang; Holman, Hoi-Ying N

    2015-01-01

    Spatially resolved infrared spectroscopy is a label-free and nondestructive analytical technique that can provide spatiotemporal information on functional groups in biomolecules of a sample by their characteristic vibrational modes. One difficulty in performing long-term FT-IR measurements on live cells is the competition between the strong IR absorption from water and the need to supply nutrients and remove waste. In this proof of principle study, we developed an open-channel membrane device that allows long-term continuous IR measurement of live, adherent mammalian cells. Composed of a gold-coated porous membrane between a feeding channel and a viewing chamber, it allows cells to be maintained on the upper membrane surface in a thin layer of fluid while media is replenished from the feeding channel below. Using this device, we monitored the spatiotemporal chemical changes in living colonies of PC12 cells under nerve growth factor (NGF) stimulation for up to 7 days using both conventional globar and high-resolution synchrotron radiation-based IR sources. We identified the primary chemical change cells undergo is an increase in glycogen that may be associated with secretion of glycoprotein to protect the cells from evaporative stress at the air-liquid interface. Analyzing the spectral maps with multivariate methods of hierarchical cluster analysis (HCA) and principal component analysis (PCA), we found that the cells at the boundary of the colony and in a localized region in the center of the colony tend to produce more glycogen and glycoprotein than cells located elsewhere in the colony and that the degree of spatial heterogeneity decreases with time. This method provides a promising approach for long-term live-cell spectromicroscopy on mammalian cell systems. PMID:25886198

  16. Medullospheres from DAOY, UW228 and ONS-76 Cells: Increased Stem Cell Population and Proteomic Modifications

    PubMed Central

    Zanini, Cristina; Ercole, Elisabetta; Mandili, Giorgia; Salaroli, Roberta; Poli, Alice; Renna, Cristiano; Papa, Valentina; Cenacchi, Giovanna; Forni, Marco

    2013-01-01

    Background Medulloblastoma (MB) is an aggressive pediatric tumor of the Central Nervous System (CNS) usually treated according to a refined risk stratification. The study of cancer stem cells (CSC) in MB is a promising approach aimed at finding new treatment strategies. Methodology/Principal Findings The CSC compartment was studied in three characterized MB cell lines (DAOY, UW228 and ONS-76) grown in standard adhesion as well as being grown as spheres, which enables expansion of the CSC population. MB cell lines, grown in adherence and as spheres, were subjected to morphologic analysis at the light and electron microscopic level, as well as cytofluorimetric determinations. Medullospheres (MBS) were shown to express increasingly immature features, along with the stem cells markers: CD133, Nestin and β-catenin. Proteomic analysis highlighted the differences between MB cell lines, demonstrating a unique protein profile for each cell line, and minor differences when grown as spheres. In MBS, MALDI-TOF also identified some proteins, that have been linked to tumor progression and resistance, such as Nucleophosmin (NPM). In addition, immunocytochemistry detected Sox-2 as a stemness marker of MBS, as well as confirming high NPM expression. Conclusions/Significance Culture conditioning based on low attachment flasks and specialized medium may provide new data on the staminal compartment of CNS tumors, although a proteomic profile of CSC is still elusive for MB. PMID:23717474

  17. HIV Medication Adherence

    MedlinePlus

    HIV Treatment HIV Medication Adherence (Last updated 3/1/2016; last reviewed 3/1/2016) Key Points Medication adherence means sticking ... exactly as prescribed. Why is adherence to an HIV regimen important? Adherence to an HIV regimen gives ...

  18. Fast, Efficient, and Gentle Transfection of Human Adherent Cells in Suspension.

    PubMed

    Agrawal, Pranav; Ingle, Nilesh P; Boyle, William S; Ward, Emily; Tolar, Jakub; Dorfman, Kevin D; Reineke, Theresa M

    2016-04-13

    We demonstrate a highly efficient method for gene delivery into clinically relevant human cell types, such as induced pluripotent stem cells (iPSCs) and fibroblasts, reducing the protocol time by one full day. To preserve cell physiology during gene transfer, we designed a microfluidic strategy, which facilitates significant gene delivery in a short transfection time (<1 min) for several human cell types. This fast, optimized and generally applicable cell transfection method can be used for rapid screening of different delivery systems and has significant potential for high-throughput cell therapy applications. PMID:27035392

  19. Response of adherent cells to mechanical perturbations of the surrounding matrix.

    PubMed

    Ben-Yaakov, Dan; Golkov, Roman; Shokef, Yair; Safran, Samuel A

    2015-02-01

    We present a generic and unified theory to explain how cells respond to perturbations of their mechanical environment such as the presence of neighboring cells, slowly applied stretch, or gradients of matrix rigidity. Motivated by experiments, we calculate the local balance of forces that give rise to a tendency for the cell to locally move or reorient, with a focus on the contribution of feedback and homeostasis to cell contractility (manifested by a fixed displacement, strain or stress) that acts on the adhesions at the cell boundary. These forces can be either reinforced or diminished by elastic stresses due to mechanical perturbations of the matrix. Our model predicts these changes and how their balance with local protrusive forces that act on the cell's leading edge either increase or decrease the tendency of the cell to locally move (toward neighboring cells or rigidity gradients) or reorient (in the direction of slowly applied stretch or rigidity gradients).

  20. Mechanisms of Pulsed Laser Microbeam Release of SU-8 Polymer “Micropallets” for the Collection and Separation of Adherent Cells

    PubMed Central

    Quinto-Su, Pedro A.; Salazar, Georgina To'a; Sims, Christopher E.; Allbritton, Nancy L.; Venugopalan, Vasan

    2008-01-01

    The release of individual polymer micropallets from glass substrates using highly focused laser pulses has been demonstrated for the efficient separation, collection, and expansion of single, adherent cells from a heterogeneous cell population. Here, we use fast-frame photography to examine the mechanism and dynamics of micropallet release produced by pulsed laser microbeam irradiation at λ = 532 nm using pulse durations ranging between 240 ps and 6 ns. The time-resolved images show the laser microbeam irradiation to result in plasma formation at the interface between the glass coverslip and the polymer micropallet. The plasma formation results in the emission of a shock wave and the ablation of material within the focal volume. Ablation products are generated at high pressure due to the confinement offered by the polymer adhesion to the glass substrate. The ablation products expand underneath the micropallet on a time scale of several hundred nanoseconds. This expansion disrupts the polymer–glass interface and accomplishes the release of the pallet from its glass substrate on the microsecond time scale (∼1.5 μs). Our experimental investigation demonstrates that the threshold energy for pallet release is constant (∼2 μJ) over a 25-fold range of pulse duration spanning the picosecond to nanosecond domain. Taken together, these results implicate that pallet release accomplished via pulsed laser microbeam irradiation is an energy-driven plasma-mediated ablation process. PMID:18489124

  1. Cystone – An ayurvedic polyherbal formulation inhibits adherence of uropathogenic E. coli and modulates H2O2-induced toxicity in NRK-52E cells

    PubMed Central

    Vidyashankar, Satyakumar; Maheshkumar, Puttanarasaiah; Patki, Pralhad S

    2010-01-01

    Gentamicin is a widely used antibiotic for the treatment of adverse urinary tract infections (UTI), which in turn causes nephrotoxicity to uroepithelial cells and hence an alternative safe herbal remedy is much desired to compensate these toxic effects. The bacterial adhesion to the uroepithelial cells is the primary step in UTI and it induces various immunogenic reactions leading to the generation of reactive oxygen species (ROS), which are detrimental to the cells survival. Inhibition of bacterial adherence to urinary tract epithelial cells has been assumed to account for the beneficial action ascribed to cystone (an ayurvedic polyherbal formulation) in the prevention of UTI. In this study, we have examined the effect of cystone on the adherence of pathogenic [2-14C]-acetate labeled Escherichia coli (MTCC-729) to rat proximal renal tubular cells (NRK-52E cells). Further, the antioxidant property of cystone was studied using hydrogen peroxide (400 μM) as a pro-oxidant in NRK-52E cells. The results showed that cystone inhibited the adherence of E. coli to NRK-52E cells significantly. Additionally cystone effectively combats the toxicity induced by H2O2 in NRK-52E cells. The cytoprotective effect of cystone is brought about by inhibiting lipid peroxidation by 36% in cells treated with cystone compared to H2O2-treated cells without cystone. The antioxidant enzymes catalase, glutathione were increased by 53% and 68% respectively and superoxide dismutase activity was increased 3-fold. The glutathione content was significantly increased by 2.4-fold in NRK-52E cells treated with cystone compared to H2O2 control group. These results suggest that cystone effectively inhibits bacterial adherence to NRK-52E cells and attenuates H2O2-induced toxicity in NRK-52E cells by inhibiting lipid peroxidation and increasing the antioxidant defense mechanism. PMID:27186087

  2. Cystone - An ayurvedic polyherbal formulation inhibits adherence of uropathogenic E. coli and modulates H2O2-induced toxicity in NRK-52E cells.

    PubMed

    Vidyashankar, Satyakumar; Maheshkumar, Puttanarasaiah; Patki, Pralhad S

    2010-01-01

    Gentamicin is a widely used antibiotic for the treatment of adverse urinary tract infections (UTI), which in turn causes nephrotoxicity to uroepithelial cells and hence an alternative safe herbal remedy is much desired to compensate these toxic effects. The bacterial adhesion to the uroepithelial cells is the primary step in UTI and it induces various immunogenic reactions leading to the generation of reactive oxygen species (ROS), which are detrimental to the cells survival. Inhibition of bacterial adherence to urinary tract epithelial cells has been assumed to account for the beneficial action ascribed to cystone (an ayurvedic polyherbal formulation) in the prevention of UTI. In this study, we have examined the effect of cystone on the adherence of pathogenic [2-(14)C]-acetate labeled Escherichia coli (MTCC-729) to rat proximal renal tubular cells (NRK-52E cells). Further, the antioxidant property of cystone was studied using hydrogen peroxide (400 μM) as a pro-oxidant in NRK-52E cells. The results showed that cystone inhibited the adherence of E. coli to NRK-52E cells significantly. Additionally cystone effectively combats the toxicity induced by H2O2 in NRK-52E cells. The cytoprotective effect of cystone is brought about by inhibiting lipid peroxidation by 36% in cells treated with cystone compared to H2O2-treated cells without cystone. The antioxidant enzymes catalase, glutathione were increased by 53% and 68% respectively and superoxide dismutase activity was increased 3-fold. The glutathione content was significantly increased by 2.4-fold in NRK-52E cells treated with cystone compared to H2O2 control group. These results suggest that cystone effectively inhibits bacterial adherence to NRK-52E cells and attenuates H2O2-induced toxicity in NRK-52E cells by inhibiting lipid peroxidation and increasing the antioxidant defense mechanism.

  3. Adherence Reduction of Campylobacter jejuni and Campylobacter coli Strains to HEp-2 Cells by Mannan Oligosaccharides and a High-Molecular-Weight Component of Cranberry Extract.

    PubMed

    Ramirez-Hernandez, Alejandra; Rupnow, John; Hutkins, Robert W

    2015-08-01

    Campylobacter infections are a leading cause of human bacterial gastroenteritis in the United States and are a major cause of diarrheal disease throughout the world. Colonization and subsequent infection and invasion of Campylobacter require that the bacteria adhere to the surface of host cells. Agents that inhibit adherence could be used prophylactically to reduce Campylobacter carriage and infection. Mannan oligosaccharides (MOS) have been used as a feed supplement in livestock animals to improve performance and to replace growth-promoting antibiotics. However, MOS and other nondigestible oligosaccharides may also prevent pathogen colonization by inhibiting adherence in the gastrointestinal tract. In addition, plant extracts, including those derived from cranberries, have been shown to have antiadherence activity against pathogens. The goal of this study was to assess the ability of MOS and cranberry fractions to serve as antiadherence agents against strains of Campylobacter jejuni and Campylobacter coli. Adherence experiments were performed using HEp-2 cells. Significant reductions in adherence of C. jejuni 29438, C. jejuni 700819, C. jejuni 3329, and C. coli 43485 were observed in the presence of MOS (up to 40 mg/ml) and with a high-molecular-weight fraction of cranberry extract (up to 3 mg/ml). However, none of the tested materials reduced adherence of C. coli BAA-1061. No additive effect in adherence inhibition was observed for an MOS-cranberry blend. These results suggest that both components, MOS and cranberry, could be used to reduce Campylobacter colonization and carriage in livestock animals and potentially limit human exposure to this pathogen.

  4. Adherence Reduction of Campylobacter jejuni and Campylobacter coli Strains to HEp-2 Cells by Mannan Oligosaccharides and a High-Molecular-Weight Component of Cranberry Extract.

    PubMed

    Ramirez-Hernandez, Alejandra; Rupnow, John; Hutkins, Robert W

    2015-08-01

    Campylobacter infections are a leading cause of human bacterial gastroenteritis in the United States and are a major cause of diarrheal disease throughout the world. Colonization and subsequent infection and invasion of Campylobacter require that the bacteria adhere to the surface of host cells. Agents that inhibit adherence could be used prophylactically to reduce Campylobacter carriage and infection. Mannan oligosaccharides (MOS) have been used as a feed supplement in livestock animals to improve performance and to replace growth-promoting antibiotics. However, MOS and other nondigestible oligosaccharides may also prevent pathogen colonization by inhibiting adherence in the gastrointestinal tract. In addition, plant extracts, including those derived from cranberries, have been shown to have antiadherence activity against pathogens. The goal of this study was to assess the ability of MOS and cranberry fractions to serve as antiadherence agents against strains of Campylobacter jejuni and Campylobacter coli. Adherence experiments were performed using HEp-2 cells. Significant reductions in adherence of C. jejuni 29438, C. jejuni 700819, C. jejuni 3329, and C. coli 43485 were observed in the presence of MOS (up to 40 mg/ml) and with a high-molecular-weight fraction of cranberry extract (up to 3 mg/ml). However, none of the tested materials reduced adherence of C. coli BAA-1061. No additive effect in adherence inhibition was observed for an MOS-cranberry blend. These results suggest that both components, MOS and cranberry, could be used to reduce Campylobacter colonization and carriage in livestock animals and potentially limit human exposure to this pathogen. PMID:26219363

  5. Porcine myelomonocytic markers and cell populations.

    PubMed

    Ezquerra, A; Revilla, C; Alvarez, B; Pérez, C; Alonso, F; Domínguez, J

    2009-03-01

    This review focuses in what is currently known about swine myeloid markers, the expression and function of these receptors in the biology of porcine myelomonocytic cells, the regulation of their expression along the different developmental stages of these cells and their utility to investigate the heterogeneity of monocyte and macrophage populations. Although the number of monoclonal antibodies recognizing surface antigens expressed on either swine granulocytes or monocytes is low compared with those available for human or mouse, they have contributed significantly to study the members of myeloid lineages in this species, allowing to discriminate different maturation stages of these cells in bone marrow and to reveal the heterogeneity of blood monocytes and tissue macrophages. Porcine myeloid cells share many similarities with humans, highlighting the relevance of the pig as a biomedical model.

  6. Comparative genomic hybridization analysis of newly established retinoblastoma cell lines of adherent growth compared with Y79 of nonadherent growth.

    PubMed

    Kim, Jeong Hun; Kim, Jin Hyoung; Yu, Young Suk; Kim, Dong Hun; Kim, Yong Kyu; Kim, Kyu-Won

    2008-08-01

    Retinoblastoma (RB) shows cytogenetic aberrations involving genes other than RB gene located on 13q14. We analyzed genomic aberration in newly established RB cell lines SNUOT-RB1 and SNUOT-RB4 of adherent growth and Y79 cell line of nonadherent growth by microarray comparative genomic hybridization. SNUOT-RB1 showed 44 significant copy number changes (gain in 11 and loss in 33, P<0.0005). SNUOT-RB4 showed 42 significant copy number changes (gain in 8 and loss in 34, P<0.0005). Y79 cell line had the greatest gain of 19.65-fold in the locus of MYCN gene 2p24.1, whereas SNUOT-RB1 and SNUOT-RB4 showed no significant gain. SNUOT-RB1 and SNUOT-RB4 gained chromosomal copy numbers commonly in chromosome 11, especially in locus 11q13, which is responsible for cancer-related genes such as CCND1, MEN1, and FGF3. Losses of copy numbers occurred in chromosomes 3, 9, 10, 11, 16, and 17. In summary, SNUOT-RB1 and SNUOT-RB4 represented similar pattern in gain and loss of chromosomal copy number changes, while different from Y79. The loss of CYLD gene of tumor suppressor gene, 16q12-q13, was only on locus of common involvement in 3 cell lines. PMID:18799932

  7. Proliferation of mutators in A cell population.

    PubMed Central

    Mao, E F; Lane, L; Lee, J; Miller, J H

    1997-01-01

    A Lac- strain of Escherichia coli that reverts by the addition of a G to a G-G-G-G-G-G sequence was used to study the proliferation of mutators in a bacterial culture. Selection for the Lac+ phenotype, which is greatly stimulated in mismatch repair-deficient strains, results in an increase in the percentage of mutators in the selected population from less than 1 per 100,000 cells to 1 per 200 cells. All the mutators detected were deficient in the mismatch repair system. Mutagenesis results in a similar increase in the percentage of mutators. Mutagenesis combined with a single selection can result in a population of more than 50% mutators when a sample of several thousand cells is grown out and selected. Mutagenesis combined with two or more successive selections can generate a population that is 100% mutator. These experiments are discussed in relation to ideas that an early step in carcinogenesis is the creation of a mutator phenotype. PMID:8990293

  8. Vertical nanopillars for in situ probing of nuclear mechanics in adherent cells.

    PubMed

    Hanson, Lindsey; Zhao, Wenting; Lou, Hsin-Ya; Lin, Ziliang Carter; Lee, Seok Woo; Chowdary, Praveen; Cui, Yi; Cui, Bianxiao

    2015-06-01

    The mechanical stability and deformability of the cell nucleus are crucial to many biological processes, including migration, proliferation and polarization. In vivo, the cell nucleus is frequently subjected to deformation on a variety of length and time scales, but current techniques for studying nuclear mechanics do not provide access to subnuclear deformation in live functioning cells. Here we introduce arrays of vertical nanopillars as a new method for the in situ study of nuclear deformability and the mechanical coupling between the cell membrane and the nucleus in live cells. Our measurements show that nanopillar-induced nuclear deformation is determined by nuclear stiffness, as well as opposing effects from actin and intermediate filaments. Furthermore, the depth, width and curvature of nuclear deformation can be controlled by varying the geometry of the nanopillar array. Overall, vertical nanopillar arrays constitute a novel approach for non-invasive, subcellular perturbation of nuclear mechanics and mechanotransduction in live cells. PMID:25984833

  9. Vertical nanopillars for in situ probing of nuclear mechanics in adherent cells

    NASA Astrophysics Data System (ADS)

    Hanson, Lindsey; Zhao, Wenting; Lou, Hsin-Ya; Lin, Ziliang Carter; Lee, Seok Woo; Chowdary, Praveen; Cui, Yi; Cui, Bianxiao

    2015-06-01

    The mechanical stability and deformability of the cell nucleus are crucial to many biological processes, including migration, proliferation and polarization. In vivo, the cell nucleus is frequently subjected to deformation on a variety of length and time scales, but current techniques for studying nuclear mechanics do not provide access to subnuclear deformation in live functioning cells. Here we introduce arrays of vertical nanopillars as a new method for the in situ study of nuclear deformability and the mechanical coupling between the cell membrane and the nucleus in live cells. Our measurements show that nanopillar-induced nuclear deformation is determined by nuclear stiffness, as well as opposing effects from actin and intermediate filaments. Furthermore, the depth, width and curvature of nuclear deformation can be controlled by varying the geometry of the nanopillar array. Overall, vertical nanopillar arrays constitute a novel approach for non-invasive, subcellular perturbation of nuclear mechanics and mechanotransduction in live cells.

  10. Vertical nanopillars for in situ probing of nuclear mechanics in adherent cells.

    PubMed

    Hanson, Lindsey; Zhao, Wenting; Lou, Hsin-Ya; Lin, Ziliang Carter; Lee, Seok Woo; Chowdary, Praveen; Cui, Yi; Cui, Bianxiao

    2015-06-01

    The mechanical stability and deformability of the cell nucleus are crucial to many biological processes, including migration, proliferation and polarization. In vivo, the cell nucleus is frequently subjected to deformation on a variety of length and time scales, but current techniques for studying nuclear mechanics do not provide access to subnuclear deformation in live functioning cells. Here we introduce arrays of vertical nanopillars as a new method for the in situ study of nuclear deformability and the mechanical coupling between the cell membrane and the nucleus in live cells. Our measurements show that nanopillar-induced nuclear deformation is determined by nuclear stiffness, as well as opposing effects from actin and intermediate filaments. Furthermore, the depth, width and curvature of nuclear deformation can be controlled by varying the geometry of the nanopillar array. Overall, vertical nanopillar arrays constitute a novel approach for non-invasive, subcellular perturbation of nuclear mechanics and mechanotransduction in live cells.

  11. A family of cell-adhering peptides homologous to fibrinogen C-termini

    SciTech Connect

    Levy-Beladev, Liron; Levdansky, Lilia; Gaberman, Elena; Friedler, Assaf; Gorodetsky, Raphael

    2010-10-08

    Research highlights: {yields} Cell-adhesive sequences homologous to fibrinogen C-termini exist in other proteins. {yields} The extended homologous cell-adhesive C-termini peptides family is termed Haptides. {yields} In membrane-like environment random coiled Haptides adopt a helical conformation. {yields} Replacing positively charged residues with alanine reduces Haptides activity. -- Abstract: A family of cell-adhesive peptides homologous to sequences on different chains of fibrinogen was investigated. These homologous peptides, termed Haptides, include the peptides C{beta}, preC{gamma}, and C{alpha}E, corresponding to sequences on the C-termini of fibrinogen chains {beta}, {gamma}, and {alpha}E, respectively. Haptides do not affect cell survival and rate of proliferation of the normal cell types tested. The use of new sensitive assays of cell adhesion clearly demonstrated the ability of Haptides, bound to inert matrices, to mediate attachment of different matrix-dependent cell types including normal fibroblasts, endothelial, and smooth muscle cells. Here we present new active Haptides bearing homologous sequences derived from the C-termini of other proteins, such as angiopoietin 1 and 2, tenascins C and X, and microfibril-associated glycoprotein-4. The cell adhesion properties of all the Haptides were found to be associated mainly with their 11 N-terminal residues. Mutated preC{gamma} peptides revealed that positively charged residues account for their attachment effect. These results suggest a mechanism of direct electrostatic interaction of Haptides with the cell membrane. The extended Haptides family may be applied in modulating adhesion of cells to scaffolds for tissue regeneration and for enhancement of nanoparticulate transfection into cells.

  12. A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

    PubMed Central

    Geletu, Mulu; Guy, Stephanie; Firth, Kevin; Raptis, Leda

    2014-01-01

    In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.  Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition. PMID:25350637

  13. AFBI assay – Aptamer Fluorescence Binding and Internalization assay for cultured adherent cells

    PubMed Central

    Thiel, William H.; Giangrande, Paloma H.

    2016-01-01

    The SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process allows for the enrichment of DNA or RNA aptamers from a complex nucleic acid library that are specific for a target molecule. The SELEX process has been adapted from identifying aptamers in vitro using recombinant target protein to cell-based methodologies (Cell-SELEX), where the targets are expressed on the surface of cells. One major advantage of Cell-SELEX is that the target molecules are maintained in a native confirmation. Additionally, Cell-SELEX may be used to discover novel therapeutic biomarkers by performing selections on diseased versus healthy cells. However, a caveat to Cell-SELEX is that testing of single aptamers identified in the selection is laborious, time-consuming, and expensive. The most frequently used methods to screen for aptamer binding and internalization on cells are flow cytometry and quantitative PCR (qPCR). While flow cytometry can directly assess binding of a fluorescently-labeled aptamer to a target, it requires significant starting material and is not easily scalable. qPCR-based approaches are highly sensitive but have non-negligible experiment-to-experiment variability due to the number of sample processing steps. Herein we describe a cell-based aptamer fluorescence binding and internalization (AFBI) assay. This assay requires minimal reagents and has few experimental steps/manipulations, thereby allowing for rapid screening of many aptamers and conditions simultaneously and direct quantitation of aptamer binding and internalization. PMID:26972784

  14. Apo A-I inhibits foam cell formation in apo E–deficient mice after monocyte adherence to endothelium

    PubMed Central

    Dansky, Hayes M.; Charlton, Sherri A.; Barlow, Courtenay B.; Tamminen, Minna; Smith, Jonathan D.; Frank, Joy S.; Breslow, Jan L.

    1999-01-01

    We have previously shown that expression of the human apo A-I transgene on the apo E–deficient background increases HDL cholesterol and greatly diminishes fatty streak lesion formation. To examine the mechanism, prelesional events in atherosclerotic plaque development were examined in 6- to 8-week-old apo E–deficient and apo E–deficient/human apo A-I transgenic mice. A quantitative assessment of subendothelial lipid deposition by freeze-fracture and deep-etch electron microscopy indicated that elevated apo A-I did not affect the distribution or amount of aortic arch subendothelial lipid deposits. Immunohistochemical staining for VCAM-1 demonstrated similar expression on endothelial cells at prelesional aortic branch sites from both apo E–deficient and apo E–deficient/human apo A-I transgenic mice. Transmission electron microscopy revealed monocytes bound to the aortic arch in mice of both genotypes, and immunohistochemical staining demonstrated that the area occupied by bound mononuclear cells was unchanged. Serum paraoxonase and aryl esterase activity did not differ between apo E–deficient and apo E–deficient/human apo A-I transgenic mice. These data suggest that increases in apo A-I and HDL cholesterol inhibit foam cell formation in apo E–deficient/human apo A-I transgenic mice at a stage following lipid deposition, endothelial activation, and monocyte adherence, without increases in HDL-associated paraoxonase. PMID:10393696

  15. Exopolysaccharides of Lactobacillus reuteri: Their influence on adherence of E. coli to epithelial cells and inflammatory response.

    PubMed

    Kšonžeková, Petra; Bystrický, Peter; Vlčková, Silvia; Pätoprstý, Vladimír; Pulzová, Lucia; Mudroňová, Dagmar; Kubašková, Terézia; Csank, Tomáš; Tkáčiková, Ľudmila

    2016-05-01

    The aim of the study was to characterize exopolysaccharides (EPS) originated from Lactobacillus reuteri strain DSM 17938 (EPS-DSM17938) and L. reuteri strain L26 Biocenol™ (EPS-L26) and evaluate their influence on adherence of enterotoxigenic Escherichia coli (ETEC) to IPEC-1 cells and proinflammatory gene expression. Both EPS were d-glucan polysaccharides with higher molecular weight (Mw), but differing in spatial conformation and elicited variable cytokine profile. EPS-DSM17938, relatively linear polysaccharide with (1→4) and (1→6) glycosidic linkages, increased IL-1β gene expression (0.1mg/mL; P<0.05), while EPS-L26, more branched polysaccharide with (1→3) and (1→6) glycosidic linkages, exerted slight but statistically significant up-regulation of NF-κB, TNF-α and IL-6 mRNA (P<0.05). The most significant finding is that preincubation of IPEC-1 cells with both EPS followed by ETEC infection inhibit ETEC adhesion on IPEC-1 cells (P<0.01) and ETEC-induced gene expression of proinflammatory cytokine IL-1β and IL-6 (P<0.01). PMID:26876991

  16. Adherence of streptococcus pyogenes, Escherichia coli, and Pseudomonas aeruginosa to fibronectin-coated and uncoated epithelial cells.

    PubMed Central

    Abraham, S N; Beachey, E H; Simpson, W A

    1983-01-01

    The relationship between the variability in the fibronectin (Fn) content on human buccal epithelial cells and the capacity of the cells to bind gram-positive (Streptococcus pyogenes) or gram-negative (Escherichia coli or Pseudomonas aeruginosa) bacteria was investigated. Adhesion experiments performed with mixtures of epithelial cells and mixed suspensions of either S. pyogenes and E. coli or S. pyogenes and P. aeruginosa exhibited three major populations of buccal cells: one of these was able to bind S. pyogenes (gram positive) but neither of the gram-negative bacteria; a second population was able to bind the gram-negative but not the gram-positive bacteria; and a third was able to bind various numbers of both types of organisms. Further adhesion experiments performed with a mixture of epithelial cells and a mixed suspension of S. pyrogens, E. coli, and fluoresceinconjugated methacrylate beads coated with immune immunoglobulin G directed against Fn revealed that the epithelial cells recognizing the gram-positive bacteria were rich in Fn, whereas those recognizing the gram-negative organisms were poor in Fn. Immunoelectron microscopy confirmed that cells of S. pyogenes bound to epithelial cells coated with Fn, whereas cells of E. coli bound to epithelial cells lacking Fn. These results suggest that Fn on the surfaces of epithelial cells may modulate the ecology of the human oropharyngeal cavity, especially with respect to the colonization of these surfaces by pathogenic gram-negative or gram-positive bacteria. Images PMID:6411621

  17. IL-6 acts on endothelial cells to preferentially increase their adherence for lymphocytes

    PubMed Central

    WATSON, C; WHITTAKER, S; SMITH, N; VORA, A J; DUMONDE, D C; BROWN, K A

    1996-01-01

    Using a quantitative monolayer adhesion assay, the current report shows that treatment of human umbilical vein endothelial cells (HUVEC) with IL-6 increases their adhesiveness for blood lymphocytes, particularly CD4+ cells, but not for polymorphonuclear cells and monocytes. This effect, which was most pronounced when using low concentrations of the cytokine (0.1–1.0 U/ml) and a short incubation period (4 h), was also apparent with microvascular endothelial cells and a hybrid endothelial cell line. Skin lesions from patients with mycosis fungoides contain high levels of IL-6, and blood lymphocytes from patients with this disorder also exhibited an enhanced adhesion to IL-6-treated HUVEC. The cytokine enhanced intercellular adhesion molecule-1 (ICAM-1) expression and induced the expression of vascular cell adhesion molecule-1 (VCAM-1) and E-selectin on endothelial cells. Antibody blocking studies demonstrated that the vascular adhesion molecules ICAM-1, VCAM-1 and E-selectin and the leucocyte integrin LFA-1 all contributed to lymphocyte binding to endothelium activated by IL-6. It is proposed that IL-6 may be involved in the recruitment of lymphocytes into non-lymphoid tissue. PMID:8697617

  18. IL-6 acts on endothelial cells to preferentially increase their adherence for lymphocytes.

    PubMed

    Watson, C; Whittaker, S; Smith, N; Vora, A J; Dumonde, D C; Brown, K A

    1996-07-01

    Using a quantitative monolayer adhesion assay, the current report shows that treatment of human umbilical vein endothelial cells (HUVEC) with IL-6 increases their adhesiveness for blood lymphocytes, particularly CD4+ cells, but not for polymorphonuclear cells and monocytes. This effect, which was most pronounced when using low concentrations of the cytokine (0.1-1.0 U/ml) and a short incubation period (4h), was also apparent with microvascular endothelial cells and a hybrid endothelial cell line. Skin lesions from patients with mycosis fungoides contain high levels of IL-6, and blood lymphocytes from patients with this disorder also exhibited an enhanced adhesion to IL-6-treated HUVEC. The cytokine enhanced intercellular adhesion molecule-1 (ICAM-1) expression and induced the expression of vascular cell adhesion molecule-1 (VCAM-1) and E-selectin on endothelial cells. Antibody blocking studies demonstrated that the vascular adhesion molecules ICAM-1, VCAM-1 and E-selectin and the leucocyte integrin LFA-1 all contributed to lymphocyte binding to endothelium activated by IL-6. It is proposed that IL-6 may be involved in the recruitment of lymphocytes into non-lymphoid tissue.

  19. Extracellular mass transport considerations for space flight research concerning suspended and adherent in vitro cell cultures

    NASA Technical Reports Server (NTRS)

    Klaus, David M.; Benoit, Michael R.; Nelson, Emily S.; Hammond, Timmothy G.

    2004-01-01

    Conducting biological research in space requires consideration be given to isolating appropriate control parameters. For in vitro cell cultures, numerous environmental factors can adversely affect data interpretation. A biological response attributed to microgravity can, in theory, be explicitly correlated to a specific lack of weight or gravity-driven motion occurring to, within or around a cell. Weight can be broken down to include the formation of hydrostatic gradients, structural load (stress) or physical deformation (strain). Gravitationally induced motion within or near individual cells in a fluid includes sedimentation (or buoyancy) of the cell and associated shear forces, displacement of cytoskeleton or organelles, and factors associated with intra- or extracellular mass transport. Finally, and of particular importance for cell culture experiments, the collective effects of gravity must be considered for the overall system consisting of the cells, their environment and the device in which they are contained. This does not, however, rule out other confounding variables such as launch acceleration, on orbit vibration, transient acceleration impulses or radiation, which can be isolated using onboard centrifuges or vibration isolation techniques. A framework is offered for characterizing specific cause-and-effect relationships for gravity-dependent responses as a function of the above parameters.

  20. Extracellular mass transport considerations for space flight research concerning suspended and adherent in vitro cell cultures.

    PubMed

    Klaus, David M; Benoit, Michael R; Nelson, Emily S; Hammond, Timmothy G

    2004-03-01

    Conducting biological research in space requires consideration be given to isolating appropriate control parameters. For in vitro cell cultures, numerous environmental factors can adversely affect data interpretation. A biological response attributed to microgravity can, in theory, be explicitly correlated to a specific lack of weight or gravity-driven motion occurring to, within or around a cell. Weight can be broken down to include the formation of hydrostatic gradients, structural load (stress) or physical deformation (strain). Gravitationally induced motion within or near individual cells in a fluid includes sedimentation (or buoyancy) of the cell and associated shear forces, displacement of cytoskeleton or organelles, and factors associated with intra- or extracellular mass transport. Finally, and of particular importance for cell culture experiments, the collective effects of gravity must be considered for the overall system consisting of the cells, their environment and the device in which they are contained. This does not, however, rule out other confounding variables such as launch acceleration, on orbit vibration, transient acceleration impulses or radiation, which can be isolated using onboard centrifuges or vibration isolation techniques. A framework is offered for characterizing specific cause-and-effect relationships for gravity-dependent responses as a function of the above parameters.

  1. The regulation of hematopoietic stem cell populations

    PubMed Central

    Mayani, Hector

    2016-01-01

    Evidence presented over the last few years indicates that the hematopoietic stem cell (HSC) compartment comprises not just one but a number of different cell populations. Based on HSCs’ proliferation and engraftment potential, it has been suggested that there are two classes of HSC, with long- and short-term engraftment potential. HSC heterogeneity seems to involve differentiation capacities as well, since it has been shown that some HSC clones are able to give rise to both myeloid and lymphoid progeny, whereas others are lymphoid deficient. It has been recognized that HSC function depends on intrinsic cell regulators, which are modulated by external signals. Among the former, we can include transcription factors and non-coding RNAs as well as epigenetic modifiers. Among the latter, cytokines and extracellular matrix molecules have been implicated. Understanding the elements and mechanisms that regulate HSC populations is of significant relevance both in biological and in clinical terms, and research in this area still has to face several complex and exciting challenges. PMID:27408695

  2. Milk digesta and milk protein fractions influence the adherence of Lactobacillus gasseri R and Lactobacillus casei FMP to human cultured cells.

    PubMed

    Volstatova, Tereza; Havlik, Jaroslav; Potuckova, Miroslava; Geigerova, Martina

    2016-08-10

    Adhesion to the intestinal epithelium is considered an important feature of probiotic bacteria, which may increase their persistence in the intestine, allowing them to exert their beneficial health effect or promote the colonisation process. However, this feature might be largely dependent on the host specificity or diet. In the present study, we investigated the effect of selected milks and milk protein fractions on the ability of selected lactobacilli to adhere to the cells of an intestinal model based on co-culture Caco-2/HT29-MTX cell lines. Most milk digesta did not significantly affect bacterial adhesion except for UHT-treated milk and sheep milk. The presence of UHT-treated milk digesta reduced the adhesion of Lactobacillus gasseri R by 61% but not that of Lactobacillus casei FMP. However, sheep milk significantly increased the adherence of L. casei FMP (P < 0.05) but not of L. gasseri R. Among the protein fractions, rennet casein (RCN) and bovine serum albumin (BSA) showed reproducible patterns and strain-specific effects on bacterial adherence. While RCN reduced the adherence of L. gasseri R to <50% compared to the control, it did not have a significant effect on L. casei FMP. In contrast, BSA reduced L. casei FMP adherence to a higher extent than that of L. gasseri R. Whey protein (WH) tended to increase the adherence of both strains by 130%-180%. Recently, interactions between the host diet and its microbiota have attracted considerable interest. Our results may explain one of the aspects of the role of milk in the development of microbiota or support of probiotic supplements. Based on our data, we conclude that the persistence of probiotic strains supplemented as part of dairy food or constitutional microbiota in the gut might be affected negatively or positively by the food matrix through complex strain or concentration dependent effects. PMID:27435508

  3. Milk digesta and milk protein fractions influence the adherence of Lactobacillus gasseri R and Lactobacillus casei FMP to human cultured cells.

    PubMed

    Volstatova, Tereza; Havlik, Jaroslav; Potuckova, Miroslava; Geigerova, Martina

    2016-08-10

    Adhesion to the intestinal epithelium is considered an important feature of probiotic bacteria, which may increase their persistence in the intestine, allowing them to exert their beneficial health effect or promote the colonisation process. However, this feature might be largely dependent on the host specificity or diet. In the present study, we investigated the effect of selected milks and milk protein fractions on the ability of selected lactobacilli to adhere to the cells of an intestinal model based on co-culture Caco-2/HT29-MTX cell lines. Most milk digesta did not significantly affect bacterial adhesion except for UHT-treated milk and sheep milk. The presence of UHT-treated milk digesta reduced the adhesion of Lactobacillus gasseri R by 61% but not that of Lactobacillus casei FMP. However, sheep milk significantly increased the adherence of L. casei FMP (P < 0.05) but not of L. gasseri R. Among the protein fractions, rennet casein (RCN) and bovine serum albumin (BSA) showed reproducible patterns and strain-specific effects on bacterial adherence. While RCN reduced the adherence of L. gasseri R to <50% compared to the control, it did not have a significant effect on L. casei FMP. In contrast, BSA reduced L. casei FMP adherence to a higher extent than that of L. gasseri R. Whey protein (WH) tended to increase the adherence of both strains by 130%-180%. Recently, interactions between the host diet and its microbiota have attracted considerable interest. Our results may explain one of the aspects of the role of milk in the development of microbiota or support of probiotic supplements. Based on our data, we conclude that the persistence of probiotic strains supplemented as part of dairy food or constitutional microbiota in the gut might be affected negatively or positively by the food matrix through complex strain or concentration dependent effects.

  4. Roles for Cell Wall Glycopeptidolipid in Surface Adherence and Planktonic Dispersal of Mycobacterium avium

    EPA Science Inventory

    The opportunistic pathogen Mycobacterium avium is a significant inhabitant of biofilms in drinking water distribution systems. M. avium expresses on its cell surface serovar-specific glycopeptidolipids (ssGPLs). Studies have implicated the core GPL in biofilm formation by M. aviu...

  5. West Nile virus adheres to human red blood cells in whole blood.

    PubMed

    Rios, Maria; Daniel, Sylvester; Chancey, Caren; Hewlett, Indira K; Stramer, Susan L

    2007-07-15

    Background. West Nile virus (WNV) is endemic in the United States. It is transmissible by blood transfusion, and the nation's blood supply is currently screened for WNV. Documented transmission of WNV infection through red blood cell (RBC) units in which the plasma co-component had a low viral load could be explained, in at least 1 instance, by cell-association of WNV; in this case, the RBC unit was released as negative by minipool nucleic acid testing (NAT) performed on plasma but was intermittently NAT-positive when subsequently tested as an individual sample. We hypothesized that a proportion of WNV bound to blood cells and was not measured by NAT performed on plasma samples. We have investigated whether WNV binds to RBCs, leading to reduction of WNV RNA detection by NAT performed on plasma samples.Methods. Equal volumes of leukoreduced RBCs and their corresponding plasma components from 20 blood donors with NAT results that were positive for WNV were tested in 5 replicates by reverse-transcriptase polymerase chain reaction TaqMan for WNV. In addition, aliquots from 8 of the RBC units were tested by infectivity assays using Vero cells.Results. The reverse-transcriptase polymerase chain reaction TaqMan assay showed that the viral load in the RBC components exceeded that in the corresponding plasma units by 1 order of magnitude. In addition, viruses associated with the RBCs were infectious in Vero cell cultures.Conclusions. These observations reinforce the notion that extraction of viral RNA from whole blood could improve assay sensitivity for blood donor screening and further reduce the residual risk of WNV transmission through transfusion.

  6. Enrichment of spermatogonial stem cells using side population in teleost.

    PubMed

    Hayashi, Makoto; Sato, Masanao; Nagasaka, Yasuhiko; Sadaie, Sakiko; Kobayashi, Satoru; Yoshizaki, Goro

    2014-07-01

    Spermatogenesis originates from a small population of spermatogonial stem cells; this population can maintain continuous sperm production throughout the life of fish via self-renewal and differentiation. Despite their biological importance, spermatogonial stem cells are not thoroughly characterized because they are difficult to distinguish from their progeny cells that become committed to differentiation. We previously established a novel technique for germ cell transplantation to identify spermatogonial stem cells based on their colonizing activity and their ability to initiate donor-derived gametogenesis in the rainbow trout (Oncorhynchus mykiss). Although spermatogonial stem cells can be retrospectively identified after transplantation, there is currently no technique to prospectively enrich for or purify spermatogonial stem cells. Here, we describe a method for spermatogonial stem cell enrichment using a side population. With optimized Hoechst 33342 staining conditions, we successfully identified side-population cells among type A spermatogonia. Side-population cells were transcriptomically and morphologically distinct from non-side-population cells. To functionally determine whether the transplantable spermatogonial stem cells were enriched in the side-population fraction, we compared the colonization activity of side-population cells with that of non-side-population cells. Colonization efficiency was significantly higher with side-population cells than with non-side-population cells or with total type A spermatogonia. In addition, side-population cells could produce billions of sperm in recipients. These results indicated that transplantable spermatogonial stem cells were enriched in the side-population fraction. This method will provide biological information that may advance our understanding of spermatogonial stem cells in teleosts. Additionally, this technique will increase the efficiency of germ cell transplantation used in surrogate broodstock

  7. Histamine increases sickle erythrocyte adherence to endothelium.

    PubMed

    Wagner, Matthew C; Eckman, James R; Wick, Timothy M

    2006-02-01

    Complications of sickle cell anaemia include vascular occlusion triggered by the adherence of sickle erythrocytes to endothelium in the postcapillary venules. Adherence can be promoted by inflammatory mediators that induce endothelial cell adhesion molecule expression and arrest flowing erythrocytes. The present study characterised the effect of histamine stimulation on the kinetics of sickle cell adherence to large vessel and microvascular endothelium under physiological flow. Increased sickle cell adherence was observed within minutes of endothelial activation by histamine and reached a maximum value within 30 min. At steady state, sickle cell adherence to histamine-stimulated endothelium was 47 +/- 4 adherent cells/mm(2), 2.6-fold higher than sickle cell adherence to unstimulated endothelial cells. Histamine-induced sickle cell adherence occurred rapidly and transiently. Studies using histamine receptor agonists and antagonists suggest that histamine-induced sickle cell adhesion depends on simultaneous stimulation of the H(2) and H(4) histamine receptors and endothelial P-selectin expression. These data show that histamine release may promote sickle cell adherence and vaso-occlusion. In vivo histamine release should be studied to determine its role in sickle complications and whether blocking of specific histamine receptors may prevent clinical complications or adverse effects from histamine release stimulated by opiate analgesic treatment.

  8. Anti-adherence potential of Enterococcus durans cells and its cell-free supernatant on plastic and stainless steel against foodborne pathogens.

    PubMed

    Amel, Ait Meddour; Farida, Bendali; Djamila, Sadoun

    2015-07-01

    It is demonstrated that numerous bacteria are able to attach to surfaces of equipment used for food handling or processing. In this study, a strain of Enterococcus durans, originally isolated from a milking machine surface, was firstly studied for its biofilm formation potential on plastic and stainless steel supports. The strain was found to be a biofilm producer either at 25, 30 or 37 °C on polystyrene microtitre plates, with a best adherence level observed at 25 °C. En. durans showed a strong adhesion to stainless steel AISI-304. Antibacterial and anti-adherence activities of En. durans were tested against four foodborne pathogens (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853 and Listeria innocua CLIP 74915) which were shown as biofilm producers on both plastic and stainless steel. En. durans cells and cell-free culture supernatant showed a significant (P < 0.05) inhibition potential of the pathogens either on solid media or in broth co-cultures. Characterization of the antibacterial substances indicated their proteinaceous nature which assigned them most probably to bacteriocins group.

  9. Anti-adherence potential of Enterococcus durans cells and its cell-free supernatant on plastic and stainless steel against foodborne pathogens.

    PubMed

    Amel, Ait Meddour; Farida, Bendali; Djamila, Sadoun

    2015-07-01

    It is demonstrated that numerous bacteria are able to attach to surfaces of equipment used for food handling or processing. In this study, a strain of Enterococcus durans, originally isolated from a milking machine surface, was firstly studied for its biofilm formation potential on plastic and stainless steel supports. The strain was found to be a biofilm producer either at 25, 30 or 37 °C on polystyrene microtitre plates, with a best adherence level observed at 25 °C. En. durans showed a strong adhesion to stainless steel AISI-304. Antibacterial and anti-adherence activities of En. durans were tested against four foodborne pathogens (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853 and Listeria innocua CLIP 74915) which were shown as biofilm producers on both plastic and stainless steel. En. durans cells and cell-free culture supernatant showed a significant (P < 0.05) inhibition potential of the pathogens either on solid media or in broth co-cultures. Characterization of the antibacterial substances indicated their proteinaceous nature which assigned them most probably to bacteriocins group. PMID:25466409

  10. Plasmodium chabaudi-Infected Erythrocytes Adhere to CD36 and Bind to Microvascular Endothelial Cells in an Organ-Specific Way

    PubMed Central

    Mota, Maria M.; Jarra, William; Hirst, Elizabeth; Patnaik, Pradeep K.; Holder, Anthony A.

    2000-01-01

    Adherence of erythrocytes infected with Plasmodium falciparum to microvascular endothelial cells (sequestration) is considered to play an important role in parasite virulence and pathogenesis. However, the real importance of sequestration for infection and disease has never been fully assessed. The absence of an appropriate in vivo model for sequestration has been a major barrier. We have examined the rodent malaria parasite Plasmodium chabaudi chabaudi AS in mice as a potential model. Erythrocytes infected with this parasite adhere in vitro to purified CD36, a critical endothelium receptor for binding P. falciparum-infected erythrocytes. P. c. chabaudi-infected erythrocytes adhere in vitro to endothelial cells in a gamma interferon-dependent manner, suggesting the involvement of additional adhesion molecules in the binding process, as is also the case with P. falciparum-infected cells. Furthermore, plasma or sera from infected and hyperimmune mice, respectively, have the ability to block binding of infected erythrocytes to endothelial cells. In vivo, erythrocytes containing mature P. c. chabaudi parasites are sequestered from the peripheral circulation. Sequestration is organ specific, occurring primarily in the liver, although intimate contact between infected erythrocytes and endothelial cells is also observed in the spleen and brain. The results are discussed in the context of the use of this model to study (i) the relationship between endothelial cell activation and the level of sequestration and (ii) the primary function of sequestration in malaria infection. PMID:10858230

  11. Involvement of the heparan sulphate-binding proteins of Helicobacter pylori in its adherence to HeLa S3 and Kato III cell lines.

    PubMed

    Guzman-Murillo, M A; Ruiz-Bustos, E; Ho, B; Ascencio, F

    2001-04-01

    To determine whether Helicobacter pylori heparan sulphate-binding proteins (HSBPs) are involved in the adherence of H. pylori to HeLa and Kato III cells, monolayers were pre-incubated with various preparations and concentrations of H. pylori HSBPs at 37 degrees C, washed and then challenged with bacteria. HSBPs did not prevent but enhanced H. pylori adherence. However, challenging cultured cells with H. pylori previously incubated with rabbit anti-HSBP IgG resulted in significant inhibition of bacterial adherence. These data demonstrate that the extracellular HSBP plays an important role in promoting H. pylori attachment to Kato III and HeLa S3 cells, that adhesion of H. pylori to Kato III and HeLa S3 cells is promoted by the presence of the 71.5-kDa extracellular HSBP and that rabbit polyclonal antibodies against this HSBP can inhibit adhesion of H. pylori to the cultured cell lines and detach cell-bound H. pylori. PMID:11289517

  12. Combinatorial control of heterogeneous cell populations

    NASA Astrophysics Data System (ADS)

    Piermarocchi, C.; Duxbury, P.; Paternostro, G.; Feala, J.; Tiziani, S.; Axelrod, J.; Chaudhury, A.; Choi, J.; McCulloch, A.; Cortes, J.

    2010-03-01

    In medicine, a recent pharmacological approach involves systematic discovery of combinatorial therapies, in which different drugs are simultaneously used to control different pathways associated with a cellular function. This control must occur with minimal response in other non-target cells exposed to treatment, i.e. it has to be selective. We have investigated the statistics of selective control of the human apoptosis (cell death) signaling network. We have built a model for a heterogeneous population of cells, characterized by a signaling network with identical topology, but having different link strengths. The control of the life/death signal is realized by acting with external perturbations, modeling the effect of drugs, on the nodes and on the signaling flow. Concepts from statistical physics and information theory, including entropy, frustration, and non-linearity have been used to characterize the general properties of selective control. This knowledge was used as a guide in designing algorithms for identifying selective perturbations. Some of these algorithms have been implemented in vitro in high throughput experiments on real cell lines where a large number of combinations of different drugs can be tested.

  13. AHCC Activation and Selection of Human Lymphocytes via Genotypic and Phenotypic Changes to an Adherent Cell Type: A Possible Novel Mechanism of T Cell Activation

    PubMed Central

    Olamigoke, Loretta; Mansoor, Elvedina; Mann, Vivek; Ellis, Ivory; Okoro, Elvis; Wakame, Koji; Fuji, Hajime; Kulkarni, Anil; Francoise Doursout, Marie; Sundaresan, Alamelu

    2015-01-01

    Active Hexose Correlated Compound (AHCC) is a fermented mushroom extract and immune supplement that has been used to treat a wide range of health conditions. It helps in augmentation of the natural immune response and affects immune cell activation and outcomes. The goal of this project was to study and understand the role and mechanisms of AHCC supplementation in the prevention of immunosuppression through T cell activation. The method described here involves “in vitro” culturing of lymphocytes, exposing them to different concentrations of AHCC (0 μg/mL, 50 μg/mL, 100 μg/mL, 250 μg/mL, and 500 μg/mL) at 0 hours. Interestingly, clumping and aggregation of the cells were seen between 24 and 72 hours of incubation. The cells lay down extracellular matrix, which become adherent, and phenotypical changes from small rounded lymphocytes to large macrophage-like, spindle shaped, elongated, fibroblast-like cells even beyond 360 hours were observed. These are probably translated from genotypic changes in the cells since the cells propagate for at least 3 to 6 generations (present observations). RNA isolated was subjected to gene array analysis. We hypothesize that cell adhesion is an activation and survival pathway in lymphocytes and this could be the mechanism of AHCC activation in human lymphocytes. PMID:26788109

  14. In vitro and in vivo properties of distinct populations of amniotic fluid mesenchymal progenitor cells

    PubMed Central

    Roubelakis, Maria G; Bitsika, Vasiliki; Zagoura, Dimitra; Trohatou, Ourania; Pappa, Kalliopi I; Makridakis, Manousos; Antsaklis, Aristidis; Vlahou, Antonia; Anagnou, Nicholas P

    2011-01-01

    Abstract Human mesenchymal progenitor cells (MPCs) are considered to be of great promise for use in tissue repair and regenerative medicine. MPCs represent multipotent adherent cells, able to give rise to multiple mesenchymal lineages such as osteoblasts, adipocytes or chondrocytes. Recently, we identified and characterized human second trimester amniotic fluid (AF) as a novel source of MPCs. Herein, we found that early colonies of AF-MPCs consisted of two morphologically distinct adherent cell types, termed as spindle-shaped (SS) and round-shaped (RS). A detailed analysis of these two populations showed that SS-AF-MPCs expressed CD90 antigen in a higher level and exhibited a greater proliferation and differentiation potential. To characterize better the molecular identity of these two populations, we have generated a comparative proteomic map of SS-AF-MPCs and RS-AF-MPCs, identifying 25 differentially expressed proteins and 10 proteins uniquely expressed in RS-AF-MPCs. Furthermore, SS-AF-MPCs exhibited significantly higher migration ability on extracellular matrices, such as fibronectin and laminin in vitro, compared to RS-AF-MPCs and thus we further evaluated SS-AF-MPCs for potential use as therapeutic tools in vivo. Therefore, we tested whether GFP-lentiviral transduced SS-AF-MPCs retained their stem cell identity, proliferation and differentiation potential. GFP-SS-AF-MPCs were then successfully delivered into immunosuppressed mice, distributed in different tissues and survived longterm in vivo. In summary, these results demonstrated that AF-MPCs consisted of at least two different MPC populations. In addition, SS-AF-MPCs, isolated based on their colony morphology and CD90 expression, represented the only MPC population that can be expanded easily in culture and used as an efficient tool for future in vivo therapeutic applications. PMID:21166769

  15. New approaches for understanding the nuclear force balance in living, adherent cells.

    PubMed

    Neelam, Srujana; Dickinson, Richard B; Lele, Tanmay P

    2016-02-01

    Cytoskeletal forces are transmitted to the nucleus to position and shape it. Linkages mediated by the LINC (linker of nucleoskeleton and cytoskeleton) complex transfer these forces to the nuclear envelope. Nuclear position and shape can be thought to be determined by a balance of cytoskeletal forces generated by microtubule motors that shear the nuclear surface, actomyosin forces that can pull, push and shear the nucleus, and intermediate filaments that may passively resist nuclear decentering and deformation. Parsing contributions of these different forces to nuclear mechanics is a very challenging task. Here we review new approaches that can be used in living cells to probe and understand the nuclear force balance.

  16. Pilus phase variation switches gonococcal adherence to invasion by caveolin-1-dependent host cell signaling.

    PubMed

    Faulstich, Michaela; Böttcher, Jan-Peter; Meyer, Thomas F; Fraunholz, Martin; Rudel, Thomas

    2013-01-01

    Many pathogenic bacteria cause local infections but occasionally invade into the blood stream, often with fatal outcome. Very little is known about the mechanism underlying the switch from local to invasive infection. In the case of Neisseria gonorrhoeae, phase variable type 4 pili (T4P) stabilize local infection by mediating microcolony formation and inducing anti-invasive signals. Outer membrane porin PorB(IA), in contrast, is associated with disseminated infection and facilitates the efficient invasion of gonococci into host cells. Here we demonstrate that loss of pili by natural pilus phase variation is a prerequisite for the transition from local to invasive infection. Unexpectedly, both T4P-mediated inhibition of invasion and PorB(IA)-triggered invasion utilize membrane rafts and signaling pathways that depend on caveolin-1-Y14 phosphorylation (Cav1-pY14). We identified p85 regulatory subunit of PI3 kinase (PI3K) and phospholipase Cγ1 as new, exclusive and essential interaction partners for Cav1-pY14 in the course of PorBIA-induced invasion. Active PI3K induces the uptake of gonococci via a new invasion pathway involving protein kinase D1. Our data describe a novel route of bacterial entry into epithelial cells and offer the first mechanistic insight into the switch from local to invasive gonococcal infection. PMID:23717204

  17. Ormocomp-modified glass increases collagen binding and promotes the adherence and maturation of human embryonic stem cell-derived retinal pigment epithelial cells.

    PubMed

    Käpylä, Elli; Sorkio, Anni; Teymouri, Shokoufeh; Lahtonen, Kimmo; Vuori, Leena; Valden, Mika; Skottman, Heli; Kellomäki, Minna; Juuti-Uusitalo, Kati

    2014-12-01

    In in vitro live-cell imaging, it would be beneficial to grow and assess human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells on thin, transparent, rigid surfaces such as cover glasses. In this study, we assessed how the silanization of glass with 3-aminopropyltriethoxysilane (APTES), 3-(trimethoxysilyl)propyl methacrylate (MAPTMS), or polymer-ceramic material Ormocomp affects the surface properties, protein binding, and maturation of hESC-RPE cells. The surface properties were studied by contact angle measurements, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and a protein binding assay. The cell adherence and proliferation were evaluated by culturing hESCRPE cells on collagen IV-coated untreated or silanized surfaces for 42 days. The Ormocomp treatment significantly increased the hydrophobicity and roughness of glass surfaces compared to the APTES and MAPTMS treatments. The XPS results indicated that the Ormocomp treatment changes the chemical composition of the glass surface by increasing the carbon content and the number of C-O/═O bonds. The protein-binding test confirmed that the Ormocomp-treated surfaces bound more collagen IV than did APTES- or MAPTMS-treated surfaces. All of the silane treatments increased the number of cells: after 42 days of culture, Ormocomp had 0.38, APTES had 0.16, MAPTMS had 0.19, and untreated glass had only 0.062, all presented as million cells cm(-2). There were no differences in cell numbers compared to smoother to rougher Ormocomp surfaces, suggesting that the surface chemistry and, more specifically, the collagen binding in combination with Ormocomp are beneficial to hESC-RPE cell culture. This study clearly demonstrates that Ormocomp treatment combined with collagen coating significantly increases hESC-RPE cell attachment compared to commonly used silanizing agents APTES and MAPTMS. Ormocomp silanization could thus enable the use of microscopic live cell imaging methods for h

  18. Performance characteristics of two automated solid-phase red cell adherence systems for pretransfusion antibody screening: a cautionary tale.

    PubMed

    Quillen, K; Caron, J; Murphy, K

    2012-01-01

    Out institution has implemented two instruments, the Galileo and the Echo, that use different solid-phase red cell adherence assays for antibody screening in pretransfusion compatibility testing.During the initial implementation of these two instruments, we noticed very different problems: falsely positive results on the Galileo, and falsely negative results and lack of reproducibility on the Echo. Comparison of falsely positive antibody screen results from approximately equivalent numbers of samples run on the Galileo and samples tested by standard manual tube technique using low-ionic-strength saline enhancement showed a false-positive rate of 1.4 percent on the Galileo (defined as a positive screen with a negative panel). Testing using the Echo identified four cases of falsely negative antibody screens, (defined as a negative screen on a patient sample subsequently shown to be positive by the same method). In addition, we note a lack of reproducibility on the Echo, which emphasizes the importance of replicate testing during validation of automated antibody screening platforms.

  19. Detection of drug-dependent, platelet-reactive antibodies by solid-phase red cell adherence assays.

    PubMed

    Leach, M F; Cooper, L K; AuBuchon, J P

    1997-06-01

    We developed a simple modification of the solid-phase red cell adherence (SPRCA) assay system that can be used to identify drug-dependent platelet antibodies (DDPAs) reactive by either the hapten or immune complex reaction mechanisms. Between January 1994 and August 1996 we tested sera from 173 patients [123 (71%) with unexplained thrombocytopenia and 50 (29%) because of poor responses to platelet transfusions not explicable by alloimmunization or the clinical situation] for DDPAs possibly associated with the receipt of 61 different drugs. We correlated positive results with patients' clinical courses. DDPAs were identified in samples from 138 (80%) of the patients tested. Antibodies reactive only by the hapten mechanism were identified in 51 (37%) of those sera exhibiting positive reactions. The clinical courses of 108 (78%) patients were evaluable. Discontinuation of the implicated drug(s) resulted in prompt (<5 d) resolution of the thrombocytopenia or improvement in response to transfusion in all of these patients. In four cases thrombocytopenia returned upon re-exposure to the implicated drug. This adaptation of SPRCA provides a simple means of investigating the possibility of DDPAs and documents a higher frequency of these antibodies than has previously been suspected.

  20. Detection of drug-dependent platelet antibodies by use of solid-phase red cell adherence techniques.

    PubMed

    Leach, M F; Cooper, L K; Aubuchon, J P

    1995-01-01

    Many drugs have been reported to cause drug-dependent thrombocytopenia, either by the immune complex or by hapten mechanisms. Testing for the presence of these platelet antibodies has not been considered feasible for transfusion services because their presence was thought to be rare, and their detection involved complex and costly methods. We have developed a new technique for detection of these antibodies that can be performed without the need for specialized and expensive instrumentation. A solid-phase red cell adherence assay was used to detect drug-dependent platelet antibodies active by either the immune complex or the hapten mechanism. Three cases were evaluated for the presence of drug-dependent platelet antibodies. Two patients presented with thrombocytopenia that could not be attributed to other causes. The third case was evaluated for the presence of drug-dependent antibodies after poor responses to platelet transfusions. In these three cases, discontinuation of the implicated drugs, i.e., porcine heparin, quinine sulfate, amoxicillin, Bactrim, and albuterol, was followed by a correction of thrombocytopenia or improved platelet transfusion response within 72 hours. This test methodology and protocol has proven very useful in avoiding transfusions with little likelihood of benefit, and in identifying drugs interfering with platelet recovery or survival. Further investigations with this technique may expand our knowledge of the capability of this technique and of the observed frequency of drug-related immunologic platelet destruction.

  1. Cell Wall-Associated Protein Antigens of Streptococcus salivarius: Purification, Properties, and Function in Adherence

    PubMed Central

    Weerkamp, Anton H.; Jacobs, Ton

    1982-01-01

    Three cell wall-associated protein antigens (antigens b, c, and d) were isolated from mutanolysin-solubilized cell walls of Streptococcus salivarius HB and purified to apparent homogeneity by a combination of ion-exchange chromatography, gel filtration, and immunoadsorption chromatography. Antigens b and c were also isolated from culture supernatants. Antigen b consisted of more than 80% protein and had an apparent molecular weight as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 320,000. Antigen c consisted of 57% protein, about 30% neutral sugar, and about 13% amino sugar, and its glycoprotein nature was confirmed by specific staining techniques. During sodium dodecyl sulfate-polyacrylamide gel electrophoresis antigen c resolved into two or more bands, depending on the source or the isolation procedure, in the molecular weight range from 220,000 to 280,000. Antigen d consisted of 95% protein and was observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as two bands with molecular weights of 129,000 and 121,000. Under nondenaturing conditions all three antigens had molecular weights in the range from 1 × 106 to 3 × 106 as determined by gel filtration. The amino acid compositions of antigens b, c, and d were characterized by low amounts of basic amino acids and relatively high levels of nonpolar amino acids. Among oral streptococcal species antigens b and c were virtually restricted to strains of S. salivarius and most often to serotype I strains. Antigen b was recognized as the factor that mediates coaggregation of S. salivarius with Veillonella strains. The purified protein retained its biological activity. Antigen c could be linked to functions relating to adhesion of the streptococci to host tissues on the basis of its absence in mutant strains and blocking by specific antisera. The purified molecule had no detectable biological activity. Antigen d could not be linked to an established adhesion function. Images

  2. Both enzymatic and non-enzymatic properties of heat-labile enterotoxin are responsible for LT-enhanced adherence of enterotoxigenic Escherichia coli to porcine IPEC-J2 cells.

    PubMed

    Fekete, Peter Z; Mateo, Kristina S; Zhang, Weiping; Moxley, Rodney A; Kaushik, Radhey S; Francis, David H

    2013-06-28

    Previous studies in piglets indicate that heat labile enterotoxin (LT) expression enhances intestinal colonization by K88 adhesin-producing enterotoxigenic Escherichia coli (ETEC) as wild-type ETEC adhered to intestinal epithelium in substantially greater numbers than did non-toxigenic constructs. Enzymatic activity of the toxin was also shown to contribute to the adhesion of ETEC and non-ETEC bacteria to epithelial cells in culture. To further characterize the contribution of LT to host cell adhesion, a nontoxigenic, K88-producing E. coli was transformed with either the gene encoding for LT holotoxin, a catalytically-attenuated form of the toxin [LT(R192G)], or LTB subunits, and resultant changes in bacterial adherence to IPEC-J2 porcine intestinal epithelial cells were measured. Strains expressing LT holotoxin or mutants were able to adhere in significantly higher numbers to IPEC-J2 cells than was an isogenic, toxin-negative construct. LT+ strains were also able to significantly block binding of a wild-type LT+ ETEC strain to IPEC-J2 cells. Adherence of isogenic strains to IPEC-J2 cells was unaltered by cycloheximide treatment, suggesting that LT enhances ETEC adherence to IPEC-J2 cells independent of host cell protein synthesis. However, pretreating IPEC-J2 cells with LT promoted adherence of negatively charged latex beads (a surrogate for bacteria which carry a negative change), which adherence was inhibited by cycloheximide, suggesting LT may induce a change in epithelial cell membrane potential. Overall, these data suggest that LT may enhance ETEC adherence by promoting an association between LTB and epithelial cells, and by altering the surface charge of the host plasma membrane to promote non-specific adherence.

  3. Both enzymatic and non-enzymatic properties of heat-labile enterotoxin are responsible for LT-enhanced adherence of enterotoxigenic Escherichia coli to porcine IPEC-J2 cells.

    PubMed

    Fekete, Peter Z; Mateo, Kristina S; Zhang, Weiping; Moxley, Rodney A; Kaushik, Radhey S; Francis, David H

    2013-06-28

    Previous studies in piglets indicate that heat labile enterotoxin (LT) expression enhances intestinal colonization by K88 adhesin-producing enterotoxigenic Escherichia coli (ETEC) as wild-type ETEC adhered to intestinal epithelium in substantially greater numbers than did non-toxigenic constructs. Enzymatic activity of the toxin was also shown to contribute to the adhesion of ETEC and non-ETEC bacteria to epithelial cells in culture. To further characterize the contribution of LT to host cell adhesion, a nontoxigenic, K88-producing E. coli was transformed with either the gene encoding for LT holotoxin, a catalytically-attenuated form of the toxin [LT(R192G)], or LTB subunits, and resultant changes in bacterial adherence to IPEC-J2 porcine intestinal epithelial cells were measured. Strains expressing LT holotoxin or mutants were able to adhere in significantly higher numbers to IPEC-J2 cells than was an isogenic, toxin-negative construct. LT+ strains were also able to significantly block binding of a wild-type LT+ ETEC strain to IPEC-J2 cells. Adherence of isogenic strains to IPEC-J2 cells was unaltered by cycloheximide treatment, suggesting that LT enhances ETEC adherence to IPEC-J2 cells independent of host cell protein synthesis. However, pretreating IPEC-J2 cells with LT promoted adherence of negatively charged latex beads (a surrogate for bacteria which carry a negative change), which adherence was inhibited by cycloheximide, suggesting LT may induce a change in epithelial cell membrane potential. Overall, these data suggest that LT may enhance ETEC adherence by promoting an association between LTB and epithelial cells, and by altering the surface charge of the host plasma membrane to promote non-specific adherence. PMID:23517763

  4. Atomic Force Microscopy Reveals a Role for Endothelial Cell ICAM-1 Expression in Bladder Cancer Cell Adherence

    PubMed Central

    Laurent, Valérie M.; Duperray, Alain; Sundar Rajan, Vinoth; Verdier, Claude

    2014-01-01

    Cancer metastasis is a complex process involving cell-cell interactions mediated by cell adhesive molecules. In this study we determine the adhesion strength between an endothelial cell monolayer and tumor cells of different metastatic potentials using Atomic Force Microscopy. We show that the rupture forces of receptor-ligand bonds increase with retraction speed and range between 20 and 70 pN. It is shown that the most invasive cell lines (T24, J82) form the strongest bonds with endothelial cells. Using ICAM-1 coated substrates and a monoclonal antibody specific for ICAM-1, we demonstrate that ICAM-1 serves as a key receptor on endothelial cells and that its interactions with ligands expressed by tumor cells are correlated with the rupture forces obtained with the most invasive cancer cells (T24, J82). For the less invasive cancer cells (RT112), endothelial ICAM-1 does not seem to play any role in the adhesion process. Moreover, a detailed analysis of the distribution of rupture forces suggests that ICAM-1 interacts preferentially with one ligand on T24 cancer cells and with two ligands on J82 cancer cells. Possible counter receptors for these interactions are CD43 and MUC1, two known ligands for ICAM-1 which are expressed by these cancer cells. PMID:24857933

  5. Non-adherent culture induces paclitaxel resistance in H460 lung cancer cells via ERK-mediated up-regulation of βIVa-tubulin.

    PubMed

    Atjanasuppat, Korakot; Lirdprapamongkol, Kriengsak; Jantaree, Phatcharida; Svasti, Jisnuson

    2015-10-23

    Circulating tumor cells (CTCs) are metastasizing epithelial cancer cells that adapt to survive when floating in bloodstream during metastasis. This condition can be mimicked in vitro by using non-adherent cell culture. The chemosensitivity of CTCs appears to correlate with the response of metastatic cancer patients to therapy, but chemoresistance is also frequently observed in advanced stage cancer patients, who have never previously received chemotherapy. We hypothesize that adaptation of epithelial cancer cells to become floating CTCs could lead to development of chemoresistance. Here, we explore whether chemoresistance is induced in epithelial cancer cells when cultured under non-adherent conditions. Increased paclitaxel-specific resistance was observed in floating cells compared to attached cells in H460, MCF-7, and HepG2 human cancer cell lines, by 15.6-, 3.9-, and 2.6-fold increases in IC50 values, respectively. qRT-PCR analysis showed that a paclitaxel-resistant β-tubulin isotype, βIVa-tubulin, was the most up-regulated gene compared with other β-tubulin isotypes in H460 floating cells, concomitant with elevated ERK activation. ERK inhibitor treatment could attenuate the up-regulation of βIVa-tubulin, and decreased the paclitaxel resistance of H460 floating cells, even though other β-tubulin isotypes were up-regulated when the ERK activation was blocked. In conclusion, we show induction of paclitaxel resistance in epithelial cancer cells, when floating in non-adherent culture, and this might occur with CTCs of cancer patients.

  6. Three-dimensional imaging of adherent cells using FIB/SEM and STEM.

    PubMed

    Villinger, Clarissa; Schauflinger, Martin; Gregorius, Heiko; Kranz, Christine; Höhn, Katharina; Nafeey, Soufi; Walther, Paul

    2014-01-01

    In this chapter we describe three different approaches for three-dimensional imaging of electron microscopic samples: serial sectioning transmission electron microscopy (TEM), scanning transmission electron microscopy (STEM) tomography, and focused ion beam/scanning electron microscopy (FIB/SEM) tomography. With these methods, relatively large volumes of resin-embedded biological structures can be analyzed at resolutions of a few nm within a reasonable expenditure of time. The traditional method is serial sectioning and imaging the same area in all sections. Another method is TEM tomography that involves tilting a section in the electron beam and then reconstruction of the volume by back projection of the images. When the scanning transmission (STEM) mode is used, thicker sections (up to 1 μm) can be analyzed. The third approach presented here is focused ion beam/scanning electron microscopy (FIB/SEM) tomography, in which a sample is repeatedly milled with a focused ion beam (FIB) and each newly produced block face is imaged with the scanning electron microscope (SEM). This process can be repeated ad libitum in arbitrary small increments allowing 3D analysis of relatively large volumes such as eukaryotic cells. We show that resolution of this approach is considerably improved when the secondary electron signal is used. However, the most important prerequisite for three-dimensional imaging is good specimen preparation. For all three imaging methods, cryo-fixed (high-pressure frozen) and freeze-substituted samples have been used.

  7. Surfactant protein D inhibits adherence of uropathogenic Escherichia coli to the bladder epithelial cells and the bacterium-induced cytotoxicity: a possible function in urinary tract.

    PubMed

    Kurimura, Yuichiro; Nishitani, Chiaki; Ariki, Shigeru; Saito, Atsushi; Hasegawa, Yoshihiro; Takahashi, Motoko; Hashimoto, Jiro; Takahashi, Satoshi; Tsukamoto, Taiji; Kuroki, Yoshio

    2012-11-16

    The adherence of uropathogenic Escherichia coli (UPEC) to the host urothelial surface is the first step for establishing UPEC infection. Uroplakin Ia (UPIa), a glycoprotein expressed on bladder urothelium, serves as a receptor for FimH, a lectin located at bacterial pili, and their interaction initiates UPEC infection. Surfactant protein D (SP-D) is known to be expressed on mucosal surfaces in various tissues besides the lung. However, the functions of SP-D in the non-pulmonary tissues are poorly understood. The purposes of this study were to investigate the possible function of SP-D expressed in the bladder urothelium and the mechanisms by which SP-D functions. SP-D was expressed in human bladder mucosa, and its mRNA was increased in the bladder of the UPEC infection model in mice. SP-D directly bound to UPEC and strongly agglutinated them in a Ca(2+)-dependent manner. Co-incubation of SP-D with UPEC decreased the bacterial adherence to 5637 cells, the human bladder cell line, and the UPEC-induced cytotoxicity. In addition, preincubation of SP-D with 5637 cells resulted in the decreased adherence of UPEC to the cells and in a reduced number of cells injured by UPEC. SP-D directly bound to UPIa and competed with FimH for UPIa binding. Consistent with the in vitro data, the exogenous administration of SP-D inhibited UPEC adherence to the bladder and dampened UPEC-induced inflammation in mice. These results support the conclusion that SP-D can protect the bladder urothelium against UPEC infection and suggest a possible function of SP-D in urinary tract.

  8. Anti-Retroviral Lectins Have Modest Effects on Adherence of Trichomonas vaginalis to Epithelial Cells In Vitro and on Recovery of Tritrichomonas foetus in a Mouse Vaginal Model

    PubMed Central

    Chatterjee, Aparajita; Ratner, Daniel M.; Ryan, Christopher M.; Johnson, Patricia J.; O’Keefe, Barry R.; Secor, W. Evan; Anderson, Deborah J.; Robbins, Phillips W.; Samuelson, John

    2015-01-01

    Trichomonas vaginalis causes vaginitis and increases the risk of HIV transmission by heterosexual sex, while Tritrichomonas foetus causes premature abortion in cattle. Our goals were to determine the effects, if any, of anti-retroviral lectins, which are designed to prevent heterosexual transmission of HIV, on adherence of Trichomonas to ectocervical cells and on Tritrichomonas infections in a mouse model. We show that Trichomonas Asn-linked glycans (N-glycans), like those of HIV, bind the mannose-binding lectin (MBL) that is part of the innate immune system. N-glycans of Trichomonas and Tritrichomonas bind anti-retroviral lectins (cyanovirin-N and griffithsin) and the 2G12 monoclonal antibody, each of which binds HIV N-glycans. Binding of cyanovirin-N appears to be independent of susceptibility to metronidazole, the major drug used to treat Trichomonas. Anti-retroviral lectins, MBL, and galectin-1 cause Trichomonas to self-aggregate and precipitate. The anti-retroviral lectins also increase adherence of ricin-resistant mutants, which are less adherent than parent cells, to ectocervical cell monolayers and to organotypic EpiVaginal tissue cells. Topical application of either anti-retroviral lectins or yeast N-glycans decreases by 40 to 70% the recovery of Tritrichomonas from the mouse vagina. These results, which are explained by a few simple models, suggest that the anti-retroviral lectins have a modest potential for preventing or treating human infections with Trichomonas. PMID:26252012

  9. High prevalence of side population in human cancer cell lines

    PubMed Central

    Boesch, Maximilian; Zeimet, Alain G.; Fiegl, Heidi; Wolf, Barbara; Huber, Julia; Klocker, Helmut; Gastl, Guenther

    2016-01-01

    Cancer cell lines are essential platforms for performing cancer research on human cells. We here demonstrate that, across tumor entities, human cancer cell lines harbor minority populations of putative stem-like cells, molecularly defined by dye extrusion resulting in the side population phenotype. These findings establish a heterogeneous nature of human cancer cell lines and argue for their stem cell origin. This should be considered when interpreting research involving these model systems. PMID:27226981

  10. The use of cell phone support for non-adherent HIV-infected youth and young adults: an initial randomized and controlled intervention trial.

    PubMed

    Belzer, Marvin E; Naar-King, Sylvie; Olson, Johanna; Sarr, Moussa; Thornton, Sarah; Kahana, Shoshana Y; Gaur, Aditya H; Clark, Leslie F

    2014-04-01

    This randomized behavioral trial examined whether youth living with HIV (YLH) receiving cell-phone support with study funded phone plans, demonstrated improved adherence and viral control during the 24 week intervention and 24 weeks post-intervention compared to controls. Monday through Friday phone calls confirmed medications were taken, provided problem-solving support, and referred to services to address adherence barriers. Of 37 participants (ages 15-24), 62 % were male and 70 % were African American. Self-reported adherence was significantly higher in the intervention group compared to the control at 24 and 48 weeks for the past month (P = 0.007) and log 10 HIV VL was significantly lower at both 24 weeks (2.82 versus 4.52 P = 0.002) and 48 weeks (3.23 versus 4.23 P = 0.043). Adherence and viral load showed medium to large effect sizes across the 48 week study. This is the first study to demonstrate sustained clinically significant reductions in HIV VL using youth friendly technology.

  11. The Use of Cell Phone Support for Non-adherent HIV-Infected Youth and Young Adults: An Initial Randomized and Controlled Intervention Trial

    PubMed Central

    Belzer, Marvin E.; Naar-King, Sylvie; Olson, Johanna; Sarr, Moussa; Thornton, Sarah; Kahana, Shoshana Y.; Gaur, Aditya H.; Clark, Leslie F.

    2014-01-01

    This randomized behavioral trial examined whether youth living with HIV (YLH) receiving cell-phone support with study funded phone plans, demonstrated improved adherence and viral control during the 24 week intervention and 24 weeks post-intervention compared to controls. Monday through Friday phone calls confirmed medications were taken, provided problem-solving support, and referred to services to address adherence barriers. Of 37 participants (ages 15–24), 62 % were male and 70 % were African American. Self-reported adherence was significantly higher in the intervention group compared to the control at 24 and 48 weeks for the past month (P = 0.007) and log 10 HIV VL was significantly lower at both 24 weeks (2.82 versus 4.52 P = 0.002) and 48 weeks (3.23 versus 4.23 P = 0.043). Adherence and viral load showed medium to large effect sizes across the 48 week study. This is the first study to demonstrate sustained clinically significant reductions in HIV VL using youth friendly technology. PMID:24271347

  12. Low adherent cancer cell subpopulations are enriched in tumorigenic and metastatic epithelial-to-mesenchymal transition-induced cancer stem-like cells

    PubMed Central

    Morata-Tarifa, Cynthia; Jiménez, Gema; García, María A.; Entrena, José M.; Griñán-Lisón, Carmen; Aguilera, Margarita; Picon-Ruiz, Manuel; Marchal, Juan A.

    2016-01-01

    Cancer stem cells are responsible for tumor progression, metastasis, therapy resistance and cancer recurrence, doing their identification and isolation of special relevance. Here we show that low adherent breast and colon cancer cells subpopulations have stem-like properties. Our results demonstrate that trypsin-sensitive (TS) breast and colon cancer cells subpopulations show increased ALDH activity, higher ability to exclude Hoechst 33342, enlarged proportion of cells with a cancer stem-like cell phenotype and are enriched in sphere- and colony-forming cells in vitro. Further studies in MDA-MB-231 breast cancer cells reveal that TS subpopulation expresses higher levels of SLUG, SNAIL, VIMENTIN and N-CADHERIN while show a lack of expression of E-CADHERIN and CLAUDIN, being this profile characteristic of the epithelial-to-mesenchymal transition (EMT). The TS subpopulation shows CXCL10, BMI-1 and OCT4 upregulation, differing also in the expression of several miRNAs involved in EMT and/or cell self-renewal such as miR-34a-5p, miR-34c-5p, miR-21-5p, miR-93-5p and miR-100-5p. Furthermore, in vivo studies in immunocompromised mice demonstrate that MDA-MB-231 TS cells form more and bigger xenograft tumors with shorter latency and have higher metastatic potential. In conclusion, this work presents a new, non-aggressive, easy, inexpensive and reproducible methodology to isolate prospectively cancer stem-like cells for subsequent biological and preclinical studies. PMID:26752044

  13. Truncated enterohemorrhagic Escherichia coli (EHEC) O157:H7 intimin (EaeA) fusion proteins promote adherence of EHEC strains to HEp-2 cells.

    PubMed Central

    McKee, M L; O'Brien, A D

    1996-01-01

    Intimin, the product of the eaeA gene in enterohemorrhagic Escherichia coli O157:H7 (EHEC), is required for intimate adherence of these organisms to tissue culture cells and formation of the attaching and effacing lesion in the gnotobiotic pig. Because of the importance of intimin in the pathogenesis of EHEC O157:H7 infection in this animal model, we began a structure-function analysis of EaeA. For this purpose, we constructed amino-terminal fusions of the intimin protein with six histidine residues to form two independent fusions. The longer fusion, RIHisEae, contained 900 of the 935 predicted amino acids and included all but the extreme amino terminus. The second fusion, RVHdHisEae, consisted of the carboxyl two-thirds of the protein. Purified extracts of either construct enhanced binding of wild-type 86-24 to HEp-2 cells and conferred HEp-2 cell adherence on 86-24eaeDelta10, an eaeA deletion mutant, and B2F1, an EHEC O91:1-121 eaeA mutant strain. When 86-24eaeDelta10 was transformed with either of the plasmids encoding the intimin fusion proteins, the transformant behaved like the wild-type parent strain and displayed localized adherence to HEp-2 cells, with positive fluorescent-actin staining. In addition, polyclonal antisera raised against RIHisEae reacted with both fusion constructs and recognized an outer membrane protein of the same mass as intimin (97 kDa) in EHEC and enteropathogenic E. coli but not E. coli K-12. The intimin-specific antisera also blocked adherence of EHEC to HEp-2 cells. Thus, intimin (i) is a 97-kDa outer membrane protein in EHEC that serves as a requisite adhesin for attachment of the bacteria to epithelial cells, even when the protein is truncated by one-third at its amino terminus and (ii) can be added exogenously to specifically facilitate HEp-2 cell adherence of EHEC but not E. coli K-12. PMID:8675331

  14. P2Y2 nucleotide receptor activation up-regulates vascular cell adhesion molecule-1 [corrected] expression and enhances lymphocyte adherence to a human submandibular gland cell line.

    PubMed

    Baker, Olga J; Camden, Jean M; Rome, Danny E; Seye, Cheikh I; Weisman, Gary A

    2008-01-01

    Sjögren's syndrome (SS) is a chronic inflammatory autoimmune disease that causes salivary and lacrimal gland tissue destruction resulting in impaired secretory function. Although lymphocytic infiltration of salivary epithelium is associated with SS, the mechanisms involved have not been adequately elucidated. Our previous studies have shown that the G protein-coupled P2Y2 nucleotide receptor (P2Y2R) is up-regulated in response to damage or stress of salivary gland epithelium, and in salivary glands of the NOD.B10 mouse model of SS-like autoimmune exocrinopathy. Additionally, we have shown that P2Y2R activation up-regulates vascular cell adhesion molecule-1 (VCAM-1) expression in endothelial cells leading to the binding of monocytes. The present study demonstrates that activation of the P2Y2R in dispersed cell aggregates from rat submandibular gland (SMG) and in human submandibular gland ductal cells (HSG) up-regulates the expression of VCAM-1. Furthermore, P2Y2R activation mediated the up-regulation of VCAM-1 expression in HSG cells leading to increased adherence of lymphocytic cells. Inhibitors of EGFR phosphorylation and metalloprotease activity abolished P2Y2R-mediated VCAM-1 expression and decreased lymphocyte binding to HSG cells. Moreover, silencing of EGFR expression abolished UTP-induced VCAM-1 up-regulation in HSG cells. These results suggest that P2Y2R activation in salivary gland cells increases the EGFR-dependent expression of VCAM-1 and the binding of lymphocytes, a pathway relevant to inflammation associated with SS.

  15. P2Y2 Nucleotide Receptor Activation Up-regulates Vascular Cell Adhesion Molecular-1 Expression and Enhances Lymphocyte Adherence to a Human Submandibular Gland Cell Line

    PubMed Central

    Baker, Olga J.; Camden, Jean M.; Rome, Danny E.; Seye, Cheikh I.; Weisman, Gary A.

    2007-01-01

    Sjögren’s syndrome (SS) is a chronic inflammatory autoimmune disease that causes salivary and lacrimal gland tissue destruction resulting in impaired secretory function. Although lymphocytic infiltration of salivary epithelium is associated with SS, the mechanisms involved have not been adequately elucidated. Our previous studies have shown that the G protein-coupled P2Y2 nucleotide receptor (P2Y2R) is up-regulated in response to damage or stress of salivary gland epithelium, and in salivary glands of the NOD.B10 mouse model of SS-like autoimmune exocrinopathy. Additionally, we have shown that P2Y2R activation up-regulates vascular cell adhesion molecule-1 (VCAM-1) expression in endothelial cells leading to the binding of monocytes. The present study demonstrates that activation of the P2Y2R in dispersed cell aggregates from rat submandibular gland (SMG) and in human submandibular gland ductal cells (HSG) up-regulates the expression of VCAM-1. Furthermore, P2Y2R activation mediated the up-regulation of VCAM-1 expression in HSG cells leading to increased adherence of lymphocytic cells. Inhibitors of EGFR phosphorylation and metalloprotease activity abolished P2Y2R-mediated VCAM-1 expression and decreased lymphocyte binding to HSG cells. Moreover, silencing of EGFR expression abolished UTP-induced VCAM-1 up-regulation in HSG cells. These results suggest that P2Y2R activation in salivary gland cells increases the EGFR-dependent expression of VCAM-1 and the binding of lymphocytes, a pathway relevant to inflammation associated with SS. PMID:17599409

  16. Unidirectional Movement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in Mucilage Extrusion, Adherence, and Ray Formation1[OPEN

    PubMed Central

    Lam, Patricia; Young, Robin; DeBolt, Seth

    2015-01-01

    CELLULOSE SYNTHASE5 (CESA5) synthesizes cellulose necessary for seed mucilage adherence to seed coat epidermal cells of Arabidopsis (Arabidopsis thaliana). The involvement of additional CESA proteins in this process and details concerning the manner in which cellulose is deposited in the mucilage pocket are unknown. Here, we show that both CESA3 and CESA10 are highly expressed in this cell type at the time of mucilage synthesis and localize to the plasma membrane adjacent to the mucilage pocket. The isoxaben resistant1-1 and isoxaben resistant1-2 mutants affecting CESA3 show defects consistent with altered mucilage cellulose biosynthesis. CESA3 can interact with CESA5 in vitro, and green fluorescent protein-tagged CESA5, CESA3, and CESA10 proteins move in a linear, unidirectional fashion around the cytoplasmic column of the cell, parallel with the surface of the seed, in a pattern similar to that of cortical microtubules. Consistent with this movement, cytological evidence suggests that the mucilage is coiled around the columella and unwinds during mucilage extrusion to form a linear ray. Mutations in CESA5 and CESA3 affect the speed of mucilage extrusion and mucilage adherence. These findings imply that cellulose fibrils are synthesized in an ordered helical array around the columella, providing a distinct structure to the mucilage that is important for both mucilage extrusion and adherence. PMID:25926481

  17. TnphoA Salmonella abortusovis mutants unable to adhere to epithelial cells and with reduced virulence in mice.

    PubMed Central

    Rubino, S; Leori, G; Rizzu, P; Erre, G; Colombo, M M; Uzzau, S; Masala, G; Cappuccinelli, P

    1993-01-01

    Salmonella abortusovis is a pathogenic bacterium highly specific to sheep, causing spontaneous abortion. In order to understand the role of genes involved in pathogenicity, we investigated S. abortusovis with the random mutagenic TnphoA transposon. A total of 95 S. abortusovis TnphoA mutants yielding alkaline phosphatase active fusion protein were obtained. In this way we created a bank of strains in order to identify any phenotypic modification which could affect the periplasmic and/or exported proteins involved in virulence. The TnphoA mutants were screened for the ability to adhere to epithelial cells: a total of 23 mutant strains lost this phenotypic feature. To detect the chromosomal TnphoA insertions, DNA was restricted by the enzyme EcoRV, which does not cleave the TnphoA sequence. Southern blotting analysis revealed the existence of four classes of integration. Colonies of adhesiveless mutants appear to be as smooth as the S. abortusovis wild type, and electrophoretic analysis indicates a normal lipopolysaccharide profile. To identify mutations affecting genes encoding for outer membrane proteins (OMPs), the alkaline phosphatase portion of the fusion proteins was revealed in TnphoA mutants by immunoblotting with specific antibodies. A mutation in OMPs was detected in seven mutants. Restriction analysis identified in four mutants a common region of 2 kb where alterations in genes coding for OMPs occur. We suggested that this region is involved in pathogenicity in mice, since a group of mutant strains has shown reduced virulence in mice and one mutant is completely avirulent. Furthermore, after mice were exposed orally to these mutants, significant protection against oral challenge with the parental virulent strain resulted. Images PMID:8386703

  18. Treatment adherence in psychoses.

    PubMed

    David, Anthony S

    2010-12-01

    A well-conducted randomised controlled trial of an intervention to improve treatment adherence in psychosis published in this issue shows beneficial effects on self- and observer-rated adherence and trends towards fewer hospital readmissions. Partial adherence is the single most important barrier to optimal treatment. National Institute for Health and Clinical Excellence guidelines on adherence need to be revised.

  19. Evaluation of 309 Environmental Chemicals Using a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity Assay

    PubMed Central

    Chandler, Kelly J.; Barrier, Marianne; Jeffay, Susan; Nichols, Harriette P.; Kleinstreuer, Nicole C.; Singh, Amar V.; Reif, David M.; Sipes, Nisha S.; Judson, Richard S.; Dix, David J.; Kavlock, Robert; Hunter, Edward S.; Knudsen, Thomas B.

    2011-01-01

    The vast landscape of environmental chemicals has motivated the need for alternative methods to traditional whole-animal bioassays in toxicity testing. Embryonic stem (ES) cells provide an in vitro model of embryonic development and an alternative method for assessing developmental toxicity. Here, we evaluated 309 environmental chemicals, mostly food-use pesticides, from the ToxCast™ chemical library using a mouse ES cell platform. ES cells were cultured in the absence of pluripotency factors to promote spontaneous differentiation and in the presence of DMSO-solubilized chemicals at different concentrations to test the effects of exposure on differentiation and cytotoxicity. Cardiomyocyte differentiation (α,β myosin heavy chain; MYH6/MYH7) and cytotoxicity (DRAQ5™/Sapphire700™) were measured by In-Cell Western™ analysis. Half-maximal activity concentration (AC50) values for differentiation and cytotoxicity endpoints were determined, with 18% of the chemical library showing significant activity on either endpoint. Mining these effects against the ToxCast Phase I assays (∼500) revealed significant associations for a subset of chemicals (26) that perturbed transcription-based activities and impaired ES cell differentiation. Increased transcriptional activity of several critical developmental genes including BMPR2, PAX6 and OCT1 were strongly associated with decreased ES cell differentiation. Multiple genes involved in reactive oxygen species signaling pathways (NRF2, ABCG2, GSTA2, HIF1A) were strongly associated with decreased ES cell differentiation as well. A multivariate model built from these data revealed alterations in ABCG2 transporter was a strong predictor of impaired ES cell differentiation. Taken together, these results provide an initial characterization of metabolic and regulatory pathways by which some environmental chemicals may act to disrupt ES cell growth and differentiation. PMID:21666745

  20. Gender differences in stem cell population are induced by pregnancy.

    PubMed

    El-Badri, Nagwa S; Groer, Maureen

    2012-10-01

    Gender differences in stem cell population have recently been identified. Blood and tissue samples from women showed consistent elevation of hematopoietic stem cell populations, mesenchymal stem cell populations and endothelial progenitor cells compared to men of similar ages. We and others have shown an increase in hematopoietic stem cell population in pregnant and multiparous women compared to nulliparous women. We propose that pregnancy exposes women to increased levels of stem cells from many sources not available for nulliparous women or for men. During pregnancy, maternal fetal microchimerism results from trafficking of fetal and maternal blood across the placenta. Physiological changes in the maternal blood cellular milieu are also recognized during pregnancy and in the early post partum due to the presence of unique pregnancy associated tissues and hormones. These include the placenta, the amniotic fluid and cord blood. These tissues are highly enriched for different populations of stem cells including hematopoietic stem cells, mesenchymal stem cells and endothelial progenitor cells. Recent studies showed accelerated healing in women affected by cardiovascular insults and stroke, in part due to faster tissue regeneration and stem cell activity. We propose that gender differences in stem cell population are caused in part due to maternal exposure to fetal and unique pregnancy associated tissues, which are significantly enriched in different stem cell populations.

  1. Adherence of Pseudomonas aeruginosa to contact lenses

    SciTech Connect

    Miller, M.J.

    1988-01-01

    The purpose of this research was to examined the interactions of P. aeruginosa with hydrogel contact lenses and other substrata, and characterize adherence to lenses under various physiological and physicochemical conditions. Isolates adhered to polystyrene, glass, and hydrogel lenses. With certain lens types, radiolabeled cells showed decreased adherence with increasing water content of the lenses, however, this correlation with not found for all lenses. Adherence to rigid gas permeable lenses was markedly greater than adherence to hydrogels. Best adherence occurred near pH 7 and at a sodium chloride concentration of 50 mM. Passive adhesion of heat-killed cells to hydrogels was lower than the adherence obtained of viable cells. Adherence to hydrogels was enhanced by mucin, lactoferrin, lysozyme, IgA, bovine serum albumin, and a mixture of these macromolecules. Adherence to coated and uncoated lenses was greater with a daily-wear hydrogel when compared with an extended-wear hydrogel of similar polymer composition. Greater adherence was attributed to a higher concentration of adsorbed macromolecules on the 45% water-content lens in comparison to the 55% water-content lens.

  2. High Acceptability of HIV Pre-exposure Prophylaxis but Challenges in Adherence and Use: Qualitative Insights from a Phase I Trial of Intermittent and Daily PrEP in At-Risk Populations in Kenya

    PubMed Central

    Van der Elst, Elisabeth Maria; Mbogua, Judie; Operario, Don; Mutua, Gaudensia; Kuo, Caroline; Mugo, Peter; Kanungi, Jennifer; Singh, Sagri; Haberer, Jessica; Priddy, Frances; Sanders, Eduard Joachim

    2013-01-01

    This paper used qualitative methods to explore experiences of men who have sex with men and female sex workers in Nairobi and Mtwapa, Kenya, who used oral preexposure prophylaxis (PrEP) for HIV prevention as part of a four-month trial of safety, acceptability and adherence. Fifty-one of 72 volunteers who took part in a randomized, placebo-controlled, blinded trial that compared daily and intermittent dosage of PrEP underwent qualitative assessments after completing the trial. Analyses identified three themes: (i) acceptability of PrEP was high, i.e. side effects were experienced early in the study but diminished over time, however characteristics of pills could improve comfort and use; (ii) social impacts such as stigma, rumors, and relationship difficulties due to being perceived as HIV positive were prevalent; (iii) adherence was challenged by complexities of daily life, in particular post-coital dosing adherence suffered from alcohol use around time of sex, mobile populations, and transactional sex work. These themes resonated across dosing regimens and gender, and while most participants favored the intermittent dosing schedule, those in the intermittent group noted particular challenges in adhering to the post-coital dose. Culturally appropriate and consistent counseling addressing these issues may be critical for PrEP effectiveness. PMID:23080358

  3. A microarray analysis of two distinct lymphatic endothelial cell populations.

    PubMed

    Schweighofer, Bernhard; Rohringer, Sabrina; Pröll, Johannes; Holnthoner, Wolfgang

    2015-06-01

    We have recently identified lymphatic endothelial cells (LECs) to form two morphologically different populations, exhibiting significantly different surface protein expression levels of podoplanin, a major surface marker for this cell type. In vitro shockwave treatment (IVSWT) of LECs resulted in enrichment of the podoplanin(high) cell population and was accompanied by markedly increased cell proliferation, as well as 2D and 3D migration. Gene expression profiles of these distinct populations were established using Affymetrix microarray analyses. Here we provide additional details about our dataset (NCBI GEO accession number GSE62510) and describe how we analyzed the data to identify differently expressed genes in these two LEC populations.

  4. Proper regulation of Cdc42 activity is required for tight actin concentration at the equator during cytokinesis in adherent mammalian cells.

    PubMed

    Zhu, Xiaodong; Wang, Junxia; Moriguchi, Kazuki; Liow, Lu Ting; Ahmed, Sohail; Kaverina, Irina; Murata-Hori, Maki

    2011-10-01

    Cytokinesis in mammalian cells requires actin assembly at the equatorial region. Although functions of RhoA in this process have been well established, additional mechanisms are likely involved. We have examined if Cdc42 is involved in actin assembly during cytokinesis. Depletion of Cdc42 had no apparent effects on the duration of cytokinesis, while overexpression of constitutively active Cdc42 (CACdc42) caused cytokinesis failure in normal rat kidney epithelial cells. Cells depleted of Cdc42 displayed abnormal cell morphology and caused a failure of tight accumulation of actin and RhoA at the equator. In contrast, in cells overexpressing CACdc42, actin formed abnormal bundles and RhoA was largely eliminated from the equator. Our results suggest that accurate regulation of Cdc42 activity is crucial for proper equatorial actin assembly and RhoA localization during cytokinesis. Notably, our observations also suggest that tight actin concentration is not essential for cytokinesis in adherent mammalian cells.

  5. Cancer stem cells and cisplatin-resistant cells isolated from non-small-lung cancer cell lines constitute related cell populations

    PubMed Central

    Lopez-Ayllon, Blanca D; Moncho-Amor, Veronica; Abarrategi, Ander; de Cáceres, Inmaculada Ibañez; Castro-Carpeño, Javier; Belda-Iniesta, Cristobal; Perona, Rosario; Sastre, Leandro

    2014-01-01

    Lung cancer is the top cause of cancer-related deceases. One of the reasons is the development of resistance to the chemotherapy treatment. In particular, cancer stem cells (CSCs), can escape treatment and regenerate the bulk of the tumor. In this article, we describe a comparison between cancer cells resistant to cisplatin and CSCs, both derived from the non-small-cell lung cancer cell lines H460 and A549. Cisplatin-resistant cells were obtained after a single treatment with the drug. CSCs were isolated by culture in defined media, under nonadherent conditions. The isolated CSCs were clonogenic, could be differentiated into adherent cells and were less sensitive to cisplatin than the original cells. Cisplatin resistant and CSCs were able to generate primary tumors and to metastasize when injected into immunodeficient Nu/Nu mice, although they formed smaller tumors with a larger latency than untreated cells. Notably, under appropriated proportions, CSCs synergized with differentiated cells to form larger tumors. CSCs also showed increased capacity to induce angiogenesis in Nu/Nu mice. Conversely, H460 cisplatin-resistant cells showed increased tendency to develop bone metastasis. Gene expression analysis showed that several genes involved in tumor development and metastasis (EGR1, COX2, MALAT1, AKAP12, ADM) were similarly induced in CSC and cisplatin-resistant H460 cells, in agreement with a close similarity between these two cell populations. Cells with the characteristic growth properties of CSCs were also isolated from surgical samples of 18 out of 44 lung cancer patients. A significant correlation (P = 0.028) was found between the absence of CSCs and cisplatin sensitivity. PMID:24961511

  6. Evaluation of 309 environmental chemicals using a mouse embryonic stem cell adherent cell differentiation and cytotoxicity assay

    EPA Science Inventory

    The vast landscape of environmental chemicals has motivated the need for alternative methods to traditional whole-animal bioassays in toxicity testing. Embryonic stem (ES) cells provide an in vitro model of embryonic development and an alternative method for assessing development...

  7. MiR-888 regulates side population properties and cancer metastasis in breast cancer cells.

    PubMed

    Huang, Shengjian; Chen, Liangbiao

    2014-08-01

    Cancer stem cells (CSCs) have recently been reported to possess properties related to cancer metastasis. However, the mechanism by which microRNAs (miRNAs) regulate these properties remains unclear. This study aims to investigate a correlation between miRNAs and the side population (SP) of human breast cancer cell line MCF-7 with CSC properties. miR-888 was found in our previous study to be up-regulated in SP cells and predicted to target E-Cadherin directly, indicating a potential role in maintaining SP properties and regulating the epithelial-mesenchymal transition (EMT) and cancer metastasis. After the over-expression of miR-888 in MCF-7 cells and knock-down of its expression in SP cells, we found that miR-888 played a role in maintaining CSC-related properties. Next, miR-888 was found to regulate the EMT process by targeting related gene expression. Lastly, MCF-7 cells over-expressing miR-888 exhibited a significant reduction in their ability to adhere to the extracellular matrix and an increased potential for migration and invasion, whereas knock-down of miR-888 expression in SP cells reversed these trends. In conclusion, miR-888 maintains SP properties and regulates EMT and metastasis in MCF-7 cells, potentially by targeting E-Cadherin expression.

  8. Combined strategies for optimal detection of the contact point in AFM force-indentation curves obtained on thin samples and adherent cells.

    PubMed

    Gavara, Núria

    2016-01-01

    Atomic Force Microscopy (AFM) is a widely used tool to study cell mechanics. Current AFM setups perform high-throughput probing of living cells, generating large amounts of force-indentations curves that are subsequently analysed using a contact-mechanics model. Here we present several algorithms to detect the contact point in force-indentation curves, a crucial step to achieve fully-automated analysis of AFM-generated data. We quantify and rank the performance of our algorithms by analysing a thousand force-indentation curves obtained on thin soft homogeneous hydrogels, which mimic the stiffness and topographical profile of adherent cells. We take advantage of the fact that all the proposed algorithms are based on sequential search strategies, and show that a combination of them yields the most accurate and unbiased results. Finally, we also observe improved performance when force-indentation curves obtained on adherent cells are analysed using our combined strategy, as compared to the classical algorithm used in the majority of previous cell mechanics studies. PMID:26891762

  9. Combined strategies for optimal detection of the contact point in AFM force-indentation curves obtained on thin samples and adherent cells

    NASA Astrophysics Data System (ADS)

    Gavara, Núria

    2016-02-01

    Atomic Force Microscopy (AFM) is a widely used tool to study cell mechanics. Current AFM setups perform high-throughput probing of living cells, generating large amounts of force-indentations curves that are subsequently analysed using a contact-mechanics model. Here we present several algorithms to detect the contact point in force-indentation curves, a crucial step to achieve fully-automated analysis of AFM-generated data. We quantify and rank the performance of our algorithms by analysing a thousand force-indentation curves obtained on thin soft homogeneous hydrogels, which mimic the stiffness and topographical profile of adherent cells. We take advantage of the fact that all the proposed algorithms are based on sequential search strategies, and show that a combination of them yields the most accurate and unbiased results. Finally, we also observe improved performance when force-indentation curves obtained on adherent cells are analysed using our combined strategy, as compared to the classical algorithm used in the majority of previous cell mechanics studies.

  10. Combined strategies for optimal detection of the contact point in AFM force-indentation curves obtained on thin samples and adherent cells

    PubMed Central

    Gavara, Núria

    2016-01-01

    Atomic Force Microscopy (AFM) is a widely used tool to study cell mechanics. Current AFM setups perform high-throughput probing of living cells, generating large amounts of force-indentations curves that are subsequently analysed using a contact-mechanics model. Here we present several algorithms to detect the contact point in force-indentation curves, a crucial step to achieve fully-automated analysis of AFM-generated data. We quantify and rank the performance of our algorithms by analysing a thousand force-indentation curves obtained on thin soft homogeneous hydrogels, which mimic the stiffness and topographical profile of adherent cells. We take advantage of the fact that all the proposed algorithms are based on sequential search strategies, and show that a combination of them yields the most accurate and unbiased results. Finally, we also observe improved performance when force-indentation curves obtained on adherent cells are analysed using our combined strategy, as compared to the classical algorithm used in the majority of previous cell mechanics studies. PMID:26891762

  11. Production of high-titer human influenza A virus with adherent and suspension MDCK cells cultured in a single-use hollow fiber bioreactor.

    PubMed

    Tapia, Felipe; Vogel, Thomas; Genzel, Yvonne; Behrendt, Ilona; Hirschel, Mark; Gangemi, J David; Reichl, Udo

    2014-02-12

    Hollow fiber bioreactors (HFBRs) have been widely described as capable of supporting the production of highly concentrated monoclonal antibodies and recombinant proteins. Only recently HFBRs have been proposed as new single-use platforms for production of high-titer influenza A virus. These bioreactors contain multiple hollow fiber capillary tubes that separate the bioreactor in an intra- and an extra-capillary space. Cells are usually cultured in the extra-capillary space and can grow to a very high cell concentration. This work describes the evaluation of the single-use hollow fiber bioreactor PRIMER HF (Biovest International Inc., USA) for production of influenza A virus. The process was setup, characterized and optimized by running a total of 15 cultivations. The HFBRs were seeded with either adherent or suspension MDCK cells, and infected with influenza virus A/PR/8/34 (H1N1), and the pandemic strain A/Mexico/4108/2009 (H1N1). High HA titers and TCID₅₀ of up to 3.87 log₁₀(HA units/100 μL) and 1.8 × 10(10)virions/mL, respectively, were obtained for A/PR/8/34 influenza strain. Influenza virus was collected by performing multiple harvests of the extra-capillary space during a virus production time of up to 12 days. Cell-specific virus yields between 2,000 and 8,000 virions/cell were estimated for adherent MDCK cells, and between 11,000 and 19,000 virions/cell for suspension MDCK.SUS2 cells. These results do not only coincide with the cell-specific virus yields obtained with cultivations in stirred tank bioreactors and other high cell density systems, but also demonstrate that HFBRs are promising and competitive single-use platforms that can be considered for commercial production of influenza virus.

  12. Investigation on cytoskeleton dynamics for no-adherent cells subjected to point-like stimuli by digital holographic microscopy and holographic optical trapping

    NASA Astrophysics Data System (ADS)

    Miccio, Lisa; Merola, Francesco; Memmolo, Pasquale; Mugnano, Martina; Fusco, Sabato; Netti, Paolo A.; Ferraro, Pietro

    2014-05-01

    Guiding, controlling and studying cellular functions are challenging themes in the biomedical field, as they are fundamental prerequisites for new therapeutic strategies from tissue regeneration to controlled drug delivery. In recent years, multidisciplinary studies in nanotechnology offer new tools to investigate important biophysical phenomena in response to the local physical characteristics of the extracellular environment, some examples are the mechanisms of cell adhesion, migration, communication and differentiation. Indeed for reproducing the features of the extracellular matrix in vitro, it is essential to develop active devices that evoke as much as possible the natural cellular environment. Our investigation is in the framework of studying and clarifying the biophysical mechanisms of the interaction between cells and the microenvironment in which they exist. We implement an optical tweezers setup to investigate cell material interaction and we use Digital Holography as non-invasive imaging technique in microscopy. We exploit Holographic Optical Tweezers arrangement in order to trap and manage functionalized micrometric latex beads to induce mechanical deformation in suspended cells. A lot of papers in literature examine the dynamics of the cytoskeleton when cells adhere on substrates and nowadays well established cell models are based on such research activities. Actually, the natural cell environment is made of a complex extracellular matrix and the single cell behavior is due to intricate interactions with the environment and are strongly correlated to the cell-cell interactions. Our investigation is devoted to understand the inner cell mechanism when it is mechanically stressed by point-like stimulus without the substrate influence.

  13. Pyruvate:ferredoxin oxidoreductase (PFO) is a surface-associated cell-binding protein in Trichomonas vaginalis and is involved in trichomonal adherence to host cells.

    PubMed

    Meza-Cervantez, Patricia; González-Robles, Arturo; Cárdenas-Guerra, Rosa Elena; Ortega-López, Jaime; Saavedra, Emma; Pineda, Erika; Arroyo, Rossana

    2011-12-01

    The Trichomonas vaginalis 120 kDa protein adhesin (AP120) is induced under iron-rich conditions and has sequence homology with pyruvate:ferredoxin oxidoreductase A (PFO A), a hydrogenosomal enzyme that is absent in humans. This homology raises the possibility that, like AP120, PFO might be localized to the parasite surface and participate in cytoadherence. Here, the cellular localization and function of PFO that was expressed under various iron concentrations was investigated using a polyclonal antibody generated against the 50 kDa recombinant C-terminal region of PFO A (anti-PFO50). In Western blot assays, this antibody recognized a 120 kDa protein band in total protein extracts, and proteins with affinity to the surface of HeLa cells from parasites grown under iron-rich conditions. In addition to localization that is typical of hydrogenosomal proteins, PFOs that were expressed under iron-rich conditions were found to localize at the surface. This localization was demonstrated using immunofluorescence and co-localization assays, as well as immunogold transmission electron microscopy. In addition to describing its enzyme activity, we describe a novel function in trichomonal host interaction for the PFO localized on the parasite surface. The anti-PFO50 antibody reduced the levels of T. vaginalis adherence to HeLa cell monolayers in a concentration-dependent manner. Thus, T. vaginalis PFO is an example of a surface-associated cell-binding protein that lacks enzyme activity and that is involved in cytoadherence. Additionally, PFO behaves like AP120 in parasites grown under iron-rich conditions. Therefore, these data suggest that AP120 and PFO A are encoded by the same gene, namely pfo a.

  14. Defining heterogeneity within bacterial populations via single cell approaches.

    PubMed

    Davis, Kimberly M; Isberg, Ralph R

    2016-08-01

    Bacterial populations are heterogeneous, which in many cases can provide a selective advantage during changes in environmental conditions. In some instances, heterogeneity exists at the genetic level, in which significant allelic variation occurs within a population seeded by a single cell. In other cases, heterogeneity exists due to phenotypic differences within a clonal, genetically identical population. A variety of mechanisms can drive this latter strategy. Stochastic fluctuations can drive differential gene expression, but heterogeneity in gene expression can also be driven by environmental changes sensed by individual cells residing in distinct locales. Utilizing multiple single cell approaches, workers have started to uncover the extent of heterogeneity within bacterial populations. This review will first describe several examples of phenotypic and genetic heterogeneity, and then discuss many single cell approaches that have recently been applied to define heterogeneity within bacterial populations. PMID:27273675

  15. Identification of multipotent mesenchymal stromal cells in the reactive stroma of a prostate cancer xenograft by side population analysis

    SciTech Connect

    Santamaria-Martinez, Albert; Barquinero, Jordi; Barbosa-Desongles, Anna; Hurtado, Antoni; Pinos, Tomas; Seoane, Joan; Poupon, Marie-France; Morote, Joan; Reventos, Jaume; Munell, Francina

    2009-10-15

    Cancer stem cells are a distinct cellular population that is believed to be responsible for tumor initiation and maintenance. Recent data suggest that solid tumors also contain another type of stem cells, the mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs), which contribute to the formation of tumor-associated stroma. The Hoechst 33342 efflux assay has proved useful to identify a rare cellular fraction, named Side Population (SP), enriched in cells with stem-like properties. Using this assay, we identified SP cells in a prostate cancer xenograft containing human prostate cancer cells and mouse stromal cells. The SP isolation, subculture and sequential sorting allowed the generation of single-cell-derived clones of murine origin that were recognized as MSC by their morphology, plastic adherence, proliferative potential, adipogenic and osteogenic differentiation ability and immunophenotype (CD45{sup -}, CD81{sup +} and Sca-1{sup +}). We also demonstrated that SP clonal cells secrete transforming growth factor {beta}1 (TGF-{beta}1) and that their inhibition reduces proliferation and accelerates differentiation. These results reveal the existence of SP cells in the stroma of a cancer xenograft, and provide evidence supporting their MSC nature and the role of TGF-{beta}1 in maintaining their proliferation and undifferentiated status. Our data also reveal the usefulness of the SP assay to identify and isolate MSC cells from carcinomas.

  16. Single cell motility and trail formation in populations of microglia

    NASA Astrophysics Data System (ADS)

    Lee, Kyoung Jin

    2009-03-01

    Microglia are a special type of glia cell in brain that has immune responses. They constitute about 20 % of the total glia population within the brain. Compared to other glia cells, microglia are very motile, constantly moving to destroy pathogens and to remove dead neurons. While doing so, they exhibit interesting body shapes, have cell-to-cell communications, and have chemotatic responses to each other. Interestingly, our recent in vitro studies show that their unusual motile behaviors can self-organize to form trails, similar to those in populations of ants. We have studied the changes in the physical properties of these trails by varying the cell population density and by changing the degree of spatial inhomogeneities (``pathogens''). Our experimental observations can be quite faithfully reproduced by a simple mathematical model involving many motile cells whose mechanical motion are driven by actin polymerization and depolymerization process within the individual cell body and by external chemical gradients.

  17. Index sorting resolves heterogeneous murine hematopoietic stem cell populations.

    PubMed

    Schulte, Reiner; Wilson, Nicola K; Prick, Janine C M; Cossetti, Chiara; Maj, Michal K; Gottgens, Berthold; Kent, David G

    2015-09-01

    Recent advances in the cellular and molecular biology of single stem cells have uncovered significant heterogeneity in the functional properties of stem cell populations. This has prompted the development of approaches to study single cells in isolation, often performed using multiparameter flow cytometry. However, many stem cell populations are too rare to test all possible cell surface marker combinations, and virtually nothing is known about functional differences associated with varying intensities of such markers. Here we describe the use of index sorting for further resolution of the flow cytometric isolation of single murine hematopoietic stem cells (HSCs). Specifically, we associate single-cell functional assay outcomes with distinct cell surface marker expression intensities. High levels of both CD150 and EPCR associate with delayed kinetics of cell division and low levels of differentiation. Moreover, cells that do not form single HSC-derived clones appear in the 7AAD(dim) fraction, suggesting that even low levels of 7AAD staining are indicative of less healthy cell populations. These data indicate that when used in combination with single-cell functional assays, index sorting is a powerful tool for refining cell isolation strategies. This approach can be broadly applied to other single-cell systems, both to improve isolation and to acquire additional cell surface marker information.

  18. Semi-quantitative monitoring of confluence of adherent mesenchymal stromal cells on calcium-phosphate granules by using widefield microscopy images.

    PubMed

    Piccinini, Filippo; Pierini, Michela; Lucarelli, Enrico; Bevilacqua, Alessandro

    2014-10-01

    The analysis of cell confluence and proliferation is essential to design biomaterials and scaffolds to use as bone substitutes in clinical applications. Accordingly, several approaches have been proposed in the literature to estimate the area of the scaffold covered by cells. Nevertheless, most of the approaches rely on sophisticated equipment not employed for routine analyses, while the rest of them usually do not provide significant statistics about the cell distribution. This research aims at studying confluence and proliferation of mesenchymal stromal cells (MSC) adherent on OSPROLIFE(®), a commercial biomaterial in the form of granules. In particular, we propose a Computer Vision approach that can routinely be employed to monitor the surface of the single granules covered by cells because only a standard widefield fluorescent microscope is required. In order to acquire significant statistics data, we analyse wide-area images built by using MicroMos v2.0, an updated version of a previously published software specific for stitching brightfield and phase-contrast images manually acquired via a widefield microscope. In particular, MicroMos v2.0 permits to build accurate "mosaics" of fluorescent images, after correcting vignetting and photo-bleaching effects, providing a consistent representation of a sample region containing numerous granules. Then, our method allows to make automatically a statistically significant estimate of the percentage of the area of the single granules covered by cells. Finally, by analysing hundreds of granules at different time intervals we also obtained reliable data regarding cell proliferation, confirming that not only MSC adhere onto the OSPROLIFE(®) granules, but even proliferate over time.

  19. A microfluidic device with removable packaging for the real time visualisation of intracellular effects of nanosecond electrical pulses on adherent cells.

    PubMed

    Dalmay, C; De Menorval, M A; Français, O; Mir, L M; Le Pioufle, B

    2012-11-21

    The biological mechanisms induced by the application of nanosecond pulsed electric fields (nsPEFs: high electrical field amplitude during very short duration) on cells remain partly misunderstood. In this context, there is an increasing need for tools that allow the delivering of such pulses with the possibility to monitor their effects in real-time. Thanks to miniaturization and technology capabilities, microtechnologies offer great potential to address this issue. We report here the design and fabrication of a microfluidic device optimized for the delivery of ultra short (10 ns) and intense (up to 280 kV cm(-1)) electrical pulses on adherent cells, and the real time monitoring of their intracellular effects. Ultra short electric field pulses (nsPEFs or nanopulses) affect both the cell membrane and the intracellular organelles of the cells. In particular, intracellular release of calcium from the endoplasmic reticulum was detected in real time using the device, after exposure of adherent cells to these nsPEFs. The high intensity and spatial homogeneity of the electric field could be achieved in the device thanks to the miniaturization and the use of thick (25 μm) electroplated electrodes, disposed on a quartz substrate whose transparency allowed real time monitoring of the nsPEFs effects. The proposed biochip is compatible with cell culture glass slides that can be placed on the chip after separate culture of several days prior to exposure. This device allows the easy exposure of almost any kind of attached cells and the monitoring in real time while exposed to nsPEFs, opening large possibilities for potential use of the developed biochips. PMID:23037002

  20. Improved assay for quantitating adherence of ruminal bacteria to cellulose.

    PubMed Central

    Rasmussen, M A; White, B A; Hespell, R B

    1989-01-01

    A quantitative technique suitable for the determination of adherence of ruminal bacteria to cellulose was developed. This technique employs adherence of cells to cellulose disks and alleviates the problem of nonspecific cell entrapment within cellulose particles. By using this technique, it was demonstrated that the adherence of Ruminococcus flavefaciens FD1 to cellulose was inhibited by formaldehyde, methylcellulose, and carboxymethyl cellulose. Adherence was unaffected by acid hydrolysates of methylcellulose, glucose, and cellobiose. PMID:2782879

  1. Antibodies Directed against Shiga-Toxin Producing Escherichia coli Serotype O103 Type III Secreted Proteins Block Adherence of Heterologous STEC Serotypes to HEp-2 Cells

    PubMed Central

    Desin, Taseen S.; Townsend, Hugh G.; Potter, Andrew A.

    2015-01-01

    Shiga toxin-producing Escherichia coli (STEC) serotype O103 is a zoonotic pathogen that is capable of causing hemorrhagic colitis and hemolytic uremic syndrome (HUS) in humans. The main animal reservoir for STEC is ruminants and hence reducing the levels of this pathogen in cattle could ultimately lower the risk of STEC infection in humans. During the process of infection, STECO103 uses a Type III Secretion System (T3SS) to secrete effector proteins (T3SPs) that result in the formation of attaching and effacing (A/E) lesions. Vaccination of cattle with STEC serotype O157 T3SPs has previously been shown to be effective in reducing shedding of STECO157 in a serotype-specific manner. In this study, we tested the ability of rabbit polyclonal sera against individual STECO103 T3SPs to block adherence of the organism to HEp-2 cells. Our results demonstrate that pooled sera against EspA, EspB, EspF, NleA and Tir significantly lowered the adherence of STECO103 relative to pre-immune sera. Likewise, pooled anti-STECO103 sera were also able to block adherence by STECO157. Vaccination of mice with STECO103 recombinant proteins induced strong IgG antibody responses against EspA, EspB, NleA and Tir but not against EspF. However, the vaccine did not affect fecal shedding of STECO103 compared to the PBS vaccinated group over the duration of the experiment. Cross reactivity studies using sera against STECO103 recombinant proteins revealed a high degree of cross reactivity with STECO26 and STECO111 proteins implying that sera against STECO103 proteins could potentially provide neutralization of attachment to epithelial cells by heterologous STEC serotypes. PMID:26451946

  2. Genetic relatedness and virulence properties of enteropathogenic Escherichia coli strains of serotype O119:H6 expressing localized adherence or localized and aggregative adherence-like patterns on HeLa cells.

    PubMed

    Garcia, Bruna G; Ooka, Tadasuke; Gotoh, Yasuhiro; Vieira, Mônica A M; Yamamoto, Denise; Ogura, Yoshitoshi; Girão, Dennys M; Sampaio, Suely C F; Melo, Alexis Bonfim; Irino, Kinue; Hayashi, Tetsuya; Gomes, Tânia A T

    2016-05-01

    Enteropathogenic Escherichia coli (EPEC) induce attaching and effacing (A/E) lesions in enterocytes and produce the bundle-forming pilus (BFP) contributing to the localized adherence (LA) pattern formation on HeLa cells. Enteroaggregative E. coli (EAEC) produce aggregative adherence (AA) on HeLa cells and form prominent biofilms. The ability to produce LA or AA is an important hallmark to classify fecal E. coli isolates as EPEC or EAEC, respectively. E. coli strains of serotype O119:H6 exhibit an LA+ phenotype and have been considered as comprising a clonal group of EPEC strains. However, we have recently identified O119:H6 EPEC strains that produce LA and an AA-like pattern concurrently (LA/AA-like+). In this study, we evaluated the relatedness of three LA/AA-like+ and three LA+ O119:H6 strains by comparing their virulence and genotypic properties. We first found that the LA/AA-like+ strains induced actin accumulation in HeLa cells (indicative of A/E lesions formation) and formed biofilms on abiotic surfaces more efficiently than the LA+ strains. MLST analysis showed that the six strains all belong to the ST28 complex. All strains carried multiple plasmids, but as plasmid profiles were highly variable, this cannot be used to differentiate LA/AA-like+ and LA+ strains. We further obtained their draft genome sequences and the complete sequences of four plasmids harbored by one LA/AA-like+ strain. Analysis of these sequences and comparison with 37 fully sequenced E. coli genomes revealed that both O119:H6 groups belong to the E. coli phylogroup B2 and are very closely related with only 58-67 SNPs found between LA/AA-like+ and LA+ strains. Search of the draft sequences of the six strains for adhesion-related genes known in EAEC and other E. coli pathotypes detected no genes specifically present in LA/AA-like+ strains. Unexpectedly however, we found that a large plasmid distinct from pEAF is responsible for the AA-like phenotype of the LA/AA-like+ strains. Although we

  3. Hyaluronic Acid-Based Hydrogels as 3D Matrices for in Vitro Evaluation of Chemotherapeutic Drugs Using Poorly Adherent Prostate Cancer Cells

    PubMed Central

    Gurski, Lisa A.; Jha, Amit K.; Zhang, Chu; Jia, Xinqiao; Farach-Carson, Mary C.

    2009-01-01

    The current investigation aimed to develop a biomimetic, three-dimensional (3D) culture system for poorly adherent bone metastatic prostate cancer cells (C4-2B) for use as an in vitro platform for anti-cancer drug screening. To this end, hyaluronic acid (HA) derivatives carrying complementary aldehyde (HAALD) and hydrazide (HAADH) groups were synthesized and characterized. In situ encapsulation of C4-2B cells was achieved by simple mixing of HAALD and HAADH in the presence of the cells. Unlike two-dimensional (2D) monolayer culture in which cells adopt an atypical spread morphology, cells residing in the HA matrix formed distinct clustered structures which grew and merged, reminiscent of real tumors. Anti-cancer drugs added to the media surrounding the cell/gel construct diffused into the gel and killed the embedded cells. The HA hydrogel system was used successfully to test the efficacy of anti-cancer drugs including camptothecin, docetaxel, and rapamycin, alone and in combination, including specificity, dose and time responses. Responses of cells to anti-neoplastics differed between the 3D HA hydrogel and 2D monolayer systems. We suggest that the data obtained from 3D HA systems is superior to that from conventional 2D monolayers as the 3D system better reflects the bone metastatic microenvironment of the cancer cells. PMID:19695694

  4. The Adherent/Invasive Escherichia coli Strain LF82 Invades and Persists in Human Prostate Cell Line RWPE-1, Activating a Strong Inflammatory Response

    PubMed Central

    Aleandri, Marta; Marazzato, Massimiliano; Conte, Antonietta L.; Ambrosi, Cecilia; Nicoletti, Mauro; Zagaglia, Carlo; Gambara, Guido; Palombi, Fioretta; De Cesaris, Paola; Ziparo, Elio; Palamara, Anna T.; Riccioli, Anna

    2016-01-01

    Adherent/invasive Escherichia coli (AIEC) strains have recently been receiving increased attention because they are more prevalent and persistent in the intestine of Crohn's disease (CD) patients than in healthy subjects. Since AIEC strains show a high percentage of similarity to extraintestinal pathogenic E. coli (ExPEC), neonatal meningitis-associated E. coli (NMEC), and uropathogenic E. coli (UPEC) strains, here we compared AIEC strain LF82 with a UPEC isolate (strain EC73) to assess whether LF82 would be able to infect prostate cells as an extraintestinal target. The virulence phenotypes of both strains were determined by using the RWPE-1 prostate cell line. The results obtained indicated that LF82 and EC73 are able to adhere to, invade, and survive within prostate epithelial cells. Invasion was confirmed by immunofluorescence and electron microscopy. Moreover, cytochalasin D and colchicine strongly inhibited bacterial uptake of both strains, indicating the involvement of actin microfilaments and microtubules in host cell invasion. Moreover, both strains belong to phylogenetic group B2 and are strong biofilm producers. In silico analysis reveals that LF82 shares with UPEC strains several virulence factors: namely, type 1 pili, the group II capsule, the vacuolating autotransporter toxin, four iron uptake systems, and the pathogenic island (PAI). Furthermore, compared to EC73, LF82 induces in RWPE-1 cells a marked increase of phosphorylation of mitogen-activated protein kinases (MAPKs) and of NF-κB already by 5 min postinfection, thus inducing a strong inflammatory response. Our in vitro data support the hypothesis that AIEC strains might play a role in prostatitis, and, by exploiting host-cell signaling pathways controlling the innate immune response, likely facilitate bacterial multiplication and dissemination within the male genitourinary tract. PMID:27600504

  5. Overshoot during phenotypic switching of cancer cell populations

    PubMed Central

    Sellerio, Alessandro L.; Ciusani, Emilio; Ben-Moshe, Noa Bossel; Coco, Stefania; Piccinini, Andrea; Myers, Christopher R.; Sethna, James P.; Giampietro, Costanza; Zapperi, Stefano; La Porta, Caterina A. M.

    2015-01-01

    The dynamics of tumor cell populations is hotly debated: do populations derive hierarchically from a subpopulation of cancer stem cells (CSCs), or are stochastic transitions that mutate differentiated cancer cells to CSCs important? Here we argue that regulation must also be important. We sort human melanoma cells using three distinct cancer stem cell (CSC) markers — CXCR6, CD271 and ABCG2 — and observe that the fraction of non-CSC-marked cells first overshoots to a higher level and then returns to the level of unsorted cells. This clearly indicates that the CSC population is homeostatically regulated. Combining experimental measurements with theoretical modeling and numerical simulations, we show that the population dynamics of cancer cells is associated with a complex miRNA network regulating the Wnt and PI3K pathways. Hence phenotypic switching is not stochastic, but is tightly regulated by the balance between positive and negative cells in the population. Reducing the fraction of CSCs below a threshold triggers massive phenotypic switching, suggesting that a therapeutic strategy based on CSC eradication is unlikely to succeed. PMID:26494317

  6. Identification of plasmid-encoded mannose-resistant hemagglutinin and HEp-2 and HeLa cell adherence factors of two diarrheagenic Escherichia coli strains belonging to an enteropathogenic serogroup.

    PubMed Central

    Pal, R; Ghose, A C

    1990-01-01

    Two Escherichia coli strains (B/M 369 and C-35) belonging to enteropathogenic serogroup O86 were isolated from patients with infantile diarrhea and studied with respect to their cellular adherence properties. Both strains exhibited adherence (Ad+) to HEp-2 and HeLa cell monolayers in vitro and expressed mannose-resistant hemagglutinating (MRHA+) activity towards human, chicken, and sheep (but not mouse, rabbit, or guinea pig) erythrocytes. Cellular adherence properties of both strains could be substantially reduced by pronase treatment and by heat treatment (100 degrees C for 5 min) of bacteria. Electron microscopic examination failed to reveal fimbria- or pilus-like structures on the bacterial cell surface. Conjugation experiments conducted with these strains suggested that both MRHA and HEp-2 and HeLa cell adherence factors were encoded by the same plasmid, with a size of 55 to 57 megadaltons (MDa). Further biochemical studies indicated that the cellular adherence factors were associated with cell surface structures of bacteria that were proteinaceous in nature. An antiserum, rendered specific for the 57-MDa plasmid (pRP201) products of B/M 369 by adsorption, reacted with both MRHA+ Ad+ strains, B/M 369 and C-35, but not with their 57- or 55-MDa plasmidless MRHA- Ad- transconjugants or with other MRHA- Ad- E. coli strains. Immunological studies showed that the absorbed antiserum recognized two proteins with subunit molecular sizes of 18 and 14.5 kDa that were present on the cell surfaces of both strains. Furthermore, the absorbed antiserum at subagglutinating dilutions did inhibit, although only partially, the MRHA and HEp-2 and HeLa cell adherence activities of both E. coli strains. All these results would indicate that some of the E. coli strains belonging to enteropathogenic serogroups express their adherence potential through factors that were hitherto unrecognized. Images PMID:1969390

  7. Serum Proteins Enhance Dispersion Stability and Influence the Cytotoxicity and Dosimetry of ZnO Nanoparticles in Suspension and Adherent Cancer Cell Models

    NASA Astrophysics Data System (ADS)

    Anders, Catherine B.; Chess, Jordan J.; Wingett, Denise G.; Punnoose, Alex

    2015-11-01

    Agglomeration and sedimentation of nanoparticles (NPs) within biological solutions is a major limitation in their use in many downstream applications. It has been proposed that serum proteins associate with the NP surface to form a protein corona that limits agglomeration and sedimentation. Here, we investigate the effect of fetal bovine serum (FBS) proteins on the dispersion stability, dosimetry, and NP-induced cytotoxicity of cationic zinc oxide nanoparticles (nZnO) synthesized via forced hydrolysis with a core size of 10 nm. Two different in vitro cell culture models, suspension and adherent, were evaluated by comparing a phosphate buffered saline (PBS) nZnO dispersion (nZnO/PBS) and an FBS-stabilized PBS nZnO dispersion (nZnO - FBS/PBS). Surface interactions of FBS on nZnO were analyzed via spectroscopic and optical techniques. Fourier transformed infrared spectroscopy (FTIR) confirmed the adsorption of negatively charged protein components on the cationic nZnO surface through the disappearance of surfaced-adsorbed carboxyl functional groups and the subsequent detection of vibrational modes associated with the protein backbone of FBS-associated proteins. Further confirmation of these interactions was noted in the isoelectric point shift of the nZnO from the characteristic pH of 9.5 to a pH of 6.1. In nZnO - FBS/PBS dispersions, the FBS reduced agglomeration and sedimentation behaviors to impart long-term improvements (>24 h) to the nZnO dispersion stability. Furthermore, mathematical dosimetry models indicate that nZnO - FBS/PBS dispersions had consistent NP deposition patterns over time unlike unstable nZnO/PBS dispersions. In suspension cell models, the stable nZnO - FBS/PBS dispersion resulted in a ~33 % increase in the NP-induced cytotoxicity for both Jurkat leukemic and Hut-78 lymphoma cancer cells. In contrast, the nZnO - FBS/PBS dispersion resulted in 49 and 71 % reductions in the cytotoxicity observed towards the adherent breast (T-47D) and prostate

  8. Binding of Streptococcus pneumoniae endopeptidase O (PepO) to complement component C1q modulates the complement attack and promotes host cell adherence.

    PubMed

    Agarwal, Vaibhav; Sroka, Magdalena; Fulde, Marcus; Bergmann, Simone; Riesbeck, Kristian; Blom, Anna M

    2014-05-30

    The Gram-positive species Streptococcus pneumoniae is a human pathogen causing severe local and life-threatening invasive diseases associated with high mortality rates and death. We demonstrated recently that pneumococcal endopeptidase O (PepO) is a ubiquitously expressed, multifunctional plasminogen and fibronectin-binding protein facilitating host cell invasion and evasion of innate immunity. In this study, we found that PepO interacts directly with the complement C1q protein, thereby attenuating the classical complement pathway and facilitating pneumococcal complement escape. PepO binds both free C1q and C1 complex in a dose-dependent manner based on ionic interactions. Our results indicate that recombinant PepO specifically inhibits the classical pathway of complement activation in both hemolytic and complement deposition assays. This inhibition is due to direct interaction of PepO with C1q, leading to a strong activation of the classical complement pathway, and results in consumption of complement components. In addition, PepO binds the classical complement pathway inhibitor C4BP, thereby regulating downstream complement activation. Importantly, pneumococcal surface-exposed PepO-C1q interaction mediates bacterial adherence to host epithelial cells. Taken together, PepO facilitates C1q-mediated bacterial adherence, whereas its localized release consumes complement as a result of its activation following binding of C1q, thus representing an additional mechanism of human complement escape by this versatile pathogen.

  9. Emergence of cytotoxic resistance in cancer cell populations: Single-cell mechanisms and population-level consequences

    NASA Astrophysics Data System (ADS)

    Lorenzi, Tommaso; Chisholm, Rebecca H.; Lorz, Alexander; Larsen, Annette K.; de Almeida, Luís Neves; Escargueil, Alexandre; Clairambault, Jean

    2016-06-01

    We formulate an individual-based model and a population model of phenotypic evolution, under cytotoxic drugs, in a cancer cell population structured by the expression levels of survival-potential and proliferation-potential. We apply these models to a recently studied experimental system. Our results suggest that mechanisms based on fundamental laws of biology can reversibly push an actively-proliferating, and drug-sensitive, cell population to transition into a weakly-proliferative and drug-tolerant state, which will eventually facilitate the emergence of more potent, proliferating and drug-tolerant cells.

  10. Krüppel-Like Factor 4 Overexpression Initiates a Mesenchymal-to-Epithelial Transition and Redifferentiation of Human Pancreatic Cells following Expansion in Long Term Adherent Culture

    PubMed Central

    Docherty, Hilary M.; McGowan, Neil W. A.; Forbes, Shareen; Heremans, Yves; Forbes, Stuart J.; Heimberg, Harry; Casey, John; Docherty, Kevin

    2015-01-01

    A replenishable source of insulin-producing cells has the potential to cure type 1 diabetes. Attempts to culture and expand pancreatic β-cells in vitro have resulted in their transition from insulin-producing epithelial cells to mesenchymal stromal cells (MSCs) with high proliferative capacity but devoid of any hormone production. The aim of this study was to determine whether the transcription factor Krüppel-like factor 4 (KLF4), could induce a mesenchymal-to-epithelial transition (MET) of the cultured cells. Islet-enriched pancreatic cells, allowed to dedifferentiate and expand in adherent cell culture, were transduced with an adenovirus containing KLF4 (Ad-Klf4). Cells were subsequently analysed for changes in cell morphology by light microscopy, and for the presence of epithelial and pancreatic markers by immunocytochemistry and quantitative RT/PCR. Infection with Ad-Klf4 resulted in morphological changes, down-regulation of mesenchymal markers, and re-expression of both epithelial and pancreatic cell markers including insulin and transcription factors specific to β-cells. This effect was further enhanced by culturing cells in suspension. However, the effects of Ad-KLf4 were transient and this was shown to be due to increased apoptosis in Klf4-expressing cells. Klf4 has been recently identified as a pioneer factor with the ability to modulate the structure of chromatin and enhance reprogramming/transdifferentiation. Our results show that Klf4 may have a role in the redifferentiation of expanded pancreatic cells in culture, but before this can be achieved the off-target effects that result in increased apoptosis would need to be overcome. PMID:26457418

  11. Extracortical origin of some murine subplate cell populations

    PubMed Central

    Pedraza, María; Hoerder-Suabedissen, Anna; Albert-Maestro, María Amparo; Molnár, Zoltán; De Carlos, Juan A.

    2014-01-01

    The subplate layer, the deepest cortical layer in mammals, has important roles in cerebral cortical development. The subplate contains heterogeneous cell populations that are morphologically diverse, with several projection targets. It is currently assumed that these cells are generated in the germinative zone of the earliest cortical neuroepithelium. Here we identify a pallial but extracortical area located in the rostromedial telencephalic wall (RMTW) that gives rise to several cell populations. Postmitotic neurons migrate tangentially from the RMTW toward the cerebral cortex. Most RMTW-derived cells are incorporated into the subplate layer throughout its rostrocaudal extension, with others contributing to the GABAergic interneuron pool of cortical layers V and VI. PMID:24778253

  12. Epithelial cell invasion and adherence directed by the enterotoxigenic Escherichia coli tib locus is associated with a 104-kilodalton outer membrane protein.

    PubMed Central

    Elsinghorst, E A; Weitz, J A

    1994-01-01

    Enterotoxigenic Escherichia coli (ETEC) is capable of invading epithelial cell lines derived from the human colon and ileocecum. Two separate loci (tia and tib) that direct noninvasive E. coli HB101 to adhere to and invade intestinal epithelial cells have previously been cosmid cloned from ETEC H10407. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cellular fractions from tib-positive HB101 shows that the tib locus directs the synthesis of a 104-kDa outer membrane protein (the TibA protein). The tib locus was subcloned to a maximum of 6.7 kb and mutagenized with transposon Tn5. Production of TibA was directly correlated with the capacity of the subclones and Tn5 mutants to invade and adhere to epithelial cells, suggesting that TibA was required for these phenotypes. The position and direction of transcription of the tibA gene were identified by complementation and in vivo T7 RNA polymerase-promoter induction experiments. The role of the tib locus in epithelial cell invasion was confirmed by the construction of chromosomal deletion derivatives in H10407. These deletion mutants invaded epithelial cells at about 15% of the parental level and were fully complemented by plasmids bearing the tib locus. The size and function of the TibA protein are similar to those of invasin from Yersinia pseudotuberculosis (103 kDa). However, a tib probe did not hybridize with the gene encoding invasin. Hybridization analyses of genomic DNA from a wide variety of pathogenic and nonpathogenic bacteria, including Salmonella, Shigella, Yersinia, and Escherichia species, indicate that the tib locus is unique to specific ETEC strains. Images PMID:8039917

  13. Epithelial cell invasion and adherence directed by the enterotoxigenic Escherichia coli tib locus is associated with a 104-kilodalton outer membrane protein.

    PubMed

    Elsinghorst, E A; Weitz, J A

    1994-08-01

    Enterotoxigenic Escherichia coli (ETEC) is capable of invading epithelial cell lines derived from the human colon and ileocecum. Two separate loci (tia and tib) that direct noninvasive E. coli HB101 to adhere to and invade intestinal epithelial cells have previously been cosmid cloned from ETEC H10407. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cellular fractions from tib-positive HB101 shows that the tib locus directs the synthesis of a 104-kDa outer membrane protein (the TibA protein). The tib locus was subcloned to a maximum of 6.7 kb and mutagenized with transposon Tn5. Production of TibA was directly correlated with the capacity of the subclones and Tn5 mutants to invade and adhere to epithelial cells, suggesting that TibA was required for these phenotypes. The position and direction of transcription of the tibA gene were identified by complementation and in vivo T7 RNA polymerase-promoter induction experiments. The role of the tib locus in epithelial cell invasion was confirmed by the construction of chromosomal deletion derivatives in H10407. These deletion mutants invaded epithelial cells at about 15% of the parental level and were fully complemented by plasmids bearing the tib locus. The size and function of the TibA protein are similar to those of invasin from Yersinia pseudotuberculosis (103 kDa). However, a tib probe did not hybridize with the gene encoding invasin. Hybridization analyses of genomic DNA from a wide variety of pathogenic and nonpathogenic bacteria, including Salmonella, Shigella, Yersinia, and Escherichia species, indicate that the tib locus is unique to specific ETEC strains. PMID:8039917

  14. In situ cell-by-cell imaging and analysis of small cell populations by mass spectrometry.

    PubMed

    Shrestha, Bindesh; Patt, Joseph M; Vertes, Akos

    2011-04-15

    Molecular imaging by mass spectrometry (MS) is emerging as a tool to determine the distribution of proteins, lipids, and metabolites in tissues. The existing imaging methods, however, mostly rely on predefined rectangular grids for sampling that ignore the natural cellular organization of the tissue. Here we demonstrate that laser ablation electrospray ionization (LAESI) MS can be utilized for in situ cell-by-cell imaging of plant tissues. The cell-by-cell molecular image of the metabolite cyanidin, the ion responsible for purple pigmentation in onion (Allium cepa) epidermal cells, correlated well with the color of cells in the tissue. Chemical imaging using single-cells as voxels reflects the spatial distribution of biochemical differences within a tissue without the distortion stemming from sampling multiple cells within the laser focal spot. Microsampling by laser ablation also has the benefit of enabling the analysis of very small cell populations for biochemical heterogeneity. For example, with a ∼30 μm ablation spot we were able to analyze 3-4 achlorophyllous cells within an oil gland on a sour orange (Citrus aurantium) leaf. To explore cell-to-cell variations within and between tissues, multivariate statistical analysis on LAESI-MS data from epidermal cells of an A. cepa bulb and a C. aurantium leaf and from human buccal epithelial cell populations was performed using the method of orthogonal projections to latent structures discriminant analysis (OPLS-DA). The OPLS-DA analysis of mass spectra, containing over 300 peaks each, provided guidance in identifying a small number of metabolites most responsible for the variance between the cell populations. These metabolites can be viewed as promising candidates for biomarkers that, however, require further verification. PMID:21388149

  15. Multiple Cancer Cell Population Dynamics in a Complex Ecology

    NASA Astrophysics Data System (ADS)

    Lin, Ke-Chih; Targa, Gonzalo; Pienta, Kenneth; Sturm, James; Austin, Robert

    We have developed a technology for study of complex ecology cancer population dynamics. The technology includes complex drug gradients, full bright field/dark field/fluorescence imaging of areas of several square millimeters and thin gas-permable membranes which allow single cell extraction and analysis. We will present results of studies of prostate cancer cell dynamics.

  16. Confirmation of positive antibody screens by solid-phase red cell adherence assay using a tube technique method with polyethylene glycol enhancement.

    PubMed

    Gammon, R R; Lake, M; Velasquez, N; Prichard, A

    2001-01-01

    Our blood bank routinely screens donors for antibodies using a solid-phase red cell adherence (SPRCA) assay. Positive results are then confirmed using a tube technique with polyethylene glycol (PEG) enhancement due to reported higher specificity than with SPRCA. Over a 5-month period, 49,084 donor serum or plasma samples were tested using the SPRCA assay. Further identification of positive samples was performed using a PEG enhancement method. Testing was performed with strict adherence to the manufacturers' inserts. Of 49,084 samples, 313 (0.64%) were positive by the SPRCA assay. Of these, 99 (31.6%) samples remained positive when tested with PEG enhancement. The remaining 214 (68.4%) were negative, giving specificity for the SPRCA assay of 99.6 percent (48,985/ 49,199). We report a high specificity for antibody screening using the SPRCA assay. However, it is cost effective to perform a confirmatory tube test with PEG enhancement because 214 SPRCA assay samples were interpreted as having a negative antibody screen, thus allowing the release of valuable blood components for transfusion.

  17. SURVIVIN as a marker for quiescent-breast cancer stem cells-An intermediate, adherent, pre-requisite phase of breast cancer metastasis.

    PubMed

    Siddharth, Sumit; Das, Sarita; Nayak, Anmada; Kundu, Chanakya Nath

    2016-10-01

    Cancer stem cells drive the metastatic cascade by undergoing epithelial to mesenchymal transition (EMT) and again mesenchymal to epithelial transition (MET). Using multiple breast cancer cell lines including cigarette smoke induced breast cancer cells and tumor derived primary cells from patient sample; we developed a breast cancer metastasis model and reported the existence of an adherent, distinct pre-metastatic phase, quiescent-breast cancer stem cells (Q-BCSCs) prior to attaining an EMT. SURVIVIN was found to be expressed in Q-BCSCs. Time dependant biphasic expression of SURVIVIN in Q-BCSCs reveals that Q-BCSCs is a pre-metastatic phase distinct from both epithelial and mesenchymal counterparts. SURVIVIN favours metastasis and up-regulates WNT/β-CATENIN pathway in a PI3 K/AKT-dependant manner for self-renewal. Knockdown of SURVIVIN in Q-BCSCs lost the metastatic property of cells by inhibiting invasion, EMT-MET, PI3 K/AKT/WNT cascade, and induced apoptosis. Thus, our data suggest the existence of a novel pre-metastatic phase (Q-BCSCs) before EMT and SURVIVIN acts as a marker for Quiescent-BCSCs. PMID:27411340

  18. SURVIVIN as a marker for quiescent-breast cancer stem cells-An intermediate, adherent, pre-requisite phase of breast cancer metastasis.

    PubMed

    Siddharth, Sumit; Das, Sarita; Nayak, Anmada; Kundu, Chanakya Nath

    2016-10-01

    Cancer stem cells drive the metastatic cascade by undergoing epithelial to mesenchymal transition (EMT) and again mesenchymal to epithelial transition (MET). Using multiple breast cancer cell lines including cigarette smoke induced breast cancer cells and tumor derived primary cells from patient sample; we developed a breast cancer metastasis model and reported the existence of an adherent, distinct pre-metastatic phase, quiescent-breast cancer stem cells (Q-BCSCs) prior to attaining an EMT. SURVIVIN was found to be expressed in Q-BCSCs. Time dependant biphasic expression of SURVIVIN in Q-BCSCs reveals that Q-BCSCs is a pre-metastatic phase distinct from both epithelial and mesenchymal counterparts. SURVIVIN favours metastasis and up-regulates WNT/β-CATENIN pathway in a PI3 K/AKT-dependant manner for self-renewal. Knockdown of SURVIVIN in Q-BCSCs lost the metastatic property of cells by inhibiting invasion, EMT-MET, PI3 K/AKT/WNT cascade, and induced apoptosis. Thus, our data suggest the existence of a novel pre-metastatic phase (Q-BCSCs) before EMT and SURVIVIN acts as a marker for Quiescent-BCSCs.

  19. Inhibition of Shigella sonnei adherence to HT-29 cells by lactobacilli from Chinese fermented food and preliminary characterization of S-layer protein involvement.

    PubMed

    Zhang, Ying-Chun; Zhang, Lan-Wei; Tuo, Yan-Feng; Guo, Chun-Feng; Yi, Hua-Xi; Li, Jing-Yan; Han, Xue; Du, Ming

    2010-10-01

    In this study, seven lactobacilli with a high degree of antagonistic activity against three pathogens and good adherence to HT-29 cells were selected. The ability of these seven lactobacilli to inhibit adhesion of Shigella sonnei to intestinal mucosa was studied on cultured HT-29 cells. Lactobacilli were added simultaneously with, before or after S. sonnei to test for their effectiveness in exclusion, competition and displacement assays, respectively. Lactobacillus paracasei subp. paracasei M5-L, Lactobacillus rhamnosus J10-L and Lactobacillus casei Q8-L all exhibited significant inhibitory activity. In order to elucidate the inhibitory functions of S-layer proteins, the S-layer proteins were removed with 5 M LiCl from the M5-L, J10-L and Q8-L strains. Under such conditions, inhibition activity was decreased in all three strains, as revealed in exclusion, competition and displacement assays. SDS-PAGE analysis confirmed the presence of S-layer proteins with dominant bands of approximately 45 kDa. Further analysis of S-layer proteins revealed that the hydrophobic amino acids accounted for 40.5%, 41.5% and 43.8% of the total amino acid for the M5-L, J10-L and Q8-L strains, respectively. These findings suggest that the M5-L, J10-L and Q8-L strains possess the ability to inhibit S. sonnei adherence to HT-29 cells, and S-layer proteins are involved in this adhesion inhibition. PMID:20600857

  20. Improving Adherence to Antiretroviral Therapy with Triggered Real Time Text Message Reminders: the China through Technology Study (CATS)

    PubMed Central

    Sabin, Lora L.; DeSilva, Mary Bachman; Gill, Christopher J.; Li, Zhong; Vian, Taryn; Wubin, Xie; Feng, Cheng; Keyi, Xu; Guanghua, Lan; Haberer, Jessica E.; Bangsberg, David R.; Yongzhen, Li; Hongyan, Lu; Gifford, Allen L.

    2015-01-01

    Background Real-time adherence monitoring is now possible through medication storage devices equipped with cellular technology. We assessed the effect of triggered cell phone reminders and counseling utilizing objective adherence data on antiretroviral (ART) adherence among Chinese HIV-infected patients. Methods We provided ART patients in Nanning, China, with a medication device (“Wisepill”) to monitor their ART adherence electronically. After 3 months, we randomized subjects within optimal (≥95%) and suboptimal (<95%) adherence strata to intervention vs. control arms. In months 4–9, intervention subjects received individualized reminders triggered by late dose-taking (no device-opening by 30 minutes past dose time), and counseling using device-generated data. Controls received no reminders or data-informed counseling. We compared post-intervention proportions achieving optimal adherence, mean adherence, and clinical outcomes. Results Of 120 subjects enrolled, 116 (96.7%) completed the trial. Pre-intervention, optimal adherence was similar in intervention vs. control arms (63.5% vs. 58.9%, respectively; p=0.60). In the last intervention month, 87.3% vs. 51.8% achieved optimal adherence (risk ratio (RR) 1.7, 95% Confidence Interval (CI) 1.3–2.2); mean adherence was 96.2% vs. 89.1% (p=0.003). Among pre-intervention suboptimal adherers, 78.3% vs. 33.3% (RR 2.4, CI 1.2–4.5) achieved optimal adherence; mean adherence was 93.3% vs. 84.7% (p=0.039). Proportions were 92.5% and 62.9% among optimal adherers, respectively (RR 1.5, CI 1.1–1.9); mean adherence was 97.8% vs. 91.7% (p=0.028). Post-intervention differences in clinical outcomes were not significant. Conclusion Real-time reminders significantly improved ART adherence in this population. This approach appears promising for managing HIV and other chronic diseases and warrants further investigation and adaptation in other settings. PMID:25886927

  1. Metabolic Differences in Microbial Cell Populations Revealed by Nanophotonic Ionization

    SciTech Connect

    Walker, Bennett; Antonakos, Cory; Retterer, Scott T; Vertes, Akos

    2013-01-01

    ellular differences are linked to cell differentiation, the proliferation of cancer and to the development of drug resistance in microbial infections. Due to sensitivity limitations, however, large- scale metabolic analysis at the single cell level is only available for cells significantly larger in volume than Saccharomyces cerevisiae (~30 fL). Here we demonstrate that by a nanophotonic ionization platform and mass spectrometry, over one hundred up to 108 metabolites, or up to 18% of the known S. cerevisiae metabolome, can be identified in very small cell populations (n < 100). Under ideal conditions, r Relative quantitation of up to 4% of the metabolites is achieved at the single cell level.

  2. Experimental depletion of different renal interstitial cell populations

    SciTech Connect

    Bohman, S.O.; Sundelin, B.; Forsum, U.; Tribukait, B.

    1988-04-01

    To define different populations of renal interstitial cells and investigate some aspects of their function, we studied the kidneys of normal rats and rats with hereditary diabetes insipidus (DI, Brattleboro) after experimental manipulations expected to alter the number of interstitial cells. DI rats showed an almost complete loss of interstitial cells in their renal papillae after treatment with a high dose of vasopressin. In spite of the lack of interstitial cells, the animals concentrated their urine to the same extent as vasopressin-treated normal rats, indicating that the renomedullary interstitial cells do not have an important function in concentrating the urine. The interstitial cells returned nearly to normal within 1 week off vasopressin treatment, suggesting a rapid turnover rate of these cells. To further distinguish different populations of interstitial cells, we studied the distribution of class II MHC antigen expression in the kidneys of normal and bone-marrow depleted Wistar rats. Normal rats had abundant class II antigen-positive interstitial cells in the renal cortex and outer medulla, but not in the inner medulla (papilla). Six days after 1000 rad whole body irradiation, the stainable cells were almost completely lost, but electron microscopic morphometry showed a virtually unchanged volume density of interstitial cells in the cortex and outer medulla, as well as the inner medulla. Thus, irradiation abolished the expression of the class II antigen but caused no significant depletion of interstitial cells.

  3. Fabrication of nanofibrous scaffold using a PLA and hagfish thread keratin composite; its effect on cell adherence, growth, and osteoblast differentiation.

    PubMed

    Kim, Beom-Su; Park, Ko Eun; Park, Won Ho; Lee, Jun

    2013-08-01

    Electrospinning is a useful method for the production of nanofibrous scaffolds in the field of tissue engineering. Keratin has been used as a biomaterial for electrospinning and can be used in a variety of biomedical applications because it is a natural protein, giving it the ability to improve cell affinity of scaffolds. In this study, keratin was extracted from hagfish slime thread (H-keratin) and blended with polylactic acid (PLA) polymer solution to construct a nanofibrous scaffold. Wool keratin (W-keratin) was used as a control for the comparison of morphological, physical, and biological properties. The results of Fourier transform infrared spectroscopy showed the presence of both W-keratin and H-keratin in the electrospun PLA/keratin. Observations with a scanning electron microscope revealed that PLA, PLA/W-keratin, and PLA/H-keratin had similar average diameters (~800 nm). Cell attachment experiments showed that MG-63 cells adhered more rapidly and spread better onto PLA/H-keratin than onto the pure PLA or PLA/W-keratin. Cell proliferation assay, DNA content, live/dead, and alkaline phosphatase activity assays showed that PLA/H-keratin scaffolds could accelerate the viability, proliferation, and osteogenesis of MG-63 cells relative to pure PLA or PLA/W-keratin nanofibrous scaffolds. These findings suggest that H-keratin can improve cellular attraction and has great potential to be used as a biomaterial in bone tissue engineering.

  4. A population of human brain cells expressing phenotypic markers of more than one lineage can be induced in vitro to differentiate into mesenchymal cells

    SciTech Connect

    Rieske, Piotr; Augelli, Brian J.; Stawski, Robert; Gaughan, John; Azizi, S. Ausim; Krynska, Barbara

    2009-02-01

    Proliferating astrocytic cells from germinal, as well as mature areas of brain parenchyma, have the characteristics of neural stem/progenitor cells and are capable of generating both neurons and glia. We previously reported that primary fetal human brain cells, designated as Normal Human Astrocytes (NHA), expressed, in addition to GFAP, Vimentin and Nestin, low levels of {beta}III-Tubulin, an early neuronal marker, and differentiated into neurons and astrocytes in vitro. Here, we showed that primary NHA cells co-express low levels of mesenchymal markers Fibronectin and Collagen-1 in culture. These cells transitioned into mesenchymal-like cells when cultured in adherent conditions in serum containing media. The mesenchymal-like derivatives of these cells were characterized based on their morphological changes, high expression of Vimentin and extracellular matrix (ECM) proteins, Collagen-1 and Fibronectin, and decline of neural markers. When incubated in osteogenic and adipogenic induction media, the mesenchymal-like cells differentiated into osteoblasts and adipocytes. Furthermore, NHA cells express markers of neural crest cells, SOX-10 and p75. These data support the idea of ectoderm-derived mesenchymal lineages. These findings suggest that a population of primitive fetal brain cells with neural/neural crest/mesenchymal phenotype, resembles the remarkable phenotypic plasticity of neural crest cells, and differentiates into adipocytes and osteocytes under the influence of environmental factors.

  5. AG490 and PF431396 Sensitive Tyrosine Kinase Control the Population Heterogeneity of Basal STAT1 Activity in Ube1l Deficient Cells.

    PubMed

    Now, Hesung; Yoo, Joo-Yeon

    2016-01-01

    A population often contains distinct sub-populations, thereby increasing the complexity of the overall heterogeneity. However, the cellular origin and biological relevance of sub-populations in cell population have not been clearly identified. Here we demonstrated the novel roles of ISGylation, which is an IFN-induced post-translational modification, controlling heterogeneity at the population level in cultured adherent cells. Without UBE1L, an E1 enzyme of ISGylation, mouse embryonic fibroblasts (MEF) exhibited low viral resistance despite high STAT1 and ISG expression compared with the wild-type MEF. We observe that Ube1l-/- MEF populations consist of two behaviorally distinguishable sub-populations with distinct basal STAT1 activity, while wild-type MEF populations are unimodal. This population heterogeneity in Ube1l knock-out cells was perturbed by tyrosine kinase inhibitors, AG490 and PF431396. In contrast, the neutralization of type I IFN did not affect population heterogeneity. Based on these results, we concluded that UBE1L functions to adjust basal immunological states with the regulation of population heterogeneity. PMID:27427993

  6. AG490 and PF431396 Sensitive Tyrosine Kinase Control the Population Heterogeneity of Basal STAT1 Activity in Ube1l Deficient Cells

    PubMed Central

    Now, Hesung; Yoo, Joo-Yeon

    2016-01-01

    A population often contains distinct sub-populations, thereby increasing the complexity of the overall heterogeneity. However, the cellular origin and biological relevance of sub-populations in cell population have not been clearly identified. Here we demonstrated the novel roles of ISGylation, which is an IFN-induced post-translational modification, controlling heterogeneity at the population level in cultured adherent cells. Without UBE1L, an E1 enzyme of ISGylation, mouse embryonic fibroblasts (MEF) exhibited low viral resistance despite high STAT1 and ISG expression compared with the wild-type MEF. We observe that Ube1l−/− MEF populations consist of two behaviorally distinguishable sub-populations with distinct basal STAT1 activity, while wild-type MEF populations are unimodal. This population heterogeneity in Ube1l knock-out cells was perturbed by tyrosine kinase inhibitors, AG490 and PF431396. In contrast, the neutralization of type I IFN did not affect population heterogeneity. Based on these results, we concluded that UBE1L functions to adjust basal immunological states with the regulation of population heterogeneity. PMID:27427993

  7. Natural cytotoxicity of haemopoietic cell populations against murine lymphoid tumours.

    PubMed Central

    Burton, R. C.; Grail, D.; Warner, N. L.

    1978-01-01

    Homozygous nude and normal mice of 3 strains, BALB/c, CBA and C57BL, were used as sources of nucleated haemopoietic "natural killer" (NK) cells. These killer cells could lyse a wide range of syngeneic and allogeneic lymphoid tumour cell lines in vitro, and it was found that cell suspensions from nude mice were always significantly more active than those from normal mice, and that the most active effector population was a polymorph-enriched peritoneal-exudate cell suspension. Eosinophils did not appear to be involved in the phenomenon, and mononuclear peritoneal-exudate cell suspensions were actually highly inhibitory. Three non-lymphoid tumours, a carcinoma, a fibrosarcoma and a mastocytoma, were totally resistant to in vitro lysis. Although all susceptible tumour cell lines were C-type virus-associated, not all of these tumours were killed by all strain sources of spleen cells, indicating a specificity of killing. PMID:656308

  8. An evaluation of a solid phase red cell adherence test for detecting platelet-associated IgG in immune thrombocytopenia.

    PubMed

    Jones, C D; Gould, L M; Lee, S

    1990-04-01

    A solid phase red cell adherence (SPRCA) test, designed for platelet cross-match testing, was evaluated to determine its efficacy in detecting platelet-associated IgG (PAIgG) in immune thrombocytopenic purpura (ITP). This new method was compared to the platelet suspension immunofluorescence (PSIF) test. Fifty-three patient samples were tested by both methods, including 17 for whom a clinical diagnosis of ITP had been made. The SPRCA method showed 65% sensitivity, whereas the PSIF method showed 53% sensitivity. The specificity for both was 100%. The SPRCA compared favorably to the PSIF test in sensitivity and cost effectiveness. The SPRCA test is rapid, easy to perform, and suitable for detecting PAIgG in ITP.

  9. Modelling Spread of Oncolytic Viruses in Heterogeneous Cell Populations

    NASA Astrophysics Data System (ADS)

    Ellis, Michael; Dobrovolny, Hana

    2014-03-01

    One of the most promising areas in current cancer research and treatment is the use of viruses to attack cancer cells. A number of oncolytic viruses have been identified to date that possess the ability to destroy or neutralize cancer cells while inflicting minimal damage upon healthy cells. Formulation of predictive models that correctly describe the evolution of infected tumor systems is critical to the successful application of oncolytic virus therapy. A number of different models have been proposed for analysis of the oncolytic virus-infected tumor system, with approaches ranging from traditional coupled differential equations such as the Lotka-Volterra predator-prey models, to contemporary modeling frameworks based on neural networks and cellular automata. Existing models are focused on tumor cells and the effects of virus infection, and offer the potential for improvement by including effects upon normal cells. We have recently extended the traditional framework to a 2-cell model addressing the full cellular system including tumor cells, normal cells, and the impacts of viral infection upon both populations. Analysis of the new framework reveals complex interaction between the populations and potential inability to simultaneously eliminate the virus and tumor populations.

  10. Rapid Cell Population Identification in Flow Cytometry Data*

    PubMed Central

    Aghaeepour, Nima; Nikolic, Radina; Hoos, Holger H.; Brinkman, Ryan R.

    2011-01-01

    We have developed flowMeans, a time-efficient and accurate method for automated identification of cell populations in flow cytometry (FCM) data based on K-means clustering. Unlike traditional K-means, flowMeans can identify concave cell populations by modelling a single population with multiple clusters. flowMeans uses a change point detection algorithm to determine the number of sub-populations, enabling the method to be used in high throughput FCM data analysis pipelines. Our approach compares favourably to manual analysis by human experts and current state-of-the-art automated gating algorithms. flowMeans is freely available as an open source R package through Bioconductor. PMID:21182178

  11. TiO2-doped phosphate glass microcarriers: A stable bioactive substrate for expansion of adherent mammalian cells

    PubMed Central

    Guedes, Joana C; Park, Jeong-Hui; Lakhkar, Nilay J; Kim, Hae-Won; Knowles, Jonathan C

    2013-01-01

    Scalable expansion of cells for regenerative cell therapy or to produce large quantities for high-throughput screening remains a challenge for bioprocess engineers. Laboratory scale cell expansion using t-flasks requires frequent passaging that exposes cells to many poorly defined bioprocess forces that can cause damage or alter their phenotype. Microcarriers offer a potential solution to scalable production, lending themselves to cell culture processes more akin to fermentation, removing the need for frequent passaging throughout the expansion period. One main problem with microcarrier expansion, however, is the difficulty in harvesting cells at the end of the process. Therefore, therapies that rely on cell delivery using biomaterial scaffolds could benefit from a microcarrier expansion system whereby the cells and microcarriers are transplanted together. In the current study, we used bioactive glass microcarriers doped with 5% TiO2 that display a controlled rate of degradation and conducted experiments to assess biocompatibility and growth of primary fibroblast cells as a model for cell therapy products. We found that the microcarriers are highly biocompatible and facilitate cell growth in a gradual controlled manner. Therefore, even without additional biofunctionalization methods, Ti-doped bioactive glass microcarriers offer potential as a cell expansion platform. PMID:22935537

  12. Cryopreservation of adherent neuronal networks.

    PubMed

    Ma, Wu; O'Shaughnessy, Thomas; Chang, Eddie

    2006-07-31

    Neuronal networks have been widely used for neurophysiology, drug discovery and toxicity testing. An essential prerequisite for future widespread application of neuronal networks is the development of efficient cryopreservation protocols to facilitate their storage and transportation. Here is the first report on cryopreservation of mammalian adherent neuronal networks. Dissociated spinal cord cells were attached to a poly-d-lysine/laminin surface and allowed to form neuronal networks. Adherent neuronal networks were embedded in a thin film of collagen gel and loaded with trehalose prior to transfer to a freezing medium containing DMSO, FBS and culture medium. This was followed by a slow rate of cooling to -80 degrees C for 24 h and then storage for up to 2 months in liquid nitrogen at -196 degrees C. The three components: DMSO, collagen gel entrapment and trehalose loading combined provided the highest post-thaw viability, relative to individual or two component protocols. The post-thaw cells with this protocol demonstrated similar neuronal and astrocytic markers and morphological structure as those detected in unfrozen cells. Fluorescent dye FM1-43 staining revealed active recycling of synaptic vesicles upon depolarizing stimulation in the post-thaw neuronal networks. These results suggest that a combination of DMSO, collagen gel entrapment and trehalose loading can significantly improve conventional slow-cooling methods in cryopreservation of adherent neuronal networks.

  13. Biocompatible micro-sized cell culture chamber for the detection of nanoparticle-induced IL8 promoter activity on a small cell population.

    PubMed

    Kohl, Yvonne; Oostingh, Gertie J; Sossalla, Adam; Duschl, Albert; von Briesen, Hagen; Thielecke, Hagen

    2011-01-01

    In most conventional in vitro toxicological assays, the response of a complete cell population is averaged, and therefore, single-cell responses are not detectable. Such averaging might result in misinterpretations when only individual cells within a population respond to a certain stimulus. Therefore, there is a need for non-invasive in vitro systems to verify the toxicity of nanoscale materials. In the present study, a micro-sized cell culture chamber with a silicon nitride membrane (0.16 mm2) was produced for cell cultivation and the detection of specific cell responses. The biocompatibility of the microcavity chip (MCC) was verified by studying adipogenic and neuronal differentiation. Thereafter, the suitability of the MCC to study the effects of nanoparticles on a small cell population was determined by using a green fluorescence protein-based reporter cell line. Interleukin-8 promoter (pIL8) induction, a marker of an inflammatory response, was used to monitor immune activation. The validation of the MCC-based method was performed using well-characterized gold and silver nanoparticles. The sensitivity of the new method was verified comparing the quantified pIL8 activation via MCC-based and standard techniques. The results proved the biocompatibility and the sensitivity of the microculture chamber, as well as a high optical quality due to the properties of Si3N4. The MCC-based method is suited for threshold- and time-dependent analysis of nanoparticle-induced IL8 promoter activity. This novel system can give dynamic information at the level of adherent single cells of a small cell population and presents a new non-invasive in vitro test method to assess the toxicity of nanomaterials and other compounds.PACS: 85.35.Be, 81.16.Nd, 87.18.Mp. PMID:21861875

  14. Biocompatible micro-sized cell culture chamber for the detection of nanoparticle-induced IL8 promoter activity on a small cell population

    NASA Astrophysics Data System (ADS)

    Kohl, Yvonne; Oostingh, Gertie J.; Sossalla, Adam; Duschl, Albert; von Briesen, Hagen; Thielecke, Hagen

    2011-08-01

    In most conventional in vitro toxicological assays, the response of a complete cell population is averaged, and therefore, single-cell responses are not detectable. Such averaging might result in misinterpretations when only individual cells within a population respond to a certain stimulus. Therefore, there is a need for non-invasive in vitro systems to verify the toxicity of nanoscale materials. In the present study, a micro-sized cell culture chamber with a silicon nitride membrane (0.16 mm2) was produced for cell cultivation and the detection of specific cell responses. The biocompatibility of the microcavity chip (MCC) was verified by studying adipogenic and neuronal differentiation. Thereafter, the suitability of the MCC to study the effects of nanoparticles on a small cell population was determined by using a green fluorescence protein-based reporter cell line. Interleukin-8 promoter (pIL8) induction, a marker of an inflammatory response, was used to monitor immune activation. The validation of the MCC-based method was performed using well-characterized gold and silver nanoparticles. The sensitivity of the new method was verified comparing the quantified pIL8 activation via MCC-based and standard techniques. The results proved the biocompatibility and the sensitivity of the microculture chamber, as well as a high optical quality due to the properties of Si3N4. The MCC-based method is suited for threshold- and time-dependent analysis of nanoparticle-induced IL8 promoter activity. This novel system can give dynamic information at the level of adherent single cells of a small cell population and presents a new non-invasive in vitro test method to assess the toxicity of nanomaterials and other compounds. PACS: 85.35.Be, 81.16.Nd, 87.18.Mp

  15. Capsule of Streptococcus equi subsp. zooepidemicus hampers the adherence and invasion of epithelial and endothelial cells and is attenuated during internalization.

    PubMed

    Xu, Bin; Pei, Xiaomeng; Su, Yiqi; Ma, Zhe; Fan, Hongjie

    2016-08-01

    Direct interaction between pathogens and host cells often is a prerequisite for colonization, infection and dissemination. Regulated production of capsular polysaccharide (CPS), which is made of hyaluronic acid, is essential for the pathogenicity of Streptococcus equi subsp. Zooepidemicus (SEZ). Here, we constructed a CPS-deleted mutant and analyzed it along with the parental wild-type strain in attachment and invasion of mammalian epithelial and endothelial cell lines. The CPS-deleted mutant exhibited significant increase in adherence and invasion by several orders of magnitude compared with the wild-type strain through quantitative analysis and electron microscopy observation. After the wild-type strain was recovered from invaded cells, its morphology was analyzed by visual methods and scanning electron microscopy, which revealed that its capsule was almost completely absent. Capsule measurements showed a similar result in which CPS production was nearly attenuated to the same extent as in the CPS-deleted mutant. qPCR assays revealed a marked reduction in the transcriptional levels of the CPS biosynthesis genes, has operon. Moreover, the repression in capsular production was stable inheritance. Our findings indicate that SEZ is a facultative intracellular bacterium, capsule attenuation in SEZ contributes to attachment and invasion in interactions with host cells, and the active regulation of capsule breakdown is controlled by SEZ during internalization. PMID:27388015

  16. The nuclear and adherent junction complex component protein ubinuclein negatively regulates the productive cycle of Epstein-Barr virus in epithelial cells.

    PubMed

    Gruffat, Henri; Lupo, Julien; Morand, Patrice; Boyer, Véronique; Manet, Evelyne

    2011-01-01

    The Epstein-Barr Virus (EBV) productive cycle is initiated by the expression of the viral trans-activator EB1 (also called Zebra, Zta, or BZLF1), which belongs to the basic leucine zipper transcription factor family. We have previously identified the cellular NACos (nuclear and adherent junction complex components) protein ubinuclein (Ubn-1) as a partner for EB1, but the function of this complex has never been studied. Here, we have evaluated the consequences of this interaction on the EBV productive cycle and find that Ubn-1 overexpression represses the EBV productive cycle whereas Ubn-1 downregulation by short hairpin RNA (shRNA) increases virus production. By a chromatin immunoprecipitation (ChIP) assay, we show that Ubn-1 blocks EB1-DNA interaction. We also show that in epithelial cells, relocalization and sequestration of Ubn-1 to the tight junctions of nondividing cells allow increased activation of the productive cycle. We propose a model in which Ubn-1 is a modulator of the EBV productive cycle: in proliferating epithelial cells, Ubn-1 is nuclear and inhibits activation of the productive cycle, whereas in differentiated cells, Ubn-1 is sequestrated to tight junctions, thereby allowing EB1 to fully function in the nucleus.

  17. Trail networks formed by populations of immune cells

    NASA Astrophysics Data System (ADS)

    Yang, Taeseok Daniel; Kwon, Tae Goo; Park, Jin-sung; Lee, Kyoung J.

    2014-02-01

    Populations of biological cells that communicate with each other can organize themselves to generate large-scale patterns. Examples can be found in diverse systems, ranging from developing embryos, cardiac tissues, chemotaxing ameba and swirling bacteria. The similarity, often shared by the patterns, suggests the existence of some general governing principle. On the other hand, rich diversity and system-specific properties are exhibited, depending on the type of involved cells and the nature of their interactions. The study on the similarity and the diversity constitutes a rapidly growing field of research. Here, we introduce a new class of self-organized patterns of cell populations that we term as ‘cellular trail networks’. They were observed with populations of rat microglia, the immune cells of the brain and the experimental evidence suggested that haptotaxis is the key element responsible for them. The essential features of the observed patterns are well captured by the mathematical model cells that actively crawl and interact with each other through a decomposing but non-diffusing chemical attractant laid down by the cells. Our finding suggests an unusual mechanism of socially cooperative long-range signaling for the crawling immune cells.

  18. The Role of Cardiac Side Population Cells in Cardiac Regeneration

    PubMed Central

    Yellamilli, Amritha; van Berlo, Jop H.

    2016-01-01

    The heart has a limited ability to regenerate. It is important to identify therapeutic strategies that enhance cardiac regeneration in order to replace cardiomyocytes lost during the progression of heart failure. Cardiac progenitor cells are interesting targets for new regenerative therapies because they are self-renewing, multipotent cells located in the heart. Cardiac side population cells (cSPCs), the first cardiac progenitor cells identified in the adult heart, have the ability to differentiate into cardiomyocytes, endothelial cells, smooth muscle cells, and fibroblasts. They become activated in response to cardiac injury and transplantation of cSPCs into the injured heart improves cardiac function. In this review, we will discuss the current literature on the progenitor cell properties and therapeutic potential of cSPCs. This body of work demonstrates the great promise cSPCs hold as targets for new regenerative strategies.

  19. The Role of Cardiac Side Population Cells in Cardiac Regeneration

    PubMed Central

    Yellamilli, Amritha; van Berlo, Jop H.

    2016-01-01

    The heart has a limited ability to regenerate. It is important to identify therapeutic strategies that enhance cardiac regeneration in order to replace cardiomyocytes lost during the progression of heart failure. Cardiac progenitor cells are interesting targets for new regenerative therapies because they are self-renewing, multipotent cells located in the heart. Cardiac side population cells (cSPCs), the first cardiac progenitor cells identified in the adult heart, have the ability to differentiate into cardiomyocytes, endothelial cells, smooth muscle cells, and fibroblasts. They become activated in response to cardiac injury and transplantation of cSPCs into the injured heart improves cardiac function. In this review, we will discuss the current literature on the progenitor cell properties and therapeutic potential of cSPCs. This body of work demonstrates the great promise cSPCs hold as targets for new regenerative strategies. PMID:27679798

  20. The Role of Cardiac Side Population Cells in Cardiac Regeneration.

    PubMed

    Yellamilli, Amritha; van Berlo, Jop H

    2016-01-01

    The heart has a limited ability to regenerate. It is important to identify therapeutic strategies that enhance cardiac regeneration in order to replace cardiomyocytes lost during the progression of heart failure. Cardiac progenitor cells are interesting targets for new regenerative therapies because they are self-renewing, multipotent cells located in the heart. Cardiac side population cells (cSPCs), the first cardiac progenitor cells identified in the adult heart, have the ability to differentiate into cardiomyocytes, endothelial cells, smooth muscle cells, and fibroblasts. They become activated in response to cardiac injury and transplantation of cSPCs into the injured heart improves cardiac function. In this review, we will discuss the current literature on the progenitor cell properties and therapeutic potential of cSPCs. This body of work demonstrates the great promise cSPCs hold as targets for new regenerative strategies.

  1. The Role of Cardiac Side Population Cells in Cardiac Regeneration.

    PubMed

    Yellamilli, Amritha; van Berlo, Jop H

    2016-01-01

    The heart has a limited ability to regenerate. It is important to identify therapeutic strategies that enhance cardiac regeneration in order to replace cardiomyocytes lost during the progression of heart failure. Cardiac progenitor cells are interesting targets for new regenerative therapies because they are self-renewing, multipotent cells located in the heart. Cardiac side population cells (cSPCs), the first cardiac progenitor cells identified in the adult heart, have the ability to differentiate into cardiomyocytes, endothelial cells, smooth muscle cells, and fibroblasts. They become activated in response to cardiac injury and transplantation of cSPCs into the injured heart improves cardiac function. In this review, we will discuss the current literature on the progenitor cell properties and therapeutic potential of cSPCs. This body of work demonstrates the great promise cSPCs hold as targets for new regenerative strategies. PMID:27679798

  2. Functional heterogeneity and heritability in CHO cell populations.

    PubMed

    Davies, Sarah L; Lovelady, Clare S; Grainger, Rhian K; Racher, Andrew J; Young, Robert J; James, David C

    2013-01-01

    In this study, we address the hypothesis that it is possible to exploit genetic/functional variation in parental Chinese hamster ovary (CHO) cell populations to isolate clonal derivatives that exhibit superior, heritable attributes for biomanufacturing--new parental cell lines which are inherently more "fit for purpose." One-hundred and ninety-nine CHOK1SV clones were isolated from a donor CHOK1SV parental population by limiting dilution cloning and microplate image analysis, followed by primary analysis of variation in cell-specific proliferation rate during extended deep-well microplate suspension culture of individual clones to accelerate genetic drift in isolated cultures. A subset of 100 clones were comparatively evaluated for transient production of a recombinant monoclonal antibody (Mab) and green fluorescent protein following transfection of a plasmid vector encoding both genes. The heritability of both cell-specific proliferation rate and Mab production was further assessed using a subset of 23 clones varying in functional capability that were subjected to cell culture regimes involving both cryopreservation and extended sub-culture. These data showed that whilst differences in transient Mab production capability were not heritable per se, clones exhibiting heritable variation in specific proliferation rate, endocytotic transfectability and N-glycan processing were identified. Finally, for clonal populations most "evolved" by extended sub-culture in vitro we investigated the relationship between cellular protein biomass content, specific proliferation rate and cell surface N-glycosylation. Rapid-specific proliferation rate was inversely correlated to CHO cell size and protein content, and positively correlated to cell surface glycan content, although substantial clone-specific variation in ability to accumulate cell biomass was evident. Taken together, our data reveal the dynamic nature of the CHO cell functional genome and the potential to evolve and

  3. Medication adherence: WHO cares?

    PubMed

    Brown, Marie T; Bussell, Jennifer K

    2011-04-01

    The treatment of chronic illnesses commonly includes the long-term use of pharmacotherapy. Although these medications are effective in combating disease, their full benefits are often not realized because approximately 50% of patients do not take their medications as prescribed. Factors contributing to poor medication adherence are myriad and include those that are related to patients (eg, suboptimal health literacy and lack of involvement in the treatment decision-making process), those that are related to physicians (eg, prescription of complex drug regimens, communication barriers, ineffective communication of information about adverse effects, and provision of care by multiple physicians), and those that are related to health care systems (eg, office visit time limitations, limited access to care, and lack of health information technology). Because barriers to medication adherence are complex and varied, solutions to improve adherence must be multifactorial. To assess general aspects of medication adherence using cardiovascular disease as an example, a MEDLINE-based literature search (January 1, 1990, through March 31, 2010) was conducted using the following search terms: cardiovascular disease, health literacy, medication adherence, and pharmacotherapy. Manual sorting of the 405 retrieved articles to exclude those that did not address cardiovascular disease, medication adherence, or health literacy in the abstract yielded 127 articles for review. Additional references were obtained from citations within the retrieved articles. This review surveys the findings of the identified articles and presents various strategies and resources for improving medication adherence.

  4. Cell population tracking and lineage construction with spatiotemporal context.

    PubMed

    Li, Kang; Miller, Eric D; Chen, Mei; Kanade, Takeo; Weiss, Lee E; Campbell, Phil G

    2008-10-01

    Automated visual-tracking of cell populations in vitro using time-lapse phase contrast microscopy enables quantitative, systematic, and high-throughput measurements of cell behaviors. These measurements include the spatiotemporal quantification of cell migration, mitosis, apoptosis, and the reconstruction of cell lineages. The combination of low signal-to-noise ratio of phase contrast microscopy images, high and varying densities of the cell cultures, topological complexities of cell shapes, and wide range of cell behaviors poses many challenges to existing tracking techniques. This paper presents a fully automated multi-target tracking system that can efficiently cope with these challenges while simultaneously tracking and analyzing thousands of cells observed using time-lapse phase contrast microscopy. The system combines bottom-up and top-down image analysis by integrating multiple collaborative modules, which exploit a fast geometric active contour tracker in conjunction with adaptive interacting multiple models (IMM) motion filtering and spatiotemporal trajectory optimization. The system, which was tested using a variety of cell populations, achieved tracking accuracy in the range of 86.9-92.5%.

  5. Cell Population Tracking and Lineage Construction with Spatiotemporal Context 1

    PubMed Central

    Li, Kang; Chen, Mei; Kanade, Takeo; Miller, Eric D.; Weiss, Lee E.; Campbell, Phil G.

    2008-01-01

    Automated visual-tracking of cell populations in vitro using phase contrast time-lapse microscopy enables quantitative, systematic and high-throughput measurements of cell behaviors. These measurements include the spatiotemporal quantification of cell migration, mitosis, apoptosis, and the construction of cell lineages. The combination of low signal-to-noise ratio of phase contrast microscopy images, high and varying densities of the cell cultures, topological complexities of cell shapes, and wide range of cell behaviors pose many challenges to existing tracking techniques. This paper presents a fully-automated multi-target tracking system that can efficiently cope with these challenges while simultaneously tracking and analyzing thousands of cells observed using time-lapse phase contrast microscopy. The system combines bottom-up and top-down image analysis by integrating multiple collaborative modules, which exploit a fast geometric active contour tracker in conjunction with adaptive interacting multiple models (IMM) motion filtering and spatiotemporal trajectory optimization. The system, which was tested using a variety of cell populations, achieved tracking accuracy in the range of 86.9%–92.5%. PMID:18656418

  6. Modeling bacterial population growth from stochastic single-cell dynamics.

    PubMed

    Alonso, Antonio A; Molina, Ignacio; Theodoropoulos, Constantinos

    2014-09-01

    A few bacterial cells may be sufficient to produce a food-borne illness outbreak, provided that they are capable of adapting and proliferating on a food matrix. This is why any quantitative health risk assessment policy must incorporate methods to accurately predict the growth of bacterial populations from a small number of pathogens. In this aim, mathematical models have become a powerful tool. Unfortunately, at low cell concentrations, standard deterministic models fail to predict the fate of the population, essentially because the heterogeneity between individuals becomes relevant. In this work, a stochastic differential equation (SDE) model is proposed to describe variability within single-cell growth and division and to simulate population growth from a given initial number of individuals. We provide evidence of the model ability to explain the observed distributions of times to division, including the lag time produced by the adaptation to the environment, by comparing model predictions with experiments from the literature for Escherichia coli, Listeria innocua, and Salmonella enterica. The model is shown to accurately predict experimental growth population dynamics for both small and large microbial populations. The use of stochastic models for the estimation of parameters to successfully fit experimental data is a particularly challenging problem. For instance, if Monte Carlo methods are employed to model the required distributions of times to division, the parameter estimation problem can become numerically intractable. We overcame this limitation by converting the stochastic description to a partial differential equation (backward Kolmogorov) instead, which relates to the distribution of division times. Contrary to previous stochastic formulations based on random parameters, the present model is capable of explaining the variability observed in populations that result from the growth of a small number of initial cells as well as the lack of it compared to

  7. Heart failure patient adherence: epidemiology, cause, and treatment.

    PubMed

    Corotto, Paul S; McCarey, Melissa M; Adams, Suzanne; Khazanie, Prateeti; Whellan, David J

    2013-01-01

    Poor adherence to therapeutic regimens is a significant impediment to improving clinical outcomes in the HF population. Typical rates of adherence to prescribed medications, low-sodium diets, and aerobic exercise programs remain lower than that needed to decrease morbidity and mortality associated with HF. Factors contributing to poor adherence include multiple comorbidities, clinical depression, and decreased cognitive functioning. HF education and programs to enhance self-management skills have improved patient quality of life but have yet to decrease mortality or rehospitalization rates significantly. Telemonitoring to improve adherence behaviors and self-management interventions within broader HF management programs have demonstrated significant clinical improvements in this population.

  8. Basic leucine zipper (bZIP) domain transcription factor MBZ1 regulates cell wall integrity, spore adherence, and virulence in Metarhizium robertsii.

    PubMed

    Huang, Wei; Shang, Yanfang; Chen, Peilin; Cen, Kai; Wang, Chengshu

    2015-03-27

    Transcription factors (TFs) containing the basic leucine zipper (bZIP) domain are widely distributed in eukaryotes and display an array of distinct functions. In this study, a bZIP-type TF gene (MBZ1) was deleted and functionally characterized in the insect pathogenic fungus Metarhizium robertsii. The deletion mutant (ΔMBZ1) showed defects in cell wall integrity, adhesion to hydrophobic surfaces, and topical infection of insects. Relative to the WT, ΔMBZ1 was also impaired in growth and conidiogenesis. Examination of putative target gene expression indicated that the genes involved in chitin biosynthesis were differentially transcribed in ΔMBZ1 compared with the WT, which led to the accumulation of a higher level of chitin in mutant cell walls. MBZ1 exhibited negative regulation of subtilisin proteases, but positive control of an adhesin gene, which is consistent with the observation of effects on cell autolysis and a reduction in spore adherence to hydrophobic surfaces in ΔMBZ1. Promoter binding assays indicated that MBZ1 can bind to different target genes and suggested the possibility of heterodimer formation to increase the diversity of the MBZ1 regulatory network. The results of this study advance our understanding of the divergence of bZIP-type TFs at both intra- and interspecific levels.

  9. Basic leucine zipper (bZIP) domain transcription factor MBZ1 regulates cell wall integrity, spore adherence, and virulence in Metarhizium robertsii.

    PubMed

    Huang, Wei; Shang, Yanfang; Chen, Peilin; Cen, Kai; Wang, Chengshu

    2015-03-27

    Transcription factors (TFs) containing the basic leucine zipper (bZIP) domain are widely distributed in eukaryotes and display an array of distinct functions. In this study, a bZIP-type TF gene (MBZ1) was deleted and functionally characterized in the insect pathogenic fungus Metarhizium robertsii. The deletion mutant (ΔMBZ1) showed defects in cell wall integrity, adhesion to hydrophobic surfaces, and topical infection of insects. Relative to the WT, ΔMBZ1 was also impaired in growth and conidiogenesis. Examination of putative target gene expression indicated that the genes involved in chitin biosynthesis were differentially transcribed in ΔMBZ1 compared with the WT, which led to the accumulation of a higher level of chitin in mutant cell walls. MBZ1 exhibited negative regulation of subtilisin proteases, but positive control of an adhesin gene, which is consistent with the observation of effects on cell autolysis and a reduction in spore adherence to hydrophobic surfaces in ΔMBZ1. Promoter binding assays indicated that MBZ1 can bind to different target genes and suggested the possibility of heterodimer formation to increase the diversity of the MBZ1 regulatory network. The results of this study advance our understanding of the divergence of bZIP-type TFs at both intra- and interspecific levels. PMID:25673695

  10. Cyclic stretching of mesangial cells up-regulates intercellular adhesion molecule-1 and leukocyte adherence: a possible new mechanism for glomerulosclerosis.

    PubMed

    Riser, B L; Varani, J; Cortes, P; Yee, J; Dame, M; Sharba, A K

    2001-01-01

    Intraglomerular hypertension is a primary causal factor in the progressive glomerulosclerosis that characterizes diabetic nephropathy or severe renal ablation. However, inflammation of the glomerular mesangium also participates in at least the early phase of these diseases. In glomerulonephritis, where inflammation is thought to be the predominant causal factor, intraglomerular hypertension is also often present. Mesangial cells (MCs) are critical in orchestrating key functions of the glomerulus including extracellular matrix metabolism, cytokine production, and interaction with leukocytes. Because MCs are subject to increased stretching when intraglomerular hypertension is present, and in glomerulonephritis MC/leukocyte interactions seem to be mediated primarily via the up-regulation of intercellular adhesion molecule-1 (ICAM-1), we examine the possibility that cyclic stretching is a stimulus for increased MC ICAM-1 activity. We demonstrate that the normal low levels of MC ICAM-1 mRNA and protein are dramatically up-regulated by even short intervals of cyclic stretch. This effect is dose- and time-dependent, and requires little amplitude and a brief period of elongation for significant induction. Stretch-induced MC ICAM-1 also leads to a marked elevation in phagocytic leukocyte adherence. This stimulated adherence is equal or greater than that induced by the inflammatory cytokine tumor necrosis factor-alpha, whereas an additive effect occurs when both are applied in combination. Our results indicate that stretch-induced ICAM-1 may provide a direct link between hypertension and inflammation in the progression of injury and glomerulosclerosis in diabetes, renal ablation, and other forms of glomerulonephritis. PMID:11141473

  11. Galectin-1 Regulates Tissue Exit of Specific Dendritic Cell Populations*

    PubMed Central

    Thiemann, Sandra; Man, Jeanette H.; Chang, Margaret H.; Lee, Benhur; Baum, Linda G.

    2015-01-01

    During inflammation, dendritic cells emigrate from inflamed tissue across the lymphatic endothelium into the lymphatic vasculature and travel to regional lymph nodes to initiate immune responses. However, the processes that regulate dendritic cell tissue egress and migration across the lymphatic endothelium are not well defined. The mammalian lectin galectin-1 is highly expressed by vascular endothelial cells in inflamed tissue and has been shown to regulate immune cell tissue entry into inflamed tissue. Here, we show that galectin-1 is also highly expressed by human lymphatic endothelial cells, and deposition of galectin-1 in extracellular matrix selectively regulates migration of specific human dendritic cell subsets. The presence of galectin-1 inhibits migration of immunogenic dendritic cells through the extracellular matrix and across lymphatic endothelial cells, but it has no effect on migration of tolerogenic dendritic cells. The major galectin-1 counter-receptor on both dendritic cell populations is the cell surface mucin CD43; differential core 2 O-glycosylation of CD43 between immunogenic dendritic cells and tolerogenic dendritic cells appears to contribute to the differential effect of galectin-1 on migration. Binding of galectin-1 to immunogenic dendritic cells reduces phosphorylation and activity of the protein-tyrosine kinase Pyk2, an effect that may also contribute to reduced migration of this subset. In a murine lymphedema model, galectin-1−/− animals had increased numbers of migratory dendritic cells in draining lymph nodes, specifically dendritic cells with an immunogenic phenotype. These findings define a novel role for galectin-1 in inhibiting tissue emigration of immunogenic, but not tolerogenic, dendritic cells, providing an additional mechanism by which galectin-1 can dampen immune responses. PMID:26216879

  12. Attachment role of gonococcal pili. Optimum conditions and quantitation of adherence of isolated pili to human cells in vitro.

    PubMed Central

    Pearce, W A; Buchanan, T M

    1978-01-01

    Gonoccocal pili facilitate attachment of virulent Neisseria gonorrhoeae to human cells. To characterize this attachment function, purified gonococcal pili isolated from four strains possessing antigenically distinct pili were radiolabeled with 125I and used to measure the attachment of pili to various human cells in vitro. Human buccal and cervical-vaginal mucosal epithealial cells, fallopian tube mucosa, and sperm bound pili in greater numbers per micrometer2 of surface area (1--10) than fetal tonsil fibroblasts, HeLa M cells, erythrocytes, or polymorphonuclear leukocytes. This cell specificity of attachment suggests a greater density of membrane pili binding sites on cells similar or identical to cells from natural sites of infection. The pili binding sites were quantitated as 1 X 10(4) per cervical-vaginal squamous cell. Pili of all antigenic types attached equally to a given cell type, implying that the attachment moiety of each pilus was similar. Attachement of gonoccocal pili to human cells occurred quickly with saturation of presumed receptor sites within 20--60 min. Attachment was temperature dependent (37 degrees greater than 20 degrees greater than 4 degrees C), and pH dependent (3.5 less than 4.5 less than 5.5 less than 7.5). Attachment was inhibited by antibody to pili (homologous pili Ab greater than heterologous Ab). The extent of possible protection against gonococcal infection due to inhibition of pili-mediated attachment might prove limited as a result of the considerable antigenic heterogeneity among pili and the observation that blockage of pili attachment is maximal only with antibody to pili of the infecting strain. Images PMID:96134

  13. Deformation measurement of individual cells in large populations using a single-cell microchamber array chip

    NASA Astrophysics Data System (ADS)

    Doh, I.; Lee, W. C.; Cho, Y.-H.; Pisano, A. P.; Kuypers, F. A.

    2012-04-01

    We analyze the deformability of individual red blood cells (RBCs) using SiCMA technology. Our approach is adequate to quickly measure large numbers of individual cells in heterogeneous populations. Individual cells are trapped in a large-scale array of micro-wells, and dielectrophoretic (DEP) force is applied to deform the cells. The simple structures of micro-wells and DEP electrodes facilitate the analysis of thousands of RBCs in parallel. This unique method allows the correlation of red cell deformation with cell surface and cytosolic characteristics to define the distribution of individual cellular characteristics in heterogeneous populations.

  14. The Stem Cell Research and the Aging of Brazilian Population.

    PubMed

    Evangelista, Laura S M; Devina, Florentin; Carvalho, Braulio F de

    2015-02-01

    The developing countries are experiencing a shift in the population profile, faster than that experienced by developed countries, especially due to the consolidation of health practices and technological advances. These social changes also imply new socioeconomic models, able to escort social demands caused by the growth of the elderly population and the decrease in young economically active population. Several countries seek for actions that do not marginalize the elderly, and invest heavily in new technologies to ensure health access and active participation of this group in society. Stem cell research may reflect at an improvement of public health, reduction in costs of hospitalization and lead to prevention and treatment of extremely debilitating illness, like the neurodegenerative diseases. Preserving the physical and mental functional capacity is of extreme importance to ensure the active participation of elderly population in society.

  15. Adherence to Insulin Therapy.

    PubMed

    Sarbacker, G Blair; Urteaga, Elizabeth M

    2016-08-01

    IN BRIEF Six million people with diabetes use insulin either alone or in combination with an oral medication. Many barriers exist that lead to poor adherence with insulin. However, there is an underwhelming amount of data on interventions to address these barriers and improve insulin adherence. Until pharmacological advancements create easier, more acceptable insulin regimens, it is imperative to involve patients in shared decision-making. PMID:27574371

  16. Adherence of Clostridium thermocellum to cellulose.

    PubMed Central

    Bayer, E A; Kenig, R; Lamed, R

    1983-01-01

    The adherence of Clostridium thermocellum, a cellulolytic, thermophilic anaerobe, to its insoluble substrate (cellulose) was studied. The adherence phenomenon was determined to be selective for cellulose. The observed adherence was not significantly affected by various parameters, including salts, pH, temperature, detergents, or soluble sugars. A spontaneous adherence-defective mutant strain (AD2) was isolated from the wild-type strain YS. Antibodies were prepared against the bacterial cell surface and rendered specific to the cellulose-binding factor (CBF) by adsorption to mutant AD2 cells. By using these CBF-specific antibodies, crossed immunoelectrophoresis of cell extracts revealed a single discrete precipitation peak in the parent strain which was absent in the mutant. This difference was accompanied by an alteration in the polypeptide profile whereby sonicates of strain YS contained a 210,000-molecular-weight band which was missing in strain AD2. The CBF antigen could be removed from cell extracts by adsorption to cellulose. A combined gel-overlay--immunoelectrophoretic technique demonstrated that the cellulose-binding properties of the CBF were accompanied by carboxymethylcellulase activity. During the exponential phase of growth, a large part of the CBF antigen and related carboxymethylcellulase activity was associated with the cells of wild-type strain YS. However, the amounts decreased in stationary-phase cells. Cellobiose-grown mutant AD2 cells lacked the cell-associated CBF, but the latter was detected in the extracellular fluid. Increased levels of CBF were observed when cells were grown on cellulose. In addition, mutant AD2 regained cell-associated CBF together with the property of cellulose adherence. The presence of the CBF antigen and related adherence characteristics appeared to be a phenomenon common to other naturally occurring strains of this species. Images PMID:6630152

  17. Single-cell and population lag times as a function of cell age.

    PubMed

    Pin, Carmen; Baranyi, József

    2008-04-01

    After inoculation, the times to the first divisions are longer and more widely distributed for those Escherichia coli single cells that spent more time in the stationary phase prior to inoculation. The second generation times are still longer than the typical generation times in the exponential phase, and this extended the apparent lag time of the cell population. The greater the variability of the single-cell interdivision intervals, the shorter are both the lag time and the doubling time of the population.

  18. γδ T Cells Shape Preimmune Peripheral B Cell Populations.

    PubMed

    Huang, Yafei; Getahun, Andrew; Heiser, Ryan A; Detanico, Thiago O; Aviszus, Katja; Kirchenbaum, Greg A; Casper, Tamara L; Huang, Chunjian; Aydintug, M Kemal; Carding, Simon R; Ikuta, Koichi; Huang, Hua; Wysocki, Lawrence J; Cambier, John C; O'Brien, Rebecca L; Born, Willi K

    2016-01-01

    We previously reported that selective ablation of certain γδ T cell subsets, rather than removal of all γδ T cells, strongly affects serum Ab levels in nonimmunized mice. This type of manipulation also changed T cells, including residual γδ T cells, revealing some interdependence of γδ T cell populations. For example, in mice lacking Vγ4(+) and Vγ6(+) γδ T cells (B6.TCR-Vγ4(-/-)/6(-/-)), we observed expanded Vγ1(+) cells, which changed in composition and activation and produced more IL-4 upon stimulation in vitro, increased IL-4 production by αβ T cells as well as spontaneous germinal center formation in the spleen, and elevated serum Ig and autoantibodies. We therefore examined B cell populations in this and other γδ-deficient mouse strains. Whereas immature bone marrow B cells remained largely unchanged, peripheral B cells underwent several changes. Specifically, transitional and mature B cells in the spleen of B6.TCR-Vγ4(-/-)/6(-/-) mice and other peripheral B cell populations were diminished, most of all splenic marginal zone (MZ) B cells. However, relative frequencies and absolute numbers of Ab-producing cells, as well as serum levels of Abs, IL-4, and BAFF, were increased. Cell transfers confirmed that these changes are directly dependent on the altered γδ T cells in this strain and on their enhanced potential of producing IL-4. Further evidence suggests the possibility of direct interactions between γδ T cells and B cells in the splenic MZ. Taken together, these data demonstrate the capability of γδ T cells of modulating size and productivity of preimmune peripheral B cell populations. PMID:26582947

  19. Inference of cell-cell interactions from population density characteristics and cell trajectories on static and growing domains.

    PubMed

    Ross, Robert J H; Yates, C A; Baker, R E

    2015-06-01

    A key feature of cell migration is how cell movement is affected by cell-cell interactions. Furthermore, many cell migratory processes such as neural crest stem cell migration [Thomas and Erickson, 2008; McLennan et al., 2012] occur on growing domains or in the presence of a chemoattractant. Therefore, it is important to study interactions between migrating cells in the context of domain growth and directed motility. Here we compare discrete and continuum models describing the spatial and temporal evolution of a cell population for different types of cell-cell interactions on static and growing domains. We suggest that cell-cell interactions can be inferred from population density characteristics in the presence of motility bias, and these population density characteristics for different cell-cell interactions are conserved on both static and growing domains. We also study the expected displacement of a tagged cell, and show that different types of cell-cell interactions can give rise to cell trajectories with different characteristics. These characteristics are conserved in the presence of domain growth, however, they are diminished in the presence of motility bias. Our results are relevant for researchers who study the existence and role of cell-cell interactions in biological systems, so far as we suggest that different types of cell-cell interactions could be identified from cell density and trajectory data.

  20. Medication adherence among transgender women living with HIV.

    PubMed

    Baguso, Glenda N; Gay, Caryl L; Lee, Kathryn A

    2016-08-01

    Medication adherence is linked to health outcomes among adults with HIV infection. Transgender women living with HIV (TWLWH) in the US report suboptimal adherence to medications and are found to have difficulty integrating HIV medication into their daily routine, but few studies explore the factors associated with medication adherence among transgender women. Thus, the purpose of this paper is to examine demographic and clinical factors related to self-reported medication adherence among transgender women. This secondary analysis is based on data collected from the Symptom and Genetic Study that included a convenience sample of 22 self-identified transgender women, 201 non-transgender men, and 72 non-transgender women recruited in northern California. Self-reported medication adherence was assessed using the AIDS Clinical Trials Group Adherence Questionnaire. Gender differences in demographic and clinical variables were assessed, as were differences between transgender women reporting high and low adherence. Transgender women had lower adherence to medications compared to non-transgender males and non-transgender females (p = .028) and were less likely to achieve viral suppression (p = .039). Within the transgender group, Black/African-Americans reported better adherence than participants who were Whites/Caucasian or other races (p = .009). Adherence among transgender women was unrelated to medication count and estrogen therapy, but consistent with other reports on the HIV population as a whole; transgender women with high adherence were more likely to achieve viral suppression compared to the transgender women with low adherence. Despite the high incidence of HIV infection in the transgender population, few studies focus on TWLWH, either in regard to their adherence to antiretroviral therapies or to their healthcare in general. To address ongoing health disparities, more studies are needed focusing on the transgender population's continuum of care in

  1. Modeling circadian clock-cell cycle interaction effects on cell population growth rates.

    PubMed

    El Cheikh, R; Bernard, S; El Khatib, N

    2014-12-21

    The circadian clock and the cell cycle are two tightly coupled oscillators. Recent analytical studies have shown counter-intuitive effects of circadian gating of the cell cycle on growth rates of proliferating cells which cannot be explained by a molecular model or a population model alone. In this work, we present a combined molecular-population model that studies how coupling the circadian clock to the cell cycle, through the protein WEE1, affects a proliferating cell population. We show that the cell cycle can entrain to the circadian clock with different rational period ratios and characterize multiple domains of entrainment. We show that coupling increases the growth rate for autonomous periods of the cell cycle around 24 h and above 48 h. We study the effect of mutation of circadian genes on the growth rate of cells and show that disruption of the circadian clock can lead to abnormal proliferation. Particularly, we show that Cry 1, Cry 2 mutations decrease the growth rate of cells, Per 2 mutation enhances it and Bmal 1 knockout increases it for autonomous periods of the cell cycle less than 21 h and decreases it elsewhere. Combining a molecular model to a population model offers new insight on the influence of the circadian clock on the growth of a cell population. This can help chronotherapy which takes benefits of physiological rhythms to improve anti-cancer efficacy and tolerance to drugs by administering treatments at a specific time of the day.

  2. Functional heterogeneity of side population cells in skeletal muscle

    SciTech Connect

    Uezumi, Akiyoshi; Ojima, Koichi; Fukada, So-ichiro; Ikemoto, Madoka; Masuda, Satoru; Miyagoe-Suzuki, Yuko; Takeda, Shin'ichi . E-mail: takeda@ncnp.go.jp

    2006-03-17

    Skeletal muscle regeneration has been exclusively attributed to myogenic precursors, satellite cells. A stem cell-rich fraction referred to as side population (SP) cells also resides in skeletal muscle, but its roles in muscle regeneration remain unclear. We found that muscle SP cells could be subdivided into three sub-fractions using CD31 and CD45 markers. The majority of SP cells in normal non-regenerating muscle expressed CD31 and had endothelial characteristics. However, CD31{sup -}CD45{sup -} SP cells, which are a minor subpopulation in normal muscle, actively proliferated upon muscle injury and expressed not only several regulatory genes for muscle regeneration but also some mesenchymal lineage markers. CD31{sup -}CD45{sup -} SP cells showed the greatest myogenic potential among three SP sub-fractions, but indeed revealed mesenchymal potentials in vitro. These SP cells preferentially differentiated into myofibers after intramuscular transplantation in vivo. Our results revealed the heterogeneity of muscle SP cells and suggest that CD31{sup -}CD45{sup -} SP cells participate in muscle regeneration.

  3. Capability of yeast derivatives to adhere enteropathogenic bacteria and to modulate cells of the innate immune system.

    PubMed

    Ganner, Anja; Schatzmayr, Gerd

    2012-07-01

    Yeast derivatives including yeast cell wall components are promising alternatives to antibiotics with respect to the promotion of health and performance in livestock, based on their capacity to bind enteropathogenic bacteria and to beneficially modulate the immune system. However, these mode(s) of action both in vitro and in vivo are still not well understood. Furthermore, standardization and reproducibility of in vitro techniques (microbiology, cell culture assays) are critical features for the application of yeast derivatives as well as for the proof of effectiveness. Yeast cell wall products are suggested as anti-adhesive agents and are thus proposed to prevent attachment of certain intestinal bacteria by providing alternative adhesion sites to enterobacteria, which contain mannose-specific type I fimbriae such as Escherichia coli or Salmonella spp. and which is well documented. Various in vitro assay techniques have become of paramount importance for biotechnological research since they allow for determination and quantification of potential mode(s) of action. However, in vitro assays may be criticized by product end users as not accurately reflecting in vivo responses. Pro and cons of different assays and their bias will be discussed specifically regarding yeast cell wall components and adhesion of enteropathogenic bacteria. Immunomodulation is a therapeutic approach intervening in auto-regulating processes of the defense system. Yeast derivatives such as beta-glucans are proposed to interact with cells of the innate immune system by receptor recognition. Controversial data in literature and mode(s) of action are reviewed and discussed here.

  4. High-Content Imaging Assays for Identifying Compounds that Generate Superoxide and Impair Mitochondrial Membrane Potential in Adherent Eukaryotic Cells.

    PubMed

    Billis, Puja; Will, Yvonne; Nadanaciva, Sashi

    2014-01-01

    Reactive oxygen species (ROS) are constantly produced in cells as a result of aerobic metabolism. When there is an excessive production of ROS and the cell's antioxidant defenses are overwhelmed, oxidative stress occurs. The superoxide anion is a type of ROS that is produced primarily in mitochondria but is also generated in other regions of the cell including peroxisomes, endoplasmic reticulum, plasma membrane, and cytosol. Here, a high-content imaging assay using the dye dihydroethidium is described for identifying compounds that generate superoxide in eukaryotic cells. A high-content imaging assay using the fluorescent dye tetramethylrhodamine methyl ester is also described to identify compounds that impair mitochondrial membrane potential in eukaryotic cells. The purpose of performing both assays is to identify compounds that (1) generate superoxide at lower concentrations than they impair mitochondrial membrane potential, (2) impair mitochondrial membrane potential at lower concentrations than they generate superoxide, (3) generate superoxide and impair mitochondrial function at similar concentrations, and (4) do not generate superoxide or impair mitochondrial membrane potential during the duration of the assays.

  5. Overexpression of the Candida albicans ALA1 Gene in Saccharomyces cerevisiae Results in Aggregation following Attachment of Yeast Cells to Extracellular Matrix Proteins, Adherence Properties Similar to Those of Candida albicans

    PubMed Central

    Gaur, Nand K.; Klotz, Stephen A.; Henderson, Ramona L.

    1999-01-01

    Candida albicans maintains a commensal relationship with human hosts, probably by adhering to mucosal tissue in a variety of physiological conditions. We show that adherence due to the C. albicans gene ALA1 when transformed into Saccharomyces cerevisiae, is comprised of two sequential steps. Initially, C. albicans rapidly attaches to extracellular matrix (ECM) protein-coated magnetic beads in small numbers (the attachment phase). This is followed by a relatively slower step in which cell-to-cell interactions predominate (the aggregation phase). Neither of these phases is observed in S. cerevisiae. However, expression of the C. albicans ALA1 gene from a low-copy vector causes S. cerevisiae transformants to attach to ECM-coated magnetic beads without appreciable aggregation. Expression of ALA1 from a high-copy vector results in both attachment and aggregation. Moreover, transcriptional fusion of ALA1 with the galactose-inducible promoters GALS, GALL, and GAL1, allowing for low, moderate, and high levels of inducible transcription, respectively, causes attachment and aggregation that correlates with the strength of the GAL promoter. The adherence of C. albicans and S. cerevisiae overexpressing ALA1 to a number of protein ligands occurs over a broad pH range, is resistant to shear forces generated by vortexing, and is unaffected by the presence of sugars, high salt levels, free ligands, or detergents. Adherence is, however, inhibited by agents that disrupt hydrogen bonds. The similarities in the adherence and aggregation properties of C. albicans and S. cerevisiae overexpressing ALA1 suggest a role in adherence and aggregation for ALA1 and ALA1-like genes in C. albicans. PMID:10531265

  6. Limits to collaborative concentration sensing in cell populations

    NASA Astrophysics Data System (ADS)

    Fancher, Sean; Mugler, Andrew

    Cells sense chemical concentrations with a precision that approaches the physical limit set by molecular diffusion. Recent experiments have vividly shown that cells can beat this limit when they communicate. We derive the physical limits to concentration sensing for cells that communicate over short distances by directly exchanging small molecules across their membranes (juxtacrine signaling), and over long distances by secreting and absorbing a diffusive messenger molecule (paracrine signaling). In the latter case, we find that the cell spacing that optimizes precision can be large, due to a tradeoff between maintaining communication strength and reducing signal cross-correlations. This leads to the surprising result that paracrine signaling allows more precise sensing than juxtacrine signaling for sufficiently large populations, even though this means that the cells are spaced far apart. We compare our results to recent experiments. This work is supported by a Grant from the Simons Foundation (376198 to A.M.).

  7. A Fine-Tuned Interaction between Trimeric Autotransporter Haemophilus Surface Fibrils and Vitronectin Leads to Serum Resistance and Adherence to Respiratory Epithelial Cells

    PubMed Central

    Singh, Birendra; Su, Yu-Ching; Al-Jubair, Tamim; Mukherjee, Oindrilla; Hallström, Teresia; Mörgelin, Matthias; Blom, Anna M.

    2014-01-01

    Haemophilus influenzae type b (Hib) escapes the host immune system by recruitment of the complement regulator vitronectin, which inhibits the formation of the membrane attack complex (MAC) by inhibiting C5b-C7 complex formation and C9 polymerization. We reported previously that Hib acquires vitronectin at the surface by using Haemophilus surface fibrils (Hsf). Here we studied in detail the interaction between Hsf and vitronectin and its role in the inhibition of MAC formation and the invasion of lung epithelial cells. The vitronectin-binding region of Hsf was defined at the N-terminal region comprising Hsf amino acids 429 to 652. Moreover, the Hsf recognition site on vitronectin consisted of the C-terminal amino acids 352 to 374. H. influenzae was killed more rapidly in vitronectin-depleted serum than in normal human serum (NHS), and increased MAC deposition was observed at the surface of an Hsf-deficient H. influenzae mutant. In parallel, Hsf-expressing Escherichia coli selectively acquired vitronectin from serum, resulting in significant inhibition of the MAC. Moreover, when vitronectin was bound to Hsf, increased bacterial adherence and internalization into epithelial cells were observed. Taking our findings together, we have defined a fine-tuned protein-protein interaction between Hsf and vitronectin that may contribute to increased Hib virulence. PMID:24664511

  8. Scalable expansion of human induced pluripotent stem cells in the defined xeno-free E8 medium under adherent and suspension culture conditions☆

    PubMed Central

    Wang, Ying; Chou, Bin-Kuan; Dowey, Sarah; He, Chaoxia; Gerecht, Sharon; Cheng, Linzhao

    2015-01-01

    Large-scale production of human induced pluripotent stem cells (hiPSCs) by robust and economic methods has been one of the major challenges for translational realization of hiPSC technology. Here we demonstrate a scalable culture system for hiPSC expansion using the E8 chemically defined and xeno-free medium under either adherent or suspension conditions. To optimize suspension conditions guided by a computational simulation, we developed a method to efficiently expand hiPSCs as undifferentiated aggregates in spinner flasks. Serial passaging of two different hiPSC lines in the spinner flasks using the E8 medium preserved their normal karyotype and expression of undifferentiated state markers of TRA-1–60, SSEA4, OCT4, and NANOG. The hiPSCs cultured in spinner flasks for more than 10 passages not only could be remained pluripotent as indicated by in vitro and in vivo assays, but also could be efficiently induced toward mesodermal and hematopoietic differentiation. Furthermore, we established a xeno-free protocol of single-cell cryopreservation and recovery for the scalable production of hiPSCs in spinner flasks. This system is the first to enable an efficient scale-up bioprocess in completely xeno-free condition for the expansion and cryopreservation of hiPSCs with the quantity and quality compliant for clinical applications. PMID:23973800

  9. In Situ Proximity Ligation Assay (PLA) Analysis of Protein Complexes Formed Between Golgi-Resident, Glycosylation-Related Transporters and Transferases in Adherent Mammalian Cell Cultures.

    PubMed

    Maszczak-Seneczko, Dorota; Sosicka, Paulina; Olczak, Teresa; Olczak, Mariusz

    2016-01-01

    In situ proximity ligation assay (PLA) is a novel, revolutionary technique that can be employed to visualize protein complexes in fixed cells and tissues. This approach enables demonstration of close (i.e., up to 40 nm) proximity between any two proteins of interest that can be detected using a pair of specific antibodies that have been raised in distinct species. Primary antibodies bound to the target proteins are subsequently recognized by two PLA probes, i.e., secondary antibodies conjugated with oligonucleotides that anneal when brought into close proximity in the presence of two connector oligonucleotides and a DNA ligase forming a circular DNA molecule. In the next step, the resulting circular DNA is amplified by a rolling circle polymerase. Finally, fluorescent oligonucleotide probes hybridize to complementary fragments of the amplified DNA molecule, forming a typical, spot-like pattern of PLA signal that reflects subcellular localization of protein complexes. Here we describe the use of in situ PLA in adherent cultures of mammalian cells in order to visualize interactions between Golgi-resident, functionally related glycosyltransferases and nucleotide sugar transporters relevant to N-glycan biosynthesis.

  10. In Situ Proximity Ligation Assay (PLA) Analysis of Protein Complexes Formed Between Golgi-Resident, Glycosylation-Related Transporters and Transferases in Adherent Mammalian Cell Cultures.

    PubMed

    Maszczak-Seneczko, Dorota; Sosicka, Paulina; Olczak, Teresa; Olczak, Mariusz

    2016-01-01

    In situ proximity ligation assay (PLA) is a novel, revolutionary technique that can be employed to visualize protein complexes in fixed cells and tissues. This approach enables demonstration of close (i.e., up to 40 nm) proximity between any two proteins of interest that can be detected using a pair of specific antibodies that have been raised in distinct species. Primary antibodies bound to the target proteins are subsequently recognized by two PLA probes, i.e., secondary antibodies conjugated with oligonucleotides that anneal when brought into close proximity in the presence of two connector oligonucleotides and a DNA ligase forming a circular DNA molecule. In the next step, the resulting circular DNA is amplified by a rolling circle polymerase. Finally, fluorescent oligonucleotide probes hybridize to complementary fragments of the amplified DNA molecule, forming a typical, spot-like pattern of PLA signal that reflects subcellular localization of protein complexes. Here we describe the use of in situ PLA in adherent cultures of mammalian cells in order to visualize interactions between Golgi-resident, functionally related glycosyltransferases and nucleotide sugar transporters relevant to N-glycan biosynthesis. PMID:27632007

  11. Transcriptional and phenotypical heterogeneity of Trypanosoma cruzi cell populations

    PubMed Central

    Seco-Hidalgo, Víctor; De Pablos, Luis Miguel; Osuna, Antonio

    2015-01-01

    Trypanosoma cruzi has a complex life cycle comprising pools of cell populations which circulate among humans, vectors, sylvatic reservoirs and domestic animals. Recent experimental evidence has demonstrated the importance of clonal variations for parasite population dynamics, survival and evolution. By limiting dilution assays, we have isolated seven isogenic clonal cell lines derived from the Pan4 strain of T. cruzi. Applying different molecular techniques, we have been able to provide a comprehensive characterization of the expression heterogeneity in the mucin-associated surface protein (MASP) gene family, where all the clonal isogenic populations were transcriptionally different. Hierarchical cluster analysis and sequence comparison among different MASP cDNA libraries showed that, despite the great variability in MASP expression, some members of the transcriptome (including MASP pseudogenes) are conserved, not only in the life-cycle stages but also among different strains of T. cruzi. Finally, other important aspects for the parasite, such as growth, spontaneous metacyclogenesis or excretion of different catabolites, were also compared among the clones, demonstrating that T. cruzi populations of cells are also phenotypically heterogeneous. Although the evolutionary strategy that sustains the MASP expression polymorphism remains unknown, we suggest that MASP clonal variability and phenotypic heterogeneities found in this study might provide an advantage, allowing a rapid response to environmental pressure or changes during the life cycle of T. cruzi. PMID:26674416

  12. 'Attached cell' antigen 28.3.7 mapping to human chromosome 15 characterises TPA-induced differentiation of the promyelocytic HL-60 cell line to give macrophage/monocyte populations.

    PubMed Central

    Blaineau, C; Avner, P; Tunnacliffe, A; Goodfellow, P

    1983-01-01

    Human cells growing in vitro attached to the substratum express a cell antigen called 28.3.7 identified by a species-specific monoclonal antibody. This antigen is not expressed on human cells growing in suspension. The antigen has a mol. wt. in reduced SDS-polyacrylamide gel electrophoresis gels of 95 000 and in human-mouse somatic cell hybrids, expression of the antigen is controlled by a gene, MIC7, mapping to human chromosome 15. The antigen functions as a marker for macrophage differentiation. In vitro differentiation of the 28.3.7 antigen-negative human promyelocytic leukaemia line HL-60 induced by phorbol ester, results in the formation of a macrophage/monocyte population and the concomitant expression of the 28.3.7 antigen on this adherent cell population. Images Fig. 1. PMID:6641710

  13. Deconstructing stem cell population heterogeneity: Single-cell analysis and modeling approaches

    PubMed Central

    Wu, Jincheng; Tzanakakis, Emmanuel S.

    2014-01-01

    Isogenic stem cell populations display cell-to-cell variations in a multitude of attributes including gene or protein expression, epigenetic state, morphology, proliferation and proclivity for differentiation. The origins of the observed heterogeneity and its roles in the maintenance of pluripotency and the lineage specification of stem cells remain unclear. Addressing pertinent questions will require the employment of single-cell analysis methods as traditional cell biochemical and biomolecular assays yield mostly population-average data. In addition to time-lapse microscopy and flow cytometry, recent advances in single-cell genomic, transcriptomic and proteomic profiling are reviewed. The application of multiple displacement amplification, next generation sequencing, mass cytometry and spectrometry to stem cell systems is expected to provide a wealth of information affording unprecedented levels of multiparametric characterization of cell ensembles under defined conditions promoting pluripotency or commitment. Establishing connections between single-cell analysis information and the observed phenotypes will also require suitable mathematical models. Stem cell self-renewal and differentiation are orchestrated by the coordinated regulation of subcellular, intercellular and niche-wide processes spanning multiple time scales. Here, we discuss different modeling approaches and challenges arising from their application to stem cell populations. Integrating single-cell analysis with computational methods will fill gaps in our knowledge about the functions of heterogeneity in stem cell physiology. This combination will also aid the rational design of efficient differentiation and reprogramming strategies as well as bioprocesses for the production of clinically valuable stem cell derivatives. PMID:24035899

  14. The surface of articular cartilage contains a progenitor cell population.

    PubMed

    Dowthwaite, Gary P; Bishop, Joanna C; Redman, Samantha N; Khan, Ilyas M; Rooney, Paul; Evans, Darrell J R; Haughton, Laura; Bayram, Zubeyde; Boyer, Sam; Thomson, Brian; Wolfe, Michael S; Archer, Charles W

    2004-02-29

    It is becoming increasingly apparent that articular cartilage growth is achieved by apposition from the articular surface. For such a mechanism to occur, a population of stem/progenitor cells must reside within the articular cartilage to provide transit amplifying progeny for growth. Here, we report on the isolation of an articular cartilage progenitor cell from the surface zone of articular cartilage using differential adhesion to fibronectin. This population of cells exhibits high affinity for fibronectin, possesses a high colony-forming efficiency and expresses the cell fate selector gene Notch 1. Inhibition of Notch signalling abolishes colony forming ability whilst activated Notch rescues this inhibition. The progenitor population also exhibits phenotypic plasticity in its differentiation pathway in an embryonic chick tracking system, such that chondroprogenitors can engraft into a variety of connective tissue types including bone, tendon and perimysium. The identification of a chondrocyte subpopulation with progenitor-like characteristics will allow for advances in our understanding of both cartilage growth and maintenance as well as provide novel solutions to articular cartilage repair. PMID:14762107

  15. Regulation of Stem Cell Populations by microRNAs

    PubMed Central

    Mathieu, Julie

    2014-01-01

    miRNAs are small non-coding RNAs that have emerged as crucial post-transcriptional regulators of gene expression. They are key players in various critical cellular processes such as proliferation, cell cycle progression, apoptosis and differentiation. Self-renewal capacity and differentiation potential are hallmarks of stem cells. The switch between self-renewal and differentiation requires rapid widespread changes in gene expression. Since miRNAs can repress the translation of many mRNA targets, they are good candidates to regulate cell fates. In the past few years, miRNAs have appeared as important new actors in stem cell development by regulating differentiation and maintenance of stem cells. In this chapter we will focus on the role of miRNAs in various stem cell populations. After an introduction on microRNA biogenesis, we will review the recent knowledge on miRNA expression and function in pluripotent cells and during the acquisition of stem cell fate. We will then brie fly examine the role of miRNAs in adult and cancer stem cells. PMID:23696365

  16. Adherence to Continuous Positive Airway Pressure Treatment for Obstructive Sleep Apnea: Implications for Future Interventions

    PubMed Central

    Weaver, Terri E.; Sawyer, Amy M.

    2010-01-01

    Adherence to continuous positive airway pressure (CPAP) treatment for obstructive sleep apnea (OSA) is a critical problem with adherence rates ranging from 30–60%. Poor adherence to CPAP is widely recognized as a significant limiting factor in treating OSA, reducing the overall effectiveness of the treatment and leaving many OSA patients at heightened risk for comorbid conditions, impaired function and quality of life. The extant literature examining adherence to CPAP provides critical insight to measuring adherence outcomes, defining optimal adherence levels, and predicting CPAP adherence. This research has revealed salient factors that are associated with or predict CPAP adherence and may guide the development of interventions to promote CPAP adherence. Over the past 10 years, intervention studies to promote CPAP adherence have incorporated a multitude of strategies including education, support, cognitive behavioral approaches, and mixed strategies. This review of the current state of science of CPAP adherence will (1) synthesize the extant literature with regard to measuring, defining, and predicting CPAP adherence, (2) review published intervention studies aimed at promoting CPAP adherence, and (3) suggest directions for future empiric study of adherence to CPAP that will have implications for translational science. Our current understanding of CPAP adherence suggests that adherence is a multi-factorial, complex clinical problem that requires similarly designed approaches to effectively address poor CPAP adherence in the OSA population. PMID:20308750

  17. Complete structure of the cell surface polysaccharide of Streptococcus oralis ATCC 10557: A receptor for lectin-mediated interbacterial adherence

    SciTech Connect

    Abeygunawardana, C.; Bush, C.A. ); Cisar, J.O. )

    1991-07-02

    Lectin-carbohydrate binding is known to play an important role in a number of different cell-cell interactions including those between certain species of oral streptococci and actinomyces that colonize teeth. The cell wall polysaccharides of Streptococcus oralis ATCC 10557, S. oralis 34, and Streptococcus mitis J22, although not identical antigenically, each function as a receptor molecule for the galactose and N-acetylgalactosamine reactive fimbrial lectins of Actinomyces viscosus and Actinomyces naeslundii. Carbohydrate analysis of the receptor polysaccharide isolated from S. oralis ATCC 10557 shows galactose (3 mol), glucose (1 mol), GalNAc (1 mol), and rhamnose (1 mol). {sup 1}H NMR spectra of the polysaccharide show that is partially O-acetylated. Analysis of the {sup 1}H NMR spectrum of the de-O-acetylated polysaccharide shows that it is composed of repeating subunits containing six monosaccharides and that the subunits are joined by a phosphodiester linkage. The {sup 1}H and {sup 13}C NMR spectra were completely assigned by two-dimensional homonuclear correlation methods and by {sup 1}H-detected heteronuclear multiple-quantum correlation ({sup 1}H({sup 13}C)HMQC). The complete {sup 1}H and {sup 13}C assignment of the native polysaccharide was carried out by the same techniques augmented by a {sup 13}C-coupled hybrid HMQC-COSY method, which is shown to be especially useful for carbohydrates in which strong coupling and overlapping peaks in the {sup 1}H spectrum pose difficulties.