Science.gov

Sample records for adherent cell lines

  1. 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

  2. A rapid and sensitive fluorometric microassay for determining cell mediated cytotoxicity to adherent growing cell lines.

    PubMed

    Krüger-Krasagakes, S; Garbe, C; Kossman, P; Orfanos, C E

    1992-11-25

    In order to measure cell mediated cytotoxicity to adherent growing cell lines in vitro more rapidly and conveniently, a fluorometric microassay was developed and results were compared with those obtained by the 51Cr release assay. The fluorometric method is based on the hydrolysis of the fluorochrome 4-methylumbelliferyl heptanoate (MUH) by intracellular esterases of viable cells. Melanoma cell monolayers were incubated with lymphokine activated killer (LAK) cells for 4 h at various effector: target (E:T) cell ratios (E:T = 16, 8, 4, 2:1). Thereafter surviving adherent melanoma cells were stained with MUH for 30 min and fluorescence was measured directly in a 96 well plate reader. For the calculation of LAK cell cytotoxicity fluorescence values were corrected for the number of nonspecifically detached tumor cells during the washes and the number of nonspecifically adherent LAK cells. Using identical target and effector cell preparations both assays showed a nearly proportional increase of percentage cytotoxicity with rising numbers of lymphocytes. Compared with the 51Cr release assay, however, higher cytotoxicity values were obtained with the fluorometric MUH microassay: 57% with MUH versus 26% with 51Cr and 39% versus 14% for cell lines StML-11 and SKMel-28, respectively (E:T ratio = 16:1). The higher cytotoxicity rates obtained with the fluorometric MUH microassay were not due to the additional 30 min staining with MUH or due to nonspecific hydrolysis of MUH by extracellular esterases released from damaged cells, as could be shown by a series of experiments. In conclusion, a simple and rapid fluorometric microassay has been developed showing reliable reproducibility and a higher sensitivity compared with the 51Cr release assay for the determination of cellular cytotoxicity to adherent growing cell lines, avoiding hazardous radioactive labels. PMID:1431156

  3. 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

  4. 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. PMID:26150966

  5. 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

  6. A mannose-specific adherence mechanism in Lactobacillus plantarum conferring binding to the human colonic cell line HT-29.

    PubMed Central

    Adlerberth, I; Ahrne, S; Johansson, M L; Molin, G; Hanson, L A; Wold, A E

    1996-01-01

    Two Lactobacillus plantarum strains of human intestinal origin, strains 299 (= DSM 6595) and 299v (= DSM 9843), have proved to be efficient colonizers of the human intestine under experimental conditions. These strains and 17 other L. plantarum strains were tested for the ability to adhere to cells of the human colonic cell line HT-29.L.plantarum 299 and 299v and nine other L. plantarum strains, including all six strains that belong to the same genetic subgroup as L. plantarum 299 and 299v, adhered to HT-29 cells in a manner that could be inhibited by methyl-alpha-D-mannoside. The ability to adhere to HT-29 cells correlated with an ability to agglutinate cells of Saccharomyces cerevisiae and erythrocytes in a mannose-sensitive manner and with adherence to D-mannose-coated agarose beads. L. plantarum 299 and 299v adhered to freshly isolated human colonic and ileal enterocytes, but the binding was not significantly inhibited by methyl-alpha-D-mannoside. Periodate treatment of HT-29 cells abolished mannose-sensitive adherence, confirming that the cell-bound receptor was of carbohydrate nature. Proteinase K treatment of the bacteria also abolished adherence, indicating that the binding involved protein structures on the bacterial cell surface. Thus, a mannose-specific adhesin has been identified in L. plantarum; this adhesin could be involved in the ability to colonize the intestine. PMID:8779562

  7. Cell surface adhesiveness of mouse sarcoma lines evaluated by latex particle adherence assay: correlation with growth behavior and electrophoretic mobility.

    PubMed

    Bubeník, J; Jandlová, T; Suhajová, E; Malkovský, M

    1979-01-01

    Using the latex particle adherence assay and five mouse sarcoma cell lines of the identical origin, etiology and genotype but differing in malignancy we attempted to correlate the degree of cell surface adhesiveness with growth behavior and electrophoretic mobility of cells. Higher tumorigenicity of four of the cell lines (Mc11--Mc14) was associated with lower cell surface adhesiveness and, conversely, lower malignancy of the fifth line (Mc15) with higher cell surface adhesiveness. No simple correlation or causal relationship was found among the electrophoretic mobility of the lines and other cellular characteristics. PMID:522921

  8. 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. PMID:25806119

  9. 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

  10. A Method to Evaluate the Efficiency of Transfection Reagents in an Adherent Zebrafish Cell Line

    PubMed Central

    Aschberger, Teresa; Pelster, Bernd

    2013-01-01

    Abstract We present a simple and robust method to evaluate the transfection efficiency of commercially available transfection reagents intended to be established for use in nonmammalian cell lines. To illustrate the method, we compare the ability of four different reagents to transfect the embryonic zebrafish cell line Z3. Z3 cells were seeded in a 96-well plate and simultaneously transfected in several variations by using minimum volumes of transfection reagent and a vector DNA encoding an amplified version of green fluorescent protein (GFP). After 24 and 48 h, transfection efficiency was determined by a dual fluorescence plate reader measurement of GFP and Hoechst 33342 fluorescence, an indicator of cell density. Of the four different reagents tested, certain variations of JetPrime™ reagent and X-tremeGene™ HP reagent produced the highest fluorescence signal per cell after 24- and 48-h incubation, respectively. The simultaneous multivariate setup enables comparing different reagent/DNA combinations at different time points well, independent of cell growth variability or seeding density. PMID:23515475

  11. 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.

  12. Robotic adherent cell injection for characterizing cell-cell communication.

    PubMed

    Liu, Jun; Siragam, Vinayakumar; Gong, Zheng; Chen, Jun; Fridman, Michael D; Leung, Clement; Lu, Zhe; Ru, Changhai; Xie, Shaorong; Luo, Jun; Hamilton, Robert M; Sun, Yu

    2015-01-01

    Compared to robotic injection of suspended cells (e.g., embryos and oocytes), fewer attempts were made to automate the injection of adherent cells (e.g., cancer cells and cardiomyocytes) due to their smaller size, highly irregular morphology, small thickness (a few micrometers thick), and large variations in thickness across cells. This paper presents a robotic system for automated microinjection of adherent cells. The system is embedded with several new capabilities: automatically locating micropipette tips; robustly detecting the contact of micropipette tip with cell culturing surface and directly with cell membrane; and precisely compensating for accumulative positioning errors. These new capabilities make it practical to perform adherent cell microinjection truly via computer mouse clicking in front of a computer monitor, on hundreds and thousands of cells per experiment (versus a few to tens of cells as state of the art). System operation speed, success rate, and cell viability rate were quantitatively evaluated based on robotic microinjection of over 4000 cells. This paper also reports the use of the new robotic system to perform cell-cell communication studies using large sample sizes. The gap junction function in a cardiac muscle cell line (HL-1 cells), for the first time, was quantified with the system. PMID:25073160

  13. Adherence of Helicobacter pylori to primary human gastrointestinal cells.

    PubMed Central

    Clyne, M; Drumm, B

    1993-01-01

    Helicobacter pylori adheres only to gastric cells in vivo. However, the organism adheres to a wide variety of nongastric cells in vitro. In this study, we have used flow cytometry to assess the adherence of H. pylori to primary epithelial cells isolated from gastric, duodenal, and colonic biopsy specimens by collagenase digestion. After incubation of bacteria and cells together and subsequent staining with a two-stage fluorescein isothiocyanate-labelled H. pylori antibody method, cells with adherent bacteria could be easily distinguished from cells without bacteria. Binding to Kato III cells (a gastric adenocarcinoma cell line) was saturable when bacteria and cells were mixed at a ratio of 250:1. Adherence to cells isolated from gastric biopsy specimens was significantly better than adherence to cells isolated from duodenal or colonic biopsy specimens. Almost 70% of gastric cells had bacteria bound, in contrast to 30% of duodenal cells and 32% of colonic cells (P < 0.0001). There was no correlation between expression of hemagglutinins by the bacteria and ability to bind to either Kato III cells or primary epithelial cells isolated from gastric biopsy specimens. In view of the strict tropism that the organism exhibits in vivo for gastric cells, the results of this study indicate that primary cells are ideal for assessing the factors that might play a role in the pathogenesis of disease caused by the organism. Images PMID:8406792

  14. Cell Lines

    PubMed Central

    Cherbas, Lucy; Gong, Lei

    2014-01-01

    We review the properties and uses of cell lines in Drosophila research, emphasizing the variety of lines, the large body of genomic and transcriptional data available for many of the lines, and the variety of ways the lines have been used to provide tools for and insights into the developmental, molecular, and cell biology of Drosophila and mammals. PMID:24434506

  15. 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

  16. 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

  17. Micropallet arrays for the separation of single, adherent cells.

    PubMed

    Salazar, Georgina To'a; Wang, Yuli; Young, Grace; Bachman, Mark; Sims, Christopher E; Li, G P; Allbritton, Nancy L

    2007-01-15

    The selection and collection of single cells from within a heterogeneous population is required to produce genetically engineered cell lines, to develop new stem cell lines, and for single-cell studies. We describe a new platform for the positive selection of single live mammalian cells while the cells remain adherent to their growth surface. Cells were grown on arrays of microfabricated, releasable elements composed of SU-8 polymer termed "cell pallets". The presence of air between the elements restricted the cells to the top surfaces of the pallets. Single pallets situated within large arrays of pallets were released on demand using a single, focused, laser pulse. The laser pulses were low in energy (2-5 muJ) and did not detach nearby, nontargeted pallets. Since the SU-8 pallets and the underlying glass substrate were optically transparent, the cells on the pallets could be visualized by microscopy before and after release. Over 90% of cells remained attached to the pallet during laser-based release. The feasibility of growing the cells from the released pallets into clonal colonies was demonstrated. The pallet array system permits adherent cells to be inspected using conventional microscopy and selected cells released for further analysis. The ability to assess cells while they remain adherent to a surface will broaden the number of attributes that can be utilized for cell separation, for example, cell shape, cytoskeletal properties, and other attributes. PMID:17222037

  18. A method for incorporating macromolecules into adherent cells

    PubMed Central

    1984-01-01

    We describe a simple method for loading exogenous macromolecules into the cytoplasm of mammalian cells adherent to tissue culture dishes. Culture medium was replaced with a thin layer of fluorescently labeled macromolecules, the cells were harvested from the substrate by scraping with a rubber policeman, transferred immediately to ice cold media, washed, and then replated for culture. We refer to the method as "scrape-loading." Viability of cells was 50-60% immediately after scrape-loading and was 90% for those cells remaining after 24 h of culture. About 40% of adherent, well-spread fibroblasts contained fluorescent molecules 18 h after scrape-loading of labeled dextrans, ovalbumin, or immunoglobulin-G. On average, 10(7) dextran molecules (70,000-mol wt) were incorporated into each fibroblast by scrape- loading in 10 mg/ml dextran. The extent of loading depended on the concentration and molecular weight of the dextrans used. A fluorescent analog of actin could also be loaded into fibroblasts where it labeled stress fibers. HeLa cells, a macrophage-like cell line, 1774A.1, and human neutrophils were all successfully loaded with dextran by scraping. The method of scrape-loading should be applicable to a broad range of adherent cell types, and useful for loading of diverse kinds of macromolecules. PMID:6201494

  19. 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

  20. 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.

  1. 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

  2. Broadening cell selection criteria with micropallet arrays of adherent cells.

    PubMed

    Wang, Yuli; Young, Grace; Aoto, Phillip C; Pai, Jeng-Hao; Bachman, Mark; Li, G P; Sims, Christopher E; Allbritton, Nancy L

    2007-10-01

    A host of technologies exists for the separation of living, nonadherent cells, with separation decisions typically based on fluorescence or immunolabeling of cells. Methods to separate adherent cells as well as to broaden the range of possible sorting criteria would be of high value and complementary to existing strategies. Cells were cultured on arrays of releasable pallets. The arrays were screened and individual cell(s)/pallets were released and collected. Conventional fluorescence and immunolabeling of cells were compatible with the pallet arrays, as were separations based on gene expression. By varying the size of the pallet and the number of cells cultured on the array, single cells or clonal colonies of cells were isolated from a heterogeneous population. Since cells remained adherent throughout the isolation process, separations based on morphologic characteristics, for example cell shape, were feasible. Repeated measurements of each cell in an array were performed permitting the selection of cells based on their temporal behavior, e.g. growth rate. The pallet array system provides the flexibility to select and collect adherent cells based on phenotypic and temporal criteria and other characteristics not accessible by alternative methods. PMID:17559133

  3. Contractile network models for adherent cells.

    PubMed

    Guthardt Torres, P; Bischofs, I B; Schwarz, U S

    2012-01-01

    Cells sense the geometry and stiffness of their adhesive environment by active contractility. For strong adhesion to flat substrates, two-dimensional contractile network models can be used to understand how force is distributed throughout the cell. Here we compare the shape and force distribution for different variants of such network models. In contrast to Hookean networks, cable networks reflect the asymmetric response of biopolymers to tension versus compression. For passive networks, contractility is modeled by a reduced resting length of the mechanical links. In actively contracting networks, a constant force couple is introduced into each link in order to model contraction by molecular motors. If combined with fixed adhesion sites, all network models lead to invaginated cell shapes, but only actively contracting cable networks lead to the circular arc morphology typical for strongly adhering cells. In this case, shape and force distribution are determined by local rather than global determinants and thus are suited to endow the cell with a robust sense of its environment. We also discuss nonlinear and adaptive linker mechanics as well as the relation to tissue shape. PMID:22400597

  4. Contractile network models for adherent cells

    NASA Astrophysics Data System (ADS)

    Guthardt Torres, P.; Bischofs, I. B.; Schwarz, U. S.

    2012-01-01

    Cells sense the geometry and stiffness of their adhesive environment by active contractility. For strong adhesion to flat substrates, two-dimensional contractile network models can be used to understand how force is distributed throughout the cell. Here we compare the shape and force distribution for different variants of such network models. In contrast to Hookean networks, cable networks reflect the asymmetric response of biopolymers to tension versus compression. For passive networks, contractility is modeled by a reduced resting length of the mechanical links. In actively contracting networks, a constant force couple is introduced into each link in order to model contraction by molecular motors. If combined with fixed adhesion sites, all network models lead to invaginated cell shapes, but only actively contracting cable networks lead to the circular arc morphology typical for strongly adhering cells. In this case, shape and force distribution are determined by local rather than global determinants and thus are suited to endow the cell with a robust sense of its environment. We also discuss nonlinear and adaptive linker mechanics as well as the relation to tissue shape.

  5. Effect of hydrostatic pressure on the viability of non-adherent HL-60 cells

    NASA Astrophysics Data System (ADS)

    Yabuki, Takahiro; Yamanoha, Banri; Shimizu, Akio

    2013-06-01

    We investigated the effect of hydrostatic pressure on the viability of non-adherent HL-60 cell line derived from leukemic cells over a high pressure range. The HL-60 cells are resistant to pressures of up to 100 MPa under pressurization for 20 min at 25°C. However, cell viability decreased markedly between 100 and 200 MPa, and almost all cells died above 200 MPa. In the case of pressures up to 25 MPa at 25°C for four days, the viability of HL-60 cells was inhibited by increasing the pressure above 20 MPa. Although high viability was observed between 1.6 and 2.0 MPa for adherent astrocytes, viability did not change over pressures up to 2.0 MPa in the case of non-adherent HL-60 cells. It is thought that the response of cells to pressure varies among cell types.

  6. 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

  7. Fluorescence assay for the detection of adherent Candida yeasts to target cells in microtest plates.

    PubMed

    Borg-von Zepelin, M; Wagner, T

    1995-01-01

    We describe an assay based on photometric analysis for the measurement of adherence of Candida species to epithelial target cells (Vero cell line). Adherent Candida cells were detected by staining the cells with the fluorescent dye Calcofluor white (CFW), which binds to chitin and glucan in the yeasts. The tests were performed on microtest plates, which were analysed automatically by fluorescence plate readers. The assay is based on the following steps: (i) coating of the microtest plates with target cells (e.g. Vero cells); (ii) infection with Candida: (iii) staining of Candida with CFW; (iv) rinsing to remove non-adherent Candida cells and unbound dye; (v) detection of adherent fluorescent Candida cells. The test was able to detect 4 x 10(4) cells ml-1. The standard deviation was +/- 8%. Day-to-day variation was +/- 10% at most. The adherence of strains of different Candida species was assayed by a standard procedure. The results confirmed the order of adherence, with C. albicans ranking first, followed by C. tropicalis, C. parapsilosis and C. glabrata. PMID:8569807

  8. RGD-functionalized spherulites as targeted vectors captured by adherent cultured cells.

    PubMed

    Chenevier, P; Delord, B; Amédée, J; Bareille, R; Ichas, F; Roux, D

    2002-12-16

    Spherulites are multilamellar vesicles consisting of concentric shells that can encapsulate small organic molecules or macromolecules. We investigate the possibility of targeting neutral spherulites to adherent culture cells by functionalizing their surface with RGD-containing ligands. The strength and specificity of association of RGD spherulites with several cell lines (EAhy 926 endothelial cell line, human umbilical vein endothelial cell (HUVEC) and human osteoprogenitor (HOP) primary cells) was studied, and the molecular interaction of RGD spherulites with the EAhy 926 cell surface was investigated. We show that, after binding to cells, spherulites are internalized. PMID:12431780

  9. 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

  10. 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

  11. Adherence of Tritrichomonas foetus to bovine vaginal epithelial cells.

    PubMed Central

    Corbeil, L B; Hodgson, J L; Jones, D W; Corbeil, R R; Widders, P R; Stephens, L R

    1989-01-01

    Adherence of Tritrichomonas foetus to bovine vaginal epithelial cells (VECs) in vitro was investigated with fresh washed bovine VECs and log-phase cultures of T. foetus. Observation under phase-contrast microscopy showed that T. foetus usually adhered first by the posterior flagellum and later by the body. Significantly more keratinized squamous epithelial cells were detected with attached parasites than nonkeratinized round epithelial cells. The optimal pH range for attachment was 6.0 to 7.5, with peak attachment at pH 6.5 for squamous VECs. Surface-reactive bovine antiserum to T. foetus prevented adherence to bovine squamous VECs. Inhibition of adherence occurred at nonagglutinating, nonimmobilizing serum dilutions. Antiserum fractions enriched for immunoglobulin G1 inhibited adherence, but fractions enriched for immunoglobulin G2 did not. The inhibitory antiserum was specific for several medium- to high-molecular-weight membrane antigens as detected in Western blots (immunoblots). The ability of surface-reactive antibodies to prevent adherence and to agglutinate and immobilize T. foetus indicates that they may be protective. Images PMID:2471692

  12. 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.

  13. Growth and adherence on stainless steel by Enterococcus faecium cells.

    PubMed

    Andrade, N J; Ajao, D B; Zottola, E A

    1998-11-01

    Enterococcus faecium isolated from Brazilian raw milk was used in this study. For growth studies, E. faecium was inoculated into 10% RSM (reconstituted skim milk) and MRS both, incubated at 6.5 and 9 degrees C for 10 days and at 30, 42, and 45 degrees C for 48 h. Cells were enumerated after spread-plating onto MRS agar and incubating at 30 degrees C for 48 h. The ability of E. faecium cells to adhere to stainless-steel chips (6 by 6 by 1 mm, AISI 304, finish #4) was investigated. MRS broth containing stainless steel chips was inoculated to an initial concentration of 10(3) or 10(6) CFU/ml of E. faecium. Adherent cells were stained with acridine orange and enumerated by epifluorescence microscopy. E. faecium grew between 6.5 and 42 degrees C in MRS and between 9 and 40 degrees C in RSM. In MRS broth with 10(6) or 10(3) CFU/ml, the g (generation time) values were 0.62 and 0.42 h and R (growth rate) values were 1.6 and 2.4 h-1. Values of R = 2.3 h-1 and g = 0.43 h were determined for E. faecium growing in RSM with 10(3) CFU/ml. In MRS broth, for samples with a starting concentration of 10(6) cells per ml, adherence to stainless-steel chips was first observed at 2 h. However, adherence was first observed at 4 h in samples with an initial concentration of 10(3) cells per ml. After 10 h of exposure the number of adherent cells was similar for all samples regardless of initial inoculum. These results indicate that E. faecium readily adheres to stainless steel. It also underscores the need to control E. faecium by using appropriate low storage temperatures and adequate sanitizing practices in the dairy industry. PMID:9829184

  14. Microchamber Device for Detection of Transporter Activity of Adherent Cells

    PubMed Central

    Tsugane, Mamiko; Uejima, Etsuko; Suzuki, Hiroaki

    2015-01-01

    We present a method to detect the transporter activity of intact adherent cells using a microchamber device. When adherent cells are seeded onto the poly-di-methyl siloxane substrate having microchambers with openings smaller than the size of a cell, the cells form a confluent layer that covers the microchambers, creating minute, confined spaces. As substances exported across the cell membrane accumulate, transporter activity can be detected by observing the fluorescence intensity increase in the microchamber. We tested the microchamber device with HeLa cells over-expressing MDR1, an ATP-binding cassette transporter, and succeeded in detecting the transport of fluorescence-conjugated paclitaxel, the anti-cancer drug, at the single-cell level. PMID:25853126

  15. Adherence, accumulation, and cell division of a natural adherent bacterial population.

    PubMed Central

    Bloomquist, C G; Reilly, B E; Liljemark, W F

    1996-01-01

    Developing dental bacterial plaques formed in vivo on enamel surfaces were examined in specimens from 18 adult volunteers during the first day of plaque formation. An intraoral model placing enamel pieces onto teeth was used to study bacterial plaque populations developing naturally to various cell densities per square millimeter of surface area of the enamel (W. F. Liljemark, C. G. Bloomquist, C. L. Bandt, B. L. Philstrom, J. E. Hinrichs, and L. F. Wolff, Oral Microbiol. Immunol. 8:5-15, 1993). Radiolabeled nucleoside incorporation was used to measure DNA synthesis concurrent with the taking of standard viable cell counts of the plaque samples. Results showed that in vivo plaque formation began with the rapid adherence of bacteria until ca. 12 to 32% of the enamel's salivary pellicle was saturated (ca. 2.5 x 10(5) to 6.3 x 10(5) cells per mm2). The pioneer adherent species were predominantly those of the "sanguis streptococci." At the above-noted density, the bacteria present on the salivary pellicle incorporated low levels of radiolabeled nucleoside per viable cell. As bacterial numbers reached densities between 8.0 x 10(5) and 2.0 x 10(6) cells per mm2, there was a small increase in the incorporation of radiolabeled nucleosides per cell. At 2.5 x 10(6) to 4.0 x 10(6) cells per mm2 of enamel surface, there was a marked increase in the incorporation of radiolabeled nucleosides per cell which appeared to be cell-density dependent. The predominant species group in developing dental plaque films during density-dependent growth was the sanguis streptococci; however, most other species present showed similar patterns of increased DNA synthesis as the density noted above approached 2.5 x 10(6) to 4.0 x 10(6) cells per mm2. PMID:8576054

  16. Silk screen based dual spin-filter module for perfusion culture of adherent and non-adherent mammalian cells.

    PubMed

    Kamthan, Shweta; Gomes, James; Roychoudhury, Pradip K

    2014-08-01

    Spin-filters have been primarily used for producing therapeutic proteins from mammalian cells. However, disposability and/or high filter clogging of the existing spin-filter systems affect the process economy and productivity. Hence, to address these drawbacks a reusable dual spin-filter module for perfusion culture of adherent and non-adherent mammalian cells was designed. Two non-woven Bombyx mori silk layers were used as filter screen; the outer layer was conducive to cell attachment whilst the inner was non-conducive. Adherent cells can be cultured either in suspended mode using its inner single module or as monolayer of cells using its dual concentric module. We achieved 30 % higher urokinase productivity as compared to the stainless-steel spin-filter during perfusion experiments of adherent human kidney cells in suspended mode. This was due to the hydrophobic and negatively-charged silk screen that allows clog-free perfusion culture for prolonged periods. PMID:24737079

  17. Initial adherence of EPEC, EHEC and VTEC to host cells

    PubMed Central

    Bardiau, Marjorie; Szalo, Mihai; Mainil, Jacques G.

    2010-01-01

    Initial adherence to host cells is the first step of the infection of enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic Escherichia coli (EHEC) and verotoxigenic Escherichia coli (VTEC) strains. The importance of this step in the infection resides in the fact that (1) adherence is the first contact between bacteria and intestinal cells without which the other steps cannot occur and (2) adherence is the basis of host specificity for a lot of pathogens. This review describes the initial adhesins of the EPEC, EHEC and VTEC strains. During the last few years, several new adhesins and putative colonisation factors have been described, especially in EHEC strains. Only a few adhesins (BfpA, AF/R1, AF/R2, Ral, F18 adhesins) appear to be host and pathotype specific. The others are found in more than one species and/or pathotype (EPEC, EHEC, VTEC). Initial adherence of EPEC, EHEC and VTEC strains to host cells is probably mediated by multiple mechanisms. PMID:20423697

  18. Automated and Online Characterization of Adherent Cell Culture Growth in a Microfabricated Bioreactor

    PubMed Central

    Jaccard, Nicolas; Macown, Rhys J.; Super, Alexandre; Griffin, Lewis D.; Veraitch, Farlan S.

    2014-01-01

    Adherent cell lines are widely used across all fields of biology, including drug discovery, toxicity studies, and regenerative medicine. However, adherent cell processes are often limited by a lack of advances in cell culture systems. While suspension culture processes benefit from decades of development of instrumented bioreactors, adherent cultures are typically performed in static, noninstrumented flasks and well-plates. We previously described a microfabricated bioreactor that enables a high degree of control on the microenvironment of the cells while remaining compatible with standard cell culture protocols. In this report, we describe its integration with automated image-processing capabilities, allowing the continuous monitoring of key cell culture characteristics. A machine learning–based algorithm enabled the specific detection of one cell type within a co-culture setting, such as human embryonic stem cells against the background of fibroblast cells. In addition, the algorithm did not confuse image artifacts resulting from microfabrication, such as scratches on surfaces, or dust particles, with cellular features. We demonstrate how the automation of flow control, environmental control, and image acquisition can be employed to image the whole culture area and obtain time-course data of mouse embryonic stem cell cultures, for example, for confluency. PMID:24692228

  19. Patterned Thermoresponsive Microgel Coatings for Noninvasive Processing of Adherent Cells.

    PubMed

    Uhlig, Katja; Wegener, Thomas; He, Jian; Zeiser, Michael; Bookhold, Johannes; Dewald, Inna; Godino, Neus; Jaeger, Magnus; Hellweg, Thomas; Fery, Andreas; Duschl, Claus

    2016-03-14

    Cultivation of adherently growing cells in artificial environments is of utmost importance in medicine and biotechnology to accomplish in vitro drug screening or to investigate disease mechanisms. Precise cell manipulation, like localized control over adhesion, is required to expand cells, to establish cell models for novel therapies and to perform noninvasive cell experiments. To this end, we developed a method of gentle, local lift-off of mammalian cells using polymer surfaces, which are reversibly and repeatedly switchable between a cell-attractive and a cell-repellent state. This property was introduced through micropatterned thermoresponsive polymer coatings formed from colloidal microgels. Patterning was obtained through automated nanodispensing or microcontact printing, making use of unspecific electrostatic interactions between microgels and substrates. This process is much more robust against ambient conditions than covalent coupling, thus lending itself to up-scaling. As an example, wound healing assays were accomplished at 37 °C with highly increased precision in microfluidic environments. PMID:26879608

  20. 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.

  1. Blasted Cell Line Names

    PubMed Central

    Wang, Jing; Byers, Lauren A.; Yordy, John S.; Liu, Wenbin; Shen, Li; Baggerly, Keith A.; Giri, Uma; Myers, Jeffrey N.; Ang, K. Kian; Story, Michael D.; Girard, Luc; Minna, John D.; Heymach, John V.; Coombes, Kevin R.

    2010-01-01

    Background: While trying to integrate multiple data sets collected by different researchers, we noticed that the sample names were frequently entered inconsistently. Most of the variations appeared to involve punctuation, white space, or their absence, at the juncture between alphabetic and numeric portions of the cell line name. Results: Reasoning that the variant names could be described in terms of mutations or deletions of character strings, we implemented a simple version of the Needleman-Wunsch global sequence alignment algorithm and applied it to the cell line names. All correct matches were found by this procedure. Incorrect matches only occured when a cell line was present in one data set but not in the other. The raw match scores tended to be substantially worse for the incorrect matches. Conclusions: A simple application of the Needleman-Wunsch global sequence alignment algorithm provides a useful first pass at matching sample names from different data sets. PMID:21082038

  2. A Prestressed Cable Network Model of the Adherent Cell Cytoskeleton

    PubMed Central

    Coughlin, Mark F.; Stamenović, Dimitrije

    2003-01-01

    A prestressed cable network is used to model the deformability of the adherent cell actin cytoskeleton. The overall and microstructural model geometries and cable mechanical properties were assigned values based on observations from living cells and mechanical measurements on isolated actin filaments, respectively. The models were deformed to mimic cell poking (CP), magnetic twisting cytometry (MTC) and magnetic bead microrheometry (MBM) measurements on living adherent cells. The models qualitatively and quantitatively captured the fibroblast cell response to the deformation imposed by CP while exhibiting only some qualitative features of the cell response to MTC and MBM. The model for CP revealed that the tensed peripheral actin filaments provide the key resistance to indentation. The actin filament tension that provides mechanical integrity to the network was estimated at ∼158 pN, and the nonlinear mechanical response during CP originates from filament kinematics. The MTC and MBM simulations revealed that the model is incomplete, however, these simulations show cable tension as a key determinant of the model response. PMID:12547813

  3. The expression of nonagglutinating fimbriae and its role in Proteus mirabilis adherence to epithelial cells.

    PubMed

    Tolson, D L; Harrison, B A; Latta, R K; Lee, K K; Altman, E

    1997-08-01

    Proteus mirabilis is a common causative agent of human urinary tract infections, especially in catheterized patients and in those patients with structural abnormalities of the urinary tract. In addition to the production of hemolysin and urease, fimbriae-mediated adherence to uroepithelial cells and kidney epithelium may be essential for virulence of P. mirabilis. A single P. mirabilis strain is capable of expressing several morphologically distinct fimbrial species, which can each be favoured by specific in vitro growth conditions. The fimbrial species reported to date include mannose-resistant/Proteus-like fimbriae, ambient temperature fimbriae, P. mirabilis fimbriae, and nonagglutinating fimbriae (NAF). Here, using intact bacteria or purified NAF as immunogens, we have generated the first reported NAF-specific monoclonal antibodies (mAbs). Bacteria expressing NAF as their only fimbrial species adhered strongly to a number of cell lines in vitro, including uroepithelial cell lines. Binding of P. mirabilis was markedly reduced following preincubation with NAF-specific mAbs and Fab fragments. The presence of NAF with highly conserved N-terminal sequences on all P. mirabilis strains so far examined, combined with the ability of both anti-NAF mAbs and purified NAF molecules to inhibit P. mirabilis adherence in vitro, suggests that NAF may contribute to the pathogenesis of P. mirabilis. PMID:9304781

  4. Evidence that extracellular components function in adherence of Actinobacillus actinomycetemcomitans to epithelial cells.

    PubMed Central

    Meyer, D H; Fives-Taylor, P M

    1993-01-01

    Extracellular microvesicles and a highly proteinaceous polymer associated with a leukotoxin-producing strain, Actinobacillus actinomycetemcomitans SUNY 75, were shown to increase adherence of other weakly adherent A. actinomycetemcomitans strains to KB epithelial cells. Images PMID:8406899

  5. Protective effects of osmolytes in cryopreserving adherent neuroblastoma (Neuro-2a) cells.

    PubMed

    Bailey, Trisha L; Wang, Mian; Solocinski, Jason; Nathan, Britto P; Chakraborty, Nilay; Menze, Michael A

    2015-12-01

    A simple method to cryopreserve adherent monolayers of neuronal cells is currently not available, but the development of this technique could facilitate numerous applications in the field of biomedical engineering, cell line development, and drug screening. However, complex tissues of some exceptional animals survive freezing in nature. These animals are known to accumulate several small molecular weight solutes prior to freezing. Following a similar strategy, we investigated the effects of osmolytes such as trehalose, proline, and sucrose as additives to the traditional cryoprotectant dimethyl sulfoxide (Me2SO) in modulating the cryopreservation outcome of mouse neuroblastoma (Neuro-2a) cells. Neuro-2a cells adhered to cell culture plates were incubated for 24 h at varying concentrations of trehalose, proline, sucrose and combinations of these compounds. Cells were cryopreserved for 24 h and cell viability post-freezing and thawing was quantified by trypan blue exclusion assay. On average, only 13.5% of adherent cells survived freezing in the presence of 10% Me2SO alone (control). Pre-incubation of cells with medium containing both trehalose and proline severely decreased cell proliferation, but increased cell recovery to about 53% of control. Furthermore, characterization using Raman microspectroscopy revealed that the addition of both trehalose and proline to 10% Me2SO substantially increased the size, and altered the nature, of ice crystals formed during freezing. Our results suggest that pre-incubation of Neuro-2a cells with trehalose and proline in combination provides cell protection along with alterations of ice structure in order to increase cell survival post-freezing. PMID:26408850

  6. Endometrial Mesenchymal Stem Cells Isolated from Menstrual Blood by Adherence

    PubMed Central

    Du, Xue; Yuan, Qing; Qu, Ye; Zhou, Yuan; Bei, Jia

    2016-01-01

    Objective. To find a convenient and efficient way to isolate MSCs from human menstrual blood and to investigate their biological characteristics, proliferative capacity, and secretion levels. Methods. MSCs were isolated from menstrual blood of 3 healthy women using adherence. Cell immunological phenotype was examined by flow cytometry; the adipogenic, osteogenic, and chondrogenic differentiation of MSCs was examined by Oil-Red-O staining, ALP staining, and Alcian Blue staining, respectively; and the secretion of cytokines, including vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1), was detected using enzyme-linked immunosorbent assay. Results. MB-MSCs were successfully isolated from human menstrual blood using adherence. They were positive for CD73, CD105, CD29, and CD44, but negative for CD31 and CD45. The differentiated MB-MSCs were positive for ALP staining, Oil-Red-O staining, and Alcian Blue staining. In addition, they could secrete antiapoptotic cytokines, such as VEGF, IGF-1, and HGF. Conclusion. It is feasible to isolate MSCs from human menstrual blood, thus avoiding invasive procedures and ethical controversies. Adherence could be a promising alternative to the density gradient centrifugation for the isolation of MSCs from menstrual blood. PMID:26681948

  7. Human placenta-derived adherent cells induce tolerogenic immune responses.

    PubMed

    Liu, Wei; Morschauser, Andrew; Zhang, Xin; Lu, Xiaohua; Gleason, Joseph; He, Shuyang; Chen, Hong-Jung; Jankovic, Vladimir; Ye, Qian; Labazzo, Kristen; Herzberg, Uri; Albert, Vivian R; Abbot, Stewart E; Liang, Bitao; Hariri, Robert

    2014-05-01

    Human placenta-derived adherent cells (PDAC cells) are a culture expanded, undifferentiated mesenchymal-like population derived from full-term placental tissue, with immunomodulatory and anti-inflammatory properties. PDA-001 (cenplacel-L), an intravenous formulation of PDAC cells, is in clinical development for the treatment of autoimmune and inflammatory diseases. To elucidate the mechanisms underlying the immunoregulatory properties of PDAC cells, we investigated their effects on immune cell populations, including T cells and dendritic cells (DC) in vitro and in vivo. PDAC cells suppressed T-cell proliferation in an OT-II T-cell adoptive transfer model, reduced the severity of myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis and ameliorated inflammation in a delayed type hypersensitivity response model. In vitro, PDAC cells suppressed T-cell proliferation and inhibited Th1 and Th17 differentiation. Analysis of tissues derived from PDAC cell-treated animals revealed diminished CD86 expression on splenic DC, suggesting that they can also modulate DC populations. Furthermore, PDAC cells modulate the differentiation and maturation of mouse bone marrow-derived DC. Similarly, human DC differentiated from CD14(+) monocytes in the presence of PDAC cells acquired a tolerogenic phenotype. These tolerogenic DC failed to induce allogeneic T-cell proliferation and differentiation toward Th1, but skewed T-cell differentiation toward Th2. Inhibition of cyclo-oxygenase-2 activity resulted in a significant, but not complete, abrogation of PDAC cells' effects on DC phenotype and function, implying a role for prostaglandin E2 in PDAC-mediated immunomodulation. This study identifies modulation of DC differentiation toward immune tolerance as a key mechanism underlying the immunomodulatory activities of PDAC cells. PMID:25505962

  8. Role of specific determinants in mannan of Candida albicans serotype A in adherence to human buccal epithelial cells.

    PubMed Central

    Miyakawa, Y; Kuribayashi, T; Kagaya, K; Suzuki, M; Nakase, T; Fukazawa, Y

    1992-01-01

    Candida albicans serotype A (C. albicans A) possesses a specific antigen, designated antigen 6, which resides in mannans on the cell surface. To determine the role of the mannan moiety of the C. albicans cell wall in adherence to buccal epithelial cells, we used antigen 6-deficient mutants which had been isolated by screening with an agglutinating monoclonal antibody against antigen 6 (MAb-6). 1H nuclear magnetic resonance spectral analysis of the purified mannans from the mutants showed a loss of the signals related to that beta-linkage of the side chains. Moreover, acetolyzed fragments of the mutant mannans showed a decreased amount of mannohexaose and mannopentaose. The mutant yeast cells exhibited significantly reduced ability to adhere both to exfoliated buccal epithelial cells and to a human buccal cell line. A number of strains of C. albicans A, C. tropicalis, and C. glabrata, all of which bear antigen 6, showed significantly higher adherence to the cell line than did those of C. albicans serotype B, which lack antigen 6. The whole mannan from the C. albicans A parent inhibited the adherence of C. albicans A to epithelial cells dose dependently, whereas mannan from a mutant strains did not. Moreover, C. albicans A treated with MAb-6 or polyclonal factor 6 serum showed reduced adherence. A close correlation was found between adhesive ability and agglutinability with MAb-6 in the C. albicans A parent, the antigenic mutants, and their spontaneous revertants. These results suggest that so far as mannan adhesion is concerned, serotype A-specific determinants are largely involved in the mechanisms of adherence of C. albicans A to human buccal epithelial cells. PMID:1375200

  9. Measles virus hemagglutinin mediates monocyte aggregation and increased adherence to measles-infected endothelial cells.

    PubMed

    Soilu-Hänninen, M; Hänninen, A; Ilonen, J; Salmi, A; Salonen, R

    1996-09-01

    The effect of measles virus (MV) infection on monocyte adhesion was studied using human peripheral blood monocytes and monocytic and endothelial cell lines. The infection of monocytic U-937 cells led to the formation of large cellular aggregates. Aggregation was independent of intercellular adhesion molecule-1 (ICAM-1)/lymphocyte function-associated antigen-1 (LFA-1), but could be inhibited by monoclonal antibodies (mAb) against the MV hemagglutinin glycoprotein (MV-H). mAb against the MV receptor, CD46, also blocked aggregation. No significant changes in the cell surface expression of adhesion molecules CD11a, CD11b, CD11c, CD18, CD54, CD44, CD49d (alpha 4-integrin) and CD62L (L-selectin) were observed on MV-infected monocytes. Infection of a human endothelial cell line, EAhy 926 (HEC), with MV led to a two-fold increase in 1CAM-1 expression and a two-fold increase in monocyte adherence to the HEC (from 22 +/- 1.6% to 42 +/- 4.8%). However, ICAM-1 mAb reduced monocyte adhesion to the control and MV-infected HEC to a similar degree, whereas anti-MV-H antibodies abolished the difference between binding to infected and control HEC. We conclude that MV hemagglutinin mediated both the homo typic aggregation in infected monocyte cultures and increased monocyte adherence to the infected endothelial cells. PMID:8884738

  10. A visual targeting system for the microinjection of unstained adherent cells.

    PubMed

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

    2013-02-01

    Automatic localization and targeting are critical steps in automating the process of microinjecting adherent cells. This process is currently performed manually by highly trained operators and is characterized as a laborious task with low success rate. Therefore, automation is desired to increase the efficiency and consistency of the operations. This research offers a contribution to this procedure through the development of a vision system for a robotic microinjection setup. Its goals are to automatically locate adherent cells in a culture dish and target them for a microinjection. Here the major concern was the achievement of an error-free targeting system to guarantee high consistency in microinjection experiments. To accomplish this, a novel visual targeting algorithm integrating different image processing techniques was proposed. This framework employed defocusing microscopy to highlight cell features and improve cell segmentation and targeting reliability. Three main image processing techniques, operating at three different focus levels in a bright field (BF) microscope, were used: an anisotropic contour completion (ACC) method, a local intensity variation background-foreground classifier, and a grayscale threshold-based segmentation. The proposed framework combined information gathered by each of these methods using a validation map and this was shown to provide reliable cell targeting results. Experiments conducted with sets of real images from two different cell lines (CHO-K1 and HEK), which contained a total of more than 650 cells, yielded flawless targeting results along with a cell detection ratio greater than 50%. PMID:23287416

  11. CLO: The cell line ontology

    PubMed Central

    2014-01-01

    Background Cell lines have been widely used in biomedical research. The community-based Cell Line Ontology (CLO) is a member of the OBO Foundry library that covers the domain of cell lines. Since its publication two years ago, significant updates have been made, including new groups joining the CLO consortium, new cell line cells, upper level alignment with the Cell Ontology (CL) and the Ontology for Biomedical Investigation, and logical extensions. Construction and content Collaboration among the CLO, CL, and OBI has established consensus definitions of cell line-specific terms such as ‘cell line’, ‘cell line cell’, ‘cell line culturing’, and ‘mortal’ vs. ‘immortal cell line cell’. A cell line is a genetically stable cultured cell population that contains individual cell line cells. The hierarchical structure of the CLO is built based on the hierarchy of the in vivo cell types defined in CL and tissue types (from which cell line cells are derived) defined in the UBERON cross-species anatomy ontology. The new hierarchical structure makes it easier to browse, query, and perform automated classification. We have recently added classes representing more than 2,000 cell line cells from the RIKEN BRC Cell Bank to CLO. Overall, the CLO now contains ~38,000 classes of specific cell line cells derived from over 200 in vivo cell types from various organisms. Utility and discussion The CLO has been applied to different biomedical research studies. Example case studies include annotation and analysis of EBI ArrayExpress data, bioassays, and host-vaccine/pathogen interaction. CLO’s utility goes beyond a catalogue of cell line types. The alignment of the CLO with related ontologies combined with the use of ontological reasoners will support sophisticated inferencing to advance translational informatics development. PMID:25852852

  12. 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

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

    PubMed

    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

  14. 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

  15. A human gallbladder adenocarcinoma cell line.

    PubMed

    Morgan, R T; Woods, L K; Moore, G E; McGavran, L; Quinn, L A; Semple, T U

    1981-06-01

    A continuous cell line, COLO 346, was established from a liver metastasis in a patient with adenocarcinoma of the gallbladder. COLO 346 grew as an adherent monolayer of pleomorphic epithelioid cells. COLO 346 cells produced esterone, but no estradiol, progesterone, or cortisol. No adrenocorticotropic hormones, beta-subunit of human chorionic gonadotropin, carcinoembryonic antigen, or alpha-fetoprotein production by the cells was detected. Cell doubling time was 36 h. Seven allelic isozymes were assayed. COLO 346 had a chromosome mode of 74 at 21 months postestablishment with 6 marker chromosomes present in 100% of the cells analyzed. COLO 346 has been in continuous culture for over 2 yr and is available to other investigators for their studies. PMID:7262900

  16. Cell shape-dependent shear stress on adherent cells in a micro-physiologic system as revealed by FEM.

    PubMed

    Pfister, C; Bozsak, C; Wolf, P; Demmel, F; Brischwein, M

    2015-05-01

    Flow-induced shear stress on adherent cells leads to biochemical signaling and mechanical responses of the cells. To determine the flow-induced shear stress on adherent cells cultured in a micro-scaled reaction chamber, we developed a suitable finite element method model. The influence of the most important parameters-cell shape, cell density, shear modulus and fluid velocity-was investigated. Notably, the cell shape strongly influences the resulting shear stress. Long and smooth cells undergo lower shear stress than more rounded cells. Changes in the curvature of the cells lead to stress peaks and single cells experience higher shear stress values than cells of a confluent monolayer. The computational results of the fluid flow simulation were validated experimentally. We also analyzed the influence of flow-induced shear stress on the metabolic activity and shape of L929, a mouse fibroblast cell line, experimentally. The results indicate that threshold stress values for continuous flow conditions cannot be transferred to quasi static flow conditions interrupted by short fluid exchange events. PMID:25856467

  17. 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 ...

  18. Factors involved in adherence of lactobacilli to human Caco-2 cells.

    PubMed Central

    Greene, J D; Klaenhammer, T R

    1994-01-01

    A quantitative assay performed with bacterial cells labelled with [3H]thymidine was used to investigate factors involved in the adherence of human isolates Lactobacillus acidophilus BG2FO4 and NCFM/N2 and Lactobacillus gasseri ADH to human Caco-2 intestinal cells. For all three strains, adherence was concentration dependent, greater at acidic pH values, and significantly greater than adherence of a control dairy isolate, Lactobacillus delbrueckii subsp. bulgaricus 1489. Adherence of L. acidophilus BG2FO4 and NCFM/N2 was decreased by protease treatment of the bacterial cells, whereas adherence of L. gasseri ADH either was not affected or was enhanced by protease treatment. Putative surface layer proteins were identified on L. acidophilus BG2FO4 and NCFM/N2 cells but were not involved in adherence. Periodate oxidation of bacterial cell surface carbohydrates significantly reduced adherence of L. gasseri ADH, moderately reduced adherence of L. acidophilus BG2FO4, and had no effect on adherence of L. acidophilus NCFM/N2. These results indicate that Lactobacillus species adhere to human intestinal cells via mechanisms which involve different combinations of carbohydrate and protein factors on the bacterial cell surface. The involvement of a secreted bridging protein, which has been proposed as the primary mediator of adherence of L. acidophilus BG2FO4 in spent culture supernatant (M.-H. Coconnier, T. R. Klaenhammer, S. Kernéis, M.-F. Bernet, and A. L. Servin, Appl. Environ. Microbiol. 58:2034-2039, 1992), was not confirmed in this study. Rather, a pH effect on Caco-2 cells contributed significantly to the adherence of this strain in spent culture supernatant.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:7811085

  19. 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

  20. 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

  1. 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.

  2. 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

  3. Traction Stresses Exerted by Adherent Cells: From Angiogenesis to Metastasis

    NASA Astrophysics Data System (ADS)

    Reinhart-King, Cynthia

    2010-03-01

    Cells exert traction stresses against their substrate that mediate their ability to sense the mechanical properties of their microenvironment. These same forces mediate cell adhesion, migration and the formation of stable cell-cell contacts during tissue formation. In this talk, I will present our data on the traction stresses generated by endothelial cells and metastatic breast cancer cells focused on understanding the processes of angiogenesis and metastasis, respectively. In the context of capillary formation, our data indicate that the mechanics of the substrate play a critical role in establishing endothelial cell-cell contacts. On more compliant substrates, endothelial cell shape and traction stresses polarize and promote the formation of stable cell-cell contacts. On stiffer substrates, traction stresses are less polarized and cell connectivity is disrupted. These data indicate that the mechanical properties of the microenvironment may drive cell connectivity and the formation of stable cell-cell contacts through the reorientation of traction stresses. In our studies of metastatic cell migration, we have found that traction stresses increase with increasing metastatic potential. We investigated three lines of varying metastatic potential (MCF10A, MCF7 and MDAMB231). MDAMB231, which are the most invasive, exert the most significant forces as measured by Traction Force Microscopy. These data present the possibility that cellular traction stress generation aids in the ability of metastatic cells to migrate through the matrix-dense tumor microenvironment. Such measurements are integral to link the mechanical and chemical microenvironment with the resulting response of the cell in health and disease.

  4. 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

  5. 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. PMID:26769129

  6. 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

  7. Cell death induced by Bothrops asper snake venom metalloproteinase on endothelial and other cell lines.

    PubMed

    Brenes, Oscar; Muñóz, Eduardo; Roldán-Rodríguez, Raquel; Díaz, Cecilia

    2010-06-01

    Two adherent cell lines, BAEC and HeLa, and non-adherent Jurkat, were treated with snake venom metalloproteinase BaP1 to determine whether cytotoxicity, previously reported for this toxin, could be mediated by the process of anoikis. It was observed that there was no correlation between the ability of this toxin to induce loss of adherence, and the cytotoxic effect, since concentrations that do not induce loss of adherence (3-6 microg/mL), were able to trigger 50% of cytotoxicity in BAEC. In the case of HeLa, where toxicity was very low (less than 20% at maximun concentrations and times of exposure), significant detachment and no toxicity was observed at concentrations of 1.5 microg/mL, showing also no correlation between both events. We also observed differences between BAEC toxicity measured by XTT reduction and DNA fragmentation determined by flow cytometry (as an indicator of apoptosis), since concentrations that induce 100% of cytotoxicity barely showed any DNA fragmentation (12% at 24h), suggesting that if apoptosis was involved, DNA damage is still not present, although chromatin condensation, another indicator of apoptosis, is observed in 40% of the cells. Inhibition of BAEC cytotoxicity by caspase inhibitors indicate that apoptosis is playing a role in this process, but other mechanisms of cell death could be participating also. Another way to determine whether the mechanism of cell death was related to anoikis was using a non-adherent cell line, which should show substrate independence. We determined by TUNEL that at 50 microg/ml BaP1 triggered 50% of apoptosis at 96 h, an effect that was seen earlier, suggesting also that if this toxin was inducing apoptosis in a non-adherent cell line, the mechanism could not be related to loss of attachment. Cell cycle arrest in S phase was also observed in Jurkat cells, an effect that could be leading to apoptosis. In conclusion, since there was no correlation between cell detachment and cytotoxicity (and apoptosis

  8. Mitogenic activation of B cells in vitro: the properties of adherent accessory cells as revealed by partition analysis.

    PubMed

    Kettman, J R; Soederberg, A; Lefkovits, I

    1986-08-15

    The requirement of B cells activated by mitogen (dextran sulfate plus lipopolysaccharide) for accessory cells was studied by partition analysis. Small numbers of splenic B cells were activated to clonal growth, as determined by visual inspection, and to immunoglobulin (Ig) synthesis, as determined by release of Ig into the culture fluid. By placing irradiated adherent cells in the periphery of the microculture wells and forcing responding cells to different areas of the well (slant experiments), it was observed that no cell contact was necessary for B cell activation, and that "promoted" contact ("Rock and Roll" experiments) does not increase the efficiency of activation. Sequential microcultures suggest that only some irradiated adherent cells act as accessory cells, but they can perform this function to more than one B cell. Attempts to perform limiting dilution analysis by varying irradiated adherent cell input showed non-single-hit behavior. When the data were rearranged, taking into account the distribution of irradiated adherent cells, then single-hit behavior with about 1 to 5% of irradiated adherent cells acting as an accessory cells for B cell clonal activation was observed. The evidence suggests that an uncommon irradiated adherent cell releases a soluble factor necessary for B cell activation and/or clonal proliferation. PMID:3488344

  9. 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

  10. Measurement of annexin V uptake and lactadherin labeling for the quantification of apoptosis in adherent Tca8113 and ACC-2 cells.

    PubMed

    Hu, T; Shi, J; Jiao, X; Zhou, J; Yin, X

    2008-09-01

    Phosphatidylserine (PS) exposure occurs during the cell death program and fluorescein-labeled lactadherin permits the detection of PS exposure earlier than annexin V in suspended cell lines. Adherent cell lines were studied for this apoptosis-associated phenomenon to determine if PS probing methods are reliable because specific membrane damage may occur during harvesting. Apoptosis was induced in the human tongue squamous carcinoma cell line (Tca8113) and the adenoid cystic carcinoma cell line (ACC-2) by arsenic trioxide. Cells were harvested with a modified procedure and labeled with lactadherin and/or annexin V. PS exposure was localized by confocal microscopy and apoptosis was quantified by flow cytometry. The detachment procedure without trypsinization did not induce cell damage. In competition binding experiments, phospholipid vesicles competed for more than 95 and 90% of lactadherin but only about 75 and 70% of annexin V binding to Tca8113 and ACC-2 cells. These data indicate that PS exposure occurs in three stages during the cell death program and that fluorescein-labeled lactadherin permitted the detection of early PS exposure. A similar pattern of PS exposure has been observed in two malignant cell lines with different adherence, suggesting that this pattern of PS exposure is common in adherent cells. Both lactadherin and annexin V could be used in adherent Tca8113 and ACC-2 cell lines when an appropriate harvesting procedure was used. Lactadherin is more sensitive than annexin V for the detection of PS exposure as the physical structure of PS in these blebs and condensed apoptotic cell surface may be more conducive to binding lactadherin than annexin V. PMID:18820763

  11. 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

  12. Interactions between Periodontal Bacteria and Human Oral Epithelial Cells: Fusobacterium nucleatum Adheres to and Invades Epithelial Cells

    PubMed Central

    Han, Yiping W.; Shi, Wenyuan; Huang, George T.-J.; Kinder Haake, Susan; Park, No-Hee; Kuramitsu, Howard; Genco, Robert J.

    2000-01-01

    Bacteria are causative agents of periodontal diseases. Interactions between oral bacteria and gingival epithelial cells are essential aspects of periodontal infections. Using an in vitro tissue culture model, a selected group of gram-negative anaerobic bacteria frequently associated with periodontal diseases, including Bacteroides forsythus, Campylobacter curvus, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia, were examined for their ability to adhere to and invade primary cultures of human gingival epithelial cells (HGEC). The effects of these bacteria on the production of interleukin-8 (IL-8), a proinflammatory chemokine, were also measured. These studies provided an initial demonstration that F. nucleatum adhered to and invaded HGEC and that this was accompanied by high levels of IL-8 secretion from the epithelial cells. The attachment and invasion characteristics of F. nucleatum were also tested using KB cells, an oral epithelial cell line. The invasion was verified by transmission electron microscopy and with metabolic inhibitors. Invasion appeared to occur via a “zipping” mechanism and required the involvement of actins, microtubules, signal transduction, protein synthesis, and energy metabolism of the epithelial cell, as well as protein synthesis by F. nucleatum. A spontaneous mutant, lam, of F. nucleatum, isolated as defective in autoagglutination, was unable to attach to or invade HGEC or KB cells, further indicating the requirement of bacterial components in these processes. Sugar inhibition assays indicated that lectin-like interactions were involved in the attachment of F. nucleatum to KB cells. Investigation of these new virulence phenotypes should improve our understanding of the role of F. nucleatum in periodontal infections. PMID:10816455

  13. 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

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

    PubMed

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

    2015-06-01

    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

  15. 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

  16. The effect of adherent and phagocytic cells on human lymphocyte PHA responsiveness.

    PubMed

    Potter, M R; Moore, M

    1977-01-01

    The effect of small numbers of adherent and phagocytic cells on the human peripheral blood lymphocyte response to PHA was examined by depleting these cells from lymphocyte preparations. Lymphocyte preparations obtained by centrifugation on Ficoll--Triosil, which contained on average 85% lymphocytes, responded well to PHA. Depletion of cells adhering to nylon fibre, giving a population containing on average 95% lymphocytes, resulted in a considerably reduced response. Depletion of cells that adhered to plastic or ingested iron powder to give populations containing on average 90% lymphocytes, also reduced the PHA response, but to a lesser extent. Reduction in PHA responsiveness correlated with increasing lymphocyte purity. The responsiveness of nylon-column-filtered cells could be restored by adding a small number of cells from a monocyte-rich population. PMID:300303

  17. Vascular cell adhesion molecule-1 and the integrin VLA-4 mediate adhesion of human B cell precursors to cultured bone marrow adherent cells.

    PubMed Central

    Ryan, D H; Nuccie, B L; Abboud, C N; Winslow, J M

    1991-01-01

    Adhesion of B cell precursors to accessory cells in the bone marrow microenvironment may be required for normal early B cell development. Human bone marrow B cell precursors adhere more avidly than mature B cells to bone marrow-derived fibroblasts. To determine the mechanism of this adhesion, expression of adhesion proteins on human B precursor cells and cell lines was measured by flow cytometry. The very late antigen (VLA) integrins VLA-4 and VLA-5 were the only adhesion proteins expressed at higher levels in B cell precursors than mature B cells. Antibodies to the alpha and beta chains of VLA-4, but not VLA-5, significantly blocked binding to bone marrow-derived fibroblasts of immature B cells and cell lines. Although fibronectin is a ligand for VLA-4, anti-fibronectin antibody and a soluble fibronectin fragment containing the VLA-4 binding domain did not block adhesion, suggesting that VLA-4 is involved in adhesion of B cell precursors, but not as a fibronectin receptor. Vascular cell adhesion molecule-1 (VCAM-1), the other known counterreceptor for VLA-4, was identified on bone marrow-derived fibroblasts, and anti-VCAM-1 significantly blocked adhesion of normal B cell precursors to bone marrow-derived fibroblasts, indicating that VLA-4/VCAM-1 interactions are important in adhesion of B cell precursors to the bone marrow microenvironment. Images PMID:1715889

  18. 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. PMID:23070491

  19. Thyroid cell lines in research on goitrogenesis.

    PubMed

    Gerber, H; Peter, H J; Asmis, L; Studer, H

    1991-12-01

    Thyroid cell lines have contributed a lot to the understanding of goitrogenesis. The cell lines mostly used in thyroid research are briefly discussed, namely the rat thyroid cell lines FRTL and FRTL-5, the porcine thyroid cell lines PORTHOS and ARTHOS, The sheep thyroid cell lines OVNIS 5H and 6H, the cat thyroid cell lines PETCAT 1 to 4 and ROMCAT, and the human thyroid cell lines FTC-133 and HTh 74. Chinese hamster ovary (CHO) cells and COS-7 cells, stably transfected with TSH receptor cDNA and expressing a functional TSH receptor, are discussed as examples for non-thyroidal cells, transfected with thyroid genes. PMID:1726925

  20. 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

  1. Further observations on the specific red cell adherence test: effects of radiation therapy

    SciTech Connect

    Richie, J.P.; Yap, W.T.

    1981-04-01

    To assess the effects of radiation therapy on the specific red cell adherence test we have evaluated 33 patients who underwent cystectomy for bladder cancer and in whom radiotherapy had been used. With this test negative tumors were found in 32 of the 33 cases. In a second series of 10 patients histologic examinations were done by biopsy before radiotherapy and by subsequent microscopic examination of the cystectomy specimen. The specific red cell adherence test results remained constant in all of these cases. These findings strongly suggest that 1) the specific red cell adherence test does remain negative after radiotherapy and 2) this test is a valuable prognosticator of the future likelihood of invasion in all patients with transitional cell carcinoma of the bladder.

  2. Effect of Lewis blood group antigen expression on bacterial adherence to COS-1 cells.

    PubMed Central

    Gaffney, R A; Schaeffer, A J; Anderson, B E; Duncan, J L

    1994-01-01

    Epithelial cells from secretor individuals demonstrate decreased bacterial adherence compared with cells from nonsecretors. Lewis blood group antigen expression is one component of the secretor/nonsecretor phenotype and several epidemiologic studies have suggested a link between Lewis blood group antigen phenotype and susceptibility to urinary tract infections. In this study, we examined the possibility that the expression of the difucosylated Lewis blood group determinants, Leb and Ley (associated with the secretor phenotype), made cells less susceptible to Escherichia coli adherence by masking receptors for pili. COS-1 cells, which do not produce Lewis (Lea, Leb, Le(x), and Ley) blood group antigens, were used as target cells for bacterial adherence. The surface blood group antigen expression pattern of the cells was then modified by cotransfection with plasmids containing DNA inserts encoding alpha (1,2)-fucosyltransferase and alpha (1,3)- and alpha (1,4)-fucosyltransferases, resulting in the expression of Leb and Ley. E. coli HB101 expressing various adhesins (type 1, PapJ96, PapIA2, PapAD110, Prs, and S) from recombinant plasmids bound equally well to untransfected cells and transfected cells expressing Lea and Le(x) (nonsecretor phenotype) and Leb and Ley (secretor phenotype) antigens. We conclude that the presence of Leb and Ley antigens on cells from secretors does not alone mask receptors for E. coli pili or hinder bacterial adherence. PMID:8005692

  3. Glucosyltransferases of Viridans Group Streptococci Modulate Interleukin-6 and Adhesion Molecule Expression in Endothelial Cells and Augment Monocytic Cell Adherence

    PubMed Central

    Yeh, Chiou-Yueh; Chen, Jen-Yang; Chia, Jean-San

    2006-01-01

    Recruitment of monocytes plays important roles during vegetation formation and endocardial inflammation in the pathogenesis of infective endocarditis (IE). Bacterial antigens or modulins can activate endothelial cells through the expression of cytokines or adhesion molecules and modulate the recruitment of leukocytes. We hypothesized that glucosyltransferases (GTFs), modulins of viridans group streptococci, may act directly to up-regulate the expression of adhesion molecules and also interleukin-6 (IL-6) to augment monocyte attachment to endothelial cells. Using primary cultured human umbilical vein endothelial cells (HUVECs) as an in vitro model, we demonstrated that GTFs (in the cell-bound or free form) could specifically modulate the expression of IL-6, and also adhesion molecules, in a dose- and time-dependent manner. Results of inhibition assays suggested that enhanced expression of adhesion molecules was dependent on the activation of nuclear factor κB (NF-κB) and extracellular signal-regulated kinase and that p38 mitogen-activated protein kinase pathways also contributed to the release of IL-6. Streptococcus-infected HUVECs or treatment with purified IL-6 plus soluble IL-6 receptor α enhanced the expression of ICAM-1 and the adherence of the monocytic cell line U937. These results suggest that streptococcal GTFs might play an important role in recruiting monocytic cells during inflammation in IE through induction of adhesion molecules and IL-6, a cytokine involved in transition from neutrophil to monocyte recruitment. PMID:16428777

  4. A Selective and Purification-Free Strategy for Labeling Adherent Cells with Inorganic Nanoparticles.

    PubMed

    Gao, Yu; Lim, Jing; Yeo, David Chen Loong; Liao, Shanshan; Lans, Malin; Wang, Yaqi; Teoh, Swee-Hin; Goh, Bee Tin; Xu, Chenjie

    2016-03-01

    Cellular labeling with inorganic nanoparticles such as magnetic iron oxide nanoparticles, quantum dots, and fluorescent silica nanoparticles is an important method for the noninvasive visualization of cells using various imaging modalities. Currently, this is mainly achieved through the incubation of cultured cells with the nanoparticles that eventually reach the intracellular compartment through specific or nonspecific internalization. This classic method is advantageous in terms of simplicity and convenience, but it suffers from issues such as difficulties in fully removing free nanoparticles (suspended in solution) and the lack of selectivity on cell types. This article reports an innovative strategy for the specific labeling of adherent cells without the concern of freely suspended nanoparticles. This method relies on a nanocomposite film that is prepared by homogeneously dispersing nanoparticles within a biodegradable polymeric film. When adherent cells are seeded on the film, they adhere, spread, and filtrate into the film through the micropores formed during the film fabrication. The pre-embedded nanoparticles are thus internalized by the cells during this infiltration process. As an example, fluorescent silica nanoparticles were homogeneously distributed within a polycaprolactone film by utilizing cryomilling and heat pressing. Upon incubation within physiological buffer, no silica nanoparticles were released from the nanocomposite film even after 20 d of incubation. However, when adherent cells (e.g., human mesenchymal stem cells) were grown on the film, they became fluorescent after 3 d, which suggests internalization of silica nanoparticles by cells. In comparison, the suspension cells (e.g., monocytes) in the medium remained nonfluorescent no matter whether there was the presence of adherent cells or not. This strategy eventually allowed the selective and concomitant labeling of mesenchymal stem cells during their harvest from bone marrow aspiration

  5. 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.

  6. 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

  7. Localized, macromolecular transport for thin, adherent, single cells via an automated, single cell electroporation biomanipulator.

    PubMed

    Sakaki, Kelly; Esmaeilsabzali, Hadi; Massah, Shabnam; Prefontaine, Gratien G; Dechev, Nikolai; Burke, Robert D; Park, Edward J

    2013-11-01

    Single cell electroporation (SCE), via microcapillary, is an effective method for molecular, transmembrane transport used to gain insight on cell processes with minimal preparation. Although possessing great potential, SCE is difficult to execute and the technology spans broad fields within cell biology and engineering. The technical complexities, the focus and expertise demanded during manual operation, and the lack of an automated SCE platform limit the widespread use of this technique, thus the potential of SCE has not been realized. In this study, an automated biomanipulator for SCE is presented. Our system is capable of delivering molecules into the cytoplasm of extremely thin cellular features of adherent cells. The intent of the system is to abstract the technical challenges and exploit the accuracy and repeatability of automated instrumentation, leaving only the focus of the experimental design to the operator. Each sequence of SCE including cell and SCE site localization, tip-membrane contact detection, and SCE has been automated. Positions of low-contrast cells are localized and "SCE sites" for microcapillary tip placement are determined using machine vision. In addition, new milestones within automated cell manipulation have been achieved. The system described herein has the capability of automated SCE of "thin" cell features less than 10 μm in thickness. Finally, SCE events are anticipated using visual feedback, while monitoring fluorescing dye entering the cytoplasm of a cell. The execution is demonstrated by inserting a combination of a fluorescing dye and a reporter gene into NIH/3T3 fibroblast cells. PMID:23771309

  8. Microfluidic Probe for Single-Cell Lysis and Analysis in Adherent Tissue Culture

    PubMed Central

    Lauffenburger, Douglas A.; Han, Jongyoon

    2014-01-01

    Single-cell analysis provides information critical to understanding key disease processes that are characterized by significant cellular heterogeneity. Few current methods allow single-cell measurement without removing cells from the context of interest, which not only destroys contextual information but also may perturb the process under study. Here we present a microfluidic probe that lyses single adherent cells from standard tissue culture and captures the contents to perform single-cell biochemical assays. We use this probe to measure kinase and housekeeping protein activities, separately or simultaneously, from single human hepatocellular carcinoma cells in adherent culture. This tool has the valuable ability to perform measurements that clarify connections between extracellular context, signals and responses, especially in cases where only a few cells exhibit a characteristic of interest. PMID:24594667

  9. Cell-mediated cytotoxicity of bovine mononuclear cells to IBRV-infected cells: dependence on Sephadex G-10 adherent cells.

    PubMed

    Campos, M; Rossi, C R

    1985-04-01

    Following intranasal inoculation of cattle with infectious bovine rhinotracheitis virus (IBRV) mononuclear cells that produced a genetically unrestricted cytotoxic response against IBRV-infected, but not against uninfected cells, were present in peripheral blood. Cytotoxicity was detected between 6 and 14 days after primary infection in a 20 h, but not in a 5 h, 51Cr-release assay. Cytotoxic activity was present in peripheral blood mononuclear cells from infected and subsequently hyperimmunized cattle for a considerably longer time. Neither natural cytotoxicity, antibody-dependent cell cytotoxicity, nor antibody produced during the assay was responsible for the cytotoxicity. However, cytotoxicity was dependent upon an adherent mononuclear cell that was partially removed by passage over nylon wool and completely removed by passage over Sephadex G-10. PMID:2408374

  10. [Ability of Staphylococcus cohnii strains to adhere to epithelial cells and solid surfaces in the hospital environment].

    PubMed

    Waldon, Edyta; Szewczyk, Eligia M

    2002-01-01

    Presented study describes abilities of staphylococci to adhere to exfoliated cheek and uroepithelial epithelium cells and to various surfaces such as plastics, glass and steel. The subject of the study were strains of Staphylococcus cohnii ssp. cohnii and Staphylococcus cohnii ssp. urealyticus isolated from Intensive Care Unit of Pediatric Hospital. Staphylococcus cohnii ssp.cohnii adhered in great number to epithelial cells. However, the adhesion differed by individual strains. We did not find relationship between slime production and adherence to epithelial cell. Most of investigated strains adhered closely to surfaces--especially of plastics and glass. This phenomenon was stronger in the presence of culture medium and phosphate buffer. PMID:12185691

  11. In vitro inhibition of Helicobacter pylori growth and adherence to gastric mucosal cells by Pycnogenol.

    PubMed

    Rohdewald, Peter; Beil, Winfried

    2008-05-01

    The emergence of antibiotic resistant H. pylori strains has necessitated the identification of alternative additive therapies for the treatment of this infection. The study tested whether a specific pine bark extract (Pycnogenol is effective in inhibiting the growth and adherence of H. pylori in vitro. Inhibition of H. pylori growth by Pycnogenol was tested in liquid medium as well as in an in vitro model by using sessile bacteria attached to AGS cells. Adherence was determined by co-incubation of gastric cells with Pycnogenol and H. pylori in vitro. Pycnogenol inhibited H. pylori growth in suspension with an MIC(50) of 12.5 microg/mL. Growth of H. pylori in infected cells was reduced to 10% of the control value by 125 microg/mL Pycnogenol. Adherence of H. pylori to gastric cells was reduced by 70% after 3 h incubation with 125 microg/mL Pycnogenol. The results show a significant, yet limited inhibition of growth and adherence of H. pylori to gastric cells by Pycnogenol. In vivo studies have to demonstrate the clinical relevance of these findings. PMID:18350522

  12. The many ways adherent cells respond to applied stretch.

    PubMed

    Sears, Candice; Kaunas, Roland

    2016-05-24

    Cells in various tissues are subjected to mechanical stress and strain that have profound effects on cell architecture and function. The specific response of the cell to applied strain depends on multiple factors, including cell contractility, spatial and temporal strain pattern, and substrate dimensionality and rigidity. Recent work has demonstrated that the cell response to applied strain depends on a complex combination of these factors, but the way these factors interact to elicit a specific response is not intuitive. We submit that an understanding of the integrated response of a cell to these factors will provide new insight into mechanobiology and contribute to the effective design of deformable engineered scaffolds meant to provide appropriate mechanical cues to the resident cells. PMID:26515245

  13. Early Warning Indicators for First-Line Virologic Failure Independent of Adherence Measures in a South African Urban Clinic

    PubMed Central

    Wu, Baohua; Hampton, Jane; Ordóñez, Claudia E.; Johnson, Brent A.; Singh, Dinesh; John, Sally; Gordon, Michelle; Hare, Anna; Murphy, Richard; Nachega, Jean; Kuritzkes, Daniel R.; del Rio, Carlos; Sunpath, Henry

    2013-01-01

    Abstract We sought to develop individual-level Early Warning Indicators (EWI) of virologic failure (VF) for clinicians to use during routine care complementing WHO population-level EWI. A case-control study was conducted at a Durban clinic. Patients after≥5 months of first-line antiretroviral therapy (ART) were defined as cases if they had VF [HIV-1 viral load (VL)>1000 copies/mL] and controls (2:1) if they had VL≤1000 copies/mL. Pharmacy refills and pill counts were used as adherence measures. Participants responded to a questionnaire including validated psychosocial and symptom scales. Data were also collected from the medical record. Multivariable logistic regression models of VF included factors associated with VF (p<0.05) in univariable analyses. We enrolled 158 cases and 300 controls. In the final multivariable model, male gender, not having an active religious faith, practicing unsafe sex, having a family member with HIV, not being pleased with the clinic experience, symptoms of depression, fatigue, or rash, low CD4 counts, family recommending HIV care, and using a TV/radio as ART reminders (compared to mobile phones) were associated with VF independent of adherence measures. In this setting, we identified several key individual-level EWI associated with VF including novel psychosocial factors independent of adherence measures. PMID:24320011

  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. 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 ...

  16. 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.

  17. A Localized Adherence-Like Pattern as a Second Pattern of Adherence of Classic Enteropathogenic Escherichia coli to HEp-2 Cells That Is Associated with Infantile Diarrhea

    PubMed Central

    Scaletsky, Isabel C. A.; Pedroso, Margareth Z.; Oliva, Carlos A. G.; Carvalho, Rozane L. B.; Morais, Mauro B.; Fagundes-Neto, Ulysses

    1999-01-01

    Escherichia coli strains that cause nonbloody diarrhea in infants are known to present three distinct patterns of adherence to epithelial cells, namely, localized (LA), diffuse (DA), and aggregative (AA) adherence. Strains with LA (typical Enteropathogenic Escherichia coli [EPEC]) are well recognized as a cause of secretory diarrhea, but the role of strains with DA (DAEC) is controversial, and strains with AA (EAEC) have been more frequently related to persistent diarrhea whereas its relationship with acute diarrhea is not well defined. To determine the relationship of the different types of E. coli adherence patterns with acute diarrhea (lasting less than 14 days) and persistent diarrhea (lasting more than 14 days) in São Paulo, Brazil, we studied stool specimens from 40 infants under 1 year of age with diarrhea and 40 age-matched control infants without any gastrointestinal symptoms. Twenty-eight (35.0%) of eighty cases yielded adherent E. coli (HEp-2 cells). Strains with localized and aggregative adherence were associated with acute and persistent diarrhea. A total of 11.2% of the adherent strains were typical EPEC serotypes and hybridized with the enteroadherence factor probe; 5.0% were EAEC and hybridized with the EAEC probe. DAEC strains were isolated from 10.0% of patients and 7.5% of controls and did not hybridize with the two probes used (daaC and AIDA-I). Strains with a localized adherence-like pattern (atypical EPEC) were found significantly more frequently (P = 0.028) in cultures from children with diarrhea (17.5%) than in controls (2.5%). PMID:10377120

  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. 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

  20. 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

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

    PubMed

    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

  2. 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

  3. Human Adrenocortical Carcinoma Cell Lines

    PubMed Central

    Wang, Tao; Rainey, William E.

    2011-01-01

    Summary The human adrenal cortex secretes mineralocorticoids, glucocorticoids and adrenal androgens. These steroids are produced from unique cell types located within the three distinct zones of the adrenal cortex. Disruption of adrenal steroid production results in a variety of diseases that can lead to hypertension, metabolic syndrome, infertility and androgen excess. The adrenal cortex is also a common site for the development of adenomas, and rarely the site for the development of carcinomas. The adenomas can lead to diseases associated with adrenal steroid excess, while the carcinomas are particularly aggressive and have a poor prognosis. In vitro cell culture models provide an important tool to examine molecular and cellular mechanisms controlling both the normal and pathologic function of the adrenal cortex. Herein we discuss the human adrenocortical cell lines and their use as model systems for adrenal studies. PMID:21924324

  4. Fibrinogen-Induced Streptococcus mutans Biofilm Formation and Adherence to Endothelial Cells

    PubMed Central

    Lombardo Bedran, Telma Blanca; Azelmat, Jabrane; Palomari Spolidorio, Denise

    2013-01-01

    Streptococcus mutans, the predominant bacterial species associated with dental caries, can enter the bloodstream and cause infective endocarditis. The aim of this study was to investigate S. mutans biofilm formation and adherence to endothelial cells induced by human fibrinogen. The putative mechanism by which biofilm formation is induced as well as the impact of fibrinogen on S. mutans resistance to penicillin was also evaluated. Bovine plasma dose dependently induced biofilm formation by S. mutans. Of the various plasma proteins tested, only fibrinogen promoted the formation of biofilm in a dose-dependent manner. Scanning electron microscopy observations revealed the presence of complex aggregates of bacterial cells firmly attached to the polystyrene support. S. mutans in biofilms induced by the presence of fibrinogen was markedly resistant to the bactericidal effect of penicillin. Fibrinogen also significantly increased the adherence of S. mutans to endothelial cells. Neither S. mutans cells nor culture supernatants converted fibrinogen into fibrin. However, fibrinogen is specifically bound to the cell surface of S. mutans and may act as a bridging molecule to mediate biofilm formation. In conclusion, our study identified a new mechanism promoting S. mutans biofilm formation and adherence to endothelial cells which may contribute to infective endocarditis. PMID:24222906

  5. Sortase inhibitor phenyl vinyl sulfone inhibits Renibacterium salmoninarum adherence and invasion of host cells.

    PubMed

    Sudheesh, Ponnerassery S; Crane, Samuel; Cain, Kenneth D; Strom, Mark S

    2007-12-13

    Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmonid fishes, is a Gram-positive diplococcobacillus belonging to the family Micrococcaceae. Analysis of the genome sequence of the bacterium demonstrated the presence of a sortase homolog (srtD), a gene specifying an enzyme found in Gram-positive bacteria and required for covalent anchoring of cell surface proteins. Interference of sortase activity is being examined as a target for therapeutic prevention of infection by several pathogenic Gram-positive bacterial species. In silico analysis identified 8 open reading frames containing sortase recognition motifs, suggesting these proteins are translocated to the bacterial cell wall. The sortase and potential sortase substrate genes are transcribed in R. salmoninarum, suggesting they encode functional proteins. Treatment of R. salmoninarum with phenyl vinyl sulfone (PVS) significantly reduced bacterial adherence to Chinook salmon fibronectin. In addition, the ability of the PVS-treated bacteria to adhere to Chinook salmon embryo cells (CHSE-214) in vitro was dramatically reduced compared to that of untreated bacteria. More importantly, PVS-treated bacteria were unable to invade and replicate within CHSE-214 cells (demonstrated by an intracellular growth assay and by light microscopy). When treated with PVS, R. salmoninarum was not cytopathic to CHSE-214 cells, whereas untreated bacteria produced cytopathology within a few days. These findings clearly show that PVS, a small molecule drug and a known sortase inhibitor, can interfere with the ability of R. salmoninarum to adhere and colonize fish cells, with a corresponding decrease in virulence. PMID:18286808

  6. Effects of cryopreservation and phenylacetate on biological characters of adherent LAK cells from patients with hepatocellular carcinoma

    PubMed Central

    Zheng, Ning; Ye, Sheng-Long; Sun, Rui-Xia; Zhao, Yan; Tang, Zhao-You

    2002-01-01

    AIM: To improve the preparation of adherent lymphokine-activated killer (A-LAK) cells and to study the effects of cryopreservation and phenylacetate (PA) on biological characters of A-LAK cells. METHODS: A-LAK cells were obtained from peripheral blood mononuclear cells (PBMCs) of the patients with hepatocellular carcinoma (HCC) by using L-phenylalanine methyl ester (PME) to deplete immunosuppressive monocytes. Proliferative activity of SMMC7721 cell line after treatment with phenylacetate (PA) was observed. A-LAK cells were treated with the supernatant of SMMC7721 cells that had been pretreated with PA. The changes of proliferation, cytotoxicity and phenotype of A-LAK cells were investigated after cryopreservation. RESULTS: The expansion of A-LAK cells (96.79 ± 69.10 folds on Day 14) was significantly higher than that of non-adherent LAK (NA-LAK) cells (22.77 ± 13.20) as well as conventional LAK cells (4.64 ± 0.91). PA significantly suppressed the growth of SMMC7721 cells, and the inhibitor ratio was 46%. The supernatant of cultured tumor cells intensively suppressed the proliferation and cytotoxicity of A-LAK cells, but the suppressive effect of the supernatant was previously decreased after treatment with PA. Impairments in proliferation and cytotoxicity of A-LAK cells immediately after thawing of cryopreservation and recovery after reincubation with IL-2 were observed. The cytotoxicity of thawed A-LAK cells on Day 5 was significantly higher than that of fresh A-LAK before freezing (54.8% ± 10.2% vs 40.5% ± 6.4%). No significant change in the percentage of lymphocyte subsets was identified in frozen A-LAK cells as compared with that in the fresh control cells. CONCLUSION: A-LAK cells can be simply prepared by using PME, and showed a synergistic anti-tumor effect with the combination of PA. Cryopreservation can increase the immunoactivities of A-LAK cells from the patients with hepatocellular carcinoma. PMID:11925598

  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. 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. PMID:25742639

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

    PubMed Central

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

    2015-01-01

    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. PMID:25742639

  10. Molluscan cells in culture: primary cell cultures and cell lines

    PubMed Central

    Yoshino, T. P.; Bickham, U.; Bayne, C. J.

    2013-01-01

    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome. PMID:24198436

  11. An in vitro clonal assay of adherent stem cells (ASC) in mouse marrow.

    PubMed

    Reincke, U; Rosenblatt, M; Hellman, S

    1984-11-01

    Hematopoietic stem cells with high proliferative capacity can be assayed when stromal bone marrow cultures are overlaid with limiting dilutions of marrow samples. This leads to hematopoietic growth after 4 weeks in a fraction of cultures, consistent with expectations based on Poisson statistics. It will be shown that monoclonal cultures are obtained that last from 2 to 15 weeks and that can generate up to several million mature granulocytes. The originating clone-forming cell is named adherent stem cell (ASC) because of its adherence to plastic or stromal surfaces. The ASC is comparable to the CFU-S in frequency, proliferative capacity and in its ability to give rise to CFU-S. As an unexpected additional finding we report that a mode of "clonal succession" was apparent in cultures which expressed more than one clone. PMID:6490726

  12. Mycoplasma pulmonis Vsa proteins and polysaccharide modulate adherence to pulmonary epithelial cells.

    PubMed

    Bolland, Jeffrey R; Dybvig, Kevin

    2012-06-01

    The Mycoplasma pulmonis Vsa proteins are a family of size- and phase-variable lipoproteins that shield the mycoplasmas from complement and modulate attachment to abiotic surfaces. Mycoplasmas producing a long Vsa protein hemadsorb poorly and yet are proficient at colonizing rats and mice. The effect of the length of the Vsa protein on the attachment of mycoplasmas to epithelial cells has not been previously explored. We find that independent of Vsa isotype, mycoplasmas producing a long Vsa protein with many tandem repeats adhere poorly to murine MLE-12 cells compared with mycoplasmas producing a short Vsa. We also find that mutants lacking the EPS-I polysaccharide of M. pulmonis exhibited decreased adherence to MLE-12 cells, even though it has been shown previously that such mutants have an enhanced ability to form a biofilm. PMID:22428866

  13. Further evidence for the existence of 'homing' receptors on murine leukemia cells which mediate adherence to normal bone marrow stromal cells.

    PubMed

    Kamenov, B; Longenecker, B M

    1985-01-01

    A significant proportion of 131IUDR-labelled cells from murine leukemia cell lines L1210 and P388, but not the L5178Y lymphoma cell line, are retained in the bone marrow (B.M.) following i.v. injection into syngeneic mice. Following this, L1210 and P388 cells grow and rapidly replace the normal hematopoietic cells of the B.M. L1210 and P388 cells, but not several lymphoma cell lines, also bind avidly to monolayers of B.M. stromal cells (Dexter cultures) and soon overgrow the cultures following rapid cell proliferation. P388 cells bound equally well to confluent monolayers of B.M., whole mouse embryo and newborn mouse kidney while L1210 cells bound well to B.M. and whole mouse embryo but showed little binding to newborn kidney monolayers. The accumulation of the two leukemia cell lines in the B.M. was constant and indistinguishable over a 48-h period. In contrast, in both spleen and liver the number of L1210 cells decreased during the same period while P388 cells were retained at a constant level. Generally there was a lack of correlation of B.M. metastasis of a cell line and its metastasis to other organs although P388 cells, but not L1210 cells, demonstrated a tremendous capacity for metastatic growth in both spleen and liver. Normal B.M. cells were fused with the syngeneic SP2/0 murine myeloma fusor line and 10 hybridomas plus the SP2/0 parent were tested for in-vitro adherence to B.M. monolayers and in-vivo metastatic behavior. The same 3 (out of 10) hybridomas showed a high level of adherence to B.M. monolayers, high levels of retention of cells in the B.M. following i.v. injection, and rapid growth and takeover of the normal B.M. In marked contrast, neither the SP2/0 parent nor the remaining 7 hybridomas show significant adherence, B.M. retention or growth in the B.M. A distinct lack of correlation of B.M. vs liver or spleen metastasis was once again noted for the hybridomas although all of the hybridomas showed much less metastatic growth in the liver than

  14. Canine PHA-stimulated adherent cell enhance interferon-gamma production and proliferation of autologous peripheral blood mononuclear cells.

    PubMed

    Ide, Kaori; Momoi, Yasuyuki; Iwasaki, Toshiroh

    2005-03-01

    Dendritic cells are specialized antigen-presenting cells with immuno-modulating functions that are attractive for clinical applications for cancer immunotherapy. This study examined immunostimulatory functions of phytohemagglutinin (PHA)-stimulated adherent cells (PHA-Ad cells) from peripheral blood mononuclear cells (PBMCs) in dogs. PHA-Ad cells enhanced interferon-gamma from autologous PBMC in vitro. PHA-Ad cells also stimulated antigen-independent proliferation of peripheral blood lymphocytes. These results suggest that PHA-Ad cells from PBMC possess a stimulatory function to evoke anti-tumour immunity and that they demonstrate potential for therapeutic applications in dogs. PMID:19379211

  15. 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

  16. Relationship between cell surface composition of Candida albicans and adherence to acrylic after growth on different carbon sources.

    PubMed Central

    McCourtie, J; Douglas, L J

    1981-01-01

    The adherence of Candida albicans to acrylic was measured in vitro after growth of the yeast to stationary phase in defined medium containing glucose, sucrose, galactose, fructose, or maltose as the carbon source. In each case, yeast adherence was proportional to the concentration of sugar in the growth medium, but equimolar concentrations of different sugars promoted adherence to different extents. In vitro adherence was further increased by the addition of divalent cations to assay mixtures but was inhibited when saliva-treated acrylic strips were used or when yeasts were suspended in mixed saliva during the assay. The rate of spheroplast formation of yeasts grown in media containing a 500 mM concentration of the different sugars correlated well with the relative adherence of the cells to acrylic. Galactose-grown yeasts were most resistant to spheroplast formation with Zymolyase-5000 and most adherent to acrylic, whereas fructose-grown organisms were least resistant to spheroplast formation and least adherent to acrylic. These results indicate that when grown to stationary phase in media containing high concentrations of certain sugars, C. albicans undergoes a change in cell surface composition which facilitates its adherence to acrylic surfaces. Electron microscopy of yeasts harvested from such media revealed the presence of an additional surface layer which may be responsible for this enhanced adherence. Images PMID:7019091

  17. Suboptimal adherence associated with virological failure and resistance mutations to first-line highly active antiretroviral therapy (HAART) in Bangalore, India

    PubMed Central

    Ekstrand, Maria L.; Shet, Anita; Chandy, Sara; Singh, Girija; Shamsundar, Ranjani; Madhavan, Vidya; Saravanan, Shanmugam; Heylen, Elsa; Kumarasamy, Nagalingeswaran

    2010-01-01

    This study was conducted to examine the relationship between adherence, viral load (VL) and resistance among outpatients receiving highly active antiretroviral therapy (HAART) in Bangalore, India. In total, 552 outpatients were recruited and VL testing was conducted for all study participants. HIV-1 genotypic resistance testing was performed for 92 participants with a VL ≥ 1000 copies/ml. Interpretation of resistance mutations was performed according to the Stanford database. Past-month adherence and treatment interruptions for >48 h were assessed via self-report. At baseline, 34 participants (6%) reported <95% past-month adherence and 110 (20%) reported a history of >48 h treatment interruptions. Combining the two adherence measures, 22% of participants were classified as ‘suboptimally adherent’. In total, 24% of study participants (n = 132) had a detectable VL. Among the 92 samples sent for resistance testing, 68% had at least one nucleoside reverse transcriptase inhibitor (NRTI) mutation, with M184V being the most common (62%) and with 48% having thymidine analogue mutations. Moreover, 72% had at least one non-nucleoside reverse transcriptase inhibitor (NNRTI) mutation and 23% had three or more NNRTI mutations. Both adherence measures were significantly associated with VL (P < 0.001). Suboptimal adherence was significantly associated with resistance mutations (P < 0.02). The findings illustrate for the first time the strong association between suboptimal adherence, treatment failure and drug resistance to first-line HAART in India. The predictive value of standard adherence measures was improved by including treatment interruption data. The observed mutations can jeopardise future treatment options, especially in light of limited access to second-line treatments. To develop effective adherence interventions, research is needed to examine culturally-specific reasons for treatment interruptions. PMID:21516199

  18. 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

  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. 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

  1. 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

  2. Effects of mononuclear phagocyte system modulating agents on Fc and C3 receptors of adherent cells.

    PubMed Central

    Hitomi, M.; Shimizu, F.

    1985-01-01

    Agents which modulate the mononuclear phagocyte system (MPS) were examined for their effects on Fc and C3 receptors of adherent cells (A-cells) as judged by rosette formation. Dextran sulphate, carrageenan, and immune complexes, known as MPS suppressants, reduced the percentage of receptor-positive A-cells, while levamisole, known as a MPS-activator, increased the percentage in vitro. The changes in the percentage of Fc receptor were parallel to those of the C3 receptor in vitro. The effects of these agents were also examined in vivo. PMID:2408651

  3. Imaging deformation of adherent cells due to shear stress using quantitative phase imaging.

    PubMed

    Eldridge, Will J; Sheinfeld, Adi; Rinehart, Matthew T; Wax, Adam

    2016-01-15

    We present a platform for detecting cellular deformations from mechanical stimuli, such as fluid shear stress, using rapid quantitative phase imaging. Rapid quantitative phase imaging was used to analyze changes in the optical path length of adherent skin cancer cells during mechanical displacement. Both the whole-cell phase displacement and the resultant shift of the cellular center of mass were calculated over the duration of the stimulus. Whole-cell phase displacement images were found to match expectation. Furthermore, center-of-mass shifts of adherent cells were found to resemble that of a one-dimensional Kelvin-Voigt (KV) viscoelastic solid. Cellular steady-state displacements from step fluid shear stimuli were found to be linearly related to the shear stress. Shear stiffness constants for cells exposed to a cytoskeletal disrupting toxin were found to be significantly lower than unexposed cells. This novel technique allows for elastographic analysis of whole-cell effective shear stiffness without the use of an exogenous force applicator, a specialized culture substrate, or tracking net perimeter movement of the cell. PMID:26766712

  4. Array of Biodegradable Microraftsfor Isolation and Implantation of Living, Adherent Cells

    PubMed Central

    Wang, Yuli; Phillips, Colleen N.; Herrera, Gabriela S.; Sims, Christopher E.; Yeh, Jen Jen; Allbritton, Nancy L.

    2013-01-01

    A new strategy for efficient sorting and implantation of viable adherent cells into animals is described. An array of biodegradable micro-structures (microrafts) was fabricated using a polydimethylsiloxane substrate for micromolding poly(lactic-co-glycolic acid) (PLGA). Screening various forms of PLGA determined that the suitability of PLGA for microraft manufacture, biocompatibility and in vitro degradation was dependent on molecular weight and lactic/glycolic ratio. Cells plated on the array selectively attached to the microrafts and could be identified by their fluorescence, morphology or other criteria. The cells were efficiently dislodged and collected from the array using a microneedle device. The platform was used to isolate specific cells from a mixed population establishing the ability to sort target cells for direct implantation. As a proof of concept, fluorescently conjugated microrafts carrying tumor cells stably expressing luciferase were isolated from an array and implanted subcutaneously into mice. In vivo bio-luminescence imaging confirmed the growth of a tumor in the recipient animals. Imaging of tissue sections from the tumors demonstrated in vivo degradation of the implanted microrafts. The process is a new strategy for isolating and delivering a small number of adherent cells for animal implantation with potential applications in tissue repair, tumor induction, in vivo differentiation of stem cells and other biomedical research. PMID:23930219

  5. Morphology and growth of murine cell lines on model biomaterials.

    PubMed

    Godek, Marisha L; Duchsherer, Nichole L; McElwee, Quinn; Grainger, David W

    2004-01-01

    All biomaterial implants are assaulted by the host "foreign body" immune response. Understanding the complex, dynamic relationship between cells, biomaterials and milieu is an important first step towards controlling this reaction. Material surface chemistry dictates protein adsorption, and thus subsequent cell interactions. The cell-implant is a microenvironment involving 1) proteins that coat the surface and 2) cells that interact with these proteins. Macrophages and fibroblasts are two cell types that interact with proteins on biomaterials surfaces and play different related, but equally important, roles in biomaterials rejection and implant failure. Growth characteristics of four murine cell lines on model biomaterials surfaces were examined. Murine monocyte-macrophages (RAW 264.7 and J774A.1), murine macrophage (IC-21) and murine fibroblast (NIH 3T3) cell lines were tested to determine whether differences exist in adhesion, proliferation, differentiation, spreading, and fusion (macrophage lineages only) on these surfaces. Differences were observed in the ability of cells to adhere to and subsequently proliferate on polymer surfaces. (Monocyte-) macrophages grew well on all surfaces tested and growth rates were measured on three representative polymer biomaterials surfaces: tissue culture polystyrene (TCPS), polystyrene, and Teflon-AF. J774A.1 cultures grown on TCPS and treated with exogenous cytokines IL-4 and GM-CSF were observed to contain multinucleate cells with unusual morphologies. Thus, (monocyte-) macrophage cell lines were found to effectively attach to and interrogate each surface presented, with evidence of extensive spreading on Teflon-AF surfaces, particularly in the IC-21 cultures. The J774A.1 line was able to proliferate and/or differentiate to more specialized cell types (multinucleate/dendritic-like cells) in the presence of soluble chemokine cues. PMID:15133927

  6. Immune adherence in renal glomeruli. Complement receptor sites on glomerular capillary epithelial cells.

    PubMed Central

    Burkholder, P. M.; Oberley, T. D.; Barber, T. A.; Beacom, A.; Koehler, C.

    1977-01-01

    Several very recent reports have indicated the presence of receptor sites for the third component of complement in human but not other vertebrate renal glomeruli. The present study constitutes a demonstration that the glomerular capillary epithelial cell bears this receptor, detectable with either EAC complexes (EAC1423b) or fluores ceinated zymosan-C3 (ZC3b) complexes, Fresh, unfixed frozen sections of normal or diseased human kidneys, mechanically isolated human glomeruli, dissociated glomerular cells, and glomeruli and golmerular cells maintained in tissue culture were examined with various EAC complexes or ZC3b and examined by phase light microscopy, fluorescence microscopy, or transmission and scanning electron microscopy. Clearly, by scanning electron microscopy it was determined that glomerular capillary epithelial cells bind the immune-adherence EAC indicator cells. Because glomeruli or glomerular epithelial cells did not bind E, EA, EACI, EAC14, or EAC142 but did bind EAC1423b or ZC3b, it is concluded that C3b (activated bound fragment of the third component of complement) is responsible for the immune-adherence reaction in glomeruli. Preliminary examination of diseased renal biopsies indicates that sclerotic glomeruli, focal segmental sclerotic or proliferative glomerular capillary lesions, and proliferative epithelial crescents are immune-adherence negative. Furthermore, a clear or consistent inverse relationship between glomerular capillary deposits of C3 which presumably might block epithelial C3 receptor sites, and immune-adherence reactivity with EAC in vitro was not as evident in this study as reported previously by other investigators. Nevertheless, it is still attractive to conceive that glomerular C3 receptor sites might be responsible for binding of antigen-antibody-complement complexes and formation of immune-complex deposits, at least on the epimembranous (subepithelial) surface of glomerular capillary walls. Inability to demonstrate this

  7. 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.

  8. Evaluation of Gelatin Microparticles as Adherent-Substrates for Mesenchymal Stem Cells in a Hydrogel Composite.

    PubMed

    Lu, Steven; Lee, Esther J; Lam, Johnny; Tabata, Yasuhiko; Mikos, Antonios G

    2016-06-01

    Due to the lack of cell-adhesive moieties in traditional synthetic hydrogels, the present work investigated the use of degradable gelatin microparticles (GMPs) as temporary adherent substrates for anchorage-dependent mesenchymal stem cells (MSCs). MSCs were seeded onto GMPs of varying crosslinking densities and sizes to investigate their role on influencing MSC differentiation and aggregation. The MSC-seeded GMPs were then encapsulated in poly(ethylene glycol)-based hydrogels and cultured in serum-free, growth factor-free osteochondral medium. Non-seeded MSCs co-encapsulated with GMPs in the hydrogels were used as a control for comparison. Over the course of 35 days, MSCs seeded on GMPs exhibited more cell-cell contacts, greater chondrogenic potential, and a down-regulation of osteogenic markers compared to the controls. Although the factors of GMP crosslinking and size had nominal influence on MSC differentiation and aggregation, GMPs demonstrate potential as an adherent-substrate for improving cell delivery from hydrogel scaffolds by facilitating cell-cell contacts and improving MSC differentiation. PMID:26935924

  9. Slow-Adhering Stem Cells Derived from Injured Skeletal Muscle Have Improved Regenerative Capacity

    PubMed Central

    Mu, Xiaodong; Xiang, Guosheng; Rathbone, Christopher R.; Pan, Haiying; Bellayr, Ian H.; Walters, Thomas J.; Li, Yong

    2011-01-01

    A wide variety of myogenic cell sources have been used for repair of injured and diseased muscle including muscle stem cells, which can be isolated from skeletal muscle as a group of slow-adhering cells on a collagen-coated surface. The therapeutic use of muscle stem cells for improving muscle regeneration is promising; however, the effect of injury on their characteristics and engraftment potential has yet to be described. In the present study, slow-adhering stem cells (SASCs) from both laceration-injured and control noninjured skeletal muscles in mice were isolated and studied. Migration and proliferation rates, multidifferentiation potentials, and differences in gene expression in both groups of cells were compared in vitro. Results demonstrated that a larger population of SASCs could be isolated from injured muscle than from control noninjured muscle. In addition, SASCs derived from injured muscle demonstrated improved migration, a higher rate of proliferation and multidifferentiation, and increased expression of Notch1, STAT3, Msx1, and MMP2. Moreover, when transplanted into dystrophic muscle in MDX/SCID mice, SASCs from injured muscle generated greater engraftments with a higher capillary density than did SASCs from control noninjured muscle. These data suggest that traumatic injury may modify stem cell characteristics through trophic factors and improve the transplantation potential of SASCs in alleviating skeletal muscle injuries and diseases. PMID:21684246

  10. 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

  11. Large-Scale Production of High-Quality Helper-Dependent Adenoviral Vectors Using Adherent Cells in Cell Factories

    PubMed Central

    Suzuki, Masataka; Cela, Racel; Clarke, Christian; Bertin, Terry K.; Mouriño, Susana

    2010-01-01

    Abstract The most efficient and widely used system for generating helper-dependent adenoviral vectors (HDAds) is the Cre/loxP system developed by Graham and co-workers (Parks, R.J., Chen, L., Anton, M., Sankar, U., Rudnicki, M.A., and Graham, F.L. [1996]. Proc. Natl. Acad. Sci. U. S. A. 93, 13565–13570). Alternative systems have been developed for HDAd production, but all are limited by the technical complexity of a three-component vector production system for reproducibly generating large quantities of adenovirus with high infectivity and low helper virus (HV) contamination. Recently, these problems were addressed by Ng and co-workers (Palmer, D., and Ng, P. [2003]. Mol Ther. 8, 846–852), who developed an improved system that combines the use of a suspension-adapted producer cell line expressing high levels of Cre recombinase, a HV resistant to mutation, and a refined purification protocol. With this system, >1 × 1013 highly infectious vector particles are easily produced without vector genome rearrangements and having very low HV contamination levels. However, the Ng system incorporates a spinner flask culture system that involves considerable time, effort, and tissue culture medium to produce HDAds. We have an alternative system to obtain comparable quantities with equivalent quality to the spinner flask approach but requiring reduced labor and lower volumes of medium. This method utilizes a 10-chamber cell factory with adherent cells to produce high infectivity of HDAds with minimal HV contamination while improving yield and reducing technical complexity, effort, and medium requirements. This system is easily translatable to the production of clinical-grade HDAds for human trials. PMID:19719388

  12. 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

  13. 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

  14. Standardized orthotopic xenografts in zebrafish reveal glioma cell-line-specific characteristics and tumor cell heterogeneity

    PubMed Central

    Welker, Alessandra M.; Jaros, Brian D.; Puduvalli, Vinay K.; Imitola, Jaime; Kaur, Balveen; Beattie, Christine E.

    2016-01-01

    ABSTRACT Glioblastoma (GBM) is a deadly brain cancer, for which few effective drug treatments are available. Several studies have used zebrafish models to study GBM, but a standardized approach to modeling GBM in zebrafish was lacking to date, preventing comparison of data across studies. Here, we describe a new, standardized orthotopic xenotransplant model of GBM in zebrafish. Dose-response survival assays were used to define the optimal number of cells for tumor formation. Techniques to measure tumor burden and cell spread within the brain over real time were optimized using mouse neural stem cells as control transplants. Applying this standardized approach, we transplanted two patient-derived GBM cell lines, serum-grown adherent cells and neurospheres, into the midbrain region of embryonic zebrafish and analyzed transplanted larvae over time. Progressive brain tumor growth and premature larval death were observed using both cell lines; however, fewer transplanted neurosphere cells were needed for tumor growth and lethality. Tumors were heterogeneous, containing both cells expressing stem cell markers and cells expressing markers of differentiation. A small proportion of transplanted neurosphere cells expressed glial fibrillary acidic protein (GFAP) or vimentin, markers of more differentiated cells, but this number increased significantly during tumor growth, indicating that these cells undergo differentiation in vivo. By contrast, most serum-grown adherent cells expressed GFAP and vimentin at the earliest times examined post-transplant. Both cell types produced brain tumors that contained Sox2+ cells, indicative of tumor stem cells. Transplanted larvae were treated with currently used GBM therapeutics, temozolomide or bortezomib, and this resulted in a reduction in tumor volume in vivo and an increase in survival. The standardized model reported here facilitates robust and reproducible analysis of glioblastoma tumor cells in real time and provides a platform for

  15. 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...

  16. 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...

  17. 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. PMID:26439917

  18. 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

  19. Experimental evidence for the role of lipids in adherence of Candida spp. to human buccal epithelial cells.

    PubMed Central

    Ghannoum, M A; Burns, G R; Elteen, K A; Radwan, S S

    1986-01-01

    Lipids extracted from Candida albicans and C. tropicalis, but not from the weakly adherent C. pseudotropicalis, significantly blocked in vitro adherence of the respective yeast cells to buccal epithelial cells. The percentage of reduction from control values ranged between 16.4 and 42.1%, depending on the species, the strain, and the solvent used for lipid extraction. The constituent lipid classes of both the acetone and chloroform-methanol extracts of C. albicans ATCC 10231 were qualitatively and quantitatively analyzed. The individual classes were isolated by preparative thin-layer chromatography and then tested for their effects on the adherence of this strain to buccal epithelial cells. Individual phospholipids, sterols, and steryl esters blocked adherence significantly (between 15.5 and 55.7% reduction). Triacylglycerols and free fatty acids showed no effect whatsoever. The same results were obtained when standard lipid samples were investigated. Images PMID:3759234

  20. 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

  1. Aspergillus fumigatus MedA governs adherence, host cell interactions and virulence

    PubMed Central

    Gravelat, Fabrice N.; Ejzykowicz, Daniele E.; Chiang, Lisa Y.; Chabot, Josée C.; Urb, Mirjam; Macdonald, K. Denyese; al-Bader, Nadia; Filler, Scott G.; Sheppard, Donald C.

    2010-01-01

    In medically important fungi, regulatory elements that control development and asexual reproduction often govern the expression of virulence traits. We therefore cloned the Aspergillus fumigatus developmental modifier MedA and characterized its role in conidiation, host cell interactions and virulence. As in the model organism Aspergillus nidulans, disruption of medA in A. fumigatus dramatically reduced conidiation. However, the conidiophore morphology was markedly different between the two species. Further, gene expression analysis suggested that MedA governs conidiation through different pathways in A. fumigatus compared to A. nidulans. The A. fumigatus ΔmedA strain was impaired in biofilm production and adherence to plastic, as well as adherence to pulmonary epithelial cells, endothelial cells and fibronectin in vitro. The ΔmedA strain also had reduced capacity to damage pulmonary epithelial cells, and stimulate pro-inflammatory cytokine mRNA and protein expression. Consistent with these results, the A. fumigatus ΔmedA strain also exhibited reduced virulence in both an invertebrate and a mammalian model of invasive aspergillosis. Collectively these results suggest that the downstream targets of A. fumigatus MedA mediate virulence, and may provide novel therapeutic targets for invasive aspergillosis. PMID:19889083

  2. The Rickettsia conorii Autotransporter Protein Sca1 Promotes Adherence to Nonphagocytic Mammalian Cells ▿ †

    PubMed Central

    Riley, Sean P.; Goh, Kenneth C.; Hermanas, Timothy M.; Cardwell, Marissa M.; Chan, Yvonne G. Y.; Martinez, Juan J.

    2010-01-01

    The pathogenesis of spotted fever group (SFG) Rickettsia species, including R. conorii and R. rickettsii, is acutely dependent on adherence to and invasion of host cells, including cells of the mammalian endothelial system. Bioinformatic analyses of several rickettsia genomes revealed the presence of a cohort of genes designated sca genes that are predicted to encode proteins with homology to autotransporter proteins of Gram-negative bacteria. Previous work demonstrated that three members of this family, rOmpA (Sca0), Sca2, and rOmpB (Sca5) are involved in the interaction with mammalian cells; however, very little was known about the function of other conserved rickettsial Sca proteins. Here we demonstrate that sca1, a gene present in nearly all SFG rickettsia genomes, is actively transcribed and expressed in R. conorii cells. Alignment of Sca1 sequences from geographically diverse SFG Rickettsia species showed that there are high degrees of sequence identity and conservation of these sequences, suggesting that Sca1 may have a conserved function. Using a heterologous expression system, we demonstrated that production of R. conorii Sca1 in the Escherichia coli outer membrane is sufficient to mediate attachment to but not invasion of a panel of cultured mammalian epithelial and endothelial cells. Furthermore, preincubation of a recombinant Sca1 peptide with host cells blocked R. conorii cell association. Together, these results demonstrate that attachment to mammalian cells can be uncoupled from the entry process and that Sca1 is involved in the adherence of R. conorii to host cells. PMID:20176791

  3. 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

  4. Trichomonas vaginalis lipophosphoglycan mutants have reduced adherence and cytotoxicity to human ectocervical cells.

    PubMed

    Bastida-Corcuera, Felix D; Okumura, Cheryl Y; Colocoussi, Angie; Johnson, Patricia J

    2005-11-01

    The extracellular human pathogen Trichomonas vaginalis is covered by a dense glycocalyx thought to play a role in host-parasite interactions. The main component of the glycocalyx is lipophosphoglycan (LPG), a polysaccharide anchored in the plasma membrane by inositol phosphoceramide. To study the role of LPG in trichomonads, we produced T. vaginalis LPG mutants by chemical mutagenesis and lectin selection and characterized them using morphological, biochemical, and functional assays. Two independently selected LPG mutants, with growth rates comparable to that of the wild-type (parent) strain, lost the ability to bind the lectins Ricinnus comunis agglutinin I (RCA120) and wheat germ agglutinin, indicating alterations in surface galactose and glucosamine residues. LPG isolated from mutants migrated faster than parent strain LPG on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting the mutants had shorter LPG molecules. Dionex high-performance anion exchange chromatography with pulsed amperometric detection analyses revealed galactosamine, glucosamine, galactose, glucose, mannose/xylose, and rhamnose as the main monosaccharides of T. vaginalis parent strain LPG. LPG from both mutants showed a reduction of galactose and glucosamine, corresponding with the reduced size of their LPG molecules and inability to bind the lectins RCA120 and wheat germ agglutinin. Mutant parasites were defective in attachment to plastic, a characteristic associated with avirulent strains of T. vaginalis. Moreover, the mutants were less adherent and less cytotoxic to human vaginal ectocervical cells in vitro than the parental strain. Finally, while parent strain LPG could inhibit the attachment of parent strain parasites to vaginal cells, LPG from either mutant could not inhibit attachment. These combined results demonstrate that T. vaginalis adherence to host cells is LPG mediated and that an altered LPG leads to reduced adherence and cytotoxicity of this parasite. PMID

  5. Trichomonas vaginalis Lipophosphoglycan Mutants Have Reduced Adherence and Cytotoxicity to Human Ectocervical Cells

    PubMed Central

    Bastida-Corcuera, Felix D.; Okumura, Cheryl Y.; Colocoussi, Angie; Johnson, Patricia J.

    2005-01-01

    The extracellular human pathogen Trichomonas vaginalis is covered by a dense glycocalyx thought to play a role in host-parasite interactions. The main component of the glycocalyx is lipophosphoglycan (LPG), a polysaccharide anchored in the plasma membrane by inositol phosphoceramide. To study the role of LPG in trichomonads, we produced T. vaginalis LPG mutants by chemical mutagenesis and lectin selection and characterized them using morphological, biochemical, and functional assays. Two independently selected LPG mutants, with growth rates comparable to that of the wild-type (parent) strain, lost the ability to bind the lectins Ricinnus comunis agglutinin I (RCA120) and wheat germ agglutinin, indicating alterations in surface galactose and glucosamine residues. LPG isolated from mutants migrated faster than parent strain LPG on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting the mutants had shorter LPG molecules. Dionex high-performance anion exchange chromatography with pulsed amperometric detection analyses revealed galactosamine, glucosamine, galactose, glucose, mannose/xylose, and rhamnose as the main monosaccharides of T. vaginalis parent strain LPG. LPG from both mutants showed a reduction of galactose and glucosamine, corresponding with the reduced size of their LPG molecules and inability to bind the lectins RCA120 and wheat germ agglutinin. Mutant parasites were defective in attachment to plastic, a characteristic associated with avirulent strains of T. vaginalis. Moreover, the mutants were less adherent and less cytotoxic to human vaginal ectocervical cells in vitro than the parental strain. Finally, while parent strain LPG could inhibit the attachment of parent strain parasites to vaginal cells, LPG from either mutant could not inhibit attachment. These combined results demonstrate that T. vaginalis adherence to host cells is LPG mediated and that an altered LPG leads to reduced adherence and cytotoxicity of this parasite. PMID

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

    PubMed Central

    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

  7. 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

  8. 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...

  9. 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. PMID:23012359

  10. 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. PMID:21797737

  11. Gain-of-Function Mutations in PDR1, a Regulator of Antifungal Drug Resistance in Candida glabrata, Control Adherence to Host Cells

    PubMed Central

    Vale-Silva, Luís; Ischer, Françoise; Leibundgut-Landmann, Salomé

    2013-01-01

    Candida glabrata is an emerging opportunistic pathogen that is known to develop resistance to azole drugs due to increased drug efflux. The mechanism consists of CgPDR1-mediated upregulation of ATP-binding cassette transporters. A range of gain-of-function (GOF) mutations in CgPDR1 have been found to lead not only to azole resistance but also to enhanced virulence. This implicates CgPDR1 in the regulation of the interaction of C. glabrata with the host. To identify specific CgPDR1-regulated steps of the host-pathogen interaction, we investigated in this work the interaction of selected CgPDR1 GOF mutants with murine bone marrow-derived macrophages and human acute monocytic leukemia cell line (THP-1)-derived macrophages, as well as different epithelial cell lines. GOF mutations in CgPDR1 did not influence survival and replication within macrophages following phagocytosis but led to decreased adherence to and uptake by macrophages. This may allow evasion from the host's innate cellular immune response. The interaction with epithelial cells revealed an opposite trend, suggesting that GOF mutations in CgPDR1 may favor epithelial colonization of the host by C. glabrata through increased adherence to epithelial cell layers. These data reveal that GOF mutations in CgPDR1 modulate the interaction with host cells in ways that may contribute to increased virulence. PMID:23460523

  12. The Atlantic Salmon MHC class II alpha and beta promoters are active in mammalian cell lines.

    PubMed

    Vestrheim, O; Lundin, M; Syed, M

    2007-01-01

    The major histocompatibility complex class II (MHCII) genes are only constitutively expressed in certain immune response cells such as B cells, macrophages, dendritic cells and other antigen presenting cells. This cell specific expression pattern and the presence of conserved regions such as the X-, X2-, Y-, and W-boxes make the MHCII promoters especially interesting as vector constructs. We tested whether the Atlantic salmon (Salmo salar L.) MHCII promoters can function in cell lines from other organisms. We found that the salmon MHCII alpha and MHCII beta promoters could drive expression of a LacZ reporter gene in adherent lymphoblast cell lines from dog (DH82) and rabbit (HybL-L). This paper shows that the promoters of Atlantic salmon MHCII alpha and beta genes can function in mammalian cell lines. PMID:17934904

  13. Characterization of the Murine Myeloid Precursor Cell Line MuMac-E8

    PubMed Central

    Fricke, Stephan; Riemschneider, Sina; Kohlschmidt, Janine; Hilger, Nadja; Fueldner, Christiane; Knauer, Jens; Sack, Ulrich; Emmrich, Frank; Lehmann, Jörg

    2014-01-01

    Starting point for the present work was the assumption that the cell line MuMac-E8 represents a murine cell population with stem cell properties. Preliminary studies already pointed to the expression of stem-cell associated markers and a self-regenerative potential of the cells. The cell line MuMac-E8 should be examined for their differential stage within stem cell hierarchy. MuMac-E8 cells were derived from a chimeric mouse model of arthritis. It could be shown that MuMac-E8 cells express mRNA of some genes associated with pluripotent stem cells (Nanog, Nucleostemin), of genes for hematopoietic markers (EPCR, Sca-1, CD11b, CD45), for the mesenchymal marker CD105 and of genes for the neural markers Pax-6 and Ezrin. In methylcellulose and May-Grünwald-Giemsa staining, hematopoietic colonies were obtained but the hematopoietic system of lethally irradiated mice could not be rescued. Osteogenic differentiation was not detectable. Thus, it became evident that MuMac-E8 represents not a stem cell line. However, MuMac-E8 cells expressed several myeloid surface markers (i.e. CD11b, F4/80, CD14, CD64), showed phagocytosis and is capable of producing nitric oxide. Thus, this cell line seems to be arrested an advanced stage of myeloid differentiation. Adherence data measured by impedance-based real-time cell analysis together with cell morphology data suggested that MuMac-E8 represents a new macrophage precursor cell line exhibiting weak adherence. This cell line is suitable as an in-vitro model for testing of macrophage functions. Moreover, it might be also useful for differentiation or reprogramming studies. PMID:25546418

  14. 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

  15. 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

  16. Cytotoxicity evaluation of silica nanoparticles using fish cell lines.

    PubMed

    Vo, Nguyen T K; Bufalino, Mary R; Hartlen, Kurtis D; Kitaev, Vladimir; Lee, Lucy E J

    2014-01-01

    Nanoparticles (NPs) have extensive industrial, biotechnological, and biomedical/pharmaceutical applications, leading to concerns over health risks to humans and biota. Among various types of nanoparticles, silica nanoparticles (SiO2 NPs) have become popular as nanostructuring, drug delivery, and optical imaging agents. SiO2 NPs are highly stable and could bioaccumulate in the environment. Although toxicity studies of SiO2 NPs to human and mammalian cells have been reported, their effects on aquatic biota, especially fish, have not been significantly studied. Twelve adherent fish cell lines derived from six species (rainbow trout, fathead minnow, zebrafish, goldfish, haddock, and American eel) were used to comparatively evaluate viability of cells by measuring metabolic impairment using Alamar Blue. Toxicity of SiO2 NPs appeared to be size-, time-, temperature-, and dose-dependent as well as tissue-specific. However, dosages greater than 100 μg/mL were needed to achieve 24 h EC50 values (effective concentrations needed to reduce cell viability by 50%). Smaller SiO2 NPs (16 nm) were relatively more toxic than larger sized ones (24 and 44 nm) and external lining epithelial tissue (skin, gills)-derived cells were more sensitive than cells derived from internal tissues (liver, brain, intestine, gonads) or embryos. Higher EC50 values were achieved when toxicity assessment was performed at higher incubation temperatures. These findings are in overall agreement with similar human and mouse cell studies reported to date. Thus, fish cell lines could be valuable for screening emerging contaminants in aquatic environments including NPs through rapid high-throughput cytotoxicity bioassays. PMID:24357037

  17. 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.

  18. Lithium attenuates lead induced toxicity on mouse non-adherent bone marrow cells.

    PubMed

    Banijamali, Mahsan; Rabbani-Chadegani, Azra; Shahhoseini, Maryam

    2016-07-01

    Lead is a poisonous heavy metal that occurs in all parts of environment and causes serious health problems in humans. The aim of the present study was to investigate the possible protective effect of lithium against lead nitrate induced toxicity in non-adherent bone marrow stem cells. Trypan blue and MTT assays represented that exposure of the cells to different concentrations of lead nitrate decreased viability in a dose dependent manner, whereas, pretreatment of the cells with lithium protected the cells against lead toxicity. Lead reduced the number and differentiation status of bone marrow-derived precursors when cultured in the presence of colony stimulating factor (CSF), while the effect was attenuated by lithium. The cells treated with lead nitrate exhibited cell shrinkage, DNA fragmentation, anion superoxide production, but lithium prevented lead action. Moreover, apoptotic indexes such as PARP cleavage and release of HMGB1 induced by lead, were protected by lithium, suggesting anti-apoptotic effect of lithium. Immunoblot analysis of histone H3K9 acetylation indicated that lithium overcame lead effect on acetylation. In conclusion, lithium efficiently reduces lead toxicity suggesting new insight into lithium action which may contribute to increased cell survival. It also provides a potentially new therapeutic strategy for lithium and a cost-effective approach to minimize destructive effects of lead on bone marrow stem cells. PMID:27259346

  19. 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.

  20. A Crosstalk Between K ras (Kirsten Rat Sarcoma Viral Oncogene Homologue) and Adherence Molecular Complex Leads to Disassociation of Cells-A Possible Contribution Towards Metastasis in Colorectal Cancer.

    PubMed

    Murtaza, Bibi Nazia; Doak, Shareen; Morgan, Claire; Nadeem, Muhammad Shahid; Al-Ghanim, Khalid A; Shakoori, Abdul Rauf

    2016-10-01

    Constitutive activation of mutant K ras (Kirsten rat sarcoma viral oncogene homologue) and disassembly of E-cadherin-catenin complex (E-cadherin, α-catenin, β-catenin, and γ-catenin) play an important role in apoptosis, differentiation, and cell proliferation. In this study, the expression pattern of K ras and E-cadherin-catenin complex has been evaluated in normal and mutant colorectal cancer cell lines with an object to determine its impact on disassociation of cells from one another. We addressed the expression analysis of K ras with reference to its association with adherence molecules in two colorectal cancer cell lines, that is, Caco-2 (wild type K ras served as a control) and DLD1 (heterozygous mutation at codon 13) at message level by qRT-PCR and translational level by western blotting. Compared to the control Caco-2 cell lines, the K ras in DLD1 cell lines showed slightly higher values while α-catenin showed a slight lower (1.3-folds), β-catenin and E-cadherin showed significantly lower expression (4.2-fold decrease). It can be inferred that a possible cross talk exists between K ras and adherent junction mediated signalling. Mutation at codon 13 (G to D) leads to the overexpression of K ras and reduced expression of adherent junction complex resulting in metastasis. J. Cell. Biochem. 117: 2340-2345, 2016. © 2016 Wiley Periodicals, Inc. PMID:26945839

  1. 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

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

    PubMed

    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

  3. 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

  4. Standards for Cell Line Authentication and Beyond

    PubMed Central

    Cole, Kenneth D.; Plant, Anne L.

    2016-01-01

    Different genomic technologies have been applied to cell line authentication, but only one method (short tandem repeat [STR] profiling) has been the subject of a comprehensive and definitive standard (ASN-0002). Here we discuss the power of this document and why standards such as this are so critical for establishing the consensus technical criteria and practices that can enable progress in the fields of research that use cell lines. We also examine other methods that could be used for authentication and discuss how a combination of methods could be used in a holistic fashion to assess various critical aspects of the quality of cell lines. PMID:27300367

  5. 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

  6. 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.

  7. Reduction of Escherichia coli adherence to uroepithelial bladder cells after consumption of cranberry juice: a double-blind randomized placebo-controlled cross-over trial.

    PubMed

    Di Martino, P; Agniel, R; David, K; Templer, C; Gaillard, J L; Denys, P; Botto, H

    2006-02-01

    To determine the efficacy of the consumption of cranberry juice versus placebo with regard to the presence of in vitro bacterial anti-adherence activity in the urine of healthy volunteers. Twenty healthy volunteers, 10 men and 10 women, were included. The study was a double-blind, randomized, placebo-controlled, and cross-over study. In addition to normal diet, each volunteer received at dinner a single dose of 750 ml of a total drink composed of: (1) 250 ml of the placebo and 500 ml of mineral water, or (2) 750 ml of the placebo, or (3) 250 ml of the cranberry juice and 500 ml of mineral water, or (4) 750 ml of the cranberry juice. Each volunteer took the four regimens successively in a randomly order, with a washout period of at least 6 days between every change in regimen. The first urine of the morning following cranberry or placebo consumption was collected and used to support bacterial growth. Six uropathogenic Escherichia coli strains (all expressing type 1 pili; three positive for the gene marker for P-fimbriae papC and three negative for papC), previously isolated from patients with symptomatic urinary tract infections, were grown in urine samples and tested for their ability to adhere to the T24 bladder cell line in vitro. There were no significant differences in the pH or specific gravity between the urine samples collected after cranberry or placebo consumption. We observed a dose dependent significant decrease in bacterial adherence associated with cranberry consumption. Adherence inhibition was observed independently from the presence of genes encoding type P pili and antibiotic resistance phenotypes. Cranberry juice consumption provides significant anti-adherence activity against different E. coli uropathogenic strains in the urine compared with placebo. PMID:16397814

  8. Curli modulates adherence of Escherichia coli O157 to bovine recto-anal junction squamous epithelial cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Our recent studies have shown that Intimin and the Locus of Enterocyte Effacement-encoded proteins do not play a role in Escherichia coli O157 (O157) adherence to the bovine recto-anal junction squamous epithelial cells (RSE) cells. Hence, to define factors that play a contributory role, we investi...

  9. The effects of disodium cromoglycate on enhanced adherence of Haemophilus influenzae to A549 cells infected with respiratory syncytial virus.

    PubMed

    Fukasawa, Chie; Ishiwada, Naruhiko; Ogita, Junko; Hishiki, Haruka; Kohno, Yoichi

    2009-08-01

    Nontypeable Haemophilus influenzae (NTHi) secondary infection often complicates respiratory syncytial virus (RSV) infections. Previous studies have revealed that RSV infections enhance NTHi adherence to airway epithelial cells. In this study, we investigated the effects of disodium cromoglycate (DSCG) and corticosteroids, which are frequently used for the treatment of wheezing often related to RSV infections, on the adherence of NTHi to RSV-infected A549 cells. DSCG inhibited enhanced adherence of NTHi to RSV-infected A549 cells, whereas dexamethasone (Dex) and fluticasone propionate (Fp) did not. DSCG suppressed the expression of ICAM-1, which is one of the NTHi receptors. Furthermore, DSCG exhibited an inhibitory effect on RSV infections. It is suggested that DSCG exerts an anti-RSV effect, and consequently attenuates the expression of NTHi receptors. PMID:19390482

  10. 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

  11. Role of the Amino-Terminal Region of Porphyromonas gingivalis Fimbriae in Adherence to Epithelial Cells

    PubMed Central

    Sojar, Hakimuddin T.; Han, Yiping; Hamada, Nobushiro; Sharma, Ashu; Genco, Robert J.

    1999-01-01

    Porphyromonas gingivalis fimbriae elicit many responses in eukaryotic cells, including mitogenicity, cytokine production, epithelial cell invasion, and cellular immune response. Specific domains of the major fimbrial protein (FimA) have been shown to be important in triggering some of these functions. The goal of the present study was to identify the domain(s) of P. gingivalis FimA responsible for specific interaction with human mucosal epithelial cells. Fimbriated P. gingivalis strains have been shown to bind to buccal epithelial cells, whereas nonfimbriated strains bind at low levels or not at all. This and other studies provide evidence that FimA mediates the adherence of P. gingivalis to oral epithelial cells. To determine the specific region(s) of P. gingivalis FimA involved in epithelial cell binding, specific antipeptide antibodies were used to inhibit the binding of iodinated purified fimbriae as well as the binding of P. gingivalis cells to epithelial cells. Antibodies directed against peptides 49 to 68 (VVMANTAGAMELVGKTLAEVK) and 69 to 90 (ALTTELTAENQEAAGLIMTAEP) were found to highly inhibit both the binding of fimbriae and the binding of P. gingivalis cells to epithelial cells. The antibody against FimA peptides 69 to 90 also reacted with P. gingivalis fimbriae in immunogold labeling and immunoblot analysis, thereby indicating that this peptide domain is exposed on the surface of fimbriae. Our results suggest that the amino-terminal domain corresponding to amino acid residues 49 to 90 of the fimbrillin protein is a major epithelial cell binding domain of P. gingivalis fimbriae. PMID:10531284

  12. Effects of pressure and temperature on the survival rate of adherent A-172 cells

    NASA Astrophysics Data System (ADS)

    Yasuhara, Ryo; Kushida, Ryo; Ishii, Shiwori; Yamanoha, Banri; Shimizu, Akio

    2013-06-01

    Preservation of cells under high pressure is an important alternative to cryopreservation. We studied the effect of temperature (4, 25, 37°C) and pressure (0.1-350 MPa) on the survival rate of A-172 glioblastoma cells. The survival rate was not changed by brief (10 min) pressurization of up to 150 MPa, but the survival rate began to decrease from 150 MPa, and most of the A-172 cells died when treated with over 200 MPa. Lengthy pressurization (4 days) at lower pressure (upto 20.1 MPa) without medium exchange showed complex results. The survival rate of cells preserved at 25°C showed two maxima at 1.6 and 20.1 MPa. After preservation, cells adhered and proliferated in the same way as normal cells when cultured at 37°C in a CO2 incubator. The other two temperatures, 4° and 37°C, showed no maximum survival rate. Therefore, a high survival rate can be maintained with high pressure treatment.

  13. The impact of environmental changes upon the microrheological response of adherent cells.

    PubMed

    Picard, C; Donald, A

    2009-10-01

    The mechanical behaviour of adherent cells cultured in vitro is known to be dependent on the mechanical properties of the substrate. We show that this mechanical behaviour is also strongly affected by the cells' environment. We focus here on the impact of temperature and pH. Experiments carried out on individual cells in a tuneable environment reveal that the intra-cellular mechanical behaviour exhibits large and fast changes when the external cell environment is changed. Fast passive microrheometry measurements allow for the precise characterisation of the transient regime observed during a temperature drop. When maintained at a non-physiological temperature, the cells reach a stabilised state distinct from the state observed in physiological conditions. The perturbation can be reversed but exhibits hysteretic behaviour when physiological conditions are restored. The transient regime observed during the recovery process is found to be different from the transient regime observed when leaving physiological conditions. A modified generalized Stokes-Einstein equation taking into account the cell activity through an effective temperature is proposed here to fit the experimental results. Excellent agreement between the model and the measurements is obtained for time lags from 10⁻³ to 1 s considered in this study. PMID:19551417

  14. 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

  15. Synergistic role of curli and cellulose in cell adherence and biofilm formation of attaching and effacing Escherichia coli and identification of Fis as a negative regulator of curli

    PubMed Central

    Saldaña, Zeus; Xicohtencatl-Cortes, Juan; Avelino, Fabiola; Phillips, Alan D.; Kaper, James B.; Puente, José L.; Girón, Jorge A.

    2009-01-01

    Summary Curli are adhesive fimbriae of Escherichia coli and Salmonella enterica. Expression of curli (csgA) and cellulose (bcsA) is co-activated by the transcriptional activator CsgD. In this study, we investigated the contribution of curli and cellulose to the adhesive properties of enterohemorragic (EHEC) O157:H7 and enteropathogenic E. coli (EPEC) O127:H6. While single mutations in csgA, csgD, or bcsA in EPEC and EHEC had no dramatic effect on cell adherence, double csgAbcsA mutants were significantly less adherent than the single mutants or wild-type strains to human colonic HT-29 epithelial cells or to cow colon tissue in vitro. Over-expression of csgD (carried on plasmid pCP994) in a csgD mutant, but not in the single csgA or bscA mutants, led to significant increase in adherence and biofilm formation in EPEC and EHEC, suggesting that synchronized over-production of curli and cellulose enhances bacterial adherence. In line with this finding, csgD transcription was activated significantly in the presence of cultured epithelial cells as compared to growth in tissue culture medium. Analysis of the influence of virulence and global regulators in the production of curli in EPEC identified Fis (factor for inversion stimulation) as a, heretofore unrecognized, negative transcriptional regulator of csgA expression. An EPEC E2348/69Δfis produced abundant amounts of curli whereas a double fiscsgD mutant yielded no detectable curli production. Our data suggest that curli and cellulose act in concert to favor host colonization, biofilm formation, and survival in different environments. PMID:19187284

  16. 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.

  17. Characterization of an ExoS Type III Translocation-Resistant Cell Line

    PubMed Central

    Rucks, Elizabeth A.; Olson, Joan C.

    2005-01-01

    Pseudomonas aeruginosa ExoS is a type III-secreted type III-secreted, bifunctional protein that causes diverse effects on eukaryotic cell function. The coculture of P. aeruginosa strains expressing ExoS with HL-60 myeloid cells revealed the cell line to be resistant to the toxic effects of ExoS. Differentiation of HL-60 cells with phorbol 12-myristate 13-acetate (TPA) rendered the cell line sensitive to ExoS. To understand the cellular basis for the alteration in sensitivity, undifferentiated and TPA-differentiated HL-60 cells were compared for differences in bacterial adherence, type III secretion induction, and ExoS translocation. These comparisons found that ExoS was translocated more efficiently in TPA-differentiated HL-60 cells than in undifferentiated cells. The studies support the ability of eukaryotic cells to influence P. aeruginosa TTS at the level of membrane translocation. PMID:15618208

  18. Cell-host, LINE and environment

    PubMed Central

    Del Re, Brunella; Giorgi, Gianfranco

    2013-01-01

    Long interspersed nuclear elements -1 (LINEs, L1s) are retroelements occupying almost 17% of the human genome. L1 retrotransposition can cause deleterious effects on the host-cell and it is generally inhibited by suppressive mechanisms, but it can occur in some specific cells during early development as well as in some tumor cells and in the presence of several environmental factors. In a recent publication we reported that extremely low frequency pulsed magnetic field can affect L1 retrotransposition in neuroblastoma cells. In this commentary we discuss the interaction between environment and L1 activity in the light of the new emerging paradigm of host-LINE relationship. PMID:23734298

  19. Lactoferrin affects the adherence and invasion of Streptococcus dysgalactiae ssp. dysgalactiae in mammary epithelial cells.

    PubMed

    O'Halloran, Fiona; Beecher, Christine; Chaurin, Valerie; Sweeney, Torres; Giblin, Linda

    2016-06-01

    Streptococcus dysgalactiae ssp. dysgalactiae is an important causative agent of bovine mastitis worldwide. Lactoferrin is an innate immune protein that is associated with many functions including immunomodulatory, antiproliferative, and antimicrobial properties. This study aimed to investigate the interactions between lactoferrin and a clinical bovine mastitis isolate, Strep. dysgalactiae ssp. dysgalactiae DPC5345. Initially a deliberate in vivo bovine intramammary challenge was performed with Strep. dysgalactiae DPC5345. Results demonstrated a significant difference in lactoferrin mRNA levels in milk cells between the control and infused quarters 7h postinfusion. Milk lactoferrin levels in the Strep. dysgalactiae DPC5345 infused quarters were significantly increased compared with control quarters at 48h postinfusion. In vitro studies demonstrated that lactoferrin had a bacteriostatic effect on the growth of Strep. dysgalactiae DPC5345 and significantly decreased the ability of the bacteria to internalize into HC-11 mammary epithelial cells. Confocal microscopy images of HC-11 cells exposed to Strep. dysgalactiae and lactoferrin further supported this effect by demonstrating reduced invasion of bacteria to HC-11 cells. The combined data suggest that a bovine immune response to Strep. dysgalactiae infection includes a significant increase in lactoferrin expression in vivo, and based on in vitro data, lactoferrin limits mammary cell invasion of this pathogen by binding to the bacteria and preventing its adherence. PMID:27016824

  20. Immune responses to an encapsulated allogeneic islet {beta}-cell line in diabetic NOD mice

    SciTech Connect

    Black, Sasha P. . E-mail: Sasha.Black@ca.crl.com; Constantinidis, Ioannis; Cui, Hong; Tucker-Burden, Carol; Weber, Collin J.; Safley, Susan A.

    2006-02-03

    Our goal is to develop effective islet grafts for treating type 1 diabetes. Since human islets are scarce, we evaluated the efficacy of a microencapsulated insulin-secreting conditionally transformed allogeneic {beta}-cell line ({beta}TC-tet) in non-obese diabetic mice treated with tetracycline to inhibit cell growth. Relatively low serum levels of tetracycline controlled proliferation of {beta}TC-tet cells without inhibiting effective control of hyperglycemia in recipients. There was no significant host cellular reaction to the allografts or host cell adherence to microcapsules, and host cytokine levels were similar to those of sham-operated controls. We conclude that encapsulated allogeneic {beta}-cell lines may be clinically relevant, because they effectively restore euglycemia and do not elicit a strong cellular immune response following transplantation. To our knowledge, this is First extensive characterization of the kinetics of host cellular and cytokine responses to an encapsulated islet cell line in an animal model of type 1 diabetes.

  1. 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

  2. Stress response of adherent cells on a polymer blend surface composed of a segmented polyurethane and MPC copolymers.

    PubMed

    Sawada, Shin-Ichi; Iwasaki, Yasuhiko; Nakabayashi, Nobuo; Ishihara, Kazuhiko

    2006-12-01

    To better understand the effect of 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymer in improving the biocompatibility of segmented polyurethane (SPU), the expression of heat shock protein (HSP) mRNA in HeLa S3 cells adhered on SPU blended with MPC copolymers was measured. Conventionally, MPC copolymers (PMEH) were synthesized by changing the feed ratios of MPC and 2-ethylhexyl methacrylate. X-ray photoelectron spectroscopic analysis of the SPU/PMEH film indicated that the surface concentration of MPC units on the SPU/PMEH film increased with an increase in PMEH composition. HeLa S3 cells were cultured on SPU/PMEH films. The number of adherent cells on the SPU/PMEH films decreased with an increase in the concentration of PMEH. When the PMEH composition was greater than 0.5 wt %, cell adhesion and proliferation decreased markedly. Expressions of HSP27 and HSP47 mRNA were detected using the reverse transcription-polymerase chain reaction (RT-PCR). After incubation for 24 h, both the HSP mRNA expressions in the HeLa S3 cells showed no significant differences among all samples. In HeLa S3 cells that adhered to the SPU film for 48 h, the expressions of HSP27 and HSP47 mRNA increased significantly when compared with those incubated for 24 h. In contrast, the two kinds of mRNA expressions decreased in the HeLa S3 cells that adhered to the SPU/PMEH films for 48 h. From these results, we concluded that PMEH was quite important in suppressing the stress response of adherent HeLa S3 cells. Therefore, SPU/PMEH blend polymers are useful as implantable biomedical materials. PMID:16758458

  3. Human placenta-derived adherent cells improve cardiac performance in mice with chronic heart failure.

    PubMed

    Chen, Hong-Jung; Chen, Chien-Hsi; Chang, Ming-Yao; Tsai, Da-Ching; Baum, Ellen Z; Hariri, Robert; Herzberg, Uri; Hsieh, Patrick C H

    2015-03-01

    Human placenta-derived adherent cells (PDACs) are a culture-expanded, undifferentiated mesenchymal-like population derived from full-term placental tissue, with immunomodulatory, anti-inflammatory, angiogenic, and neuroprotective properties. PDA-001 (cenplacel-L), an intravenous formulation of PDAC cells, is in clinical development for the treatment of autoimmune and inflammatory diseases. We tested the therapeutic effects of PDA-001 in mice with chronic heart failure (CHF). Three weeks after transaortic constriction surgery to induce CHF, the mice underwent direct intramyocardial (IM) or i.v. injection of PDA-001 at a high (0.5 × 10(6) cells per mouse), medium (0.5 × 10(5) cells per mouse), or low (0.5 × 10(4) cells per mouse) dose. The mice were sacrificed 4 weeks after treatment. Echocardiography and ventricular catheterization showed that IM injection of PDA-001 significantly improved left ventricular systolic and diastolic function compared with injection of vehicle or i.v. injection of PDA-001. IM injection of PDA-001 also decreased cardiac fibrosis, shown by trichrome staining in the vicinity of the injection sites. Low-dose treatment showed the best improvement in cardiac performance compared with the medium- and high-dose groups. In another independent study to determine the mechanism of action with bromodeoxyuridine labeling, the proliferation rates of endothelial cells and cardiomyocytes were significantly increased by low or medium IM dose PDA-001. However, no surviving PDA-001 cells were detected in the heart 1 month after injection. In vivo real-time imaging consistently revealed that the PDA-001 cells were detectable only within 2 days after IM injection of luciferase-expressing PDA-001. Together, these results have demonstrated the cardiac therapeutic potential of PDA-001, likely through a paracrine effect. PMID:25673767

  4. Human Cardiac-Derived Adherent Proliferating Cells Reduce Murine Acute Coxsackievirus B3-Induced Myocarditis

    PubMed Central

    Miteva, Kapka; Haag, Marion; Peng, Jun; Savvatis, Kostas; Becher, Peter Moritz; Seifert, Martina; Warstat, Katrin; Westermann, Dirk; Ringe, Jochen; Sittinger, Michael; Schultheiss, Heinz-Peter

    2011-01-01

    Background Under conventional heart failure therapy, inflammatory cardiomyopathy typically has a progressive course, indicating a need for alternative therapeutic strategies to improve long-term outcomes. We recently isolated and identified novel cardiac-derived cells from human cardiac biopsies: cardiac-derived adherent proliferating cells (CAPs). They have similarities with mesenchymal stromal cells, which are known for their anti-apoptotic and immunomodulatory properties. We explored whether CAPs application could be a novel strategy to improve acute Coxsackievirus B3 (CVB3)-induced myocarditis. Methodology/Principal Findings To evaluate the safety of our approach, we first analyzed the expression of the coxsackie- and adenovirus receptor (CAR) and the co-receptor CD55 on CAPs, which are both required for effective CVB3 infectivity. We could demonstrate that CAPs only minimally express both receptors, which translates to minimal CVB3 copy numbers, and without viral particle release after CVB3 infection. Co-culture of CAPs with CVB3-infected HL-1 cardiomyocytes resulted in a reduction of CVB3-induced HL-1 apoptosis and viral progeny release. In addition, CAPs reduced CD4 and CD8 T cell proliferation. All CAPs-mediated protective effects were nitric oxide- and interleukin-10-dependent and required interferon-γ. In an acute murine model of CVB3-induced myocarditis, application of CAPs led to a decrease of cardiac apoptosis, cardiac CVB3 viral load and improved left ventricular contractility parameters. This was associated with a decline in cardiac mononuclear cell activity, an increase in T regulatory cells and T cell apoptosis, and an increase in left ventricular interleukin-10 and interferon-γ mRNA expression. Conclusions We conclude that CAPs are a unique type of cardiac-derived cells and promising tools to improve acute CVB3-induced myocarditis. PMID:22174827

  5. 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

  6. CsrRS and environmental pH regulate group B streptococcus adherence to human epithelial cells and extracellular matrix.

    PubMed

    Park, Su Eun; Jiang, Shengmei; Wessels, Michael R

    2012-11-01

    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

  7. 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

  8. Difference in Membrane Repair Capacity Between Cancer Cell Lines and a Normal Cell Line.

    PubMed

    Frandsen, Stine Krog; McNeil, Anna K; Novak, Ivana; McNeil, Paul L; Gehl, Julie

    2016-08-01

    Electroporation-based treatments and other therapies that permeabilize the plasma membrane have been shown to be more devastating to malignant cells than to normal cells. In this study, we asked if a difference in repair capacity could explain this observed difference in sensitivity. Membrane repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique, providing a sensitive index of repair capacity. The normal primary cell line of all tested cell lines exhibited the slowest rate of dye entry after laser disruption and lowest level of dye uptake. Significantly, more rapid dye uptake and a higher total level of dye uptake occurred in six of the seven tested cancer cell lines (p < 0.05) as well as the immortalized cell line (p < 0.001). This difference in sensitivity was also observed when a viability assay was performed one day after plasma membrane permeabilization by electroporation. Viability in the primary normal cell line (98 % viable cells) was higher than in the three tested cancer cell lines (81-88 % viable cells). These data suggest more effective membrane repair in normal, primary cells and supplement previous explanations why electroporation-based therapies and other therapies permeabilizing the plasma membrane are more effective on malignant cells compared to normal cells in cancer treatment. PMID:27312328

  9. Enrichment and characterization of cancer stem cells from a human non-small cell lung cancer cell line.

    PubMed

    Zhao, Changhong; Setrerrahmane, Sarra; Xu, Hanmei

    2015-10-01

    Tumor cells from the same origin comprise different cell populations. Among them, cancer stem cells (CSCs) have higher tumorigenicity. It is necessary to enrich CSCs to determine an effective way to suppress and eliminate them. In the present study, using the non-adhesive culture system, tumor spheres were successfully generated from human A549 non-small cell lung cancer (NSCLC) cell line within 2 weeks. Compared to A549 adherent cells, sphere cells had a higher self-renewal ability and increased resistance to cytotoxic drugs. Sphere cells were more invasive and expressed stem cell markers including octamer‑binding transcription factor 4 (Oct4) and sex-determining region Y-box 2 (Sox2) at high levels. CD133, a disputed marker of lung CSCs, was also upregulated. Tumor sphere cells showed higher tumorigenic ability in vivo, indicating that more CSCs were enriched in the sphere cells. More blood vessels were formed in the tumor generated by sphere cells suggesting the interaction between CSCs and blood vessel. A reliable model of enriching CSCs from the human A549 NSCLC cell line was established that was simple and cost-effective compared to other methods. PMID:26239272

  10. Hematopoietic Cancer Cell Lines Can Support Replication of Sabin Poliovirus Type 1

    PubMed Central

    van Eikenhorst, Gerco; de Gruijl, Tanja D.; van der Pol, Leo A.; Bakker, Wilfried A. M.

    2015-01-01

    Viral vaccines can be produced in adherent or in suspension cells. The objective of this work was to screen human suspension cell lines for the capacity to support viral replication. As the first step, it was investigated whether poliovirus can replicate in such cell lines. Sabin poliovirus type 1 was serially passaged on five human cell lines, HL60, K562, KG1, THP-1, and U937. Sabin type 1 was capable of efficiently replicating in three cell lines (K562, KG1, and U937), yielding high viral titers after replication. Expression of CD155, the poliovirus receptor, did not explain susceptibility to replication, since all cell lines expressed CD155. Furthermore, we showed that passaged virus replicated more efficiently than parental virus in KG1 cells, yielding higher virus titers in the supernatant early after infection. Infection of cell lines at an MOI of 0.01 resulted in high viral titers in the supernatant at day 4. Infection of K562 with passaged Sabin type 1 in a bioreactor system yielded high viral titers in the supernatant. Altogether, these data suggest that K562, KG1, and U937 cell lines are useful for propagation of poliovirus. PMID:25815312

  11. Using Propensity to Succeed Modeling to Increase Utilization and Adherence in a Nurse HealthLine Telephone Triage Program.

    PubMed

    Navratil-Strawn, Jessica L; Hawkins, Kevin; Hartley, Stephen K; Wells, Timothy S; Ozminkowski, Ronald J; Migliori, Richard J; Yeh, Charlotte S

    2016-01-01

    Propensity to succeed modeling was used to identify characteristics associated with higher utilization of a telephone triage program and adherence to nurse recommendations among callers. Characteristics significantly associated with calling the telephone triage service and engaging in triage services were being female and having an elevated health risk score. Callers most likely to adhere to nurse recommendations were younger than 85 years of age, had called on a weekday, and had received a recommendation to seek care at an emergency department or a doctor's office visit. Additional analyses suggest the propensity to succeed modeling is stable and valid. PMID:27232680

  12. Prevalence of Escherichia coli strains with localized, diffuse, and aggregative adherence to HeLa cells in infants with diarrhea and matched controls.

    PubMed Central

    Gomes, T A; Blake, P A; Trabulsi, L R

    1989-01-01

    To determine the possible role of Escherichia coli strains with three different patterns of adherence to HeLa cells in causing diarrhea in infants in São Paulo, Brazil, we studied stool specimens from 100 infants up to 1 year of age with acute diarrheal illnesses and 100 age-matched control infants without recent diarrhea. E. coli with localized adherence to HeLa cells was much more common in patients (23%) than in controls (2%) (P less than 0.0001) and was detected more frequently than rotavirus (19%) was in patients, even though the study was conducted during the coldest months of the year. Most (80%) of the E. coli colonies with localized adherence were of traditional enteropathogenic E. coli serotypes. Little difference was found between patients and controls in the rate of isolation of E. coli with diffuse adherence (31 and 32%, respectively) or aggregative adherence (10 and 8%, respectively). A genetic probe used to detect a plasmid-mediated adhesin which confers expression of localized adherence proved to be 100% sensitive and 99.9% specific in detecting E. coli with localized adherence to HeLa cells. Although E. coli strains with localized adherence have now been shown to be enteric pathogens in several parts of the world, the role of strains showing diffuse adherence and aggregative adherence is still uncertain. PMID:2563383

  13. Characterization of Binding of Candida albicans to Small Intestinal Mucin and Its Role in Adherence to Mucosal Epithelial Cells

    PubMed Central

    de Repentigny, Louis; Aumont, Francine; Bernard, Karine; Belhumeur, Pierre

    2000-01-01

    In order to approximate and adhere to mucosal epithelial cells, Candida must traverse the overlying mucus layer. Interactions of Candida species with mucin and human buccal epithelial cells (BECs) were thus investigated in vitro. Binding of the Candida species to purified small intestinal mucin showed a close correlation with their hierarchy of virulence. Significant differences (P < 0.05) were found among three categories of Candida species adhering highly (C. dubliniensis, C. tropicalis, and C. albicans), moderately (C. parapsilosis and C. lusitaniae) or weakly (C. krusei and C. glabrata) to mucin. Adherence of C. albicans to BECs was quantitatively inhibited by graded concentrations of mucin. However, inhibition of adherence was reversed by pretreatment of mucin with pronase or C. albicans secretory aspartyl proteinase Sap2p but not with sodium periodate. Saturable concentration- and time-dependent binding of mucin to C. albicans was abrogated by pronase or Sap2p treatment of mucin but was unaffected by β-mercaptoethanol, sodium periodate, neuraminidase, lectins, or potentially inhibitory sugars. Probing of membrane blots of the mucin with C. albicans revealed binding of the yeast to the 66-kDa cleavage product of the 118-kDa C-terminal glycopeptide of mucin. Although no evidence was found for the participation of C. albicans cell surface mannoproteins in specific receptor-ligand binding to mucin, inhibition of binding by p-nitrophenol (1 mM) and tetramethylurea (0.36 M) revealed that hydrophobic interactions are involved in adherence of C. albicans to mucin. These results suggest that C. albicans may both adhere to and enzymatically degrade mucins by the action of Saps, and that both properties may act to modulate Candida populations in the oral cavity and gastrointestinal tract. PMID:10816460

  14. 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...

  15. Adherence of Candida albicans to human buccal epithelial cells: host-induced protein synthesis and signaling events.

    PubMed Central

    Bailey, A; Wadsworth, E; Calderone, R

    1995-01-01

    The synthesis of proteins by Candida albicans was studied following adherence of blastoconidia to human buccal epithelial cells (HBEC). Initially, labeling of HBEC, C. albicans, and HBEC-C. albicans with [35S]methionine was performed. After a 3-h incubation and prior to labeling with [35S]methionine, the cultures were treated with cycloheximide to prevent HBEC protein synthesis. The HBEC-C. albicans mixture as well as C. albicans and HBEC incubated separately were extracted with beta-mercaptoethanol (beta-ME). These extracts as well as the cell residue (solubilized by boiling with sodium dodecyl sulfate [SDS]) were examined by SDS-polyacrylamide gel electrophoresis and autoradiography. In comparison to cultures of C. albicans incubated without HBEC, proteins with molecular masses of approximately 52 to 56 kDa from beta-ME extracts and from SDS-solubilized cells were observed only from adhering cultures. In addition, unlabeled beta-ME extracts were electrotransferred to nitrocellulose and immunoblotted with antiphosphotyrosine antibodies to determine whether cell signaling events were occurring during adherence. Proteins with molecular masses of 54 and 60 kDa were recognized only in mixed cultures of C. albicans and HBEC. These data indicate that following adherence of C. albicans to HBEC, new Candida proteins are expressed. Further, these events are accompanied by the expression of signal proteins, presumably of Candida origin. PMID:7822023

  16. Automatic segmentation and quantification of fluorescing microspheres adhering to capillary endothelial cells in the rat lung

    NASA Astrophysics Data System (ADS)

    Albert, Thomas A.; Fingar, Victor H.; Taber, Scott W.; Wieman, Thomas J.

    1995-05-01

    Adhesion molecules present in the cellular membrane of the endothelium provide sites of leukocyte adherence as a first step in the process of leukocyte migration into the interstitium. New evidence suggests the same adhesion proteins may be responsible for the spread of metastatic tumors by providing a location for tumor cell attachment. A method was sought to quantitate the degree of adhesion molecule expression in the pulmonary capillary endothelium using a recently developed animal model which allows for viewing the lung surface in vivo. Videoimages of the pulmonary vascular system were gathered using this new lung chamber technique. A fully automated digital image processing and analysis (DIPA) system was also developed to estimate the level of intercellular adhesion molecule-1 (ICAM-1) expression on the capillary endothelial cells in these videoimages. Fluorescent microspheres were immunologically bound to the ICAM-1 molecules present on the endothelial cell surface. The DIPA system then located and quantified the fluorescent spots present in the videoimages. The ability of this system to locate and measure the fluorescence was compared with human measurements of the same images.

  17. The cell-cell junctions of mammalian testes: I. The adhering junctions of the seminiferous epithelium represent special differentiation structures.

    PubMed

    Domke, Lisa M; Rickelt, Steffen; Dörflinger, Yvette; Kuhn, Caecilia; Winter-Simanowski, Stefanie; Zimbelmann, Ralf; Rosin-Arbesfeld, Rina; Heid, Hans; Franke, Werner W

    2014-09-01

    The seminiferous tubules and the excurrent ducts of the mammalian testis are physiologically separated from the mesenchymal tissues and the blood and lymph system by a special structural barrier to paracellular translocations of molecules and particles: the "blood-testis barrier", formed by junctions connecting Sertoli cells with each other and with spermatogonial cells. In combined biochemical as well as light and electron microscopical studies we systematically determine the molecules located in the adhering junctions of adult mammalian (human, bovine, porcine, murine, i.e., rat and mouse) testis. We show that the seminiferous epithelium does not contain desmosomes, or "desmosome-like" junctions, nor any of the desmosome-specific marker molecules and that the adhering junctions of tubules and ductules are fundamentally different. While the ductules contain classical epithelial cell layers with E-cadherin-based adherens junctions (AJs) and typical desmosomes, the Sertoli cells of the tubules lack desmosomes and "desmosome-like" junctions but are connected by morphologically different forms of AJs. These junctions are based on N-cadherin anchored in cytoplasmic plaques, which in some subforms appear thick and dense but in other subforms contain only scarce and loosely arranged plaque structures formed by α- and β-catenin, proteins p120, p0071 and plakoglobin, together with a member of the striatin family and also, in rodents, the proteins ZO-1 and myozap. These N-cadherin-based AJs also include two novel types of junctions: the "areae adhaerentes", i.e., variously-sized, often very large cell-cell contacts and small sieve-plate-like AJs perforated by cytoplasm-to-cytoplasm channels of 5-7 nm internal diameter ("cribelliform junctions"). We emphasize the unique character of this epithelium that totally lacks major epithelial marker molecules and structures such as keratin filaments and desmosomal elements as well as EpCAM- and PERP-containing junctions. We also

  18. Refractory lining for electrochemical cell

    DOEpatents

    Blander, Milton; Cook, Glenn M.

    1987-01-01

    Apparatus for processing a metallic fluid containing iron oxide, container for a molten metal including an electrically conductive refractory disposed for contact with the molten metal which contains iron oxide, an electrolyte in the form of a basic slag on top of the molten metal, an electrode in the container in contcat with the slag electrically separated from the refractory, and means for establishing a voltage across the refractory and the electrode to reduce iron oxide to iron at the surface of the refractory in contact with the iron oxide containing fluid. A process is disclosed for refining an iron product containing not more than about 10% by weight oxygen and not more than about 10% by weight sulfur, comprising providing an electrolyte of a slag containing one or more of calcium oxide, magnesium oxide, silica or alumina, providing a cathode of the iron product in contact with the electrolyte, providing an anode in contact with the electrolyte electrically separated from the cathode, and operating an electrochemical cell formed by the anode, the cathode and the electrolyte to separate oxygen or sulfur present in the iron product therefrom.

  19. Platelet activating factor amplifies human neutrophil adherence to bovine endothelial cells: evidence for a lipoxygenase dependent mechanism.

    PubMed

    Damtew, B; Spagnuolo, P J

    1992-10-01

    Platelet activating factor (PAF) is a potent lipid mediator that induces the release of leukotrienes and prostaglandins from various cells and tissues. We examined the capacity of PAF alone and in combination with soluble stimuli to enhance eicosanoid synthesis and adherence of human neutrophils. Neutrophils were preincubated with PAF and washed before exposure to the soluble stimuli F-Met-Leu-Phe (FMLP), calcium ionophore A23187, and phorbol myristate acetate. Preincubation of neutrophils with 1 microM PAF enhanced the release of both LTB4 and LTC4 in response to each of the three agonists, in contrast with the unprimed neutrophils. Priming was specific for PAF since lyso-PAF was inactive. Priming concentrations of PAF also augmented the adherence of neutrophils to endothelium in the presence of the soluble agonists A23187, phorbol myristate acetate, and FMLP. The priming effect of PAF on eicosanoid release and neutrophil adherence was shown to have similar time- and dose-dependent effects. Further, the priming effects of PAF on adherence could be reversed by preincubation of neutrophils with the lipoxygenase inhibitors nordihydroguiaretic acid and 5,8,11,14-ETYA but not by preincubation with the cyclooxygenase inhibitor indomethacin. These data demonstrate that PAF amplifies neutrophil adherence to endothelium through a lipoxygenase dependent mechanism. PMID:1330924

  20. Role of M3 protein in the adherence and internalization of an invasive Streptococcus pyogenes strain by epithelial cells.

    PubMed

    Eyal, Osnat; Jadoun, Jeries; Bitler, Arcady; Skutelski, Ehud; Sela, Shlomo

    2003-10-15

    Streptococcus pyogenes utilizes multiple mechanisms for adherence to and internalization by epithelial cells. One of the molecules suggested of being involved in adherence and internalization is the M protein. Although strains of the M3 serotype form the second largest group isolated from patients with severe invasive diseases and fatal infections, not much information is known regarding the interactions of M3 protein with mammalian cells. In this study we have constructed an emm3 mutant of an invasive M3 serotype (SP268), and demonstrated that the M3 protein is involved in both adherence to and internalization by HEp-2 cells. Fibronectin promoted both adherence and internalization of SP268 in an M3-independent pathway. Utilizing speB and speB/emm3 double mutants, it was found that M3 protein is not essential for the maturation of SpeB, as was reported for the M1 protein. Increased internalization efficiency observed in both the speB and emm3/speB mutants suggested that inhibition of S. pyogenes internalization by SpeB is not related to the presence of an intact M3 protein. Thus, other proteins in SP268, which serve as targets for SpeB activity, have a prominent role in the internalization process. PMID:14522456

  1. 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

  2. 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

  3. Umbelliprenin Induces Apoptosis in CLL Cell Lines

    PubMed Central

    Ziai, Seyed Ali; Gholami, Omid; Iranshahi, Mehrdad; Zamani, Amir Hassan; Jeddi-Tehrani, Mahmood

    2012-01-01

    Chronic lymphocytic leukemia (CLL) remains an incurable disease that requires innovative new approaches to improve therapeutic outcome. Many Ferula species, including F. asa-foetida, synthesize terpenyloxy coumarins. One of these coumarins is umbelliprenin, which has been implicated with induction of apoptosis in some cancer cell lines. In this study induction of apoptosis by umbelliprenin on Jurkat T-CLL and Raji B-CLL cell lines was studied. In this regard, cells were incubated with various concentrations of umbelliprenin in-vitro for different times and assayed for apoptosis with annexin V–FITC/PI double staining flowcytometry method. Results showed that umbelliprenin induced apoptosis in leukemic cells in a dose- and time-dependent manner and that CLL cells were more susceptible to umbelliprenin induced cell death than normal peripheral blood mononuclear cell (PBMCs). Moreover, we study the induction of apoptosis in Jurkat cells by umbelliprenin in the presence of interleukin 4 (IL-4) as an agent that causes resistance to apoptosis in CLL cells, was also student. We showed that IL-4 can not reduce apoptotic effect of umbelliprenin. The preferential toxicity of umbelliprenin for CLL cells, supports the hypothesis that oral administration of umbelliprenin in the form of foods or folk medicines containing this coumarin, might enhance protection against the development of CLL in man with little side effects. In conclusion, umbelliprenin may be an effective therapeutic agent in the treatment of CLL, and thus clinical studies with umbelliprenin may be appropriate. PMID:24250490

  4. 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...

  5. Fibronectin-binding protein of Streptococcus pyogenes: sequence of the binding domain involved in adherence of streptococci to epithelial cells.

    PubMed Central

    Talay, S R; Valentin-Weigand, P; Jerlström, P G; Timmis, K N; Chhatwal, G S

    1992-01-01

    The sequence of the fibronectin-binding domain of the fibronectin-binding protein of Streptococcus pyogenes (Sfb protein) was determined, and its role in streptococcal adherence was investigated by use of an Sfb fusion protein in adherence studies. A 1-kb DNA fragment coding for the binding domain of Sfb protein was cloned into the expression vector pEX31 to produce an Sfb fusion protein consisting of the N-terminal part of MS2 polymerase and a C-terminal fragment of the streptococcal protein. Induction of the vector promoter resulted in hyperexpression of fibronectin-binding fusion protein in the cytoplasm of the recombinant Escherichia coli cells. Sequence determination of the cloned 1-kb fragment revealed an in-frame reading frame for a 268-amino-acid peptide composed of a 37-amino-acid sequence which is completely repeated three times and incompletely repeated a fourth time. Cloning of one repeat into pEX31 resulted in expression of small fusion peptides that show fibronectin-binding activity, indicating that one repeat contains at least one binding domain. Each repeat exhibits two charged domains and shows high homology with the 38-amino-acid D3 repeat of the fibronectin-binding protein of Staphylococcus aureus. Sequence comparison with other streptococcal ligand-binding surface proteins, including M protein, failed to reveal significant homology, which suggests that Sfb protein represents a novel type of functional protein in S. pyogenes. The Sfb fusion protein isolated from the cytoplasm of recombinant cells was purified by fast protein liquid chromatography. It showed a strong competitive inhibition of fibronectin binding to S. pyogenes and of the adherence of bacteria to cultured epithelial cells. In contrast, purified streptococcal lipoteichoic acid showed only a weak inhibition of fibronectin binding and streptococcal adherence. These results demonstrate that Sfb protein is directly involved in the fibronectin-mediated adherence of S. pyogenes to

  6. Expression and function of a putative cell surface receptor for fibronectin in hamster and human cell lines

    SciTech Connect

    Brown, P.J.; Juliano, R.L.

    1986-10-01

    The authors have previously reported the use of monoclonal antibodies to identify a 140-kD cell surface glycoprotein in mammalian cells that is specifically involved in fibronectin-mediated cell adhesion. They now report the purification of this molecule using immunoaffinity chromatography and the subsequent generation of polyclonal antibodies that selectively immunoprecipitate 140-kD putative fibronectin receptor glycoprotein (gp140) extracted from rodent or human cells; these antibodies also specifically block fibronectin-mediated cell adhesion but not adhesion mediated by other factors in serum. Expression of gp140-like molecules was detected on the surfaces of several adherent human cell lines (HDF, WISH, and EFC) but not on erythrocytes; however, gp140 was also detected on a nonadherent human lymphoid line (DAUDI). Analysis of gp140 on nonreducing SDS gels revealed two closely migrating bands. Protease digestion and peptide mapping suggests that the two bnads are closely related polypeptides.

  7. Effects of teicoplanin on cell number of cultured cell lines

    PubMed Central

    Kashkolinejad-Koohi, Tahere; Saadat, Iraj

    2015-01-01

    Teicoplanin is a glycopeptide antibiotic with a wide variation in human serum half-life. It is also a valuable alternative of vancomycin. There is however no study on its effect on cultured cells. The aim of the present study was to test the effect of teicoplanin on cultured cell lines CHO, Jurkat E6.1 and MCF-7. The cultured cells were exposed to teicoplanin at final concentrations of 0–11000 μg/ml for 24 hours. To determine cell viability, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was performed. At low concentrations of teicoplanin the numbers of cultured cells (due to cell proliferation) were increased in the three cell lines examined. The maximum cell proliferation rates were observed at concentrations of 1000, 400, and 200 μg/ml of teicoplanin for CHO, MCF-7 and Jurkat cell lines, respectively. Cell toxicity was observed at final concentrations over 2000, 6000, and 400 μg/ml of teicoplanin for CHO, MCF-7 and Jurkat cell lines, respectively. A dose-dependent manner of cell toxicity was observed. Our present findings indicated that teicoplanin at clinically used concentrations induced cell proliferation. It should therefore be used cautiously, particularly in children, pregnant women and patients with cancer.

  8. Development of a cell line from the American eel brain expressing endothelial cell properties.

    PubMed

    Bloch, Sophia R; Vo, Nguyen T K; Walsh, Sarah K; Chen, Cici; Lee, Lucy E J; Hodson, Peter V; Bols, Niels C

    2016-04-01

    A cell line (eelB) was developed from the outgrowth of adherent cells from brain explants of the American eel, Anguilla rostrata (Lesueur). EelB cells have been grown routinely in L-15 with 10% fetal bovine serum (FBS), undergone over 100 passages, and cryopreserved successfully. The cells from late-passage cultures (>45) were polygonal, formed capillary-like structures (CLS) on Matrigel, and stained immunocytochemically for von Willebrand factor (vWF) and for three tight junction proteins, zonula occludens-1 (ZO-1), claudin 3, and claudin 5. These results suggest that eelB is an endothelial cell line, one of the few from fish and the first from the brain. Despite this, eelB did not respond to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with the induction of CYP1A protein. The cells from early-passage cultures (<20) had more varied shapes and did not form CLS on Matrigel. Only cells from early-passage cultures formed in suspension three-dimensional aggregates that had some cells expressing alkaline phosphatase and nestin. These cells are thought to be neural stem cells and the aggregates neurospheres. The emergence of endothelial-like cells upon the continued subcultivation of cells from early-passage cultures that had neural stem cells has been described previously for mammals, but this is a first for teleosts. Remarkably, cells from all passage levels were stained strongly for senescence-associated β-galactosidase (SA β-Gal) activity. PMID:26714751

  9. Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line

    PubMed Central

    Sluch, Valentin M.; Davis, Chung-ha O.; Ranganathan, Vinod; Kerr, Justin M.; Krick, Kellin; Martin, Russ; Berlinicke, Cynthia A.; Marsh-Armstrong, Nicholas; Diamond, Jeffrey S.; Mao, Hai-Quan; Zack, Donald J.

    2015-01-01

    Retinal ganglion cell (RGC) injury and cell death from glaucoma and other forms of optic nerve disease is a major cause of irreversible vision loss and blindness. Human pluripotent stem cell (hPSC)-derived RGCs could provide a source of cells for the development of novel therapeutic molecules as well as for potential cell-based therapies. In addition, such cells could provide insights into human RGC development, gene regulation, and neuronal biology. Here, we report a simple, adherent cell culture protocol for differentiation of hPSCs to RGCs using a CRISPR-engineered RGC fluorescent reporter stem cell line. Fluorescence-activated cell sorting of the differentiated cultures yields a highly purified population of cells that express a range of RGC-enriched markers and exhibit morphological and physiological properties typical of RGCs. Additionally, we demonstrate that aligned nanofiber matrices can be used to guide the axonal outgrowth of hPSC-derived RGCs for in vitro optic nerve-like modeling. Lastly, using this protocol we identified forskolin as a potent promoter of RGC differentiation. PMID:26563826

  10. Characterization of functional mannose receptor in a continuous hybridoma cell line

    PubMed Central

    2012-01-01

    Background The mannose receptor is the best described member of the type I transmembrane C-type lectins; however much remains unanswered about the biology of the receptor. One difficulty has been the inability to consistently express high levels of a functional full length mannose receptor cDNA in mammalian cells. Another difficulty has been the lack of a human macrophage cell line expressing a fully functional receptor. Commonly used human macrophage cell lines such as U937, THP-1, Mono-Mac and HL60 do not express the mannose receptor. We have developed a macrophage hybridoma cell line (43MR cells) created by fusion of U937 cells with primary human monocyte-derived macrophages, resulting in a non-adherent cell line expressing several properties of primary macrophages. The purpose of this study was to identify and select mannose receptor-expressing cells using fluorescence-activated cell sorting and to characterize the expression and function of the receptor. Results In the current study we show that the mannose receptor found on this novel cell has endocytic characteristics consistent with and similar to the mannose receptor found on the surface of monocyte-derived human macrophages and rat bone marrow-derived macrophages. In addition, we demonstrate that these cells engage and internalize pathogen particles such as S. aureus and C. albicans. We further establish the transfectability of these cells via the introduction of a plasmid expressing influenza A hemagglutinin. Conclusions The 43MR cell line represents the first naturally expressed MR-positive cell line derived from a human macrophage background. This cell line provides an important cell model for other researchers for the study of human MR biology and host-pathogen interactions. PMID:22967244

  11. In Vitro Analysis of Breast Cancer Cell Line Tumourspheres and Primary Human Breast Epithelia Mammospheres Demonstrates Inter- and Intrasphere Heterogeneity

    PubMed Central

    Vargas, Ana Cristina; Keith, Patricia; Reid, Lynne; Wockner, Leesa; Amiri, Marjan Askarian; Sarkar, Debina; Simpson, Peter T.; Clarke, Catherine; Schmidt, Chris W.; Reynolds, Brent A.

    2013-01-01

    Mammosphere and breast tumoursphere culture have gained popularity as in vitro assays for propagating and analysing normal and cancer stem cells. Whether the spheres derived from different sources or parent cultures themselves are indeed single entities enriched in stem/progenitor cells compared to other culture formats has not been fully determined. We surveyed sphere-forming capacity across 26 breast cell lines, immunophenotyped spheres from six luminal- and basal-like lines by immunohistochemistry and flow cytometry and compared clonogenicity between sphere, adherent and matrigel culture formats using in vitro functional assays. Analyses revealed morphological and molecular intra- and inter-sphere heterogeneity, consistent with adherent parental cell line phenotypes. Flow cytometry showed sphere culture does not universally enrich for markers previously associated with stem cell phenotypes, although we found some cell-line specific changes between sphere and adherent formats. Sphere-forming efficiency was significantly lower than adherent or matrigel clonogenicity and constant over serial passage. Surprisingly, self-renewal capacity of sphere-derived cells was similar/lower than other culture formats. We observed significant correlation between long-term-proliferating-cell symmetric division rates in sphere and adherent cultures, suggesting functional overlap between the compartments sustaining them. Experiments with normal primary human mammary epithelia, including sorted luminal (MUC1+) and basal/myoepithelial (CD10+) cells revealed distinct luminal-like, basal-like and mesenchymal entities amongst primary mammospheres. Morphological and colony-forming-cell assay data suggested mammosphere culture may enrich for a luminal progenitor phenotype, or induce reversion/relaxation of the basal/mesenchymal in vitro selection occurring with adherent culture. Overall, cell line tumourspheres and primary mammospheres are not homogenous entities enriched for stem cells

  12. Butyrate modulates bacterial adherence on LS174T human colorectal cells by stimulating mucin secretion and MAPK signaling pathway

    PubMed Central

    Jung, Tae-Hwan; Park, Jeong Hyeon; Han, Kyoung-Sik

    2015-01-01

    BACKGROUND/OBJECTIVES Fermentation of dietary fiber results in production of various short chain fatty acids in the colon. In particular, butyrate is reported to regulate the physical and functional integrity of the normal colonic mucosa by altering mucin gene expression or the number of goblet cells. The objective of this study was to investigate whether butyrate modulates mucin secretion in LS174T human colorectal cells, thereby influencing the adhesion of probiotics such as Lactobacillus and Bifidobacterium strains and subsequently inhibiting pathogenic bacteria such as E. coli. In addition, possible signaling pathways involved in mucin gene regulation induced by butyrate treatment were also investigated. MATERIALS/METHODS Mucin protein content assay and periodic acid-Schiff (PAS) staining were performed in LS174T cells treated with butyrate at various concentrations. Effects of butyrate on the ability of probiotics to adhere to LS174T cells and their competition with E. coli strains were examined. Real time polymerase chain reaction for mucin gene expression and Taqman array 96-well fast plate-based pathway analysis were performed on butyrate-treated LS174T cells. RESULTS Treatment with butyrate resulted in a dose-dependent increase in mucin protein contents in LS174T cells with peak effects at 6 or 9 mM, which was further confirmed by PAS staining. Increase in mucin protein contents resulted in elevated adherence of probiotics, which subsequently reduced the adherent ability of E. coli. Treatment with butyrate also increased transcriptional levels of MUC3, MUC4, and MUC12, which was accompanied by higher gene expressions of signaling kinases and transcription factors involved in mitogen-activated protein kinase (MAPK) signaling pathways. CONCLUSIONS Based on our results, butyrate is an effective regulator of modulation of mucin protein production at the transcriptional and translational levels, resulting in changes in the adherence of gut microflora. Butyrate

  13. Breast cancer cell lines: friend or foe?

    PubMed Central

    Burdall, Sarah E; Hanby, Andrew M; Lansdown, Mark RJ; Speirs, Valerie

    2003-01-01

    The majority of breast cancer research is conducted using established breast cancer cell lines as in vitro models. An alternative is to use cultures established from primary breast tumours. Here, we discuss the pros and cons of using both of these models in translational breast cancer research. PMID:12631387

  14. TRANSFECTION OF INSECT CELL LINES USING POLYETHYLENIMINE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Insect cell lines have been widely used in recombinant baculovirus expression systems and transient gene expression studies. Critical to these applications have been the transfection of foreign DNA. This has been widely done using labor intensive and cytotoxic liposome-based transfection reagents....

  15. Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines

    SciTech Connect

    Felthaus, O.; Ettl, T.; Gosau, M.; Driemel, O.; Brockhoff, G.; Reck, A.; Zeitler, K.; Hautmann, M.; Reichert, T.E.; Schmalz, G.; Morsczeck, C.

    2011-04-01

    Research highlights: {yields} Four oral squamous cancer cell lines (OSCCL) were analyzed for cancer stem cells (CSCs). {yields} Single cell derived colonies of OSCCL express CSC-marker CD133 differentially. {yields} Monoclonal cell lines showed reduced sensitivity for Paclitaxel. {yields} In situ CD133{sup +} cells are slow cycling (Ki67-) indicating a reduced drug sensitivity. {yields} CD133{sup +} and CSC-like cells can be obtained from single colony forming cells of OSCCL. -- Abstract: Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simple method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133{sup +} cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.

  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. Plasmids in Yersinia enterocolitica serotypes O:3 and O:9: correlation with epithelial cell adherence in vitro.

    PubMed Central

    Vesikari, T; Nurmi, T; Mäki, M; Skurnik, M; Sundqvist, C; Granfors, K; Grönroos, P

    1981-01-01

    Human isolates of Yersinia enterocolitica serotypes O:3 (biotype 4) and O:9 (biotype 3) harbored plasmids sized approximately 47 and 44 megadaltons, respectively. No such plasmids were found in "apathogenic" strains of Y. enterocolitica belonging to biotype 1. There was a positive correlation among the presence of plasmid, autoagglutination, and adherence to and toxicity for HEp-2 cell cultures; all of these properties were lost by culturing at 37 degrees C in the absence of calcium. Strains of Y. enterocolitica O:3 and O:9 cured of the plasmids showed increased invasiveness in the HEp-2 cell culture model, but no invasiveness in guinea pig eye. It is suggested that the plasmids of Y. enterocolitica primarily determine epithelial cell adherence, but may also be associated with other pathogenic properties. Images PMID:7287174

  19. 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...

  20. Characterization of small spheres derived from various solid tumor cell lines: are they suitable targets for T cells?

    PubMed

    Busse, Antonia; Letsch, Anne; Fusi, Alberto; Nonnenmacher, Anika; Stather, David; Ochsenreither, Sebastian; Regenbrecht, Christian R A; Keilholz, Ulrich

    2013-08-01

    T cell based immunotherapy has been investigated in a variety of malignancies and analyses have been mostly founded on in vitro data with tumor cell monolayers. However, three-dimensional (3D) culture models might mimic more closely the 'in vivo' conditions than 2D monolayers. Therefore, we analyzed the expression of tumor-associated antigens (TAA) and of molecules involved in antigen processing and presentation (APM) in tumor spheres, which served as an in vitro model for micrometastasis which might be enriched in tumor propagating cancer stem cells. For enrichment of sphere cells 12 human solid tumor cell lines were cultured in serum-free medium. Expression of a variety of TAA and APM were analyzed by RT-PCR and/or flow cytometry and compared to expression in corresponding adherent bulk cells grown in regular growth medium. Compared to adherent cells, spheres showed equal or higher mRNA expression levels of LMP2, LMP7 and MECL-1, of TAP1 and TAP2 transporters and, surprisingly, also of TAA including differentiation antigens. However, downregulation or loss of HLA-I and HLA-II molecules in spheres was observed in 8 of 10 and 1 of 2 cell lines, respectively, and was unresponsive to stimulation with IFN-γ. Although tumor spheres express TAA and molecules of intracellular antigen processing, they are defective in antigen presentation due to downregulation of HLA surface expression which may lead to immune evasion. PMID:23519726

  1. Antiproliferative efficacy of Tabernaemontana divaricata against HEP2 cell line and Vero cell line

    PubMed Central

    Kumar, Arvind; Selvakumar, S.

    2015-01-01

    Background: Laryngeal cancer may also be called cancer of the larynx or laryngeal carcinoma. Conventional plants are a precious source of novel anticancer agents and are still in performance better role in health concern. The study was intended to estimation of the anticancer activity of the chloroformic extract of Tabernaemontana divaricata on the human epidermoid larynx carcinoma cell line (Hep 2). Materials and Method: The aerial parts (leaves, stem, and flowers) of T. divaricata were tested for its inhibitory effect in 96 microplate formats against Hep 2 cell line. The anticancer activity of samples on Hep 2 and Vero was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and various enzymatic parameters like catalase, reduced glutathione (GSH), GSH peroxidase, and superoxide anion scavenging activity. Viable cells were determined by the absorbance at 540 nm. Measurements were performed, and the concentration required for a 50% inhibition of viability (IC50) was determined graphically. The effect of the samples on the proliferation of Hep 2 and Vero cells was expressed as the % cell viability. Results: The extract on Hep 2 cell line up to 7.8 μg/ml and that IC50 value on Hep 2 cell line was 112 μg whereas 94 μg for Vero cell line. Hence, T. divaricata has lesser significant action on Vero cell line. Conclusion: Medicinal plant drug discovery continues to provide new and important leads against various pharmacological targets including cancer. Our results clearly indicate the anticancer property of the medicinal plant T. divaricata against the human laryngeal carcinoma cell lines (Hep 2 cell line). PMID:26109773

  2. Characterization of swine testicular cell line as immature porcine Sertoli cell line.

    PubMed

    Ma, Changping; Song, Huibin; Guan, Kaifeng; Zhou, Jiawei; Xia, Xuanyan; Li, Fenge

    2016-04-01

    Swine testicular (ST) cell line is isolated from swine fetal testes and has been widely used in biomedical research fields related to pig virus infection. However, the potential benefit and utilization of ST cells in boar reproductive studies has not been fully explored. As swine fetal testes mainly contain multiple types of cells such as Leydig cells, Sertoli cells, gonocytes, and peritubular myoid cells, it is necessary to clarify the cell type of ST cell line. In this study, we identified ST cell line was a collection of Sertoli cells by analyzing the unique morphological characteristic with satellite karyosomes and determining the protein expression of two markers (androgen-binding protein, ABP; Fas ligand, FASL) of Sertoli cells. Then ST cells were further confirmed to be immature Sertoli cells by examining the expression of three markers (anti-Mullerian hormone, AMH; keratin 18, KRT18; follicle-stimulating hormone receptor, FSHR). In conclusion, ST cells are a collection of immature Sertoli cells which can be good experimental materials for the researches involved in Sertoli cell functions and maturation, or even in boar reproductions. PMID:26744029

  3. 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

  4. Effect of eicosapentaenoic acid on the proliferation and incidence of apoptosis in the colorectal cell line HT29.

    PubMed

    Clarke, R G; Lund, E K; Latham, P; Pinder, A C; Johnson, I T

    1999-12-01

    Fish oil has been shown to reduce the induction of colorectal cancer in animal models by a mechanism which may involve suppression of mitosis, increased apoptosis, or both. We used the human colonic adenocarcinoma cell line HT29 to explore the effects of the long-chain n-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) on cell proliferation and death in vitro. Cells were cultured in media containing EPA at 5, 10, and 15 microg/mL. Cell number and thymidine incorporation were used to quantify proliferation, and cell cycle effects were studied using flow cytometry. Gel electrophoresis, annexin-V binding, and morphological criteria were used to characterize apoptosis. Adherent cells and freely floating detached cells were treated as two distinct populations. In the presence of EPA at 10 and 15 microg/mL there was a marked reduction in the growth rate of adherent HT29 colonies, owing to an increased detachment of adherent cells. After treatment with 10 or 15 microg/mL EPA the proportion of adherent cells in S-phase increased, indicating either a block in late S-phase or early G2. Floating cells showed evidence of extensive DNA cleavage, but the proportion of floating cells with sub GO DNA content declined on treatment with 10 or 15 microg/mL EPA even though the number of floating cells increased. We conclude that EPA does not inhibit mitosis of adherent cells, but increases the rate at which they become detached from the substrate, probably at an early stage in the initiation of apoptosis. This mechanism may be analogous to "anoikis," or induction of apoptosis in response to loss of cell contact, and may contribute to the anticarcinogenic effects of fish oil in vivo. PMID:10652988

  5. Cloning of a genetic determinant from Clostridium difficile involved in adherence to tissue culture cells and mucus.

    PubMed Central

    Karjalainen, T; Barc, M C; Collignon, A; Trollé, S; Boureau, H; Cotte-Laffitte, J; Bourlioux, P

    1994-01-01

    Our laboratory has previously shown that Clostridium difficile adherence to Caco-2 cells is greatly enhanced after heat shock at 60 degrees C and that it is mediated by a proteinaceous surface component. The experiments described here show that C. difficile could adhere to several types of tissue culture cells (Vero, HeLa, and KB) after heat shock. The type of culture medium (liquid or solid, with or without blood) had little effect on adhesion. To clone the adhesin gene, polyclonal antibodies against C. difficile heated at 60 degrees C were used to screen a genomic library of C. difficile constructed in lambda ZapII. Ten positive clones were identified in the library, one of which (pCL6) agglutinated several types of erythrocytes in the presence of mannose. In Western blots (immunoblots), this clone expressed in Escherichia coli a 40- and a 27-kDa protein; a 27-kDa protein has been previously identified in the surface extracts of heat-shocked C. difficile as a possible adhesin. The clone adhered to Vero, Caco-2, KB, and HeLa cells; the adherence was blocked by anti-C. difficile antibodies, by a surface extract of C. difficile, and by mucus isolated from axenic mice. Furthermore, the clone could attach ex vivo to intestinal mucus isolated from axenic mice. Preliminary studies on the receptor moieties implicated in C. difficile adhesion revealed that glucose and galactose could partially block adhesion to tissue culture cells, as did di- or trisaccharides containing these sugars, suggesting that the adhesin is a lectin. In addition, N-acetylgalactosamine, a component of mucus, and gelatin partially impeded cell attachment. Images PMID:7927694

  6. 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

  7. Characterization of Three-Dimensional Retinal Tissue Derived from Human Embryonic Stem Cells in Adherent Monolayer Cultures.

    PubMed

    Singh, Ratnesh K; Mallela, Ramya K; Cornuet, Pamela K; Reifler, Aaron N; Chervenak, Andrew P; West, Michael D; Wong, Kwoon Y; Nasonkin, Igor O

    2015-12-01

    Stem cell-based therapy of retinal degenerative conditions is a promising modality to treat blindness, but requires new strategies to improve the number of functionally integrating cells. Grafting semidifferentiated retinal tissue rather than progenitors allows preservation of tissue structure and connectivity in retinal grafts, mandatory for vision restoration. Using human embryonic stem cells (hESCs), we derived retinal tissue growing in adherent conditions consisting of conjoined neural retina and retinal pigment epithelial (RPE) cells and evaluated cell fate determination and maturation in this tissue. We found that deriving such tissue in adherent conditions robustly induces all eye field genes (RX, PAX6, LHX2, SIX3, SIX6) and produces four layers of pure populations of retinal cells: RPE (expressing NHERF1, EZRIN, RPE65, DCT, TYR, TYRP, MITF, PMEL), early photoreceptors (PRs) (coexpressing CRX and RCVRN), inner nuclear layer neurons (expressing CALB2), and retinal ganglion cells [RGCs, expressing BRN3B and Neurofilament (NF) 200]. Furthermore, we found that retinal progenitors divide at the apical side of the hESC-derived retinal tissue (next to the RPE layer) and then migrate toward the basal side, similar to that found during embryonic retinogenesis. We detected synaptogenesis in hESC-derived retinal tissue, and found neurons containing many synaptophysin-positive boutons within the RGC and PR layers. We also observed long NF200-positive axons projected by RGCs toward the apical side. Whole-cell recordings demonstrated that putative amacrine and/or ganglion cells exhibited electrophysiological responses reminiscent of those in normal retinal neurons. These responses included voltage-gated Na(+) and K(+) currents, depolarization-induced spiking, and responses to neurotransmitter receptor agonists. Differentiation in adherent conditions allows generation of long and flexible pieces of 3D retinal tissue suitable for isolating transplantable slices of tissue

  8. A human gallbladder adenocarcinoma cell line.

    PubMed

    Johzaki, H; Iwasaki, H; Nishida, T; Isayama, T; Kikuchi, M

    1989-12-01

    A cell strain (FU-GBC-1) was established from cancerous ascites of a 68-year-old male patient with well-differentiated adenocarcinoma of the gallbladder. By light and electron microscopy, the cultured cells showed the morphologic features of adenocarcinoma characterized by gland-like structures, intracellular microcystic spaces, and mucous production. Immunoperoxidase stains showed that FU-GBC-1 cells expressed several epithelial tumor antigens including CA 19-9, carcinoembryonic antigen (CEA), and epithelial membrane antigen (EMA). The cell strain has been in continuous culture up to passage 44 for 1 1/2 years, with the population doubling time of 120 hours. The cytogenetic analysis by a G-band technique showed a constant loss of chromosome Y in FU-GBC-1 cells. The modal chromosome number at passage 12 was 82 with a range of 77 to 85. Flow cytometry with an ethidium bromide technique additionally confirmed aneuploid DNA content (4C) in the cultured cells at passage 12 and 35. Inoculation of FU-GBC-1 cells into the dermis of BALB/c nude mice produced transplantable adenocarcinoma identical to the original tumor. Because no continuous cell lines of the well-differentiated type of gallbladder adenocarcinoma have been reported in the literature currently, the newly established cell strain we report may yield a useful system for studying the morphologic and biologic characteristics of gallbladder adenocarcinoma. PMID:2680052

  9. Isolation of a protein-containing cell surface component from Streptococcus sanguis which affects its adherence to saliva-coated hydroxyapatite.

    PubMed Central

    Liljemark, W F; Bloomquist, C G

    1981-01-01

    The isolation and partial characterization of a protein-containing cell surface component from Streptococcus sanguis which blocks the adherence of this microbe to saliva-coated hydroxyapatite are described. Several methods of extraction were attempted. Sonication of whole cells and cell walls proved to be the most successful and yielded biologically active adherence-blocking components. The adherence-blocking ability of these components was effective in intraspecies blocking experiments. The extract obtained from cell walls of S. sanguis was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and shown to contain one major and two to three minor bands when stained with Coomassie blue. The molecular weight of the major band was estimated to be 70,000 to 90,000. Gel filtration of the sonified cell wall extract on 10% agarose yielded two active adherence-blocking peaks, the void volume and a second peak. Images PMID:6273317

  10. Development, characterization, and optimization of a new suspension chicken-induced pluripotent stem cell line for the production of Newcastle disease vaccine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Traditionally, substrates for production of vaccines have been embryonated eggs or adherent cell culture. The daunting challenge of scaling up these technologies in the face of an outbreak has been a limitation for industrial applicability. Suspension cell lines are better suited in many ways to e...

  11. 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

  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. Hydrodynamic Determinants of Cell Necrosis and Molecular Delivery Produced by Pulsed Laser Microbeam Irradiation of Adherent Cells

    PubMed Central

    Compton, Jonathan L.; Hellman, Amy N.; Venugopalan, Vasan

    2013-01-01

    Time-resolved imaging, fluorescence microscopy, and hydrodynamic modeling were used to examine cell lysis and molecular delivery produced by picosecond and nanosecond pulsed laser microbeam irradiation in adherent cell cultures. Pulsed laser microbeam radiation at λ = 532 nm was delivered to confluent monolayers of PtK2 cells via a 40×, 0.8 NA microscope objective. Using laser microbeam pulse durations of 180–1100 ps and pulse energies of 0.5–10.5 μJ, we examined the resulting plasma formation and cavitation bubble dynamics that lead to laser-induced cell lysis, necrosis, and molecular delivery. The cavitation bubble dynamics are imaged at times of 0.5 ns to 50 μs after the pulsed laser microbeam irradiation, and fluorescence assays assess the resulting cell viability and molecular delivery of 3 kDa dextran molecules. Reductions in both the threshold laser microbeam pulse energy for plasma formation and the cavitation bubble energy are observed with decreasing pulse duration. These energy reductions provide for increased precision of laser-based cellular manipulation including cell lysis, cell necrosis, and molecular delivery. Hydrodynamic analysis reveals critical values for the shear-stress impulse generated by the cavitation bubble dynamics governs the location and spatial extent of cell necrosis and molecular delivery independent of pulse duration and pulse energy. Specifically, cellular exposure to a shear-stress impulse J≳0.1 Pa s ensures cell lysis or necrosis, whereas exposures in the range of 0.035≲J≲0.1 Pa s preserve cell viability while also enabling molecular delivery of 3 kDa dextran. Exposure to shear-stress impulses of J≲0.035 Pa s leaves the cells unaffected. Hydrodynamic analysis of these data, combined with data from studies of 6 ns microbeam irradiation, demonstrates the primacy of shear-stress impulse in determining cellular outcome resulting from pulsed laser microbeam irradiation spanning a nearly two

  15. Hydrodynamic determinants of cell necrosis and molecular delivery produced by pulsed laser microbeam irradiation of adherent cells.

    PubMed

    Compton, Jonathan L; Hellman, Amy N; Venugopalan, Vasan

    2013-11-01

    Time-resolved imaging, fluorescence microscopy, and hydrodynamic modeling were used to examine cell lysis and molecular delivery produced by picosecond and nanosecond pulsed laser microbeam irradiation in adherent cell cultures. Pulsed laser microbeam radiation at λ = 532 nm was delivered to confluent monolayers of PtK2 cells via a 40×, 0.8 NA microscope objective. Using laser microbeam pulse durations of 180-1100 ps and pulse energies of 0.5-10.5 μJ, we examined the resulting plasma formation and cavitation bubble dynamics that lead to laser-induced cell lysis, necrosis, and molecular delivery. The cavitation bubble dynamics are imaged at times of 0.5 ns to 50 μs after the pulsed laser microbeam irradiation, and fluorescence assays assess the resulting cell viability and molecular delivery of 3 kDa dextran molecules. Reductions in both the threshold laser microbeam pulse energy for plasma formation and the cavitation bubble energy are observed with decreasing pulse duration. These energy reductions provide for increased precision of laser-based cellular manipulation including cell lysis, cell necrosis, and molecular delivery. Hydrodynamic analysis reveals critical values for the shear-stress impulse generated by the cavitation bubble dynamics governs the location and spatial extent of cell necrosis and molecular delivery independent of pulse duration and pulse energy. Specifically, cellular exposure to a shear-stress impulse J≳0.1 Pa s ensures cell lysis or necrosis, whereas exposures in the range of 0.035≲J≲0.1 Pa s preserve cell viability while also enabling molecular delivery of 3 kDa dextran. Exposure to shear-stress impulses of J≲0.035 Pa s leaves the cells unaffected. Hydrodynamic analysis of these data, combined with data from studies of 6 ns microbeam irradiation, demonstrates the primacy of shear-stress impulse in determining cellular outcome resulting from pulsed laser microbeam irradiation spanning a nearly two-orders-of-magnitude range of

  16. 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

  17. Serum Proteins Enhance Dispersion Stability and Influence the Cytotoxicity and Dosimetry of ZnO Nanoparticles in Suspension and Adherent Cancer Cell Models.

    PubMed

    Anders, Catherine B; Chess, Jordan J; Wingett, Denise G; Punnoose, Alex

    2015-12-01

    (LNCaP) cancer cell lines, respectively. Presence of FBS in the NP dispersions also increased the reactive oxygen species generation. These observations indicate that the improved dispersion stability leads to increased NP bioavailability for suspension cell models and reduced NP sedimentation onto adherent cell layers resulting in more accurate in vitro toxicity assessments. PMID:26577392

  18. 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

    (LNCaP) cancer cell lines, respectively. Presence of FBS in the NP dispersions also increased the reactive oxygen species generation. These observations indicate that the improved dispersion stability leads to increased NP bioavailability for suspension cell models and reduced NP sedimentation onto adherent cell layers resulting in more accurate in vitro toxicity assessments.

  19. 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.

  20. 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

  1. Mechanism of Adherence of Streptococcus mutans to Smooth Surfaces I. Roles of Insoluble Dextran-Levan Synthetase Enzymes and Cell Wall Polysaccharide Antigen in Plaque Formation

    PubMed Central

    Mukasa, Hidehiko; Slade, Hutton D.

    1973-01-01

    The mechanism of adherence of Streptococcus mutans to smooth glass surfaces has been studied. The results with both viable and heat-killed cells showed that the process required (i) the synthesis of a water-insoluble dextran-levan polymer by cell-bound enzymes and (ii) the participation of a binding site on the surface of the S. mutans cell. Synthesis of the polymer from sucrose in the presence of the cells was required for adherence, and indicates that an “active” form of the polymer was required. Polymer synthesized by cell-free S. mutans enzymes when added to S. mutans cells did not produce adherence. Purified antibody globulin, specific for the a-d site in the polysaccharide S. mutans group a antigen, completely inhibited adherence. Antibody to the second antigen present in the polysaccharide molecule, the a antigen, did not inhibit adherence. The evidence indicates that adherence did not require an antigenic binding site which might be common to all S. mutans strains. The orientation of the synthetase enzyme(s), antigenic binding site, and dextran-levan polymer on the cell surface is under study. Images PMID:4582634

  2. Role of flagella in adherence, internalization, and translocation of Campylobacter jejuni in nonpolarized and polarized epithelial cell cultures.

    PubMed Central

    Grant, C C; Konkel, M E; Cieplak, W; Tompkins, L S

    1993-01-01

    Previous studies of Campylobacter jejuni have suggested that flagellin is an adhesin for epithelial cells and that motility is a virulence factor of this bacterium. The role of flagella in the interactions of C. jejuni with nonpolarized and polarized epithelial cells was examined with flagellar mutants. Flagellated, nonmotile (flaA flaB+ Mot-) and nonflagellated, nonmotile (flaA flaB Mot-) mutants of C. jejuni were constructed by in vivo homologous recombination and gene replacement techniques. Both classes of mutants were found to adhere to cells of human epithelial origin (INT 407) equally well; however, on the basis of the percentage of the inoculum internalized, internalization of the flaA flaB Mot- mutants was decreased by factors ranging from approximately 30 to 40 compared with the parent. The flaA flaB+ Mot- mutant was internalized by the INT 407 cells at levels six- to sevenfold higher than the flaA flaB Mot- mutants. Both classes of mutants, unlike the parent, were unable to translocate across polarized Caco-2 monolayers. These results indicate that flagella are not involved in C. jejuni adherence to epithelial cells but that they do play a role in internalization. Furthermore, the results suggest that either the motility of C. jejuni or the product of flaA is essential for the bacterium to cross polarized epithelial cell monolayers. Images PMID:8478066

  3. A Clark-type oxygen chip for in situ estimation of the respiratory activity of adhering cells.

    PubMed

    Wu, Ching-Chou; Luk, Hsiang-Ning; Lin, Yen-Ting Tsai; Yuan, Chia-Yin

    2010-04-15

    A Clark-type oxygen chip consisting of a polydimethylsiloxane (PDMS) reservoir containing an amino group-modified PDMS oxygen-permeable membrane (OPM) and a glass substrate containing a three-electrode detector has been constructed by using microfabrication techniques, and it is utilized for in situ measurement of the respiration activity of adhering cells. Use of the alginate sol electrolyte and the electroplating Ag/AgCl pseudo-reference electrode can effectively diminish the crosstalk between the electrochemical electrodes and supply a stable potential for the detection of dissolved oxygen, respectively. The Clark-type oxygen chips possess only 1.00% residual current, response time of 13.4s and good linearity with a correlation coefficient of 0.9933. The modification of amino groups for the OPM obviously facilitates the adhesion of HeLa cells onto the PDMS OPM surface and allows the cells to spread after 2h of incubation. The oxygen consumption of the cells in the cell-adhesion process increases with the adhesion time, and the increment of cellular oxygen consumption per minute reaches a maximum after 30 min of incubation. Moreover, the change in the respiration activity of adhering HeLa cells stimulated by the high concentration of glucose or propofol anaesthetic can be monitored in real time with the Clark-type oxygen chip. PMID:20188913

  4. Display of Cell Surface Sites for Fibronectin Assembly Is Modulated by Cell Adherence to 1F3 and C-Terminal Modules of Fibronectin

    PubMed Central

    Zhang, Qinghong; Annis, Douglas S.; Erickson, Harold P.; Mosher, Deane F.

    2009-01-01

    Background Fibronectin-null cells assemble soluble fibronectin shortly after adherence to a substrate coated with intact fibronectin but not when adherent to the cell-binding domain of fibronectin (modules 7F3-10F3). Interactions of adherent cells with regions of adsorbed fibronectin other than modules 7F3-10F3, therefore, are required for early display of the cell surface sites that initiate and direct fibronectin assembly. Methodology/Principal Findings To identify these regions, coatings of proteolytically derived or recombinant pieces of fibronectin containing modules in addition to 7F3-10F3 were tested for effects on fibronectin assembly by adherent fibronectin-null fibroblasts. Pieces as large as one comprising modules 2F3-14F3, which include the heparin-binding and cell adhesion domains, were not effective in supporting fibronectin assembly. Addition of module 1F3 or the C-terminal modules to modules 2F3-14F3 resulted in some activity, and addition of both 1F3 and the C-terminal modules resulted in a construct, 1F3-C, that best mimicked the activity of a coating of intact fibronectin. Constructs 1F3-C V0, 1F3-C V64, and 1F3-C Δ(V15F310F1) were all able to support fibronectin assembly, suggesting that 1F3 through 11F1 and/or 12F1 were important for activity. Coatings in which the active parts of 1F3-C were present in different proteins were much less active than intact 1F3-C. Conclusions These results suggest that 1F3 acts together with C-terminal modules to induce display of fibronectin assembly sites on adherent cells. PMID:19119318

  5. Establishment and Biological Characterization of a Panel of Glioblastoma Multiforme (GBM) and GBM Variant Oncosphere Cell Lines

    PubMed Central

    Binder, Zev A.; Wilson, Kelli M.; Salmasi, Vafi; Orr, Brent A.; Eberhart, Charles G.; Siu, I-Mei; Lim, Michael; Weingart, Jon D.; Quinones-Hinojosa, Alfredo; Bettegowda, Chetan; Kassam, Amin B.; Olivi, Alessandro; Brem, Henry; Riggins, Gregory J.; Gallia, Gary L.

    2016-01-01

    Objective Human tumor cell lines form the basis of the majority of present day laboratory cancer research. These models are vital to studying the molecular biology of tumors and preclinical testing of new therapies. When compared to traditional adherent cell lines, suspension cell lines recapitulate the genetic profiles and histologic features of glioblastoma multiforme (GBM) with higher fidelity. Using a modified neural stem cell culture technique, here we report the characterization of GBM cell lines including GBM variants. Methods Tumor tissue samples were obtained intra-operatively and cultured in neural stem cell conditions containing growth factors. Tumor lines were characterized in vitro using differentiation assays followed by immunostaining for lineage-specific markers. In vivo tumor formation was assayed by orthotopic injection in nude mice. Genetic uniqueness was confirmed via short tandem repeat (STR) DNA profiling. Results Thirteen oncosphere lines derived from GBM and GBM variants, including a GBM with PNET features and a GBM with oligodendroglioma component, were established. All unique lines showed distinct genetic profiles by STR profiling. The lines assayed demonstrated a range of in vitro growth rates. Multipotency was confirmed using in vitro differentiation. Tumor formation demonstrated histologic features consistent with high grade gliomas, including invasion, necrosis, abnormal vascularization, and high mitotic rate. Xenografts derived from the GBM variants maintained histopathological features of the primary tumors. Conclusions We have generated and characterized GBM suspension lines derived from patients with GBMs and GBM variants. These oncosphere cell lines will expand the resources available for preclinical study. PMID:27028405

  6. 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

  7. Permanently Blocked Stem Cells Derived from Breast Cancer Cell Lines

    PubMed Central

    Sajithlal, Gangadharan B.; Rothermund, Kristi; Zhang, Fang; Dabbs, David J.; Latimer, Jean J.; Grant, Stephen G.; Prochownik, Edward V.

    2016-01-01

    Cancer stem cells (CSCs) are thought to be resistant to standard chemotherapeutic drugs and the inimical conditions of the tumor microenvironment. Obtaining CSCs in sufficient quantities and maintaining their undifferentiated state have been major hurdles to their further characterization and to the identification of new pharmaceuticals that preferentially target these cells. We describe here the tagging of CSC-like populations from four human breast cancer cell lines with green fluorescent protein (GFP) under the control of the Oct3/4 stem cell-specific promoter. As expected, GFP was expressed by the CSC-enriched populations. An unanticipated result, however, was that these cells remained blocked in a CSC-like state and tended to be resistant to chemotherapeutic drugs as well as acidotic and hypoxic conditions. These CSC-like cells possessed several other in vitro attributes of CSCs and were able to reproducibly generate tumors in immuno-compromised mice from as few as 100 cells. Moreover, the tumors derived from these cells were comprised almost exclusively of pure CSCs. The ability of the Oct3/4 promoter to block CSC differentiation underscores its potential general utility for obtaining highly purified CSC populations, although the mechanism by which it does so remains undefined and subject to further study. Nonetheless, such stable cell lines should be extremely valuable tools for studying basic questions pertaining to CSC biology and for the initial identification of novel CSC-specific chemotherapeutic agents, which can then be verified in primary CSCs. PMID:20506227

  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. Effect of shear stress and of transmural pressure on cAMP-dependent responses of cells adhering to a biomaterial

    NASA Astrophysics Data System (ADS)

    Chotard-Ghodsnia, R.; Drochon, A.; Faucheux, N.; Nagel, M.-D.; Grebe, R.

    2002-02-01

    Biomaterials used in some bioreactors are porous and exposed to normal and tangential flow of physiological fluid. Flow-induced forces may influence the morphological and biochemical responses of cells adhering to these materials. The objective of this work is to examine the capacity of mechanical stress to cause changes in cell morphology via the cAMP pathway (cyclic adenosine monophosphate). This second messenger is known to modulate cell morphology in static conditions. In classical flow devices, cells are submitted to only tangential stresses. We designed a new flow system, a Hele-Shaw cell with a porous bottom wall, in order to take into account the influence of a transmural pressure. This flow chamber allows to follow up continuously the shape changes of cells that are adherent to a porous biomaterial (polyacrylonitrile) and are exposed to controlled levels of shear stress or transmural pressure. Mouse Swiss 3T3 fibroblasts exposed to a 1.1-Pa shear stress, as well as those exposed to a 84-mm Hg transmural pressure, round up (up to 50%) in a few minutes. If the cAMP pathway is inhibited when a mechanical stress is applied, cell rounding is significantly prevented. These observations suggest that flow-induced cell shape changes are cAMP-dependent. This conclusion is supported by an increased cAMP accumulation measured in cells under mechanical stress when compared to static experiments. Our in vitro flow system is thus useful to study the influence of transmural pressure or shear stress on the early morphological and biochemical responses of cells in contact with a biomaterial.

  10. Adherent invasive Escherichia coli strains from patients with Crohn's disease survive and replicate within macrophages without inducing host cell death.

    PubMed

    Glasser, A L; Boudeau, J; Barnich, N; Perruchot, M H; Colombel, J F; Darfeuille-Michaud, A

    2001-09-01

    Escherichia coli strains recovered from Crohn's disease (CD) lesions are able to adhere to and invade cultured intestinal epithelial cells. We analyzed the behavior within macrophages of adherent invasive E. coli (AIEC) strains isolated from patients with CD. All the 15 AIEC strains tested were able to replicate extensively within J774-A1 cells: the numbers of intracellular bacteria increased 2.2- to 74.2-fold at 48 h over that at 1 h postinfection. By use of murine peritoneal macrophages and human monocyte-derived-macrophages, the reference AIEC strain LF82 was confirmed to be able to survive intracellularly. Transmission electron micrographs of AIEC LF82-infected macrophages showed that at 24 h postinfection, infected cells harbored large vacuoles containing numerous bacteria, as a result of the fusion of several vacuoles occurring after 8 h postinfection. No lactate dehydrogenase (LDH) release, no sign of DNA fragmentation or degradation, and no binding to fluorescein isothlocyanate-labeled annexin V were observed with LF82-infected J774-A1 cells, even after 24 h postinfection. LF82-infected J774-A1 cells secreted 2.7-fold more tumor necrosis factor alpha (TNF-alpha) than cells stimulated with 1 microg of lipopolysaccharide (LPS)/ml. No release of interleukin-1beta was observed with LPS-prestimulated J774-A1 cells infected with AIEC LF82. These findings showed that (i) AIEC strains are able to survive and to replicate within macrophages, (ii) AIEC LF82 replication does not induce any cell death of the infected cells, and (iii) LF82-infected J774-A1 cells release high levels of TNF-alpha. These properties could be related to some features of CD and particularly to granuloma formation, one of the hallmarks of CD lesions. PMID:11500426

  11. The DNA damage/repair cascade in glioblastoma cell lines after chemotherapeutic agent treatment.

    PubMed

    Annovazzi, Laura; Caldera, Valentina; Mellai, Marta; Riganti, Chiara; Battaglia, Luigi; Chirio, Daniela; Melcarne, Antonio; Schiffer, Davide

    2015-01-01

    Therapeutic resistance in glioblastoma multiforme (GBM) has been linked to a subpopulation of cells with stem cell-like properties, the glioma stem cells (GSCs), responsible for cancer progression and recurrence. This study investigated the in vitro cytotoxicity of three chemotherapeutics, temozolomide (TMZ), doxorubicin (Dox) and paclitaxel (PTX) on glioma cell lines, by analyzing the molecular mechanisms leading to DNA repair and cell resistance, or to cell death. The drugs were tested on 16 GBM cell lines, grown as neurospheres (NS) or adherent cells (AC), by studying DNA damage occurrence by Comet assay, the expression by immunofluorescence and western blotting of checkpoint/repair molecules and apoptosis. The three drugs were able to provoke a genotoxic injury and to inhibit dose- and time-dependently cell proliferation, more evidently in AC than in NS. The first cell response to DNA damage was the activation of the damage sensors (p-ATM, p-53BP1, γ-H2AX), followed by repair effectors; the expression of checkpoint/repair molecules appeared higher in NS than in AC. The non-homologous repair pathway (NHEJ) seemed more involved than the homologous one (HR). Apoptosis occurred after long treatment times, but only a small percentage of cells in NS underwent death, even at high drug concentration, whereas most cells survived in a quiescent state and resumed proliferation after drug removal. In tumor specimens, checkpoint/repair proteins were constitutively expressed in GBMs, but not in low-grade gliomas. PMID:25892134

  12. EXAFS studies of prostate cancer cell lines

    NASA Astrophysics Data System (ADS)

    Czapla, J.; Kwiatek, W. M.; Lekki, J.; Kisiel, A.; Steininger, R.; Goettlicher, J.

    2013-04-01

    Sulphur plays a vital role in every human organism. It is known, that sulphur-bearing compounds, such as for example cysteine and glutathione, play critical roles in development and progression of many diseases. Any alteration in sulphur's biochemistry could become a precursor of serious pathological conditions. One of such condition is prostate cancer, the most frequently diagnosed malignancy in the western world and the second leading cause of cancer related death in men. The purpose of presented studies was to examine what changes occur in the nearest chemical environment of sulphur in prostate cancer cell lines in comparison to healthy cells. The Extended X-ray Absorption Fine Structure (EXAFS) spectroscopy was used, followed by theoretical calculations. The results of preliminary analysis is presented.

  13. A Cell Line Resource Derived from Honey Bee (Apis mellifera) Embryonic Tissues

    PubMed Central

    Goblirsch, Michael J.; Spivak, Marla S.; Kurtti, Timothy J.

    2013-01-01

    A major hindrance to the study of honey bee pathogens or the effects of pesticides and nutritional deficiencies is the lack of controlled in vitro culture systems comprised of honey bee cells. Such systems are important to determine the impact of these stress factors on the developmental and cell biology of honey bees. We have developed a method incorporating established insect cell culture techniques that supports sustained growth of honey bee cells in vitro. We used honey bee eggs mid to late in their embryogenesis to establish primary cultures, as these eggs contain cells that are progressively dividing. Primary cultures were initiated in modified Leibovitz’s L15 medium and incubated at 32°C. Serial transfer of material from several primary cultures was maintained and has led to the isolation of young cell lines. A cell line (AmE-711) has been established that is composed mainly of fibroblast-type cells that form an adherent monolayer. Most cells in the line are diploid (2n = 32) and have the Apis mellifera karyotype as revealed by Giemsa stain. The partial sequence for the mitochondrial-encoded cytochrome c oxidase subunit I (Cox 1) gene in the cell line is identical to those from honey bee tissues and a consensus sequence for A. mellifera. The population doubling time is approximately 4 days. Importantly, the cell line is continuously subcultured every 10–14 days when split at a 1:3 ratio and is cryopreserved in liquid nitrogen. The cell culture system we have developed has potential application for studies aimed at honey bee development, genetics, pathogenesis, transgenesis, and toxicology. PMID:23894551

  14. 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

  15. EnP1, a Microsporidian Spore Wall Protein That Enables Spores To Adhere to and Infect Host Cells In Vitro▿

    PubMed Central

    Southern, Timothy R.; Jolly, Carrie E.; Lester, Melissa E.; Hayman, J. Russell

    2007-01-01

    Microsporidia are spore-forming fungal pathogens that require the intracellular environment of host cells for propagation. We have shown that spores of the genus Encephalitozoon adhere to host cell surface glycosaminoglycans (GAGs) in vitro and that this adherence serves to modulate the infection process. In this study, a spore wall protein (EnP1; Encephalitozoon cuniculi ECU01_0820) from E. cuniculi and Encephalitozoon intestinalis is found to interact with the host cell surface. Analysis of the amino acid sequence reveals multiple heparin-binding motifs, which are known to interact with extracellular matrices. Both recombinant EnP1 protein and purified EnP1 antibody inhibit spore adherence, resulting in decreased host cell infection. Furthermore, when the N-terminal heparin-binding motif is deleted by site-directed mutagenesis, inhibition of adherence is ablated. Our transmission immunoelectron microscopy reveals that EnP1 is embedded in the microsporidial endospore and exospore and is found in high abundance in the polar sac/anchoring disk region, an area from which the everting polar tube is released. Finally, by using a host cell binding assay, EnP1 is shown to bind host cell surfaces but not to those that lack surface GAGs. Collectively, these data show that given its expression in both the endospore and the exospore, EnP1 is a microsporidian cell wall protein that may function both in a structural capacity and in modulating in vitro host cell adherence and infection. PMID:17557882

  16. Detection algorithm for the validation of human cell lines.

    PubMed

    Eltonsy, Névine; Gabisi, Vivian; Li, Xuesong; Russe, K Blair; Mills, Gordon B; Stemke-Hale, Katherine

    2012-09-15

    Cell lines are an important tool in understanding all aspects of cancer growth, development, metastasis and tumor cell death. There has been a dramatic increase in the number of cell lines and diversity of the cancers they represent; however, misidentification and cross-contamination of cell lines can lead to erroneous conclusions. One method that has gained favor for authenticating cell lines is the use of short tandem repeats (STR) to generate a unique DNA profile. The challenge in validating cell lines is the requirement to compare the large number of existing STR profiles against cell lines of interest, particularly when considering that the profiles of many cell lines have drifted over time and original samples are not available. We report here methods that analyze the variations and the proportional changes extracted from tetra-nucleotide repeat regions in the STR analysis. This technique allows a paired match between a target cell line and a reference database of cell lines to find cell lines that match within a user designated percentage cut-off quality matrix. Our method accounts for DNA instability and can suggest whether the target cell lines are misidentified or unstable. PMID:22419365

  17. B-lymphocyte responses to trinitrophenyl-conjugated Ficoll: requirement for T lymphocytes and Ia-bearing adherent cells.

    PubMed Central

    Letvin, N L; Benacerraf, B; Germain, R N

    1981-01-01

    These studies were done to characterize the cellular requirements for B-lymphocyte responses to the haptenated polysaccharide trinitrophenyl-conjugated Ficoll. By using an in vitro microculture system, it was demonstrated that hapten-specific anti-trinitrophenyl-conjugated Ficoll plaque-forming cell responses by B lymphocytes require Ia-bearing adherent accessory cells and Thy 1+ Lyt 1+2- nylon wool-nonadherent (T) lymphocytes. Such T cells could be primed in vivo with nonderivatized Ficoll to show carrier-specific helper cell function for derivatized Ficoll responses in vitro. The implications of these findings for our understanding of b-lymphocyte triggering by so-called T-independent antigens are discussed. PMID:6975477

  18. 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

  19. Personalized chemotherapy profiling using cancer cell lines from selectable mice

    PubMed Central

    Kamiyama, Hirohiko; Rauenzahn, Sherri; Shim, Joong Sup; Karikari, Collins A.; Feldmann, Georg; Hua, Li; Kamiyama, Mihoko; Schuler, F. William; Lin, Ming-Tseh; Beaty, Robert M.; Karanam, Balasubramanyam; Liang, Hong; Mullendore, Michael E.; Mo, Guanglan; Hidalgo, Manuel; Jaffee, Elizabeth; Hruban, Ralph H.; Jinnah, H. A.; Roden, Richard B. S.; Jimeno, Antonio; Liu, Jun O.; Maitra, Anirban; Eshleman, James R.

    2013-01-01

    Purpose High-throughput chemosensitivity testing of low-passage cancer cell lines can be used to prioritize agents for personalized chemotherapy. However, generating cell lines from primary cancers is difficult, because contaminating stromal cells overgrow the malignant cells. Experimental Design We produced a series of hypoxanthine phosphoribosyl transferase (hprt)-null immunodeficient mice. During growth of human cancers in these mice, hprt-null murine stromal cells replace their human counterparts. Results Pancreatic and ovarian cancers explanted from these mice were grown in selection media to produce pure human cancer cell lines. We screened one cell line with a 3,131-drug panel and identified seventy-seven FDA approved drugs with activity, including two novel drugs to which the cell line was uniquely sensitive. Xenografts of this carcinoma were selectively responsive to both drugs. Conclusion Chemotherapy can be personalized using patient-specific cell lines derived in biochemically selectable mice. PMID:23340293

  20. 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.

  1. 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

  2. Inhibition of Streptococcus pneumoniae adherence to human epithelial cells in vitro by the probiotic Lactobacillus rhamnosus GG

    PubMed Central

    2013-01-01

    Background Colonization of the nasopharynx by Streptococcus pneumoniae is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. Probiotic bacteria can influence disease outcomes through various mechanisms, including inhibition of pathogen colonization. Here, we examine the effect of the probiotic Lactobacillus rhamnosus GG (LGG) on S. pneumoniae colonization of human epithelial cells using an in vitro model. We investigated the effects of LGG administered before, at the same time as, or after the addition of S. pneumoniae on the adherence of four pneumococcal isolates. Results LGG significantly inhibited the adherence of all the pneumococcal isolates tested. The magnitude of inhibition varied with LGG dose, time of administration, and the pneumococcal isolate used. Inhibition was most effective when a higher dose of LGG was administered prior to establishment of pneumococcal colonization. Mechanistic studies showed that LGG binds to epithelial cells but does not affect pneumococcal growth or viability. Administration of LGG did not lead to any significant changes in host cytokine responses. Conclusions These findings demonstrate that LGG can inhibit pneumococcal colonization of human epithelial cells in vitro and suggest that probiotics could be used clinically to prevent the establishment of pneumococcal carriage. PMID:23561014

  3. Gemcitabine induces cell senescence in human pancreatic cancer cell lines.

    PubMed

    Song, Yao; Baba, Tomohisa; Mukaida, Naofumi

    2016-08-26

    Patients with pancreatic ductal adenocarcinoma (PDAC) commonly require chemotherapy because they frequently develop metastatic disease or locally advanced tumors. Gemcitabine, an analogue of cytosine arabinoside, is commonly used for PDAC treatment. We observed that gemcitabine induced senescence phenotypes characterized by enhanced senescence-associated β-galactosidase (SA β-Gal) staining and increased expression of senescence-associated molecules in two human pancreatic cancer cell lines, Miapaca-2 and Panc-1, which exhibit resistance to gemcitabine but not L3.pl cells with a high sensitivity to gemcitabine. Gemcitabine-induced cell senescence can be inhibited by reactive oxygen species inhibitor, N-acetyl cysteine. Although gemcitabine also enhanced CXCL8 expression, anti-CXCL8 antibody failed to reduce gemcitabine-induced increases in SA β-Gal-positive cell numbers. These observations would indicate that cell senescence can proceed independently of CXCL8 expression, a characteristic feature of senescence-associated secretion phenotype. PMID:27311854

  4. Derivation of three new human embryonic stem cell lines.

    PubMed

    Bradley, Cara K; Chami, Omar; Peura, Teija T; Bosman, Alexis; Dumevska, Biljana; Schmidt, Uli; Stojanov, Tomas

    2010-04-01

    Human embryonic stem cells are pluripotent cells capable of extensive self-renewal and differentiation to all cells of the embryo proper. Here, we describe the derivation and characterization of three Sydney IVF human embryonic stem cell lines not already reported elsewhere, designated SIVF001, SIVF002, and SIVF014. The cell lines display typical compact colony morphology of embryonic stem cells, have stable growth rates over more than 40 passages and are cytogenetically normal. Furthermore, the cell lines express pluripotency markers including Nanog, Oct4, SSEA3 and Tra-1-81, and are capable of generating teratoma cells derived from each of the three germ layers in immunodeficient mice. These experiments show that the cell lines constitute pluripotent stem cell lines. PMID:20198447

  5. DNA profiling and characterization of animal cell lines.

    PubMed

    Stacey, Glyn N; Byrne, Ed; Hawkins, J Ross

    2014-01-01

    The history of the culture of animal cell lines is littered with published and much unpublished experience with cell lines that have become switched, mislabelled, or cross-contaminated during laboratory handling. To deliver valid and good quality research and to avoid waste of time and resources on such rogue lines, it is vital to perform some kind of qualification for the provenance of cell lines used in research and particularly in the development of biomedical products. DNA profiling provides a valuable tool to compare different sources of the same cells and, where original material or tissue is available, to confirm the correct identity of a cell line. This chapter provides a review of some of the most useful techniques to test the identity of cells in the cell culture laboratory and gives methods which have been used in the authentication of cell lines. PMID:24297409

  6. 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.

  7. Neuronal cell lines as model dorsal root ganglion neurons

    PubMed Central

    Yin, Kathleen; Baillie, Gregory J

    2016-01-01

    Background Dorsal root ganglion neuron-derived immortal cell lines including ND7/23 and F-11 cells have been used extensively as in vitro model systems of native peripheral sensory neurons. However, while it is clear that some sensory neuron-specific receptors and ion channels are present in these cell lines, a systematic comparison of the molecular targets expressed by these cell lines with those expressed in intact peripheral neurons is lacking. Results In this study, we examined the expression of RNA transcripts in the human neuroblastoma-derived cell line, SH-SY5Y, and two dorsal root ganglion hybridoma cell lines, F-11 and ND7/23, using Illumina next-generation sequencing, and compared the results with native whole murine dorsal root ganglions. The gene expression profiles of these three cell lines did not resemble any specific defined dorsal root ganglion subclass. The cell lines lacked many markers for nociceptive sensory neurons, such as the Transient receptor potential V1 gene, but expressed markers for both myelinated and unmyelinated neurons. Global gene ontology analysis on whole dorsal root ganglions and cell lines showed similar enrichment of biological process terms across all samples. Conclusions This paper provides insights into the receptor repertoire expressed in common dorsal root ganglion neuron-derived cell lines compared with whole murine dorsal root ganglions, and illustrates the limits and potentials of these cell lines as tools for neuropharmacological exploration. PMID:27130590

  8. Cell line banks and their role in cancer research.

    PubMed

    Hay, R J; Reid, Y A; McClintock, P R; Chen, T R; Macy, M L

    1996-01-01

    The utility of centralized cell banks in providing reference cultures for cancer research is reviewed. Procedures applied at The American Type Culture Collection in development, maintenance and expansion of such a resource are discussed for example, with emphasis on human tumor cell lines. The various categories of cell-line holdings are explained, and status with regard both to the numbers of lines available and distribution experienced are documented. The locations of other national cell repositories plus contact data are provided. PMID:8806094

  9. The anti-inflammatory drug nimesulide inhibits neutrophil adherence to and migration across monolayers of cytokine-activated endothelial cells.

    PubMed

    Dapino, P; Ottonello, L; Dallegri, F

    1994-01-01

    Neutrophil migration through the microvascular endothelium represents a fundamental event for the cell accumulation at sites of tissue injury. Owing to their capacity to modify the structural and functional characteristics of endothelial cells, inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF alpha) play a pivotal role in directing circulating neutrophils away from the bloodstream to the interstitial tissue. In order to study neutrophil transendothelial migration, human umbilical vein endothelial cells were grown to confluence on the polycarbonate filter of two-compartment migration chambers. Pretreatment of the endothelial cell monolayers with TNF alpha for 4 h resulted in rapid migration of approximately 50% of subsequently added neutrophils across the layers. In contrast, < 10% of added neutrophils penetrated untreated endothelial monolayers. Using TNF alpha-treated endothelium, neutrophil transmigration was inhibited by the methane sulfonanilide anti-inflammatory drug nimesulide. Moreover, neutrophil adherence to TNF alpha-treated endothelial monolayers, cultured in microtiter wells, was markedly reduced by nimesulide. A linear correlation between the drug-dependent inhibition of neutrophil transmigration and neutrophil adherence was found. Finally, nimesulide did not interfere with the TNF alpha ability to convert resting endothelium into a pro-adhesive and pro-locomotory cell layer. The data suggest that nimesulide reduces neutrophil transendothelial migration primarily by limiting the cell anchorage to the TNF alpha-activated endothelium. Therefore, the drug has the potential to down-regulate neutrophil extravasation and, in turn, the burden of neutrophil oxidants and proteases leading to tissue injury at sites of inflammation. PMID:7824814

  10. Group A Streptococcus Adheres to Pharyngeal Epithelial Cells with Salivary Proline-rich Proteins via GrpE Chaperone Protein*

    PubMed Central

    Murakami, Jumpei; Terao, Yutaka; Morisaki, Ichijiro; Hamada, Shigeyuki; Kawabata, Shigetada

    2012-01-01

    Group A Streptococcus pyogenes (GAS) is an important human pathogen that frequently causes pharyngitis. GAS organisms can adhere to and invade pharyngeal epithelial cells, which are overlaid by salivary components. However, the role of salivary components in GAS adhesion to pharyngeal cells has not been reported precisely. We collected human saliva and purified various salivary components, including proline-rich protein (PRP), statherin, and amylase, and performed invasion assays. The GAS-HEp-2 association ratio (invasion/adhesion ratio) and invasion ratio of GAS were increased significantly with whole human saliva and PRP, while the anti-PRP antibody inhibited the latter. GAS strain NY-5, which lacks M and F proteins on the cell surface, was promoted to cohere with HEp-2 cells by whole human saliva and PRP. The 28-kDa protein of GAS bound to PRP and was identified as GrpE, a chaperone protein, whereas the N-terminal of GrpE was found to bind to PRP. A GrpE-deficient mutant of GAS strain B514Sm, TR-45, exhibited a reduced ability to adhere to and invade HEp-2 cells. Microscopic observations showed the GrpE was mainly expressed on the surface of the cell division site of GAS. Furthermore, GrpE-deficient mutants of GAS and Streptococcus pneumoniae showed an elongated morphology as compared with the wild type. Taken together, this is the first study to show an interaction between salivary PRP and GAS GrpE, which plays an important role in GAS infection on the pharynx, whereas the expression of GrpE on the surface of GAS helps to maintain morphology. PMID:22566698

  11. 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

  12. 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

  13. Cardiomyocyte marker expression in a human lymphocyte cell line using mouse cardiomyocyte extract.

    PubMed

    Vojdani, Zahra; Tavakolinejad, Sima; Talaei-Khozani, Tahereh; Esmaeilpour, Tahereh; Rasooli, Manuchehr

    2011-03-01

    Cell transplantation shows potential for the treatment of cardiac diseases. Embryonic stem cells, cord blood and mesenchymal stem cells have been suggested as sources for transplantation therapy. Because of some technical limitations with the use of stem cells, transdifferentiation of fully differentiated cells is a potentially useful alternative. We investigated whether human peripheral blood cells could transdifferentiate into cardiomyocyte. Transdifferentiation was induced in a human B lymphocyte cell line (Raji). Cardiomyocyte extract was prepared from adult mouse cardiomyocytes. The cells were treated with 5-aza-2-deoxycytidine and trichostatin A, permeabilized with streptolysin O, and exposed to the mouse cardiomyocyte extract. They were cultured for 10 days, 3 weeks and 4 weeks. Cardiomyocyte markers were detected with immunohistochemistry and flow cytometry. Immunocytochemistry revealed that some cells expressed myosin heavy chain, α-actinin and cardiac troponin T after 3 and 4 weeks. Flow cytometry confirmed these data. In cells exposed to trichostatin A and 5-aza-2-deoxycytidine and permeabilized in the presence of the cardiomyocyte extract, troponin T expression was seen in 3.53% of the cells and 3.11% of them expressed α-actinin. After exposure to the cardiomyocyte extract, some permeabilized cells adhered to the plate loosely; however, the morphology did not change significantly, and they continued to show a rounded shape after 4 weeks. Our treated lymphocytes expressed cardiomyocyte markers. Our results suggest that lymphocytes may be useful in future research as a source of cells for reprogramming procedures. PMID:21547694

  14. 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

  15. High-molecular-mass lipopolysaccharides are involved in Actinobacillus pleuropneumoniae adherence to porcine respiratory tract cells.

    PubMed Central

    Paradis, S E; Dubreuil, D; Rioux, S; Gottschalk, M; Jacques, M

    1994-01-01

    Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia. The major adhesin of A. pleuropneumoniae has been identified as the lipopolysaccharides (LPSs) (M. Bélanger, D. Dubreuil, J. Harel, C. Girard, and M. Jacques, Infect. Immun. 58:3523-3530, 1990). Using immunoelectron microscopy and flow cytometry, we showed in the present study that LPSs were well exposed at the surface of this encapsulated microorganism. Immunolocalization with porcine lung and tracheal frozen sections showed that extracted LPS bound to the lung mesenchyme and vascular endothelium and to the tracheal epithelium, respectively. Inhibition of adherence of A. pleuropneumoniae with extracted LPS was also performed with lung and tracheal frozen sections. Acid hydrolysis of LPS revealed that the active component of LPS was not lipid A but the polysaccharides. LPSs from A. pleuropneumoniae serotypes 1 and 2 were separated by chromatography on Sephacryl S-300 SF, in the presence of sodium deoxycholate, according to their molecular masses. The adherence-inhibitory activity was found in the high-molecular-mass fractions. These high-molecular-mass fractions contained 2-keto-3-deoxyoctulosonic acid and neutral sugars, and they were recognized by a monoclonal antibody directed against A. pleuropneumoniae O antigen but not recognized by a monoclonal antibody against capsular antigen. Images PMID:8039902

  16. 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

  17. Development and characterization of a new human hepatic cell line.

    PubMed

    Ramboer, Eva; De Craene, Bram; De Kock, Joey; Berx, Geert; Rogiers, Vera; Vanhaecke, Tamara; Vinken, Mathieu

    2015-01-01

    The increasing demand and hampered use of primary human hepatocytes for research purposes have urged scientists to search for alternative cell sources, such as immortalized hepatic cell lines. The aim of this study was to develop a human hepatic cell line using the combined overexpression of TERT and the cell cycle regulators cyclin D1 and mutant isoform CDK4R24C. Following transduction of adult human primary hepatocytes with the selected immortalization genes, cell growth was triggered and a cell line was established. When cultured under appropriate conditions, the cell line expressed several hepatocytic markers and liver-enriched transcription factors at the transcriptional and/or translational level, secreted liver-specific proteins and showed glycogen deposition. These results suggest that the immortalization strategy applied to primary human hepatocytes could generate a novel hepatic cell line that seems to retain some key hepatic characteristics. PMID:26869867

  18. Development and characterization of a new human hepatic cell line

    PubMed Central

    Ramboer, Eva; De Craene, Bram; De Kock, Joey; Berx, Geert; Rogiers, Vera; Vanhaecke, Tamara; Vinken, Mathieu

    2015-01-01

    The increasing demand and hampered use of primary human hepatocytes for research purposes have urged scientists to search for alternative cell sources, such as immortalized hepatic cell lines. The aim of this study was to develop a human hepatic cell line using the combined overexpression of TERT and the cell cycle regulators cyclin D1 and mutant isoform CDK4R24C. Following transduction of adult human primary hepatocytes with the selected immortalization genes, cell growth was triggered and a cell line was established. When cultured under appropriate conditions, the cell line expressed several hepatocytic markers and liver-enriched transcription factors at the transcriptional and/or translational level, secreted liver-specific proteins and showed glycogen deposition. These results suggest that the immortalization strategy applied to primary human hepatocytes could generate a novel hepatic cell line that seems to retain some key hepatic characteristics. PMID:26869867

  19. Differential signaling of the GnRH receptor in pituitary gonadotrope cell lines and prostate cancer cell lines

    PubMed Central

    Sviridonov, Ludmila; Dobkin-Bekman, Masha; Shterntal, Boris; Przedecki, Fiorenza; Formishell, Linor; Kravchook, Shani; Navi, Liat Rahamim-Ben; Bar-Lev, Tali Hana; Kazanietz, Marcelo G.; Yao, Zhong; Seger, Rony; Naor, Zvi

    2014-01-01

    The GnRH receptor (GnRHR) mediates the pituitary functions of GnRH, as well as its anti-proliferative effects in sex hormone-dependent cancer cells. Here we compare the signaling of GnRHR in pituitary gonadotrope cell lines vs. prostate cancer cell lines. We first noticed that the expression level of PKCα, PKCβII and PKCε is much higher in αT3-1 and LβT2 gonadotrope cell lines vs. LNCaP and DU-145 cell lines, while the opposite is seen for PKCδ. Activation of PKCα, PKCβII and PKCε by GnRH is relatively transient in αT3-1 and LβT2 gonadotrope cell lines and more prolonged in LNCaP and DU-145 cell lines. On the otherhand, the activation and re-distribution of the above PKCs by PMA was similar for both gonadotrope cell lines and prostate cancer cell lines. Activation of ERK1/2 by GnRH and PMA was robust in the gonadotrope cell lines, with a smaller effect observed in the prostate cancer cell lines. The Ca2+ ionophore A23187 stimulated ERK1/2 in gonadotrope cell lines but not in prostate cancer cell lines. GnRH, PMA and A23187 stimulated JNK activity in gonadotrope cell lines, with a more sustained effect in prostate cancer cell lines. Sustained activation of p38 was observed for PMA and A23187 in Du-145 cells, while p38 activation by GnRH, PMA and A23187 in LβT2 cells was transient. Thus, differential expression and re-distribution of PKCs by GnRH and the transient vs. the more sustained nature of the activation of the PKC-MAPK cascade by GnRH in gonadotrope cell lines vs. prostate cancer cell lines respectively, may provide the mechanistic basis for the cell context-dependent differential biological responses observed in GnRH interaction with pituitary gonadotropes vs. prostate cancer cells. PMID:23380421

  20. Impact on disinfection efficiency of cell load and of planktonic/adherent/detached state: case of Hafnia alvei inactivation by plasma activated water.

    PubMed

    Kamgang-Youbi, Georges; Herry, Jean-Marie; Brisset, Jean-Louis; Bellon-Fontaine, Marie-Noëlle; Doubla, Avaly; Naïtali, Murielle

    2008-12-01

    This paper describes the effects of initial microbial concentration and planktonic/adherent/detached states on the efficiency of plasma-activated water. This disinfecting solution was obtained by treating distilled water with an atmospheric pressure plasma produced by gliding electric discharges in humid air. The inactivation kinetics of planktonic cells of Hafnia alvei (selected as a bacterial model) were found to be of the first order. They were influenced by the initial microbial concentration. Efficiency decreased when the initial viable population N(0) increased, and the inactivation rate k(max) was linearly modified as a function of Log(10) (N(0)). This relation was used to compare planktonic, adherent, and detached cells independently from the level of population. Bacteria adhering to stainless steel and high-density polyethylene were also sensitive to treatment, but at a lower rate than their free-living counterparts. Moreover, cells detached from these solid substrates exhibited an inactivation rate lower than that of planktonic cells but similar to adherent bacteria. This strongly suggests the induction of a physiological modification to bacteria during the adhesion step, rendering adherent--and further detached--bacteria less susceptible to the treatment, when compared to planktonic bacteria. PMID:18769918

  1. 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

  2. Alpha-2 Heremans Schmid Glycoprotein (AHSG) Modulates Signaling Pathways in Head and Neck Squamous Cell Carcinoma Cell Line SQ20B

    SciTech Connect

    Thompson, Pamela D.; Sakwe, Amos; Koumangoye, Rainelli; Yarbrough, Wendell G.; Ochieng, Josiah; Marshall, Dana R.

    2014-02-15

    This study was performed to identify the potential role of Alpha-2 Heremans Schmid Glycoprotein (AHSG) in Head and Neck Squamous Cell Carcinoma (HNSCC) tumorigenesis using an HNSCC cell line model. HNSCC cell lines are unique among cancer cell lines, in that they produce endogenous AHSG and do not rely, solely, on AHSG derived from serum. To produce our model, we performed a stable transfection to down-regulate AHSG in the HNSCC cell line SQ20B, resulting in three SQ20B sublines, AH50 with 50% AHSG production, AH20 with 20% AHSG production and EV which is the empty vector control expressing wild-type levels of AHSG. Utilizing these sublines, we examined the effect of AHSG depletion on cellular adhesion, proliferation, migration and invasion in a serum-free environment. We demonstrated that sublines EV and AH50 adhered to plastic and laminin significantly faster than the AH20 cell line, supporting the previously reported role of exogenous AHSG in cell adhesion. As for proliferative potential, EV had the greatest amount of proliferation with AH50 proliferation significantly diminished. AH20 cells did not proliferate at all. Depletion of AHSG also diminished cellular migration and invasion. TGF-β was examined to determine whether levels of the TGF-β binding AHSG influenced the effect of TGF-β on cell signaling and proliferation. Whereas higher levels of AHSG blunted TGF-β influenced SMAD and ERK signaling, it did not clearly affect proliferation, suggesting that AHSG influences on adhesion, proliferation, invasion and migration are primarily due to its role in adhesion and cell spreading. The previously reported role of AHSG in potentiating metastasis via protecting MMP-9 from autolysis was also supported in this cell line based model system of endogenous AHSG production in HNSCC. Together, these data show that endogenously produced AHSG in an HNSCC cell line, promotes in vitro cellular properties identified as having a role in tumorigenesis. Highlights: • Head

  3. Butyrate downregulates α2β1 integrin: a possible role in the induction of apoptosis in colorectal cancer cell lines

    PubMed Central

    Buda, A; Qualtrough, D; Jepson, M A; Martines, D; Paraskeva, C; Pignatelli, M

    2003-01-01

    Background: Integrins mediate cell matrix adhesion and regulate cell growth and survival. In colonic epithelial cells, α2β1 integrin controls glandular differentiation and proliferation. Butyrate stimulates differentiation and induces apoptosis in vitro. Aims: We investigated whether butyrate induction of apoptosis was associated with perturbation of integrin mediated cell matrix adhesion. Methods: Three colonic cancer cell lines (SW1222, SW620, LS174T) were studied. Adhesion to extracellular matrix proteins, expression of α2β1 integrin, and apoptosis were studied in adherent cells after treatment with 4 mM butyrate. Results: Butyrate decreased the attachment to type I collagen in SW620 cells and type I and IV collagen in LS174T cells. The decreased cell attachment was associated with downregulation of α2β1 integrin and increased apoptosis in adherent cells. No changes in α2β1 expression or matrix adhesion were seen in SW1222 cells, which were also found to be less sensitive to butyrate induction of apoptosis. Downregulation of α2β1 integrin preceded the detection of apoptosis. Conclusion: Apoptosis induced by butyrate is associated with downregulation of expression and functional activity of α2β1 integrin. Perturbation of cell matrix adhesion may be a novel mechanism by which butyrate induces apoptosis in colorectal cancer cells. PMID:12692060

  4. Analysis of three marine fish cell lines by rapd assay.

    PubMed

    Guo, H R; Zhang, S C; Tong, S L; Xiang, J H

    2001-01-01

    We tested the applicability of the random amplified polymorphic deoxyribonucleic acid (RAPD) analysis for identification of three marine fish cell lines FG, SPH, and RSBF, and as a possible tool to detect cross-contamination. Sixty commercial 10-mer RAPD primers were tested on the cell lines and on samples collected from individual fish. The results obtained showed that the cell lines could be identified to the correspondent species on the basis of identical patterns produced by 35-48% of the primers tested; the total mean similarity indices for cell lines versus correspondent species of individual fish ranged from 0.825 to 0.851, indicating the existence of genetic variation in these cell lines in relation to the species of their origin. Also, four primers, which gave a monomorphic band pattern within species/line, but different among the species/line, were obtained. These primers can be useful for identification of these cell lines and for characterization of the genetic variation of these cell lines in relation to the species of their origin. This supported the use of RAPD analysis as an effective tool in species identification and cross-contamination test among different cell lines. PMID:11573817

  5. Establishment and characterization of a new cell line of canine inflammatory mammary cancer: IPC-366.

    PubMed

    Caceres, Sara; Peña, Laura; de Andres, Paloma J; Illera, Maria J; Lopez, Mirtha S; Woodward, Wendy A; Reuben, James M; Illera, Juan C

    2015-01-01

    Canine inflammatory mammary cancer (IMC) shares epidemiologic, histopathological and clinical characteristics with the disease in humans and has been proposed as a natural model for human inflammatory breast cancer (IBC). The aim of this study was to characterize a new cell line from IMC (IPC-366) for the comparative study of both IMC and IBC. Tumors cells from a female dog with clinical IMC were collected. The cells were grown under adherent conditions. The growth, cytological, ultrastructural and immunohistochemical (IHC) characteristics of IPC-366 were evaluated. Ten female Balb/SCID mice were inoculated with IPC-366 cells to assess their tumorigenicity and metastatic potential. Chromosome aberration test and Karyotype revealed the presence of structural aberration, numerical and neutral rearrangements, demonstrating a chromosomal instability. Microscopic examination of tumor revealed an epithelial morphology with marked anysocytosis. Cytological and histological examination of smears and ultrathin sections by electron microscopy revealed that IPC-366 is formed by highly malignant large round or polygonal cells characterized by marked atypia and prominent nucleoli and frequent multinucleated cells. Some cells had cytoplasmic empty spaces covered by cytoplasmic membrane resembling capillary endothelial cells, a phenomenon that has been related to s vasculogenic mimicry. IHC characterization of IPC-366 was basal-like: epithelial cells (AE1/AE3+, CK14+, vimentin+, actin-, p63-, ER-, PR-, HER-2, E-cadherin, overexpressed COX-2 and high Ki-67 proliferation index (87.15 %). At 2 weeks after inoculating the IPC-366 cells, a tumor mass was found in 100 % of mice. At 4 weeks metastases in lung and lymph nodes were found. Xenograph tumors maintained the original IHC characteristics of the female dog tumor. In summary, the cell line IPC-366 is a fast growing malignant triple negative cell line model of inflammatory mammary carcinoma that can be used for the comparative

  6. Establishment and Characterization of a New Cell Line of Canine Inflammatory Mammary Cancer: IPC-366

    PubMed Central

    Caceres, Sara; Peña, Laura; de Andres, Paloma J.; Illera, Maria J.; Lopez, Mirtha S.; Woodward, Wendy A.; Reuben, James M.; Illera, Juan C.

    2015-01-01

    Canine inflammatory mammary cancer (IMC) shares epidemiologic, histopathological and clinical characteristics with the disease in humans and has been proposed as a natural model for human inflammatory breast cancer (IBC). The aim of this study was to characterize a new cell line from IMC (IPC-366) for the comparative study of both IMC and IBC. Tumors cells from a female dog with clinical IMC were collected. The cells were grown under adherent conditions. The growth, cytological, ultrastructural and immunohistochemical (IHC) characteristics of IPC-366 were evaluated. Ten female Balb/SCID mice were inoculated with IPC-366 cells to assess their tumorigenicity and metastatic potential. Chromosome aberration test and Karyotype revealed the presence of structural aberration, numerical and neutral rearrangements, demonstrating a chromosomal instability. Microscopic examination of tumor revealed an epithelial morphology with marked anysocytosis. Cytological and histological examination of smears and ultrathin sections by electron microscopy revealed that IPC-366 is formed by highly malignant large round or polygonal cells characterized by marked atypia and prominent nucleoli and frequent multinucleated cells. Some cells had cytoplasmic empty spaces covered by cytoplasmic membrane resembling capillary endothelial cells, a phenomenon that has been related to s vasculogenic mimicry. IHC characterization of IPC-366 was basal-like: epithelial cells (AE1/AE3+, CK14+, vimentin+, actin-, p63-, ER-, PR-, HER-2, E-cadherin, overexpressed COX-2 and high Ki-67 proliferation index (87.15 %). At 2 weeks after inoculating the IPC-366 cells, a tumor mass was found in 100 % of mice. At 4 weeks metastases in lung and lymph nodes were found. Xenograph tumors maintained the original IHC characteristics of the female dog tumor. In summary, the cell line IPC-366 is a fast growing malignant triple negative cell line model of inflammatory mammary carcinoma that can be used for the comparative

  7. A new permanent cell line derived from the bank vole (Myodes glareolus) as cell culture model for zoonotic viruses

    PubMed Central

    2011-01-01

    Background Approximately 60% of emerging viruses are of zoonotic origin, with three-fourths derived from wild animals. Many of these zoonotic diseases are transmitted by rodents with important information about their reservoir dynamics and pathogenesis missing. One main reason for the gap in our knowledge is the lack of adequate cell culture systems as models for the investigation of rodent-borne (robo) viruses in vitro. Therefore we established and characterized a new cell line, BVK168, using the kidney of a bank vole, Myodes glareolus, the most abundant member of the Arvicolinae trapped in Germany. Results BVK168 proved to be of epithelial morphology expressing tight junctions as well as adherence junction proteins. The BVK168 cells were analyzed for their infectability by several arbo- and robo-viruses: Vesicular stomatitis virus, vaccinia virus, cowpox virus, Sindbis virus, Pixuna virus, Usutu virus, Inkoo virus, Puumalavirus, and Borna disease virus (BDV). The cell line was susceptible for all tested viruses, and most interestingly also for the difficult to propagate BDV. Conclusion In conclusion, the newly established cell line from wildlife rodents seems to be an excellent tool for the isolation and characterization of new rodent-associated viruses and may be used as in vitro-model to study properties and pathogenesis of these agents. PMID:21729307

  8. Establishment and characterization of unique human gallbladder cancer cell lines.

    PubMed

    Ghosh, Mila; Koike, Naoto; Yanagimoto, Go; Tsunoda, Shin-Ichi; Kaul, Sunil; Hirano, Takashi; Emura, Fabian; Kashiwagi, Hironobu; Kawamoto, Toru; Ohkohchi, Nobuhiro; Saijo, Kaoru; Ohno, Tadao; Miwa, Masanao; Todoroki, Takeshi

    2004-05-01

    Gallbladder cancer has a dismal prognosis. Understanding the disease at the biological, genetic, molecular, cellular, and clinical level is essential for effective diagnostics and therapeutics. However, the currently established gallbladder cell lines are insufficient for better understanding and further research. The aim of our present study was to establish and characterize human gallbladder cancer cell lines. We established 5 cell lines from resected specimens of gallbladder cancers. These cell lines revealed typical tumor histopathological characteristics. We examined growth characteristics and the colony-forming ability of established cell lines in terms of their cell cycle parameters, expression of tumor markers (carcinoembryonic antigen; CEA, carbohydrated antigen 19-9; CA19-9, MUC-1 and c-kit) and the oncogene c-erbB2 by flow cytometer. Comparative genomic hybridization (CGH) analysis with specific gene probes was performed to detect changes in the gene copy numbers. Human origin of cell lines was confirmed by chromosomal analysis. Cells maintained differentiation characteristics of the original tumors. The doubling time of different cell lines varied from 30 to 96 h. All 5 cell lines formed colonies in the colony forming assays and expressed CEA, CA19-9, MUC-1 and the oncogene c-erbB2 and showed chromosomal aneuploidy. CGH analysis demonstrated gain of chromosomal region bearing SRC, RAB1, and PAP in all cell lines and hTERT in 4 cell lines. These newly established cell lines might serve as a useful model for studying the molecular pathogenesis of gallbladder cancer. Furthermore, they may serve as a model for testing new therapeutics against gallbladder cancer. These chromosomal aberrations and imbalances provide a starting point for molecular analyses of genomic regions and genes in gallbladder carcinogenesis. PMID:15067341

  9. Continuous human cell lines and method of making same

    DOEpatents

    Stampfer, M.R.

    1985-07-01

    Substantially genetically stable continuous human cell lines derived from normal human mammary epithelial cells (HMEC) and processes for making and using the same. In a preferred embodiment, the cell lines are derived by treating normal human mammary epithelial tissue with a chemical carcinogen such as benzo(a)pyrene. The novel cell lines serve as useful substrates for elucidating the potential effects of a number of toxins, carcinogens and mutagens as well as of the addition of exogenous genetic material. The autogenic parent cells from which the cell lines are derived serve as convenient control samples for testing. The cell lines are not neoplastically transformed, although they have acquired several properties which distinguish them from their normal progenitors. 2 tabs.

  10. Continuous human cell lines and method of making same

    DOEpatents

    Stampfer, Martha R.

    1989-01-01

    Substantially genetically stable continuous human cell lines derived from normal human mammary epithelial cells (HMEC) and processes for making and using the same. In a preferred embodiment, the cell lines are derived by treating normal human mammary epithelial tissue with a chemical carcinogen such as benzo[a]pyrene. The novel cell lines serve as useful substrates for elucidating the potential effects of a number of toxins, carcinogens and mutagens as well as of the addition of exogenous genetic material. The autogenic parent cells from which the cell lines are derived serve as convenient control samples for testing. The cell lines are not neoplastically transformed, although they have acquired several properties which distinguish them from their normal progenitors.

  11. Adhering heat-killed human Lactobacillus acidophilus, strain LB, inhibits the process of pathogenicity of diarrhoeagenic bacteria in cultured human intestinal cells.

    PubMed

    Coconnier, M H; Bernet, M F; Chauvière, G; Servin, A L

    1993-12-01

    Heat-killed L. acidophilus, strain LB, was tested for its ability to adhere in vitro onto human enterocyte-like Caco-2 and muco-secreting HT29-MTX cells in culture. The heat-killed LB bacteria exhibited a high adhesive property. A diffuse pattern of adhesion was observed to the undifferentiated cells, the apical brush border of the enterocytic cells, and to the mucus layer that covered the surface of the mucus-secreting cells. The inhibitory effect of heat-killed LB organisms against the human intestinal Caco-2 cell-adhesion and cell-invasion by a large variety of diarrhoeagenic bacteria was investigated. The following dose-dependent inhibitions were obtained: (i) against the cell-association of enterotoxigenic, diffusely-adhering and enteropathogenic Escherichia coli, Listeria monocytogenes, Yersinia pseudotuberculosis, and Salmonella typhimurium; (ii) against the cell-invasion by enteropathogenic Escherichia coli, Yersinia pseudotuberculosis, Listeria monocytogenes and Salmonella typhimurium. PMID:8188996

  12. Lactobacillus acidophilus LA 1 binds to cultured human intestinal cell lines and inhibits cell attachment and cell invasion by enterovirulent bacteria.

    PubMed Central

    Bernet, M F; Brassart, D; Neeser, J R; Servin, A L

    1994-01-01

    Four human Lactobacillus acidophilus strains were tested for their ability to adhere onto human enterocyte like Caco-2 cells in culture. The LA 1 strain exhibited a high calcium independent adhesive property. This adhesion onto Caco-2 cells required a proteinaceous adhesion promoting factor, which was present in the spent bacterial broth culture supernatant. LA 1 strain also strongly bound to the mucus secreted by the homogeneous cultured human goblet cell line HT29-MTX. The inhibitory effect of LA 1 organisms against Caco-2 cell adhesion and cell invasion by a large variety of diarrhoeagenic bacteria was investigated. As a result, the following dose dependent inhibitions were obtained: (a) against the cell association of enterotoxigenic, diffusely adhering and enteropathogenic Escherichia coli, and Salmonella typhimurium; (b) against the cell invasion by enteropathogenic Escherichia coli, Yersinia pseudotuberculosis, and Salmonella typhimurium. Incubations of L acidophilus LA 1 before and together with enterovirulent E coli were more effective than incubation after infection by E coli. Images Figure 1 Figure 2 PMID:8174985

  13. Lactobacillus acidophilus LA 1 binds to cultured human intestinal cell lines and inhibits cell attachment and cell invasion by enterovirulent bacteria.

    PubMed

    Bernet, M F; Brassart, D; Neeser, J R; Servin, A L

    1994-04-01

    Four human Lactobacillus acidophilus strains were tested for their ability to adhere onto human enterocyte like Caco-2 cells in culture. The LA 1 strain exhibited a high calcium independent adhesive property. This adhesion onto Caco-2 cells required a proteinaceous adhesion promoting factor, which was present in the spent bacterial broth culture supernatant. LA 1 strain also strongly bound to the mucus secreted by the homogeneous cultured human goblet cell line HT29-MTX. The inhibitory effect of LA 1 organisms against Caco-2 cell adhesion and cell invasion by a large variety of diarrhoeagenic bacteria was investigated. As a result, the following dose dependent inhibitions were obtained: (a) against the cell association of enterotoxigenic, diffusely adhering and enteropathogenic Escherichia coli, and Salmonella typhimurium; (b) against the cell invasion by enteropathogenic Escherichia coli, Yersinia pseudotuberculosis, and Salmonella typhimurium. Incubations of L acidophilus LA 1 before and together with enterovirulent E coli were more effective than incubation after infection by E coli. PMID:8174985

  14. The pursuit of ES cell lines of domesticated ungulates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In contrast to differentiated cells, embryonic stem cells (ESC) maintain an undifferentiated state, have the ability to self-renew, and exhibit pluripotency, i.e., they can give rise to most if not all somatic cell types and to the germ cells, egg and sperm. These characteristics make ES cell lines...

  15. A Serine-Threonine Kinase (StkP) Regulates Expression of the Pneumococcal Pilus and Modulates Bacterial Adherence to Human Epithelial and Endothelial Cells In Vitro

    PubMed Central

    Herbert, Jenny A.; Mitchell, Andrea M.; Mitchell, Timothy J.

    2015-01-01

    The pneumococcal serine threonine protein kinase (StkP) acts as a global regulator in the pneumococcus. Bacterial mutants deficient in StkP are less virulent in animal models of infection. The gene for this regulator is located adjacent to the gene for its cognate phosphatase in the pneumococcal genome. The phosphatase dephosphorylates proteins phosphorylated by StkP and has been shown to regulate a number of key pneumococcal virulence factors and to modulate adherence to eukaryotic cells. The role of StkP in adherence of pneumococci to human cells has not previously been reported. In this study we show StkP represses the pneumococcal pilus, a virulence factor known to be important for bacterial adhesion. In a serotype 4 strain regulation of the pilus by StkP modulates adherence to human brain microvascular endothelial cells (HBMEC) and human lung epithelial cells. This suggests that the pneumococcal pilus may play a role in adherence during infections such as meningitis and pneumonia. We show that regulation of the pilus occurs at the population level as StkP alters the number of pili-positive cells within a single culture. As far as we are aware this is the first gene identified outside of the pilus islet that regulates the biphasic expression of the pilus. These findings suggest StkPs role in cell division may be linked to regulation of expression of a cell surface adhesin. PMID:26090876

  16. 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

  17. Establishment of human colon cancer cell lines from fresh tumors versus xenografts: comparison of success rate and cell line features.

    PubMed

    Dangles-Marie, Virginie; Pocard, Marc; Richon, Sophie; Weiswald, Louis-Bastien; Assayag, Franck; Saulnier, Patrick; Judde, Jean-Gabriel; Janneau, Jean-Louis; Auger, Nathalie; Validire, Pierre; Dutrillaux, Bernard; Praz, Françoise; Bellet, Dominique; Poupon, Marie-France

    2007-01-01

    Obtaining representative human colon cancer cell lines from fresh tumors is technically difficult. Using 32 tumor fragments from patients with colon cancer, the present study shows that prior xenograft leads to more efficient cell line establishment compared with direct establishment from fresh tumors (P < 0.05). From 26 tumor specimens, we successfully established 20 tumor xenografts in nude mice (77%); among 19 of these xenografts, 9 (47%) led to cell lines, including four from liver metastases. Only 3 of 31 tumor specimens (9.7%) grew immediately in vitro, and all were derived from primary tumors. To compare major phenotypic and genotypic characteristics of human colon cancer cell lines derived from the same tumor fragment using two protocols, the two pairs of cell lines obtained from 2 of 32 tumor fragments were extensively studied. They displayed similar morphology and were able to form compact spheroids. Chemosensitivity to 5-fluorouracil, CPT11, and L-OHP differed between cell lines obtained from patient tumors and those derived from xenografts. Matched cell lines shared a common core of karyotype alterations and distinctive additional chromosomal aberrations. Expression levels of genes selected for their role in oncogenesis evaluated by real-time quantitative PCR were found to be statistically correlated whatever the in vitro culture model used. In conclusion, xenotransplantation in mice of tumor fragments before establishment of cell lines enables generation of more novel human cancer cell lines for investigation of colon cancer cell biology, opening up the opportunity of reproducing the diversity of this disease. PMID:17210723

  18. Adherence of Bordetella bronchiseptica to hamster lung fibroblasts.

    PubMed Central

    Plotkin, B J; Bemis, D A

    1984-01-01

    The adherence of Bordetella bronchiseptica smooth-, intermediate-, and rough-phase isolates to hamster lung fibroblasts (HLF) (Don line) was characterized by competitive inhibition studies and enzyme and chemical treatments of both the bacteria and the HLF. The adherence of the rough- and intermediate-phase isolates (n = 13) was altered by coincubation of the bacteria and HLF with cationic chelators, including EGTA and citrate. EGTA inhibition of the adherence of the rough- and intermediate-phase isolates could be overcome by the addition of Ca2+, Mn2+, Cd2+, or Sr2+ to the reaction mixture. In addition, citrate released bound bacteria from the HLF. Although the adherence of the smooth-phase isolates (n = 4) was unaltered by cationic chelators, binding was inhibited by N-acetylated amino sugars, with N-acetylglucosamine inhibiting 98% of the adherence of the smooth-phase isolates. Homogenization, protease K, and heat treatment (60 min, 60 degrees C) of the bacteria also resulted in a loss of adherence. It was concluded that B. bronchiseptica can adhere to HLF by at least two mechanisms and that the ligand responsible appears to be a proteinacious, heat labile cell surface component. PMID:6437989

  19. Mitigation of Lethal Radiation Syndrome in Mice by Intramuscular Injection of 3D Cultured Adherent Human Placental Stromal Cells.

    PubMed

    Gaberman, Elena; Pinzur, Lena; Levdansky, Lilia; Tsirlin, Maria; Netzer, Nir; Aberman, Zami; Gorodetsky, Raphael

    2013-01-01

    Exposure to high lethal dose of ionizing radiation results in acute radiation syndrome with deleterious systemic effects to different organs. A primary target is the highly sensitive bone marrow and the hematopoietic system. In the current study C3H/HeN mice were total body irradiated by 7.7 Gy. Twenty four hrs and 5 days after irradiation 2×10(6) cells from different preparations of human derived 3D expanded adherent placental stromal cells (PLX) were injected intramuscularly. Treatment with batches consisting of pure maternal cell preparations (PLX-Mat) increased the survival of the irradiated mice from ∼27% to 68% (P<0.001), while cell preparations with a mixture of maternal and fetal derived cells (PLX-RAD) increased the survival to ∼98% (P<0.0001). The dose modifying factor of this treatment for both 50% and 37% survival (DMF50 and DMF37) was∼1.23. Initiation of the more effective treatment with PLX-RAD injection could be delayed for up to 48 hrs after irradiation with similar effect. A delayed treatment by 72 hrs had lower, but still significantly effect (p<0.05). A faster recovery of the BM and improved reconstitution of all blood cell lineages in the PLX-RAD treated mice during the follow-up explains the increased survival of the cells treated irradiated mice. The number of CD45+/SCA1+ hematopoietic progenitor cells within the fast recovering population of nucleated BM cells in the irradiated mice was also elevated in the PLX-RAD treated mice. Our study suggests that IM treatment with PLX-RAD cells may serve as a highly effective "off the shelf" therapy to treat BM failure following total body exposure to high doses of radiation. The results suggest that similar treatments may be beneficial also for clinical conditions associated with severe BM aplasia and pancytopenia. PMID:23823334

  20. Mitigation of Lethal Radiation Syndrome in Mice by Intramuscular Injection of 3D Cultured Adherent Human Placental Stromal Cells

    PubMed Central

    Gaberman, Elena; Pinzur, Lena; Levdansky, Lilia; Tsirlin, Maria; Netzer, Nir; Aberman, Zami; Gorodetsky, Raphael

    2013-01-01

    Exposure to high lethal dose of ionizing radiation results in acute radiation syndrome with deleterious systemic effects to different organs. A primary target is the highly sensitive bone marrow and the hematopoietic system. In the current study C3H/HeN mice were total body irradiated by 7.7 Gy. Twenty four hrs and 5 days after irradiation 2×106 cells from different preparations of human derived 3D expanded adherent placental stromal cells (PLX) were injected intramuscularly. Treatment with batches consisting of pure maternal cell preparations (PLX-Mat) increased the survival of the irradiated mice from ∼27% to 68% (P<0.001), while cell preparations with a mixture of maternal and fetal derived cells (PLX-RAD) increased the survival to ∼98% (P<0.0001). The dose modifying factor of this treatment for both 50% and 37% survival (DMF50 and DMF37) was∼1.23. Initiation of the more effective treatment with PLX-RAD injection could be delayed for up to 48 hrs after irradiation with similar effect. A delayed treatment by 72 hrs had lower, but still significantly effect (p<0.05). A faster recovery of the BM and improved reconstitution of all blood cell lineages in the PLX-RAD treated mice during the follow-up explains the increased survival of the cells treated irradiated mice. The number of CD45+/SCA1+ hematopoietic progenitor cells within the fast recovering population of nucleated BM cells in the irradiated mice was also elevated in the PLX-RAD treated mice. Our study suggests that IM treatment with PLX-RAD cells may serve as a highly effective “off the shelf” therapy to treat BM failure following total body exposure to high doses of radiation. The results suggest that similar treatments may be beneficial also for clinical conditions associated with severe BM aplasia and pancytopenia. PMID:23823334

  1. High incidence of TERT mutation in brain tumor cell lines.

    PubMed

    Johanns, Tanner M; Fu, Yujie; Kobayashi, Dale K; Mei, Yu; Dunn, Ian F; Mao, Diane D; Kim, Albert H; Dunn, Gavin P

    2016-07-01

    TERT promoter gene mutations are highly recurrent in malignant glioma. However, little information exists regarding their presence in experimental brain tumor models. To better characterize systems in which TERT mutation studies could be appropriately modeled experimentally, the TERT promoter was examined by conventional sequencing in primary brain tumor initiating cells (BTIC), two matched recurrent BTIC lines, a panel of established malignant glioma cell lines, and two meningioma cell lines. Telomerase gene expression was examined by quantitative PCR. We found that all glioblastoma BTIC lines harbored a TERT mutation, which was retained in two patient-matched recurrent BTIC. The TERT C228T or C250T mutation was found in 33/35 (94 %) of established malignant glioma cell lines and both meningioma cell lines examined. Brain tumor cell lines expressed variably high telomerase levels. Thus, a high percentage of glioma cell lines, as well as two meningioma cell lines, harbors TERT mutations. These data characterize tractable, accessible models with which to further explore telomerase biology in these tumor types. PMID:26960334

  2. The alkaloid Berberine inhibits the growth of Anoikis-resistant MCF-7 and MDA-MB-231 breast cancer cell lines by inducing cell cycle arrest.

    PubMed

    Kim, J B; Yu, J-H; Ko, E; Lee, K-W; Song, A K; Park, S Y; Shin, I; Han, W; Noh, D Y

    2010-05-01

    Berberine is a pure phenanthren alkaloid isolated from the roots and bark of herbal plants such as Berberis, Hydrastis canadensis and Coptis chinensis. Berberine has been established to inhibit the growth of breast cancer cells, but its effects on the drug resistance and anoikis-resistance of breast cancer cells have yet to be elucidated. Anoikis, or detachment-induced apoptosis, may prevent cancer progression and metastasis by blocking signals necessary for survival of localized cancer cells. Resistance to anoikis is regarded as a prerequisite for metastasis; however, little is known about the role of berberine in anoikis-resistance. We established anoikis-resistant cells from the breast cancer cell lines MCF-7 and MDA-MB-231 by culturing them on a Poly-Hema substratum. We then investigated the effects of berberine on the growth of these cells. The anoikis-resistant cells had a reduced growth rate and were more invasive than their respective adherent cell lines. The effect of berberine on growth was compared to that of doxorubicine, which is a drug commonly used to treat breast cancer, in both the adherent and anoikis-resistant cell lines. Berberine promoted the growth inhibition of anoikis-resistant cells to a greater extent than doxorubicine treatment. Treatment with berberine-induced cell cycle arrest at G0/G1 in the anoikis-resistant MCF-7 and MDA-MB-231 cells as compared to untreated control cells. In summary, these results revealed that berberine can efficiently inhibit growth by inducing cell cycle arrest in anoikis-resistant MCF-7 and MDA-MB-231 cells. Further analysis of these phenotypes is essential for understanding the effect of berberine on anoikis-resistant breast cancer cells, which would be relevant for the therapeutic targeting of breast cancer metastasis. PMID:19800775

  3. Polyphenolic Profile and Targeted Bioactivity of Methanolic Extracts from Mediterranean Ethnomedicinal Plants on Human Cancer Cell Lines.

    PubMed

    Pollio, Antonino; Zarrelli, Armando; Romanucci, Valeria; Di Mauro, Alfredo; Barra, Federica; Pinto, Gabriele; Crescenzi, Elvira; Roscetto, Emanuela; Palumbo, Giuseppe

    2016-01-01

    The methanol extracts of the aerial part of four ethnomedicinal plants of Mediterranean region, two non-seed vascular plants, Equisetum hyemale L. and Phyllitis scolopendrium (L.) Newman, and two Spermatophyta, Juniperus communis L. (J. communis) and Cotinus coggygria Scop. (C. coggygria), were screened against four human cells lines (A549, MCF7, TK6 and U937). Only the extracts of J. communis and C. coggygria showed marked cytotoxic effects, affecting both cell morphology and growth. A dose-dependent effect of these two extracts was also observed on the cell cycle distribution. Incubation of all the cell lines in a medium containing J. communis extract determined a remarkable accumulation of cells in the G2/M phase, whereas the C. coggygria extract induced a significant increase in the percentage of G1 cells. The novelty of our findings stands on the observation that the two extracts, consistently, elicited coherent effects on the cell cycle in four cell lines, independently from their phenotype, as two of them have epithelial origin and grow adherent and two are lymphoblastoid and grow in suspension. Even the expression profiles of several proteins regulating cell cycle progression and cell death were affected by both extracts. LC-MS investigation of methanol extract of C. coggygria led to the identification of twelve flavonoids (compounds 1-11, 19) and eight polyphenols derivatives (12-18, 20), while in J. communis extract, eight flavonoids (21-28), a α-ionone glycoside (29) and a lignin (30) were found. Although many of these compounds have interesting individual biological activities, their natural blends seem to exert specific effects on the proliferation of cell lines either growing adherent or in suspension, suggesting potential use in fighting cancer. PMID:27023497

  4. In vitro inhibition of calcium oxalate crystallization and crystal adherence to renal tubular epithelial cells by Terminalia arjuna.

    PubMed

    Mittal, A; Tandon, S; Singla, S K; Tandon, C

    2016-04-01

    Urolithiasis is a multifactorial disease and remains a public health problem around the world. Of all types of renal stones, calcium oxalate (CaOx) is the most common composition formed in the urinary system of the patients with urolithiasis. The present study is aimed at evaluating the antiurolithiatic properties of the Tris-Cl extract (TE) of Terminalia arjuna (T. arjuna). The antilithiatic activity of TE of T. arjuna was investigated on nucleation, aggregation, and growth of the CaOx crystals, as well as its protective potency was tested on oxalate-induced cell injury of NRK-52E renal epithelial cells. Also, in vitro antioxidant activity of TE T. arjuna bark was also determined. The TE of T. arjuna exhibited a concentration-dependent inhibition of nucleation and growth of CaOx crystals. Inhibition of aggregation of CaOx crystals remains constant. When NRK-52E cells were injured by exposure to oxalate for 48 h, the TE prevented the cells from injury and CaOx crystal adherence resulting in increased cell viability in a dose-dependent manner. The TE also scavenged the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals with an IC50 at 51.72 µg/mL. The results indicated that T. arjuna is a potential candidate for phytotherapy against urolithiasis as it attains the ability to inhibit CaOx crystallization and scavenge DPPH free radicals in vitro along with a cytoprotective role. PMID:26424092

  5. Authentication of the R06E Fruit Bat Cell Line

    PubMed Central

    Jordan, Ingo; Munster, Vincent J.; Sandig, Volker

    2012-01-01

    Fruit bats and insectivorous bats are believed to provide a natural reservoir for a wide variety of infectious diseases. Several lines of evidence, including the successful isolation of infectious viruses, indicate that Marburg virus and Ravn virus have found a major reservoir in colonies of the Egyptian rousette (Rousettus aegyptiacus). To facilitate molecular studies on virus-reservoir host interactions and isolation of viruses from environmental samples, we established cell lines from primary cells of this animal. The cell lines were given to several laboratories until we realized that a contamination with Vero cells in one of the cultures had occurred. Here we describe a general diagnostic procedure for identification of cross-species contamination with the focus on Vero and Rousettus cell lines, and summarize newly discovered properties of the cell lines that may pertain to pathogen discovery. PMID:22754654

  6. 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 ...

  7. Human Rhabdomyosarcoma Cell Lines for Rhabdomyosarcoma Research: Utility and Pitfalls

    PubMed Central

    Hinson, Ashley R. P.; Jones, Rosanne; Crose, Lisa E. S.; Belyea, Brian C.; Barr, Frederic G.; Linardic, Corinne M.

    2013-01-01

    Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence. Despite intergroup clinical trials conducted in Europe and North America, outcomes for high risk patients with this disease have not significantly improved in the last several decades, and survival of metastatic or relapsed disease remains extremely poor. Accrual into new clinical trials is slow and difficult, so in vitro cell-line research and in vivo xenograft models present an attractive alternative for preclinical research for this cancer type. Currently, 30 commonly used human RMS cell lines exist, with differing origins, karyotypes, histologies, and methods of validation. Selecting an appropriate cell line for RMS research has important implications for outcomes. There are also potential pitfalls in using certain cell lines including contamination with murine stromal cells, cross-contamination between cell lines, discordance between the cell line and its associated original tumor, imposter cell lines, and nomenclature errors that result in the circulation of two or more presumed unique cell lines that are actually from the same origin. These pitfalls can be avoided by testing for species-specific isoenzymes, microarray analysis, assays for subtype-specific fusion products, and short tandem repeat analysis. PMID:23882450

  8. 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

  9. 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

  10. Establishment of the first humpback whale fibroblast cell lines and their application in chemical risk assessment.

    PubMed

    Burkard, Michael; Whitworth, Deanne; Schirmer, Kristin; Nash, Susan Bengtson

    2015-10-01

    This paper reports the first successful derivation and characterization of humpback whale fibroblast cell lines. Primary fibroblasts were isolated from the dermal connective tissue of skin biopsies, cultured at 37 °C and 5% CO2 in the standard mammalian medium DMEM/F12 supplemented with 10% fetal bovine serum (FBS). Of nine initial biopsies, two cell lines were established from two different animals and designated HuWa1 and HuWa2. The cells have a stable karyotype with 2n=44, which has commonly been observed in other baleen whale species. Cells were verified as being fibroblasts based on their spindle-shaped morphology, adherence to plastic and positive immunoreaction to vimentin. Population doubling time was determined to be ∼41 h and cells were successfully cryopreserved and thawed. To date, HuWa1 cells have been propagated 30 times. Cells proliferate at the tested temperatures, 30, 33.5 and 37 °C, but show the highest rate of proliferation at 37 °C. Short-term exposure to para,para'-dichlorodiphenyldichloroethylene (p,p'-DDE), a priority compound accumulating in southern hemisphere humpback whales, resulted in a concentration-dependent loss of cell viability. The effective concentration which caused a 50% reduction in HuWa1 cell viability (EC50 value) was approximately six times greater than the EC50 value for the same chemical measured with human dermal fibroblasts. HuWa1 exposed to a natural, p,p'-DDE-containing, chemical mixture extracted from whale blubber showed distinctively higher sensitivity than to p,p'-DDE alone. Thus, we provide the first cytotoxicological data for humpback whales and with establishment of the HuWa cell lines, a unique in vitro model for the study of the whales' sensitivity and cellular response to chemicals and other environmental stressors. PMID:26363275

  11. 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

  12. Non-Viral Transfection Methods Optimized for Gene Delivery to a Lung Cancer Cell Line

    PubMed Central

    Salimzadeh, Loghman; Jaberipour, Mansooreh; Hosseini, Ahmad; Ghaderi, Abbas

    2013-01-01

    Background Mehr-80 is a newly established adherent human large cell lung cancer cell line that has not been transfected until now. This study aims to define the optimal transfection conditions and effects of some critical elements for enhancing gene delivery to this cell line by utilizing different non-viral transfection Procedures. Methods In the current study, calcium phosphate (CaP), DEAE-dextran, superfect, electroporation and lipofection transfection methods were used to optimize delivery of a plasmid construct that expressed Green Fluorescent Protein (GFP). Transgene expression was detected by fluorescent microscopy and flowcytometry. Toxicities of the methods were estimated by trypan blue staining. In order to evaluate the density of the transfected gene, we used a plasmid construct that expressed the Stromal cell-Derived Factor-1 (SDF-1) gene and measured its expression by real-time PCR. Results Mean levels of GFP-expressing cells 48 hr after transfection were 8.4% (CaP), 8.2% (DEAE-dextran), 4.9% (superfect), 34.1% (electroporation), and 40.1% (lipofection). Lipofection had the highest intense SDF-1 expression of the analyzed methods. Conclusion This study has shown that the lipofection and electroporation methods were more efficient at gene delivery to Mehr-80 cells. The quantity of DNA per transfection, reagent concentration, and incubation time were identified as essential factors for successful transfection in all of the studied methods. PMID:23799175

  13. 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). PMID:25604950

  14. AB241. Cancer stem cell-like side population cells in clear cell renal cell carcinoma cell line 769P

    PubMed Central

    Huang, Bin; Wang, Dao-Hu; Chen, Jun-Xing; Qiu, Shao-Peng

    2016-01-01

    Background Although cancers are widely considered to be maintained by stem cells, the existence of stem cells in renal cell carcinoma (RCC) has seldom been reported, in part due to the lack of unique surface markers. We here identified cancer stem cell-like cells with side population (SP) phenotype in five human RCC cell lines. Methods We here identified cancer stem cell-like cells with side population (SP) phenotype in five human RCC cell lines. Results Flow cytometry analysis revealed that 769P, a human clear cell RCC cell line, contained the largest amount of SP cells among five cell lines. These 769P SP cells possessed characteristics of proliferation, self-renewal, and differentiation, as well as strong resistance to chemotherapy and radiotherapy that were possibly related to the ABCB1 transporter. In vivo experiments with serial tumor transplantation in mice also showed that 769P SP cells formed tumors in NOD/SCID mice. Conclusions Taken together, these results indicate that 769P SP cells have the properties of cancer stem cells, which may play important roles in tumorigenesis and therapy-resistance of RCC.

  15. Permissiveness of human hepatoma cell lines for HCV infection

    PubMed Central

    2012-01-01

    Background Although primary and established human hepatoma cell lines have been evaluated for hepatitis C virus (HCV) infection in vitro, thus far only Huh7 cells have been found to be highly permissive for infectious HCV. Since our understanding of the HCV lifecycle would benefit from the identification of additional permissive cell lines, we assembled a panel of hepatic and non-hepatic cell lines and assessed their ability to support HCV infection. Here we show infection of the human hepatoma cell lines PLC/PRF/5 and Hep3B with cell culture-derived HCV (HCVcc), albeit to lower levels than that achieved in Huh7 cells. To better understand the reduced permissiveness of PLC and Hep3B cells for HCVcc infection, we performed studies to evaluate the ability of each cell line to support specific steps of the viral lifecycle (i.e. entry, replication, egress and spread). Results We found that while the early events in HCV infection (i.e. entry plus replication initiation) are cumulatively equivalent or only marginally reduced in PLC and Hep3B cells, later steps of the viral life cycle such as steady-state replication, de novo virus production and/or spread are impaired to different degrees in PLC and Hep3B cultures compared to Huh7 cell cultures. Interestingly, we also observed that interferon stimulated gene (i.e. ISG56) expression was significantly and differentially up-regulated in PLC and Hep3B cells following viral infection. Conclusions We conclude that the restrictions observed later during HCV infection in these cell lines could in part be attributed to HCV-induced innate signaling. Nevertheless, the identification of two new cell lines capable of supporting authentic HCVcc infection, even at reduced levels, expands the current repertoire of cell lines amendable for the study of HCV in vitro and should aid in further elucidating HCV biology and the cellular determinants that modulate HCV infection. PMID:22273112

  16. Regulated expression of erythropoietin by two human hepatoma cell lines

    SciTech Connect

    Goldberg, M.A.; Glass, G.A.; Cunningham, J.M.; Bunn, H.F.

    1987-11-01

    The development of a cell culture system that produces erythropoietin (Epo) in a regulated manner has been the focus of much effort. The authors have screened multiple renal and hepatic cell lines for either constitutive or regulated expression of Epo. Only the human hepatoma cell lines, Hep3B and HepG2, made significant amounts of Epo as measured both by radioimmunoassay and in vitro bioassay (as much as 330 milliunits per 10/sup 6/ cells in 24 hr). The constitutive production of Epo increased dramatically as a function of cell density in both cell lines. At cell densities < 3.3 x 10/sup 5/ cells per cm/sup 2/, there was little constitutive release of Epo in the medium. With Hep3B cells grown at low cell densities, a mean 18-fold increase in Epo expression was seen in response to hypoxia and a 6-fold increase was observed in response to incubation in medium containing 50 ..mu..M cobalt(II) chloride. At similar low cell densities, Epo production in HepG2 cells could be enhanced an average of about 3-fold by stimulation with either hypoxia or cobalt(II) chloride. Upon such stimulation, both cell lines demonstrated markedly elevated levels of Epo mRNA. Hence, both Hep3B and HepG2 cell lines provide an excellent in vitro system in which to study the physiological regulation of Epo expression.

  17. Derivation of the human embryonic stem cell line RCM1.

    PubMed

    De Sousa, P A; Tye, B J; Sneddon, S; Bruce, K; Dand, P; Russell, G; Collins, D M; Greenshields, A; McDonald, K; Bradburn, H; Gardner, J; Downie, J M; Courtney, A; Brison, D R

    2016-03-01

    The human embryonic stem cell line RCM-1 was derived from a failed to fertilise egg undergoing parthenogenetic stimulation. The cell line shows normal pluripotency marker expression and differentiation to three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available. PMID:27346018

  18. Trichloroethylene toxicity in a human hepatoma cell line

    SciTech Connect

    Thevenin, E.; McMillian, J.

    1994-12-31

    The experiments conducted in this study were designed to determine the usefullness of hepatocyte cultures and a human hepatoma cell line as model systems for assessing human susceptibility to hepatocellular carcinoma due to exposure to trichloroethylene. The results from these studies will then be analyzed to determine if human cell lines can be used to conduct future experiments of this nature.

  19. GREG cells, a dysferlin-deficient myogenic mouse cell line

    SciTech Connect

    Humphrey, Glen W.; Mekhedov, Elena; Blank, Paul S.; Morree, Antoine de; Pekkurnaz, Gulcin; Nagaraju, Kanneboyina; Zimmerberg, Joshua

    2012-01-15

    The dysferlinopathies (e.g. LGMD2b, Myoshi myopathy) are progressive, adult-onset muscle wasting syndromes caused by mutations in the gene coding for dysferlin. Dysferlin is a large ({approx} 200 kDa) membrane-anchored protein, required for maintenance of plasmalemmal integrity in muscle fibers. To facilitate analysis of dysferlin function in muscle cells, we have established a dysferlin-deficient myogenic cell line (GREG cells) from the A/J mouse, a genetic model for dysferlinopathy. GREG cells have no detectable dysferlin expression, but proliferate normally in growth medium and fuse into functional myotubes in differentiation medium. GREG myotubes exhibit deficiencies in plasma membrane repair, as measured by laser wounding in the presence of FM1-43 dye. Under the wounding conditions used, the majority ({approx} 66%) of GREG myotubes lack membrane repair capacity, while no membrane repair deficiency was observed in dysferlin-normal C2C12 myotubes, assayed under the same conditions. We discuss the possibility that the observed heterogeneity in membrane resealing represents genetic compensation for dysferlin deficiency.

  20. The effects of oncolytic reovirus in canine lymphoma cell lines.

    PubMed

    Hwang, C C; Umeki, S; Igase, M; Coffey, M; Noguchi, S; Okuda, M; Mizuno, T

    2016-08-01

    Reovirus is a potent oncolytic virus in many human neoplasms that has reached phase II and III clinical trials. Our laboratory has previously reported the oncolytic effects of reovirus in canine mast cell tumour (MCT). In order to further explore the potential of reovirus in veterinary oncology, we tested the susceptibility of reovirus in 10 canine lymphoma cell lines. Reovirus-induced cell death, virus replication and infectivity were confirmed in four cell lines with variable levels of susceptibility. The level of Ras activation varied among the cell lines with no correlation with reovirus susceptibility. Reovirus-susceptible cell lines underwent apoptosis as proven by propidium iodide (PI) staining, Annexin V-FITC/PI assay, cleavage of PARP and inhibition of cell death by caspase inhibitor. A single intratumoral injection of reovirus suppressed the growth of canine lymphoma subcutaneous tumour in NOD/SCID mice. Unlike canine MCT, canine lymphoma is less susceptible to reovirus. PMID:25319493

  1. Use of designed experiments for the improvement of pre-analytical workflow for the quantification of intracellular nucleotides in cultured cell lines.

    PubMed

    Machon, Christelle; Bordes, Claire; Cros-Perrial, Emeline; Clement, Yohann; Jordheim, Lars Petter; Lanteri, Pierre; Guitton, Jérôme

    2015-07-31

    The present study is focused on the development of a pre-analytical strategy for the quantification of intracellular nucleotides from cultured cell lines. Different protocols, including cell recovery, nucleotide extraction and purification, were compared on a panel of nucleoside mono-, di- and triphosphates from four cell lines (adherent and suspension cells). The quantification of nucleotides was performed using a validated technique with on-line solid-phase extraction coupled with liquid chromatography-triple quadrupole tandem mass spectrometry (LC-MS/MS). Designed experiments were implemented to investigate, in a rigorous and limited-testing experimental approach, the influence of several operating parameters. Results showed that the technique used to harvest adherent cells drastically affected the amounts of intracellular nucleotides. Scraping cells was deleterious because of a major leakage (more than 70%) of intracellular nucleotides during scraping. Moreover, some other tested conditions should be avoided, such as using pure methanol as extraction solvent (decrease over 50% of intracellular nucleotides extracted from NCI-H292 cells) or adding a purification step with chloroform. Designed experiments allowed identifying an interaction between the percentage of methanol and the presence of chloroform. The mixture methanol/water (70/30, v/v) was considered as the best compromise according to the nucleoside mono-, di-, or triphosphates and the four cell lines studied. This work highlights the importance of pre-analytical step combined with the cell lines studied associated to sensitive and validated assay for the quantification of nucleotides in biological matrices. PMID:26094139

  2. CACO-2 CELL LINES IN DRUG DISCOVERY- AN UPDATED PERSPECTIVE

    PubMed Central

    Kumar, Kalyan K.V; Karnati, Swathi; Reddy, Mamatha B; Chandramouli, R

    2010-01-01

    Cell lines are the invitro models used for the drug permeability studies in the preclinical and clinical phases of the drug discovery. Cell line models are simple and quick to use and avoids the usage of animal models for pharmacological and toxicological studies and hence cost effective, produce reliable and reproducible results for understanding and evaluating the permeability characteristics of the potential lead drug candidates. Different cell line models used in the drug permeability studies, their characteristics has been summarized emphasizing on CACO-2. By virtue of its merits, CACO-2 cell line development, transport experiments, automated assays, optimization of experimental conditions and mechanistic uses of CACO-2 cell lines dealt comprehensively in the following context. PMID:24825967

  3. Rapidly Self-Renewing Human Multipotent Marrow Stromal Cells (hMSC) Express Sialyl Lewis X and Actively Adhere to Arterial Endothelium in a Chick Embryo Model System

    PubMed Central

    McFerrin, Harris E.; Olson, Scott D.; Gutschow, Miriam V.; Semon, Julie A.; Sullivan, Deborah E.; Prockop, Darwin J.

    2014-01-01

    Background There have been conflicting observations regarding the receptors utilized by human multipotent mesenchymal bone marrow stromal cells (hMSC) to adhere to endothelial cells (EC). To address the discrepancies, we performed experiments with cells prepared with a standardized, low-density protocol preserving a sub-population of small cells that are rapidly self-renewing. Methods Sialyl Lewis X (SLeX) and α4 integrin expression were determined by flow cytometry. Fucosyltransferase expression was determined by quantitative realtime RT-PCR. Cell adhesion assays were carried out with a panel of endothelial cells from arteries, veins and the microvasculature in vitro. In vivo experiments were performed to determine single cell interactions in the chick embryo chorioallantoic membrane (CAM). The CAM is a well-characterized respiratory organ allowing for time-lapse image acquisition of large numbers of cells treated with blocking antibodies against adhesion molecules expressed on hMSC. Results hMSC expressed α4 integrin, SLeX and fucosyltransferase 4 and adhered to human EC from arteries, veins and the microvasculature under static conditions in vitro. In vivo, hMSC rolled on and adhered to arterioles in the chick embryo CAM, whereas control melanoma cells embolized. Inhibition of α4 integrin and/or SLeX with blocking antibodies reduced rolling and adhesion in arterioles and increased embolism of hMSC. Conclusions The results demonstrated that rapidly self-renewing hMSC were retained in the CAM because they rolled on and adhered to respiratory arteriolar EC in an α4 integrin- and SLeX-dependent manner. It is therefore important to select cells based on their cell adhesion receptor profile as well as size depending on the intended target of the cell and the injection route. PMID:25144321

  4. Human Placenta-Derived Adherent Cells Prevent Bone loss, Stimulate Bone formation, and Suppress Growth of Multiple Myeloma in Bone

    PubMed Central

    Li, Xin; Ling, Wen; Pennisi, Angela; Wang, Yuping; Khan, Sharmin; Heidaran, Mohammad; Pal, Ajai; Zhang, Xiaokui; He, Shuyang; Zeitlin, Andy; Abbot, Stewart; Faleck, Herbert; Hariri, Robert; Shaughnessy, John D.; van Rhee, Frits; Nair, Bijay; Barlogie, Bart; Epstein, Joshua; Yaccoby, Shmuel

    2011-01-01

    Human placenta has emerged as a valuable source of transplantable cells of mesenchymal and hematopoietic origin for multiple cytotherapeutic purposes, including enhanced engraftment of hematopoietic stem cells, modulation of inflammation, bone repair, and cancer. Placenta-derived adherent cells (PDACs) are mesenchymal-like stem cells isolated from postpartum human placenta. Multiple myeloma is closely associated with induction of bone disease and large lytic lesions, which are often not repaired and are usually the sites of relapses. We evaluated the antimyeloma therapeutic potential, in vivo survival, and trafficking of PDACs in the severe combined immunodeficiency (SCID)–rab model of medullary myeloma-associated bone loss. Intrabone injection of PDACs into non-myelomatous and myelomatous implanted bone in SCID-rab mice promoted bone formation by stimulating endogenous osteoblastogenesis, and most PDACs disappeared from bone within 4 weeks. PDACs inhibitory effects on myeloma bone disease and tumor growth were dose-dependent and comparable with those of fetal human mesenchymal stem cells (MSCs). Intrabone, but not subcutaneous, engraftment of PDACs inhibited bone disease and tumor growth in SCID-rab mice. Intratumor injection of PDACs had no effect on subcutaneous growth of myeloma cells. A small number of intravenously injected PDACs trafficked into myelomatous bone. Myeloma cell growth rate in vitro was lower in coculture with PDACs than with MSCs from human fetal bone or myeloma patients. PDACs also promoted apoptosis in osteoclast precursors and inhibited their differentiation. This study suggests that altering the bone marrow microenvironment with PDAC cytotherapy attenuates growth of myeloma and that PDAC cytotherapy is a promising therapeutic approach for myeloma osteolysis. PMID:21732484

  5. Acute Shear Stress Direction Dictates Adherent Cell Remodeling and Verifies Shear Profile of Spinning Disc Assays

    PubMed Central

    Fuhrmann, Alexander; Engler, Adam J.

    2015-01-01

    Several methods have been developed to quantify population level changes in cell attachment strength given its large heterogeneity. One such method is the rotating disc chamber or “spinning disc” in which a range of shear forces are applied to attached cells to quantify detachment force, i.e. attachment strength, which can be heterogeneous within cell populations. However, computing the exact force vectors that act upon cells is complicated by complex flow fields and variable cell morphologies. Recent observations suggest that cells may remodel their morphology and align during acute shear exposure, but contrary to intuition, shear is not orthogonal to the radial direction. Here we theoretically derive the magnitude and direction of applied shear and demonstrate that cells, under certain physiological conditions, align in this direction within minutes. Shear force magnitude is also experimentally verified which validates that for spread cells shear forces and not torque or drag dominate in this assay, and demonstrates that the applied force per cell area is largely independent of initial morphology. These findings suggest that direct quantified comparison of the effects of shear on a wide array of cell types and conditions can be made with confidence using this assay without the need for computational or numerical modeling. PMID:25619322

  6. Derivation of Genea057 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-01-01

    The Genea057 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype and female allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea057 was demonstrated with 97% of cells expressing Nanog, 81% Oct4, 75% Tra1-60 and 97% SSEA4, a PluriTest Pluripotency score of 27.59 and Novelty score of 1.32. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345782

  7. Derivation of Genea042 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea042 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype and female allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea042 was demonstrated with 81% of cells expressing Nanog, 95% Oct4, 53% Tra1-60 and 97% SSEA4, a PluriTest Pluripotency score of 30.06, Novelty score of 1.24 and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345994

  8. Derivation of Genea002 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Bosman, Alexis; McKernan, Robert; Goel, Divya; Peura, Teija; Schmidt, Uli

    2016-01-01

    The Genea002 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype by CGH and male Allele pattern through STR analysis. Pluripotency of Genea002 was demonstrated with 75% of cells expressing Nanog, 93% Oct4, 83% Tra1-60 and 98% SSEA4, a Pluritest pluripotency score of 24.55, Novelty score of 1.39, teratomas with tissues from all embryonic germ layers and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345802

  9. Derivation of Genea052 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea052 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea052 was demonstrated with 85% of cells expressing Nanog, 87% Oct4, 60% Tra1-60 and 97% SSEA4, a PluriTest Pluripotency score of 27.21, Novelty score of 1.2 and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345996

  10. Derivation of human embryonic stem cell line Genea023.

    PubMed

    Dumevska, Biljana; Bosman, Alexis; McKernan, Robert; Goel, Divya; Schmidt, Uli; Peura, Teija

    2016-03-01

    The Genea023 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea023 was demonstrated with 85% of cells expressed Nanog, 98% Oct4, 55% Tra1-60 and 98% SSEA4, gave a Pluritest Pluripotency score of 42.76, Novelty of 1.23, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination. PMID:27346015

  11. Derivation of Genea015 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea015 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male Allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea015 was demonstrated with 80% of cells expressing Nanog, 97% Oct4, 75% Tra1-60 and 98% SSEA4, a PluriTest Pluripotency score of 29.52, Novelty score of 1.3 and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27346028

  12. Derivation of human embryonic stem cell line Genea022.

    PubMed

    Dumevska, Biljana; Bosman, Alexis; McKernan, Robert; Schmidt, Uli; Peura, Teija

    2016-03-01

    The Genea022 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea022 was demonstrated with 84% of cells expressed Nanog, 98% Oct4, 55% Tra1-60 and 97% SSEA4, gave a Pluritest Pluripotency score of 42.95, Novelty of 1.23, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination. PMID:27346017

  13. Derivation of Genea047 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea047 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype and female allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea047 was demonstrated with 88% of cells expressing Nanog, 95% Oct4, 59% Tra1-60 and 99% SSEA4, a PluriTest Pluripotency score of 30.86, Novelty score of 1.23 and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345995

  14. Derivation of Genea043 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-01-01

    The Genea043 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea043 was demonstrated with 92% of cells expressing Nanog, 95% Oct4, 61% Tra1-60 and 99% SSEA4, a PluriTest Pluripotency score of 31.74, Novelty score of 1.2 and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345801

  15. Derivation of Genea016 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Peura, Teija; Schmidt, Uli

    2016-01-01

    The Genea016 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype and female Allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea016 was demonstrated with 77% of cells expressing Nanog, 95% Oct4, 53% Tra1-60 and 98% SSEA4, a PluriTest Pluripotency score of 28.4, Novelty score of 1.37 and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345780

  16. Regulatory networks define phenotypic classes of human stem cell lines

    PubMed Central

    Müller, Franz-Josef; Laurent, Louise C.; Kostka, Dennis; Ulitsky, Igor; Williams, Roy; Lu, Christina; Park, In-Hyun; Rao, Mahendra S.; Shamir, Ron; Schwartz, Philip H.; Schmidt, Nils O.; Loring, Jeanne F.

    2008-01-01

    Stem cells are defined as self-renewing cell populations that can differentiate into multiple distinct cell types. However, hundreds of different human cell lines from embryonic, fetal, and adult sources have been called stem cells, even though they range from pluripotent cells, typified by embryonic stem cells, which are capable of virtually unlimited proliferation and differentiation, to adult stem cell lines, which can generate a far more limited repertory of differentiated cell types. The rapid increase in reports of new sources of stem cells and their anticipated value to regenerative medicine1, 2 have highlighted the need for a general, reproducible method for classification of these cells3. We report here the creation and analysis of a database of global gene expression profiles (“Stem Cell Matrix”) that enables the classification of cultured human stem cells in the context of a wide variety of pluripotent, multipotent, and differentiated cell types. Using an unsupervised clustering method4, 5 to categorize a collection of ~150 cell samples, we discovered that pluripotent stem cell lines group together, while other cell types, including brain-derived neural stem cell lines, are very diverse. Using further bioinformatic analysis6 we uncovered a protein-protein network (“PluriNet”) that is shared by the pluripotent cells (embryonic stem cells, embryonal carcinomas, and induced pluripotent cells). Analysis of published data showed that the PluriNet appears to be a common characteristic of pluripotent cells, including mouse ES and iPS cells and human oocytes. Our results offer a new strategy for classifying stem cells and support the idea that pluripotence and self-renewal are under tight control by specific molecular networks. PMID:18724358

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. A functional assay for gap junctional examination; electroporation of adherent cells on indium-tin oxide.

    PubMed

    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

  2. AFBI assay - Aptamer Fluorescence Binding and Internalization assay for cultured adherent cells.

    PubMed

    Thiel, William H; Giangrande, Paloma H

    2016-07-01

    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

  3. Recombinant protein production from stable mammalian cell lines and pools.

    PubMed

    Hacker, David L; Balasubramanian, Sowmya

    2016-06-01

    We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced. PMID:27322762

  4. Functional calcium imaging in zebrafish lateral-line hair cells.

    PubMed

    Zhang, Q X; He, X J; Wong, H C; Kindt, K S

    2016-01-01

    Sensory hair-cell development, function, and regeneration are fundamental processes that are challenging to study in mammalian systems. Zebrafish are an excellent alternative model to study hair cells because they have an external auxiliary organ called the lateral line. The hair cells of the lateral line are easily accessible, which makes them suitable for live, function-based fluorescence imaging. In this chapter, we describe methods to perform functional calcium imaging in zebrafish lateral-line hair cells. We compare genetically encoded calcium indicators that have been used previously to measure calcium in lateral-line hair cells. We also outline equipment required for calcium imaging and compare different imaging systems. Lastly, we discuss how to set up optimal imaging parameters and how to process and visualize calcium signals. Overall, using these methods, in vivo calcium imaging is a powerful tool to examine sensory hair-cell function in an intact organism. PMID:27263415

  5. Novel human bronchial epithelial cell lines for cystic fibrosis research

    PubMed Central

    Fulcher, M. L.; Gabriel, S. E.; Olsen, J. C.; Tatreau, J. R.; Gentzsch, M.; Livanos, E.; Saavedra, M. T.; Salmon, P.; Randell, S. H.

    2009-01-01

    Immortalization of human bronchial epithelial (hBE) cells often entails loss of differentiation. Bmi-1 is a protooncogene that maintains stem cells, and its expression creates cell lines that recapitulate normal cell structure and function. We introduced Bmi-1 and the catalytic subunit of telomerase (hTERT) into three non-cystic fibrosis (CF) and three ΔF508 homozygous CF primary bronchial cell preparations. This treatment extended cell life span, although not as profoundly as viral oncogenes, and at passages 14 and 15, the new cell lines had a diploid karyotype. Ussing chamber analysis revealed variable transepithelial resistances, ranging from 200 to 1,200 Ω·cm2. In the non-CF cell lines, short-circuit currents were stimulated by forskolin and inhibited by CFTR(inh)-172 at levels mostly comparable to early passage primary cells. CF cell lines exhibited no forskolin-stimulated current and minimal CFTR(inh)-172 response. Amiloride-inhibitable and UTP-stimulated currents were present, but at lower and higher amplitudes than in primary cells, respectively. The cells exhibited a pseudostratified morphology, with prominent apical membrane polarization, few apoptotic bodies, numerous mucous secretory cells, and occasional ciliated cells. CF and non-CF cell lines produced similar levels of IL-8 at baseline and equally increased IL-8 secretion in response to IL-1β, TNF-α, and the Toll-like receptor 2 agonist Pam3Cys. Although they have lower growth potential and more fastidious growth requirements than viral oncogene transformed cells, Bmi-1/hTERT airway epithelial cell lines will be useful for several avenues of investigation and will help fill gaps currently hindering CF research and therapeutic development. PMID:18978040

  6. Antibody against the Carboxyl Terminus of Intimin α Reduces Enteropathogenic Escherichia coli Adherence to Tissue Culture Cells and Subsequent Induction of Actin Polymerization

    PubMed Central

    Carvalho, Humberto M.; Teel, Louise D.; Kokai-Kun, John F.; O'Brien, Alison D.

    2005-01-01

    The C-terminal third of intimin binds to its translocated receptor (Tir) to promote attaching and effacing lesion formation during infection with enteropathogenic Escherichia coli (EPEC). We observed that the adherence of EPEC strains to HEp-2 cells was reduced and that actin polymerization was blocked by antibody raised against the C-terminal third of intimin α. PMID:15784601

  7. 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...

  8. 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

  9. Invitro study of adherent mandibular osteoblast-like cells on carrier materials.

    PubMed

    Turhani, D; Weissenböck, M; Watzinger, E; Yerit, K; Cvikl, B; Ewers, R; Thurnher, D

    2005-07-01

    Augmentation of the craniofacial region is necessary for many aesthetic and reconstructive procedures. Tissue engineering offers a new option to supplement existing treatment regimens. In this procedure, materials composed of hydroxyapatite (HA), of synthetic or natural origin, are used as scaffolds. The aim of this study was to evaluate the effects of three HA materials on cultured human osteoblasts in vitro. Explant cultures of cells from human alveolar bone were established. Human osteoblasts were cultured on the surface of HA calcified from red algae (C GRAFT/Algipore), deproteinized bovine HA (Bio-Oss) and bovine HA carrying the cell binding peptide P-15 (Pep Gen P-15). Cultured cells were evaluated with respect to cell attachment, proliferation and differentiation. Cells were cultured for 6 and 21 days under osteogenic differentiation conditions, and tissue-culture polystyrene dishes were used as control. The ability of cells to proliferate and form extracellular matrix on these scaffolds was assessed by a DNA quantification assay, protein synthesis analysis and by scanning electron microscopical examination. Osteogenic differentiation was screened by the expression of alkaline phosphatase. The osteoblastic phenotype of the cells was monitored using mRNA levels of the bone-related proteins including osteocalcin, osteopontin and collagen Type I. We found that cells cultured on C GRAFT/Algipore) and Pep Gen P-15 showed a continuous increase in DNA content and protein synthesis. Cells cultured on Bio-Oss showed a decrease in DNA content from Day 6 (P < 0.05) to Day 21 (P < 0.0001) and protein synthesis on Day 21 (P < 0.005). Alkaline phosphatase activity increased in cells grown on C GRAFT/Algipore and Pep Gen P-15 in contrast to cells grown on Bio-Oss, in which the lowest levels of activity could be observed on Day 21 (P < 0.05). Reverse transcriptase polymerase chain reaction analysis confirmed the osteoblastic phenotype of the cells grown on all three

  10. Generation and characterization of human insulin-releasing cell lines

    PubMed Central

    Labriola, Leticia; Peters, Maria G; Krogh, Karin; Stigliano, Iván; Terra, Letícia F; Buchanan, Cecilia; Machado, Marcel CC; Joffé, Elisa Bal de Kier; Puricelli, Lydia; Sogayar, Mari C

    2009-01-01

    Background The in vitro culture of insulinomas provides an attractive tool to study cell proliferation and insulin synthesis and secretion. However, only a few human beta cell lines have been described, with long-term passage resulting in loss of insulin secretion. Therefore, we set out to establish and characterize human insulin-releasing cell lines. Results We generated ex-vivo primary cultures from two independent human insulinomas and from a human nesidioblastosis, all of which were cultured up to passage number 20. All cell lines secreted human insulin and C-peptide. These cell lines expressed neuroendocrine and islets markers, confirming the expression profile found in the biopsies. Although all beta cell lineages survived an anchorage independent culture, none of them were able to invade an extracellular matrix substrate. Conclusion We have established three human insulin-releasing cell lines which maintain antigenic characteristics and insulin secretion profiles of the original tumors. These cell lines represent valuable tools for the study of molecular events underlying beta cell function and dysfunction. PMID:19545371

  11. Sialidases Affect the Host Cell Adherence and Epsilon Toxin-Induced Cytotoxicity of Clostridium perfringens Type D Strain CN3718

    PubMed Central

    Li, Jihong; Sayeed, Sameera; Robertson, Susan; Chen, Jianming; McClane, Bruce A.

    2011-01-01

    Clostridium perfringens type B or D isolates, which cause enterotoxemias or enteritis in livestock, produce epsilon toxin (ETX). ETX is exceptionally potent, earning it a listing as a CDC class B select toxin. Most C. perfringens strains also express up to three different sialidases, although the possible contributions of those enzymes to type B or D pathogenesis remain unclear. Type D isolate CN3718 was found to carry two genes (nanI and nanJ) encoding secreted sialidases and one gene (nanH) encoding a cytoplasmic sialidase. Construction in CN3718 of single nanI, nanJ and nanH null mutants, as well as a nanI/nanJ double null mutant and a triple sialidase null mutant, identified NanI as the major secreted sialidase of this strain. Pretreating MDCK cells with NanI sialidase, or with culture supernatants of BMC206 (an isogenic CN3718 etx null mutant that still produces sialidases) enhanced the subsequent binding and cytotoxic effects of purified ETX. Complementation of BMC207 (an etx/nanH/nanI/nanJ null mutant) showed this effect is mainly attributable to NanI production. Contact between BMC206 and certain mammalian cells (e.g., enterocyte-like Caco-2 cells) resulted in more rapid sialidase production and this effect involved increased transcription of BMC206 nanI gene. BMC206 was shown to adhere to some (e.g. Caco-2 cells), but not all mammalian cells, and this effect was dependent upon sialidase, particularly NanI, expression. Finally, the sialidase activity of NanI (but not NanJ or NanH) could be enhanced by trypsin. Collectively these in vitro findings suggest that, during type D disease originating in the intestines, trypsin may activate NanI, which (in turn) could contribute to intestinal colonization by C. perfringens type D isolates and also increase ETX action. PMID:22174687

  12. Investigation of Radiosensitivity Gene Signatures in Cancer Cell Lines

    PubMed Central

    Hall, John S.; Iype, Rohan; Senra, Joana; Taylor, Janet; Armenoult, Lucile; Oguejiofor, Kenneth; Li, Yaoyong; Stratford, Ian; Stern, Peter L.; O’Connor, Mark J.; Miller, Crispin J.; West, Catharine M. L.

    2014-01-01

    Intrinsic radiosensitivity is an important factor underlying radiotherapy response, but there is no method for its routine assessment in human tumours. Gene signatures are currently being derived and some were previously generated by expression profiling the NCI-60 cell line panel. It was hypothesised that focusing on more homogeneous tumour types would be a better approach. Two cell line cohorts were used derived from cervix [n = 16] and head and neck [n = 11] cancers. Radiosensitivity was measured as surviving fraction following irradiation with 2 Gy (SF2) by clonogenic assay. Differential gene expression between radiosensitive and radioresistant cell lines (SF2 median) was investigated using Affymetrix GeneChip Exon 1.0ST (cervix) or U133A Plus2 (head and neck) arrays. There were differences within cell line cohorts relating to tissue of origin reflected by expression of the stratified epithelial marker p63. Of 138 genes identified as being associated with SF2, only 2 (1.4%) were congruent between the cervix and head and neck carcinoma cell lines (MGST1 and TFPI), and these did not partition the published NCI-60 cell lines based on SF2. There was variable success in applying three published radiosensitivity signatures to our cohorts. One gene signature, originally trained on the NCI-60 cell lines, did partially separate sensitive and resistant cell lines in all three cell line datasets. The findings do not confirm our hypothesis but suggest that a common transcriptional signature can reflect the radiosensitivity of tumours of heterogeneous origins. PMID:24466029

  13. 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.

  14. 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

  15. Radiosensitivity of hepatoma cell lines and human normal liver cell lines exposed to 12C6+ ions

    NASA Astrophysics Data System (ADS)

    Jing, X.; Yang, J.; Li, W.; Guo, C.; Dang, B.; Wang, J.; Zhou, L.; Wei, W.; Gao, Q.

    AIM To investigate the radiosensitivity of hepatoma cell lines and human normal liver cell lines METHODS Accelerated carbon ions by heavy ion research facility in Lanzhou HIRFL have high LET We employed it to study the radiosensitivity of hepatoma cell lines SMMC-7721 and human normal liver cell lines L02 using premature chromosome condensation technique PCC Cell survive was documented by a colony assay Chromatid breaks were measured by counting the number of chromatid breaks and isochromatid breaks immediately after prematurely chromosome condensed by Calyculin-A RESULTS The survival curve of the two cell lines presented a good linear relationship and the survival fraction of L02 is higher than that of SMMC-7721 Additionally the two types of G 2 phase chromosome breaks chromatid breaks and isochromatid breaks of L02 are lower than that of SMMC-7721 CONCLUSION Human normal liver cell line have high radioresistance than that of hepatoma cell line It imply that it is less damage to normal organs when radiotherapy to hepatoma

  16. Development and characterization of a largemouth bass cell line.

    PubMed

    Getchell, Rodman G; Groocock, Geoffrey H; Cornwell, Emily R; Schumacher, Vanessa L; Glasner, Lindsay I; Baker, Barry J; Frattini, Stephen A; Wooster, Gregory A; Bowser, Paul R

    2014-09-01

    Abstract The development and characterization of a new cell line, derived from the ovary of Largemouth Bass Micropterus salmoides, is described. Gonad tissue was collected from Largemouth Bass that were electrofished from Oneida Lake, New York. The tissue was processed and grown in culture flasks at approximately 22°C for more than 118 passages during an 8-year period from 2004 to 2011. The identity of these cells as Largemouth Bass origin was confirmed by sequencing a portion of the cytochrome b gene. Growth rate at three different temperatures was documented. The cell line was susceptible to Largemouth Bass virus (LMBV) and its replication was compared with that of Bluegill Lepomis macrochirus fry (BF-2), one of the cell lines recommended for LMBV isolation by the American Fisheries Society Fish Health Section Blue Book. Quantitative PCR results from the replication trial showed the BF-2 cell line produced approximately 10-fold more LMBV copies per cell than the new Largemouth Bass cell line after 6 d, while the titration assay showed similar quantities in each cell line after 1 week. Received February 18, 2014; accepted April 16, 2014. PMID:25229492

  17. Antiproliferative effect of isopentenylated coumarins on several cancer cell lines.

    PubMed

    Kawaii, S; Tomono, Y; Ogawa, K; Sugiura, M; Yano, M; Yoshizawa, Y; Ito, C; Furukawa, H

    2001-01-01

    33 coumarins, mainly the simple isopentenylated coumarins and derived pyrano- and furanocoumarins, were examined for their antiproliferative activity towards several cancer and normal human cell lines. The pyrano- and furanocoumarins showed strong activity against the cancer cell lines, whereas they had weak antiproliferative activity against the normal human cell lines. The decreasing rank order of potency was osthenone (10), clausarin (25), clausenidin (26), dentatin (24), nordentatin (23), imperatorin (29), seselin (27), xanthyletin (21), suberosin (17), phebalosin (8) and osthol (12). The structure-activity relationship established from the results revealed that the 1,1-dimethylallyl and isopentenyl groups have an important role for antiproliferative activity. PMID:11497276

  18. Establishment and Characterization of Rat Portal Myofibroblast Cell Lines

    PubMed Central

    Fausther, Michel; Goree, Jessica R.; Lavoie, Élise G.; Graham, Alicia L.; Sévigny, Jean; Dranoff, Jonathan A.

    2015-01-01

    The major sources of scar-forming myofibroblasts during liver fibrosis are activated hepatic stellate cells (HSC) and portal fibroblasts (PF). In contrast to well-characterized HSC, PF remain understudied and poorly defined. This is largely due to the facts that isolation of rodent PF for functional studies is technically challenging and that PF cell lines had not been established. To address this, we have generated two polyclonal portal myofibroblast cell lines, RGF and RGF-N2. RGF and RGF-N2 were established from primary PF isolated from adult rat livers that underwent culture activation and subsequent SV40-mediated immortalization. Specifically, Ntpdase2/Cd39l1-sorted primary PF were used to generate the RGF-N2 cell line. Both cell lines were functionally characterized by RT-PCR, immunofluorescence, immunoblot and bromodeoxyuridine-based proliferation assay. First, immortalized RGF and RGF-N2 cells are positive for phenotypic myofibroblast markers alpha smooth muscle actin, type I collagen alpha-1, tissue inhibitor of metalloproteinases-1, PF-specific markers elastin, type XV collagen alpha-1 and Ntpdase2/Cd39l1, and mesenchymal cell marker ecto-5’-nucleotidase/Cd73, while negative for HSC-specific markers desmin and lecithin retinol acyltransferase. Second, both RGF and RGF-N2 cell lines are readily transfectable using standard methods. Finally, RGF and RGF-N2 cells attenuate the growth of Mz-ChA-1 cholangiocarcinoma cells in co-culture, as previously demonstrated for primary PF. Immortalized rat portal myofibroblast RGF and RGF-N2 cell lines express typical markers of activated PF-derived myofibroblasts, are suitable for DNA transfection, and can effectively inhibit cholangiocyte proliferation. Both RGF and RGF-N2 cell lines represent novel in vitro cellular models for the functional studies of portal (myo)fibroblasts and their contribution to the progression of liver fibrosis. PMID:25822334

  19. 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

  20. Cancer stem cells and cisplatin-resistant cells isolated from non-small-lung cancer cell lines constitute related cell populations.

    PubMed

    Lopez-Ayllon, Blanca D; Moncho-Amor, Veronica; Abarrategi, Ander; Ibañez de Cáceres, Inmaculada; Castro-Carpeño, Javier; Belda-Iniesta, Cristobal; Perona, Rosario; Sastre, Leandro

    2014-10-01

    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

  1. Immunosuppression associated with the development of chronic infections with Rickettsia tsutsugamushi: adherent suppressor cell activity and macrophage activation.

    PubMed Central

    Jerrells, T R

    1985-01-01

    Measures of general immunocompetency such as lymphocyte responses to mitogens and alloantigens and the ability to produce antibody to T-dependent and T-independent antigens were evaluated during the development of chronic infections with Rickettsia tsutsugamushi resulting from subcutaneous infection of BALB/c mice. It was found that a transient immunosuppression was demonstrable regardless of the infecting strain of rickettsiae; however, the immunosuppression produced by the Karp and Kato strains was more pronounced and longer lived. As a marked splenomegaly resulting from inflammatory macrophage influx accompanied this immunosuppression, mitogen- and antigen-induced lymphocyte proliferation was also evaluated after adherent cell depletion or in the presence of indomethacin, and both treatments significantly improved the responses. Isolated splenic macrophages were shown to suppress the responses of lymphocytes from naive mice as well as to exhibit parameters of activation including tumor cell cytolysis and cytostasis and the ability to inhibit the replication of R. tsutsugamushi in vitro. These data suggest an association between macrophage activation involved in rickettsial clearance and a transient immunosuppression. PMID:2931378

  2. Oxaliplatin induces different cellular and molecular chemoresistance patterns in colorectal cancer cell lines of identical origins

    PubMed Central

    2013-01-01

    Background Cancer cells frequently adopt cellular and molecular alterations and acquire resistance to cytostatic drugs. Chemotherapy with oxaliplatin is among the leading treatments for colorectal cancer with a response rate of 50%, inducing intrastrand cross-links on the DNA. Despite of this drug’s efficiency, resistance develops in nearly all metastatic patients. Chemoresistance being of crucial importance for the drug’s clinical efficiency this study aimed to contribute to the identification and description of some cellular and molecular alterations induced by prolonged oxaliplatin therapy. Resistance to oxaliplatin was induced in Colo320 (Colo320R) and HT-29 (HT-29R) colorectal adenocarcinoma cell lines by exposing the cells to increasing concentrations of the drug. Alterations in morphology, cytotoxicity, DNA cross-links formation and gene expression profiles were assessed in the parental and resistant variants with microscopy, MTT, alkaline comet and pangenomic microarray assays, respectively. Results Morphology analysis revealed epithelial-to-mesenchymal transition in the resistant vs parental cells suggesting alterations of the cells’ adhesion complexes, through which they acquire increased invasiveness and adherence. Cytotoxicity measurements demonstrated resistance to oxaliplatin in both cell lines; Colo320 being more sensitive than HT-29 to this drug (P < 0.001). The treatment with oxaliplatin caused major DNA cross-links in both parental cell lines; in Colo320R small amounts of DNA cross-links were still detectable, while in HT-29R not. We identified 441 differentially expressed genes in Colo320R and 613 in HT-29R as compared to their parental counterparts (at least 1.5 -fold up- or down- regulation, p < 0.05). More disrupted functions and pathways were detected in HT-29R cell line than in Colo320R, involving genes responsible for apoptosis inhibition, cellular proliferation and epithelial-to-mesenchymal transition. Several upstream

  3. Differential pattern of integrin receptor expression in differentiated and anaplastic thyroid cancer cell lines.

    PubMed

    Hoffmann, S; Maschuw, K; Hassan, I; Reckzeh, B; Wunderlich, A; Lingelbach, S; Zielke, A

    2005-09-01

    Adhesion of tumor cells to the extracellular matrix (ECM) is a crucial step for the development of metastatic disease and is mediated by specific integrin receptor molecules (IRM). The pattern of metastatic spread differs substantially among the various histotypes of thyroid cancer (TC). However, IRM have only occasionally been characterized in TC until now. IRM expression was investigated in 10 differentiated (FTC133, 236, 238, HTC, HTC TSHr, XTC, PTC4.0/4.2, TPC1, Kat5) and two anaplastic TC cell lines (ATC, C643, Hth74), primary cultures of normal thyroid tissue (Thy1,3), and thyroid cancer specimens (TCS). Expression of 16 IRM (beta1-4, beta7, alpha1-6, alphaV, alphaIIb, alphaL, alphaM, alphaX) and of four IRM heterodimers (alpha2beta1, alpha5beta1, alphaVbeta3, alphaVbeta5), was analyzed by fluorescent-activated cell sorter (FACS) and immunohistochemical staining. Thyroid tumor cell adhesion to ECM proteins and their IRM expression in response to thyrotropin (TSH) was assessed. Follicular TC cell lines presented high levels of integrins alpha2, alpha3, alpha5, beta1, beta3 and low levels of alpha1, whereas papillary lines expressed a heterogenous pattern of IRM, dominated by alpha5 and beta1. ATC mainly displayed integrins alpha2, alpha3, alpha5, alpha6, beta1 and low levels of alpha1, alpha4 and alphaV. Integrin heterodimers correlated with monomer expression. Evaluation of TCS largely confirmed these results with few exceptions, namely alpha4, alpha6, and beta3. The ability of TC cell lines to adhere to purified ECM proteins correlated with IRM expression. TSH induced TC cell adhesion in a dose-dependent fashion, despite an unchanged array of IRM expression or level of a particular IRM. Thyroid carcinoma cell lines of different histogenetic background display profoundly different patterns of IRM expression that appear to correlate with tumor aggressiveness. In vitro adhesion to ECM proteins and IRM expression concur. Finally, TSH-stimulated adhesion of

  4. Human papillomavirus in vulvar and vaginal carcinoma cell lines.

    PubMed Central

    Hietanen, S.; Grénman, S.; Syrjänen, K.; Lappalainen, K.; Kauppinen, J.; Carey, T.; Syrjänen, S.

    1995-01-01

    A number of reports associate human papillomavirus (HPV) with cervical cancer and cancer cell lines derived from this tumour type. Considerably fewer reports have focused on the role of HPV in carcinomas from other sites of female anogenital squamous epithelia. In this study we have tested for the presence of HPV in eight low-passage vulvar carcinoma cell lines and one extensively passaged cell line, A431. One cell line from a primary vaginal carcinoma was included. The presence of the HPV was evaluated by the polymerase chain reaction (PCR), by Southern blot analysis and by two-dimensional gel electrophoresis. General primer-mediated PCR was applied by using primers from the L1 region, E1 region and HPV 16 E7 region. Southern blot hybridisation was performed under low-stringency conditions (Tm = -35 degrees C) using a whole genomic HPV 6/16/18 probe mixture and under high stringency conditions (Tm = -18 degrees C) with the whole genomic probes of HPV 16 and 33. HPV 16 E6-E7 mRNA was assessed by ribonuclease protection assay (RPA). HPV was found in only one vulvar carcinoma cell line, UM-SCV-6. The identified type, HPV 16, was integrated in the cell genome and could be amplified with all primers used. Also E6-E7 transcripts were found in these cells. Five original tumour biopsies were available from the HPV-negative cell lines for in situ hybridisation. All these were HPV negative with both the HPV 6/16/18 screening probe mixture under low stringency and the HPV 16 probe under high stringency. The results indicate that vulvar carcinoma cell lines contain HPV less frequently than cervical carcinoma cell lines and suggest that a significant proportion of vulvar carcinomas may evolve by an HPV-independent mechanism. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7599042

  5. CHARACTERIZATION OF A SPONTANEOUSLY TRANSFORMED CHICKEN MONONUCLEAR CELL LINE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We describe the characterization of a spontaneously transformed chicken monocytic cell line that developed as a single colony of cells in a heterophil culture that was inadvertently left in the incubator over a period of 25 days. These cells, hitherto named HTC, grow efficiently at both 37 C or 41 C...

  6. [The effects of actovegin on cell proliferation of permanent lines].

    PubMed

    Gulevskiĭ, A K; Trifonova, A V; Lavrik, A A

    2008-01-01

    The influence of Actovegin on proliferation activity and mitotic regimen of cells of permanent lines PK-15-IEKVM and BHK-21 clone 13/04 was investigated. Addition of Actovegin into growth media containing bovine serums of different components and concentrations stimulates cell proliferation. Conclusion has been made that Actovegin can be used in cell culture biotechnology. PMID:18411759

  7. Characterization of an epithelial cell line from bovine mammary gland.

    PubMed

    German, Tania; Barash, Itamar

    2002-05-01

    Elucidation of the bovine mammary gland's unique characteristics depends on obtaining an authentic cell line that will reproduce its function in vitro. Representative clones from bovine mammary cell populations, differing in their attachment capabilities, were cultured. L-1 cells showed strong attachment to the plate, whereas H-7 cells detached easily. Cultures established from these clones were nontumorigenic upon transplantation to an immunodeficient host; they exhibited the epithelial cell characteristics of positive cytokeratin but not smooth muscle actin staining. Both cell lines depended on fetal calf serum for proliferation. They exhibited distinct levels of differentiation on Matrigel in serum-free, insulin-supplemented medium on the basis of their organization and beta-lactoglobulin (BLG) secretion. H-7 cells organized into mammospheres, whereas L-1 cells arrested in a duct-like morphology. In both cell lines, prolactin activated phosphorylation of the signal transducer and activator of transcription, Stat5-a regulator of milk protein gene transcription, and of PHAS-I-an inhibitor of translation initiation in its nonphosphorylated form. De novo synthesis and secretion of BLG were detected in differentiated cultures: in L-1 cells, BLG was dependent on lactogenic hormones for maximal induction but was less stringently controlled than was beta-casein in the mouse CID-9 cell line. L-1 cells also encompassed a near-diploid chromosomal karyotype and may serve as a tool for studying functional characteristics of the bovine mammary gland. PMID:12418925

  8. Apoptotic effect of noscapine in breast cancer cell lines.

    PubMed

    Quisbert-Valenzuela, Edwin O; Calaf, Gloria M

    2016-06-01

    Cancer is a public health problem in the world and breast cancer is the most frequently cancer in women. Approximately 15% of the breast cancers are triple-negative. Apoptosis regulates normal growth, homeostasis, development, embryogenesis and appropriate strategy to treat cancer. Bax is a protein pro-apoptotic enhancer of apoptosis in contrast to Bcl-2 with antiapoptotic properties. Initiator caspase-9 and caspase-8 are features of intrinsic and extrinsic apoptosis pathway, respectively. NF-κB is a transcription factor known to be involved in the initiation and progression of breast cancer. Noscapine, an alkaloid derived from opium is used as antitussive and showed antitumor properties that induced apoptosis in cancer cell lines. The aim of the present study was to determine the apoptotic effect of noscapine in breast cancer cell lines compared to breast normal cell line. Three cell lines were used: i) a control breast cell line MCF-10F; ii) a luminal-like adenocarcinoma triple-positive breast cell line MCF-7; iii) breast cancer triple-negative cell line MDA-MB-231. Our results showed that noscapine had lower toxicity in normal cells and was an effective anticancer agent that induced apoptosis in breast cancer cells because it increases Bax gene and protein expression in three cell lines, while decreases Bcl-xL gene expression, and Bcl-2 protein expression decreased in breast cancer cell lines. Therefore, Bax/Bcl-2 ratio increased in the three cell lines. This drug increased caspase-9 gene expression in breast cancer cell lines and caspase-8 gene expression increased in MCF-10F and MDA-MB-231. Furthermore, it increased cleavage of caspase-8, suggesting that noscapine-induced apoptosis is probably due to the involvement of extrinsic and intrinsic apoptosis pathways. Antiapoptotic gene and protein expression diminished and proapoptotic gene and protein expression increased noscapine-induced expression, probably due to decrease in NF-κB gene and protein expression

  9. Molecular profiling reveals primary mesothelioma cell lines recapitulate human disease.

    PubMed

    Chernova, T; Sun, X M; Powley, I R; Galavotti, S; Grosso, S; Murphy, F A; Miles, G J; Cresswell, L; Antonov, A V; Bennett, J; Nakas, A; Dinsdale, D; Cain, K; Bushell, M; Willis, A E; MacFarlane, M

    2016-07-01

    Malignant mesothelioma (MM) is an aggressive, fatal tumor strongly associated with asbestos exposure. There is an urgent need to improve MM patient outcomes and this requires functionally validated pre-clinical models. Mesothelioma-derived cell lines provide an essential and relatively robust tool and remain among the most widely used systems for candidate drug evaluation. Although a number of cell lines are commercially available, a detailed comparison of these commercial lines with freshly derived primary tumor cells to validate their suitability as pre-clinical models is lacking. To address this, patient-derived primary mesothelioma cell lines were established and characterized using complementary multidisciplinary approaches and bioinformatic analysis. Clinical markers of mesothelioma, transcriptional and metabolic profiles, as well as the status of p53 and the tumor suppressor genes CDKN2A and NF2, were examined in primary cell lines and in two widely used commercial lines. Expression of MM-associated markers, as well as the status of CDKN2A, NF2, the 'gatekeeper' in MM development, and their products demonstrated that primary cell lines are more representative of the tumor close to its native state and show a degree of molecular diversity, thus capturing the disease heterogeneity in a patient cohort. Molecular profiling revealed a significantly different transcriptome and marked metabolic shift towards a greater glycolytic phenotype in commercial compared with primary cell lines. Our results highlight that multiple, appropriately characterised, patient-derived tumor cell lines are required to enable concurrent evaluation of molecular profiles versus drug response. Furthermore, application of this approach to other difficult-to-treat tumors would generate improved cellular models for pre-clinical evaluation of novel targeted therapies. PMID:26891694

  10. Molecular profiling reveals primary mesothelioma cell lines recapitulate human disease

    PubMed Central

    Chernova, T; Sun, X M; Powley, I R; Galavotti, S; Grosso, S; Murphy, F A; Miles, G J; Cresswell, L; Antonov, A V; Bennett, J; Nakas, A; Dinsdale, D; Cain, K; Bushell, M; Willis, A E; MacFarlane, M

    2016-01-01

    Malignant mesothelioma (MM) is an aggressive, fatal tumor strongly associated with asbestos exposure. There is an urgent need to improve MM patient outcomes and this requires functionally validated pre-clinical models. Mesothelioma-derived cell lines provide an essential and relatively robust tool and remain among the most widely used systems for candidate drug evaluation. Although a number of cell lines are commercially available, a detailed comparison of these commercial lines with freshly derived primary tumor cells to validate their suitability as pre-clinical models is lacking. To address this, patient-derived primary mesothelioma cell lines were established and characterized using complementary multidisciplinary approaches and bioinformatic analysis. Clinical markers of mesothelioma, transcriptional and metabolic profiles, as well as the status of p53 and the tumor suppressor genes CDKN2A and NF2, were examined in primary cell lines and in two widely used commercial lines. Expression of MM-associated markers, as well as the status of CDKN2A, NF2, the ‘gatekeeper' in MM development, and their products demonstrated that primary cell lines are more representative of the tumor close to its native state and show a degree of molecular diversity, thus capturing the disease heterogeneity in a patient cohort. Molecular profiling revealed a significantly different transcriptome and marked metabolic shift towards a greater glycolytic phenotype in commercial compared with primary cell lines. Our results highlight that multiple, appropriately characterised, patient-derived tumor cell lines are required to enable concurrent evaluation of molecular profiles versus drug response. Furthermore, application of this approach to other difficult-to-treat tumors would generate improved cellular models for pre-clinical evaluation of novel targeted therapies. PMID:26891694

  11. Global Conservation of Protein Status between Cell Lines and Xenografts.

    PubMed

    Biau, Julian; Chautard, Emmanuel; Court, Frank; Pereira, Bruno; Verrelle, Pierre; Devun, Flavien; De Koning, Leanne; Dutreix, Marie

    2016-08-01

    Common preclinical models for testing anticancer treatment include cultured human tumor cell lines in monolayer, and xenografts derived from these cell lines in immunodeficient mice. Our goal was to determine how similar the xenografts are compared with their original cell line and to determine whether it is possible to predict the stability of a xenograft model beforehand. We studied a selection of 89 protein markers of interest in 14 human cell cultures and respective subcutaneous xenografts using the reverse-phase protein array technology. We specifically focused on proteins and posttranslational modifications involved in DNA repair, PI3K pathway, apoptosis, tyrosine kinase signaling, stress, cell cycle, MAPK/ERK signaling, SAPK/JNK signaling, NFκB signaling, and adhesion/cytoskeleton. Using hierarchical clustering, most cell culture-xenograft pairs cluster together, suggesting a global conservation of protein signature. Particularly, Akt, NFkB, EGFR, and Vimentin showed very stable protein expression and phosphorylation levels highlighting that 4 of 10 pathways were highly correlated whatever the model. Other proteins were heterogeneously conserved depending on the cell line. Finally, cell line models with low Akt pathway activation and low levels of Vimentin gave rise to more reliable xenograft models. These results may be useful for the extrapolation of cell culture experiments to in vivo models in novel targeted drug discovery. PMID:27567954

  12. Listeria monocytogenes listeriolysin O and phosphatidylinositol-specific phospholipase C affect adherence to epithelial cells.

    PubMed

    Krawczyk-Balska, Agata; Bielecki, Jacek

    2005-09-01

    Listeria monocytogenes, a foodborn intracellular animal and human pathogen, produces several exotoxins contributing to virulence. Among these are listeriolysin O (LLO), a pore-forming cholesterol-dependent hemolysin, and a phosphatidylinositol-specific phospholipase C (PI-PLC). LLO is known to play an important role in the escape of bacteria from the primary phagocytic vacuole of macrophages, and PI-PLC supports this process. Evidence is accumulating that LLO and PI-PLC are multifunctional virulence factors with many important roles in the host-parasite interaction other than phagosomal membrane disruption. LLO and PI-PLC may induce a number of host cell responses by modulating signal transduction of infected cells via intracellular Ca2+ levels and the metabolism of phospholipids. This would result in the activation of host phospholipase C and protein kinase C. In the present study, using Bacillus sub tilis strains expressing LLO, PI-PLC, and simultaneously LLO and PI-PLC, we show that LLO and PI-PLC enhance bacterial binding to epithelial cells Int407, with LLO being necessary and PI-PLC playing an accessory role. The results of this work suggest that these two listerial proteins act on epithelial cells prior to internalization. PMID:16391652

  13. Specific adherence of Borrelia burgdorferi extracellular vesicles to human endothelial cells in culture.

    PubMed Central

    Shoberg, R J; Thomas, D D

    1993-01-01

    Borrelia burgdorferi produces extracellular vesicles which contain some of the outer surface proteins of the bacterium (e.g., OspA and OspB). Borrelial vesicles, isolated by differential centrifugation and filtration, were tested for the ability to bind to cultured human umbilical vein endothelial (HUVE) cells in culture. The recently described lipoprotein OspD was expressed on vesicles. Vesicles exhibited differential expression of OspB and OspD in a relationship with passage number and medium serum supplement type, respectively. Qualitative immunoblotting analyses demonstrated dose-dependent, passage number-dependent adsorption of vesicles by HUVE cells. This adsorption was demonstrated to be dependent upon a borrelial component of the vesicle and not due to the presence of minor contamination with intact spirochetes. Quantitative experiments examining inhibition of B. burgdorferi-HUVE association as a function of prior vesicle-HUVE association demonstrated dependence upon (i) a borrelial component(s) in the vesicle, (ii) low passage number, and (iii) vesicle protein concentration. However, vesicle pretreatment of the HUVE cell monolayer was not requisite for this inhibition. Vesicles from highly passaged borrelias were noninhibitory for B. burgdorferi-HUVE cell association, regardless of the serum used to supplement the medium. The use of vesicles as a tool for studying B. burgdorferi pathogenesis and/or physiology is proposed. Images PMID:8359911

  14. 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...

  15. Development of cystic fibrosis and noncystic fibrosis airway cell lines.

    PubMed

    Zabner, Joseph; Karp, Phil; Seiler, Michael; Phillips, Stacia L; Mitchell, Calista J; Saavedra, Mimi; Welsh, Michael; Klingelhutz, Aloysius J

    2003-05-01

    In this study, we utilized the reverse transcriptase component of telomerase, hTERT, and human papillomavirus type 16 (HPV-16) E6 and E7 genes to transform normal and cystic fibrosis (CF) human airway epithelial (HAE) cells. One cell line, designated NuLi-1 (normal lung, University of Iowa), was derived from HAE of normal genotype; three cell lines, designated CuFi (cystic fibrosis, University of Iowa)-1, CuFi-3, and CuFi-4, were derived from HAE of various CF genotypes. When grown at the air-liquid interface, the cell lines were capable of forming polarized differentiated epithelia that exhibited transepithelial resistance and maintained the ion channel physiology expected for the genotypes. The CF transmembrane conductance regulator defect in the CuFi cell lines could be corrected by infecting from the basolateral surface using adenoviral vectors. Using nuclear factor-kappaB promoter reporter constructs, we also demonstrated that the NuLi and CuFi cell lines retained nuclear factor-kappaB responses to lipopolysaccharide. These cell lines should therefore be useful as models for studying ion physiology, therapeutic intervention for CF, and innate immunity. PMID:12676769

  16. Strategies for selecting recombinant CHO cell lines for cGMP manufacturing: improving the efficiency of cell line generation.

    PubMed

    Porter, Alison J; Racher, Andrew J; Preziosi, Richard; Dickson, Alan J

    2010-01-01

    Transfectants with a wide range of cellular phenotypes are obtained during the process of cell line generation. For the successful manufacture of a therapeutic protein, a means is required to identify a cell line with desirable growth and productivity characteristics from this phenotypically wide-ranging transfectant population. This identification process is on the critical path for first-in-human studies. We have stringently examined a typical selection strategy used to isolate cell lines suitable for cGMP manufacturing. One-hundred and seventy-five transfectants were evaluated as they progressed through the different assessment stages of the selection strategy. High producing cell lines, suitable for cGMP manufacturing, were identified. However, our analyses showed that the frequency of isolation of the highest producing cell lines was low and that ranking positions were not consistent between each assessment stage, suggesting that there is potential to improve upon the strategy. Attempts to increase the frequency of isolation of the 10 highest producing cell lines, by in silico analysis of alternative selection strategies, were unsuccessful. We identified alternative strategies with similar predictive capabilities to the typical selection strategy. One alternate strategy required fewer cell lines to be progressed at the assessment stages but the stochastic nature of the models means that cell line numbers are likely to change between programs. In summary, our studies illuminate the potential for improvement to this and future selection strategies, based around use of assessments that are more informative or that reduce variance, paving the way to improved efficiency of generation of manufacturing cell lines. PMID:20623584

  17. 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

  18. Surface charge characteristics of cells from malignant cell lines and normal cell lines of the human hematopoietic system.

    PubMed

    Marikovsky, Y; Ben-Bassat, H; Leibovich, S J; Cividalli, L; Fischler, H; Danon, D

    1979-02-01

    Cells from malignant and normal lines of human hematopoietic origin were studied for their surface charge characteristics with the use of the following criteria: 1) the electron microscopic appearance of cell membranes after labeling with cationized ferritin (CF) either before or after glutaraldehyde fixation, 2) electrophoretic mobility, 3) total sialic acid content, and 4) agglutinability with poly-L-lysine (PLL). CF induced a time-dependent redistribution of surface receptors in unfixed malignant cells but not in unfixed normal cells. After 10 seconds of labeling with CF, both normal and malignant unfixed cells showed a uniform and even labeling pattern. After 5 minutes of labeling, malignant cells exhibited a highly pronounced pattern of clusters and patches, as distinct from a random and even pattern exhibited by normal cells. Both normal and malignant cells after fixation exhibited an equivalent random and even labeling pattern with CF, independent of the duration of labeling. The malignant cells studied possessed less sialic acid, had a lower electric mobility, and were agglutinated more readily with PLL than were the normal cells. PMID:310907

  19. Effects of ethanol on an intestinal epithelial cell line

    SciTech Connect

    Nano, J.L.; Cefai, D.; Rampal, P. )

    1990-02-01

    The effect of exposure of an intestinal epithelial cell line to various concentrations of ethanol (217 mM (1%) to 652 mM (3%)) during 24, 48, and 72 hr was investigated in vitro using a rat intestinal epithelial cell line (IRD 98). Incubation of these cells in the presence of ethanol significantly decreased cell growth. This inhibition was accompanied by a strong increase in cellular protein. Stimulation of specific disaccharidases, gamma-glutamyl transferase, and aminopeptidase activities by ethanol was dose- and time-dependent. Ethanol induces a change in the relative proportions of the different lipid classes synthesized; triglycerides, fatty acids, and cholesterol esters were preferentially synthethysed. Our findings show that cell lines are good models for investigation of the effects of ethanol, and that alcohol considerably modifies the functions of intestinal epithelial cells.

  20. MORPHOMETRIC SUBTYPING FOR A PANEL OF BREAST CANCER CELL LINES

    SciTech Connect

    Han, Ju; Chang, Hang; Fontenay, Gerald; Wang, Nicholas J.; Gray, Joe W.; Parvin, Bahram

    2009-05-08

    A panel of cell lines of diverse molecular background offers an improved model system for high-content screening, comparative analysis, and cell systems biology. A computational pipeline has been developed to collect images from cell-based assays, segment individual cells and colonies, represent segmented objects in a multidimensional space, and cluster them for identifying distinct subpopulations. While each segmentation strategy can vary for different imaging assays, representation and subpopulation analysis share a common thread. Application of this pipeline to a library of 41 breast cancer cell lines is demonstrated. These cell lines are grown in 2D and imaged through immunofluorescence microscopy. Subpopulations in this panel are identified and shown to correlate with previous subtyping literature that was derived from transcript data.

  1. Factors influencing human leukocyte adherence in vitro.

    PubMed

    Stepniewicz, W; Tchórzewski, H; Luciak, M

    1983-01-01

    Studies were performed on factors influencing leucocyte adherence in vitro. Blood condensation was found to increase leukocyte adherence. Addition of heparin, dextran or ethanol caused a significant reduction of white blood cell count in blood samples in comparison with blood mixed with sodium EDTA or ACD solution. This suggests the existence of two granulocyte subpopulations; viz, rapidly adhering and slowly adhering. Heparin enhanced granulocyte adherence, while dextran and ethanol decreased it. Five-day storage of ACD blood led to a decrease in granulocyte adherence, while addition of heparin or histamine to ACD blood prevented this change to occur. The glucose concentration of 1,000 mg/dl augmented granulocyte adherence, while higher glucose concentrations induced its progressive fall below the control values. There was no significant change of lymphocyte adherence during the experiments. PMID:6194070

  2. DNA Fingerprinting of the NCI-60 Cell Line Panel

    PubMed Central

    Lorenzi, Philip L.; Reinhold, William C.; Varma, Sudhir; Hutchinson, Amy A.; Pommier, Yves; Chanock, Stephen J.; Weinstein, John N.

    2009-01-01

    The National Cancer Institute’s NCI-60 cell line panel, the most extensively characterized set of cells in existence and a public resource, is frequently used as a screening tool for drug discovery. Since many laboratories around the world rely on data from the NCI-60 cells, confirmation of their genetic identities represents an essential step in validating results from them. Given the consequences of cell line contamination or misidentification, quality control measures should routinely include DNA fingerprinting. We have, therefore, used standard DNA microsatellite short tandem repeats to profile the NCI-60, and the resulting DNA fingerprints are provided here as a reference. Consistent with previous reports, the fingerprints suggest that several NCI-60 lines have common origins: the melanoma lines MDA-MB-435, MDA-N, and M14; the central nervous system lines U251 and SNB-19; the ovarian lines OVCAR-8 and OVCAR-8/ADR (also called NCI/ADR); and the prostate lines DU-145, DU-145 (ATCC), and RC0.1. Those lines also demonstrate that the ability to connect two fingerprints to the same origin is not affected by stable transfection or by the development of multidrug resistance. As expected, DNA fingerprints were not able to distinguish different tissues-of-origin. The fingerprints serve principally as a barcodes. PMID:19372543

  3. DNA fingerprinting of the NCI-60 cell line panel.

    PubMed

    Lorenzi, Philip L; Reinhold, William C; Varma, Sudhir; Hutchinson, Amy A; Pommier, Yves; Chanock, Stephen J; Weinstein, John N

    2009-04-01

    The National Cancer Institute's NCI-60 cell line panel, the most extensively characterized set of cells in existence and a public resource, is frequently used as a screening tool for drug discovery. Because many laboratories around the world rely on data from the NCI-60 cells, confirmation of their genetic identities represents an essential step in validating results from them. Given the consequences of cell line contamination or misidentification, quality control measures should routinely include DNA fingerprinting. We have, therefore, used standard DNA microsatellite short tandem repeats to profile the NCI-60, and the resulting DNA fingerprints are provided here as a reference. Consistent with previous reports, the fingerprints suggest that several NCI-60 lines have common origins: the melanoma lines MDA-MB-435, MDA-N, and M14; the central nervous system lines U251 and SNB-19; the ovarian lines OVCAR-8 and OVCAR-8/ADR (also called NCI/ADR); and the prostate lines DU-145, DU-145 (ATCC), and RC0.1. Those lines also show that the ability to connect two fingerprints to the same origin is not affected by stable transfection or by the development of multidrug resistance. As expected, DNA fingerprints were not able to distinguish different tissues-of-origin. The fingerprints serve principally as a barcodes. PMID:19372543

  4. 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

  5. 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

    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

  6. Cold storage and cryopreservation of tick cell lines

    PubMed Central

    2010-01-01

    Background Tick cell lines are now available from fifteen ixodid and argasid species of medical and veterinary importance. However, some tick cell lines can be difficult to cryopreserve, and improved protocols for short- and long-term low temperature storage will greatly enhance their use as tools in tick and tick-borne pathogen research. In the present study, different protocols were evaluated for cold storage and cryopreservation of tick cell lines derived from Rhipicephalus (Boophilus) decoloratus, Rhipicephalus (Boophilus) microplus, Ixodes ricinus and Ixodes scapularis. For short-term cold storage, cells were kept under refrigeration at 6°C for 15, 30 and 45 days. For cryopreservation in liquid nitrogen, use of a sucrose-phosphate-glutamate freezing buffer (SPG) as cryoprotectant was compared with dimethylsulfoxide (DMSO) supplemented with sucrose. Cell viability was determined by the trypan blue exclusion test and cell morphology was evaluated in Giemsa-stained cytocentrifuge smears. Results Cold storage at 6°C for up to 30 days was successful in preserving R. (B.) microplus, R. (B.) decoloratus, I. ricinus and I. scapularis cell lines; lines from the latter three species could be easily re-cultivated after 45 days under refrigeration. While cell lines from all four tick species cryopreserved with 6% DMSO were successfully resuscitated, the R. (B.) decoloratus cells did not survive freezing in SPG and of the other three species, only the R. (B.) microplus cells resumed growth during the observation period. Conclusions This constitutes the first report on successful short-term refrigeration of cells derived from R. (B.) decoloratus, R. (B.) microplus, and I. ricinus, and use of SPG as an alternative to DMSO for cryopreservation, thus making an important contribution to more reliable and convenient tick cell culture maintenance. PMID:20388200

  7. Lateral flagella are required for increased cell adherence, invasion and biofilm formation by Aeromonas spp.

    PubMed

    Gavín, Rosalina; Merino, Susana; Altarriba, Maria; Canals, Rocío; Shaw, Jonathan G; Tomás, Juan M

    2003-07-15

    Two types of flagella are responsible for motility in mesophilic Aeromonas strains. A polar unsheathed flagellum is expressed constitutively that allows the bacterium to swim in liquid environments and, in media where the polar flagellum is unable to propel the cell, Aeromonas express peritrichous lateral flagella. Recently, Southern blot analysis using a DNA probe based on the Aeromonas caviae Sch3N lateral flagellin gene sequence showed a good correlation between strains positive for the DNA probe, swarming motility and the presence of lateral flagella by microscopy. Here, we conclude that the easiest method for the detection of the lateral flagellin gene(s) is by PCR (polymerase chain reaction); this showed good correlation with swarming motility and the presence of lateral flagella. This was despite the high degree of DNA heterogeneity found in Aeromonas gene sequences. Furthermore, by reintroducing the laf (lateral flagella) genes into several mesophilic lateral-flagella-negative Aeromonas wild-type strains, we demonstrate that this surface structure enhances the adhesion to and invasion of HEp-2 cells and the capacity for biofilm formation in vitro. These results, together with previous data obtained using Laf- mutants, demonstrate that lateral flagella production is a pathogenic feature due to its enhancement of the interaction with eukaryotic cell surfaces. PMID:12855171

  8. Chemical stimulation of adherent cells by localized application of acetylcholine from a microfluidic system.

    PubMed

    Zibek, Susanne; Hagmeyer, Britta; Stett, Alfred; Stelzle, Martin

    2010-01-01

    Chemical stimulation of cells is inherently cell type selective in contrast to electro-stimulation. The availability of a system for localized application of minute amounts of chemical stimulants could be useful for dose related response studies to test new compounds. It could also bring forward the development of a novel type of neuroprostheses. In an experimental setup microdroplets of an acetylcholine solution were ejected from a fluidic microsystem and applied to the bottom of a nanoporous membrane. The solution traveled through the pores to the top of the membrane on which TE671 cells were cultivated. Calcium imaging was used to visualize cellular response with temporal and spatial resolution. Experimental demonstration of chemical stimulation for both threshold gated stimulation as well as accumulated dose-response was achieved by either employing acetylcholine as chemical stimulant or applying calcein uptake, respectively. Numerical modeling and simulation of transport mechanisms involved were employed to gain a theoretical understanding of the influence of pore size, concentration of stimulant and droplet volume on the spatial-temporal distribution of stimulant and on the cellular response. Diffusion, pressure driven flow and evaporation effects were taken into account. Fast stimulation kinetic is achieved with pores of 0.82 μm diameter, whereas sustained substance delivery is obtained with nanoporous membranes. In all cases threshold concentrations ranging from 0.01 to 0.015 μM acetylcholine independent of pore size were determined. PMID:21151808

  9. Chemical Stimulation of Adherent Cells by Localized Application of Acetylcholine from a Microfluidic System

    PubMed Central

    Zibek, Susanne; Hagmeyer, Britta; Stett, Alfred; Stelzle, Martin

    2010-01-01

    Chemical stimulation of cells is inherently cell type selective in contrast to electro-stimulation. The availability of a system for localized application of minute amounts of chemical stimulants could be useful for dose related response studies to test new compounds. It could also bring forward the development of a novel type of neuroprostheses. In an experimental setup microdroplets of an acetylcholine solution were ejected from a fluidic microsystem and applied to the bottom of a nanoporous membrane. The solution traveled through the pores to the top of the membrane on which TE671 cells were cultivated. Calcium imaging was used to visualize cellular response with temporal and spatial resolution. Experimental demonstration of chemical stimulation for both threshold gated stimulation as well as accumulated dose–response was achieved by either employing acetylcholine as chemical stimulant or applying calcein uptake, respectively. Numerical modeling and simulation of transport mechanisms involved were employed to gain a theoretical understanding of the influence of pore size, concentration of stimulant and droplet volume on the spatial-temporal distribution of stimulant and on the cellular response. Diffusion, pressure driven flow and evaporation effects were taken into account. Fast stimulation kinetic is achieved with pores of 0.82 μm diameter, whereas sustained substance delivery is obtained with nanoporous membranes. In all cases threshold concentrations ranging from 0.01 to 0.015 μM acetylcholine independent of pore size were determined. PMID:21151808

  10. Pharmacogenomic agreement between two cancer cell line data sets.

    PubMed

    2015-12-01

    Large cancer cell line collections broadly capture the genomic diversity of human cancers and provide valuable insight into anti-cancer drug response. Here we show substantial agreement and biological consilience between drug sensitivity measurements and their associated genomic predictors from two publicly available large-scale pharmacogenomics resources: The Cancer Cell Line Encyclopedia and the Genomics of Drug Sensitivity in Cancer databases. PMID:26570998

  11. Measles virus persistence in an immortalized murine macrophage cell line.

    PubMed

    Goldman, M B; Buckthal, D J; Picciotto, S; O'Bryan, T A; Goldman, J N

    1995-02-20

    Persistent infection with the Edmonston strain of measles virus (MV) has been established in IC-21 cells, an immortalized murine macrophage cell line. Persistence was established immediately without syncytia formation or cytopathic effects. MV was expressed in the majority of the cells as evidenced by immunofluorescence microscopy, flow cytometry, infectious centers assays, and limiting dilution analysis. Hemagglutinin (H) and phosphoprotein expressed in persistently infected IC-21 cells had retarded migration in SDS-PAGE gels when compared to these proteins expressed in Vero cells. H protein differences were also found between freshly infected IC-21 cells and persistently infected IC-21 cells passaged for over 2 years. Six sublines of IC-21 cells, infected at different times, have maintained these characteristics for 2 years of passage. During this time period the intensity of immunofluorescence and the number of infectious virus particles recoverable fluctuated in five of the six cell lines. In one cell line virus expression remained at a consistent high level. The ability to establish a persistent MV infection in murine macrophages allows studies using a cell important in disseminating the infection. It facilitates experiments on immunological aspects of viral immunity by enabling cell mixing experiments with histocompatible cell populations and by making available the wide array of cellular and humoral reagents in the mouse. PMID:7871720

  12. Reliable in vitro studies require appropriate ovarian cancer cell lines.

    PubMed

    Jacob, Francis; Nixdorf, Sheri; Hacker, Neville F; Heinzelmann-Schwarz, Viola A

    2014-01-01

    Ovarian cancer is the fifth most common cause of cancer death in women and the leading cause of death from gynaecological malignancies. Of the 75% women diagnosed with locally advanced or disseminated disease, only 30% will survive five years following treatment. This poor prognosis is due to the following reasons: limited understanding of the tumor origin, unclear initiating events and early developmental stages of ovarian cancer, lack of reliable ovarian cancer-specific biomarkers, and drug resistance in advanced cases. In the past, in vitro studies using cell line models have been an invaluable tool for basic, discovery-driven cancer research. However, numerous issues including misidentification and cross-contamination of cell lines have hindered research efforts. In this study we examined all ovarian cancer cell lines available from cell banks. Hereby, we identified inconsistencies in the reporting, difficulties in the identification of cell origin or clinical data of the donor patients, restricted ethnic and histological type representation, and a lack of tubal and peritoneal cancer cell lines. We recommend that all cell lines should be distributed via official cell banks only with strict guidelines regarding the minimal available information required to improve the quality of ovarian cancer research in future. PMID:24936210

  13. Reliable in vitro studies require appropriate ovarian cancer cell lines

    PubMed Central

    2014-01-01

    Ovarian cancer is the fifth most common cause of cancer death in women and the leading cause of death from gynaecological malignancies. Of the 75% women diagnosed with locally advanced or disseminated disease, only 30% will survive five years following treatment. This poor prognosis is due to the following reasons: limited understanding of the tumor origin, unclear initiating events and early developmental stages of ovarian cancer, lack of reliable ovarian cancer-specific biomarkers, and drug resistance in advanced cases. In the past, in vitro studies using cell line models have been an invaluable tool for basic, discovery-driven cancer research. However, numerous issues including misidentification and cross-contamination of cell lines have hindered research efforts. In this study we examined all ovarian cancer cell lines available from cell banks. Hereby, we identified inconsistencies in the reporting, difficulties in the identification of cell origin or clinical data of the donor patients, restricted ethnic and histological type representation, and a lack of tubal and peritoneal cancer cell lines. We recommend that all cell lines should be distributed via official cell banks only with strict guidelines regarding the minimal available information required to improve the quality of ovarian cancer research in future. PMID:24936210

  14. Metronidazole Decreases Viability of DLD-1 Colorectal Cancer Cell Line

    PubMed Central

    Sadowska, Anna; Krętowski, Rafał; Szynaka, Beata; Cechowska-Pasko, Marzanna

    2013-01-01

    Abstract The aim of our study was to evaluate the impact of metronidazole (MTZ) on DLD-1 colorectal cancer cell (CRC) line. Toxicity of MTZ was determined by MTT test. Cells were incubated with MTZ used in different concentrations for 24, 48, and 72 hours. The effect of MTZ on DNA synthesis was measured as [3H]-thymidine incorporation. The morphological changes in human DLD-1 cell line were defined by transmission electron microscope OPTON 900. The influence of MTZ on the apoptosis of DLD-1 cell lines was detected by flow cytometry and fluorescence microscopy, while cell concentration, volume, and diameter were displayed by Scepter Cell Counter from Millipore. Our results show that cell viability was diminished in all experimental groups in comparison with the control, and the differences were statistically significant. We did not find any significant differences in [3H]-thymidine incorporation in all experimental groups and times of observation. Cytofluorimetric assays demonstrated a statistically significant increase of apoptotic rate in MTZ concentrations 10 and 50 μg/mL after 24 hours; 0.1, 10, 50, and 250 μg/mL after 48 hours; and in all concentrations after 72 hours compared with control groups. In the ultrastructural studies, necrotic or apoptotic cells were occasionally seen. In conclusion, MTZ affects human CRC cell line viability. The reduction of cell viability was consistent with the apoptotic test. PMID:23777253

  15. Genotypes and immunophenotypes of Hodgkin's disease-derived cell lines.

    PubMed

    Drexler, H G; Leber, B F; Norton, J; Yaxley, J; Tatsumi, E; Hoffbrand, A V; Minowada, J

    1988-06-01

    This report describes the geno- and immunophenotypic analysis of the Hodgkin's disease-derived cell lines HDLM-2, KM-H2, and L-428. The lines were all positive for the antigens CD15 (Leu-M1), CD30 (Ki-1), Hefi-1 (antigen detected by a monoclonal antibody produced against L-428), HLA class I and II, and activation/proliferation markers. The cells from all 3 cell lines lacked almost all cell lineage-associated/specific markers: HDLM-2 was only CD2+, KM-H2 was only CD9+ and CD21+, and L-428 was negative for all the specific markers tested. Genomic analysis of HDLM-2 cells revealed monoclonal rearrangements of T cell receptor beta and gamma loci and germ line configuration of immunoglobulin genes. Immunoglobulin heavy chain genes were rearranged in KM-H2 and L-428. These data suggest a possible lymphoid origin for HDLM-2, KM-H2, and L-428. Although the data presented do not provide formal proof of a lymphoid nature of Hodgkin and Reed-Sternberg cells and do not unequivocally exclude a derivation from other hematopoietic cells, extrapolation of the results from the in vitro cultures to the in vivo situation suggests a lymphoid (T or B cell) origin of these cells. PMID:3131596

  16. 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

  17. Inducible human immunodeficiency virus type 1 packaging cell lines.

    PubMed Central

    Yu, H; Rabson, A B; Kaul, M; Ron, Y; Dougherty, J P

    1996-01-01

    Packaging cell lines are important tools for transferring genes into eukaryotic cells. Human immunodeficiency virus type 1 (HIV-1)-based packaging cell lines are difficult to obtain, in part owing to the problem that some HIV-1 proteins are cytotoxic in a variety of cells. To overcome this, we have developed an HIV-1-based packaging cell line which has an inducible expression system. The tetracycline-inducible expression system was utilized to control the expression of the Rev regulatory protein, which in turn controls the expression of the late proteins including Gag, Pol, and Env. Western blotting (immunoblotting) demonstrated that the expression of p24gag and gp120env from the packaging cells peaked on days 6 and 7 postinduction. Reverse transcriptase activity could be detected by day 4 after induction and also peaked on days 6 and 7. Defective vector virus could be propagated, yielding titers as high as 7 x 10(3) CFU/ml, while replication-competent virus was not detectable at any time. Thus, the cell line should enable the transfer of specific genes into CD4+ cells and should be a useful tool for studying the biology of HIV-1. We have also established an inducible HIV-1 Env-expressing cell line which could be used to propagate HIV-1 vectors that require only Env in trans. The env-minus vector virus titer produced from the Env-expressing cells reached 2 x 10(4) CFU/ml. The inducible HIV-1 Env-expressing cell line should be a useful tool for the study of HIV-1 Env as well. PMID:8676479

  18. 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

  19. Surface-Mediated Stimuli Responsive Delivery of Organic Molecules from Porous Carriers to Adhered Cells.

    PubMed

    Ergün, Bahar; De Cola, Luisa; Galla, Hans-Joachim; Kehr, Nermin Seda

    2016-07-01

    The alternating layer-by-layer deposition of oppositely charged polyelectrolytes on fluorescence-dye-(Hst)-loaded zeolites L ((Hst) Zeo-PSS/PLL) is described. The arrays and nanocomposite (NC) hydrogels of (Hst) Zeo-PSS/PLL are prepared. The subsequent cell experiments show the potential application of arrays and NC hydrogels of (Hst) Zeo-PSS/PLL as alternative 2D- and 3D-surfaces, respectively, for 2D- and 3D-surface-mediated controlled organic molecules delivery applications. PMID:27114067

  20. Haemophilus influenzae Type f Hijacks Vitronectin Using Protein H To Resist Host Innate Immunity and Adhere to Pulmonary Epithelial Cells.

    PubMed

    Al-Jubair, Tamim; Mukherjee, Oindrilla; Oosterhuis, Sharon; Singh, Birendra; Su, Yu-Ching; Fleury, Christophe; Blom, Anna M; Törnroth-Horsefield, Susanna; Riesbeck, Kristian

    2015-12-15

    The incidence of invasive Haemophilus influenzae type b (Hib) disease has significantly decreased since the introduction of an efficient vaccine against Hib. However, in contrast to Hib, infections caused by H. influenzae serotype f (Hif) are emerging. We recently did a whole genome sequencing of an invasive Hif isolate, and reported that Hif interacts with factor H by expressing protein H (PH). In this study, upon screening with various human complement regulators, we revealed that PH is also a receptor for vitronectin (Vn), an abundant plasma protein that regulates the terminal pathway of the human complement system in addition to being a component of the extracellular matrix. Bacterial Vn binding was significantly reduced when the lph gene encoding PH was deleted in an invasive Hif isolate. The dissociation constant (KD) of the interaction between recombinant PH and Vn was 2.2 μM, as revealed by Biolayer interferometry. We found that PH has different regions for simultaneous interaction with both Vn and factor H, and that it recognized the C-terminal part of Vn (aa 352-362). Importantly, PH-dependent Vn binding resulted in better survival of the wild-type Hif or PH-expressing Escherichia coli when exposed to human serum. Finally, we observed that PH mediated an increased bacterial adherence to alveolar epithelial cells in the presence of Vn. In conclusion, our study reveals that PH most likely plays an important role in Hif pathogenesis by increasing serum resistance and adhesion to the airways. PMID:26538390

  1. Xenotropic retrovirus Bxv1 in human pancreatic β cell lines

    PubMed Central

    Kirkegaard, Jeannette S.; Ingvarsen, Signe; Diedisheim, Marc; Bricout-Neveu, Emilie; Grønborg, Mads; Frogne, Thomas; Scharfmann, Raphael; Madsen, Ole D.; Rescan, Claude; Albagli, Olivier

    2016-01-01

    It has been reported that endogenous retroviruses can contaminate human cell lines that have been passaged as xenotransplants in immunocompromised mice. We previously developed and described 2 human pancreatic β cell lines (EndoC-βH1 and EndoC-βH2) that were generated in this way. Here, we have shown that B10 xenotropic virus 1 (Bxv1), a xenotropic endogenous murine leukemia virus (MuLV), is present in these 2 recently described cell lines. We determined that Bxv1 was also present in SCID mice that were used for in vivo propagation of EndoC-βH1/2 cells, suggesting that contamination occurred during xenotransplantation. EndoC-βH1/2 cells released Bxv1 particles that propagated to human 293T and Mus dunni cells. Mobilization assays demonstrated that Bxv1 transcomplements defective MuLV-based retrovectors. In contrast, common rodent β cell lines, rat INS-1E and RIN-5F cells and mouse MIN6 and βTC3 cells, displayed either no or extremely weak xenotropic helper activity toward MuLV-based retrovectors, although xenotropic retrovirus sequences and transcripts were detected in both mouse cell lines. Bxv1 propagation from EndoC-βH1/2 to 293T cells occurred only under optimized conditions and was overall poorly efficient. Thus, although our data imply that MuLV-based retrovectors should be cautiously used in EndoC-βH1/2 cells, our results indicate that an involuntary propagation of Bxv1 from these cells can be easily avoided with good laboratory practices. PMID:26901817

  2. Functional and phenotypic characterization of a protein from Lactobacillus acidophilus involved in cell morphology, stress tolerance and adherence to intestinal cells.

    PubMed

    O'Flaherty, Sarah J; Klaenhammer, Todd R

    2010-11-01

    Structural components of the cell surface have an impact on some of the beneficial attributes of probiotic bacteria. In silico analysis of the L. acidophilus NCFM genome sequence revealed the presence of a putative cell surface protein that was predicted to be a myosin cross-reactive antigen (MCRA). As MCRAs are conserved among many probiotic bacteria, we used the upp-based counterselective gene replacement system, designed recently for use in L. acidophilus, to determine the functional role of this gene (LBA649) in L. acidophilus NCFM. Phenotypic assays were undertaken with the parent strain (NCK1909) and deletion mutant (NCK2015) to assign a function for this gene. The growth of NCK2015 (ΔLBA649) was reduced in the presence of lactate, acetate, porcine bile and salt. Adhesion of NCK2015 to Caco-2 cells was substantially reduced for both stationary-phase (∼45 % reduction) and exponential-phase cells (∼50 % reduction). Analysis of NCK2015 by scanning electron microscopy revealed a longer cell morphology after growth in MRS broth compared to NCK1909. These results indicate a role for LBA649 in stress tolerance, cell wall division and adherence to Caco-2 cells. PMID:20829293

  3. 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

  4. The comparison of the effect of endodontic irrigation on cell adherence to root canal dentin.

    PubMed

    Ring, Karla C; Murray, Peter E; Namerow, Kenneth N; Kuttler, Sergio; Garcia-Godoy, Franklin

    2008-12-01

    The purpose of this study was to compare the effect of 10 different endodontic irrigation and chelating treatments on dental pulp stem cell (DPSC) attachment to root canal surfaces. Thirty-eight extracted human nondiseased single-canal teeth were cleaned and shaped using ProTaper and ProFile rotary instrumentation (Tulsa Dentsply, Tulsa, OK). The irrigation treatments investigated were 6% sodium hypochlorite, 2% chlorhexidine gluconate, Aquatine Endodontic Cleanser, and Morinda citrifolia juice. The irrigation treatments were used in conjunction with EDTA or MTAD. The instrumented teeth were immediately placed in cell culture with confluent DPSCs for 1 week. The number of attached DPSCs appeared to be correlated with the cytotoxicity of the root canal irrigating solution (analysis of variance, p < 0.0001). The presence or absence of the smear layer had little influence on DPSC activity (chi-square, p > 0.05). The results suggest that biocompatible irrigants are needed to promote DPSC attachment to root canal dentin, which is essential to accomplish some regenerative endodontic therapies. PMID:19026877

  5. Experimental Adaptation of Rotaviruses to Tumor Cell Lines

    PubMed Central

    Guerrero, Carlos A.; Guerrero, Rafael A.; Silva, Elver; Acosta, Orlando; Barreto, Emiliano

    2016-01-01

    A number of viruses show a naturally extended tropism for tumor cells whereas other viruses have been genetically modified or adapted to infect tumor cells. Oncolytic viruses have become a promising tool for treating some cancers by inducing cell lysis or immune response to tumor cells. In the present work, rotavirus strains TRF-41 (G5) (porcine), RRV (G3) (simian), UK (G6-P5) (bovine), Ym (G11-P9) (porcine), ECwt (murine), Wa (G1-P8), Wi61 (G9) and M69 (G8) (human), and five wild-type human rotavirus isolates were passaged multiple times in different human tumor cell lines and then combined in five different ways before additional multiple passages in tumor cell lines. Cell death caused by the tumor cell-adapted isolates was characterized using Hoechst, propidium iodide, 7-AAD, Annexin V, TUNEL, and anti-poly-(ADP ribose) polymerase (PARP) and -phospho-histone H2A.X antibodies. Multiple passages of the combined rotaviruses in tumor cell lines led to a successful infection of these cells, suggesting a gain-of-function by the acquisition of greater infectious capacity as compared with that of the parental rotaviruses. The electropherotype profiles suggest that unique tumor cell-adapted isolates were derived from reassortment of parental rotaviruses. Infection produced by such rotavirus isolates induced chromatin modifications compatible with apoptotic cell death. PMID:26828934

  6. Exometabolom analysis of breast cancer cell lines: Metabolic signature

    PubMed Central

    Willmann, Lucas; Erbes, Thalia; Halbach, Sebastian; Brummer, Tilman; Jäger, Markus; Hirschfeld, Marc; Fehm, Tanja; Neubauer, Hans; Stickeler, Elmar; Kammerer, Bernd

    2015-01-01

    Cancer cells show characteristic effects on cellular turnover and DNA/RNA modifications leading to elevated levels of excreted modified nucleosides. We investigated the molecular signature of different subtypes of breast cancer cell lines and the breast epithelial cell line MCF-10A. Prepurification of cell culture supernatants was performed by cis-diol specific affinity chromatography using boronate-derivatized polyacrylamide gel. Samples were analyzed by application of reversed phase chromatography coupled to a triple quadrupole mass spectrometer. Collectively, we determined 23 compounds from RNA metabolism, two from purine metabolism, five from polyamine/methionine cycle, one from histidine metabolism and two from nicotinate and nicotinamide metabolism. We observed major differences of metabolite excretion pattern between the breast cancer cell lines and MCF-10A, just as well as between the different breast cancer cell lines themselves. Differences in metabolite excretion resulting from cancerous metabolism can be integrated into altered processes on the cellular level. Modified nucleosides have great potential as biomarkers in due consideration of the heterogeneity of breast cancer that is reflected by the different molecular subtypes of breast cancer. Our data suggests that the metabolic signature of breast cancer cell lines might be a more subtype-specific tool to predict breast cancer, rather than a universal approach. PMID:26293811

  7. Effect of dehydrodidemnin B on human colon carcinoma cell lines.

    PubMed

    Lobo, C; García-Pozo, S G; Núñez de Castro, I; Alonso, F J

    1997-01-01

    Didemnins are cytotoxic agents belonging to a depsipeptide family isolated from marine tunicates. In the present study, a new member, dehydrodidemnin B (DDB), isolated from the mediterranean tunicate Aplidium albicans, was used. The effect of the drug on human colon cultured cell lines was tested using multiple approaches: proliferation studies, long term survival after three hours of exposure to DDB by means of a clonogenic assay and the decrease of the protooncogen, ornithine decarboxylase, activity. A dehydrodidemnin B concentration of 10(-8) M completely inhibited cell growth. The IC50 obtained using the MTT proliferation test, indicated that the most proliferative cell line (CT-2) was the most sensitive to the drug. Using a clonogenic assay a clear dose-response was obtained for the three cell lines used; HT-29 cell line showed the minimum survival after 3 hours of dehydrodidemnin B treatment. A dose-dependent decrease in ornithine decarboxylase activity was also observed in three cell lines assayed. The data presented indicate that the dehydrodidemnin B is a potent cytotoxic agent on rapidly dividing human colon cancer cells. PMID:9066673

  8. Transcription profiles of non-immortalized breast cancer cell lines

    PubMed Central

    Fernandez-Cobo, Mariana; Holland, James F; Pogo, Beatriz GT

    2006-01-01

    Background Searches for differentially expressed genes in tumours have made extensive use of array technology. Most samples have been obtained from tumour biopsies or from established tumour-derived cell lines. Here we compare cultures of non-immortalized breast cancer cells, normal non-immortalized breast cells and immortalized normal and breast cancer cells to identify which elements of a defined set of well-known cancer-related genes are differentially expressed. Methods Cultures of cells from pleural effusions or ascitic fluids from breast cancer patients (MSSMs) were used in addition to commercially-available normal breast epithelial cells (HMECs), established breast cancer cell lines (T-est) and established normal breast cells (N-est). The Atlas Human Cancer 1.2 cDNA expression array was employed. The data obtained were analysed using widely-available statistical and clustering software and further validated through real-time PCR. Results According to Significance Analysis of Microarray (SAM) and AtlasImage software, 48 genes differed at least 2-fold in adjusted intensities between HMECs and MSSMs (p < 0.01). Some of these genes have already been directly linked with breast cancer, metastasis and malignant progression, whilst others encode receptors linked to signal transduction pathways or are otherwise related to cell proliferation. Fifty genes showed at least a 2.5-fold difference between MSSMs and T-est cells according to AtlasImage, 2-fold according to SAM. Most of these classified as genes related to metabolism and cell communication. Conclusion The expression profiles of 1176 genes were determined in finite life-span cultures of metastatic breast cancer cells and of normal breast cells. Significant differences were detected between the finite life-span breast cancer cell cultures and the established breast cancer cell lines. These data suggest caution in extrapolating information from established lines for application to clinical cancer research. PMID

  9. Characterisation of thyroid medullary carcinoma TT cell line.

    PubMed

    Zabel, M; Grzeszkowiak, J

    1997-01-01

    TT cell line is the best known stabilized cell line derived from the human medullary thyroid carcinoma. The ultrastructural characteristics of these cells include well developed rough endoplasmic reticulum, a prominent Golgi apparatus and a considerable number of secretory granules. Numerous hormones were immunocytochemically demonstrated in TT cells of which calcitonin and calcitonin gene-related peptide (CGRP) are the products of the same gene but an alternative RNA processing. TT cells were found to produce some other hormones as well, namely ACTH, neurotensin, enkephalin, PTHrP, gastrin-releasing peptide (GRP), serotonin but also functional proteins of the chromogranin group, synaptophysin, NSE, calbindin and tyrosine hydroxylase. Some marker proteins have been detected in the cytosol (CEA) and in the cytoskeleton (alpha-tubulin, cytokeratin). The influence of numerous factors on the secretory activity of these cells has been demonstrated so far, including effects of 1,25-dihydroxycholecalciferol, glucocorticoids, sex steroids, cAMP, gastrin-releasing peptide, sodium butyrate, phorbol esters, ionomycin and forskolin. The investigators performed on the TT cell line demonstrate that this is the most reliable model system for the human parafollicular cells developed so far, in comparison to other cell lines derived from the medullary carcinoma of the thyroid. PMID:9046062

  10. 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

  11. Adherence of non-O157 Shiga-toxin Escherichia coli to bovine recto-anal junction squamous epithelial cells appears to be mediated by mechanisms distinct from those used by O157

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study presents evidence that the pattern of adherence of clinically relevant non-O157 Shiga-toxin producing Escherichia coli (STEC) to bovine recto-anal junction squamous epithelial cells (RSE) is similar to that of O157, although the mechanisms of adherence appear to be distinct. Our results f...

  12. Proteins other than the Locus of Enterocyte Effacement-encoded proteins may contribute to Escherichia coli O157:H7 adherence to bovine rectoanal junction stratified squamous epithelial cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study, the Type III Secretion System (TTSS) proteins considered critical for Escherichia coli O157 (O157) adherence to the follicle-associated epithelial (FAE) cells at the bovine recto-anal junction (RAJ), did not appear to contribute to O157 adherence to the RAJ squamous epithelial (RSE) ...

  13. Colonization of human epithelial cell lines by Corynebacterium ulcerans from human and animal sources.

    PubMed

    Hacker, Elena; Ott, Lisa; Hasselt, Kristin; Mattos-Guaraldi, Ana Luiza; Tauch, Andreas; Burkovski, Andreas

    2015-08-01

    Corynebacterium ulcerans is an emerging pathogen transmitted by a zoonotic pathway to humans. Despite rising numbers of infections and potentially fatal outcomes, data on the colonization of the human host are lacking up to now. In this study, adhesion of two C. ulcerans isolates to human epithelial cells, invasion of host cells and the function of two putative virulence factors with respect to these processes were investigated. C. ulcerans strains BR-AD22 and 809 were able to adhere to Detroit562 and HeLa cells, and invade these epithelial cell lines with a rate comparable to other pathogens as shown by scanning electron microscopy, fluorescence microscopy and replication assays. Infection led to detrimental effects on the cells as deduced from measurements of transepithelial resistance. Mutant strains of putative virulence factors phospholipase D and DIP0733 homologue CULC22_00609 generated in this study showed no influence on colonization under the experimental conditions tested. The data presented here indicate a high infectious potential of this emerging pathogen. PMID:26066797

  14. Suppression of BCG cell wall induced delayed-type hypersensitivity by BCG pre-treatment. I. Induction of adherent suppressor cells by live BCG injection and their characterization.

    PubMed Central

    Kato, K; Yamamoto, K I; Kakinuma, M; Ishihara, C; Azuma, I

    1981-01-01

    Previous injections of live Bacillus Calmette-Guérin (BCG) in mice produced a suppression of delayed-type hypersensitivity (DTH) induced by oil-treated BCG cell walls (CW). This phenomenon was analysed by the macrophage migration inhibition (MI) test in which peritoneal exudate cells (PEC) from live BCG-injected mice were mixed with PEC from BCG CW-immunized mice, with the result that the former cells suppressed the MI activity in the latter. We considered the Mi test to be a reliable method for demonstrating the existence of suppressor cells induced by the injection of live BCG. Moreover, we found that the adherent cells of PEC possessed a suppressive effect which was retained even after treatment with either anti-mouse Ig or anti-brain associated theta (BA theta) antigen; that the PEC from mice injected with live BCG on at least the 12th day before cell harvesting showed the suppression; and that the suppression operated across the H-2 barrier. PMID:6450731

  15. Characterization of a human ovarian teratocarcinoma-derived cell line.

    PubMed

    Zeuthen, J; Nørgaard, J O; Avner, P; Fellous, M; Wartiovaara, J; Vaheri, A; Rosén, A; Giovanella, B C

    1980-01-15

    A cell line (PA I), derived from human ovarian teratocarcinoma cells, was obtained by culturing ascitic fluid cells from a patient with recurrence of malignant ovarian teratoma. During early passages the cultured cells showed a variable morphology, a long doubling time, and a low plating efficiency (2%). After about 50 passages in vitro, a cell population which was more homogeneous and resembled embryonal carcinoma cells were obtained. These cells had a shorter doubling time (26 h), and increased plating efficiency (77%). The early-passage cells were aneuploid (P 24) whereas the late-passage cells had a normal diploid karyotype with one balanced translocation between chromosomes No. 15 and No. 20 (P 224). Details of the karyotype suggest that the cells are heterozygous, i.e. derived from a stage before the first meiotic division. One of the two X chromosomes were inactive, and the cells expressed HLA antigens (A28 and B12), and beta 2-microglobulin. Expression of F9 antigen, characteristic of two-cell and later preimplantation embryos, was absent, while expression of PCC4 antigen, expressed also by blastocysts, was present. This finding suggests that the line might express some embryonic characteristics. The PA I cell line maintained in monolayer cultures showed several characteristics of malignant cells. The proportion of malignant cells increased with successive passages in vitro. The late-passage cells represented a fairly homogenous population of malignant cells similar to embryonal carcinoma cells. Late-passage PA I cells, when seeded under conditions that prevented attachment of cells to the substratum, formed embryoid bodies consisting of an inner core of cells similar to embryonal carcinoma cells, surrounded by a rind of endoderm-like cells. These two cell layers were separated by a basement membrane-like structure containing fibronectin. The core embryonal carcinoma cells expressed high alkaline phosphatase activity whereas the endoderm-like cells had low

  16. Three-dimensional cultured glioma cell lines

    NASA Technical Reports Server (NTRS)

    Gonda, Steve R. (Inventor); Marley, Garry M. (Inventor)

    1991-01-01

    Three-dimensional glioma spheroids were produced in vitro with size and histological differentiation previously unattained. The spheroids were grown in liquid media suspension in a Johnson Space Center (JSC) Rotating Wall Bioreactor without using support matrices such as microcarrier beads. Spheroid volumes of greater than 3.5 cu mm and diameters of 2.5 mm were achieved with a viable external layer or rim of proliferating cells, a transitional layer beneath the external layer with histological differentiation, and a degenerative central region with a hypoxic necrotic core. Cell debris was evident in the degenerative central region. The necrotics centers of some of the spheroids had hyaline droplets. Granular bodies were detected predominantly in the necrotic center.

  17. Cell Line Modeling to Study Biomarker Panel in Prostate Cancer

    PubMed Central

    NickKholgh, Bita; Fang, Xiaolan; Winters, Shira M.; Raina, Anvi; Pandya, Komal S.; Gyabaah, Kenneth; Fino, Nora; Balaji, K.C.

    2016-01-01

    BACKGROUND African–American men with prostate cancer (PCa) present with higher-grade and -stage tumors compared to Caucasians. While the disparity may result from multiple factors, a biological basis is often strongly suspected. Currently, few well-characterized experimental model systems are available to study the biological basis of racial disparity in PCa. We report a validated in vitro cell line model system that could be used for the purpose. METHODS We assembled a PCa cell line model that included currently available African–American PCa cell lines and LNCaP (androgen-dependent) and C4-2 (castration-resistant) Caucasian PCa cells. The utility of the cell lines in studying the biological basis of variance in a malignant phenotype was explored using a multiplex biomarker panel consisting of proteins that have been proven to play a role in the progression of PCa. The panel expression was evaluated by Western blot and RT-PCR in cell lines and validated in human PCa tissues by RT-PCR. As proof-of-principle to demonstrate the utility of our model in functional studies, we performed MTS viability assays and molecular studies. RESULTS The dysregulation of the multiplex biomarker panel in primary African–American cell line (E006AA) was similar to metastatic Caucasian cell lines, which would suggest that the cell line model could be used to study an inherent aggressive phenotype in African–American men with PCa. We had previously demonstrated that Protein kinase D1 (PKD1) is a novel kinase that is down regulated in advanced prostate cancer. We established the functional relevance by over expressing PKD1, which resulted in decreased proliferation and epithelial mesenchymal transition (EMT) in PCa cells. Moreover, we established the feasibility of studying the expression of the multiplex biomarker panel in archived human PCa tissue from African–Americans and Caucasians as a prelude to future translational studies. CONCLUSION We have characterized a novel in

  18. 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

  19. Measurement of Acetylcholine from Cell Lines

    PubMed Central

    Lau, Jamie K.; Brown, Kathleen C.; Dasgupta, Piyali

    2016-01-01

    Cigarette smoking is the leading risk factor for the development of lung cancer. It is estimated that smoking is associated with 80–90% of lung cancer cases throughout the world (see References 1 and 2). The addictive component of cigarette smoke is nicotine. Our published data shows that nicotine promotes the production of acetylcholine (ACh) in human bronchioalveolar carcinoma cells (BACs) (Lau et al., 2013). ACh functions as a growth factor in human BACs. The following protocol is based on a published protocol by (Song et al., 2003), with some modifications (Lau et al., 2013; Song et al., 2008; Song et al., 2003; Sekhon et al., 2003). An important point to remember is that fetal bovine serum (FBS) contains a high amount of acetylcholine (ACh). Therefore, cells must be cultured in serum-free medium to measure ACh in the culture supernatant. Two aliquots of the culture supernatant are used for analysis. This protocol measures the total choline in the cell supernatent under two conditions: 1) After treatment with acetylcholinesterase (AChE), which converts the ACh to choline (also called the total choline sample) and 2) after measuring the amount of free choline in the sample. The concentration of ACh in the sample calculated by subtracting the free choline from the total choline.

  20. Steroid hormone secretion in inflammatory breast cancer cell lines.

    PubMed

    Illera, Juan Carlos; Caceres, Sara; Peña, Laura; de Andres, Paloma J; Monsalve, Beatriz; Illera, Maria J; Woodward, Wendy A; Reuben, James M; Silvan, Gema

    2015-12-01

    Inflammatory breast carcinoma (IBC) is a special type of breast cancer with a poor survival rate. Though several IBC cell lines have been established, recently a first IMC cell line was established. The aims of this study were: (1) to validate a highly sensitive, reliable, accurate and direct amplified enzyme immunoassay (EIA) to measure several cell-secreted steroid hormones: progesterone (P4), androstenedione (A4), testosterone (T), 17β-estradiol (E2) and estrone sulfate (SO4E1) in the culture medium. (2) To assess whether hormone production profile by IPC-366 cells validates the IMC model for human IBC. We validated a non-competitive amplified EIA for inflammatory breast cancer cell lines based on the results of accuracy, precision, sensitivity and parallelism. The low detection limits of the technique were: P4=13.2 pg/well, A4=2.3 pg/well, T=11.4 pg/well, E2=1.9 pg/well and SO4E1=4.5 pg/well. Intra- and inter-assay coefficient of variation percentages were <10%. The mean recovery rate of hormone added to the culture medium was >90%. In all hormones studied SUM149 have higher levels (1.4 times, but not significant) than IPC-366, and the correlation index between SUM149 and IPC-366 concentrations were >97%. We can coclude that cells of both cell lines, IPC-366 and SUM149, are capable to produce steroid hormone in culture media. The presented EIA methodology is very valuable for the detection of steroid production in culture media and could be used in hormone regulation studies and therapeutic agents in cell lines of inflammatory and non-inflammatory mammary carcinoma or other cancer cell lines in preclinical studies. PMID:26495931

  1. Embryonic germ cell lines and their derivation from mouse primordial germ cells.

    PubMed

    Labosky, P A; Barlow, D P; Hogan, B L

    1994-01-01

    When primordial germ cells of the mouse are cultured on feeder layers with the addition of the polypeptide signalling molecules leukaemia inhibitory factor, Steel factor and basic fibroblast growth factor they give rise to cells that resemble undifferentiated blastocyst-derived embryonic stem cells. These primordial germ cell-derived embryonic germ cells (EG cells) can be induced to differentiate extensively in culture and also form teratocarcinomas when injected into nude mice. Additionally, they contribute to chimeras when injected into host blastocysts. We have derived multiple EG cell lines from 8.5 days post coitum (dpc) embryos of C57BL/6 inbred mice. Four independent EG cell lines with normal male karyotypes have formed chimeras (up to 70% coat colour chimerism) when injected into BALB/c host blastocysts. Chimeric mice from all four cell lines are fertile, but only those from one line have transmitted coat colour markers through the germline. Studies have also been carried out to determine whether gonadal primordial germ cells can give rise to pluripotent EG cells. Germ cells from gonads of 15.5 dpc C57BL/6 embryos and newborn mice failed to produce EG cell lines. EG cell lines capable of forming teratocarcinomas and coat colour chimeras have been established from primordial germ cells of 12.5 dpc genital ridges. We are currently testing the genomic imprinting status of the insulin-like growth factor type 2 receptor gene (Igf2r) in our different EG cell lines. PMID:7835148

  2. MOLECULAR AND CYTOGENETIC ANALYSIS OF LUNG TUMOR CELL LINES

    EPA Science Inventory

    We have measured the levels of amplification of oncogenes and tumor marker genes or other genes of interest in nine human lung tumor cell lines in comparison to normal human bronchial epithelial cells or normal blood lymphocytes to test the hypothesis that aberrant amplification ...

  3. 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

  4. Solid Oxide Fuel Cell Systems PVL Line

    SciTech Connect

    Susan Shearer - Stark State College; Gregory Rush - Rolls-Royce Fuel Cell Systems

    2012-05-01

    In July 2010, Stark State College (SSC), received Grant DE-EE0003229 from the U.S. Department of Energy (DOE), Golden Field Office, for the development of the electrical and control systems, and mechanical commissioning of a unique 20kW scale high-pressure, high temperature, natural gas fueled Stack Block Test System (SBTS). SSC worked closely with subcontractor, Rolls-Royce Fuel Cell Systems (US) Inc. (RRFCS) over a 13 month period to successfully complete the project activities. This system will be utilized by RRFCS for pre-commercial technology development and training of SSC student interns. In the longer term, when RRFCS is producing commercial products, SSC will utilize the equipment for workforce training. In addition to DOE Hydrogen, Fuel Cells, and Infrastructure Technologies program funding, RRFCS internal funds, funds from the state of Ohio, and funding from the DOE Solid State Energy Conversion Alliance (SECA) program have been utilized to design, develop and commission this equipment. Construction of the SBTS (mechanical components) was performed under a Grant from the State of Ohio through Ohio's Third Frontier program (Grant TECH 08-053). This Ohio program supported development of a system that uses natural gas as a fuel. Funding was provided under the Department of Energy (DOE) Solid-state Energy Conversion Alliance (SECA) program for modifications required to test on coal synthesis gas. The subject DOE program provided funding for the electrical build, control system development and mechanical commissioning. Performance testing, which includes electrical commissioning, was subsequently performed under the DOE SECA program. Rolls-Royce Fuel Cell Systems is developing a megawatt-scale solid oxide fuel cell (SOFC) stationary power generation system. This system, based on RRFCS proprietary technology, is fueled with natural gas, and operates at elevated pressure. A critical success factor for development of the full scale system is the capability to

  5. Heterozygous Embryonic Stem Cell Lines Derived from Nonhuman Primate Parthenotes

    PubMed Central

    Dighe, Vikas; Clepper, Lisa; Pedersen, Darlene; Byrne, James; Ferguson, Betsy; Gokhale, Sumita; Penedo, M. Cecilia T.; Wolf, Don; Mitalipov, Shoukhrat

    2009-01-01

    Monoparental parthenotes represent a potential source of histocompatible stem cells that should be isogenic with the oocyte donor and therefore suitable for use in cell or tissue replacement therapy. We generated five rhesus monkey parthenogenetic embryonic stem cell (PESC) lines with stable, diploid female karyotypes that were morphologically indistinguishable from biparental controls, expressed key pluripotent markers, and generated cell derivatives representative of all three germ layers following in vivo and in vitro differentiation. Interestingly, high levels of heterozygosity were observed at the majority of loci that were polymorphic in the oocyte donors. Some PESC lines were also heterozygous in the major histocompatibility complex region, carrying haplotypes identical to those of the egg donor females. Expression analysis revealed transcripts from some imprinted genes that are normally expressed from only the paternal allele. These results indicate that limitations accompanying the potential use of PESC-derived phenotypes in regenerative medicine, including aberrant genomic imprinting and high levels of homozygosity, are cell line-dependent and not always present. PESC lines were derived in high enough yields to be practicable, and their derivatives are suitable for autologous transplantation into oocyte donors or could be used to establish a bank of histocompatible cell lines for a broad spectrum of patients. PMID:18192229

  6. Establishment and characterization of duck embryo epithelial (DEE) cell line and its use as a new approach toward DHAV-1 propagation and vaccine development.

    PubMed

    Wang, Wenxiu; Said, Abdelrahman; Wang, Yan; Fu, Qiang; Xiao, Yueqiang; Lv, Sufang; Shen, Zhiqiang

    2016-02-01

    The primary cell culture was derived from duck embryonic tissue, digested with collagenase type I. The existence of cell colonies with epithelial-like morphology, named duck embryo epithelial (DEE), were purified and optimally maintained at 37°C in M199 medium supplemented with 5% fetal bovine serum. The purified cells were identified as epithelial cell line by detecting Keratin-18 expression using immunofluorescence assay. Our findings demonstrated that DEE cell line can be propagated in culture with (i) a great capacity to adhere, (ii) a great proliferation activity, and (iii) a population doubling time of approximately 18h. Chromosomal features of the DEE cell line were remained constant after the 50th passage. Further characterizations of DEE cell line showed that cell line can normally be grown even after several passages and never converted to tumorigenic cells either in vitro or in vivo study. Susceptibility of DEE cell line was determined for transfection and duck hepatitis A type 1 virus (DHAV-1)-infection. Interestingly, the 50% egg lethal dose (ELD50) of the propagated virus in DEE cell line was higher than ELD50 of the propagated virus in embryonated eggs. Finally, DEE cell line was evaluated to be used as a candidate for DHAV-1 vaccine development. Our results showed that the propagated DHAV-1 vaccine strain SDE in DEE cell line was able to protect ducklings against DHAV-1 challenge. Taken together, our findings suggest that the DEE cell line can serve as a valuable tool for DHAV-1 propagation and vaccine production. PMID:26739426

  7. Germ line development: lessons learned from pluripotent stem cells.

    PubMed

    Martínez-Arroyo, Ana M; Medrano, Jose V; Remohí, José; Simón, Carlos

    2014-10-01

    Current knowledge about mammalian germ line development is mainly based on the mouse model and little is known about how this fundamental process occurs in humans. This review summarizes our current knowledge of genetic and epigenetic germ line development in mammals, mainly focusing on primordial germ cell (PGC) specification events, comparing the differences between mouse and human models. We also emphasize the knowledge derived from the most successful strategies used to generate germ cell-like cells in vitro in both models and major obstacles to obtaining bona fide in vitro-derived gametes are considered. PMID:25461452

  8. 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

  9. Derivation of human embryonic stem cell line Genea019.

    PubMed

    Dumevska, Biljana; Peura, Teija; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea019 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype, female Allele pattern and unaffected Htt CAG repeat length, compared to HD affected sibling Genea020. Pluripotency of Genea019 was demonstrated with 75% of cells expressing Nanog, 89% Oct4, 48% Tra1-60 and 85% SSEA4, a Pluritest Pluripotency score of 22.97, Novelty score of 1.42, tri-lineage teratoma formation and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27346002

  10. Guidelines for the use of cell lines in biomedical research

    PubMed Central

    Geraghty, R J; Capes-Davis, A; Davis, J M; Downward, J; Freshney, R I; Knezevic, I; Lovell-Badge, R; Masters, J R W; Meredith, J; Stacey, G N; Thraves, P; Vias, M

    2014-01-01

    Cell-line misidentification and contamination with microorganisms, such as mycoplasma, together with instability, both genetic and phenotypic, are among the problems that continue to affect cell culture. Many of these problems are avoidable with the necessary foresight, and these Guidelines have been prepared to provide those new to the field and others engaged in teaching and instruction with the information necessary to increase their awareness of the problems and to enable them to deal with them effectively. The Guidelines cover areas such as development, acquisition, authentication, cryopreservation, transfer of cell lines between laboratories, microbial contamination, characterisation, instability and misidentification. Advice is also given on complying with current legal and ethical requirements when deriving cell lines from human and animal tissues, the selection and maintenance of equipment and how to deal with problems that may arise. PMID:25117809

  11. Guidelines for the use of cell lines in biomedical research.

    PubMed

    Geraghty, R J; Capes-Davis, A; Davis, J M; Downward, J; Freshney, R I; Knezevic, I; Lovell-Badge, R; Masters, J R W; Meredith, J; Stacey, G N; Thraves, P; Vias, M

    2014-09-01

    Cell-line misidentification and contamination with microorganisms, such as mycoplasma, together with instability, both genetic and phenotypic, are among the problems that continue to affect cell culture. Many of these problems are avoidable with the necessary foresight, and these Guidelines have been prepared to provide those new to the field and others engaged in teaching and instruction with the information necessary to increase their awareness of the problems and to enable them to deal with them effectively. The Guidelines cover areas such as development, acquisition, authentication, cryopreservation, transfer of cell lines between laboratories, microbial contamination, characterisation, instability and misidentification. Advice is also given on complying with current legal and ethical requirements when deriving cell lines from human and animal tissues, the selection and maintenance of equipment and how to deal with problems that may arise. PMID:25117809

  12. Structure and function of sinusoidal lining cells in the liver.

    PubMed

    Wisse, E; Braet, F; Luo, D; De Zanger, R; Jans, D; Crabbé, E; Vermoesen, A

    1996-01-01

    , interleukin 1, interleukin 6, and a series of eicosanoids (28) and become cytotoxic against tumor cells [e.g., colon carcinoma cells (19, 22, 48)]. Toxic secretory products can cause necrosis of the liver parenchyma, which constitutes a crucial factor in liver transplantation (55). Pit cells possess characteristic azurophylic granules and display a high level of spontaneous cytolytic activity against various tumor cells, identifying themselves as natural killer cells (10). The number and cytotoxicity of pit cells can be considerably enhanced with biological response modifiers, such as Zymosan or interleukin 2 (8). Pit cell proliferation occurs within the liver, but recent evidence indicates that blood large granular lymphocytes develop into pit cells in 2 steps involving high- and low-density pit cells (88). Kupffer cells control the motility, adherence, viability, and cytotoxicity of pit cells (89), whereas cytotoxicity against tumor cells is synergistically enhanced (80, 81). PMID:8839287

  13. Comparative study of the damage produced by acute ethanol and acetaldehyde treatment in a human fetal hepatic cell line.

    PubMed

    Olivares, I P; Bucio, L; Souza, V; Cárabez, A; Gutiérrez-Ruiz, M C

    1997-06-27

    The effects of acute ethanol and acetaldehyde treatment on cell proliferation, cell adhesion capacity, neutral red incorporation into lysosomes, glutathione content, protein sulfhydryl compounds, lipid peroxidation, inner mitochondrial membrane integrity (MTT test), lactate dehydrogenase activity (LDH) and ultrastructural alterations were investigated in a human fetal hepatic cell line (WRL-68 cells). WRL-68 cells were used, due to the fact that, although this cell line expresses some hepatic characteristics, it does not express alcohol dehydrogenase or cytochrome P450 activity, so it could be a good model to study the effect of the toxic agents per se. Cells were exposed during 120 min with 200 mM ethanol or 10 mM acetaldehyde. Under these conditions, cells presented 100% viability and no morphological alteration was observed by light microscopy. Acetaldehyde-treated cells reduced their proliferative capacity drastically while the ethanol-treated ones presented no difference with control cells. Cell adhesion to substrate, measured as time required to adhere to the substrate and time required to detach from the substrate, was diminished in acetaldehyde WRL-68-treated cells. Cytotoxicity measures as neutral red and MTT test showed that acetaldehyde-treated cells presented more damage than ethanol-treated ones. Cellular respiratory capacity was compromised by acetaldehyde treatment due to 40% less oxygen consumption than control cells. Lipid peroxidation values, measured as malondialdehyde production, were higher in ethanol-treated WRL-68 cells (127%) than in acetaldehyde-treated ones (60%) to control cell values. Lactate dehydrogenase activity (LDH) in extracellular media of ethanol-treated cells presented the highest values. GSH content was reduced 95% and thiol protein content was diminished severely in acetaldehyde-treated cells. Transmission electron microscopy showed more ultrastructural alterations in cells treated with acetaldehyde. The results indicate that

  14. Female Sex Bias in Human Embryonic Stem Cell Lines

    PubMed Central

    Ben-Yosef, Dalit; Amit, Ami; Malcov, Mira; Frumkin, Tsvia; Ben-Yehudah, Ahmi; Eldar, Ido; Mey-Raz, Nava; Azem, Foad; Altarescu, Gheona; Renbaum, Paul; Beeri, Rachel; Varshaver, Irit; Eldar-Geva, Talia; Epsztejn-Litman, Silvina; Levy-Lahad, Ephrat

    2012-01-01

    The factors limiting the rather inefficient derivation of human embryonic stem cells (HESCs) are not fully understood. The aim of this study was to analyze the sex ratio in our 42 preimplantation genetic diagnosis (PGD)-HESC lines, in an attempt to verify its affect on the establishment of HESC lines. The ratio between male and female PGD-derived cell lines was compared. We found a significant increase in female cell lines (76%). This finding was further confirmed by a meta-analysis for combining the results of all PGD-derived HESC lines published to date (148) and all normal karyotyped HESC lines derived from spare in vitro fertilization embryos worldwide (397). Further, gender determination of embryos demonstrated that this difference originates from the actual derivation process rather than from unequal representation of male and female embryos. It can therefore be concluded that the clear-cut tendency for female preponderance is attributed to suboptimal culture conditions rather than from a true gender imbalance in embryos used for derivation of HESC lines. We propose a mechanism in which aberrant X chromosome inactivation and/or overexpression of critical metabolic X-linked genes might explain this sex dimorphism. PMID:21585244

  15. Female sex bias in human embryonic stem cell lines.

    PubMed

    Ben-Yosef, Dalit; Amit, Ami; Malcov, Mira; Frumkin, Tsvia; Ben-Yehudah, Ahmi; Eldar, Ido; Mey-Raz, Nava; Azem, Foad; Altarescu, Gheona; Renbaum, Paul; Beeri, Rachel; Varshaver, Irit; Eldar-Geva, Talia; Epsztejn-Litman, Silvina; Levy-Lahad, Ephrat; Eiges, Rachel

    2012-02-10

    The factors limiting the rather inefficient derivation of human embryonic stem cells (HESCs) are not fully understood. The aim of this study was to analyze the sex ratio in our 42 preimplantation genetic diagnosis (PGD)-HESC lines, in an attempt to verify its affect on the establishment of HESC lines. The ratio between male and female PGD-derived cell lines was compared. We found a significant increase in female cell lines (76%). This finding was further confirmed by a meta-analysis for combining the results of all PGD-derived HESC lines published to date (148) and all normal karyotyped HESC lines derived from spare in vitro fertilization embryos worldwide (397). Further, gender determination of embryos demonstrated that this difference originates from the actual derivation process rather than from unequal representation of male and female embryos. It can therefore be concluded that the clear-cut tendency for female preponderance is attributed to suboptimal culture conditions rather than from a true gender imbalance in embryos used for derivation of HESC lines. We propose a mechanism in which aberrant X chromosome inactivation and/or overexpression of critical metabolic X-linked genes might explain this sex dimorphism. PMID:21585244

  16. Generation of Mouse iNKT Cell Lines

    PubMed Central

    Li, Xiangming; Tsuji, Moriya; Schneck, Jonathan; Webb, Tonya J.

    2016-01-01

    Natural killer T (NKT) cells bridge the innate and adaptive arms of the immune system, and manipulating their effector functions can have therapeutic significances in the treatment of autoimmunity, transplant biology, infectious disease and cancer. This important lymphocyte subset regulates the immune system through their potent cytokine production following the recognition of lipid antigen present in the context of the MHC class I-like CD1d molecule, in addition their ability to directly mediate cytotoxicity. Here, we describe a method of expanding mouse invariant NKT (iNKT) cell lines from mononuclear cells isolated from the thymus, spleen, or liver using bone marrow derived dendritic cells. These iNKT cell lines can be used study their co-signaling requirements, cytokine profiles and cytotoxic functions which will greatly enhance our knowledge of iNKT cell biology.

  17. JNK Inhibition Inhibits Lateral Line Neuromast Hair Cell Development

    PubMed Central

    Cai, Chengfu; Lin, Jinchao; Sun, Shaoyang; He, Yingzi

    2016-01-01

    JNK signaling is known to play a role in regulating cell behaviors such as cell cycle progression, cell proliferation, and apoptosis, and recent studies have suggested important roles for JNK signaling in embryonic development. However, the precise function of JNK signaling in hair cell development remains poorly studied. In this study, we used the small molecule JNK inhibitor SP600125 to examine the effect of JNK signaling abrogation on the development of hair cells in the zebrafish lateral line neuromast. Our results showed that SP600125 reduced the numbers of both hair cells and supporting cells in neuromasts during larval development in a dose-dependent manner. Additionally, JNK inhibition strongly inhibited the proliferation of neuromast cells, which likely explains the decrease in the number of differentiated hair cells in inhibitor-treated larvae. Furthermore, western blot and in situ analysis showed that JNK inhibition induced cell cycle arrest through induction of p21 expression. We also showed that SP600125 induced cell death in developing neuromasts as measured by cleaved caspase-3 immunohistochemistry, and this was accompanied with an induction of p53 gene expression. Together these results indicate that JNK might be an important regulator in the development of hair cells in the lateral line in zebrafish by controlling both cell cycle progression and apoptosis. PMID:26903805

  18. Establishment, Immortalisation and Characterisation of Pteropid Bat Cell Lines

    PubMed Central

    Crameri, Gary; Todd, Shawn; Grimley, Samantha; McEachern, Jennifer A.; Marsh, Glenn A.; Smith, Craig; Tachedjian, Mary; De Jong, Carol; Virtue, Elena R.; Yu, Meng; Bulach, Dieter; Liu, Jun-Ping; Michalski, Wojtek P.; Middleton, Deborah; Field, Hume E.; Wang, Lin-Fa

    2009-01-01

    Background Bats are the suspected natural reservoir hosts for a number of new and emerging zoonotic viruses including Nipah virus, Hendra virus, severe acute respiratory syndrome coronavirus and Ebola virus. Since the discovery of SARS-like coronaviruses in Chinese horseshoe bats, attempts to isolate a SL-CoV from bats have failed and attempts to isolate other bat-borne viruses in various mammalian cell lines have been similarly unsuccessful. New stable bat cell lines are needed to help with these investigations and as tools to assist in the study of bat immunology and virus-host interactions. Methodology/Findings Black flying foxes (Pteropus alecto) were captured from the wild and transported live to the laboratory for primary cell culture preparation using a variety of different methods and culture media. Primary cells were successfully cultured from 20 different organs. Cell immortalisation can occur spontaneously, however we used a retroviral system to immortalise cells via the transfer and stable production of the Simian virus 40 Large T antigen and the human telomerase reverse transcriptase protein. Initial infection experiments with both cloned and uncloned cell lines using Hendra and Nipah viruses demonstrated varying degrees of infection efficiency between the different cell lines, although it was possible to infect cells in all tissue types. Conclusions/Significance The approaches developed and optimised in this study should be applicable to bats of other species. We are in the process of generating further cell lines from a number of different bat species using the methodology established in this study. PMID:20011515

  19. Tools for Targeted Genome Engineering of Established Drosophila Cell Lines

    PubMed Central

    Cherbas, Lucy; Hackney, Jennifer; Gong, Lei; Salzer, Claire; Mauser, Eric; Zhang, Dayu; Cherbas, Peter

    2015-01-01

    We describe an adaptation of φC31 integrase–mediated targeted cassette exchange for use in Drosophila cell lines. Single copies of an attP-bounded docking platform carrying a GFP-expression marker, with or without insulator elements flanking the attP sites, were inserted by P-element transformation into the Kc167 and Sg4 cell lines; each of the resulting docking-site lines carries a single mapped copy of one of the docking platforms. Vectors for targeted substitution contain a cloning cassette flanked by attB sites. Targeted substitution occurs by integrase-mediated substitution between the attP sites (integrated) and the attB sites (vector). We describe procedures for isolating cells carrying the substitutions and for eliminating the products of secondary off-target events. We demonstrate the technology by integrating a cassette containing a Cu2+-inducible mCherry marker, and we report the expression properties of those lines. When compared with clonal lines made by traditional transformation methods, which lead to the illegitimate insertion of tandem arrays, targeted insertion lines give more uniform expression, lower basal expression, and higher induction ratios. Targeted substitution, though intricate, affords results that should greatly improve comparative expression assays—a major emphasis of cell-based studies. PMID:26450921

  20. Non-targeted radiation effects in vertebrate cell lines

    NASA Astrophysics Data System (ADS)

    Ryan, Lorna

    Radiation effects, such as bystander effects, hyper radiosensitivity/induced radioresistance (HRS/IRR) and adaptive response that are not related to direct DNA damage are now accepted. However the inter-relationship between them and the possible impact on the scientific basis for radiation protection are highly controversial. This thesis attempts to elucidate the mechanisms of some of these well known but little understood effects. Each paper examines some aspect of bystander effects, adaptive responses and HRS/IRR in an effort to understand how they vary with cell type, dose and time of exposure to single or multiple doses. All the effects involve non-linear dose effect curves and are mainly evident following low doses. Overall findings of the thesis include (1) A clear difference was observed between radioresistant, tumorigenic cell lines with mutant p53 gene expression, and radiosensitive, more normal, cell lines with wild type p53. In general death inducing bystander responses are induced in normal cell populations exposed to low doses of radiation while survival inducing IRR and adaptive responses are seen in the radioresistant tumorigenic cell lines. (2) A cohort of fish cell lines which demonstrated survival promoting bystander effects, also did not show a protective adaptive responses. (3) Adaptive responses traditionally occur when a large challenge dose is given 4--6hrs following low (10--100mGy) priming doses but this thesis shows that for the epithelial cell lines tested, the size of the priming dose (range 0.1--2Gy) does not appear to alter the size of the recovery response. Additionally increased survival could be detected in some cases when the challenge dose was given within one hour of the priming dose. The overall conclusion is that cell lines induce either a bystander response or a protective/adaptive response depending on genetic background and other factors. Care is needed in the interpretation of data generated from only one or two cell lines

  1. Artificial islets from hybrid spheroids of three pancreatic cell lines.

    PubMed

    Jo, Y H; Jang, I J; Nemeno, J G; Lee, S; Kim, B Y; Nam, B M; Yang, W; Lee, K M; Kim, H; Takebe, T; Kim, Y S; Lee, J I

    2014-05-01

    Pancreatic islets have been the focus of recent studies exploring the pathologic mechanisms of diabetes mellitus as well as more effective and radical treatments for this disease. Islet transplantation is a promising therapeutic strategy; however, isolation of pancreatic islets for this purpose has been challenging, because the technique is time consuming and technically difficult, and tissue handling can be variable. Pseudo-islets can be used as an alternative to naïve islets, but require cellular sources or artificial materials. In this study, pancreas-derived cells were used to generate pseudo-islets. Because the pancreas is composed of a variety of cell types, namely α cells, β cells, δ cells, and other pancreatic cells that perform different functions, we used 3 different cell lines-NIT-1 (a β-cell line), α TC1 clone 6 (an α-cell line), and TGP52 (a pancreatic epithelial-like cell line)-which we cocultured in nonadhesive culture plates to produce hybrid cellular spheroids. These pseudo-islets had an oval shape and were morphologically similar to naïve islets; additionally, they expressed and secreted the pancreatic hormones insulin, glucagon, and somatostatin, as confirmed by reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay. The results demonstrate that pseudo-islets that mimic naïve islets can be successfully generated by a coculture method. These artificial islets can potentially be used for in vitro tests related to diabetes mellitus, specifically, in drug discovery or for investigating pathology. Moreover, they can be useful for examining basic questions pertaining to cell-cell interactions and tissue development. PMID:24815150

  2. 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

  3. Comparative proteomic profiling of Hodgkin lymphoma cell lines.

    PubMed

    Vergara, D; Simeone, P; De Matteis, S; Carloni, S; Lanuti, P; Marchisio, M; Miscia, S; Rizzello, A; Napolitano, R; Agostinelli, C; Maffia, M

    2016-01-01

    Classical Hodgkin lymphoma (cHL) is a malignancy with complex pathogenesis. The hallmark of the disease is the presence of large mononucleated Hodgkin and bi- or multinucleated Reed/Sternberg (H/RS) cells. The origin of HRS cells in cHL is controversial as these cells show the coexpression of markers of several lineages. Using a proteomic approach, we compared the protein expression profile of cHL models of T- and B-cell derivation to find proteins differentially expressed in these cell lines. A total of 67 proteins were found differentially expressed between the two cell lines including metabolic proteins and proteins involved in the regulation of the cytoskeleton and/or cell migration, which were further validated by western blotting. Additionally, the expression of selected B- and T-cell antigens was also assessed by flow cytometry to reveal significant differences in the expression of different surface markers. Bioinformatics analysis was then applied to our dataset to find enriched pathways and networks, and to identify possible key regulators. In the present study, a proteomic approach was used to compare the protein expression profiles of two cHL cell lines. The identified proteins and/or networks, many of which not previously related to cHL, may be important to better define the pathogenesis of the disease, to identify novel diagnostic markers, and to design new therapeutic strategies. PMID:26588820