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Sample records for adherent cells sac

  1. Transdifferentiation of mouse visceral yolk sac cells into parietal yolk sac cells in vitro.

    PubMed

    Yagi, Shinomi; Tagawa, Yoh-Ichi; Shiojiri, Nobuyoshi

    2016-02-19

    The mouse embryonic yolk sac is an extraembryonic membrane that consists of a visceral yolk sac (VYS) and parietal yolk sac (PYS), and functions in hematopoietic-circulation in the fetal stage. The present study was undertaken to examine the normal development of both murine VYS and PYS tissues using various molecular markers, and to establish a novel VYS cell culture system in vitro for analyzing differentiation potentials of VYS cells. RT-PCR and immunohistochemical analyses of gene expression in VYS and PYS tissues during development revealed several useful markers for their identification: HNF1β, HNF4α, Cdh1 (E-cadherin), Krt8 and Krt18 for VYS epithelial cells, and Stra6, Snail1, Thbd and vimentin for PYS cells. PYS cells exhibited mesenchymal characteristics in gene expression and morphology. When VYS cells at 11.5 days of gestation were cultured in vitro for 7 days, the number of HNF1β-, HNF4α-, E-cadherin- and cytokeratin-positive VYS epithelial cells was significantly reduced and, instead, Stra6-and vimentin-positive PYS-like cells increased with culture. RT-PCR analyses also demonstrated that gene expression of VYS markers decreased, whereas that of PYS markers increased in the primary culture of VYS cells. These data indicate that VYS epithelial cells rapidly transdifferentiate into PYS cells having mesenchymal characteristics in vitro, which may provide a culture system suitable for studying molecular mechanisms of VYS transdifferentiation into PYS cells and also epithelial-mesenchymal transition. PMID:26820538

  2. 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.

  3. The PIKfyve–ArPIKfyve–Sac3 triad in human breast cancer: Functional link between elevated Sac3 phosphatase and enhanced proliferation of triple negative cell lines

    SciTech Connect

    Ikonomov, Ognian C. Filios, Catherine Sbrissa, Diego Chen, Xuequn Shisheva, Assia

    2013-10-18

    Highlights: •We assess PAS complex proteins and phosphoinositide levels in breast cancer cells. •Sac3 and ArPIKfyve are markedly elevated in triple-negative breast cancer cells. •Sac3 silencing inhibits proliferation in triple-negative breast cancer cell lines. •Phosphoinositide profiles are altered in breast cancer cells. •This is the first evidence linking high Sac3 with breast cancer cell proliferation. -- Abstract: The phosphoinositide 5-kinase PIKfyve and 5-phosphatase Sac3 are scaffolded by ArPIKfyve in the PIKfyve–ArPIKfyve–Sac3 (PAS) regulatory complex to trigger a unique loop of PtdIns3P–PtdIns(3,5)P{sub 2} synthesis and turnover. Whereas the metabolizing enzymes of the other 3-phosphoinositides have already been implicated in breast cancer, the role of the PAS proteins and the PtdIns3P–PtdIns(3,5)P{sub 2} conversion is unknown. To begin elucidating their roles, in this study we monitored the endogenous levels of the PAS complex proteins in cell lines derived from hormone-receptor positive (MCF7 and T47D) or triple-negative breast cancers (TNBC) (BT20, BT549 and MDA-MB-231) as well as in MCF10A cells derived from non-tumorigenic mastectomy. We report profound upregulation of Sac3 and ArPIKfyve in the triple negative vs. hormone-sensitive breast cancer or non-tumorigenic cells, with BT cell lines showing the highest levels. siRNA-mediated knockdown of Sac3, but not that of PIKfyve, significantly inhibited proliferation of BT20 and BT549 cells. In these cells, knockdown of ArPIKfyve had only a minor effect, consistent with a primary role for Sac3 in TNBC cell proliferation. Intriguingly, steady-state levels of PtdIns(3,5)P{sub 2} in BT20 and T47D cells were similar despite the 6-fold difference in Sac3 levels between these cell lines. However, steady-state levels of PtdIns3P and PtdIns5P, both regulated by the PAS complex, were significantly reduced in BT20 vs. T47D or MCF10A cell lines, consistent with elevated Sac3 affecting directly or

  4. Ovarian mixed germ cell tumor with yolk sac and teratomatous components in a dog.

    PubMed

    Robinson, Nicholas A; Manivel, J Carlos; Olson, Erik J

    2013-05-01

    Mixed germ cell tumors of the ovary have rarely been reported in veterinary species. A 3-year-old intact female Labrador Retriever dog was presented for lethargy, abdominal distention, and a midabdominal mass. An exploratory laparotomy revealed a large (23 cm in diameter) left ovarian tumor and multiple small (2-3 cm in diameter) pale tan masses on the peritoneum and abdominal surface of the diaphragm. Histological examination of the left ovary revealed a mixed germ cell tumor with a yolk sac component with rare Schiller-Duval bodies and a teratomatous component comprised primarily of neural differentiation. The abdominal metastases were solely comprised of the yolk sac component. The yolk sac component was diffusely immunopositive for cytokeratin with scattered cells reactive for α-fetoprotein and placental alkaline phosphatase. Within the teratomatous component, the neuropil was diffusely immunopositive for S100, neuron-specific enolase, and neurofilaments with a few glial fibrillary acidic protein immunopositive cells. Ovarian germ cell tumors may be pure and consist of only 1 germ cell element or may be mixed and include more than 1 germ cell element, such as teratoma and yolk sac tumor. PMID:23604259

  5. Cells of the connective tissue differentiate and migrate into pollen sacs

    NASA Astrophysics Data System (ADS)

    Iqbal, M. C. M.; Wijesekara, Kolitha B.

    2002-01-01

    In angiosperms, archesporial cells in the anther primordium undergo meiosis to form haploid pollen, the sole occupants of anther sacs. Anther sacs are held together by a matrix of parenchyma cells, the connective tissue. Cells of the connective tissue are not known to differentiate. We report the differentiation of parenchyma cells in the connective tissue of two Gordonia species into pollen-like structures (described as pseudopollen), which migrate into the anther sacs before dehiscence. Pollen and pseudopollen were distinguishable by morphology and staining. Pollen were tricolpate to spherical while pseudopollen were less rigid and transparent with a ribbed surface. Both types were different in size, shape, staining and surface architecture. The ratio of the number of pseudopollen to pollen was 1:3. During ontogeny in the connective tissue, neither cell division nor tetrad formation was observed and hence pseudopollen were presumed to be diploid. Only normal pollen germinated on a germination medium. Fixed preparations in time seemed to indicate that pseudopollen migrate from the connective tissue into the anther sac.

  6. 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

  7. Target cells for avian myeloblastosis virus in embryonic yolk sac and relationship of cell differentiation to cell transformation.

    PubMed Central

    Boettiger, D; Durban, E

    1984-01-01

    The yolk sac of the 12-day chicken embryo retains the blast stage progenitors to cells of the myeloid lineages with a very low level of contamination by more mature myeloid cells which have begun to express the characteristic myeloid cell markers. Both in vivo and in vitro experiments have supported the hypothesis that target cells for the BAI-A strain of avian myeloblastosis virus are contained within the myeloid lineages. An assay system for avian myeloblastosis virus was developed which utilizes this yolk sac cell system and which appears to be more sensitive than previous published assays. In addition, the kinetics of a liquid culture transformation system is presented in which at least 4% of the yolk sac cell population was transformed in a relatively synchronous fashion at 2 days after infection. The morphological transformation preceded an increased rate of cell proliferation. Cell separation procedures provided a 10- to 20-fold enrichment of target cells and demonstrated that the target cell population copurifies with macrophage colony-forming cells which are the committed progenitors to the macrophage lineage. In combination with earlier work, this work demonstrated that cells committed to the macrophage lineage at all stages of differentiation may serve as target cells for infection by avian myeloblastosis virus. PMID:6699939

  8. 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

  9. 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

  10. Plant-derived SAC domain of PAR-4 (Prostate Apoptosis Response 4) exhibits growth inhibitory effects in prostate cancer cells

    PubMed Central

    Sarkar, Shayan; Jain, Sumeet; Rai, Vineeta; Sahoo, Dipak K.; Raha, Sumita; Suklabaidya, Sujit; Senapati, Shantibhusan; Rangnekar, Vivek M.; Maiti, Indu B.; Dey, Nrisingha

    2015-01-01

    The gene Par-4 (Prostate Apoptosis Response 4) was originally identified in prostate cancer cells undergoing apoptosis and its product Par-4 showed cancer specific pro-apoptotic activity. Particularly, the SAC domain of Par-4 (SAC-Par-4) selectively kills cancer cells leaving normal cells unaffected. The therapeutic significance of bioactive SAC-Par-4 is enormous in cancer biology; however, its large scale production is still a matter of concern. Here we report the production of SAC-Par-4-GFP fusion protein coupled to translational enhancer sequence (5′ AMV) and apoplast signal peptide (aTP) in transgenic Nicotiana tabacum cv. Samsun NN plants under the control of a unique recombinant promoter M24. Transgene integration was confirmed by genomic DNA PCR, Southern and Northern blotting, Real-time PCR, and Nuclear run-on assays. Results of Western blot analysis and ELISA confirmed expression of recombinant SAC-Par-4-GFP protein and it was as high as 0.15% of total soluble protein. In addition, we found that targeting of plant recombinant SAC-Par-4-GFP to the apoplast and endoplasmic reticulum (ER) was essential for the stability of plant recombinant protein in comparison to the bacterial derived SAC-Par-4. Deglycosylation analysis demonstrated that ER-targeted SAC-Par-4-GFP-SEKDEL undergoes O-linked glycosylation unlike apoplast-targeted SAC-Par-4-GFP. Furthermore, various in vitro studies like mammalian cells proliferation assay (MTT), apoptosis induction assays, and NF-κB suppression suggested the cytotoxic and apoptotic properties of plant-derived SAC-Par-4-GFP against multiple prostate cancer cell lines. Additionally, pre-treatment of MAT-LyLu prostate cancer cells with purified SAC-Par-4-GFP significantly delayed the onset of tumor in a syngeneic rat prostate cancer model. Taken altogether, we proclaim that plant made SAC-Par-4 may become a useful alternate therapy for effectively alleviating cancer in the new era. PMID:26500666

  11. 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.

  12. Rat visceral yolk sac cells: viability and expression of cell markers during maternal diabetes

    PubMed Central

    Aires, M.B.; Santos, J.R.A.; Souza, K.S.; Farias, P.S.; Santos, A.C.V.; Fioretto, E.T.; Maria, D.A.

    2015-01-01

    The function of the visceral yolk sac (VYS) is critical for embryo organogenesis until final fetal development in rats, and can be affected by conditions such as diabetes. In view of the importance of diabetes during pregnancy for maternal and neonatal health, the objective of this study was to assess fetal weight, VYS cell markers, and viability in female Wistar rats (200-250 g) with induced diabetes (alloxan, 37 mg/kg) on the 8th gestational day (gd 8). At gd 15, rats from control (n=5) and diabetic (n=5) groups were anesthetized and laparotomized to remove the uterine horns for weighing of fetuses and collecting the VYS. Flow cytometry was used for characterizing VYS cells, and for determining mitochondrial activity, cell proliferation, DNA ploidy, cell cycle phases, and caspase-3 activity. Fetal weight was reduced in the diabetic group. Expression of the cell markers CD34, VEGFR1, CD115, CD117, CD14, CCR2, CD90, CD44, STRO-1, OCT3/4, and Nanog was detected in VYS cells in both groups. In the diabetic group, significantly decreased expression of CD34 (P<0.05), CCR2 (P<0.001), and OCT3/4 (P<0.01), and significantly increased expression of CD90 (P<0.05), CD117 (P<0.01), and CD14 (P<0.05) were observed. VYS cells with inactive mitochondria, activated caspase-3, and low proliferation were present in the rats with diabetes. Severe hyperglycemia caused by maternal diabetes had negative effects on pregnancy, VYS cell viability, and the expression of cell markers. PMID:26176314

  13. Mixed ovarian germ cell tumor composed of immature teratoma, yolk sac tumor and embryonal carcinoma.

    PubMed

    Wang, Ying; Zhou, Feng; Qian, Zhida; Qing, Jiale; Zhao, Mengdam; Huang, Lili

    2014-11-01

    We report the case of a 19-year-old woman experiencing lower abdominal distension and pain. Laboratory tests indicated elevated serum levels of Alpha-Fetoprotein (AFP) and human Chorionic Gonadotropin (hCG). A large mass was detected in the abdomen by physical examination and by transvaginal ultrasonography. Exploratory laparotomy was performed, and a smooth-surfaced, spherical, solid tumor was found on the left ovary, measuring 11.5 x 9.9 x 6.9 cm. Histological evaluation revealed that the tumor consisted of a combination of immature teratoma, Yolk Sac Tumor, and embryonal carcinoma; this is a very rare combination in mixed germ cell tumors. PMID:25518772

  14. 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

  15. 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

  16. 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.

  17. Enlarging cells initiating apomixis in Hieracium praealtum transition to an embryo sac program prior to entering mitosis.

    PubMed

    Okada, Takashi; Hu, Yingkao; Tucker, Matthew R; Taylor, Jennifer M; Johnson, Susan D; Spriggs, Andrew; Tsuchiya, Tohru; Oelkers, Karsten; Rodrigues, Julio C M; Koltunow, Anna M G

    2013-09-01

    Hieracium praealtum forms seeds asexually by apomixis. During ovule development, sexual reproduction initiates with megaspore mother cell entry into meiosis and formation of a tetrad of haploid megaspores. The sexual pathway ceases when a diploid aposporous initial (AI) cell differentiates, enlarges, and undergoes mitosis, forming an aposporous embryo sac that displaces sexual structures. Embryo and endosperm development in aposporous embryo sacs is fertilization independent. Transcriptional data relating to apomixis initiation in Hieracium spp. ovules is scarce and the functional identity of the AI cell relative to other ovule cell types is unclear. Enlarging AI cells with undivided nuclei, early aposporous embryo sacs containing two to four nuclei, and random groups of sporophytic ovule cells not undergoing these events were collected by laser capture microdissection. Isolated amplified messenger RNA samples were sequenced using the 454 pyrosequencing platform and comparatively analyzed to establish indicative roles of the captured cell types. Transcriptome and protein motif analyses showed that approximately one-half of the assembled contigs identified homologous sequences in Arabidopsis (Arabidopsis thaliana), of which the vast majority were expressed during early Arabidopsis ovule development. The sporophytic ovule cells were enriched in signaling functions. Gene expression indicative of meiosis was notably absent in enlarging AI cells, consistent with subsequent aposporous embryo sac formation without meiosis. The AI cell transcriptome was most similar to the early aposporous embryo sac transcriptome when comparing known functional annotations and both shared expressed genes involved in gametophyte development, suggesting that the enlarging AI cell is already transitioning to an embryo sac program prior to mitotic division. PMID:23864557

  18. Isolation and characterization of mesenchymal stem cells from the yolk sacs of bovine embryos.

    PubMed

    Mançanares, C A F; Oliveira, V C; Oliveira, L J; Carvalho, A F; Sampaio, R V; Mançanares, A C F; Souza, A F; Perecin, F; Meirelles, F V; Miglino, M A; Ambrósio, C E

    2015-10-01

    The yolk sac (YS) represents a promising source of stem cells for research because of the hematopoietic and mesenchymal cell niches that are present in this structure during the development of the embryo. In this study, we report on the isolation and characterization of YS tissue and mesenchymal stem cells (MSCs) derived from bovine YSs. Our results show that the YS is macroscopically located in the exocoelomic cavity in the ventral portion of the embryo and consists of a transparent membrane formed by a central sac-like portion and two ventrally elongated projections. Immunohistochemistry analyses were positive for OCT4, CD90, CD105, and CD44 markers in the YS of both gestational age groups. The MSCs of bovine YS were isolated using enzymatic digestion and were grown in vitro for at least 11 passages to verify their capacity to proliferate. These cells were also subjected to immunophenotypic characterization that revealed the presence of CD90, CD105, and CD79 and the absence of CD45, CD44, and CD79, which are positive and negative markers of MSCs, respectively. To prove their multipotency, the cells were induced to differentiate into three cell types, chondrocytes, osteoblasts, and adipocytes, which were stained with tissue-specific dyes (chondrogenic: Alcian Blue, osteogenic: Alizarin Red, and adipogenic: Oil Red O) to confirm differentiation. Gene expression analyses showed no differences in the patterns of gene expression between the groups or passages tested, with the exception of the expression of SOX2, which was slightly different in the G1P3 group compared to the other groups. Our results suggest that YS tissue from bovines can be used as a source of MSCs, which makes YS tissue-derived cells an interesting option for cell therapy and regenerative medicine. PMID:26143361

  19. Squamous cell carcinoma of the anal sac in a spotted hyena (Crocuta crocuta).

    PubMed

    Goodnight, Andrea L; Traslavina, Ryan P; Emanuelson, Karen; Affolter, Verena K; Gaffney, Patricia M; Vernau, William; Williams, Colette; Wu, Connie I-kuan; Sturges, Beverly K; Lowenstine, Linda J

    2013-12-01

    A 25-yr-old spayed female spotted hyena (Crocuta crocuta) developed intermittent right pelvic limb lameness that persisted following conservative medical therapy. No obvious musculoskeletal lesions were noted on initial physical exam; however, spinal radiography was suspicious for possible intervertebral degenerative joint disease or discospondylitis. Despite prolonged medical therapy, the lameness progressed to minimal weight bearing and marked muscle atrophy of the right pelvic limb. Electromyography showed spontaneous activity in the muscles of right sciatic nerve distribution. Sensory and motor nerve conduction velocities in the right tibial and peroneal nerves were undetectable and markedly reduced, respectively. A magnetic resonance imaging (MRI) scan revealed a large, space-occupying mass on the right side of the sacrum and pelvis. Antemortem fine-needle aspiration of the mass and postmortem histopathology resulted in diagnosis of a high-grade squamous cell carcinoma of the anal sac. Squamous cell carcinoma of the anal sac is very rare in domestic dogs and previously unreported in spotted hyenas. PMID:24450071

  20. 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

  1. SALL4 and SF-1 are sensitive and specific markers for distinguishing granulosa cell tumors from yolk sac tumors.

    PubMed

    Bai, Shuting; Wei, Shi; Ziober, Amy; Yao, Yuan; Bing, Zhanyong

    2013-04-01

    Granulosa cell tumors are classified as juvenile and adult types. They may be misinterpreted as a yolk sac tumor when they exhibit a "reticular" growth pattern and contain prominent mitotic activity. In this study, the authors performed immunohistochemical stains for SALL4 and steroidogenic factor-1 (SF-1) on 27 cases of yolk sac tumors and 24 granulosa cell tumors. Nuclear stains for both antibodies were considered as positive and the intensity of staining was graded as negative, weak, moderate, and strong. All the yolk sac tumors were positive for SALL4 (100%) with moderate to strong grade staining and negative for SF-1 (100%). In contrast, all the granulosa cell tumors were positive for SF-1 (85% moderate to strong grade staining and 15% weak staining) and negative for SALL4 (100%). The difference was significant (P < .01, Student's t test). This result indicates that these 2 markers could be used to distinguish these 2 tumors in a difficult situation. PMID:22832114

  2. 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

  3. Distal regeneration involves the age dependent activity of branchial sac stem cells in the ascidian Ciona intestinalis

    PubMed Central

    2014-01-01

    Abstract Tunicates have high capacities for regeneration but the underlying mechanisms and their relationship to life cycle progression are not well understood. Here we investigate the regeneration of distal structures in the ascidian tunicate Ciona intestinalis. Analysis of regenerative potential along the proximal−distal body axis indicated that distal organs, such as the siphons, their pigmented sensory organs, and the neural complex, could only be replaced from body fragments containing the branchial sac. Distal regeneration involves the formation of a blastema composed of cells that undergo cell proliferation prior to differentiation and cells that differentiate without cell proliferation. Both cell types originate in the branchial sac and appear in the blastema at different times after distal injury. Whereas the branchial sac stem cells are present in young animals, they are depleted in old animals that have lost their regeneration capacity. Thus Ciona adults contain a population of age‐related stem cells located in the branchial sac that are a source of precursors for distal body regeneration. PMID:25893097

  4. Maternal-fetal immunoglobulin transport: Studies on the binding, internalization, and release of IgG by chick yolk sac tissue and cultured cells

    SciTech Connect

    Donaldson, J.G.

    1988-01-01

    Immunoglobulin G (IgG) is transported from the yolk across the endodermal cells of the yolk sac and into the fetal circulation during chick embryonic development, thus providing the chick with passive immunity until it becomes immunocompetent. Saturable, Fc-specific receptors are present on the endodermal cells and are believed to mediate this transfer. In this study, IgG receptors were shown to be present on the yolk sac endodermal cells throughout the 21 days of development, although most of the transport occurs during the last 3 days prior to hatching. Fluorescently conjugated IgG was internalized by a receptor mechanism into small apical vesicles in yolk sac endoderm throughout, but cells from 19 day yolk sacs internalized more conjugate than those from 14 day yolk sacs. This was confirmed and quantitated by assaying the internalization of {sup 125}I-IgG into yolk sac tissue. IgG was internalized by a receptor mediated mechanism, reaching a steady state level after 1 to 2 hours. Although both ages of yolk sac tissue possessed the same number of surface IgG receptors, as measured by equilibrium binding assays at 4{degree}C, 19 day yolk sac had the capacity to internalize six times as much IgG by a receptor mechanism as 14 day yolk sac.

  5. Integrated Phase Array Antenna/Solar Cell System for Flexible Access Communication (IA/SAC)

    NASA Technical Reports Server (NTRS)

    Clark, E. B.; Lee, R. Q.; Pal, A. T.; Wilt, D. M.; McElroy, B. D.; Mueller, C. H.

    2005-01-01

    This paper describes recent efforts to integrate advanced solar cells with printed planar antennas. Several previous attempts have been reported in the literature, but this effort is unique in several ways. It uses Gallium Arsenide (GaAs) multi-junction solar cell technology. The solar cells and antennas will be integrated onto a common GaAs substrate. When fully implemented, IA/SAC will be capable of dynamic beam steering. In addition, this program targets the X-band (8 - 12 GHz) and higher frequencies, as compared to the 2.2 - 2.9 GHz arrays targeted by other organizations. These higher operating frequencies enable a greater bandwidth and thus higher data transfer rates. The first phase of the effort involves the development of 2 x 2 cm GaAs Monolithically Integrated Modules (MIM) with integrated patch antennas on the opposite side of the substrate. Subsequent work will involve the design and development of devices having the GaAs MIMs and the antennas on the same side of the substrate. Results from the phase one efforts will be presented.

  6. Maize EMBRYO SAC family peptides interact differentially with pollen tubes and fungal cells

    PubMed Central

    Woriedh, Mayada; Merkl, Rainer; Dresselhaus, Thomas

    2015-01-01

    EMBRYO SAC1-4 (ES1-4) peptides belong to the defensin subgroup of cysteine-rich peptides known to mediate pollen tube burst in Zea mays (maize). ES1-4 are reported here to also be capable of inhibiting germination and growth of the maize fungal pathogens Fusarium graminearum and Ustilago maydis at higher concentrations. Dividing the peptides into smaller pieces showed that a 15-amino-acid peptide located in a highly variable loop region lacking similarity to other defensins or defensin-like peptides binds to maize pollen tube surfaces, causing swelling prior to burst. This peptide fragment and a second conserved neighbouring fragment showed suppression of fungal germination and growth. The two peptides caused swelling of fungal cells, production of reactive oxygen species, and finally the formation of big vacuoles prior to burst at high peptide concentration. Furthermore, peptide fragments were found to bind differently to fungal cells. In necrotrophic F. graminearum, a peptide fragment named ES-d bound only at cell surfaces whereas the peptide ES-c bound at cell surfaces and also accumulated inside cells. Conversely, in biotrophic U. maydis, both peptide fragments accumulated inside cells, but, if applied at higher concentration, ES-c but not ES-d accumulated mainly in vacuoles. Mapping of peptide interaction sites identified amino acids differing in pollen tube burst and fungal response reactions. In summary, these findings indicate that residues targeting pollen tube burst in maize are specific to the ES family, while residues targeting fungal growth are conserved within defensins and defensin-like peptides. PMID:26071527

  7. Integrated Antenna/Solar Array Cell (IA/SAC) System for Flexible Access Communications

    NASA Technical Reports Server (NTRS)

    Lee, Ricard Q.; Clark, Eric B.; Pal, Anna Maria T.; Wilt, David M.; Mueller, Carl H.

    2004-01-01

    Present satellite communications systems normally use separate solar cells and antennas. Since solar cells generally account for the largest surface area of the spacecraft, co-locating the antenna and solar cells on the same substrate opens the possibility for a number of data-rate-enhancing communications link architecture that would have minimal impact on spacecraft weight and size. The idea of integrating printed planar antenna and solar array cells on the same surface has been reported in the literature. The early work merely attempted to demonstrate the feasibility by placing commercial solar cells besides a patch antenna. Recently, Integrating multiple antenna elements and solar cell arrays on the same surface was reported for both space and terrestrial applications. The application of photovoltaic solar cell in a planar antenna structure where the radiating patch antenna is replaced by a Si solar cell has been demonstrated in wireless communication systems (C. Bendel, J. Kirchhof and N. Henze, 3rd Would Photovotaic Congress, Osaka, Japan, May 2003). Based on a hybrid approach, a 6x1 slot array with circularly polarized crossdipole elements co-located on the same surface of the solar cells array has been demonstrated (S. Vaccaro, J. R. Mosig and P. de Maagt, IEEE Trans. Ant. and Propag., Vol. 5 1, No. 8, Aug. 2003). Amorphous silicon solar cells with about 5-10% efficiency were used in these demonstrations. This paper describes recent effort to integrate advanced solar cells with printed planar antennas. Compared to prior art, the proposed WSAC concept is unique in the following ways: 1) Active antenna element will be used to achieve dynamic beam steering; 2) High efficiency (30%) GaAs multi-junction solar cells will be used instead of Si, which has an efficiency of about 15%; 3) Antenna and solar cells are integrated on a common GaAs substrate; and 4) Higher data rate capability. The IA/SAC is designed to operate at X-band (8-12 GH) and higher frequencies

  8. Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos.

    PubMed

    Lin, Han Jen; Wang, Siou Huei; Pan, Yu Hui; Ding, Shih-Torng

    2016-01-01

    We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient utilization by avian embryos during development. Fertilized Japanese quail eggs were incubated at 37 °C for 5 days and then yolk sac membranes (YSM) were collected to establish the EEC culture system. We isolated the embryonic endoderm layer from YSM, and sliced the membrane into 2 - 3 mm pieces and partially digested with collagenase before seeding in 24-well culture plates. The EECs proliferate out of the tissue and are ready for cell culture studies. We found that the EECs had typical characteristics of YSM in vivo, for example, accumulation of lipid droplets, expression of sterol O-acyltransferase and lipoprotein lipase. The partial digestion treatment significantly increased the successful rate of EEC culture. Utilizing the EECs, we demonstrated that the expression of SOAT1 was regulated by the cAMP dependent protein kinase A related pathway. This primary Japanese quail EEC culture system is a useful tool to study embryonic lipid transportation and to clarify the role of genes involved in mediating nutrient utilization in YSM during avian embryonic development. PMID:27022687

  9. 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

  10. 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

  11. Sustainable complete remission in recurrence yolk sac tumor patient treated with tandem high-dose chemotherapy and autologous stem cell.

    PubMed

    Abdullah, N A; Wang, P N; Huang, K G; Adlan, A S; Casanova, J

    2013-01-01

    A 21-year-old lady diagnosed with Stage 3 ovarian yolk sac tumor (YST) underwent primary cytoreductive fertility sparing surgery, followed by conventional courses of platinum-based chemotherapy and etoposide. Recurrence at cul-da-sac was noted after a short period of remission and secondary debulking performed followed by four cycles of conventional chemotherapy. The patient's disease progressed despite courses of treatments. A joint team management including a hematologist was commenced following the failure of conventional chemotherapies. Two cycles of high-dose chemotherapy (HDCT) with ifosfamide/cisplatin/etoposide (ICE) regimen, followed by autologous stem cell transplantation (ASCT) were given. With this salvage treatment, she remained in complete remission and disease-free for more than 30 months, while maintaining her reproductive function. These approaches appear to be effective as a salvage treatment in selected cases of patients with ovarian germ cell tumor, especially those who failed primary conventional chemotherapy. PMID:23781595

  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. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. A Genetic Mosaic Analysis With a Repressible Cell Marker Screen to Identify Genes Involved in Tracheal Cell Migration During Drosophila Air Sac Morphogenesis

    PubMed Central

    Chanut-Delalande, Hélène; Jung, Alain C.; Lin, Li; Baer, Magdalena M.; Bilstein, Andreas; Cabernard, Clemens; Leptin, Maria; Affolter, Markus

    2007-01-01

    Branching morphogenesis of the Drosophila tracheal system relies on the fibroblast growth factor receptor (FGFR) signaling pathway. The Drosophila FGF ligand Branchless (Bnl) and the FGFR Breathless (Btl/FGFR) are required for cell migration during the establishment of the interconnected network of tracheal tubes. However, due to an important maternal contribution of members of the FGFR pathway in the oocyte, a thorough genetic dissection of the role of components of the FGFR signaling cascade in tracheal cell migration is impossible in the embryo. To bypass this shortcoming, we studied tracheal cell migration in the dorsal air sac primordium, a structure that forms during late larval development. Using a mosaic analysis with a repressible cell marker (MARCM) clone approach in mosaic animals, combined with an ethyl methanesulfonate (EMS)-mutagenesis screen of the left arm of the second chromosome, we identified novel genes implicated in cell migration. We screened 1123 mutagenized lines and identified 47 lines displaying tracheal cell migration defects in the air sac primordium. Using complementation analyses based on lethality, mutations in 20 of these lines were genetically mapped to specific genomic areas. Three of the mutants were mapped to either the Mhc or the stam complementation groups. Further experiments confirmed that these genes are required for cell migration in the tracheal air sac primordium. PMID:17603108

  3. 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.

  4. 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

  5. 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

  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. Immunocytochemical and biochemical characterization of the Heymann nephritis antigenic complex in rat L2 yolk sac cells.

    PubMed Central

    Lundstrom, M.; Orlando, R. A.; Saedi, M. S.; Woodward, L.; Kurihara, H.; Farquhar, M. G.

    1993-01-01

    Heymann nephritis in the rat is the most widely used model of human membranous glomerulonephritis. Glycoprotein (gp)330, a large (M(r) > 550,000) membrane-associated glycoprotein, has been identified as the main antigen in this autoimmune disease. Studies of gp330 and receptor-associated protein (RAP), its 44-kd subunit, have been restricted largely to rat kidney, as no stable cultured cell line has been available that expresses gp330. We have recently identified a rat yolk sac carcinoma cell line (L2) that expresses both gp330 and RAP. In this report, we have carried out detailed morphological, immunocytochemical, and biochemical studies characterizing the biosynthesis and localization of gp330 and RAP in the L2 rat yolk sac cell line. At the electron microscope level, the L2 cells are seen to be attached by cell junctions, and their predominant morphological features include extensive networks of rough endoplasmic reticulum (ER) and numerous clathrin-coated pits found on the cell membrane. By immunocytochemistry, gp330 was localized primarily to clathrin-coated pits at the cell surface, whereas RAP was localized predominantly to the lumen of the rough ER. Pulse-chase experiments indicated that gp330 spends a prolonged time maturing in the ER of L2 cells, as transport of gp330 to the Golgi complex (based on acquisition of endoglycosidase H resistance) is slow (t1/2 = 90 to 120 minutes). Gp330 reached the L2 cell surface beginning at 2 hours after synthesis, where it could be detected by cell surface immunoprecipitation. RAP was found to be an N-linked glycoprotein, and it remained endoglycosidase H-sensitive up to 4 hours after synthesis. Co-precipitation and co-sedimentation experiments demonstrated that gp330 and RAP form a large heterodimer (M(r) approximately 669,000) immediately after biosynthesis and are further assembled into a large hetero-oligomer in the ER. These findings demonstrate that the localization and the kinetics of assembly of gp330 and RAP

  8. 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

  9. 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

  10. Comparative morphology of the marrow sac.

    PubMed

    Bi, L X; Simmons, D J; Hawkins, H K; Cox, R A; Mainous, E G

    2000-12-01

    Electron microscopic techniques have been used to profile the morphologies of marrow sacs in different laboratory species. These structures all comprise a condensed layer of overlapping fibroblast-like stromal cells and apparently confine the medullary and endosteal osteoblast/lining cells to separate histiotypic compartments. There were some variations in the morphology of the sac cells in the different species. In rats, cats, and sheep, scanning electron microscopy (SEM) showed a seamless arrangement of marrow sac cells which resembled a thin, flat simple squamous epithelium; they displayed few intercellular cytoplasmic processes. In the rabbit and pigeon, the sac comprised a more woven, multilayered fabric of broadly elongate flat fibroblast-like cells which displayed numerous intercellular processes. Transmission electron microscopy (TEM) showed that all marrow sac cells were attenuated with elongated nuclei, a few small round mitochondria, and a sparse rough endoplasmic reticulum. In the majority of animals, the sac was one to two cell layers thick. The rabbit and pigeon sacs were multilayered, and never less than three to four cells deep. The cell layers were not closely apposed. Tight or gap junctions were absent at the points of intercellular contact. These morphological results suggest that marrow sacs are common elements of the vertebrate skeleton with species specific morphologies. PMID:11074407

  11. 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

  12. 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

  13. Trophoblast-decidual cell interactions and establishment of maternal blood circulation in the parietal yolk sac placenta of the rat.

    PubMed

    Welsh, A O; Enders, A C

    1987-02-01

    Implantation sites from rats were studied on days 6, 7, and 8 of pregnancy to determine the sequence of events in the formation of blood spaces in the trophoblast that is part of the parietal wall of the yolk sac placenta and to determine how trophoblast gains access to maternal blood. The maternal blood flowing through these spaces is the source of nutrients that reach the embryo via the visceral endoderm. Tissues were prepared for light microscopy, scanning electron microscopy, and transmission electron microscopy. Trophoblast blood spaces are derived from the lateral intercellular spaces of trophoblast cells and are present in a collapsed condition until day 8, when maternal vessels are tapped by trophoblast. These spaces then contain circulating maternal blood, and trophoblast cells reflect adaptations for metabolic exchange including thinning of trophoblast covering Reichert's membrane and the appearance of numerous fenestrations, with and without diaphragms, in the areas where trophoblast is attenuated. Between days 6 and 7 decidual cells appear to form a barrier between the maternal circulation and trophoblast. On day 7, however, decidual cell processes penetrate the residual uterine luminal epithelial basal lamina, and then the decidual cells that are juxtaposed to trophoblast undergo degradative changes that resemble apoptosis. There is condensation of cytoplasmic contents, fragmentation of the cells, and phagocytosis of the fragments by trophoblast. Some decidual cells are interposed between endothelial cells in the walls of maternal vessels as early as day 7. Trophoblast may gain access to the maternal vessels by replacing decidual cells or by direct imposition of trophoblast cell processes between endothelial cells. PMID:3578838

  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. Aneuploidy generates proteotoxic stress and DNA damage concurrently with p53-mediated post-mitotic apoptosis in SAC-impaired cells.

    PubMed

    Ohashi, Akihiro; Ohori, Momoko; Iwai, Kenichi; Nakayama, Yusuke; Nambu, Tadahiro; Morishita, Daisuke; Kawamoto, Tomohiro; Miyamoto, Maki; Hirayama, Takaharu; Okaniwa, Masanori; Banno, Hiroshi; Ishikawa, Tomoyasu; Kandori, Hitoshi; Iwata, Kentaro

    2015-01-01

    The molecular mechanism responsible that determines cell fate after mitotic slippage is unclear. Here we investigate the post-mitotic effects of different mitotic aberrations--misaligned chromosomes produced by CENP-E inhibition and monopolar spindles resulting from Eg5 inhibition. Eg5 inhibition in cells with an impaired spindle assembly checkpoint (SAC) induces polyploidy through cytokinesis failure without a strong anti-proliferative effect. In contrast, CENP-E inhibition causes p53-mediated post-mitotic apoptosis triggered by chromosome missegregation. Pharmacological studies reveal that aneuploidy caused by the CENP-E inhibitor, Compound-A, in SAC-attenuated cells causes substantial proteotoxic stress and DNA damage. Polyploidy caused by the Eg5 inhibitor does not produce this effect. Furthermore, p53-mediated post-mitotic apoptosis is accompanied by aneuploidy-associated DNA damage response and unfolded protein response activation. Because Compound-A causes p53 accumulation and antitumour activity in an SAC-impaired xenograft model, CENP-E inhibitors could be potential anticancer drugs effective against SAC-impaired tumours. PMID:26144554

  16. 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 ...

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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

  2. Mitochondria-rich cell activity in the yolk-sac membrane of tilapia (Oreochromis mossambicus) larvae acclimatized to different ambient chloride levels.

    PubMed

    Lin, Li-Yih; Hwang, Pung-Pung

    2004-03-01

    Mitochondria-rich cells (MRCs) in the yolk-sac membrane of tilapia (Oreochromis mossambicus) larvae were examined by Na+/K(+)-ATPase immunocytochemistry and vital staining for glycoproteins following acclimation to high (7.5-7.9 mmol l(-1)), normal (0.48-0.52 mmol l(-1)) or low (0.002-0.007 mmol l(-1)) ambient Cl- levels. With a combination of concanavalin-A (Con-A)-Texas-Red conjugate staining (larvae exposed to the dye in vivo in the water) and a monoclonal antibody raised against Na+/K(+)-ATPase, MRCs were easily recognized and presumed to be active when Con-A-positive (i.e. with their apical membrane in contact with the water) or inactive when Con-A-negative. The proportion of active cells gradually increased during a 48-h acclimation to low-Cl- medium but decreased during acclimation to high-Cl- medium. Total densities of MRCs did not change when ambient chloride levels were altered. Furthermore, in live larvae exposed to changes in ambient Cl-, yolk-sac MRCs, vitally stained with DASPEI and subsequently traced in time, did not significantly alter turnover. The polymorphism of the apical membrane compartment of the MRCs represents structural modification of the active MRCs. Yolk-sac pavement cells labeled with the membrane marker FM1-43 (fluorescent lipophilic tracer) were shown to cover active MRCs in larvae transferred from normal to high ambient Cl- levels, thereby inactivating the MRCs. PMID:15010484

  3. 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

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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

  19. Primary Nasopharngeal Yolk Sac Tumor: A Case Report

    PubMed Central

    Arumugam, Dhanalakshmi; Chidambaram, Lalitha; Boj, Sudha; Marudasalam, Sumathi

    2016-01-01

    Yolk sac tumour also known as primitive endodermal tumour is the most common malignant germ cell tumour (GCT) in the paediatric age group. Most common sites of involvement are ovaries and testes, but rarely can occur in the extragonadal sites. In the head and neck region, yolk sac tumours have been reported in the nasopharynx, sinonasal tract, orbit, ear and parotid gland. Nasopharynx is an uncommon site for yolk sac tumour and very few cases of nasopharngeal pure yolk sac tumour have been reported so far. Yolk sac tumours are highly malignant and have a poor prognosis. This is a case of pure GCT in a three-year-old female child who presented with a rapidly growing nasopharyngeal mass. Histopathological examination followed by immunohistochemistry and serum AFP study clinched the diagnosis of yolk sac tumour. The tumour responded well to chemotherapy as evidenced by decrease in serum AFP levels. PMID:27437234

  20. Primary Nasopharngeal Yolk Sac Tumor: A Case Report.

    PubMed

    Arumugam, Dhanalakshmi; Thandavarayan, Pavithra; Chidambaram, Lalitha; Boj, Sudha; Marudasalam, Sumathi

    2016-05-01

    Yolk sac tumour also known as primitive endodermal tumour is the most common malignant germ cell tumour (GCT) in the paediatric age group. Most common sites of involvement are ovaries and testes, but rarely can occur in the extragonadal sites. In the head and neck region, yolk sac tumours have been reported in the nasopharynx, sinonasal tract, orbit, ear and parotid gland. Nasopharynx is an uncommon site for yolk sac tumour and very few cases of nasopharngeal pure yolk sac tumour have been reported so far. Yolk sac tumours are highly malignant and have a poor prognosis. This is a case of pure GCT in a three-year-old female child who presented with a rapidly growing nasopharyngeal mass. Histopathological examination followed by immunohistochemistry and serum AFP study clinched the diagnosis of yolk sac tumour. The tumour responded well to chemotherapy as evidenced by decrease in serum AFP levels. PMID:27437234

  1. 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

  2. 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.

  3. [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

  4. 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

  5. The chicken yolk sac IgY receptor, a mammalian mannose receptor family member, transcytoses IgY across polarized epithelial cells.

    PubMed

    Tesar, Devin B; Cheung, Evelyn J; Bjorkman, Pamela J

    2008-04-01

    In mammals the transfer of passive immunity from mother to young is mediated by the MHC-related receptor FcRn, which transports maternal IgG across epithelial cell barriers. In birds, maternal IgY in egg yolk is transferred across the yolk sac to passively immunize chicks during gestation and early independent life. The chicken yolk sac IgY receptor (FcRY) is the ortholog of the mammalian phospholipase A2 receptor, a mannose receptor family member, rather than an FcRn or MHC homolog. FcRn and FcRY both exhibit ligand binding at the acidic pH of endosomes and ligand release at the slightly basic pH of blood. Here we show that FcRY expressed in polarized mammalian epithelial cells functioned in endocytosis, bidirectional transcytosis, and recycling of chicken FcY/IgY. Confocal immunofluorescence studies demonstrated that IgY binding and endocytosis occurred at acidic but not basic pH, mimicking pH-dependent uptake of IgG by FcRn. Colocalization studies showed FcRY-mediated internalization via clathrin-coated pits and transport involving early and recycling endosomes. Disruption of microtubules partially inhibited apical-to-basolateral and basolateral-to-apical transcytosis, but not recycling, suggesting the use of different trafficking machinery. Our results represent the first cell biological evidence of functional equivalence between FcRY and FcRn and provide an intriguing example of how evolution can give rise to systems in which similar biological requirements in different species are satisfied utilizing distinct protein folds. PMID:18256279

  6. 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

  7. 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

  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. 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 ...

  10. 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.

  11. In vitro potential modulation of baicalin and baicalein on P-glycoprotein activity and expression in Caco-2 cells and rat gut sacs.

    PubMed

    Miao, Qing; Wang, Zhiyong; Zhang, Yuanyuan; Miao, Peipei; Zhao, Yuanyuan; Zhang, Yujie; Ma, Shuangcheng

    2016-09-01

    Context Previous studies have shown that Scutellariae Radix, the dried root of Scutellaria baicalensis Georgi (Labiatae), has a certain inhibitory effect on P-glycoprotein (P-gp), but the effects of its main active constituents on P-gp are still ambiguous. Objectives In vitro studies were performed to investigate the effects of its main active constituents (baicalin and its aglycone, baicalein) on the activity and expression of P-gp in intestine using Caco-2 cells and rat gut sacs. Materials and methods In Caco-2 cell experiments, the effects of baicalin and baicalein on P-gp activity were investigated using a P-gp substrate, rhodamine 123 and non-substrate fluorescein Na, by determining their intracellular fluorescence accumulation, and their effects on P-gp expression were determined using flow cytometry. In addition, rat gut sac model was selected to investigate the effects of baicalin and baicalein on the transport of verapamil, a classical P-gp substrate. The gut sacs of male Sprague-Dawley rats were filled with 0.4 mL the test solution contained verapamil (0.2575 mg/mL) and the drugs [baicalin and baicalein, at concentrations of 1/8 IC50 (59.875, 41.5 μg/mL), 1/4 IC50 (119.75, 83 μg/mL) and 1/2 IC50 (239.5, 166 μg/mL)], and then incubated in Tyrode's solution for a period of time. After termination of the incubation, the incubated solution was processed for the subsequent detection. Results According to the results of MTT assay, the IC50 values of verapamil, baicalin and baicalein were 104, 479, 332 μg/mL, respectively. The obtained results from the two models were confirmed mutually. As a result, baicalin exhibited no obvious effect on intracellular accumulation of Rh-123, and almost had no effect on P-gp expression and verapamil transportation, while baicalein significantly increased intracellular accumulation of Rh-123 (p < 0.01), down-regulated P-gp expression (p < 0.01) and increased the transport of verapamil (p < 0

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. Prognosis of hepatoid yolk sac tumor in women: what's up, Doc?

    PubMed

    Rittiluechai, Kristsanamon; Wilcox, Rebecca; Lisle, Jennifer; Everett, Elise; Wallace, H James; Verschraegen, Claire F

    2014-04-01

    Ovarian yolk sac tumors are highly malignant germ cell tumors that commonly occur in young women. The hepatoid yolk sac tumor is a variant form of yolk sac tumor in which there has been extensive tumor differentiation to early liver tissue. Hepatoid differentiation is traditionally considered to signify a poor prognosis. This review focuses on diagnostic criteria and establishes the optimal treatment for patients with hepatoid yolk sac tumor. Immunohistochemical stains are useful for distinguishing hepatoid yolk sac tumor from the other hepatoid-appearing tumors. With a multidisciplinary treatment approach using platinum-based regimens, the outcome is similar to those of any yolk sac tumor. PMID:24462393

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. On the robustness of SAC silencing in closed mitosis

    NASA Astrophysics Data System (ADS)

    Ruth, Donovan; Liu, Jian

    Mitosis equally partitions sister chromatids to two daughter cells. This is achieved by properly attaching these chromatids via their kinetochores to microtubules that emanate from the spindle poles. Once the last kinetochore is properly attached, the spindle microtubules pull the sister chromatids apart. Due to the dynamic nature of microtubules, however, kinetochore-microtubule attachment often goes wrong. When this erroneous attachment occurs, it locally activates an ensemble of proteins, called the spindle assembly checkpoint proteins (SAC), which halts the mitotic progression until all the kinetochores are properly attached by spindle microtubules. The timing of SAC silencing thus determines the fidelity of chromosome segregation. We previously established a spatiotemporal model that addresses the robustness of SAC silencing in open mitosis for the first time. Here, we focus on closed mitosis by examining yeast mitosis as a model system. Though much experimental work has been done to study the SAC in cells undergoing closed mitosis, the processes responsible are not well understood. We leverage and extend our previous model to study SAC silencing mechanism in closed mitosis. We show that a robust signal of the SAC protein accumulation at the spindle pole body can be achieved. This signal is a nonlinear increasing function of number of kinetochore-microtubule attachments, and can thus serve as a robust trigger to time the SAC silencing. Together, our mechanism provides a unified framework across species that ensures robust SAC silencing and fidelity of chromosome segregation in mitosis. Intramural research program in NHLBI at NIH.

  11. 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

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

    NASA Technical Reports Server (NTRS)

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

    2003-01-01

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

  13. 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

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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

  9. Recent developments in SAC2000

    SciTech Connect

    Goldstein, P.; Dodge, D.; Firpo, M

    1997-07-01

    Before discussing recent developments in SAC2000, I will summarize what SAC2000 is/does. SAC2000 is the rebirth and evolution of Lawrence Livermore National Laboratory`s (LLNL`s) Seismic Analysis Code (SAC) developed during the 1980`s for a variety of geophysical applications. Primary funding for the development of SAC2000 has been through the LLNL as part of the Department of Energy`s (DOE`s) CTBT R&D program. The primary development goals for SAC2000 have been to meet the seismic signal processing and analysis needs of the DOE CTBT R&D teams and the rest of the CTBT R&D community. SAC2000`s strengths include its ability to process a diverse range of data types, its extensive, well documented signal processing capabilities (both on-line and on the web at http://www-ep.es.llnl.gov/tvp/sac.html), its macro language, and its ability to do both branch and interactive processing. Its extensive usage (over 200 institutions worldwide) had also made it much easier for researchers to develop collaborative research projects. SAC2000`s extensive signal processing capabilities include: data inspection, signal correction, and quality control, unary and binary data operations, travel-time analysis, spectral analysis including high-resolution spectral estimation, spectrograms and binary sonograms, and array and three-component analysis. Recent development in SAC2000 include: enhanced compatibility with the CSS3.0 database schema, complete compatibility with the widely used SEED data format instrument responses, map making capabilities via an interface to GMT, a new three component polarization and phase identification tool, an external interface that allows users to define their own commands, and an interface to MATLAB that allows the user to use MATLAB commands and scripts on SAC data from within SAC2000. We have also implemented a number of commands to enhance user efficiency and numerous improvements and enhancements to many individual SAC commands. Current development in SAC

  10. 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

  11. 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

  12. 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...

  13. 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...

  14. 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

  15. 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

  16. 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

  17. 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

  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. 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

  1. 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

  2. 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

  3. Primary omental yolk sac tumor.

    PubMed

    Lim, Seon Hwa; Kim, Yon Hee; Yim, Ga Won; Nam, Eun Ji; Kim, Young Tae; Kim, Sunghoon

    2013-11-01

    Extra-ovarian yolk sac tumor arising in the omentum is extremely rare. As yolk sac tumor originated from the omentum has been rarely reported, its clinical information is very limited. The authors encountered a case of yolk sac tumor originated from the omentum, and reported the case herein. A 32-year-old woman was presented with developed low abdominal distension for a month. Magnetic resonance imaging findings were suggestive of ovarian malignancy with ascites and peritoneal seeding nodules. Explorative laparotomy was performed and then the findings from frozen biopsy of omentum were suggestive of poorly differentiated tumor though whether it was primary or metastatic was uncertain. Thus, staging laparotomy were performed. Histopathology confirmed that the tumor was a yolk sac tumor of omentum origin. Then, 6 cycles of postoperative adjuvant chemotherapy at intervals of 3 weeks were performed using bleomycin, etoposide, and cisplatin regimen. Four-year outpatient follow-up thereafter showed no relapse. PMID:24396822

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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...

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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.

  4. 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...

  5. 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

  6. 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

  7. Transgenic manipulation of plant embryo sacs tracked through cell-type-specific fluorescent markers: cell labeling, cell ablation, and adventitious embryos.

    PubMed

    Lawit, Shai J; Chamberlin, Mark A; Agee, April; Caswell, Eric S; Albertsen, Marc C

    2013-06-01

    Expression datasets relating to the Arabidopsis female gametophyte have enabled the creation of a tool set which allows simultaneous visual tracking of each specific cell type (egg, synergids, central cell, and antipodals). This cell-specific, fluorescent labeling tool-set functions from gametophyte cellularization through fertilization and early embryo development. Using this system, cell fates were tracked within Arabidopsis ovules following molecular manipulations, such as the ablation of the egg and/or synergids. Upon egg cell ablation, it was observed that a synergid can switch its developmental fate to become egg/embryo-like upon loss of the native egg. Also, manipulated was the fate of the somatic ovular cells, which can become egg- and embryo-like, reminiscent of adventitious embryony. These advances represent initial steps toward engineering synthetic apomixis resulting in seed derived wholly from the maternal plant. The end goal of applied apomixis research, fixing important agronomic traits such as hybrid vigor, would be a key benefit to agricultural productivity. PMID:23539301

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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

  13. Haemangiopericytoma of the lacrimal sac.

    PubMed

    Watanabe, Akihide; Wu, Albert; Sun, Michelle T; Inatani, Masaru; Katori, Nobutada; Selva, Dinesh

    2016-08-01

    Haemangiopericytomas (HPCs) are rare tumours which infrequently occur in the lacrimal sac. Only 8 cases of lacrimal sac HPC have previously been reported. The authors report 2 additional cases presenting clinically with epiphora and a mass. One case recurred 3 times during an 18-year period. The other case did not recur during 51 months of follow-up. The tumours showed immunohistochemical features consistent with a diagnosis of HPC. The authors recommend wide excision for these tumours and careful long-term follow-up to detect recurrence which is not uncommon. PMID:27322416

  14. 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.

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. Many postchemotherapy sarcomatous tumors in patients with testicular germ cell tumors are sarcomatoid yolk sac tumors: a study of 33 cases.

    PubMed

    Howitt, Brooke E; Magers, Martin J; Rice, Kevin R; Cole, Cristina D; Ulbright, Thomas M

    2015-02-01

    Sarcomatoid neoplasms in patients with testicular germ cell tumors (TGCTs) may show diverse lineages and are usually attributed to "transformation" of teratoma, although origin from yolk sac tumor (YST) has also been suggested. We evaluated 33 sarcomatoid tumors from 23 TGCT patients that lacked specific features of a defined sarcoma subtype for a number of features, including: atypia (mild, moderate, severe), cellularity, tumor necrosis, mitotic index, stromal vascularity, cell profile (spindle or epithelioid), and stromal quality (myxoid and/or fibrous). Immunohistochemical staining analyses directed against cytokeratin (AE1/AE3), SALL4, glypican-3 (GPC3), α-fetoprotein (AFP), p63, glial fibrillary acidic protein (GFAP), CD34, MUC4, smooth muscle actin (SMA), desmin, caldesmon, and myogenin were performed. Staining intensity (0=negative, 1=weak, 2=moderate, 3=strong) and extent (0=<1%, 1=1% to 10%, 2=10% to 50%, 3=>50%) were scored. Tumor grade based on the French sarcoma grading system was assessed, with grades 2-3 considered high grade. Tumors with at least moderate intensity and >10% (+) cells for both AE1/AE3 and GPC3 were considered to be sarcomatoid YST (SYST); 22 tumors from 14 patients (ages 18 to 38 y, mean 27 y) met these criteria and were the focus of this study. All SYSTs occurred after chemotherapy (3 to 132 mo after TGCT diagnosis; mean 42.5 mo, median 30.5 mo). They had spindled (100%; 19 predominant) and epithelioid cells (77%; 3 predominant) in myxoid to fibrous stroma. Thirteen exhibited at least focally severe nuclear atypia. Distinctive tumor "ringlets" and intercellular basement membrane deposits (parietal YST differentiation) were common. In addition to positivity for AE1/AE3 and GPC3, 15/22 were SALL4 (+), 10/22 were at least focally CD34 (+), and 2/22 were focally p63 (+). Fifty percent exhibited smooth muscle differentiation as evidenced by desmin (8/19), caldesmon (2/4), and/or SMA (4/6) reactivity. AFP, MUC4, GFAP, and myogenin were

  1. Cul-de-Sac Kids

    ERIC Educational Resources Information Center

    Hochschild, Thomas R., Jr.

    2013-01-01

    Previous research indicates that adults who live on cul-de-sac streets are more likely to have positive experiences with neighbors than residents of other street types (Brown and Werner, 1985; Hochschild Jr, 2011; Mayo Jr, 1979; Willmott, 1963). The present research ascertains whether street design has an impact on children's neighborhood…

  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. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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...

  12. 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

  13. 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.

  14. ZBTB16: a novel sensitive and specific biomarker for yolk sac tumor.

    PubMed

    Xiao, Guang-Qian; Li, Faqian; Unger, Pamela D; Katerji, Hani; Yang, Qi; McMahon, Loralee; Burstein, David E

    2016-06-01

    Although the function of zinc finger and BTB domain containing 16 (ZBTB16) in spermatogenesis is well documented, expression of ZBTB16 in germ cell tumors has not yet been studied. The aim of this study was to investigate the immunohistochemical expression and diagnostic utility of ZBTB16 in germ cell tumors. A total of 67 adult germ cell tumors were studied (62 testicular germ cell tumors, 2 ovarian yolk sac tumors, 1 mediastinal yolk sac tumor, and 2 retroperitoneal metastatic yolk sac tumors). The 62 testicular primary germ cell tumors are as follows: 34 pure germ cell tumors (20 seminomas, 8 embryonal carcinomas, 2 teratomas, 1 choriocarcinoma, 1 carcinoid, and 2 spermatocytic tumors) and 28 mixed germ cell tumors (composed of 13 embryonal carcinomas, 15 yolk sac tumors, 15 teratomas, 7 seminomas, and 3 choriocarcinomas in various combinations). Thirty-five cases contained germ cell neoplasia in situ. Yolk sac tumor was consistently reactive for ZBTB16. Among the 15 testicular yolk sac tumors in mixed germ cell tumors, all displayed moderate to diffuse ZBTB16 staining. ZBTB16 reactivity was present regardless of the histologic patterns of yolk sac tumor and ZBTB16 was able to pick up small foci of yolk sac tumor intermixed/embedded in other germ cell tumor subtype elements. Diffuse ZBTB16 immunoreactivity was also observed in 2/2 metastatic yolk sac tumors, 1/1 mediastinal yolk sac tumor, 2/2 ovarian yolk sac tumors, 2/2 spermatocytic tumors, 1/1 carcinoid, and the spermatogonial cells. All the other non-yolk sac germ cell tumors were nonreactive, including seminoma (n=27), embryonal carcinoma (n=21), teratoma (n=17), choriocarcinoma (n=4), and germ cell neoplasia in situ (n=35). The sensitivity and specificity of ZBTB16 in detecting yolk sac tumor among the germ cell tumors was 100% (20/20) and 96% (66/69), respectively. In conclusion, ZBTB16 is a highly sensitive and specific marker for yolk sac tumor. PMID:26916077

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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...

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. The annular hematoma of the shrew yolk-sac placenta.

    PubMed

    King, B F; Enders, A C; Wimsatt, W A

    1978-05-01

    The annular hematoma of the shrew, Blarina brevicauda, is a specialized portion of the yolk-sac wall. In this study, we have examined the fine structure of the different cellular components of the anular hematoma. Small pieces of the gestation sacs from seven pregnant shrews were fixed in glutaraldehyde and osmium tetroxide and processed for transmission electron microscopy. In the area of the trophoblastic curtain, the maternal capillary endothelial cells were hypertrophied and syncytial trophoblast surrounded the capillaries. Cellular trophoblast covered part of the luminal surface of the curtain region, whereas masses of apparently degenerating syncytium were present on other areas of the surface. Maternal erythrocytes, released into the uterine lumen from the curtain region, were phagocytized and degraded by the columnar cells of the trophoblastic annulus. No evidence of iron or pigment accumulation was evident in the parietal endodermal cells underlying the annular trophoblast. Parietal endodermal cells were characterized by cuboidal shape, widely dilated intercellular spaces, and cytoplasm containing granular endoplasmic reticulum. Endodermal cells of the visceral yolk-sac accumulated large numbers of electron-dense granules as well as glycogen in their cytoplasm. Hemopoietic areas and vitelline capillaries were found subjacent to the visceral endoderm. The various portions of the yolk-sac wall of Blarina appear to perform complementary functions which are probably important in maternal-fetal iron transfer. PMID:677046

  8. 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

  9. 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...

  10. Spatiotemporal control of phosphatidylinositol 4-phosphate by Sac2 regulates endocytic recycling

    PubMed Central

    Hsu, FoSheng; Hu, Fenghua

    2015-01-01

    It is well established that the spatial- and temporal-restricted generation and turnover of phosphoinositides (PIs) by a cascade of PI-metabolizing enzymes is a key regulatory mechanism in the endocytic pathway. Here, we demonstrate that the Sac1 domain–containing protein Sac2 is a PI 4-phosphatase that specifically hydrolyzes phosphatidylinositol 4-phosphate in vitro. We further show that Sac2 colocalizes with early endosomal markers and is recruited to transferrin (Tfn)-containing vesicles during endocytic recycling. Exogenous expression of the catalytically inactive mutant Sac2C458S resulted in altered cellular distribution of Tfn receptors and delayed Tfn recycling. Furthermore, genomic ablation of Sac2 caused a similar perturbation on Tfn and integrin recycling as well as defects in cell migration. Structural characterization of Sac2 revealed a unique pleckstrin-like homology Sac2 domain conserved in all Sac2 orthologues. Collectively, our findings provide evidence for the tight regulation of PIs by Sac2 in the endocytic recycling pathway. PMID:25869669

  11. Endolymphatic sac involvement in bacterial meningitis.

    PubMed

    Møller, Martin Nue; Brandt, Christian; Østergaard, Christian; Caye-Thomasen, Per

    2015-04-01

    The commonest sequelae of bacterial meningitis are related to the inner ear. Little is known about the inner ear immune defense. Evidence suggests that the endolymphatic sac provides some protection against infection. A potential involvement of the endolymphatic sac in bacterial meningitis is largely unaccounted for, and thus the object of the present study. A well-established adult rat model of Streptococcus pneumoniae meningitis was employed. Thirty adult rats were inoculated intrathecally with Streptococcus pneumoniae and received no additional treatment. Six rats were sham-inoculated. The rats were killed when reaching terminal illness or on day 7, followed by light microscopy preparation and PAS-Alcian blue staining. The endolymphatic sac was examined for bacterial invasion and leukocyte infiltration. Neither bacteria nor leukocytes infiltrated the endolymphatic sac during the first days. Bacteria invaded the inner ear through the cochlear aquaduct. On days 5-6, the bacteria invaded the endolymphatic sac through the endolymphatic duct subsequent to invasion of the vestibular endolymphatic compartment. No evidence of direct bacterial invasion of the sac through the meninges was found. Leukocyte infiltration of the sac occurred prior to bacterial invasion. During meningitis, bacteria do not invade the endolymphatic sac through the dura, but solely through the endolymphatic duct, following the invasion of the vestibular system. Leukocyte infiltration of the sac occurs prior to, as well as concurrent with bacterial invasion. The findings support the endolymphatic sac as part of an innate immune defense system protecting the inner ear from infection. PMID:24452771

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. Performance Analysis of Wavelength Multiplexed Sac Ocdma Codes in Beat Noise Mitigation in Sac Ocdma Systems

    NASA Astrophysics Data System (ADS)

    Alhassan, A. M.; Badruddin, N.; Saad, N. M.; Aljunid, S. A.

    2013-07-01

    In this paper we investigate the use of wavelength multiplexed spectral amplitude coding (WM SAC) codes in beat noise mitigation in coherent source SAC OCDMA systems. A WM SAC code is a low weight SAC code, where the whole code structure is repeated diagonally (once or more) in the wavelength domain to achieve the same cardinality as a higher weight SAC code. Results show that for highly populated networks, the WM SAC codes provide better performance than SAC codes. However, for small number of active users the situation is reversed. Apart from their promising improvement in performance, these codes are more flexible and impose less complexity on the system design than their SAC counterparts.

  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. 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

  20. Primary yolk sac tumor of the gluteus: a case report and literature review.

    PubMed

    Li, Bo; Jiang, Qianqian; Zhang, Shitai; Zhou, Yang; Zhang, Qing-Fu; OuYang, Ling

    2016-01-01

    Yolk sac tumor (YST) is a common malignant primitive germ cell tumor that often exhibits differentiation into endodermal structures. They most commonly occur in childhood and adolescence and are rare after the age of 40 years. Derived from the yolk sac during the embryonic period, YSTs can occur in the gonads and germ cells because the tumor cells migrate from the yolk sac toward the gonads. Here, we present a rare case of primary gluteus YST in a 3-year-old girl. She received BEP chemotherapy (bleomycin + etoposide + cisplatin) after surgical resection. There was no evidence of recurrence 7 months after primary treatment. PMID:27536133

  1. Primary yolk sac tumor of the gluteus: a case report and literature review

    PubMed Central

    Li, Bo; Jiang, Qianqian; Zhang, Shitai; Zhou, Yang; Zhang, Qing-Fu; OuYang, Ling

    2016-01-01

    Yolk sac tumor (YST) is a common malignant primitive germ cell tumor that often exhibits differentiation into endodermal structures. They most commonly occur in childhood and adolescence and are rare after the age of 40 years. Derived from the yolk sac during the embryonic period, YSTs can occur in the gonads and germ cells because the tumor cells migrate from the yolk sac toward the gonads. Here, we present a rare case of primary gluteus YST in a 3-year-old girl. She received BEP chemotherapy (bleomycin + etoposide + cisplatin) after surgical resection. There was no evidence of recurrence 7 months after primary treatment. PMID:27536133

  2. 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

  3. 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

  4. 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

  5. Pituitary carcinoma with endolymphatic sac metastasis.

    PubMed

    Balili, Irida; Sullivan, Steven; Mckeever, Paul; Barkan, Ariel

    2014-06-01

    Pituitary carcinoma is characterized by the presence of a metastatic lesion(s) in a location non-contiguous with the original pituitary tumor. The mechanism(s) of malignant transformation are not known. A 15 year-old male was diagnosed in 1982 with a pituitary macroadenoma and acromegaly (random GH 67 ng/ml and no suppression by oral glucose). His prolactin was normal between 18 and 23 ng/ml. Transcranial resection in July 1983 was followed by radiation therapy. The tumor was immunopositive for GH and prolactin. The proliferation MIB-1 index was 0-1%. With aqueous Octreotide 100 mcg 4× daily both GH and IGF-1 became normal. The patient was lost to follow-up and was treated by his local physician. In 2001, his IGF-1 level was 1271 ng/ml, and his random GH was 1.8-2.4 ng/ml by ILMA despite progressive increase in the dose of Sandostatin LAR to 140 mg/month in divided doses. Prolactin remained normal or minimally increased between 15 and 25 ng/ml. In 2009 he was diagnosed with the tumor in the location of left endolymphatic sac. Histological examination showed low grade pituitary carcinoma strongly immunopositive for prolactin but negative for GH. MIB-1 antibody labeled 0-5% cells. In 2012 endoscopic resection of the pituitary tumor remnant was attempted. Immunohistochemical stains were strongly immunopositive for both prolactin and GH, similar to his original pituitary tumor. The MIB-1 proliferation index was low from 0 to 1%. To our knowledge this is the first case of pituitary carcinoma in the endolymphatic sac region. The dichotomy between the cell population of the pituitary lesion (GH/prolactin producing) and the metastasis (purely prolactin-producing) may suggest that the metastatic pituitary lesion derived from a clone distinct from the original one. PMID:23645293

  6. 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.

  7. 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

  8. 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

  9. Giant prosthetic reinforcement of the visceral sac.

    PubMed

    Wantz, G E

    1989-11-01

    One hundred and seventy-nine patients with 237 hernias of the groin who were at high risk for recurrence after classic hernioplasty were operated upon; the procedure of giant prosthetic reinforcement of the visceral sac (GPRVS) was used. The patients in this series had predominantly recurrent and re-recurrent hernias. However, a few were obese with bilateral primary direct hernias and some had associated connective tissue disorders, such as Marfan and Ehlers-Danlos syndromes. GPRVS eliminates hernias of the groin by rendering the peritoneum inextensible by placing, in the preperitoneal space, a large prosthesis that extends far beyond the borders of the myopectineal orifice. The myopectineal orifice is the weak spot at which all hernias of the groin begin and is bounded by the rectus, oblique abdominal and iliopsoas muscles and the pectin of the pubis. In bilateral GPRVS, the peritoneum of both groins is reinforced with a single prosthesis inserted in the preperitoneal space through the midline. In unilateral GPRVS, the mesh envelops the peritoneum of a single groin. This simplifies the operation and makes it suitable for surgical centers that perform outpatient operations. The prosthesis with the best physical characteristics for GPRVS is Mersilene (polyester fiber). Unsutured prostheses of polypropylene and Teflon (polytetrafluoroethylene) may not adhere at the far edges, leading to a failure and recurrence. The over-all recurrence rate in this series of problem hernias was 3.7 per cent, which is extremely good. However, the rate becomes outstanding if recurrences resulting from meshes unsuitable for GPRVS are excluded. PMID:2814751

  10. Development of the endolymphatic sac in chick embryos, with reference to the degradation of otoconia

    NASA Technical Reports Server (NTRS)

    Yoshihara, T.; Kaname, H.; Narita, N.; Ishii, T.; Igarashi, M.; Fermin, C. D.

    1992-01-01

    The endolymphatic sac of chick embryos (from embryonic day 7 to 1-day-old chicks) was studied light- and electron-microscopically. At stage 30-31 (embryonic day 7-7.5), the epithelial cells of the endolymphatic sac were cuboidal to columnar in shape. Microvilli were relatively well developed. The intercellular space was wide. In the endolymphatic space of the endolymphatic sac, varying shapes and sizes of otoconia-like bodies were often observed. Intracytoplasmic phagosomes containing these bodies were rarely found. After stage 37 (embryonic day 11), otoconia-like bodies in the endolymphatic sac decreased in number and size. They were almost the same as the otoconia in the macular organs, ultrastructurally. These findings indicate that the endolymphatic sac of the chick embryos may possess the function of otoconial degradation and removal of calcium from otoconia.

  11. 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

  12. 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

  13. 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

  14. 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

  15. Placentation in mammals: Definitive placenta, yolk sac, and paraplacenta.

    PubMed

    Carter, A M; Enders, A C

    2016-07-01

    An overview is given of variations in placentation with particular focus on yolk sac, paraplacenta, and other structures important to histotrophic nutrition. The placenta proper varies in general shape, internal structure, and the number of tissues in the interhemal barrier. Yolk sac membranes persist to term in insectivores, colugos, rodents, and lagomorphs. In the latter two orders, they are of known importance for maternal-fetal transfer of antibodies, vitamins, lipids, and proteins. The detached yolk sac of bats is also active throughout gestation. A vascular paraplacenta, or smooth chorioallantois, has known functions in ruminants and carnivores and is found in several other orders of mammal where its function has yet to be explored. In human gestation, the chorion (avascular chorioallantois) is important for hormone synthesis. The true chorion of squirrels and hedgehogs is avascular but may nevertheless allow transfer from mother to fetus through the exocelom. Hemophagous areas with columnar trophoblast are paraplacental structures in carnivores and elephants but occur also within the placenta as in hyenas and moles. In shrews, it is the yolk sac that ingests and processes red cells. Areolas and chorionic vesicles are other structures important for absorption of uterine secretions and ingestion of cellular debris. In conclusion, we find that paraplacental structures, while showing less variation than the placenta proper, contribute not just to the integrity of overall placentation, but in various ways to maternal-fetal interrelationships. PMID:27155730

  16. 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

  17. 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.

  18. 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

  19. 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

  20. Evaluation of an anal sac adenocarcinoma tumor in a Spitz dog

    PubMed Central

    Javanbakht, Javad; Tavassoli, Abbas; Sabbagh, Atefeh; Hassan, Mehdy Aghamohammmad; Samakkhah, Shohreh Alian; Shafiee, Radmehr; Lakzian, Ali; Ghalee, Vahideh Rahmani; Gharebagh, Sonia Shoja

    2013-01-01

    A 9-year-old emasculated male Spitz with tenesmus and constipation had a subcutaneous mass at the left ventral aspect of the anus with history of polyuria and polydipsia. A complete blood cell count, serum biochemistry panel, and urinalysis (cystocentesis sample) were evaluated. Abnormalities in the serum biochemistry panel included a mildly elevated serum cholesterol concentration (7.28 mmol/L; reference interval, 2.70–5.94 mmol/L), increased serum alkaline phosphatase activity (184 U/L; reference interval, 9–90 U/L), alanine transaminase (122 U/L; reference interval, 5–60 U/L) activity and aspartate aminotransferase (80 U/L; reference interval, 5–55 U/L) activity, severe increased total calcium concentration (16.3 mg/dL; reference interval, 8.2–12.4 mg/dL or 9.3–11.4 mg/dL), and decreased total calcium concentration (3.4 mg/dL, reference interval, 2.5–5.6mg/dL). Furthermore, testing revealed an increased intact parathyroid hormone concentration (38.6 pmol/L; reference interval, 3–17 pmol/L). On cytologic and histopathologic examinations, various types of cells were observed. Most of the cells were oval to polygonal and had elliptical or elongate nuclei and a moderate amount of pale to basophilic cytoplasm. The remaining cells had round to oval nuclei and pale to basophilic cytoplasm. Cells of both types were loosely adhered to each other and were arranged in rosette-like structures. Both neoplastic cell types had fine homogenous chromatin and either a small indistinct nucleolus or no visible nucleolus. Mild anisokaryosis and anisocytosis were observed. Histologically, the mass consists of glandular structures formed by cuboidal cells admixed with bundles of spindle cells. Based on location and histologic features, the final diagnosis was adenocarcinoma of the apocrine gland of the anal sac, which should be included as a cytologic differential diagnosis when spindle cells and typical epithelial cells are observed in masses in the region of the anal

  1. 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

  2. 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

  3. 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

  4. Established and potential physiological roles of bicarbonate-sensing soluble adenylyl cyclase (sAC) in aquatic animals.

    PubMed

    Tresguerres, Martin; Barott, Katie L; Barron, Megan E; Roa, Jinae N

    2014-03-01

    Soluble adenylyl cyclase (sAC) is a recently recognized source of the signaling molecule cyclic AMP (cAMP) that is genetically and biochemically distinct from the classic G-protein-regulated transmembrane adenylyl cyclases (tmACs). Mammalian sAC is distributed throughout the cytoplasm and it may be present in the nucleus and inside mitochondria. sAC activity is directly stimulated by HCO3(-), and sAC has been confirmed to be a HCO3(-) sensor in a variety of mammalian cell types. In addition, sAC can functionally associate with carbonic anhydrases to act as a de facto sensor of pH and CO2. The two catalytic domains of sAC are related to HCO3(-)-regulated adenylyl cyclases from cyanobacteria, suggesting the cAMP pathway is an evolutionarily conserved mechanism for sensing CO2 levels and/or acid/base conditions. Reports of sAC in aquatic animals are still limited but are rapidly accumulating. In shark gills, sAC senses blood alkalosis and triggers compensatory H(+) absorption. In the intestine of bony fishes, sAC modulates NaCl and water absorption. And in sea urchin sperm, sAC may participate in the initiation of flagellar movement and in the acrosome reaction. Bioinformatics and RT-PCR results reveal that sAC orthologs are present in most animal phyla. This review summarizes the current knowledge on the physiological roles of sAC in aquatic animals and suggests additional functions in which sAC may be involved. PMID:24574382

  5. Established and potential physiological roles of bicarbonate-sensing soluble adenylyl cyclase (sAC) in aquatic animals

    PubMed Central

    Tresguerres, Martin; Barott, Katie L.; Barron, Megan E.; Roa, Jinae N.

    2014-01-01

    Soluble adenylyl cyclase (sAC) is a recently recognized source of the signaling molecule cyclic AMP (cAMP) that is genetically and biochemically distinct from the classic G-protein-regulated transmembrane adenylyl cyclases (tmACs). Mammalian sAC is distributed throughout the cytoplasm and it may be present in the nucleus and inside mitochondria. sAC activity is directly stimulated by HCO3−, and sAC has been confirmed to be a HCO3− sensor in a variety of mammalian cell types. In addition, sAC can functionally associate with carbonic anhydrases to act as a de facto sensor of pH and CO2. The two catalytic domains of sAC are related to HCO3−-regulated adenylyl cyclases from cyanobacteria, suggesting the cAMP pathway is an evolutionarily conserved mechanism for sensing CO2 levels and/or acid/base conditions. Reports of sAC in aquatic animals are still limited but are rapidly accumulating. In shark gills, sAC senses blood alkalosis and triggers compensatory H+ absorption. In the intestine of bony fishes, sAC modulates NaCl and water absorption. And in sea urchin sperm, sAC may participate in the initiation of flagellar movement and in the acrosome reaction. Bioinformatics and RT-PCR results reveal that sAC orthologs are present in most animal phyla. This review summarizes the current knowledge on the physiological roles of sAC in aquatic animals and suggests additional functions in which sAC may be involved. PMID:24574382

  6. 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

  7. 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.

  8. 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

  9. Histological and histochemical analyses of the cuttlebone sac of the golden cuttlefish Sepia esculenta

    NASA Astrophysics Data System (ADS)

    Zheng, Xiaodong; Xiao, Shu; Wang, Zhaoping; Wang, Rucai

    2007-10-01

    The secretion function of mantle is closely related to shell formation in some bivalves and gastropods. Up to now, few researches have been reported for cuttlebone formation in the class Cephalopoda. In this study, the structure and secretion function of cuttlebone sac of the golden cuttlefish Sepia esculenta was analyzed using the histological and histochemical methods. The results showed that high and columnar cells located in sac epithelium, and flat cells existed near the base membrane. A lot of fibroblasts were found in the lateral mantle collective tissue. Some mucus, mucopolysaccharide and alkaline phosphatase (ALP) were found in the sac. The ultrastructural characteristics of Quasi-connective-tissue-calcium cells (QCTCC) were observed using a transmission electron microscope (TEM). The relationship between cuttlebone sac secretion function and shell formation was discussed.

  10. 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.

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. The First Transmembrane Domain of Lipid Phosphatase SAC1 Promotes Golgi Localization

    PubMed Central

    Wang, Jinzhi; Chen, Juxing; Enns, Caroline A.; Mayinger, Peter

    2013-01-01

    The lipid phosphatase Sac1 cycles between endoplasmic reticulum and cisternal Golgi compartments. In proliferating mammalian cells, a canonical dilysine motif at the C-terminus of Sac1 is required for coatomer complex-I (COP-I)-binding and continuous retrieval to the ER. Starvation triggers accumulation of Sac1 at the Golgi. The mechanism responsible for Golgi retention of Sac1 is unknown. Here we show that the first of the two transmembrane regions in human SAC1 (TM1) functions in Golgi localization. A minimal construct containing only TM1 and the adjacent flanking sequences is concentrated at the Golgi. Transplanting TM1 into transferrin receptor 2 (TfR2) induces Golgi accumulation of this normally plasma membrane and endosomal protein, indicating that TM1 is sufficient for Golgi localization. In addition, we determined that the N-terminal cytoplasmic domain of SAC1 also promotes Golgi localization, even when TM1 is mutated or absent. We conclude that the distribution of SAC1 within the Golgi is controlled via both passive membrane thickness-dependent partitioning of TM1 and a retention mechanism that requires the N-terminal cytoplasmic region. PMID:23936490

  17. SACS: Spitzer Archival Cluster Survey

    NASA Astrophysics Data System (ADS)

    Stern, Daniel

    Emerging from the cosmic web, galaxy clusters are the most massive gravitationally bound structures in the universe. Thought to have begun their assembly at z > 2, clusters provide insights into the growth of large-scale structure as well as the physics that drives galaxy evolution. Understanding how and when the most massive galaxies assemble their stellar mass, stop forming stars, and acquire their observed morphologies in these environments remain outstanding questions. The redshift range 1.3 < z < 2 is a key epoch in this respect: elliptical galaxies start to become the dominant population in cluster cores, and star formation in spiral galaxies is being quenched. Until recently, however, this redshift range was essentially unreachable with available instrumentation, with clusters at these redshifts exceedingly challenging to identify from either ground-based optical/nearinfrared imaging or from X-ray surveys. Mid-infrared (MIR) imaging with the IRAC camera on board of the Spitzer Space Telescope has changed the landscape. High-redshift clusters are easily identified in the MIR due to a combination of the unique colors of distant galaxies and a negative k-correction in the 3-5 μm range which makes such galaxies bright. Even 90-sec observations with Spitzer/IRAC, a depth which essentially all extragalactic observations in the archive achieve, is sufficient to robustly detect overdensities of L* galaxies out to z~2. Here we request funding to embark on a ambitious scientific program, the “SACS: Spitzer Archival Cluster Survey”, a comprehensive search for the most distant galaxy clusters in all Spitzer/IRAC extragalactic pointings available in the archive. With the SACS we aim to discover ~2000 of 1.3 < z < 2.5 clusters, thus provide the ultimate catalog for high-redshift MIR selected clusters: a lasting legacy for Spitzer. The study we propose will increase by more than a factor of 10 the number of high-redshift clusters discovered by all previous surveys

  18. 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

  19. 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

  20. 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

  1. Sac3, an Snf1-like serine/threonine kinase that positively and negatively regulates the responses of Chlamydomonas to sulfur limitation.

    PubMed Central

    Davies, J P; Yildiz, F H; Grossman, A R

    1999-01-01

    The Sac3 gene product of Chlamydomonas positively and negatively regulates the responses of the cell to sulfur limitation. In wild-type cells, arylsulfatase activity is detected only during sulfur limitation. The sac3 mutant expresses arylsulfatase activity even when grown in nutrient-replete medium, which suggests that the Sac3 protein has a negative effect on the induction of arylsulfatase activity. In contrast to its effect on arylsulfatase activity, Sac3 positively regulates the high-affinity sulfate transport system-the sac3 mutant is unable to fully induce high-affinity sulfate transport during sulfur limitation. We have complemented the sac3 mutant and cloned a cDNA copy of the Sac3 gene. The deduced amino acid sequence of the Sac3 gene product is similar to the catalytic domain of the yeast Snf1 family of serine/threonine kinases and is therefore classified as a Snf1-related kinase (SnRK). Specifically, Sac3 falls within the SnRK2 subfamily of kinases from vascular plants. In addition to the 11 subdomains common to Snf1-like serine/threonine kinases, Sac3 and the plant kinases have two additional subdomains and a highly acidic C-terminal region. The role of Sac3 in the signal transduction system that regulates the responses of Chlamydomonas to sulfur limitation is discussed. PMID:10368187

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. A Rare Case of Primary Anterior Mediastinal Yolk Sac Tumor in an Elderly Adult Male

    PubMed Central

    Nakhla, Sammy G.; Sundararajan, Srinath

    2016-01-01

    Mediastinal germ cell tumors are extragonadal germ cell tumors (EGGCTs) commonly seen in children and young adults. They are more common in men. Clinically they are classified as teratomas, seminomas, and nonseminomatous germ cell tumors. Primary mediastinal yolk sac neoplasm is an extremely rare tumor. We present here a very rare case of primary yolk sac tumor of the anterior mediastinum in a 73-year-old male. Mediastinal germ cell tumors have a worse prognosis than gonadal germ cell tumors. Chemotherapy followed by adjuvant surgery improves overall response in EGGCTs. However, comorbidities can render treatment with chemotherapy and surgery challenging in elderly patients. PMID:27144043

  10. 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

  11. Endothelial cell migration during murine yolk sac vascular remodeling occurs by means of a Rac1 and FAK activation pathway in vivo

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The molecular mechanism(s) controlling cell migration during vascular morphogenesis in vivo remain largely undefined. To address this within a physiological context, we used retinaldehyde dehydrogenase-2 (Raldh2) null mouse embryos and demonstrate that retinoic acid (RA) deficiency results in abnorm...

  12. 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 ...

  13. Endometriosis in a spigelian hernia sac: an unexpected finding.

    PubMed

    Moris, Demetrios; Michalinos, Adamantios; Vernadakis, Spiridon

    2015-01-01

    Describes the existence of endometrioma in a spigelian hernia sac. Spigelian Hernia is a rare ventral hernia, presenting difficulties in diagnosis and carrying a high incarceration and obstruction risk. Endometriomas occur due to implantation of endometrial cells into a surgical wound, most often after a cesarean delivery. A 37-year-old woman presented to our department with persistent abdominal pain, exacerbating during menses, and vomiting for 2 days. Physical examination revealed a mass-like lesion in the border between the left-upper and left-lower quadrant. Ultrasound examination was inconclusive and a computed tomography scan of the abdomen revealed an abdominal wall mass. During surgery, a spigelian hernia was found 5 to 7 cm above a previous cesarean incision. Tissue like "chocolate cysts" was present at the hernia sac. Hernia was repaired while tissue was excised and sent for histological examination that confirmed the diagnosis. Spigelian hernia is a hernia presenting difficulties in diagnosis and treatment. Endometrioma in a spigelian hernia sac is a rare diagnosis, confirmed only histologically. Clinical suspicion can be posed only through symptoms and thorough investigation. PMID:25594648

  14. Endometriosis in a Spigelian Hernia Sac: An Unexpected Finding

    PubMed Central

    Moris, Demetrios; Michalinos, Adamantios; Vernadakis, Spiridon

    2015-01-01

    Describes the existence of endometrioma in a spigelian hernia sac. Spigelian Hernia is a rare ventral hernia, presenting difficulties in diagnosis and carrying a high incarceration and obstruction risk. Endometriomas occur due to implantation of endometrial cells into a surgical wound, most often after a cesarean delivery. A 37-year-old woman presented to our department with persistent abdominal pain, exacerbating during menses, and vomiting for 2 days. Physical examination revealed a mass-like lesion in the border between the left-upper and left-lower quadrant. Ultrasound examination was inconclusive and a computed tomography scan of the abdomen revealed an abdominal wall mass. During surgery, a spigelian hernia was found 5 to 7 cm above a previous cesarean incision. Tissue like “chocolate cysts” was present at the hernia sac. Hernia was repaired while tissue was excised and sent for histological examination that confirmed the diagnosis. Spigelian hernia is a hernia presenting difficulties in diagnosis and treatment. Endometrioma in a spigelian hernia sac is a rare diagnosis, confirmed only histologically. Clinical suspicion can be posed only through symptoms and thorough investigation. PMID:25594648

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. A Surprise in the Lacrimal Sac

    PubMed Central

    Yuksel, Nilay; Akcay, Emine; Kilicarslan, Aydan; Ozen, Umut; Ozturk, Faruk

    2016-01-01

    To present a case with recurrent dacryocystitis as an unusual complication of medial orbital wall fracture repair with cartilage tissue graft. A 20-year-old male had facial trauma and underwent surgery to reconstruct right medial orbital wall fracture. During follow–up, he presented with continuous epiphora, mucopurulent discharge from the right eye. A thorough history taking indicated that medial orbital fracture was reconstructed with postauricular cartilage. We planned a standard external dacryocystorhinostomy (DCR). During the creation of lacrimal sac flaps, hard tissue was noted in the lacrimal sac. This tissue was excised and sent for pathological examination. The pathological examination revealed cartilage tissue. There were no further ipsilateral symptoms or complications after DCR. In patients with lacrimal system injury related to orbital wall fracture, iatrogenic foreign bodies in the lacrimal sac should be considered in patients with recurrent dacryocystitis who had reconstructive surgery for facial and orbital trauma. PMID:27555715

  2. SAC-B, Argentine scientific satellite

    NASA Technical Reports Server (NTRS)

    Gulich, J. M.; White, C.

    1994-01-01

    The project and the missions of the Argentine scientific satellite, SAC-B, are summarized. SAC-B is an international cooperative project between NASA and the Secretariat of State of Science and Technology of the Argentine Republic. The objective of SAC-B is to advance the study of solar physics and astrophysics through the examination of solar flares, gamma ray burst sources and the diffuse soft X-ray cosmic background. The scientific payload comprises an instrument to measure the temporal evolution of X-ray emissions from solar flares as well as nonsolar gamma ray bursts, a combined soft X-ray and gamma ray burst detector, a diffuse X-ray background detector, and an energetic neutral atoms detector.

  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. 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

  7. 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.

  8. Phosphoregulatory protein 14-3-3 facilitates SAC1 transport from the endoplasmic reticulum

    PubMed Central

    Bajaj Pahuja, Kanika; Wang, Jinzhi; Blagoveshchenskaya, Anastasia; Lim, Lillian; Madhusudhan, M. S.; Mayinger, Peter; Schekman, Randy

    2015-01-01

    Most secretory cargo proteins in eukaryotes are synthesized in the endoplasmic reticulum and actively exported in membrane-bound vesicles that are formed by the cytosolic coat protein complex II (COPII). COPII proteins are assisted by a variety of cargo-specific adaptor proteins required for the concentration and export of secretory proteins from the endoplasmic reticulum (ER). Adaptor proteins are key regulators of cargo export, and defects in their function may result in disease phenotypes in mammals. Here we report the role of 14-3-3 proteins as a cytosolic adaptor in mediating SAC1 transport in COPII-coated vesicles. Sac1 is a phosphatidyl inositol-4 phosphate (PI4P) lipid phosphatase that undergoes serum dependent translocation between the endoplasmic reticulum and Golgi complex and controls cellular PI4P lipid levels. We developed a cell-free COPII vesicle budding reaction to examine SAC1 exit from the ER that requires COPII and at least one additional cytosolic factor, the 14-3-3 protein. Recombinant 14-3-3 protein stimulates the packaging of SAC1 into COPII vesicles and the sorting subunit of COPII, Sec24, interacts with 14-3-3. We identified a minimal sorting motif of SAC1 that is important for 14-3-3 binding and which controls SAC1 export from the ER. This LS motif is part of a 7-aa stretch, RLSNTSP, which is similar to the consensus 14-3-3 binding sequence. Homology models, based on the SAC1 structure from yeast, predict this region to be in the exposed exterior of the protein. Our data suggest a model in which the 14-3-3 protein mediates SAC1 traffic from the ER through direct interaction with a sorting signal and COPII. PMID:26056309

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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...

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

    PubMed

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

    2016-05-01

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

  16. 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

  17. 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

  18. Histopathology after Endolymphatic Sac Surgery for Meniere’s Syndrome

    PubMed Central

    Chung, Jong Woo; Fayad, Jose; Linthicum, Fred; Ishiyama, Akira; Merchant, Saumil N.

    2011-01-01

    Background The putative goal of sac surgery in Meniere’s syndrome is to promote the flow of endolymph from the labyrinth to the endolymphatic sac, and thereby relieving hydrops. There is scant published histopathological data whether sac surgery actually accomplishes this goal. Objective To determine if sac surgery relieves hydrops by examining the histopathologic changes in temporal bones obtained from individuals who had undergone sac surgery during life for Meniere’s syndrome. Methods Temporal bones were examined from 15 patients who had sac surgery. Data was collected on presence and severity of hydrops, histology of the sac, and whether the procedure relieved vertigo. Results The surgery failed to expose the sac in 5 cases; 4 of the 5 had relief from vertigo. The sac was exposed, but the shunt failed to reach the lumen of the sac in 8 cases; 4 of the 8 had relief from vertigo. The shunt was successfully placed within the lumen of the sac in two cases; both cases failed to experience relief from vertigo. Endolymphatic hydrops was present in all 15 cases. Conclusion Endolymphatic sac surgery does not relieve hydrops in patients with Meniere’s syndrome. Yet, sac surgery relieves vertigo in some patients, but the mechanism of such symptomatic relief remains unknown. PMID:21436748

  19. 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

  20. 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.

  1. 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

  2. Milk digesta and milk protein fractions influence the adherence of Lactobacillus gasseri R and Lactobacillus casei FMP to human cultured cells.

    PubMed

    Volstatova, Tereza; Havlik, Jaroslav; Potuckova, Miroslava; Geigerova, Martina

    2016-08-10

    Adhesion to the intestinal epithelium is considered an important feature of probiotic bacteria, which may increase their persistence in the intestine, allowing them to exert their beneficial health effect or promote the colonisation process. However, this feature might be largely dependent on the host specificity or diet. In the present study, we investigated the effect of selected milks and milk protein fractions on the ability of selected lactobacilli to adhere to the cells of an intestinal model based on co-culture Caco-2/HT29-MTX cell lines. Most milk digesta did not significantly affect bacterial adhesion except for UHT-treated milk and sheep milk. The presence of UHT-treated milk digesta reduced the adhesion of Lactobacillus gasseri R by 61% but not that of Lactobacillus casei FMP. However, sheep milk significantly increased the adherence of L. casei FMP (P < 0.05) but not of L. gasseri R. Among the protein fractions, rennet casein (RCN) and bovine serum albumin (BSA) showed reproducible patterns and strain-specific effects on bacterial adherence. While RCN reduced the adherence of L. gasseri R to <50% compared to the control, it did not have a significant effect on L. casei FMP. In contrast, BSA reduced L. casei FMP adherence to a higher extent than that of L. gasseri R. Whey protein (WH) tended to increase the adherence of both strains by 130%-180%. Recently, interactions between the host diet and its microbiota have attracted considerable interest. Our results may explain one of the aspects of the role of milk in the development of microbiota or support of probiotic supplements. Based on our data, we conclude that the persistence of probiotic strains supplemented as part of dairy food or constitutional microbiota in the gut might be affected negatively or positively by the food matrix through complex strain or concentration dependent effects. PMID:27435508

  3. 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

  4. 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

  5. Ultrastructure of the endolymphatic sac in the larva of the japanese red-bellied newt Cynops pyrrhogaster

    NASA Technical Reports Server (NTRS)

    Gao, W.; Wiederhold, M.; Hejl, R.

    1998-01-01

    The ultrastructure of the endolymphatic sac (ES) of the late stage larva of the Japanese red-bellied newt, Cynops pyrrhogaster (stage 57), was examined by light and transmission electron microscopy. The two endolymphatic sacs are located at the dorsal-medial side of the otic vesicle on the dorsal-lateral side of the midbrain in the cranial cavity. The wall of the sac is composed of a layer of cubical epithelial cells with loose, interposed intercellular spaces. The sac contains a large luminal cavity, in which endolymph and numerous otoconia are present. The epithelial cells of different portions of the sac have a similar structure. These cells contain an abundance of cytoplasmic organelles, including ribosomes, Golgi complexes, and numerous vesicles. Two types of vesicles are found in the epithelial cells: the "floccular" vesicle and the "granular" vesicle. The floccular vesicles are located in the supra- and lateral-nuclear cytoplasm and contain floccular material. The granular vesicles have a fine granular substance and are usually situated apposed to the apical cell membrane. The granular vesicles are suggested to be secreted into the lumen, while the floccular vesicles are thought to be absorbed from the lumen and conveyed to the intercellular spaces by the epithelial cells. The apical surfaces of the epithelial cells bear numerous microvilli. Apparently floating cells, which bear long microvilli on the free surfaces, are observed in the lumen of the ES. Based on the fine structure, the function of the endolymphatic sac of the newt Cynops pyrrhogaster is discussed.

  6. Surveillance Analysis Computer System (SACS) software requirements specification (SRS)

    SciTech Connect

    Glasscock, J.A.; Flanagan, M.J.

    1995-09-01

    This document is the primary document establishing requirements for the Surveillance Analysis Computer System (SACS) Database, an Impact Level 3Q system. The purpose is to provide the customer and the performing organization with the requirements for the SACS Project.

  7. 60. SAC emblem on side of missile, front lawn, building ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    60. SAC emblem on side of missile, front lawn, building 500, looking west - Offutt Air Force Base, Strategic Air Command Headquarters & Command Center, Headquarters Building, 901 SAC Boulevard, Bellevue, Sarpy County, NE

  8. 13. SAC command center, weather center, underground structure, building 501, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    13. SAC command center, weather center, underground structure, building 501, undated - Offutt Air Force Base, Strategic Air Command Headquarters & Command Center, Command Center, 901 SAC Boulevard, Bellevue, Sarpy County, NE

  9. 63. Aerial view of SAC command post construction, looking west ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    63. Aerial view of SAC command post construction, looking west - Offutt Air Force Base, Strategic Air Command Headquarters & Command Center, Headquarters Building, 901 SAC Boulevard, Bellevue, Sarpy County, NE

  10. 67. Aerial view of SAC command post, building 500, looking ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    67. Aerial view of SAC command post, building 500, looking northeast, undated - Offutt Air Force Base, Strategic Air Command Headquarters & Command Center, Headquarters Building, 901 SAC Boulevard, Bellevue, Sarpy County, NE

  11. 62. Aerial view of SAC command post, building 500, looking ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    62. Aerial view of SAC command post, building 500, looking east - Offutt Air Force Base, Strategic Air Command Headquarters & Command Center, Headquarters Building, 901 SAC Boulevard, Bellevue, Sarpy County, NE

  12. 3. Threequarter view of building 500 looking southeast from SAC ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    3. Three-quarter view of building 500 looking southeast from SAC Boulevard - Offutt Air Force Base, Strategic Air Command Headquarters & Command Center, Headquarters Building, 901 SAC Boulevard, Bellevue, Sarpy County, NE

  13. 68. Aerial view of SAC command post, building 500, looking ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    68. Aerial view of SAC command post, building 500, looking northeast, spring, 1957 - Offutt Air Force Base, Strategic Air Command Headquarters & Command Center, Headquarters Building, 901 SAC Boulevard, Bellevue, Sarpy County, NE

  14. 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

  15. 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

  16. 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...

  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. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. Quantitative assessment of gestational sac shape: the gestational sac shape score

    PubMed Central

    Deter, R.L.; Li, J.; Lee, W.; Liu, S.; Romero, R.

    2012-01-01

    Objective To develop a quantitative method for characterizing gestational sac shape. Methods Twenty first-trimester gestational sacs in normal pregnancies were studied with three-dimensional (3D) ultrasonography. The 3D coordinates of surface-point sets were obtained for each sac using 30-, 15- and six-slice sampling. Cubic spline interpolation was used with the 15- and six-slice surface-point samples to generate coordinates for those 30-slice surface points not measured. Interpolated and measured values, the latter from the 30-slice sample, were compared and the percent error calculated. Cubic spline interpolation was used to determine the coordinates of a standard surface-point sample (3660) for each sac in each slice sample. These coordinate data were used to give each sac a standard configuration by moving its center of gravity to the origin, aligning its inertial axes along the coordinate axes and converting its volume to 1.0 mL. In this form, a volume shape descriptor could be generated for each sac that was then transformed into a vector containing only shape information. The 20 shape vectors of each slice sample were subjected to principal components analysis, and principal component scores (PCSs) calculated. The first four PCSs were used to define a gestational sac shape score (GSSS-30, GSSS-15 or GSSS-6) for each sac in a given slice sample. The characteristics of each set of GSSSs were determined and those for the GSSS-15 and GSSS-6 were compared with the GSSS-30 characteristics. Results Cubic spline interpolations were very accurate in most cases, with means close to 0%, and approximately 95% of the errors being less than 10%. GSSS-30 accounted for 67.6% of the shape variance, had a mean of zero and an SD of 1.1, was normally distributed and was not related to menstrual age (R = −0.16, P = 0.51). GSSS-15 and GSSS-6 had essentially the same characteristics. No significant differences between individual GSSS-30 values and those for GSSS-15 or GSSS-6

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors.

    PubMed

    Gomez Perdiguero, Elisa; Klapproth, Kay; Schulz, Christian; Busch, Katrin; Azzoni, Emanuele; Crozet, Lucile; Garner, Hannah; Trouillet, Celine; de Bruijn, Marella F; Geissmann, Frederic; Rodewald, Hans-Reimer

    2015-02-26

    Most haematopoietic cells renew from adult haematopoietic stem cells (HSCs), however, macrophages in adult tissues can self-maintain independently of HSCs. Progenitors with macrophage potential in vitro have been described in the yolk sac before emergence of HSCs, and fetal macrophages can develop independently of Myb, a transcription factor required for HSC, and can persist in adult tissues. Nevertheless, the origin of adult macrophages and the qualitative and quantitative contributions of HSC and putative non-HSC-derived progenitors are still unclear. Here we show in mice that the vast majority of adult tissue-resident macrophages in liver (Kupffer cells), brain (microglia), epidermis (Langerhans cells) and lung (alveolar macrophages) originate from a Tie2(+) (also known as Tek) cellular pathway generating Csf1r(+) erythro-myeloid progenitors (EMPs) distinct from HSCs. EMPs develop in the yolk sac at embryonic day (E) 8.5, migrate and colonize the nascent fetal liver before E10.5, and give rise to fetal erythrocytes, macrophages, granulocytes and monocytes until at least E16.5. Subsequently, HSC-derived cells replace erythrocytes, granulocytes and monocytes. Kupffer cells, microglia and Langerhans cells are only marginally replaced in one-year-old mice, whereas alveolar macrophages may be progressively replaced in ageing mice. Our fate-mapping experiments identify, in the fetal liver, a sequence of yolk sac EMP-derived and HSC-derived haematopoiesis, and identify yolk sac EMPs as a common origin for tissue macrophages. PMID:25470051

  13. 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

  14. What is inside the hernia sac?

    PubMed Central

    Virgínia, Ana Araújo; Santos, Cláudia; Contente, Helena; Branco, Cláudia

    2016-01-01

    Most ovarian inguinal hernias occur in children and are frequently associated with congenital genitalia defects. The authors present the case of a multiparous 89-year-old woman, without any genitalia defect, who was brought to the emergency department with an irreducible inguinal hernia. The patient was proposed for emergency surgery during which we encountered an ovary and a fallopian tube inside the hernial sac. An oophorosalpingectomy and a Lichtenstein procedure were carried out and the postoperative period was uneventful. This case shows that, even though it is rare, a hernial sac may contain almost any intra-abdominal organ, including those least frequent such as the appendix, an ovary or the fallopian tubes. PMID:27511751

  15. 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...

  16. 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) ...

  17. Aquarius/SAC-D Mission Overview

    NASA Technical Reports Server (NTRS)

    Sen, Amit; Kim, Yunjin; Caruso, Daniel; Lagerloef, Gary; Colomb, Raul; Yueh, Simon; LeVine, David

    2006-01-01

    Aquarius/SAC-D is a cooperative international mission developed between the National Aeronautics and Space Administration (NASA) of United States of America (USA) and the Comision Nacional de Actividades Espaciales (CONAE) of Argentina. The overall mission objective is to contribute to the understanding of the total Earth system and the consequences of the natural and man-made changes in the environment of the planet. Major themes are: ocean surface salinity, water cycle, climate, natural hazards and cryosphere.

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. Developmental expression of otoconin-22 in the bullfrog endolymphatic sac and inner ear.

    PubMed

    Yaoi, Yuichi; Onda, Tomoaki; Hidaka, Yoshie; Yajima, Shinya; Suzuki, Masakazu; Tanaka, Shigeyasu

    2004-05-01

    In amphibians, calcium carbonate crystals are present in the endolymphatic sac and the inner ear. The formation of these crystals is considered to be facilitated by a protein called otoconin-22. We examined the spatial and temporal expression of otoconin-22 during the development of the bullfrog (Rana catesbeiana) using RT-PCR, in situ hybridization (ISH), and immunofluorescence techniques. By RT-PCR, otoconin-22 mRNA was first detected in embryos at Shumway stage 20, and this expression pattern continues in late stages. The first otoconin-22 mRNA-positive reaction was detected in stage 22 embryos in the placode of the endolymphatic sac. Otoconin-22 protein was observed in the epithelial cells of the endolymphatic sac at stage 24. On the other hand, a whole-mount ISH technique showed the first expression of otoconin-22 mRNA in the inner ear, in addition to the endolymphatic sac, at the mid-phase of Shumway stage 25. We discuss the role of otoconin-22 in the formation of calcium carbonate crystals in the endolymphatic sac and inner ear. PMID:15100243

  4. 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

  5. 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

  6. Pollen tubes introduce Raspberry bushy dwarf virus into embryo sacs during fertilization processes.

    PubMed

    Isogai, Masamichi; Yoshida, Tetu; Shimura, Takuya; Yoshikawa, Nobuyuki

    2015-10-01

    We developed a fertilization method in which pollen tubes entered into embryo sacs without any need to contact surrounding female sporophytic cells by using Torenia fournieri (Torenia) plants under the condition of hindering movement of the virus from a stigma, which is the first infection site leading to systemic infection. When RBDV-infected Torenia pollen grains were used for the developed fertilization method, the virus was transmitted to the seeds by pollen tubes germinating from them. On the other hand, no seeds were infected with the virus when Torenia plants were pollinated with healthy Torenia pollen grains in combination with RBDV-infected raspberry pollen grains, which caused the virus infection in the stigma by penetration of their pollen tubes arrested in its style. Our results indicate that vertical transmission of RBDV by pollen occurs in the transport of the virus into embryo sacs by pollen tubes reaching the embryo sacs. PMID:26176979

  7. New development of the yolk sac theory in diabetic embryopathy: molecular mechanism and link to structural birth defects.

    PubMed

    Dong, Daoyin; Reece, E Albert; Lin, Xue; Wu, Yanqing; AriasVillela, Natalia; Yang, Peixin

    2016-02-01

    Maternal diabetes mellitus is a significant risk factor for structural birth defects, including congenital heart defects and neural tube defects. With the rising prevalence of type 2 diabetes mellitus and obesity in women of childbearing age, diabetes mellitus-induced birth defects have become an increasingly significant public health problem. Maternal diabetes mellitus in vivo and high glucose in vitro induce yolk sac injuries by damaging the morphologic condition of cells and altering the dynamics of organelles. The yolk sac vascular system is the first system to develop during embryogenesis; therefore, it is the most sensitive to hyperglycemia. The consequences of yolk sac injuries include impairment of nutrient transportation because of vasculopathy. Although the functional relationship between yolk sac vasculopathy and structural birth defects has not yet been established, a recent study reveals that the quality of yolk sac vasculature is related inversely to embryonic malformation rates. Studies in animal models have uncovered key molecular intermediates of diabetic yolk sac vasculopathy, which include hypoxia-inducible factor-1α, apoptosis signal-regulating kinase 1, and its inhibitor thioredoxin-1, c-Jun-N-terminal kinases, nitric oxide, and nitric oxide synthase. Yolk sac vasculopathy is also associated with abnormalities in arachidonic acid and myo-inositol. Dietary supplementation with fatty acids that restore lipid levels in the yolk sac lead to a reduction in diabetes mellitus-induced malformations. Although the role of the human yolk in embryogenesis is less extensive than in rodents, nevertheless, human embryonic vasculogenesis is affected negatively by maternal diabetes mellitus. Mechanistic studies have identified potential therapeutic targets for future intervention against yolk sac vasculopathy, birth defects, and other complications associated with diabetic pregnancies. PMID:26432466

  8. 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

  9. Evaluation of 309 environmental chemicals using a mouse embryonic stem cell adherent cell differentiation and cytotoxicity assay.

    PubMed

    Chandler, Kelly J; Barrier, Marianne; Jeffay, Susan; Nichols, Harriette P; Kleinstreuer, Nicole C; Singh, Amar V; Reif, David M; Sipes, Nisha S; Judson, Richard S; Dix, David J; Kavlock, Robert; Hunter, Edward S; Knudsen, Thomas B

    2011-01-01

    The vast landscape of environmental chemicals has motivated the need for alternative methods to traditional whole-animal bioassays in toxicity testing. Embryonic stem (ES) cells provide an in vitro model of embryonic development and an alternative method for assessing developmental toxicity. Here, we evaluated 309 environmental chemicals, mostly food-use pesticides, from the ToxCast™ chemical library using a mouse ES cell platform. ES cells were cultured in the absence of pluripotency factors to promote spontaneous differentiation and in the presence of DMSO-solubilized chemicals at different concentrations to test the effects of exposure on differentiation and cytotoxicity. Cardiomyocyte differentiation (α,β myosin heavy chain; MYH6/MYH7) and cytotoxicity (DRAQ5™/Sapphire700™) were measured by In-Cell Western™ analysis. Half-maximal activity concentration (AC₅₀) values for differentiation and cytotoxicity endpoints were determined, with 18% of the chemical library showing significant activity on either endpoint. Mining these effects against the ToxCast Phase I assays (∼500) revealed significant associations for a subset of chemicals (26) that perturbed transcription-based activities and impaired ES cell differentiation. Increased transcriptional activity of several critical developmental genes including BMPR2, PAX6 and OCT1 were strongly associated with decreased ES cell differentiation. Multiple genes involved in reactive oxygen species signaling pathways (NRF2, ABCG2, GSTA2, HIF1A) were strongly associated with decreased ES cell differentiation as well. A multivariate model built from these data revealed alterations in ABCG2 transporter was a strong predictor of impaired ES cell differentiation. Taken together, these results provide an initial characterization of metabolic and regulatory pathways by which some environmental chemicals may act to disrupt ES cell growth and differentiation. PMID:21666745

  10. Evaluation of 309 Environmental Chemicals Using a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity Assay

    PubMed Central

    Chandler, Kelly J.; Barrier, Marianne; Jeffay, Susan; Nichols, Harriette P.; Kleinstreuer, Nicole C.; Singh, Amar V.; Reif, David M.; Sipes, Nisha S.; Judson, Richard S.; Dix, David J.; Kavlock, Robert; Hunter, Edward S.; Knudsen, Thomas B.

    2011-01-01

    The vast landscape of environmental chemicals has motivated the need for alternative methods to traditional whole-animal bioassays in toxicity testing. Embryonic stem (ES) cells provide an in vitro model of embryonic development and an alternative method for assessing developmental toxicity. Here, we evaluated 309 environmental chemicals, mostly food-use pesticides, from the ToxCast™ chemical library using a mouse ES cell platform. ES cells were cultured in the absence of pluripotency factors to promote spontaneous differentiation and in the presence of DMSO-solubilized chemicals at different concentrations to test the effects of exposure on differentiation and cytotoxicity. Cardiomyocyte differentiation (α,β myosin heavy chain; MYH6/MYH7) and cytotoxicity (DRAQ5™/Sapphire700™) were measured by In-Cell Western™ analysis. Half-maximal activity concentration (AC50) values for differentiation and cytotoxicity endpoints were determined, with 18% of the chemical library showing significant activity on either endpoint. Mining these effects against the ToxCast Phase I assays (∼500) revealed significant associations for a subset of chemicals (26) that perturbed transcription-based activities and impaired ES cell differentiation. Increased transcriptional activity of several critical developmental genes including BMPR2, PAX6 and OCT1 were strongly associated with decreased ES cell differentiation. Multiple genes involved in reactive oxygen species signaling pathways (NRF2, ABCG2, GSTA2, HIF1A) were strongly associated with decreased ES cell differentiation as well. A multivariate model built from these data revealed alterations in ABCG2 transporter was a strong predictor of impaired ES cell differentiation. Taken together, these results provide an initial characterization of metabolic and regulatory pathways by which some environmental chemicals may act to disrupt ES cell growth and differentiation. PMID:21666745

  11. Adherence of human mesenchymal stem cells on Ti and TiO2 nano-columnar surfaces fabricated by glancing angle sputter deposition

    NASA Astrophysics Data System (ADS)

    Motemani, Yahya; Greulich, Christina; Khare, Chinmay; Lopian, Michael; Buenconsejo, Pio John S.; Schildhauer, Thomas A.; Ludwig, Alfred; Köller, Manfred

    2014-02-01

    The interaction of human mesenchymal stem cells (hMSCs) with Ti and TiO2 nano-columnar surfaces fabricated using glancing angle sputter deposition was investigated. The adherence and proliferation of hMSCs on different nano-columnar surfaces, including vertical columns, slanted columns and chevrons, were examined with calcein-acetoxymethyl ester fluorescence staining and scanning electron microscopy. For comparison, adherence of hMSCs on compact, dense films was also studied. After 24 h and 7 days, adherent and viable cells were observed on both, Ti nano-columns as well as dense Ti films, which confirms the biocompatibility of these nanostructures. Very small pseudopodia with width of approximately 20-35 nm and length varying from 20 to 200 nm were observed between the nano-columns, independent of the type of the nano-columnar morphology. Large inter-column spacing and effectively increased surface area make these nanostructures promising candidates for bio-functionalization or drug loading on the surface of Ti-based implants.

  12. Increased protein secretion and adherence to HeLa cells by Shigella spp. following growth in the presence of bile salts.

    PubMed Central

    Pope, L M; Reed, K E; Payne, S M

    1995-01-01

    Growth of Shigella spp. in the presence of the bile salt deoxycholate or chenodeoxycholate enhanced the bacterial invasion of HeLa cells. Growth in the presence of other structurally similar bile salts or detergents had little or no effect. Deoxycholate-enhanced invasion was not observed when bacteria were exposed to deoxycholate at low temperatures or when chloramphenicol was added to the growth medium, indicating that bacterial growth and protein synthesis are required. Increased invasion is associated with the presence of an intact Shigella virulence plasmid and is correlated with increased secretion of a set of proteins, including the Ipa proteins, to the outer membrane and into the growth medium. The increased invasion induced by the bile salts appears to be due to increased adherence. The enhanced adherence was specific to Shigella spp., since the enteroinvasive Escherichia coli strains tested did not exhibit the effect in response to growth in bile salts. PMID:7642302

  13. Proper Regulation of Cdc42 Activity is Required for Tight Actin Concentration at the Equator during Cytokinesis in Adherent Mammalian Cells

    PubMed Central

    Zhu, Xiaodong; Wang, Junxia; Moriguchi, Kazuki; Liow, Lu Ting; Ahmed, Sohail; Kaverina, Irina; Murata-Hori, Maki

    2012-01-01

    Cytokinesis in mammalian cells requires actin assembly at the equatorial region. Although functions of RhoA in this process have been well established, additional mechanisms are likely involved. We have examined if Cdc42 is involved in actin assembly during cytokinesis. Depletion of Cdc42 had no apparent effects on the duration of cytokinesis, while overexpression of constitutively active Cdc42 (CACdc42) caused cytokinesis failure in normal rat kidney epithelial cells. Cells depleted of Cdc42 displayed abnormal cell morphology and caused a failure of tight accumulation of actin and RhoA at the equator. In contrast, in cells overexpressing CACdc42, actin formed abnormal bundles and RhoA was largely eliminated from the equator. Our results suggest that accurate regulation of Cdc42 activity is crucial for proper equatorial actin assembly and RhoA localization during cytokinesis. Notably, our observations also suggest that tight actin concentration is not essential for cytokinesis in adherent mammalian cells. PMID:21763307

  14. Ovarian Yolk Sac Tumor With High-Grade Serous Carcinoma in a 62-Year-Old Woman.

    PubMed

    McCarthy, Whitney A; Masand, Ramya P

    2016-06-01

    Ovarian yolk sac tumors are germ cell tumors that usually present in children and young women. Rarely, these tumors can arise in older women, usually in conjunction with surface epithelial tumors, suggesting divergent differentiation from the latter. The combination of mixed ovarian yolk sac tumor and high-grade serous carcinoma is rare, with only one case documented in the literature. We present a case of mixed ovarian yolk sac tumor and high-grade serous carcinoma in a postmenopausal woman, including a brief discussion of the immunohistochemical findings and differential diagnosis. Despite the rarity of mixed ovarian yolk sac tumor and surface epithelial tumors, it is important to recognize the biphasic nature of the tumor, which should prompt a thorough immunohistochemical evaluation. The therapeutic and prognostic implications of proper diagnosis cannot be overemphasized. PMID:26782153

  15. Evaluation of 309 environmental chemicals using a mouse embryonic stem cell adherent cell differentiation and cytotoxicity assay

    EPA Science Inventory

    The vast landscape of environmental chemicals has motivated the need for alternative methods to traditional whole-animal bioassays in toxicity testing. Embryonic stem (ES) cells provide an in vitro model of embryonic development and an alternative method for assessing development...

  16. Progressive myoclonus epilepsy associated with SACS gene mutations.

    PubMed

    Nascimento, Fábio A; Canafoglia, Laura; Aljaafari, Danah; Muona, Mikko; Lehesjoki, Anna-Elina; Berkovic, Samuel F; Franceschetti, Silvana; Andrade, Danielle M

    2016-08-01

    Pathogenic variants in the SACS gene (OMIM #604490) cause autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS). ARSACS is a neurodegenerative early-onset progressive disorder, originally described in French Canadians, but later observed elsewhere.(1) Whole-exome sequencing of a large group of patients with unclassified progressive myoclonus epilepsies (PMEs) identified 2 patients bearing SACS gene mutations.(2) We detail the PME clinical features associated with SACS mutations and suggest the inclusion of the SACS gene in diagnostic screening of PMEs. PMID:27433545

  17. Whisker Formation on SAC305 Soldered Assemblies

    NASA Astrophysics Data System (ADS)

    Meschter, S.; Snugovsky, P.; Bagheri, Z.; Kosiba, E.; Romansky, M.; Kennedy, J.; Snugovsky, L.; Perovic, D.

    2014-11-01

    This article describes the results of a whisker formation study on SAC305 assemblies, evaluating the effects of lead-frame materials and cleanliness in different environments: low-stress simulated power cycling (50-85°C thermal cycling), thermal shock (-55°C to 85°C), and high temperature/high humidity (85°C/85% RH). Cleaned and contaminated small outline transistors, large leaded quad flat packs (QFP), plastic leaded chip carrier packages, and solder balls with and without rare earth elements (REE) were soldered to custom designed test boards with Sn3Ag0.5Cu (SAC305) solder. After assembly, all the boards were cleaned, and half of them were recontaminated (1.56 µg/cm2 Cl-). Whisker length, diameter, and density were measured. Detailed metallurgical analysis on components before assembly and on solder joints before and after testing was performed. It was found that whiskers grow from solder joint fillets, where the thickness is less than 25 µm, unless REE was present. The influence of lead-frame and solder ball material, microstructure, cleanliness, and environment on whisker characteristics is discussed. This article provides detailed metallurgical observations and select whisker length data obtained during this multiyear testing program.

  18. Combined strategies for optimal detection of the contact point in AFM force-indentation curves obtained on thin samples and adherent cells.

    PubMed

    Gavara, Núria

    2016-01-01

    Atomic Force Microscopy (AFM) is a widely used tool to study cell mechanics. Current AFM setups perform high-throughput probing of living cells, generating large amounts of force-indentations curves that are subsequently analysed using a contact-mechanics model. Here we present several algorithms to detect the contact point in force-indentation curves, a crucial step to achieve fully-automated analysis of AFM-generated data. We quantify and rank the performance of our algorithms by analysing a thousand force-indentation curves obtained on thin soft homogeneous hydrogels, which mimic the stiffness and topographical profile of adherent cells. We take advantage of the fact that all the proposed algorithms are based on sequential search strategies, and show that a combination of them yields the most accurate and unbiased results. Finally, we also observe improved performance when force-indentation curves obtained on adherent cells are analysed using our combined strategy, as compared to the classical algorithm used in the majority of previous cell mechanics studies. PMID:26891762

  19. Combined strategies for optimal detection of the contact point in AFM force-indentation curves obtained on thin samples and adherent cells

    PubMed Central

    Gavara, Núria

    2016-01-01

    Atomic Force Microscopy (AFM) is a widely used tool to study cell mechanics. Current AFM setups perform high-throughput probing of living cells, generating large amounts of force-indentations curves that are subsequently analysed using a contact-mechanics model. Here we present several algorithms to detect the contact point in force-indentation curves, a crucial step to achieve fully-automated analysis of AFM-generated data. We quantify and rank the performance of our algorithms by analysing a thousand force-indentation curves obtained on thin soft homogeneous hydrogels, which mimic the stiffness and topographical profile of adherent cells. We take advantage of the fact that all the proposed algorithms are based on sequential search strategies, and show that a combination of them yields the most accurate and unbiased results. Finally, we also observe improved performance when force-indentation curves obtained on adherent cells are analysed using our combined strategy, as compared to the classical algorithm used in the majority of previous cell mechanics studies. PMID:26891762

  20. 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

  1. Urokinase receptor-dependent and -independent p56/59(hck) activation state is a molecular switch between myelomonocytic cell motility and adherence.

    PubMed Central

    Chiaradonna, F; Fontana, L; Iavarone, C; Carriero, M V; Scholz, G; Barone, M V; Stoppelli, M P

    1999-01-01

    Anchorage-independent myelomonocytic cells acquire adherence within minutes of differentiation stimuli, such as the proteolytically inactive N-terminal fragment of urokinase binding to its cognate glycosylphosphatidylinositol (GPI)-anchored receptor. Here, we report that urokinase-treated differentiating U937 monocyte-like cells exhibit a rapid and transient inhibition of p56/59(hck) and p55(fgr) whereas no changes in the activity of other Src family kinases, such as p53/56(lyn) and p59(fyn) were observed. U937 transfectants expressing a kinase-defective (Lys267 to Met) p56/59(hck) variant exhibit enhanced adhesiveness and a marked F-actin redistribution in thin protruding structures. Conversely, urokinase as well as expression of wild-type or constitutively active (Tyr499 to Phe) p56/59(hck) stimulates the directional migration of uninduced U937 cells. Accordingly, expression of constitutively active or kinase inactive p56/59(hck) selectively prevents urokinase receptor-dependent induction of either adhesion or motility, indicating that a specific activation state of p56/59(hck) is required for each cell response. In conclusion, modulation of the intracellular p56/59(hck) tyrosine kinase activity switches cell motility towards adherence, providing a mutually exclusive mechanism to regulate these properties during monocyte/macrophage differentiation in vivo. PMID:10357814

  2. Combined strategies for optimal detection of the contact point in AFM force-indentation curves obtained on thin samples and adherent cells

    NASA Astrophysics Data System (ADS)

    Gavara, Núria

    2016-02-01

    Atomic Force Microscopy (AFM) is a widely used tool to study cell mechanics. Current AFM setups perform high-throughput probing of living cells, generating large amounts of force-indentations curves that are subsequently analysed using a contact-mechanics model. Here we present several algorithms to detect the contact point in force-indentation curves, a crucial step to achieve fully-automated analysis of AFM-generated data. We quantify and rank the performance of our algorithms by analysing a thousand force-indentation curves obtained on thin soft homogeneous hydrogels, which mimic the stiffness and topographical profile of adherent cells. We take advantage of the fact that all the proposed algorithms are based on sequential search strategies, and show that a combination of them yields the most accurate and unbiased results. Finally, we also observe improved performance when force-indentation curves obtained on adherent cells are analysed using our combined strategy, as compared to the classical algorithm used in the majority of previous cell mechanics studies.

  3. Carbon source-induced reprogramming of the cell wall proteome and secretome modulates the adherence and drug resistance of the fungal pathogen Candida albicans

    PubMed Central

    Ene, Iuliana V; Heilmann, Clemens J; Sorgo, Alice G; Walker, Louise A; de Koster, Chris G; Munro, Carol A; Klis, Frans M; Brown, Alistair J P

    2012-01-01

    The major fungal pathogen Candida albicans can occupy diverse microenvironments in its human host. During colonization of the gastrointestinal or urogenital tracts, mucosal surfaces, bloodstream, and internal organs, C. albicans thrives in niches that differ with respect to available nutrients and local environmental stresses. Although most studies are performed on glucose-grown cells, changes in carbon source dramatically affect cell wall architecture, stress responses, and drug resistance. We show that growth on the physiologically relevant carboxylic acid, lactate, has a significant impact on the C. albicans cell wall proteome and secretome. The regulation of cell wall structural proteins (e.g. Cht1, Phr1, Phr2, Pir1) correlated with extensive cell wall remodeling in lactate-grown cells and with their increased resistance to stresses and antifungal drugs, compared with glucose-grown cells. Moreover, changes in other proteins (e.g. Als2, Gca1, Phr1, Sap9) correlated with the increased adherence and biofilm formation of lactate-grown cells. We identified mating and pheromone-regulated proteins that were exclusive to lactate-grown cells (e.g. Op4, Pga31, Pry1, Scw4, Yps7) as well as mucosa-specific and other niche-specific factors such as Lip4, Pga4, Plb5, and Sap7. The analysis of the corresponding null mutants confirmed that many of these proteins contribute to C. albicans adherence, stress, and antifungal drug resistance. Therefore, the cell wall proteome and secretome display considerable plasticity in response to carbon source. This plasticity influences important fitness and virulence attributes known to modulate the behavior of C. albicans in different host microenvironments during infection. PMID:22997008

  4. Large-scale enrichment of mobilized CD34+ peripheral blood hematopoietic progenitors by removal of nylon wool-adherent mature cells.

    PubMed

    Di Nicola, M; Siena, S; Bregni, M; Ravagnani, F; Vitello, F; Belli, N; Dodero, A; Magni, M; Bonadonna, G; Gianni, A M

    1994-12-01

    With the aim of facilitating the ex vivo manipulation of peripheral blood hematopoietic progenitors (CPCs = circulating progenitor cells) collected by leukapheresis, we removed polymorphonuclear cells and monocytes that naturally adhere to nylon wool fibers. Leukapheresed cells harvested at the time of hematopoietic recovery after cancer therapy with high-dose cyclophosphamide plus hematopoietic growth factors were incubated with nylon wool fibers for 1 h at 37 degrees C. Evaluation of the cells non-adherent to the nylon wool in all experiments (n = 14) showed that the median recovery of nucleated cells and CPCs detected as CD34+ cells, CFU-GM and BFU-E was 16.4% (range 4.8%-34.0%), 60.0% (range 30.8-80.8%), 60.9% (range 33.4-74.5%) and 65.5% (range 30.8-69.2%), respectively. Therefore exposure to the nylon wool determined a selective removal of mature cells and a complementary enrichment of CPCs. The wide range of results depended on the significantly different cell compositions of the unmanipulated leukaphereses. The latter from patients receiving rhG-CSF (n = 10) comprised a median of 88.5% (range 77.8-93.8%) and 11.5% (range 6.2-22.2%) polymorphonuclear and mononuclear cells, respectively. In contrast, leukaphereses from patients receiving rhGM-CSF or PIXY321 (n = 4) comprised a median of 71.1% (range 55.4-85.0%) and 28.9% (range 15.0-44.6%) polymorphonuclear and mononuclear cells, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7536068

  5. Production of high-titer human influenza A virus with adherent and suspension MDCK cells cultured in a single-use hollow fiber bioreactor.

    PubMed

    Tapia, Felipe; Vogel, Thomas; Genzel, Yvonne; Behrendt, Ilona; Hirschel, Mark; Gangemi, J David; Reichl, Udo

    2014-02-12

    Hollow fiber bioreactors (HFBRs) have been widely described as capable of supporting the production of highly concentrated monoclonal antibodies and recombinant proteins. Only recently HFBRs have been proposed as new single-use platforms for production of high-titer influenza A virus. These bioreactors contain multiple hollow fiber capillary tubes that separate the bioreactor in an intra- and an extra-capillary space. Cells are usually cultured in the extra-capillary space and can grow to a very high cell concentration. This work describes the evaluation of the single-use hollow fiber bioreactor PRIMER HF (Biovest International Inc., USA) for production of influenza A virus. The process was setup, characterized and optimized by running a total of 15 cultivations. The HFBRs were seeded with either adherent or suspension MDCK cells, and infected with influenza virus A/PR/8/34 (H1N1), and the pandemic strain A/Mexico/4108/2009 (H1N1). High HA titers and TCID₅₀ of up to 3.87 log₁₀(HA units/100 μL) and 1.8 × 10(10)virions/mL, respectively, were obtained for A/PR/8/34 influenza strain. Influenza virus was collected by performing multiple harvests of the extra-capillary space during a virus production time of up to 12 days. Cell-specific virus yields between 2,000 and 8,000 virions/cell were estimated for adherent MDCK cells, and between 11,000 and 19,000 virions/cell for suspension MDCK.SUS2 cells. These results do not only coincide with the cell-specific virus yields obtained with cultivations in stirred tank bioreactors and other high cell density systems, but also demonstrate that HFBRs are promising and competitive single-use platforms that can be considered for commercial production of influenza virus. PMID:24269322

  6. Hydroxy decenoic acid down regulates gtfB and gtfC expression and prevents Streptococcus mutans adherence to the cell surfaces

    PubMed Central

    2012-01-01

    Background 10-Hydroxy-2-decenoic acid, an unsaturated fatty acid is the most active and unique component to the royal jelly that has antimicrobial properties. Streptococcus mutans is associated with pathogenesis of oral cavity, gingivoperiodontal diseases and bacteremia following dental manipulations. In the oral cavity, S. mutans colonize the soft tissues including tongue, palate, and buccal mucosa. When considering the role of supragingival dental plaque in caries, the proportion of acid producing bacteria (particularly S. mutans), has direct relevance to the pathogenicity of the plaque. The genes that encode glucosyltransferases (gtfs) especially gtfB and gtfC are important in S. mutans colonization and pathogenesis. This study investigated the hydroxy-decenoic acid (HDA) effects on gtfB and gtfC expression and S. mutans adherence to cells surfaces. Methods Streptococcus mutans was treated by different concentrations of HPLC purified HDA supplied by Iran Beekeeping and Veterinary Association. Real time RT-PCR and western blot assays were conducted to evaluate gtfB and gtfC genes transcription and translation before and after HDA treatment. The bacterial attachment to the cell surfaces was evaluated microscopically. Results 500 μg ml-1 of HDA inhibited gtfB and gtfC mRNA transcription and its expression. The same concentration of HDA decreased 60% the adherence of S. mutans to the surface of P19 cells. Conclusion Hydroxy-decenoic acid prevents gtfB and gtfC expression efficiently in the bactericide sub-concentrations and it could effectively reduce S. mutans adherence to the cell surfaces. In the future, therapeutic approaches to affecting S. mutans could be selective and it’s not necessary to put down the oral flora completely. PMID:22839724

  7. Gonadoblastoma and hepatoid and endometrioid-like yolk sac tumor: an update.

    PubMed

    Ulbright, Thomas M

    2014-07-01

    Dr Robert E. Scully greatly advanced our understanding of germ cell neoplasia to the extent that it is difficult to narrow the discussion of his contributions to this topic so that it can be covered in a brief article. This article accordingly focuses on some of the recent developments concerning 2 of his major contributions in this area-the gonadoblastoma (GB) and variant morphologies of yolk sac tumor. GB was defined by Dr Scully in 1953 and its features elaborated in detail by him in 1970. This neoplasm occurred in young patients who often displayed phenotypic sex ambiguities and frequently presented with primary amenorrhea. It was bilateral in 40%, and consisted of circumscribed nests of small sex cord cells and germinoma-like cells admixed with round deposits of eosinophilic, hyaline, often calcified material. These nests were set in a spindle cell gonadal stroma with Leydig-like or lutein-like cells. Because of his work we now understand that this precursor to invasive germ cell tumors occurs in patients with a specific form of disorder of sex development, namely gonadal dysgenesis, and only in those who have a particular portion of the Y chromosome, the GB locus/TSPY gene, within the gonadal tissue. An essential element to the development of GB appears to be a defect in the genetic pathway that leads to the development of Sertoli cells. Improperly formed Sertoli cells predispose to "delayed maturation" of the gonocytes of the gonad and predispose them to undergo malignant transformation. "Undifferentiated gonadal tissue" has been proposed as the precursor to the development of GB and consists of an unorganized mixture of apparently non-neoplastic germ cells, germ cells with delayed maturation, and neoplastic germ cells with sex cord cells and gonadal stroma. Two variant morphologies of yolk sac tumor were also recognized by Dr Scully. In the hepatoid variant features similar to hepatocellular carcinoma occurred, although primitive glandular foci and lack of

  8. Efficient entry of cell-penetrating peptide nona-arginine into adherent cells involves a transient increase in intracellular calcium

    PubMed Central

    Melikov, Kamran; Hara, Ann; Yamoah, Kwabena; Zaitseva, Elena; Zaitsev, Eugene; Chernomordik, Leonid V.

    2015-01-01

    Understanding the mechanism of entry of cationic peptides such as nona-arginine (R9) into cells remains an important challenge to their use as efficient drug-delivery vehicles. At nanomolar to low micromolar R9 concentrations and at physiological temperature, peptide entry involves endocytosis. In contrast, at a concentration ≥10 μM, R9 induces a very effective non-endocytic entry pathway specific for cationic peptides. We found that a similar entry pathway is induced at 1–2 μM concentrations of R9 if peptide application is accompanied by a rapid temperature drop to 15°C. Both at physiological and at sub-physiological temperatures, this entry mechanism was inhibited by depletion of the intracellular ATP pool. Intriguingly, we found that R9 at 10–20 μM and 37°C induces repetitive spikes in intracellular Ca2+ concentration. This Ca2+ signalling correlated with the efficiency of the peptide entry. Pre-loading cells with the Ca2+ chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) inhibited both Ca2+ spikes and peptide entry, suggesting that an increase in intracellular Ca2+ precedes and is required for peptide entry. One of the hallmarks of Ca2+ signalling is a transient cell-surface exposure of phosphatidylserine (PS), a lipid normally residing only in the inner leaflet of the plasma membrane. Blocking the accessible PS with the PS-binding domain of lactadherin strongly inhibited non-endocytic R9 entry, suggesting the importance of PS externalization in this process. To conclude, we uncovered a novel mechanistic link between calcium signalling and entry of cationic peptides. This finding will enhance our understanding of the properties of plasma membrane and guide development of future drug-delivery vehicles. PMID:26272944

  9. Immunohistochemical expression of SALL4 in hepatocellular carcinoma, a potential pitfall in the differential diagnosis of yolk sac tumors.

    PubMed

    Gonzalez-Roibon, Nilda; Katz, Betina; Chaux, Alcides; Sharma, Rajni; Munari, Enrico; Faraj, Sheila F; Illei, Peter B; Torbenson, Michael; Netto, George J

    2013-07-01

    SALL4 is a transcription factor that serves as a marker of yolk sac tumor. Yolk sac tumor and hepatocellular carcinoma share histologic, serologic, and immunohistochemical features. Previous studies have shown lack of SALL4 expression in hepatocellular carcinoma, suggesting utility in this differential diagnosis. Sixty-nine samples of hepatocellular carcinoma were retrieved from surgical pathology archives and used to construct 9 tissue microarrays. A germ cell tumor tissue microarray containing 10 yolk sac tumors was used for comparison. Extent, intensity, and pattern of nuclear SALL4 expression were assessed in each spot. Mean percentage of expression was calculated for each tumor and used during analysis. Optimal discriminatory extent of expression cutoff was determined by receiver operating characteristic curve analysis. Other potential discriminatory markers including Hep Par1 were also evaluated. Forty-six percent (32/69) of hepatocellular carcinoma and all yolk sac tumors revealed at least focal expression of SALL4. A unique punctuate/clumped pattern of nuclear staining was present in 94% (30/32) of hepatocellular carcinoma, whereas all yolk sac tumors displayed a diffuse finely granular nuclear staining pattern. A 25% extent of SALL4 expression cutoff was found to be optimal for the distinction of yolk sac tumor from hepatocellular carcinoma yielding a sensitivity of 100%, specificity of 92.8%, and a positive predictive value of 66.6% for yolk sac tumor diagnosis. The addition of Hep Par1 increased the specificity (99%) and positive predictive value (90%). This is the first report of SALL4 expression in hepatocellular carcinoma. Our finding should be taken into consideration in the differential diagnosis of hepatocellular carcinoma and yolk sac tumor. The unique punctuate/clumped pattern seen in hepatocellular carcinoma cases could be of further discriminatory value. PMID:23347651

  10. Investigation on cytoskeleton dynamics for no-adherent cells subjected to point-like stimuli by digital holographic microscopy and holographic optical trapping

    NASA Astrophysics Data System (ADS)

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

    2014-05-01

    Guiding, controlling and studying cellular functions are challenging themes in the biomedical field, as they are fundamental prerequisites for new therapeutic strategies from tissue regeneration to controlled drug delivery. In recent years, multidisciplinary studies in nanotechnology offer new tools to investigate important biophysical phenomena in response to the local physical characteristics of the extracellular environment, some examples are the mechanisms of cell adhesion, migration, communication and differentiation. Indeed for reproducing the features of the extracellular matrix in vitro, it is essential to develop active devices that evoke as much as possible the natural cellular environment. Our investigation is in the framework of studying and clarifying the biophysical mechanisms of the interaction between cells and the microenvironment in which they exist. We implement an optical tweezers setup to investigate cell material interaction and we use Digital Holography as non-invasive imaging technique in microscopy. We exploit Holographic Optical Tweezers arrangement in order to trap and manage functionalized micrometric latex beads to induce mechanical deformation in suspended cells. A lot of papers in literature examine the dynamics of the cytoskeleton when cells adhere on substrates and nowadays well established cell models are based on such research activities. Actually, the natural cell environment is made of a complex extracellular matrix and the single cell behavior is due to intricate interactions with the environment and are strongly correlated to the cell-cell interactions. Our investigation is devoted to understand the inner cell mechanism when it is mechanically stressed by point-like stimulus without the substrate influence.

  11. Laparoscopic management of hydatid cyst in the lesser sac

    PubMed Central

    Sahoo, Manash Ranjan; Kumar, Saurabh; Panda, Srikanta; Shameel, P. Ahammed

    2016-01-01

    Hydatid cyst is a disease caused by Echinococcus granulosus. Various anatomical location of hydatid cyst has been described in literature. Liver is the most common site of hydatid cyst and lungs are the second most common site. Hydatid cyst of lesser sac is a rare entity. Here we present a rare case report of laparoscopic management of hydatid cyst in lesser sac. PMID:27073313

  12. The pathogenic potential of Helicobacter cinaedi isolated from non-human sources: adherence, invasion and translocation ability in polarized intestinal epithelial Caco-2 cells in vitro.

    PubMed

    Taniguchi, Takako; Yamazaki, Wataru; Saeki, Yuji; Takajo, Ichiro; Okayama, Akihiko; Hayashi, Tetsuya; Misawa, Naoaki

    2016-05-01

    Helicobacter cinaedi infection has been recognized as an increasingly important emerging disease in humans. Infection with H. cinaedi causes bacteremia, cellulitis and enteritis. H. cinaedi has been isolated from non-human sources, including dogs, cats and rodents; however, it remains unclear whether animal strains are pathogenic in humans and as zoonotic pathogens. In this study, H. cinaedi isolates were recovered from a dog and a hamster, and the ability of these isolates to adhere to, invade and translocate across polarized human intestinal epithelial Caco-2 cells was examined in vitro. To better understand the pathogenic potential of animal H. cinaedi isolates, these results were compared with those for a human strain that was isolated from a patient with bacteremia. The animal and human strains adhered to and invaded Caco-2 cells, but to a lesser degree than the C. jejuni 81-176 strain, which was used as a control. The integrity of tight junctions was monitored by measuring transepithelial electrical resistance (TER) with a membrane insert system. The TER values for all H. cinaedi strains did not change during the experimental periods compared with those of the controls; however, translocation of H. cinaedi from the apical side to the basolateral side was confirmed by cultivation and H. cinaedi-specific PCR, suggesting that the H. cinaedi strains translocated by transcellular route. This study demonstrated that H. cinaedi strains of animal origin might have a pathogenic potential in human epithelial cells as observed in a translocation assay in vitro with a human isolate. PMID:26685883

  13. The pathogenic potential of Helicobacter cinaedi isolated from non-human sources: adherence, invasion and translocation ability in polarized intestinal epithelial Caco-2 cells in vitro

    PubMed Central

    TANIGUCHI, Takako; YAMAZAKI, Wataru; SAEKI, Yuji; TAKAJO, Ichiro; OKAYAMA, Akihiko; HAYASHI, Tetsuya; MISAWA, Naoaki

    2015-01-01

    Helicobacter cinaedi infection has been recognized as an increasingly important emerging disease in humans. Infection with H. cinaedi causes bacteremia, cellulitis and enteritis. H. cinaedi has been isolated from non-human sources, including dogs, cats and rodents; however, it remains unclear whether animal strains are pathogenic in humans and as zoonotic pathogens. In this study, H. cinaedi isolates were recovered from a dog and a hamster, and the ability of these isolates to adhere to, invade and translocate across polarized human intestinal epithelial Caco-2 cells was examined in vitro. To better understand the pathogenic potential of animal H. cinaedi isolates, these results were compared with those for a human strain that was isolated from a patient with bacteremia. The animal and human strains adhered to and invaded Caco-2 cells, but to a lesser degree than the C. jejuni 81–176 strain, which was used as a control. The integrity of tight junctions was monitored by measuring transepithelial electrical resistance (TER) with a membrane insert system. The TER values for all H. cinaedi strains did not change during the experimental periods compared with those of the controls; however, translocation of H. cinaedi from the apical side to the basolateral side was confirmed by cultivation and H. cinaedi-specific PCR, suggesting that the H. cinaedi strains translocated by transcellular route. This study demonstrated that H. cinaedi strains of animal origin might have a pathogenic potential in human epithelial cells as observed in a translocation assay in vitro with a human isolate. PMID:26685883

  14. Improved assay for quantitating adherence of ruminal bacteria to cellulose.

    PubMed Central

    Rasmussen, M A; White, B A; Hespell, R B

    1989-01-01

    A quantitative technique suitable for the determination of adherence of ruminal bacteria to cellulose was developed. This technique employs adherence of cells to cellulose disks and alleviates the problem of nonspecific cell entrapment within cellulose particles. By using this technique, it was demonstrated that the adherence of Ruminococcus flavefaciens FD1 to cellulose was inhibited by formaldehyde, methylcellulose, and carboxymethyl cellulose. Adherence was unaffected by acid hydrolysates of methylcellulose, glucose, and cellobiose. PMID:2782879

  15. Stretch induced endothelin-1 secretion by adult rat astrocytes involves calcium influx via stretch-activated ion channels (SACs)

    SciTech Connect

    Ostrow, Lyle W.; Suchyna, Thomas M.; Sachs, Frederick

    2011-06-24

    Highlights: {yields} Endothelin-1 expression by adult rat astrocytes correlates with cell proliferation. {yields} Stretch-induced ET-1 is inhibited by GsMtx-4, a specific inhibitor of Ca{sup 2+} permeant SACs. {yields} The less specific SAC inhibitor streptomycin also inhibits ET-1 secretion. {yields} Stretch-induced ET-1 production depends on a calcium influx. {yields} SAC pharmacology may provide a new class of therapeutic agents for CNS pathology. -- Abstract: The expression of endothelins (ETs) and ET-receptors is often upregulated in brain pathology. ET-1, a potent vasoconstrictor, also inhibits the expression of astrocyte glutamate transporters and is mitogenic for astrocytes, glioma cells, neurons, and brain capillary endothelia. We have previously shown that mechanical stress stimulates ET-1 production by adult rat astrocytes. We now show in adult astrocytes that ET-1 production is driven by calcium influx through stretch-activated ion channels (SACs) and the ET-1 production correlates with cell proliferation. Mechanical stimulation using biaxial stretch (<20%) of a rubber substrate increased ET-1 secretion, and 4 {mu}M GsMTx-4 (a specific inhibitor of SACs) inhibited secretion by 30%. GsMTx-4 did not alter basal ET-1 levels in the absence of stretch. Decreasing the calcium influx by lowering extracellular calcium also inhibited stretch-induced ET-1 secretion without effecting ET-1 secretion in unstretched controls. Furthermore, inhibiting SACs with the less specific inhibitor streptomycin also inhibited stretch-induced ET-1 secretion. The data can be explained with a simple model in which ET-1 secretion depends on an internal Ca{sup 2+} threshold. This coupling of mechanical stress to the astrocyte endothelin system through SACs has treatment implications, since all pathology deforms the surrounding parenchyma.

  16. Differential air sac pressures in diving tufted ducks Aythya fuligula.

    PubMed

    Boggs, D F; Butler, P J; Wallace, S E

    1998-09-01

    The air in the respiratory system of diving birds contains a large proportion of the body oxygen stores, but it must be in the lungs for gas exchange with blood to occur. To test the hypothesis that locomotion induces mixing of air sac air with lung air during dives, we measured differential pressures between the interclavicular and posterior thoracic air sacs in five diving tufted ducks Aythya fuligula. The peak differential pressure between posterior thoracic and interclavicular air sacs, 0.49+/-0.13 kPa (mean +/- s.d.), varied substantially during underwater paddling as indicated by gastrocnemius muscle activity. These data support the hypothesis that locomotion, perhaps through associated abdominal muscle activity, intermittently compresses the posterior air sacs more than the anterior ones. The result is differential pressure fluctuations that might induce the movement of air between air sacs and through the lungs during dives. PMID:9716518

  17. Antibodies Directed against Shiga-Toxin Producing Escherichia coli Serotype O103 Type III Secreted Proteins Block Adherence of Heterologous STEC Serotypes to HEp-2 Cells

    PubMed Central

    Desin, Taseen S.; Townsend, Hugh G.; Potter, Andrew A.

    2015-01-01

    Shiga toxin-producing Escherichia coli (STEC) serotype O103 is a zoonotic pathogen that is capable of causing hemorrhagic colitis and hemolytic uremic syndrome (HUS) in humans. The main animal reservoir for STEC is ruminants and hence reducing the levels of this pathogen in cattle could ultimately lower the risk of STEC infection in humans. During the process of infection, STECO103 uses a Type III Secretion System (T3SS) to secrete effector proteins (T3SPs) that result in the formation of attaching and effacing (A/E) lesions. Vaccination of cattle with STEC serotype O157 T3SPs has previously been shown to be effective in reducing shedding of STECO157 in a serotype-specific manner. In this study, we tested the ability of rabbit polyclonal sera against individual STECO103 T3SPs to block adherence of the organism to HEp-2 cells. Our results demonstrate that pooled sera against EspA, EspB, EspF, NleA and Tir significantly lowered the adherence of STECO103 relative to pre-immune sera. Likewise, pooled anti-STECO103 sera were also able to block adherence by STECO157. Vaccination of mice with STECO103 recombinant proteins induced strong IgG antibody responses against EspA, EspB, NleA and Tir but not against EspF. However, the vaccine did not affect fecal shedding of STECO103 compared to the PBS vaccinated group over the duration of the experiment. Cross reactivity studies using sera against STECO103 recombinant proteins revealed a high degree of cross reactivity with STECO26 and STECO111 proteins implying that sera against STECO103 proteins could potentially provide neutralization of attachment to epithelial cells by heterologous STEC serotypes. PMID:26451946

  18. Antibodies Directed against Shiga-Toxin Producing Escherichia coli Serotype O103 Type III Secreted Proteins Block Adherence of Heterologous STEC Serotypes to HEp-2 Cells.

    PubMed

    Desin, Taseen S; Townsend, Hugh G; Potter, Andrew A

    2015-01-01

    Shiga toxin-producing Escherichia coli (STEC) serotype O103 is a zoonotic pathogen that is capable of causing hemorrhagic colitis and hemolytic uremic syndrome (HUS) in humans. The main animal reservoir for STEC is ruminants and hence reducing the levels of this pathogen in cattle could ultimately lower the risk of STEC infection in humans. During the process of infection, STECO103 uses a Type III Secretion System (T3SS) to secrete effector proteins (T3SPs) that result in the formation of attaching and effacing (A/E) lesions. Vaccination of cattle with STEC serotype O157 T3SPs has previously been shown to be effective in reducing shedding of STECO157 in a serotype-specific manner. In this study, we tested the ability of rabbit polyclonal sera against individual STECO103 T3SPs to block adherence of the organism to HEp-2 cells. Our results demonstrate that pooled sera against EspA, EspB, EspF, NleA and Tir significantly lowered the adherence of STECO103 relative to pre-immune sera. Likewise, pooled anti-STECO103 sera were also able to block adherence by STECO157. Vaccination of mice with STECO103 recombinant proteins induced strong IgG antibody responses against EspA, EspB, NleA and Tir but not against EspF. However, the vaccine did not affect fecal shedding of STECO103 compared to the PBS vaccinated group over the duration of the experiment. Cross reactivity studies using sera against STECO103 recombinant proteins revealed a high degree of cross reactivity with STECO26 and STECO111 proteins implying that sera against STECO103 proteins could potentially provide neutralization of attachment to epithelial cells by heterologous STEC serotypes. PMID:26451946

  19. Hyaluronic Acid-Based Hydrogels as 3D Matrices for in Vitro Evaluation of Chemotherapeutic Drugs Using Poorly Adherent Prostate Cancer Cells

    PubMed Central

    Gurski, Lisa A.; Jha, Amit K.; Zhang, Chu; Jia, Xinqiao; Farach-Carson, Mary C.

    2009-01-01

    The current investigation aimed to develop a biomimetic, three-dimensional (3D) culture system for poorly adherent bone metastatic prostate cancer cells (C4-2B) for use as an in vitro platform for anti-cancer drug screening. To this end, hyaluronic acid (HA) derivatives carrying complementary aldehyde (HAALD) and hydrazide (HAADH) groups were synthesized and characterized. In situ encapsulation of C4-2B cells was achieved by simple mixing of HAALD and HAADH in the presence of the cells. Unlike two-dimensional (2D) monolayer culture in which cells adopt an atypical spread morphology, cells residing in the HA matrix formed distinct clustered structures which grew and merged, reminiscent of real tumors. Anti-cancer drugs added to the media surrounding the cell/gel construct diffused into the gel and killed the embedded cells. The HA hydrogel system was used successfully to test the efficacy of anti-cancer drugs including camptothecin, docetaxel, and rapamycin, alone and in combination, including specificity, dose and time responses. Responses of cells to anti-neoplastics differed between the 3D HA hydrogel and 2D monolayer systems. We suggest that the data obtained from 3D HA systems is superior to that from conventional 2D monolayers as the 3D system better reflects the bone metastatic microenvironment of the cancer cells. PMID:19695694

  20. Genetic relatedness and virulence properties of enteropathogenic Escherichia coli strains of serotype O119:H6 expressing localized adherence or localized and aggregative adherence-like patterns on HeLa cells.

    PubMed

    Garcia, Bruna G; Ooka, Tadasuke; Gotoh, Yasuhiro; Vieira, Mônica A M; Yamamoto, Denise; Ogura, Yoshitoshi; Girão, Dennys M; Sampaio, Suely C F; Melo, Alexis Bonfim; Irino, Kinue; Hayashi, Tetsuya; Gomes, Tânia A T

    2016-05-01

    Enteropathogenic Escherichia coli (EPEC) induce attaching and effacing (A/E) lesions in enterocytes and produce the bundle-forming pilus (BFP) contributing to the localized adherence (LA) pattern formation on HeLa cells. Enteroaggregative E. coli (EAEC) produce aggregative adherence (AA) on HeLa cells and form prominent biofilms. The ability to produce LA or AA is an important hallmark to classify fecal E. coli isolates as EPEC or EAEC, respectively. E. coli strains of serotype O119:H6 exhibit an LA+ phenotype and have been considered as comprising a clonal group of EPEC strains. However, we have recently identified O119:H6 EPEC strains that produce LA and an AA-like pattern concurrently (LA/AA-like+). In this study, we evaluated the relatedness of three LA/AA-like+ and three LA+ O119:H6 strains by comparing their virulence and genotypic properties. We first found that the LA/AA-like+ strains induced actin accumulation in HeLa cells (indicative of A/E lesions formation) and formed biofilms on abiotic surfaces more efficiently than the LA+ strains. MLST analysis showed that the six strains all belong to the ST28 complex. All strains carried multiple plasmids, but as plasmid profiles were highly variable, this cannot be used to differentiate LA/AA-like+ and LA+ strains. We further obtained their draft genome sequences and the complete sequences of four plasmids harbored by one LA/AA-like+ strain. Analysis of these sequences and comparison with 37 fully sequenced E. coli genomes revealed that both O119:H6 groups belong to the E. coli phylogroup B2 and are very closely related with only 58-67 SNPs found between LA/AA-like+ and LA+ strains. Search of the draft sequences of the six strains for adhesion-related genes known in EAEC and other E. coli pathotypes detected no genes specifically present in LA/AA-like+ strains. Unexpectedly however, we found that a large plasmid distinct from pEAF is responsible for the AA-like phenotype of the LA/AA-like+ strains. Although we

  1. 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

    Agglomeration and sedimentation of nanoparticles (NPs) within biological solutions is a major limitation in their use in many downstream applications. It has been proposed that serum proteins associate with the NP surface to form a protein corona that limits agglomeration and sedimentation. Here, we investigate the effect of fetal bovine serum (FBS) proteins on the dispersion stability, dosimetry, and NP-induced cytotoxicity of cationic zinc oxide nanoparticles (nZnO) synthesized via forced hydrolysis with a core size of 10 nm. Two different in vitro cell culture models, suspension and adherent, were evaluated by comparing a phosphate buffered saline (PBS) nZnO dispersion (nZnO/PBS) and an FBS-stabilized PBS nZnO dispersion (nZnO - FBS/PBS). Surface interactions of FBS on nZnO were analyzed via spectroscopic and optical techniques. Fourier transformed infrared spectroscopy (FTIR) confirmed the adsorption of negatively charged protein components on the cationic nZnO surface through the disappearance of surfaced-adsorbed carboxyl functional groups and the subsequent detection of vibrational modes associated with the protein backbone of FBS-associated proteins. Further confirmation of these interactions was noted in the isoelectric point shift of the nZnO from the characteristic pH of 9.5 to a pH of 6.1. In nZnO - FBS/PBS dispersions, the FBS reduced agglomeration and sedimentation behaviors to impart long-term improvements (>24 h) to the nZnO dispersion stability. Furthermore, mathematical dosimetry models indicate that nZnO - FBS/PBS dispersions had consistent NP deposition patterns over time unlike unstable nZnO/PBS dispersions. In suspension cell models, the stable nZnO - FBS/PBS dispersion resulted in a ~33 % increase in the NP-induced cytotoxicity for both Jurkat leukemic and Hut-78 lymphoma cancer cells. In contrast, the nZnO - FBS/PBS dispersion resulted in 49 and 71 % reductions in the cytotoxicity observed towards the adherent breast (T-47D) and prostate

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

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

    Agglomeration and sedimentation of nanoparticles (NPs) within biological solutions is a major limitation in their use in many downstream applications. It has been proposed that serum proteins associate with the NP surface to form a protein corona that limits agglomeration and sedimentation. Here, we investigate the effect of fetal bovine serum (FBS) proteins on the dispersion stability, dosimetry, and NP-induced cytotoxicity of cationic zinc oxide nanoparticles (nZnO) synthesized via forced hydrolysis with a core size of 10 nm. Two different in vitro cell culture models, suspension and adherent, were evaluated by comparing a phosphate buffered saline (PBS) nZnO dispersion (nZnO/PBS) and an FBS-stabilized PBS nZnO dispersion (nZnO - FBS/PBS). Surface interactions of FBS on nZnO were analyzed via spectroscopic and optical techniques. Fourier transformed infrared spectroscopy (FTIR) confirmed the adsorption of negatively charged protein components on the cationic nZnO surface through the disappearance of surfaced-adsorbed carboxyl functional groups and the subsequent detection of vibrational modes associated with the protein backbone of FBS-associated proteins. Further confirmation of these interactions was noted in the isoelectric point shift of the nZnO from the characteristic pH of 9.5 to a pH of 6.1. In nZnO - FBS/PBS dispersions, the FBS reduced agglomeration and sedimentation behaviors to impart long-term improvements (>24 h) to the nZnO dispersion stability. Furthermore, mathematical dosimetry models indicate that nZnO - FBS/PBS dispersions had consistent NP deposition patterns over time unlike unstable nZnO/PBS dispersions. In suspension cell models, the stable nZnO - FBS/PBS dispersion resulted in a ~33 % increase in the NP-induced cytotoxicity for both Jurkat leukemic and Hut-78 lymphoma cancer cells. In contrast, the nZnO - FBS/PBS dispersion resulted in 49 and 71 % reductions in the cytotoxicity observed towards the adherent breast (T-47D) and prostate

  3. Portrait of Candida albicans Adherence Regulators

    PubMed Central

    Finkel, Jonathan S.; Xu, Wenjie; Huang, David; Hill, Elizabeth M.; Desai, Jigar V.; Woolford, Carol A.; Nett, Jeniel E.; Taff, Heather; Norice, Carmelle T.; Andes, David R.; Lanni, Frederick; Mitchell, Aaron P.

    2012-01-01

    Cell-substrate adherence is a fundamental property of microorganisms that enables them to exist in biofilms. Our study focuses on adherence of the fungal pathogen Candida albicans to one substrate, silicone, that is relevant to device-associated infection. We conducted a mutant screen with a quantitative flow-cell assay to identify thirty transcription factors that are required for adherence. We then combined nanoString gene expression profiling with functional analysis to elucidate relationships among these transcription factors, with two major goals: to extend our understanding of transcription factors previously known to govern adherence or biofilm formation, and to gain insight into the many transcription factors we identified that were relatively uncharacterized, particularly in the context of adherence or cell surface biogenesis. With regard to the first goal, we have discovered a role for biofilm regulator Bcr1 in adherence, and found that biofilm regulator Ace2 is a major functional target of chromatin remodeling factor Snf5. In addition, Bcr1 and Ace2 share several target genes, pointing to a new connection between them. With regard to the second goal, our findings reveal existence of a large regulatory network that connects eleven adherence regulators, the zinc-response regulator Zap1, and approximately one quarter of the predicted cell surface protein genes in this organism. This limited yet sensitive glimpse of mutant gene expression changes had thus defined one of the broadest cell surface regulatory networks in C. albicans. PMID:22359502

  4. Krüppel-Like Factor 4 Overexpression Initiates a Mesenchymal-to-Epithelial Transition and Redifferentiation of Human Pancreatic Cells following Expansion in Long Term Adherent Culture

    PubMed Central

    Docherty, Hilary M.; McGowan, Neil W. A.; Forbes, Shareen; Heremans, Yves; Forbes, Stuart J.; Heimberg, Harry; Casey, John; Docherty, Kevin

    2015-01-01

    A replenishable source of insulin-producing cells has the potential to cure type 1 diabetes. Attempts to culture and expand pancreatic β-cells in vitro have resulted in their transition from insulin-producing epithelial cells to mesenchymal stromal cells (MSCs) with high proliferative capacity but devoid of any hormone production. The aim of this study was to determine whether the transcription factor Krüppel-like factor 4 (KLF4), could induce a mesenchymal-to-epithelial transition (MET) of the cultured cells. Islet-enriched pancreatic cells, allowed to dedifferentiate and expand in adherent cell culture, were transduced with an adenovirus containing KLF4 (Ad-Klf4). Cells were subsequently analysed for changes in cell morphology by light microscopy, and for the presence of epithelial and pancreatic markers by immunocytochemistry and quantitative RT/PCR. Infection with Ad-Klf4 resulted in morphological changes, down-regulation of mesenchymal markers, and re-expression of both epithelial and pancreatic cell markers including insulin and transcription factors specific to β-cells. This effect was further enhanced by culturing cells in suspension. However, the effects of Ad-KLf4 were transient and this was shown to be due to increased apoptosis in Klf4-expressing cells. Klf4 has been recently identified as a pioneer factor with the ability to modulate the structure of chromatin and enhance reprogramming/transdifferentiation. Our results show that Klf4 may have a role in the redifferentiation of expanded pancreatic cells in culture, but before this can be achieved the off-target effects that result in increased apoptosis would need to be overcome. PMID:26457418

  5. 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

  6. 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

  7. Myoblast cytonemes mediate Wg signaling from the wing imaginal disc and Delta-Notch signaling to the air sac primordium

    PubMed Central

    Huang, Hai; Kornberg, Thomas B

    2015-01-01

    The flight muscles, dorsal air sacs, wing blades, and thoracic cuticle of the Drosophila adult function in concert, and their progenitor cells develop together in the wing imaginal disc. The wing disc orchestrates dorsal air sac development by producing decapentaplegic and fibroblast growth factor that travel via specific cytonemes in order to signal to the air sac primordium (ASP). Here, we report that cytonemes also link flight muscle progenitors (myoblasts) to disc cells and to the ASP, enabling myoblasts to relay signaling between the disc and the ASP. Frizzled (Fz)-containing myoblast cytonemes take up Wingless (Wg) from the disc, and Delta (Dl)-containing myoblast cytonemes contribute to Notch activation in the ASP. Wg signaling negatively regulates Dl expression in the myoblasts. These results reveal an essential role for cytonemes in Wg and Notch signaling and for a signal relay system in the myoblasts. DOI: http://dx.doi.org/10.7554/eLife.06114.001 PMID:25951303

  8. [Ovarian yolk sac tumour: general review].

    PubMed

    Even, Caroline; Lhommé, Catherine; Duvillard, Pierre; Morice, Philippe; Balleyguier, Corinne; Pautier, Patricia; Troalen, Frédéric; de La Motte Rouge, Thibault

    2011-08-01

    Ovarian yolk sac tumour (OYST) is a very rare malignancy arising most often in young women. Preoperative clinical, biological (alpha-foetoprotein) and radiological findings should help to establish the diagnosis of OYST, in order to propose adequate surgical treatment. The aim of surgery is to remove the primary tumour, to obtain an accurate histological diagnosis and to assess the disease extent. In young women, fertility-sparing surgery should be performed, in order to preserve the possibility of pregnancy later on. Chemotherapy has substantially modified the prognosis of these tumours, and practically all patients will be cured. The overall 5-year survival rate is 94% when patients are treated with BEP chemotherapy. Depending on the clinical situation, two to four cycles of the BEP regimen should be administered after surgery. Identification of prognostic factors may help to propose risk-adapted treatment in order to increase the cure rate in patients with a poor prognosis and to decrease toxicity in patients with a low risk of relapse. Fertility preservation represents a major objective in women treated for OYSTs. PMID:21708513

  9. NbHSWP11, a microsporidia Nosema bombycis protein, localizing in the spore wall and membranes, reduces spore adherence to host cell BME.

    PubMed

    Yang, Donglin; Dang, Xiaoqun; Peng, Pai; Long, Mengxian; Ma, Cheng; Qin, Junjie Jia Guowei; Wu, Haijing; Liu, Tie; Zhou, Xiaowei; Pan, Guoqing; Zhou, Zeyang

    2014-10-01

    Microsporidia are obligate intracellular parasites, and a derivative of fungi, which harbor a rigid spore wall to resist adverse environmental pressures. The spore wall protein, which is thought to be the first and direct protein interacting with the host cell, may play a key role in the process of microsporidia infection. In this study, we report a protein, NbHSWP11, with a dnaJ domain. The protein also has 6 heparin-binding motifs which are known to interact with extracellular glycosaminoglycans. Syntenic analysis indicated that gene loci of Nbhswp11 are conserved and syntenic between Nosema bombycis and Nosema ceranae. Phylogenetic tree analysis showed that Nbhswp11 clusters with fungal dnaJ proteins and has 98% identity with an N. bombycis dnaJ protein. Nbhswp11 was transcribed throughout the entire life stages, and gradually increased during 1-7 days, in a silkworm that was infected by N. bombycis, as determined by reverse-transcription PCR (RT-PCR). The recombinant protein NbHSWP11 (rSWP11-HIS) was obtained and purified using gene cloning and prokaryotic expression. Western blotting analysis displayed NbHSWP11 expressed in the total mature spore proteins and spore coat proteins. Indirect immunofluorescence assay revealed NbHSWP11 located at the spore wall of mature spores and the spore coats. Furthermore, immune electron microscopy showed that NbHSWP11 localized in the cytoplasm of the sporont. Within the developmental process of N. bombycis, a portion of NbHSWP11 is targeted to the spore wall of sporoblasts and mature spores. However, most of NbHSWP11 distributes on the membraneous structures of the sporoblast and mature spore. In addition, using a host cell binding assay, native protein NbHSWP11 in the supernatant of total soluble mature spore proteins is shown to bind to the host cell BmE surface. Finally, an antibody blocking assay showed that purified rabbit antibody of NbHSWP11 inhibits spore adherence and decreases the adherence rate of spores by 20

  10. Escherichia coli O157 adherence to the bovine recto-anal junction (RAJ) squamous epithelial cells is mediated by adhesins other than those encoded by genes on the Locus of Enterocyte Attachment (LEE) pathogenicity island

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli O157 (O157) persist in the gastrointestinal tracts (GIT) of bovine reservoirs primarily by adhering to the mucocutaneous, recto-anal junction (RAJ), comprising of both follicle-associated-epithelial (FAE) cells proximally and stratified squamous epithelial (RSE) cells distally. Whe...

  11. IgG1 antimycobacterial antibodies can reverse the inhibitory effect of pentoxifylline on tumour necrosis factor alpha (TNF-α) secreted by mycobacterial antigen-stimulated adherent cells

    PubMed Central

    THAKURDAS, S M; HASAN, Z; HUSSAIN, R

    2004-01-01

    Chronic inflammation associated with cachexia, weight loss, fever and arthralgia is the hallmark of advanced mycobacterial diseases. These symptoms are attributed to the chronic stimulation of tumour necrosis factor (TNF)-α. Mycobacterial components directly stimulate adherent cells to secrete TNF-α. We have shown recently that IgG1 antimycobacterial antibodies play a role in augmenting TNF-α in purified protein derivative (PPD)-stimulated adherent cells from non-BCG-vaccinated donors. We now show that IgG1 antibodies can also augment TNF-α expression in stimulated adherent cells obtained from BCG-vaccinated donors and this augmentation is not linked to interleukin (IL)-10 secretion. In addition IgG1 antimycobacterial antibodies can reverse the effect of TNF-α blockers such as pentoxifylline and thalidomide. These studies therefore have clinical implications for anti-inflammatory drug treatments which are used increasingly to alleviate symptoms associated with chronic inflammation. PMID:15086397

  12. A YadA-like autotransporter, Hag 1, in Veillonella atypica is a Multivalent Hemagglutinin Involved in Adherence to Oral Streptococci, Porphyromonas gingivalis, and Human Oral Buccal Cells

    PubMed Central

    Merritt, Justin; Qi, Fengxia

    2015-01-01

    Dental biofilm development is a sequential process, and adherence between microbes and the salivary pellicle (adhesion) as well as among different microbes (co-adhesion or coaggregation) plays a critical role in building a biofilm community. The Veillonella species are among the most predominant species in the oral cavity and coaggregate with many initial, early, middle and late colonizers. Similar to oral fusobacteria, they are also considered bridging species in biofilm development. However, the mechanism of this ability has yet to be reported, due to the previous lack of a genetic transformation system in the entire genus. In this study, we used our recently discovered transformable Veillonella strain, V. atypica OK5, to probe the mechanism of coaggregation between Veillonella species and other oral bacteria. By insertional inactivation of all 8 putative hemagglutinin genes, we identified one gene, hag1, which is involved in V. atypica coaggregation with the initial colonizers Streptococcus gordonii, Streptococcus oralis and Streptococcus cristatus, and the periodontal pathogen Porphyromonas gingivalis. The hag1 mutant also abolished adherence to human buccal cells. Inhibition assays using various chemical or physiological treatments suggest different mechanisms being involved in coaggregation with different partners. The entire hag1 gene was sequenced and shown to be the largest known bacterial hemagglutinin gene. PMID:25440509

  13. Inhibition of Shigella sonnei adherence to HT-29 cells by lactobacilli from Chinese fermented food and preliminary characterization of S-layer protein involvement.

    PubMed

    Zhang, Ying-Chun; Zhang, Lan-Wei; Tuo, Yan-Feng; Guo, Chun-Feng; Yi, Hua-Xi; Li, Jing-Yan; Han, Xue; Du, Ming

    2010-10-01

    In this study, seven lactobacilli with a high degree of antagonistic activity against three pathogens and good adherence to HT-29 cells were selected. The ability of these seven lactobacilli to inhibit adhesion of Shigella sonnei to intestinal mucosa was studied on cultured HT-29 cells. Lactobacilli were added simultaneously with, before or after S. sonnei to test for their effectiveness in exclusion, competition and displacement assays, respectively. Lactobacillus paracasei subp. paracasei M5-L, Lactobacillus rhamnosus J10-L and Lactobacillus casei Q8-L all exhibited significant inhibitory activity. In order to elucidate the inhibitory functions of S-layer proteins, the S-layer proteins were removed with 5 M LiCl from the M5-L, J10-L and Q8-L strains. Under such conditions, inhibition activity was decreased in all three strains, as revealed in exclusion, competition and displacement assays. SDS-PAGE analysis confirmed the presence of S-layer proteins with dominant bands of approximately 45 kDa. Further analysis of S-layer proteins revealed that the hydrophobic amino acids accounted for 40.5%, 41.5% and 43.8% of the total amino acid for the M5-L, J10-L and Q8-L strains, respectively. These findings suggest that the M5-L, J10-L and Q8-L strains possess the ability to inhibit S. sonnei adherence to HT-29 cells, and S-layer proteins are involved in this adhesion inhibition. PMID:20600857

  14. 79. Sac digital network (Sacdin), summary fault indicator at top, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    79. Sac digital network (Sacdin), summary fault indicator at top, south side - Ellsworth Air Force Base, Delta Flight, Launch Control Facility, County Road CS23A, North of Exit 127, Interior, Jackson County, SD

  15. Automatic segmentation and classification of gestational sac based on mean sac diameter using medical ultrasound image

    NASA Astrophysics Data System (ADS)

    Khazendar, Shan; Farren, Jessica; Al-Assam, Hisham; Sayasneh, Ahmed; Du, Hongbo; Bourne, Tom; Jassim, Sabah A.

    2014-05-01

    Ultrasound is an effective multipurpose imaging modality that has been widely used for monitoring and diagnosing early pregnancy events. Technology developments coupled with wide public acceptance has made ultrasound an ideal tool for better understanding and diagnosing of early pregnancy. The first measurable signs of an early pregnancy are the geometric characteristics of the Gestational Sac (GS). Currently, the size of the GS is manually estimated from ultrasound images. The manual measurement involves multiple subjective decisions, in which dimensions are taken in three planes to establish what is known as Mean Sac Diameter (MSD). The manual measurement results in inter- and intra-observer variations, which may lead to difficulties in diagnosis. This paper proposes a fully automated diagnosis solution to accurately identify miscarriage cases in the first trimester of pregnancy based on automatic quantification of the MSD. Our study shows a strong positive correlation between the manual and the automatic MSD estimations. Our experimental results based on a dataset of 68 ultrasound images illustrate the effectiveness of the proposed scheme in identifying early miscarriage cases with classification accuracies comparable with those of domain experts using K nearest neighbor classifier on automatically estimated MSDs.

  16. Topographical and mechanical characterization of living eukaryotic cells on opaque substrates: development of a general procedure and its application to the study of non-adherent lymphocytes

    NASA Astrophysics Data System (ADS)

    Daza, Rafael; Cruces, Julia; Arroyo-Hernández, María; Marí-Buyé, Núria; De la Fuente, Mónica; Plaza, Gustavo R.; Elices, Manuel; Pérez-Rigueiro, José; Guinea, Gustavo V.

    2015-04-01

    The mechanical behavior of living murine T-lymphocytes was assessed by atomic force microscopy (AFM). A robust experimental procedure was developed to overcome some features of lymphocytes, in particular their spherical shape and non-adherent character. The procedure included the immobilization of the lymphocytes on amine-functionalized substrates, the use of hydrodynamic effects on the deflection of the AFM cantilever to monitor the approaching, and the use of the jumping mode for obtaining the images. Indentation curves were analyzed according to Hertz’s model for contact mechanics. The calculated values of the elastic modulus are consistent both when considering the results obtained from a single lymphocyte and when comparing the curves recorded from cells of different specimens.

  17. Increased S-Nitrosylation and Proteasomal Degradation of Caspase-3 during Infection Contribute to the Persistence of Adherent Invasive Escherichia coli (AIEC) in Immune Cells

    PubMed Central

    Dunne, Karl A.; Allam, Amr; McIntosh, Anne; Houston, Stephanie A.; Cerovic, Vuk; Goodyear, Carl S.; Roe, Andrew J.; Beatson, Scott A.; Milling, Simon W.; Walker, Daniel; Wall, Daniel M.

    2013-01-01

    Adherent invasive Escherichia coli (AIEC) have been implicated as a causative agent of Crohn’s disease (CD) due to their isolation from the intestines of CD sufferers and their ability to persist in macrophages inducing granulomas. The rapid intracellular multiplication of AIEC sets it apart from other enteric pathogens such as Salmonella Typhimurium which after limited replication induce programmed cell death (PCD). Understanding the response of infected cells to the increased AIEC bacterial load and associated metabolic stress may offer insights into AIEC pathogenesis and its association with CD. Here we show that AIEC persistence within macrophages and dendritic cells is facilitated by increased proteasomal degradation of caspase-3. In addition S-nitrosylation of pro- and active forms of caspase-3, which can inhibit the enzymes activity, is increased in AIEC infected macrophages. This S-nitrosylated caspase-3 was seen to accumulate upon inhibition of the proteasome indicating an additional role for S-nitrosylation in inducing caspase-3 degradation in a manner independent of ubiquitination. In addition to the autophagic genetic defects that are linked to CD, this delay in apoptosis mediated in AIEC infected cells through increased degradation of caspase-3, may be an essential factor in its prolonged persistence in CD patients. PMID:23861899

  18. 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

  19. Altered callose deposition during embryo sac formation of multi-pistil mutant (mp1) in Medicago sativa.

    PubMed

    Zhou, H C; Jin, L; Li, J; Wang, X J

    2016-01-01

    Whether callose deposition is the cause or result of ovule sterility in Medicago sativa remains controversial, because it is unclear when and where changes in callose deposition and dissolution occur during fertile and sterile embryo sac formation. Here, alfalfa spontaneous multi-pistil mutant (mp1) and wild-type plants were used to compare the dynamics of callose deposition during embryo sac formation using microscopy. The results showed that both mutant and wild-type plants experienced megasporogenesis and megagametogenesis, and there was no significant difference during megasporogenesis. In contrast to the wild-type plants, in which the mature embryo sac was observed after three continuous cycles of mitosis, functional megaspores of mutant plants developed abnormally after the second round of mitosis, leading to degeneration of synergid, central, and antipodal cells. Callose deposition in both mutant and wild-type plants was first observed in the walls of megasporocytes, and then in the megaspore tetrad walls. After meiosis, the callose wall began to degrade as the functional megaspore underwent mitosis, and almost no callose was observed in the mature embryo sac in wild-type plants. However, callose deposition was observed in mp1 plants around the synergid, and increased with the development of the embryo sac, and was mainly deposited at the micropylar end. Our results indicate that synergid, central, and antipodal cells, which are surrounded by callose, may degrade owing to lack of nutrition. Callose accumulation around the synergid and at the micropylar end may hinder signals required for the pollen tube to enter the embryo sac, leading to abortion. PMID:27323128

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

    PubMed Central

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

    1983-01-01

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

  1. Contributions of NanI Sialidase to Caco-2 Cell Adherence by Clostridium perfringens Type A and C Strains Causing Human Intestinal Disease

    PubMed Central

    Li, Jihong

    2014-01-01

    Previous studies showed that Clostridium perfringens type D animal disease strain CN3718 uses NanI sialidase for adhering to enterocyte-like Caco-2 cells. The current study analyzed whether NanI is similarly important when type A and C human intestinal disease strains attach to Caco-2 cells. A PCR survey determined that the nanI gene was absent from typical type A food poisoning (FP) strains carrying a chromosomal enterotoxin (CPE) gene or the genetically related type C Darmbrand (Db) strains. However, the nanI gene was present in type A strains from healthy humans, type A strains causing CPE-associated antibiotic-associated diarrhea (AAD) or sporadic diarrhea (SD), and type C Pig-Bel strains. Consistent with NanI sialidase being the major C. perfringens sialidase when produced, FP and Db strains had little supernatant sialidase activity compared to other type A or C human intestinal strains. All type A and C human intestinal strains bound to Caco-2 cells, but NanI-producing strains had higher attachment levels. When produced, NanI can contribute to host cell attachment of human intestinal disease strains, since a nanI null mutant constructed in type A SD strain F4969 had lower Caco-2 cell adhesion than wild-type F4969 or a complemented strain. Further supporting a role for NanI in host cell attachment, sialidase inhibitors reduced F4969 adhesion to Caco-2 cells. Collectively, these results suggest that NanI may contribute to the intestinal attachment and colonization needed for the chronic diarrhea of CPE-associated AAD and SD, but this sialidase appears to be dispensable for the acute pathogenesis of type A FP or type C enteritis necroticans. PMID:25135687

  2. Intraoperative Sac Pressure Measurement During Endovascular Abdominal Aortic Aneurysm Repair

    SciTech Connect

    Ishibashi, Hiroyuki; Ishiguchi, Tsuneo; Ohta, Takashi; Sugimoto, Ikuo; Iwata, Hirohide; Yamada, Tetsuya; Tadakoshi, Masao; Hida, Noriyuki; Orimoto, Yuki; Kamei, Seiji

    2010-10-15

    PurposeIntraoperative sac pressure was measured during endovascular abdominal aortic aneurysm repair (EVAR) to evaluate the clinical significance of sac pressure measurement.MethodsA microcatheter was placed in an aneurysm sac from the contralateral femoral artery, and sac pressure was measured during EVAR procedures in 47 patients. Aortic blood pressure was measured as a control by a catheter from the left brachial artery.ResultsThe systolic sac pressure index (SPI) was 0.87 {+-} 0.10 after main-body deployment, 0.63 {+-} 0.12 after leg deployment (P < 0.01), and 0.56 {+-} 0.12 after completion of the procedure (P < 0.01). Pulse pressure was 55 {+-} 21 mmHg, 23 {+-} 15 mmHg (P < 0.01), and 16 {+-} 12 mmHg (P < 0.01), respectively. SPI showed no significant differences between the Zenith and Excluder stent grafts (0.56 {+-} 0.13 vs. 0.54 {+-} 0.10, NS). Type I endoleak was found in seven patients (15%), and the SPI decreased from 0.62 {+-} 0.10 to 0.55 {+-} 0.10 (P = 0.10) after fixing procedures. Type II endoleak was found in 12 patients (26%) by completion angiography. The SPI showed no difference between type II endoleak positive and negative (0.58 {+-} 0.12 vs. 0.55 {+-} 0.12, NS). There were no significant differences between the final SPI of abdominal aortic aneurysms in which the diameter decreased in the follow-up and that of abdominal aortic aneurysms in which the diameter did not change (0.53 {+-} 0.12 vs. 0.57 {+-} 0.12, NS).ConclusionsSac pressure measurement was useful for instant hemodynamic evaluation of the EVAR procedure, especially in type I endoleaks. However, on the basis of this small study, the SPI cannot be used to reliably predict sac growth or regression.

  3. A primary pure yolk sac tumor of the lung exhibiting CDX-2 immunoreactivity and increased serum levels of alkaline phosphatase intestinal isoenzyme.

    PubMed

    Pelosi, Giuseppe; Petrella, Francesco; Sandri, Maria Teresa; Spaggiari, Lorenzo; Galetta, Domenico; Viale, Giuseppe

    2006-07-01

    Malignant extragonadal germ cell tumors primary to the lung are quite uncommon lesions, but pure yolk sac tumor is even more exceptional. This is believed to be the first reported case of yolk sac tumor of the lung in which an intense and diffuse immunoreactivity for CDX2, a marker of intestinal differentiation reportedly expressed also in gonadal yolk sac tumor, was associated with increased serum levels of the alkaline phosphatase intestinal isoform. Nine months after radical surgery and adjuvant chemotherapy, the patient is alive and well without evidence of recurrent or metastatic disease and with serum levels of the alkaline phosphatase intestinal isoform within normal limits. The pathologist should be aware of yolk sac tumor arising in the lung and that alkaline phosphatase intestinal isoform could become an additional serum marker for such a tumor. PMID:16959714

  4. Basic Leucine Zipper (bZIP) Domain Transcription Factor MBZ1 Regulates Cell Wall Integrity, Spore Adherence, and Virulence in Metarhizium robertsii *

    PubMed Central

    Huang, Wei; Shang, Yanfang; Chen, Peilin; Cen, Kai; Wang, Chengshu

    2015-01-01

    Transcription factors (TFs) containing the basic leucine zipper (bZIP) domain are widely distributed in eukaryotes and display an array of distinct functions. In this study, a bZIP-type TF gene (MBZ1) was deleted and functionally characterized in the insect pathogenic fungus Metarhizium robertsii. The deletion mutant (ΔMBZ1) showed defects in cell wall integrity, adhesion to hydrophobic surfaces, and topical infection of insects. Relative to the WT, ΔMBZ1 was also impaired in growth and conidiogenesis. Examination of putative target gene expression indicated that the genes involved in chitin biosynthesis were differentially transcribed in ΔMBZ1 compared with the WT, which led to the accumulation of a higher level of chitin in mutant cell walls. MBZ1 exhibited negative regulation of subtilisin proteases, but positive control of an adhesin gene, which is consistent with the observation of effects on cell autolysis and a reduction in spore adherence to hydrophobic surfaces in ΔMBZ1. Promoter binding assays indicated that MBZ1 can bind to different target genes and suggested the possibility of heterodimer formation to increase the diversity of the MBZ1 regulatory network. The results of this study advance our understanding of the divergence of bZIP-type TFs at both intra- and interspecific levels. PMID:25673695

  5. Cyclic stretching of mesangial cells up-regulates intercellular adhesion molecule-1 and leukocyte adherence: a possible new mechanism for glomerulosclerosis.

    PubMed

    Riser, B L; Varani, J; Cortes, P; Yee, J; Dame, M; Sharba, A K

    2001-01-01

    Intraglomerular hypertension is a primary causal factor in the progressive glomerulosclerosis that characterizes diabetic nephropathy or severe renal ablation. However, inflammation of the glomerular mesangium also participates in at least the early phase of these diseases. In glomerulonephritis, where inflammation is thought to be the predominant causal factor, intraglomerular hypertension is also often present. Mesangial cells (MCs) are critical in orchestrating key functions of the glomerulus including extracellular matrix metabolism, cytokine production, and interaction with leukocytes. Because MCs are subject to increased stretching when intraglomerular hypertension is present, and in glomerulonephritis MC/leukocyte interactions seem to be mediated primarily via the up-regulation of intercellular adhesion molecule-1 (ICAM-1), we examine the possibility that cyclic stretching is a stimulus for increased MC ICAM-1 activity. We demonstrate that the normal low levels of MC ICAM-1 mRNA and protein are dramatically up-regulated by even short intervals of cyclic stretch. This effect is dose- and time-dependent, and requires little amplitude and a brief period of elongation for significant induction. Stretch-induced MC ICAM-1 also leads to a marked elevation in phagocytic leukocyte adherence. This stimulated adherence is equal or greater than that induced by the inflammatory cytokine tumor necrosis factor-alpha, whereas an additive effect occurs when both are applied in combination. Our results indicate that stretch-induced ICAM-1 may provide a direct link between hypertension and inflammation in the progression of injury and glomerulosclerosis in diabetes, renal ablation, and other forms of glomerulonephritis. PMID:11141473

  6. Attachment role of gonococcal pili. Optimum conditions and quantitation of adherence of isolated pili to human cells in vitro.

    PubMed Central

    Pearce, W A; Buchanan, T M

    1978-01-01

    Gonoccocal pili facilitate attachment of virulent Neisseria gonorrhoeae to human cells. To characterize this attachment function, purified gonococcal pili isolated from four strains possessing antigenically distinct pili were radiolabeled with 125I and used to measure the attachment of pili to various human cells in vitro. Human buccal and cervical-vaginal mucosal epithealial cells, fallopian tube mucosa, and sperm bound pili in greater numbers per micrometer2 of surface area (1--10) than fetal tonsil fibroblasts, HeLa M cells, erythrocytes, or polymorphonuclear leukocytes. This cell specificity of attachment suggests a greater density of membrane pili binding sites on cells similar or identical to cells from natural sites of infection. The pili binding sites were quantitated as 1 X 10(4) per cervical-vaginal squamous cell. Pili of all antigenic types attached equally to a given cell type, implying that the attachment moiety of each pilus was similar. Attachement of gonoccocal pili to human cells occurred quickly with saturation of presumed receptor sites within 20--60 min. Attachment was temperature dependent (37 degrees greater than 20 degrees greater than 4 degrees C), and pH dependent (3.5 less than 4.5 less than 5.5 less than 7.5). Attachment was inhibited by antibody to pili (homologous pili Ab greater than heterologous Ab). The extent of possible protection against gonococcal infection due to inhibition of pili-mediated attachment might prove limited as a result of the considerable antigenic heterogeneity among pili and the observation that blockage of pili attachment is maximal only with antibody to pili of the infecting strain. Images PMID:96134

  7. The sac mutants of Chlamydomonas reinhardtii reveal transcriptional and posttranscriptional control of cysteine biosynthesis.

    PubMed

    Ravina, Cristina G; Chang, Chwenn-In; Tsakraklides, George P; McDermott, Jeffery P; Vega, Jose M; Leustek, Thomas; Gotor, Cecilia; Davies, John P

    2002-12-01

    Algae and vascular plants are cysteine (Cys) prototrophs. They are able to import, reduce, and assimilate sulfate into Cys, methionine, and other organic sulfur-containing compounds. Characterization of genes encoding the enzymes required for Cys biosynthesis from the unicellular green alga Chlamydomonas reinhardtii reveals that transcriptional and posttranscriptional mechanisms regulate the pathway. The derived amino acid sequences of the C. reinhardtii genes encoding 5'-adenylylsulfate (APS) reductase and serine (Ser) acetyltransferase are orthologous to sequences from vascular plants. The Cys biosynthetic pathway of C. reinhardtii is regulated by sulfate availability. The steady-state level of transcripts and activity of ATP sulfurylase, APS reductase, Ser acetyltransferase, and O-acetyl-Ser (thiol) lyase increase when cells are deprived of sulfate. The sac1 mutation, which impairs C. reinhardtii ability to acclimate to sulfur-deficient conditions, prevents the increase in accumulation of the transcripts encoding these enzymes and also prevents the increase in activity of all the enzymes except APS reductase. The sac2 mutation, which does not affect accumulation of APS reductase transcripts, blocks the increase in APS reductase activity. These results suggest that APS reductase activity is regulated posttranscriptionally in a SAC2-dependent process. PMID:12481091

  8. The anuran vocal sac: a tool for multimodal signalling

    PubMed Central

    Starnberger, Iris; Preininger, Doris; Hödl, Walter

    2014-01-01

    Although in anurans the predominant mode of intra- and intersexual communication is vocalization, modalities used in addition to or instead of acoustic signals range from seismic and visual to chemical. In some cases, signals of more than one modality are produced through or by the anuran vocal sac. However, its role beyond acoustics has been neglected for some time and nonacoustic cues such as vocal sac movement have traditionally been seen as an epiphenomenon of sound production. The diversity in vocal sac coloration and shape found in different species is striking and recently its visual properties have been given a more important role in signalling. Chemosignals seem to be the dominant communication mode in newts, salamanders and caecilians and certainly play a role in the aquatic life phase of anurans, but airborne chemical signalling has received less attention. There is, however, increasing evidence that at least some terrestrial anuran species integrate acoustic, visual and chemical cues in species recognition and mate choice and a few secondarily mute anuran species seem to fully rely on volatile chemical cues produced in glands on the vocal sac. Within vertebrates, frogs in particular are suitable organisms for investigating multimodal communication by means of experiments, since they are tolerant of disturbance by observers and can be easily manipulated under natural conditions. Thus, the anuran vocal sac might be of great interest not only to herpetologists, but also to behavioural biologists studying communication systems. PMID:25389375

  9. The anuran vocal sac: a tool for multimodal signalling.

    PubMed

    Starnberger, Iris; Preininger, Doris; Hödl, Walter

    2014-11-01

    Although in anurans the predominant mode of intra- and intersexual communication is vocalization, modalities used in addition to or instead of acoustic signals range from seismic and visual to chemical. In some cases, signals of more than one modality are produced through or by the anuran vocal sac. However, its role beyond acoustics has been neglected for some time and nonacoustic cues such as vocal sac movement have traditionally been seen as an epiphenomenon of sound production. The diversity in vocal sac coloration and shape found in different species is striking and recently its visual properties have been given a more important role in signalling. Chemosignals seem to be the dominant communication mode in newts, salamanders and caecilians and certainly play a role in the aquatic life phase of anurans, but airborne chemical signalling has received less attention. There is, however, increasing evidence that at least some terrestrial anuran species integrate acoustic, visual and chemical cues in species recognition and mate choice and a few secondarily mute anuran species seem to fully rely on volatile chemical cues produced in glands on the vocal sac. Within vertebrates, frogs in particular are suitable organisms for investigating multimodal communication by means of experiments, since they are tolerant of disturbance by observers and can be easily manipulated under natural conditions. Thus, the anuran vocal sac might be of great interest not only to herpetologists, but also to behavioural biologists studying communication systems. PMID:25389375

  10. ELECTRON MICROSCOPIC OBSERVATIONS OF THE DEVELOPMENT OF COXIELLA BURNETII IN THE CHICK YOLK SAC1

    PubMed Central

    Anacker, R. L.; Fukushi, K.; Pickens, E. G.; Lackman, D. B.

    1964-01-01

    Anacker, R. L. (Rocky Mountain Laboratory, Hamilton, Mont.), K. Fukushi, E. G. Pickens, and D. B. Lackman. Electron microscopic observations of the development of Coxiella burnetii in the chick yolk sac. J. Bacteriol. 88:1130–1138. 1964.—Yolk sac material, obtained daily over a period of 1 week from embryos inoculated with seed of phase I Coxiella burnetii strain Ohio 314 containing 250 units of penicillin, was examined by electron microscopy and other techniques for the presence of rickettsiae. The concentration of rickettsiae in the yolk sac, as determined by electron microscopy, light microscopy, the complement-fixation test, recovery of organisms, and mouse infectivity, was low for the first 3 days, increased rapidly 3 to 5 days after infection, and then remained relatively constant. Rickettsiae in 3- to 7-day cultures, when observed by electron microscopy, had dense fibrillar centers surrounded by less-dense cytoplasmic material containing granules approximately 15 mμ in diameter. The whole was enclosed by multiple external layers. Many appeared to be in various stages of binary fission, and one form which contained a cross-wall was observed. These forms readily combined with ferritin-labeled specific antibody. In rare instances, several kinds of ”atypical” forms which did not combine with ferritin-labeled antibody were found in the cytoplasm of yolk-sac cells 4 to 5 days after inoculation; it is not certain whether these forms are artifacts or normal stages in the maturation of C. burnetii. These atypical forms were not observed in subsequent experiments in which embryonated eggs were inoculated with doses of penicillin varying from 0 to 4,000 units per egg. Images PMID:14219028

  11. Adherence of Clostridium thermocellum to cellulose.

    PubMed Central

    Bayer, E A; Kenig, R; Lamed, R

    1983-01-01

    The adherence of Clostridium thermocellum, a cellulolytic, thermophilic anaerobe, to its insoluble substrate (cellulose) was studied. The adherence phenomenon was determined to be selective for cellulose. The observed adherence was not significantly affected by various parameters, including salts, pH, temperature, detergents, or soluble sugars. A spontaneous adherence-defective mutant strain (AD2) was isolated from the wild-type strain YS. Antibodies were prepared against the bacterial cell surface and rendered specific to the cellulose-binding factor (CBF) by adsorption to mutant AD2 cells. By using these CBF-specific antibodies, crossed immunoelectrophoresis of cell extracts revealed a single discrete precipitation peak in the parent strain which was absent in the mutant. This difference was accompanied by an alteration in the polypeptide profile whereby sonicates of strain YS contained a 210,000-molecular-weight band which was missing in strain AD2. The CBF antigen could be removed from cell extracts by adsorption to cellulose. A combined gel-overlay--immunoelectrophoretic technique demonstrated that the cellulose-binding properties of the CBF were accompanied by carboxymethylcellulase activity. During the exponential phase of growth, a large part of the CBF antigen and related carboxymethylcellulase activity was associated with the cells of wild-type strain YS. However, the amounts decreased in stationary-phase cells. Cellobiose-grown mutant AD2 cells lacked the cell-associated CBF, but the latter was detected in the extracellular fluid. Increased levels of CBF were observed when cells were grown on cellulose. In addition, mutant AD2 regained cell-associated CBF together with the property of cellulose adherence. The presence of the CBF antigen and related adherence characteristics appeared to be a phenomenon common to other naturally occurring strains of this species. Images PMID:6630152

  12. Giant dacryocystomucopyocele in an adult: a review of lacrimal sac enlargements with clinical and histopathologic differential diagnoses.

    PubMed

    Perry, Lynn J P; Jakobiec, Frederick A; Zakka, Fouad R; Rubin, Peter A D

    2012-09-01

    Dacryocystocele is an umbrella term that refers to any diffuse, centrifugal enlargement of the lacrimal sac that results from combined proximal and distal obstructions in the tear drainage system. In adults, the presence of mucus in the cyst's contents leads to the modified term of dacryocystomucocele. If infection supervenes, which almost always occurs in protracted cases and adds the clinical dimension of a dacryocystitis, then a dacryocystomucopyocele is created. Dacryocystocele and its congeners are much rarer in adults than in children. We describe a 95-year-old woman with an acquired, enormous dacryocystomucopyocele, larger than any previously reported, that developed over 25 years and produced globe displacement with an associated conspicuous enlargement of the nasolacrimal duct. The aspirated sac fluid was mucopurulent and harbored low-virulence bacterial organisms of the Prevotella and Petosteptococcus species. In infants, dacryocystoceles are transitory as the result of spontaneously reversible factors. In adults, secondary proximal irreversible fibrotic strictures or bony changes around the nasolacrimal duct typically arise from chronic inflammation or low grade infection. Other possible causations of duct obstruction, in addition to florid mucosal edema, include encroachment on the duct by enlarged contiguous ethmoid air cells; a sinus mucocele or sinusitis; idiopathic, post-traumatic or dysplastic bony remodeling of the wall of the duct; and a neoplasm-all of which require some form of surgical intervention, typically dacryocystorhinostomy. The differential diagnosis of medial canthal swellings centered on the lacrimal sac spans malformations, diverticula, dermoid/epidermoid cysts, sac inflammations/infections causing swelling without generalized sac enlargement, encephaloceles and primary epithelial tumors, as well as extrinsic tumors impinging on the sac. PMID:22784678

  13. Enhancing adherence through education.

    PubMed

    Smrtka, Jennifer; Caon, Christina; Saunders, Carol; Becker, Brenda L; Baxter, Nancy

    2010-10-01

    The treatment of multiple sclerosis (MS) has advanced greatly since the introduction of disease-modifying therapies (DMTs) in the early 1990s. Although the DMTs have exhibited significant efficacy in relapsing-remitting MS and other forms of the disease, the degree of benefit depends heavily on patient adherence to recommended regimens. This article addresses some of the most pressing areas of unmet need in educating advanced-practice nurses, neurologists, patients, and support care partners regarding strategies that can overcome obstacles to adherence. The observations presented here are based on clinical experience with real-life cognitive, psychosocial, and cultural impediments to adherence. The article also explores the ways in which adherence may be affected by emerging therapies for MS (such as oral agents) as well as the educational needs that will arise with the further evolution of MS care. PMID:21049830

  14. Adherence to Insulin Therapy.

    PubMed

    Sarbacker, G Blair; Urteaga, Elizabeth M

    2016-08-01

    IN BRIEF Six million people with diabetes use insulin either alone or in combination with an oral medication. Many barriers exist that lead to poor adherence with insulin. However, there is an underwhelming amount of data on interventions to address these barriers and improve insulin adherence. Until pharmacological advancements create easier, more acceptable insulin regimens, it is imperative to involve patients in shared decision-making. PMID:27574371

  15. Coccoid and spiral Helicobacter pylori differ in their abilities to adhere to gastric epithelial cells and induce interleukin-8 secretion.

    PubMed Central

    Cole, S P; Cirillo, D; Kagnoff, M F; Guiney, D G; Eckmann, L

    1997-01-01

    Helicobacter pylori exists as an actively dividing spiral form and a nonculturable, but viable, metabolizing coccoid form. Both forms are present in the stomach, but their relative pathophysiologic significances are unknown. Here we show that the coccoid form of H. pylori, in contrast to the spiral form, binds poorly to gastric epithelial cells and induces little, if any, interleukin-8 secretion by these cells. PMID:9009355

  16. 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

  17. Fast frequency hopping codes applied to SAC optical CDMA network

    NASA Astrophysics Data System (ADS)

    Tseng, Shin-Pin

    2015-06-01

    This study designed a fast frequency hopping (FFH) code family suitable for application in spectral-amplitude-coding (SAC) optical code-division multiple-access (CDMA) networks. The FFH code family can effectively suppress the effects of multiuser interference and had its origin in the frequency hopping code family. Additional codes were developed as secure codewords for enhancing the security of the network. In considering the system cost and flexibility, simple optical encoders/decoders using fiber Bragg gratings (FBGs) and a set of optical securers using two arrayed-waveguide grating (AWG) demultiplexers (DeMUXs) were also constructed. Based on a Gaussian approximation, expressions for evaluating the bit error rate (BER) and spectral efficiency (SE) of SAC optical CDMA networks are presented. The results indicated that the proposed SAC optical CDMA network exhibited favorable performance.

  18. SAC-C mission, an example of international cooperation

    NASA Astrophysics Data System (ADS)

    Colomb, F. R.; Alonso, C.; Hofmann, C.; Nollmann, I.

    2004-01-01

    The SAC-C is an international Earth observing satellite mission developed as a partnership between CONAE and NASA, with additional support in instrumentation and satellite development from the Danish DSRI, the Italian ASI, the French CNES and the Brazilian INPE. The SAC-C satellite was successfully launched by a Delta II rocket on November 21, 2000, from Vandenberg AFB, California, USA. The purpose of this mission is to carry out observations which bear interest both for the USA and Argentina, thus contributing effectively to NASA's Earth Science Program and to CONAE's National Space Program. The inclusion of SAC-C in the "morning constellation", jointly with NASA satellites Landsat 7, EO 1 and Terra, is another example of an important international cooperation which strengthens the output of any single mission.

  19. Aquarius and the Aquarius/SAC-D Mission

    NASA Technical Reports Server (NTRS)

    LeVine, D. M.; Lagerloef, G. S. E.; Torrusio, S.

    2010-01-01

    Aquarius is a combination L-band radiometer and scatterometer designed to map the salinity field at the ocean surface from space. It will be flown on the Aquarius/SAC-D mission, a partnership between the USA space agency (NASA) and Argentine space agency (CONAE). The mission is composed of two parts: (a) The Aquarius instrument being developed as part of NASA.s Earth System Science Pathfinder (ESSP) program; and (b) SAC-D the fourth spacecraft service platform in the CONAE Satellite de Aplicaciones Cientificas (SAC) program. The primary focus of the mission is to monitor the seasonal and interannual variations of the salinity field in the open ocean. The mission also meets the needs of the Argentine space program for monitoring the environment and for hazard detection and includes several instruments related to these goals.

  20. The Collagen-Binding Protein Cnm Is Required for Streptococcus mutans Adherence to and Intracellular Invasion of Human Coronary Artery Endothelial Cells

    PubMed Central

    Abranches, Jacqueline; Miller, James H.; Martinez, Alaina R.; Simpson-Haidaris, Patricia J.; Burne, Robert A.; Lemos, José A.

    2011-01-01

    Streptococcus mutans is considered the primary etiologic agent of dental caries, a global health problem that affects 60 to 90% of the population, and a leading causative agent of infective endocarditis. It can be divided into four different serotypes (c, e, f, and k), with serotype c strains being the most common in the oral cavity. In this study, we demonstrate that in addition to OMZ175 and B14, three other strains (NCTC11060, LM7, and OM50E) of the less prevalent serotypes e and f are able to invade primary human coronary artery endothelial cells (HCAEC). Invasive strains were also significantly more virulent than noninvasive strains in the Galleria mellonella (greater wax worm) model of systemic disease. Interestingly, the invasive strains carried an additional gene, cnm, which was previously shown to bind to collagen and laminin in vitro. Inactivation of cnm rendered the organisms unable to invade HCAEC and attenuated their virulence in G. mellonella. Notably, the cnm knockout strains did not adhere to HCAEC as efficiently as the parental strains did, indicating that the loss of the invasion phenotype observed for the mutants was linked to an adhesion defect. Comparisons of the invasive strains and their respective cnm mutants did not support a correlation between biofilm formation and invasion. Thus, Cnm is required for S. mutans invasion of endothelial cells and possibly represents an important virulence factor of S. mutans that may contribute to cardiovascular infections and pathologies. PMID:21422186

  1. 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

  2. Human Placenta-Derived Adherent Cell Treatment of Experimental Stroke Promotes Functional Recovery after Stroke in Young Adult and Older Rats

    PubMed Central

    Shehadah, Amjad; Chen, Jieli; Pal, Ajai; He, Shuyang; Zeitlin, Andrew; Cui, Xu; Zacharek, Alex; Cui, Yisheng; Roberts, Cynthia; Lu, Mei; Hariri, Robert; Chopp, Michael

    2014-01-01

    Background Human Placenta-Derived Adherent Cells (PDAC®) are a novel mesenchymal-like cell population derived from normal human placental tissue. PDA-001 is a clinical formulation of PDAC® developed for intravenous administration. In this study, we investigated the efficacy of PDA-001 treatment in a rat model of transient middle cerebral artery occlusion (MCAo) in young adult (2–3 month old) and older rats (10–12 months old). Methods To evaluate efficacy and determine the optimal number of transplanted cells, young adult Wistar rats were subjected to MCAo and treated 1 day post MCAo with 1×106, 4×106 or 8×106 PDA-001 cells (i.v.), vehicle or cell control. 4×106 or 8×106 PDA-001 cells were also tested in older rats after MCAo. Treatment response was evaluated using a battery of functional outcome tests, consisting of adhesive-removal test, modified Neurological Severity Score (mNSS) and foot-fault test. Young adult rats were sacrificed 56 days after MCAo, older rats were sacrificed 29 days after MCAo, and lesion volumes were measured using H&E. Immunohistochemical stainings for bromodeoxyuridine (BrdU) and von Willebrand Factor (vWF), and synaptophysin were performed. Results In young adult rats, treatment with 4×106 PDA-001 cells significantly improved functional outcome after stroke (p<0.05). In older rats, significant functional improvement was observed with PDA-001 cell therapy in both of the 4×106 and 8×106 treatment groups. Functional benefits in young adult and older rats were associated with significant increases in the number of BrdU immunoreactive endothelial cells, vascular density and perimeter in the ischemic brain, as well as significantly increased synaptophysin expression in the ischemic border zone (p<0.05). Conclusion PDA-001 treatment significantly improved functional outcome after stroke in both young adult and older rats. The neurorestorative effects induced by PDA-001 treatment may be related to increased vascular density and

  3. An advanced cone-and-plate reactor for the in vitro-application of shear stress on adherent cells.

    PubMed

    Dreyer, Lutz; Krolitzki, Benjamin; Autschbach, Rüdiger; Vogt, Peter; Welte, Tobias; Ngezahayo, Anaclet; Glasmacher, Birgit

    2011-01-01

    Endothelial cells (ECs) are permanently exposed to the blood flow and the resulting shear stress, its magnitude varying with the EC site in the blood stream. Along with other mechanical stimuli like vessel wall stretching or hydrostatic blood pressure, this shear stress modulates the endothelial cell function, morphology and gene expression. Here, we describe our improved cone-and-plate reactor that applies up to 10 dyn/cm(2) uniform wall shear stress on a defined, ring-shaped region on a culture dish. At the same time, a hydrostatic pressure of up to 195 mmHg can be applied by increasing the atmospheric pressure in the incubator box. Gas composition can be controlled additionally, used for maintaining CO2-homeostasis or inducing hypoxic conditions. For better comparability, six cone-and-plate systems can be used at the same time at different rotational velocities. The effects on cell morphology, cytoskeleton and cell alignment can be monitored during application using a laser scanning microscope. Flow conditions have been studied and a sufficient area of uniform wall shear stress could be shown. To exceed 10 dyn/cm2, we suggest an increase in medium viscosity. PMID:22214709

  4. The influence of the air plasma jet on early adherent events of L929 fibroblasts on cell culture polystyrene plate

    NASA Astrophysics Data System (ADS)

    Lee, Jung-Hwan; Kwon, Jae-Sung; Om, Ji-Yeon; Kim, Yong-Hee; Choi, Eun-Ha; Kim, Kwang-Mahn; Kim, Kyoung-Nam

    2012-10-01

    Recently, atmospheric pressure plasma was applied to biological field. The aim of this study was to identify whether the air plasma jet increases fibroblast early attachment under moving motion on the cell culture polystyrene plate. Polystyrene plate was treated with plasma jet using compressed air. After 2 minutes of treatment, L929 was seeded on polystyrene plate as well as on untreated plate. Cells were allowed to attach for 4 hours under 70 RPM. FE-SEM, confocal microscopy and RT-PCR were used to evaluate characters of cells. The results suggested that plasma treatment on the polystyrene plate altered surface energy without change of roughness. In occasion of treatment plate, attached L292 were significantly found but not found on untreated surface. Also, despite the small area of treated center by the flame of the plasma jet, cells were also attached on round surface of the area covered by the flame, which suggests that the effect was not only due to the jet flame but perhaps due to the jet interacting with surrounding atmosphere. In the light of this study, the air plasma jet could be useful for early attachment of L292 on the polystyrene plate under moving motion and can be applied to biomaterials.

  5. Reproducible expansion and characterization of mouse neural stem/progenitor cells in adherent cultures derived from the adult subventricular zone

    PubMed Central

    Theus, Michelle H.; Ricard, Jerome; Liebl, Daniel J.

    2012-01-01

    Endogenous neural stem/progenitor cells (NSPCs) residing in the subventricular zone (SVZ) of the adult mouse forebrain have been shown to enhance their neurogenic potential in response to CNS injury. Mechanisms involved in regulating adult neurogenesis under naïve or stressed conditions can be studied using a monolayer cell-culture system of the nestin-expressing NSPC lineage to analyze proliferation, survival and differentiation. Here, we describe a protocol for the expansion of NSPCs for studies aimed at understanding the functional role of NSPCs in maintaining adult neurogenic processes. In this unit, we outline in detail the procedures for: (1) isolation, maintenance and culture of the NSPC component of the SVZ niche from the lateral wall of the lateral ventricle; (2) characterization of NSPC functions by examining proliferation, survival and differentiation; and (3) efficient siRNA transfection methods in 96-well format. PMID:22415840

  6. Complete structure of the cell surface polysaccharide of Streptococcus oralis ATCC 10557: A receptor for lectin-mediated interbacterial adherence

    SciTech Connect

    Abeygunawardana, C.; Bush, C.A. ); Cisar, J.O. )

    1991-07-02

    Lectin-carbohydrate binding is known to play an important role in a number of different cell-cell interactions including those between certain species of oral streptococci and actinomyces that colonize teeth. The cell wall polysaccharides of Streptococcus oralis ATCC 10557, S. oralis 34, and Streptococcus mitis J22, although not identical antigenically, each function as a receptor molecule for the galactose and N-acetylgalactosamine reactive fimbrial lectins of Actinomyces viscosus and Actinomyces naeslundii. Carbohydrate analysis of the receptor polysaccharide isolated from S. oralis ATCC 10557 shows galactose (3 mol), glucose (1 mol), GalNAc (1 mol), and rhamnose (1 mol). {sup 1}H NMR spectra of the polysaccharide show that is partially O-acetylated. Analysis of the {sup 1}H NMR spectrum of the de-O-acetylated polysaccharide shows that it is composed of repeating subunits containing six monosaccharides and that the subunits are joined by a phosphodiester linkage. The {sup 1}H and {sup 13}C NMR spectra were completely assigned by two-dimensional homonuclear correlation methods and by {sup 1}H-detected heteronuclear multiple-quantum correlation ({sup 1}H({sup 13}C)HMQC). The complete {sup 1}H and {sup 13}C assignment of the native polysaccharide was carried out by the same techniques augmented by a {sup 13}C-coupled hybrid HMQC-COSY method, which is shown to be especially useful for carbohydrates in which strong coupling and overlapping peaks in the {sup 1}H spectrum pose difficulties.

  7. Graphene oxide sheets-based platform for induced pluripotent stem cells culture: toxicity, adherence, growth and application

    NASA Astrophysics Data System (ADS)

    Durán, Marcela; Andrade, Patricia F.; Durán, Nelson; Luzo, Angela C. M.; Fávaro, Wagner J.

    2015-05-01

    It was prepared the graphene oxide (GO) sheets by suspension of GO in ultrapure deionized water or in Pluronic F-68 using a ultrasonicator bath. Total characterization of GO sheets was carried out. The results on suspension of GO in water showed excellent growth and cell adhesion. GO/Pluronic F-68 platform for the growth and adhesion of adipose-derived stem cells (ASCs) that exhibits excellent properties for these processes. GO in water suspension exhibited an inhibition of the cell growth over 5 μg/mL In vivo study with GO suspended in water (100 μg/mL) on Fisher 344 rats via i.p. administration showed low toxicity. Despite GO particle accumulates in the intraperitoneal cavity, this fact did not interfere with the final absorption of GO. The AST (aspartate aminotransferase) and ALT (alanine aminotransferase) levels (liver function) did not differ statistically in all experimental groups. Also, creatinine and urea levels (renal function) did not differ statistically in all experimental groups. Taking together, the data suggest the great potential of graphene oxide sheets as platform to ACSs, as well as, new material for treatment several urological diseases.

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

    PubMed

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

    2002-07-01

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

  9. Adherence to Sublingual Immunotherapy.

    PubMed

    Incorvaia, Cristoforo; Mauro, Marina; Leo, Gualtiero; Ridolo, Erminia

    2016-02-01

    Adherence is a major issue in any medical treatment. Allergen immunotherapy (AIT) is particularly affected by a poor adherence because a flawed application prevents the immunological effects that underlie the clinical outcome of the treatment. Sublingual immunotherapy (SLIT) was introduced in the 1990s, and the early studies suggested that adherence and compliance to such a route of administration was better than the traditional subcutaneous route. However, the recent data from manufacturers revealed that only 13% of patients treated with SLIT reach the recommended 3-year duration. Therefore, improved adherence to SLIT is an unmet need that may be achieved by various approaches. The utility of patient education and accurate monitoring during the treatment was demonstrated by specific studies, while the success of technology-based tools, including online platforms, social media, e-mail, and a short message service by phone, is currently considered to improve the adherence. This goal is of pivotal importance to fulfill the object of SLIT that is to modify the natural history of allergy, ensuring a long-lasting clinical benefit, and a consequent pharmaco-economic advantage, when patients complete at least a 3-year course of treatment. PMID:26758865

  10. Interaction and assembly of two novel proteins in the spore wall of the microsporidian species Nosema bombycis and their roles in adherence to and infection of host cells.

    PubMed

    Yang, Donglin; Pan, Guoqing; Dang, Xiaoqun; Shi, Yawei; Li, Chunfeng; Peng, Pai; Luo, Bo; Bian, Maofei; Song, Yue; Ma, Cheng; Chen, Jie; Ma, Zhengang; Geng, Lina; Li, Zhi; Tian, Rui; Wei, Cuifang; Zhou, Zeyang

    2015-04-01

    Microsporidia are obligate intracellular parasites with rigid spore walls that protect against various environmental pressures. Despite an extensive description of the spore wall, little is known regarding the mechanism by which it is deposited or the role it plays in cell adhesion and infection. In this study, we report the identification and characterization of two novel spore wall proteins, SWP7 and SWP9, in the microsporidian species Nosema bombycis. SWP7 and SWP9 are mainly localized to the exospore and endospore of mature spores and the cytoplasm of sporonts, respectively. In addition, a portion of SWP9 is targeted to the spore wall of sporoblasts earlier than SWP7 is. Both SWP7 and SWP9 are specifically colocalized to the spore wall in mature spores. Furthermore, immunoprecipitation, far-Western blotting, unreduced SDS-PAGE, and yeast two-hybrid data demonstrated that SWP7 interacted with SWP9. The chitin binding assay showed that, within the total spore protein, SWP9 and SWP7 can bind to the deproteinated chitin spore coats (DCSCs) of N. bombycis. However, binding of the recombinant protein rSWP7-His to the DCSCs is dependent on the combination of rSWP9-glutathione S-transferase (GST) with the DCSCs. Finally, rSWP9-GST, anti-SWP9, and anti-SWP7 antibodies decreased spore adhesion and infection of the host cell. In conclusion, SWP7 and SWP9 may have important structural capacities and play significant roles in modulating host cell adherence and infection in vitro. A possible major function of SWP9 is as a scaffolding protein that supports other proteins (such as SWP7) that form the integrated spore wall of N. bombycis. PMID:25605761

  11. Interaction and Assembly of Two Novel Proteins in the Spore Wall of the Microsporidian Species Nosema bombycis and Their Roles in Adherence to and Infection of Host Cells

    PubMed Central

    Yang, Donglin; Pan, Guoqing; Dang, Xiaoqun; Shi, Yawei; Li, Chunfeng; Peng, Pai; Luo, Bo; Bian, Maofei; Song, Yue; Ma, Cheng; Chen, Jie; Ma, Zhengang; Geng, Lina; Li, Zhi; Tian, Rui; Wei, Cuifang

    2015-01-01

    Microsporidia are obligate intracellular parasites with rigid spore walls that protect against various environmental pressures. Despite an extensive description of the spore wall, little is known regarding the mechanism by which it is deposited or the role it plays in cell adhesion and infection. In this study, we report the identification and characterization of two novel spore wall proteins, SWP7 and SWP9, in the microsporidian species Nosema bombycis. SWP7 and SWP9 are mainly localized to the exospore and endospore of mature spores and the cytoplasm of sporonts, respectively. In addition, a portion of SWP9 is targeted to the spore wall of sporoblasts earlier than SWP7 is. Both SWP7 and SWP9 are specifically colocalized to the spore wall in mature spores. Furthermore, immunoprecipitation, far-Western blotting, unreduced SDS-PAGE, and yeast two-hybrid data demonstrated that SWP7 interacted with SWP9. The chitin binding assay showed that, within the total spore protein, SWP9 and SWP7 can bind to the deproteinated chitin spore coats (DCSCs) of N. bombycis. However, binding of the recombinant protein rSWP7-His to the DCSCs is dependent on the combination of rSWP9–glutathione S-transferase (GST) with the DCSCs. Finally, rSWP9-GST, anti-SWP9, and anti-SWP7 antibodies decreased spore adhesion and infection of the host cell. In conclusion, SWP7 and SWP9 may have important structural capacities and play significant roles in modulating host cell adherence and infection in vitro. A possible major function of SWP9 is as a scaffolding protein that supports other proteins (such as SWP7) that form the integrated spore wall of N. bombycis. PMID:25605761

  12. Abnormalities of polymorphonuclear leukocyte function associated with a heritable deficiency of high molecular weight surface glycoproteins (GP138): common relationship to diminished cell adherence.

    PubMed Central

    Anderson, D C; Schmalstieg, F C; Arnaout, M A; Kohl, S; Tosi, M F; Dana, N; Buffone, G J; Hughes, B J; Brinkley, B R; Dickey, W D

    1984-01-01

    Investigations of polymorphonuclear leukocyte (PMN) function were performed in a 5-yr-old white female with delayed umbilical cord separation, impaired pus formation, and a severe defect of PMN chemotaxis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated an almost total deficiency of a high molecular weight glycoprotein(s) (GP138) in the granule and membrane fractions of the patient's cells, and NaB3H4-galactose oxidase labeling demonstrated the absence of a major glycoprotein complex on the surface of her PMNs. Monoclonal antibodies (MAb) were employed in flow cytometry experiments to demonstrate that two previously characterized glycoproteins (Mo1 and LFA1) were undetectable on the surface of the patient's PMNs and monocytes. Immunoprecipitation of 125I-labeled patient cells with subunit specific MAbs confirmed that the alpha-subunits of Mo1 (155 kD) and LFA1 (177 kD) and their common beta-subunit (94 kD) were totally deficient. Functional analyses of patient PMNs demonstrated severe impairment of adherence- and adhesion-dependent cell functions including spreading, aggregation, orientation in chemotactic gradients, antibody-dependent cellular cytotoxicity, and phagocytosis of particles (Oil-Red-0-paraffin, zymosan) selectively opsonized with C3-derived ligands. Patient PMNs demonstrated a normal capacity to rosette with IgG or C3b-coated sheep erythrocytes, but rosette formation with C3bi-coated erythrocytes was profoundly diminished. Adhesion-independent functions including shape change, N-formyl-methionyl-leucyl-3H-phenylalanine binding, and O-2 generation or secretion elicited by soluble stimuli were normal. Membrane fluidity, surface charge, and microtubule assembly were also normal. These findings provide new evidence that critical PMN surface glycoproteins are required to facilitate multiple adhesion-dependent cellular functions of the inflammatory response. Images PMID:6746906

  13. Medium-throughput ESR detection of superoxide production in undetached adherent cells using cyclic nitrone spin traps.

    PubMed

    Abbas, K; Hardy, M; Poulhès, F; Karoui, H; Tordo, P; Ouari, O; Peyrot, F

    2015-01-01

    Spin trapping with cyclic nitrones coupled to electron spin resonance (ESR) is recognized as a specific method of detection of oxygen free radicals in biological systems, especially in culture cells. In this case, the detection is usually performed on cell suspensions, which is however unsuitable when adhesion influences free radical production. Here, we performed ESR detection of superoxide with four spin traps (5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide, DEPMPO; 5-diisopropoxyphosphoryl-5-methyl-1-pyrroline N-oxide, DIPPMPO; (4R*, 5R*)-5-(diisopropyloxyphosphoryl)-5-methyl-4-[({[2-(triphenylphosphonio)ethyl]carbamoyl}oxy)methyl]pyrroline N-oxide bromide, Mito-DIPPMPO; and 6-monodeoxy-6-mono-4-[(5-diisopropoxyphosphoryl-5-methyl-1-pyrroline-N-oxide)-ethylenecarbamoyl-(2,3-di-O-methyl) hexakis (2,3,6-tri-O-methyl)]-β-cyclodextrin, CD-DIPPMPO) directly on RAW 264.7 macrophages cultured on microscope coverslip glasses after phorbol 12-myristate 13-acetate (PMA) stimulation. Distinct ESR spectra were obtained with each spin trap using this method. CD-DIPPMPO, a recently published phosphorylated cyclic nitrone bearing a permethylated β-cyclodextrin moiety, was confirmed as the most specific spin trap of the superoxide radical, with exclusive detection of the superoxide adduct. ESR detection performed on cells attached to coverslips represents significant advances over other methods in terms of simplicity, speed, and measurement under near-physiological conditions. It thus opens the way for numerous applications, such as medium-throughput screening of antioxidants and reactive oxygen species (ROS)-modulating agents. PMID:25968949

  14. Type IV Pili in Francisella tularensis: Roles of pilF and pilT in Fiber Assembly, Host Cell Adherence, and Virulence ▿

    PubMed Central

    Chakraborty, Subhra; Monfett, Michael; Maier, Tamara M.; Benach, Jorge L.; Frank, Dara W.; Thanassi, David G.

    2008-01-01

    Francisella tularensis, a highly virulent facultative intracellular bacterium, is the causative agent of tularemia. Genome sequencing of all F. tularensis subspecies revealed the presence of genes that could encode type IV pili (Tfp). The live vaccine strain (LVS) expresses surface fibers resembling Tfp, but it was not established whether these fibers were indeed Tfp encoded by the pil genes. We show here that deletion of the pilF putative Tfp assembly ATPase in the LVS resulted in a complete loss of surface fibers. Disruption of the pilT putative disassembly ATPase also caused a complete loss of pili, indicating that pilT functions differently in F. tularensis than in model Tfp systems such as those found in Pseudomonas aeruginosa and Neisseria spp. The LVS pilF and pilT mutants were attenuated for virulence in a mouse model of tularemia by the intradermal route. Furthermore, although absence of pili had no effect on the ability of the LVS to replicate intracellularly, the pilF and pilT mutants were defective for adherence to macrophages, pneumocytes, and hepatocytes. This work confirms that the surface fibers expressed by the LVS are encoded by the pil genes and provides evidence that the Francisella pili contribute to host cell adhesion and virulence. PMID:18426883

  15. Unusual presentation of Enterobius vermicularis in conjunctival sac.

    PubMed

    Mallick, Sanjay Kumar; Sengupta, Ranadeep; Banerjee, Arup Kumar

    2015-10-01

    We report an unusual case of extraintestinal infection with adult Enterobius vermicularis worms in the conjunctival sac of a two-and-a-half year old boy from Alipurduar, West Bengal, India. Only two other similar cases have been reported in the English literature, one from Assam, India in 1976, and the other from Illinois and California in 2011. PMID:25540166

  16. Combination Chemotherapy in Treating Young Patients With Recurrent or Resistant Malignant Germ Cell Tumors

    ClinicalTrials.gov

    2016-04-12

    Childhood Extracranial Germ Cell Tumor; Childhood Extragonadal Germ Cell Tumor; Childhood Malignant Ovarian Germ Cell Tumor; Childhood Malignant Testicular Germ Cell Tumor; Ovarian Choriocarcinoma; Ovarian Embryonal Carcinoma; Ovarian Yolk Sac Tumor; Recurrent Childhood Malignant Germ Cell Tumor; Recurrent Malignant Testicular Germ Cell Tumor; Recurrent Ovarian Germ Cell Tumor; Testicular Choriocarcinoma; Testicular Choriocarcinoma and Embryonal Carcinoma; Testicular Choriocarcinoma and Yolk Sac Tumor; Testicular Embryonal Carcinoma; Testicular Embryonal Carcinoma and Yolk Sac Tumor; Testicular Yolk Sac Tumor

  17. Comparative adherence of granulocytes to endothelial monolayers and nylon fiber.

    PubMed

    MacGregor, R R; Macarak, E J; Kefalides, N A

    1978-03-01

    Adherence of granulocytes to tissue culture monolayers of endothelium averaged 26.2 +/- 1.3% SEM, which was similar to their adherence on 50-mg nylon fiber columns (27.7 +/- 3.6%). In contrast, adherence to epithelial cells, fibroblasts, kidney cells, and plastic Petri dishes without monolayers was only 12.4, 9.9, 11.1, and 4.3%, respectively. Cyclic nucleotides and adherence-modifying plasma factors induced changes of adherence to endothelium similar to those in nylon fiber columns. Adherence of granulocytes in whole blood was the same as for purified granulocytes in Hank's balanced salt solution. Exposure of endothelial monolayers to 0.18% trypsin for 10 min reduced subsequent granulocyte adherence to 25.2% of control values. Incubation of trypsin-treated monolayers with nutrient medium for 4 h did not improve adherence, but values returned to normal or above by 24 h, with or without serum proteins present in the nutrient medium. The similarity of granulocyte adherence to nylon fiber and to endothelial monolayers in vitro suggests that results with the nylon fiber assay reflect in vivo granulocyte-endothelium interaction. Furthermore, the endothelial monolayer offers a new model for studying this cell-cell relationship in vitro. PMID:641148

  18. Yolk sac tumor of the external auditory canal: a case report and literature review.

    PubMed

    Shi, Huijuan; Tang, Qionglan; Zhen, Tiantian; Li, Hui; Zhang, Fenfen; Han, Anjia

    2015-01-01

    We report one case of yolk sac tumor of the ear and review the literature. The patient was a 9-month boy who scratched his right ear repeatedly one month ago. Computed tomography scan showed an irregular elongated mass image measuring 42×16 mm was found in the right external auditory canal. The tumor was located underneath of the epidermis with ulceration. Mild or moderate atypical round or oval tumor cells were arranged in nest and reticular pattern around vesicular or cystic spaces. Tumor cells had abundant eosinophilic or clear cytoplasm and marked nucleoli. Mitotic figures were about 7/10 HPF. Poorly formed Schiller-Duvall body was occasionally present. The stroma was loose and rich in capillaries. Hyaline globules could be found in the stroma. Immunohistochemistry staining showed that tumor cells were positive for cytokeratin, SALL4, glypican-3, focal positive for EMA, vimentin, CD10, and CD34, but negative for a-fetoprotein, HCG, PLAP. The serum α-fetoprotein was 664.60 ng/mL (normal, ≤ 25 ng/mL). Yolk sac tumor of the ear is extremely rare, especially α-fetoprotein negative expression in our case. The differential diagnosis includes embryonal rhabdomyosarcoma, paraganglioma, myoepithelioma, carcinoma of skin appendages, and metastatic renal cell carcinoma. PMID:26823835

  19. Adherence to treatment in adolescents

    PubMed Central

    Taddeo, Danielle; Egedy, Maud; Frappier, Jean-Yves

    2008-01-01

    Health care professionals must be alert to the high prevalence of low adherence to treatment during adolescence. Low adherence increases morbidity and medical complications, contributes to poorer quality of life and an overuse of the health care system. Many different factors have an impact on adherence. However, critical factors to consider in teens are their developmental stage and challenges, emotional issues and family dysfunction. Direct and indirect methods have been described to assess adherence. Eliciting an adherence history is the most useful way for clinicians to evaluate adherence, and could be the beginning of a constructive dialogue with the adolescent. Interventions to improve adherence are multiple – managing mental health issues appropriately, building a strong relationship, customizing the treatment regimen if possible, empowering the adolescent to deal with adherence issues, providing information, ensuring family and peer support, and motivational enhancement therapy. Evaluation of adherence at regular intervals should be an important aspect of health care for adolescents. PMID:19119348

  20. Primary orbital yolk sac tumor: report of a case and review of literature.

    PubMed

    Kamal, Saurabh; Kaliki, Swathi; Sreedhar, Ani; Mishra, Dilip K

    2016-06-01

    Germ cell tumor can affect extragonadal sites. Teratoma is a well-recognized extragonadal tumor in the orbit. Primary yolk sac tumor (YST) or endodermal sinus tumor of orbit is rare and only few cases have been reported in the literature. Its clinical presentation may mimic many common pediatric orbital conditions, and delay in diagnosis affects ocular morbidity and mortality. In the past orbital YST has been treated with multimodal therapy including surgery, systemic chemotherapy, and radiotherapy. Herein we describe a case of primary orbital YST and reviewed the literature for similar cases. The review aims to describe the clinical presentation, imaging features, histopathological characteristics, and management of orbital YST. PMID:26481249

  1. 38 CFR Appendix A to Part 41 - Data Collection Form (Form SF-SAC)

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 2 2010-07-01 2010-07-01 false Data Collection Form (Form SF-SAC) A Appendix A to Part 41 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS.... Appendix A to Part 41—Data Collection Form (Form SF-SAC) Note: Data Collection Form SF-SAC and...

  2. Teratogenic effects of amniotic sac puncture: a mouse model.

    PubMed Central

    MacIntyre, D J; Chang, H H; Kaufman, M H

    1995-01-01

    The possibility of an association between chorionic villus sampling (cvs) and limb abnormalities has prompted a review of the relevant experimental data. Although a vascular aetiology is favoured by many at present, the possibility exists that a proportion of cases may be caused by oligohydramnios secondary to inadvertent amniotic sac puncture. A mouse model of amniotic puncture syndrome has been developed to study the craniofacial and limb abnormalities produced by this procedure. Pregnant mice were anaesthetised and a laparotomy performed. One uterine horn was exteriorised, and the amniotic sacs punctured through the wall of the uterus with either a 21 gauge or a 25 gauge needle. The conceptuses in the contralateral uterine horn acted as controls. The mice were all killed on d 19 of pregnancy (day of finding a vaginal plug = d 1 of pregnancy) by cervical dislocation, and the morphological features of the embryos examined in detail. In a preliminary study, amniotic sac puncture was carried out on d 12, 13, 14, 15 or 16 of pregnancy, with either a 21 or a 25 gauge needle. Since the highest rates of palatal defects and limb deformities were observed following amniotic sac puncture using a 21 gauge needle, when this procedure was carried out on either d 13 or 14 of pregnancy, the main study was undertaken using a 21 gauge needle on these two days of pregnancy. Of 102 embryos in which amniotic sac puncture was carried out on d 13, 53% survived to d 19. Of the latter, 35% had a cleft palate, 61% had one or more morphologically abnormal limbs, and 43% had an abnormal tail. When amniotic sac puncture was carried out on d 14 of pregnancy, of 83 embryos subjected to this procedure, 81% survived to d 19. Of the latter, 27% had a cleft palate, 39% had one or more morphologically abnormal limbs, and 19% had an abnormal tail. In the controls, of 86 and 61 embryos isolated respectively from the d 13 and 14 mice, the survival rates were 97 and 90%, respectively. Palatal, limb

  3. The Escherichia coli O157:H7 cattle immunoproteome includes outer membrane protein A (OmpA), a modulator of adherence to bovine rectoanal junction squamous epithelial (RSE) cells.

    PubMed

    Kudva, Indira T; Krastins, Bryan; Torres, Alfredo G; Griffin, Robert W; Sheng, Haiqing; Sarracino, David A; Hovde, Carolyn J; Calderwood, Stephen B; John, Manohar

    2015-06-01

    Building on previous studies, we defined the repertoire of proteins comprising the immunoproteome (IP) of Escherichia coli O157:H7 (O157) cultured in DMEM supplemented with norepinephrine (O157 IP), a β-adrenergic hormone that regulates E. coli O157 gene expression in the gastrointestinal tract, using a variation of a novel proteomics-based platform proteome mining tool for antigen discovery, called "proteomics-based expression library screening" (PELS; Kudva et al., 2006). The E. coli O157 IP (O157-IP) comprised 91 proteins, and included those identified previously using proteomics-based expression library screening, and also proteins comprising DMEM and bovine rumen fluid proteomes. Outer membrane protein A (OmpA), a common component of the above proteomes, and reportedly a contributor to E. coli O157 adherence to cultured HEp-2 epithelial cells, was interestingly found to be a modulator rather than a contributor to E. coli O157 adherence to bovine rectoanal junction squamous epithelial cells. Our results point to a role for yet to be identified members of the O157-IP in E. coli O157 adherence to rectoanal junction squamous epithelial cells, and additionally implicate a possible role for the outer membrane protein A regulator, TdcA, in the expression of such adhesins. Our observations have implications for the development of efficacious vaccines for preventing E. coli O157 colonization of the bovine gastrointestinal tract. PMID:25643951

  4. The Escherichia coli O157:H7 cattle immuno-proteome includes outer membrane protein A (OmpA), a modulator of adherence to bovine recto-anal junction squamous epithelial (RSE) cells

    PubMed Central

    Kudva, Indira T.; Krastins, Bryan; Torres, Alfredo G.; Griffin, Robert W.; Sheng, Haiqing; Sarracino, David A.; Hovde, Carolyn J.; Calderwood, Stephen B.; John, Manohar

    2015-01-01

    SUMMARY Building on previous studies, we defined the repertoire of proteins comprising the immuno-proteome of E. coli O157:H7 (O157) cultured in DMEM supplemented with norepinephrine (NE; O157 immuno-proteome), a β-adrenergic hormone that regulates E. coli O157 gene expression in the gastrointestinal tract, using a variation of a novel proteomics-based platform proteome mining tool for antigen discovery, called Proteomics-based Expression Library Screening (PELS; Kudva et al., 2006). The E. coli O157 immuno-proteome (O157-IP) comprised 91 proteins, and included those identified previously using PELS, and also proteins comprising DMEM- and bovine rumen fluid- proteomes. Outer membrane protein A (OmpA), a common component of the above proteomes, and reportedly a contributor to E. coli O157 adherence to cultured Hep-2 epithelial cells, was interestingly found to be a modulator rather than a contributor to E. coli O157 adherence to bovine recto-anal junction squamous epithelial (RSE) cells. Our results point to a role for yet to be identified members of the O157-IP in E. coli O157 adherence to RSE-cells, and additionally implicate a possible role for the OmpA regulator, TdcA, in the expression of such adhesins. Our observations have implications for development of efficacious vaccines for preventing E. coli O157 colonization of the bovine gastrointestinal tract. PMID:25643951

  5. A large mobility of hydrophilic molecules at the outmost layer controls the protein adsorption and adhering behavior with the actin fiber orientation of human umbilical vein endothelial cells (HUVEC).

    PubMed

    Kakinoki, Sachiro; Seo, Ji-Hun; Inoue, Yuuki; Ishihara, Kazuhiko; Yui, Nobuhiko; Yamaoka, Tetsuji

    2013-01-01

    Adhesion behaviors of human umbilical vein endothelial cells (HUVECs) are interestingly affected by the mobility of hydrophilic chains on the material surfaces. Surfaces with different molecular mobilities were prepared using ABA-type block copolymers consisting polyrotaxane (PRX) or poly(ethylene glycol) (PEG) central block (A block), and amphiphilic anchoring B blocks of poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB). Two different molecular mobilities of the PRX chains were designed by using normal α-cyclodextrin (α-CD) or α-CD whose hydroxyl groups were converted to methoxy groups in a given ratio to improve its molecular mobility (PRX-PMB and OMe-PRX-PMB). The surface mobility of these materials was assessed as the mobility factor (Mf), which is measured by quartz crystal microbalance with dissipation monitoring system. HUVECs adhered on OMe-PRX-PMB surface much more than PRX-PMB and PMB-block-PEG-block-PMB (PEG-PMB) surfaces. These different HUVEC adhesions were correlated with the density of cell-binding site of adsorbed fibronectin. In addition, the alignment of the actin cytoskeleton of adhered HUVECs was strongly suppressed on the PEG-PMB, PRX-PMB, and OMe-PRX-PMB in response to the increased Mf value. Remarkably, the HUVECs adhered on the OMe-PRX-PMB surface with much less actin organization. We concluded that not only the cell adhesion but also the cellular function are regulated by the molecular mobility of the outmost material surfaces. PMID:23796033

  6. Efficacy of Several Pesticide Products on Brown Widow Spider (Araneae: Theridiidae) Egg Sacs and Their Penetration Through the Egg Sac Silk.

    PubMed

    Vetter, Richard S; Tarango, Jacob; Campbell, Kathleen A; Tham, Christine; Hayashi, Cheryl Y; Choe, Dong-Hwan

    2016-02-01

    Information on pesticide effects on spiders is less common than for insects; similar information for spider egg sacs is scarcer in the open literature. Spider egg sacs are typically covered with a protective silk layer. When pesticides are directly applied to egg sacs, the silk might prevent active ingredients from reaching the eggs, blocking their insecticidal effect. We investigated the impact of six water-based pesticide sprays and four oil-based aerosol products against egg sacs of brown widow spiders, Latrodectus geometricus C. L. Koch. All water-based spray products except one failed to provide significant mortality to egg sacs, resulting in successful spiderling emergence from treated egg sacs at a similar rate to untreated egg sacs. In contrast to water-based sprays, oil-based aerosols provided almost complete control, with 94-100% prevention of spiderling emergence. Penetration studies using colored pesticide products indicated that oil-based aerosols were significantly more effective in penetrating egg sac silk than were the water-based sprays, delivering the active ingredients on most (>99%) of the eggs inside the sac. The ability of pesticides to penetrate spider egg sac silk and deliver lethal doses of active ingredients to the eggs is discussed in relation to the chemical nature of egg sac silk proteins. Our study suggests that pest management procedures primarily relying on perimeter application of water-based sprays might not provide satisfactory control of brown widow spider eggs. Determination of the most effective active ingredients and carrier characteristics warrant further research to provide more effective control options for spider egg sacs. PMID:26530954

  7. A sperm-specific Na+/H+ exchanger (sNHE) is critical for expression and in vivo bicarbonate regulation of the soluble adenylyl cyclase (sAC)

    PubMed Central

    Wang, Dan; Hu, Jie; Bobulescu, I. Alexandru; Quill, Timothy A.; McLeroy, Paul; Moe, Orson W.; Garbers, David L.

    2007-01-01

    We previously identified a sperm-specific Na+/H+ exchanger (sNHE) principally localized to the flagellum. Disruption of the sNHE gene in mice resulted in absolute male infertility associated with a complete loss of sperm motility. Here, we show that the sNHE-null spermatozoa fail to develop the cAMP-dependent protein tyrosine phosphorylation that coincides with the functional maturation occurring upon incubation in capacitating conditions in vitro. Both the sperm motility defect and the lack of induced protein tyrosine phosphorylation are rescued by the addition of cell-permeable cAMP analogs, suggesting that cAMP metabolism is impaired in spermatozoa lacking sNHE. Our analyses of the bicarbonate-dependent soluble adenylyl cyclase (sAC) signaling pathway in sNHE-null sperm cells reveal that sNHE is required for the expression of full-length sAC, and that it is important for the bicarbonate stimulation of sAC activity in spermatozoa. Furthermore, both codependent expression and coimmunoprecipitation experiments indicate that sNHE and sAC associate with each other. Thus, these two proteins appear to be components of a signaling complex at the sperm flagellar plasma membrane. We propose that the formation of this complex efficiently modulates intracellular pH and bicarbonate levels through the rapid and effective control of sAC and sNHE activities to facilitate sperm motility regulation. PMID:17517652

  8. Escherichia coli O157:H7 Strains That Persist in Feedlot Cattle Are Genetically Related and Demonstrate an Enhanced Ability To Adhere to Intestinal Epithelial Cells

    PubMed Central

    Carlson, Brandon A.; Nightingale, Kendra K.; Mason, Gary L.; Ruby, John R.; Choat, W. Travis; Loneragan, Guy H.; Smith, Gary C.; Sofos, John N.; Belk, Keith E.

    2009-01-01

    A longitudinal study was conducted to investigate the nature of Escherichia coli O157:H7 colonization of feedlot cattle over the final 100 to 110 days of finishing. Rectal fecal grab samples were collected from an initial sample population of 788 steers every 20 to 22 days and microbiologically analyzed to detect E. coli O157:H7. The identities of presumptive colonies were confirmed using a multiplex PCR assay that screened for gene fragments unique to E. coli O157:H7 (rfbE and fliCh7) and other key virulence genes (eae, stx1, and stx2). Animals were classified as having persistent shedding (PS), transient shedding (TS), or nonshedding (NS) status if they consecutively shed the same E. coli O157:H7 genotype (based on the multiplex PCR profile), exhibited variable E. coli O157 shedding, or never shed morphologically typical E. coli O157, respectively. Overall, 1.0% and 1.4% of steers were classified as PS and NS animals, respectively. Characterization of 132 E. coli O157:H7 isolates from PS and TS animals by pulsed-field gel electrophoresis (PFGE) typing yielded 32 unique PFGE types. One predominant PFGE type accounted for 53% of all isolates characterized and persisted in cattle throughout the study. Isolates belonging to this predominant and persistent PFGE type demonstrated an enhanced (P < 0.0001) ability to adhere to Caco-2 human intestinal epithelial cells compared to isolates belonging to less common PFGE types but exhibited equal virulence expression. Interestingly, the attachment efficacy decreased as the genetic divergence from the predominant and persistent subtype increased. Our data support the hypothesis that certain E. coli O157:H7 strains persist in feedlot cattle, which may be partially explained by an enhanced ability to colonize the intestinal epithelium. PMID:19617387

  9. Interferon-Gamma and Nitric Oxide Synthase 2 Mediate the Aggregation of Resident Adherent Peritoneal Exudate Cells: Implications for the Host Response to Pathogens

    PubMed Central

    Chandrasekar, Bhagawat S.; Yadav, Shikha; Victor, Emmanuel S.; Majumdar, Shamik; Deobagkar-Lele, Mukta; Wadhwa, Nitin; Podder, Santosh; Das, Mrinmoy; Nandi, Dipankar

    2015-01-01

    Interferon-gamma (Ifnγ), a key macrophage activating cytokine, plays pleiotropic roles in host immunity. In this study, the ability of Ifnγ to induce the aggregation of resident mouse adherent peritoneal exudate cells (APECs), consisting primarily of macrophages, was investigated. Cell-cell interactions involve adhesion molecules and, upon addition of Ifnγ, CD11b re-localizes preferentially to the sites of interaction on APECs. A functional role of CD11b in enhancing aggregation is demonstrated using Reopro, a blocking reagent, and siRNA to Cd11b. Studies with NG-methyl-L-arginine (LNMA), an inhibitor of Nitric oxide synthase (Nos), NO donors, e.g., S-nitroso-N-acetyl-DL-penicillamine (SNAP) or Diethylenetriamine/nitric oxide adduct (DETA/NO), and Nos2-/- mice identified Nitric oxide (NO) induced by Ifnγ as a key regulator of aggregation of APECs. Further studies with Nos2-/- APECs revealed that some Ifnγ responses are independent of NO: induction of MHC class II and CD80. On the other hand, Nos2 derived NO is important for other functions: motility, phagocytosis, morphology and aggregation. Studies with cytoskeleton depolymerizing agents revealed that Ifnγ and NO mediate the cortical stabilization of Actin and Tubulin which contribute to aggregation of APECs. The biological relevance of aggregation of APECs was delineated using infection experiments with Salmonella Typhimurium (S. Typhimurium). APECs from orally infected, but not uninfected, mice produce high amounts of NO and aggregate upon ex vivo culture in a Nos2-dependent manner. Importantly, aggregated APECs induced by Ifnγ contain fewer intracellular S. Typhimurium compared to their single counterparts post infection. Further experiments with LNMA or Reopro revealed that both NO and CD11b are important for aggregation; in addition, NO is bactericidal. Overall, this study elucidates novel roles for Ifnγ and Nos2 in regulating Actin, Tubulin, CD11b, motility and morphology during the aggregation

  10. Investigation of sulfonated aromatic compound (SAC) modification to nylon film. 2. Study of SAC sorption isotherm and atomic force microscopic characterization of nylon surfaces

    SciTech Connect

    Zhang, J.; Watson, B.A.; Keown, R.W.; Malone, C.P.; Barteau, M.A.

    1995-08-01

    Nylon 6 and nylon 66 films have been treated with aqueous sulfonated aromatic compound (SAC) solutions at concentrations ranging from 0.005 to 1.0 wt%. SAC uptakes at different treatment concentrations were measured and found to follow a BET isotherm. The surface morphologies of nylon film samples, including the original and SAC-treated films, have been characterized by atomic force microscopy (AFM). For untreated nylon 6 and nylon 66 films, AFM images show a randomly distributed fibrillar surface structure. Characteristic widths of fibrils in the nylon 66 and 6 films were 150-225 and 75-150 nm, respectively. For SAC-treated nylon films, the AFM images revealed that the surfaces of the films became covered with nodule-like features having a diameter range of 25-60 nm. AFM analysis provides evidence that SAC treatment deposited a surface coating on nylon films. AFM images of SAC-treated nylon films suggest a mechanism for stain resistance in which the SAC first forms a thin coating on the nylon via bondings between attractive groups in the SAC and nylon polymers. After treatment at increased SAC concentration, the surface is covered with nodule-like deposits which likely serve as a physical barrier to dye permeation. 20 refs., 8 figs.

  11. SAC-C Mission and the Morning Constellation

    NASA Astrophysics Data System (ADS)

    Colomb, F. R.; Alonso, C.; Hofmann, C.; Frulla, L.; Nollmann, I.; Milovich, J.; Kuba, J.; Ares, F.; Kalemkarian, M.

    2002-01-01

    SAC-C is an international Earth observing satellite mission conceived as a partnership between CONAE and NASA, but with additional support in instrumentation and satellite development from the Danish DSRI, the Italian ASI, the French CNES and the Brazilian INPE. A Delta II rocket successfully launched it on November 21st, 2000, from Vandenberg AFB, California, USA. SAC-C has been designed primarily to fulfill the requirements of countries with large extension of territory or scarcely populated like Argentina. Its design is a good compromise between resolution and swath width that makes SAC-C an appropriate tool for global and high dynamic phenomena studies. There are ten instruments on board of SAC-C that will perform different studies, the Multispectral Medium Resolution Scanner (MMRS), provided by CONAE, Argentina, will help in the studies about desertification processes evaluation and their evolution in time (i.e., Patagonia, Argentina), to identify and predict agriculture production, to monitor flood areas and to make studies in coastal and fluvial areas. The MMRS will be associated with a High Resolution Technological Camera (HRTC), also provided by CONAE that will permit improvement in the MMRS resolution in the areas where it will be required. A High Sensitivity Technological Camera (HSTC) is also included in the mission. SAC-C also carries instruments to monitor the condition and dynamics of the terrestrial and marine biosphere and environment (GPS OccuLtation and Passive reflection Experiment (GOLPE)) from NASA/JPL. The Magnetic Mapping Payload, (MMP) developed by the Danish Space Research Institute helps to better understand the Earth's magnetic field and related Sun -Earth interactions .Italian Star Tracker (IST) and Italian Navigation Experiment (INES) developed by the Italian Space Agency, constitute a technological payload that will permit testing a fully autonomous system for attitude and orbit determination. Influence of space radiation on advanced

  12. Spectral Amplitude Coding (SAC)-OCDMA Network with 8DPSK

    NASA Astrophysics Data System (ADS)