Nanopattern-induced changes in morphology and motility of smooth muscle cells

Cells are known to be surrounded by nanoscale topography in their natural extracellular environment. The cell behavior, including morphology, proliferation, and motility of bovine pulmonary artery smooth muscle cells (SMC) were studied on poly(methyl methacrylate) (PMMA) and poly(dimethylsiloxane) (PDMS) surfaces comprising nanopatterned gratings with 350 nm linewidth, 700 nm pitch, and 350 nm depth. More than 90% of the cells aligned to the gratings, and were significantly elongated compared to the SMC cultured on non-patterned surfaces. The nuclei were also elongated and aligned. Proliferation of the cells was significantly reduced on the nanopatterned surfaces. The polarization of microtubule organizing centers (MTOC), which are associated with cell migration, of SMC cultured on nanopatterned surfaces showed a preference towards the axis of cell alignment in an in vitro wound healing assay. In contrast, the MTOC of SMC on non-patterned surfaces preferentially polarized towards the wound edge. It is proposed that this nanoimprinting technology will provide a valuable platform for studies in cell-substrate interactions and for development of medical devices with nanoscale features.     

Discordant quantitative detection of putative biomarkers in nodal micrometastases of colorectal cancer: biological and clinical implications

AIMS: Nodal expression of the carcinoembryonic antigen (CEA), cytokeratin 20 (CK20), and guanylyl cyclase C (GCC) genes was measured in tandem in patients with colorectal cancer (CRC) to assess whether there would be sufficient agreement between these markers in their ability to detect micrometastasis to qualify one of them as a universal marker, and whether frozen and paraffin wax embedded tissues would yield similar results. METHODS: One hundred and seventy five frozen lymph nodes (FT) and 158 formalin fixed, paraffin wax embedded lymph nodes (PET) from 28 CRC cases were analysed using gene specific quantitative real time polymerase chain reaction, carried out on the LightCycler system with SYBR Green chemistry. RESULTS: There was significant disparity in positive detection of the three biomarkers in FT versus PET, with notable agreement achieved only for CEA (66.6%) in FT versus PET in Dukes' B disease, and between CK20 and GCC (44.6%) in FT, also in Dukes' B disease. One patient with full concordance in all three tumour markers with both tissue types suffered a relapse and died within two years of follow up. CONCLUSIONS: There was considerable discordance in the positive detection of the three tumour markers in both tissue types (FT versus PET). This brings into question whether using a single tumour marker to detect micrometastasis in one tissue type (FT or PET) is adequately representative, and challenges the concept of universal markers for molecular CRC metastatic detection. Multiple tumour markers would predict more accurately the metastatic potential of Dukes' B CRCs.     

Expression of superoxide dismutase and xanthine oxidase in myometrium, fetal membranes and placenta during normal human pregnancy and parturition

Superoxide, an agent which attenuates the half-life of nitric oxide, is metabolized and synthesized by superoxide dismutase (SOD) and xanthine oxidase, respectively. Over the last few years much work has focused on the role of nitric oxide in human parturition. The aim of this study was to determine whether the onset of human parturition is associated with a change in the expression of copper/zinc superoxide dismutase (Cu/Zn SOD), manganese superoxide dismutase (Mn SOD) or xanthine oxidase within the uterus. Samples of myometrium, placenta, decidua and fetal membranes were obtained from women before and after the onset of labour at term. Immunocytochemistry was used to localize Cu/Zn SOD, Mn SOD and xanthine oxidase and measure SOD enzyme activity. Cu/Zn and Mn SOD-like immunoreactivity was detected in syncytiotrophoblast cells, villous stromal cells and endothelial cells of blood vessels in the placenta. In the myometrium Cu/Zn and Mn SOD were localized to myocytes and endothelial cells and to some vascular smooth muscle cells. In the fetal membranes we observed staining for Cu/Zn SOD and Mn SOD in the amnion, chorion, extravillous trophoblast and decidua. There was no difference in SOD enzyme activity or staining intensity for SOD between different cell types before and during labour. Xanthine oxidase immunoreactivity was identified in each of the tissues examined and again there was no difference in immunostaining in tissues obtained from women delivered before or after the onset of labour. These results show that the pregnant uterus is capable of both synthesizing and degrading superoxide and suggest that superoxide dismutase and xanthine oxidase may play a role in the maintenance of uterine quiescence during pregnancy, but not in the initiation of parturition.      

High density of immobilized galactose ligand enhances hepatocyte attachment and function

Galactosylated surface is an attractive substrate for hepatocyte culture because of the specific interaction between the galactose ligand and the asialoglycoprotein receptor on hepatocytes. In this study, we described a scheme to achieve high density of immobilized galactose ligands on polyethylene terephthalate (PET) surface by first surface-grafting polyacrylic acid on plasma-pretreated PET film under UV irradiation, followed by conjugation of a galactose derivative (1-O-(6'-aminohexyl)-D-galactopyranoside) to the grafted polyacrylic acid chains. A high galactose density of 513 nmol/cm(2) on the PET surface was used in this study to investigate the behavior of cultured hepatocyte. This engineered substrate showed high affinity to fluorescein isothiocyanate-lectin binding. Primary rat hepatocytes, when seeded at a density of 2 x 10(5) cells/cm(2), attached to the galactosylated PET substrate at a similar efficiency compared with collagen-coated substrate. The hepatocytes spontaneously formed aggregates 1 day after cell seeding and showed better maintenance of albumin secretion and urea synthesis functions than those cultured on collagen-coated surface.     

Endothelin receptor expression in human decidua

The endothelins are signalling peptides that act via two receptors, ET(A) and ET(B). In the human endometrium, endothelin receptors have been demonstrated in glands and stroma and have been shown to vary during the course of the menstrual cycle. The present study was undertaken to determine whether or not expression of endothelin receptors changes during pregnancy or after administration of exogenous progestagens. The expression of the receptors was correlated with the appearance of basement membrane components during decidualization of the endometrial stroma. Decidual specimens (n = 15) were obtained during the first trimester of pregnancy and 10 at term. Sixteen pairs of endometrial biopsies were obtained from women with menorrhagia before and after exposure to exogenous progestagens. A total of 15 hysterectomy specimens were used as controls for the expression of stromal basement membrane proteins in the absence of decidualization. Autoradiography was carried out with selective ligands for ET(A) ([125I]-PD 151242) and ET(B) ([125I]-BQ3020). The distribution of ligand binding was then compared with the distribution of laminin alpha2 light chain and collagen IV. ET(A), ET(B), laminin alpha2 light chain, and collagen IV were expressed in stromal decidual cells in the first trimester of pregnancy. ET(B) was also found on endometrial glandular epithelium. Quantitative macro-autoradiography and multiple regression analysis demonstrated a highly significant positive correlation (P < 0.001) between expression of ET(B) and laminin alpha2 light chain. In the third trimester qualitative examination suggested a reduction of ET(A) in the stroma. Progestagen-induced decidua exhibited a similar pattern to that found in first trimester decidua. This study has demonstrated up-regulation of ET(B) during the progesterone- dependent process of decidualization and suggests a paracrine or autocrine role for endothelins in the decidua.      

Organisation and molecular analysis of repeated DNA sequences in the rice blast fungus Magnaporthe grisea

The distribution of a previously described repeated DNA sequence present as a 1.3-kb PstI fragment in the genome of the rice blast fungus Magnaporthe grisea was analysed by carrying out DNA fingerprint analysis of 36 isolates including rice, non-rice and laboratory strains. The analysis of various higher-molecular-weight PstI fragments with homology to the 1.3-kb repeat revealed that these may arise predominantly from transposon insertions or point mutations. Analysis of a 5.1-kb derivative revealed both a point mutation at a PstI site and an insertion of a putative transposable element which caused an increase in molecular weight from 1.3 to 5.1 kb. Another repeat element of 1.4 kb was identified and found to exist in association with the 1.3-kb repeat. Both 1.3- and 1.4-kb elements were found to be parts of MGR583 (Hamer et al. 1989), a LINE-like element. These elements were present in a high copy number in all the rice and a majority of non-rice pathogens indicating that MGR583 is not a host-specific sequence as reported earlier. Our results suggest that repeated DNA elements in M. grisea have amplified independently of one another and further indicate that different isolates of M. grisea may have evolved from several distinct lines of origin. Received: 12 April / 12 November 1996     

Interaction of group A streptococcal peptidoglycan polysaccharide with human polymorphonuclear leukocytes: implications for pathogenesis of chronic inflammation.

Injection of sterile aqueous preparations of the peptidoglycan polysaccharide of group A streptococci produces chronic inflammation in several animal models. Accordingly, the effect of peptidoglycan and group A-specific polysaccharide (PG-APS) polymers on human polymorphonuclear neutrophil oxidative metabolism was studied with the supposition that this interaction may contribute to the inflammation observed. PG-APS in concentrations of 1.0 to 100 micrograms/ml stimulated oxygen consumption and hexose monophosphate shunt activity in the presence of 10% normal serum in a dose-related manner. Stimulation did not occur in serum-free media and was reduced in media with heat-treated serum. The stimulation of hexose monophosphate shunt activity by PG-APS opsonized with normal serum (bound complement components) and the activated supernatant from which PG-APS had been removed by centrifugation (presumably containing the soluble complement component, C5a) demonstrated 79 and 75%, respectively, of the stimulation with PG-APS in the presence of 10% normal serum. PG-APS triggered release of O2- (3.4 +/- 2.0 nmol by 10(6) human polymorphonuclear neutrophils over 30 min), which was significantly enhanced (9.6 +/- 2.9 nmol O2-) by treatment of cells with cytochalasin B. These results show that PG-APS interacts with serum in such a fashion as to activate human polymorphonuclear neutrophil metabolism and increase secretion of O2-.     

Selection of cytotoxic T lymphocytes against autologous human melanoma from lymph nodes with metastatic melanoma using repeatedin vitro sensitization

Our goal was to determine the cytotoxic activity of effector cells in lymph nodes with metastatic melanoma. Lymphocytes contained within tumor cells from metastatic lymph nodes of two patients were allowed to proliferate in recombinant IL-2 (rIL-2, 100-1,000 units/ml) after 14–21 days of culture. Each set of lymphocytes showed cytotoxicity against autologous melanoma (AM, mean 72%) at effector to target ratio of 201 and K562 cells (mean 60%) using 4-h chromium-51 release assay. Using unlabeled AM and K562, each AM could partially block the activity against K562, but K562 could not block the activity against AM. These activated lymphocytes underwentin vitro sensitization (IVS) with irradiated AM cells and rIL-2 at 2-week intervals. After repeated IVS over about 50 days, each patient's lymphocytes showed cytotoxicity against AM (mean 54%) but not K562 (mean 5%,P < 0.001). These results indicate that different cytotoxic effector cells were present in the early and late phase of lymphocyte tumor culture. Repeated IVS resulted in the selection of specific cytotoxic T lymphocytes. Cold target inhibition assay demonstrated that melanoma cells contained common and individual AM-associated antigen in addition to K562-associated antigens.This work was supported by Biomedical Research Support Grant of the University of Arizona (no. 2S07 RR05675-20), the Elsa U. Pardee Foundation Grant, partly by the Arizona Chronic Disease Research Commission and partly by CA23074 from the National Institutes of Health, Bethesda, 20892, U.S.A.Recipient of the American Cancer Society Clinical Oncology Career Award, 1987–90.     

Adhesion contact dynamics of HepG2 cells on galactose-immobilized substrates

The specific recognition between asialoglycoprotein receptor and galactose ligand at cell-substrate interfaces has been shown to mediate hepatocyte adhesion and maintain liver specific functions of hepatocytes. Conventionally, the success of hepatocyte attachment on engineered tissue scaffold is inferred from the degree of two-dimensional cell spreading that is measured by transmitted light microscopy. However, the actual contact mechanics and adhesion strength of hepatocytes during two-dimensional cell spreading has not been elucidated due to lack of biophysical probe. In this study, a novel biophysical technique known as confocal reflectance interference contrast microscopy (C-RICM) in conjunction with phase contrast microscopy is utilized to probe the adhesion dynamics, contact mechanics and two-dimensional spreading kinetics of HepG2 cells on galactose immobilized and collagen gel coated substrates. C-RICM demonstrates that HepG2 cells form strong adhesion contacts with both galactose-immobilized surfaces and collagen gel coated substrates. Moreover, HepG2 cells maintain their compact shapes in the presence of asialoglycoprotein receptor-mediated recognition while they become exceedingly spread under integrin-mediated adhesion on collagen gel coated substrate. The initial rate of adhesion contact formation and the steady-state adhesion energy of HepG2 cell population are highest on substrate conjugated with galactose ligand via a longer spacer. The adhesion dynamics and final adhesion energy of HepG2 cells depends both on the type of ligand-receptor interaction and the length of spacer between the ligand and substrate. Most importantly, new biophysical insights into the initial hepatocyte attachment that are critical for hepatocyte culture are provided through the decomposition of two-dimensional spreading and adhesion contact formation on bio-functional substrates.     

Galactosylated PVDF membrane promotes hepatocyte attachment and functional maintenance

One of the major challenges in BLAD design is to develop functional substrates suitable for hepatocyte attachment and functional maintenance. In the present study, we designed a poly(vinylidene difluoride) (PVDF) surface coated with galactose-tethered Pluronic polymer. The galactose-derived Pluronic F68 (F68-Gal) was adsorbed on PVDF membrane through hydrophobic-hydrophobic interaction between PVDF and the polypropylene oxide segment in Pluronic. The galactose density on the modified PVDF surface increased with the concentration of the F68-Gal solution, reaching 15.4 nmol galactosyl groups per cm2 when a 1 mg/ml of F68-Gal solution was used. The adsorbed F68-Gal remained relatively stable in culture medium. Rat hepatocytes attachment efficiency on F68-Gal modified PVDF membrane was similar to that on collagen-coated surface. The attached hepatocytes on PVDF/F68-Gal membrane self-assembled into multi-cellular spheroids after 1 day of culture. These attached hepatocytes in spheroids exhibited higher cell functions such as albumin synthesis and P450 1A1 detoxification function compared to unmodified PVDF membrane and collagen-coated surface. These results suggest the potential of this galactose-immobilized PVDF membrane as a suitable substrate for hepatocyte culture.