Roles of hepatocyte growth factor/scatter factor and the met receptor in the early development of the metanephros.

Several lines of evidence suggest that hepatocyte growth factor/scatter factor (HGF/SF), a soluble protein secreted by embryo fibroblasts and several fibroblast lines, may elicit morphogenesis in adjacent epithelial cells. We investigated the role of HGF/SF and its membrane receptor, the product of the c-met protooncogene, in the early development of the metanephric kidney. At the inception of the mouse metanephros at embryonic day 11, HGF/SF was expressed in the mesenchyme, while met was expressed in both the ureteric bud and the mesenchyme, as assessed by reverse transcription PCR, in situ hybridization, and immunohistochemistry. To further investigate the expression of met in renal mesenchyme, we isolated 13 conditionally immortal clonal cell lines from transgenic mice expressing a temperature-sensitive mutant of the SV-40 large T antigen. Five had the HGF/SF+/met+ phenotype and eight had the HGF/SF-/met+ phenotype. None had the HGF/SF+/met- nor the HGF/SF-/met- phenotypes. Thus the renal mesenchyme contains cells that express HGF/SF and met or met alone. When metanephric rudiments were grown in serum-free organ culture, anti-HGF/SF antibodies (a) inhibited the differentiation of metanephric mesenchymal cells into the epithelial precursors of the nephron; (b) increased cell death within the renal mesenchyme; and (c) perturbed branching morphogenesis of the ureteric bud. These data provide the first demonstration for coexpression of the HGF/SF and met genes in mesenchymal cells during embryonic development and also imply an autocrine and/or paracrine role for HGF/SF and met in the survival of the renal mesenchyme and in the mesenchymal-epithelial transition that occurs during nephrogenesis. They also confirm the postulated paracrine role of HGF/SF in the branching of the ureteric bud.

A pH-sensitive, neurogenic pathway mediates disease severity in a model of post-ERCP pancreatitis.

BACKGROUND: Endoscopic retrograde cholangiopancreatography (ERCP) has a high risk of pancreatitis although the underlying mechanisms are unclear. Transient receptor potential vanilloid 1 (TRPV1) is a cation channel expressed on C and Adelta fibres of primary sensory neurons and is activated by low pH. TRPV1 activation causes release of inflammatory mediators that produce oedema and neutrophil infiltration. We previously demonstrated that neurogenic factors contribute to the pathogenesis of pancreatitis. Resiniferatoxin (RTX) is a TRPV1 agonist that, in high doses, defunctionalises C and Adelta fibres. When we discovered that the pH of radio-opaque contrast solutions used for ERCP was 6.9, we hypothesised that low pH may contribute to the development of contrast-induced pancreatitis via activation of TRPV1. METHODS: Rats underwent equal pressure pancreatic ductal injection of contrast solutions at varying pH with or without RTX. RESULTS: Contrast solution (pH 6.9) injected into the pancreatic duct caused a significant increase in pancreatic oedema, serum amylase, neutrophil infiltration, and histological damage. Solutions of pH 7.3 injected at equal pressure caused little damage. The severity of the pancreatitis was significantly increased by injection of solutions at pH 6.0. To determine if the effects of low pH were mediated by TRPV1, RTX was added to the contrast solutions. At pH levels of 6.0 and 6.9, RTX significantly reduced the severity of pancreatitis. CONCLUSIONS: Contrast solutions with low pH contribute to the development of pancreatitis through a TRPV1-dependent mechanism. It is possible that increasing the pH of contrast solution and/or adding an agent that inhibits primary sensory nerve activation may reduce the risk of post-ERCP pancreatitis.

Immortalization and transformation are associated with specific alterations in choline metabolism.

Analysis of transformed, immortalized, and primary rat Schwann cells by high-resolution proton nuclear magnetic resonance spectroscopy reveals that immortalization of Schwann cells (by SV40 large T antigen) induced a decrease in sn-glycero-3-phosphocholine (GPCho), whereas H-ras alone, which is known to cause growth arrest in these cells, induced a marked increase in GPCho and a decrease in phosphocholine (PCho). An increase of PCho was found only in cells fully transformed by both oncogenes together. Moreover, we examined 11 human tumor cell lines, all of which expressed a PCho:GPCho ratio similar to that of fully transformed rat Schwann cells. Importantly, neither the absolute levels of PCho nor the ratio of PCho:GPCho were correlated with the rate of cell division across a range of normal (primary cultures) and transformed cells. Thus, raised PCho:GPCho ratios may serve as an indicator of multiple oncogenic lesions and malignancy in noninvasive tumor investigations.

Cell type-specific fingerprinting of meningioma and meningeal cells by proton nuclear magnetic resonance spectroscopy.

We compared the properties of six human meningiomas with normal rat meningeal cells using cell culture techniques, high resolution in vitro 1H-NMR (nuclear magnetic resonance) spectroscopy, and chromatographic analysis. Cell cultures were immunocytochemically characterized at all stages with specific antibodies. Quantitative and qualitative metabolite assessments in cell extracts were obtained from 1H-NMR spectra and chromatographic analysis. Human meningioma cells expressed a characteristic spectrum of metabolites including free amino acids, compounds related to membrane phospholipid metabolism, energy metabolites, and other intermediary products. These spectral characteristics, although different in some respects, were strikingly similar to the ones of rat meningeal cells. Particularly, several metabolites that allow discrimination between meningeal cells and other cell types of the central nervous system were preserved in meningiomas. These similarities suggest that the regulation of intracellular levels of such metabolites is so intrinsic to the identity of cell type as to be conserved across species and through transformation. Additionally, human meningioma cultures expressed some spectroscopic characteristics that enabled them to be clearly distinguished from primary rat meningeal cultures. Thus, human meningiomas may be both specifically recognizable by 1H-NMR spectroscopy and also distinguishable from normal rat meningeal tissue. Our results raise the eventual possibility of using NMR in the noninvasive diagnosis of brain tumors in vivo.

Retinoblastoma gene deletions in human glioblastomas.

The retinoblastoma susceptibility gene, RB, is the best characterised of the tumour suppressor genes, or 'anti-oncogenes'. Abnormal function of the RB protein is thought to result in loss of an inhibitory effect on cell growth, and thus contribute towards the development of certain human cancers. One group of human cancers of particular interest in relationship to retinoblastoma gene function are the gliomas, which are central nervous system tumours thought to originate from the neuroectoderm, the embryological tissue which also gives rise to retinoblastomas. We have therefore examined a group of benign and malignant gliomas for evidence of structural alterations of the RB gene. Four out of nine (44%) glioblastomas, the most malignant gliomas, showed loss of heterozygosity of a locus within this gene. In addition, one of these hemizygous tumours showed deletion of part of the RB protein-coding region, and this abnormality was also present in cells cultured from the tumour. These findings suggest that RB gene abnormalities may contribute to the development of glioblastomas.

A randomized trial comparing heparin initiation 6 h or 24 h after pacemaker or defibrillator implantation.

OBJECTIVES: The purpose of this randomized study was to evaluate the prevalence of pocket hematomas in patients treated with heparin 6 h or 24 h after pacemaker or defibrillator implantation. BACKGROUND: The risks of pocket hematoma and need for evacuation after device implantation have not been defined in patients who require anticoagulation. METHODS: Forty-nine consecutive patients with an indication for anticoagulation with heparin after implantable defibrillator or pacemaker implantation were randomized to receive intravenous heparin either 6 h (n = 26) or 24 h (n = 23) postoperatively. Both groups also received warfarin on a daily basis starting the evening of surgery. Twenty-eight patients who received postoperative warfarin alone and 115 patients who did not receive anticoagulation were followed up in a study registry. RESULTS: A pocket hematoma developed in 6 of 26 patients (22%) who were treated with intravenous heparin 6 h postoperatively, as compared with 4 of 23 patients (17%) who were treated with intravenous heparin 24 h postoperatively (p = 0.7). In total, a pocket hematoma developed in 10 of 49 patients (20%) treated with heparin, 1 of 28 patients (4%) treated with warfarin alone and 2 of 115 (2%) patients who received no anticoagulation (p < 0.001). CONCLUSIONS: Intravenous heparin initiation 6 h or 24 h after pacemaker or defibrillator implantation is associated with a 20% prevalence of pocket hematoma formation. Warfarin therapy or no anticoagulation is associated with only a 2% to 4% risk of pocket hematoma formation.

Cellular transplants as sources for therapeutic agents.

Soluble factors normally produced by cells of the human body are of increasing importance as potential therapeutic agents. Although considerable progress has been made in understanding the etiology and pathogenesis of disease, in developing animal models and newer experimental therapeutics, few discoveries have been translated into clinically effective ways of delivering the multiple therapeutic agents obtained from living mammalian cells. This review examines the use of transplanted cells as alternatives to conventional delivery systems to deliver a variety of protein based therapeutic agents. The chapter begins with a set of questions to establish the complexity and challenges of this form of drug delivery. The following section focuses the discussion on our understanding of genetic engineering, tissue engineering, and some areas of developmental biology as they relate to the development of this nascent field. Much of the discussion has a neuro/endocrine emphasis. The chapter ends by listing the basic ingredients needed to push the use of transplanted cells toward medical practice and some general comments about future developments.

Recovery of spatial learning by grafts of a conditionally immortalized hippocampal neuroepithelial cell line into the ischaemia-lesioned hippocampus.

Transient global cerebral ischaemia in rats causes relatively circumscribed and specific damage to the CA1 pyramidal cells of the dorsal hippocampus, along with a cognitive deficit manifest as difficulties in the performance of a range of spatial learning and memory tasks. Our previous studies have shown that restoration of behavioural performance in ischaemic rats by neural grafts taken relatively late in fetal development occurs only after local replacement of cells homotypic to those lost through the ischaemic insult. This lesion-plus-behaviour model therefore offers a powerful means for establishing whether multipotent embryonic neuroepithelial cells will engraft the damaged CA1, develop into appropriate neuronal phenotypes and produce behavioural recovery. Here we report that, in rats subjected to 15 min of global cerebral ischaemia, intrahippocampal implants of a conditionally immortal, multipotent cell line, directly derived from the embryonic day 14 hippocampal neuroepithelium of the H-2Kb-tsA58 transgenic mouse, selectively repopulated the lesioned CA1 pyramidal layer and restored ischaemia-induced deficits in acquisition of a hidden platform location in the Morris water maze.

Rat neural antigen-2 (RAN-2): a cell surface antigen on astrocytes, ependymal cells, Müller cells and lepto-meninges defined by a monoclonal antibody.

We have immunized mice with enriched populations of cultured rat astrocytes and fused their spleen cells with NS-1 myeloma cells to generate antibody-secreting hybridomas. We have isolated two stable hybridoma clones which secrete monoclonal IgG2 antibodies that react with the surface of the great majority of rat astrocytes in culture. We have studied one of these antibodies in indirect immunofluorescence assays and show that it binds to the surface of rat ependymal cells, retinal Müller cells and leptomeningeal cells as well as to astrocytes, but not to cultured neurones, oligodendrocytes, Schwann cells, microglia or various non-neural cells. The antigen defined by this monoclonal antibody is protease-sensitive and rat-specific and we have called it rat neural antigen-2 (Ran-2). We also show that isolated rat ependymal cells and cultured rat Müller cells do not express other neural cell-type-specific markers, such as tetanus toxin receptors, rat neural antigen-1 (Ran-1), galactocerebroside or glial fibrillary acidic protein (GFAP). Nor do these cells express cell surface Fc receptors for IgG, phagocytose latex beads or make detectable amounts of the Thy-1 or fibronectin glycoproteins.

Direct effects of neostigmine on aneural myotube cultures.

Long term (24--96 h) treatment of a mouse-derived myogenic cell line (G8) with meostigmine or physostigmine markedly reduces binding of alpha-bungarotoxin (alpha-BuTx) to these cells. Protein synthesis in these cultures is markedly reduced and cell morphology degenerates. Myotubes maintain slightly hyperpolarised resting membrane potentials, and are able to respond to iontophoretic acetylcholine (ACh) application with overshooting action potentials. Prolonged exposure to neostigmine also inhibits protein synthesis in a fibroblastic cell line (B82). The results suggest that degenerative changes at the neuromuscular junction associated with chronic neostigmine treatment in vivo are due to a direct action of the anticholinesterase on the muscle, rather than to altered intracleft ACh levels [2] or to presynaptic effects of the anticholinesterase [15].

Molecular genetic study showing that the IN/157 'oligodendroglioma' cell line has been contaminated by rhabdomyosarcoma (RD) cells.

The IN/157 cell line was originally isolated from a human oligodendroglioma biopsy and has been used in recent years to study aspects of glioma cell biology. We established that IN/157 cells carry a relatively infrequent mutation at position three of codon 61 of the N-ras gene, suggesting that such a mutation may have contributed towards the genesis of the original tumour. However, the mutation was not detectable within the original paraffin-embedded glioma biopsy from which the cell line was supposedly derived. We thus considered the possibility that the cells had been contaminated by another cell line and, by means of DNA fingerprinting, have demonstrated that the contaminating cell line is the rhabdomyosarcoma line RD. We feel that this study makes several important points regarding experiments which make use of cell lines. We discuss the possible implications of contamination events with regard to erroneous conclusions about the biology of the cell lines and tumour types from which they supposedly derive. We also suggest ways in which future contamination-related errors can be minimized.

Regulation of acetylcholine receptor levels by a cholinergic agonist in mouse muscle cell cultures.

The effects of continuous exposure to carbamylcholine (CbCho) on regulation and stabilization of acetylcholine receptors (AcChoR) were studied in cell cultures of G8, a continuous mouse muscle cell line. Exposure of cultures to 10-100 muM CbCho for 24-48 hr produced a 30-50% reduction in (125)I-labeled alpha-bungarotoxin binding. CbCho was not found to alter cell morphology, protein metabolism, or amino acid incorporation. Electrophysiological experiments demonstrated a 75% reduction in the maximum sensitivity of the myotubes to iontophoretic application of acetylcholine (AcCho). The reduction in AcCho sensitivity appeared to represent a true loss of functional receptors because there were no changes in the passive electrical properties of the cells or in the AcCho reversal potential and because receptor desensitization appeared not to be involved. Tetrodotoxin had no effect on receptor levels, either alone or in combination with CbCho. Receptor degradation in control cells could be described kinetically as a first-order process with a half-time of 19.2 hr; turnover rate in receptors remaining after prolonged exposure to CbCho was indistinguishable from that in control cells. We conclude that a receptor-active ligand can exert negative control over AcChoR levels and that prolonged exposure to an AcCho analog is not sufficient to induce a stable population of receptors in these cells.

Characterization of cortical astrocytes on materials of differing surface chemistry.

The behavior of cortical astrocytes was evaluated on a number of medically relevant materials of differing physicochemical properties. This study describes cell attachment, DNA synthesis, production of extracellular matrix (ECM) proteins, and neuronal interactions of perinatal rat astrocytes in vitro. The number of attached astrocytes initially differed among the materials, decreasing with increasing material hydrophobicity. In contrast, the rate of DNA synthesis increased with increasing material hydrophobicity. With the exception of only one material, astrocytes reached confluence by 12 days in culture on all the materials tested. Furthermore, the expression of characteristic ECM proteins and the fundamental ability of astrocytes to support neuronal attachment and growth was qualitatively identical between populations of astrocytes on different materials. The ability of astrocytes to colonize different surfaces initially was mediated via adsorbed serum proteins, as reducing the capacity of a model surface to adsorb proteins inhibited astrocyte colonization for up to 2 weeks in culture. We propose that astrocytes are relatively insensitive to differences in surface chemistries so long as the proteins necessary for cellular attachment are capable of adsorbing to the material to some extent. It seems likely that the ability of astrocytes to produce and remodel a matrix creates a surface environment that eventually becomes similar regardless of the surface chemistry of the underlying material.

Prednisone-neostigmine interactions at cholinergic junctions.

The effect of corticosteroid (prednisone) and/or chronic anticholinesterase (neostigmine) treatment on alpha-bungarotoxin binding was examined in the diaphragms of male rats. In endplate regions of the diaphragm, prednisone treatment had no effect on the density of toxin binding sites, either when given alone or when administered in conjunction with neostigmine while neostigmine was observed to reduce specific binding to less than half after one week.

Generation of osteoclast-inductive and osteoclastogenic cell lines from the H-2KbtsA58 transgenic mouse.

The development of osteoclastic cell lines would greatly facilitate analysis of the cellular and molecular biology of bone resorption. Several cell lines have previously been reported to be capable of osteoclastic differentiation. However, such cell lines form at best only occasional excavations, suggesting that osteoclastic differentiation is either incomplete or that osteoclasts represent a very small proportion of the cells present. We have used the recently developed H-2KbtsA58 transgenic mouse, in which the interferon-inducible major mouse histocompatibility complex H-2Kb promoter drives the temperature-sensitive (ts) immortalizing gene of simian virus 40 (tsA58), to develop cell lines from bone marrow with high efficiency. Bone marrow cells were incubated with gamma interferon at 33 degrees C, then cloned, and expanded. The cell lines were characterized at 39.5 degrees C in the absence of gamma interferon. First, stromal cell lines were established that induced osteclast formation (resorption of bone slices) when cocultured with hemopoietic spleen cells. Some of the stromal cell lines so generated were able to resorb approximately 30 mm2/cm2 of bone surface. We then established cell lines of hemopoietic origin, several of which possess osteoclastic potential. When these osteoclast-precursor cell lines were cocultured with stromal cell lines, extensive bone resorption was observed. Osteoclast formation did not occur if the precursor cell lines were incubated on bone slices without stromal cells; osteoclast formation was also dependent upon the presence of 1 alpha,25-dihydroxyvitamin D3. These cell lines represent a model for osteoclast formation and a valuable resource for identification of the mechanisms and factors that regulate osteoclast differentiation and function.

Establishment of conditionally immortalized epithelial cell lines from both colon and small intestine of adult H-2Kb-tsA58 transgenic mice.

Intestinal mucosal cells have proved difficult to culture in vitro. Many attempts have been made to develop long-term cultures of these cells either by direct culturing or by attempting to immortalize these cells by using a range of transforming viral genes, but with little success. The recent development of a transgenic mouse bearing a temperature-sensitive mutation of the simian virus 40 large tumor antigen gene (tsA58) has enabled us to initiate conditionally immortalized cultures of epithelial cells from both small intestinal and colonic mucosa of adult mice. Crypts were isolated from either the small intestines or colons of young adult mice and cultured at the permissive temperature (33 degrees C) in medium containing conditioned medium from a human colon carcinoma cell line, LIM1863. Crypts from both tissues yielded cultures of epithelial cells that have now been in culture for more than 12 months with regular passaging. The epithelial nature of the cells has been confirmed by staining with anti-keratin antibodies. The intestinal origin of the cells was demonstrated by the ability of the cells to synthesize low levels of both brush border peptidases and a disaccharidase. The levels of expression of these enzymes were modulated by the addition of sodium butyrate or phorbol myristate acetate to the medium, which resulted in an increase in the synthesis of the peptidases and a decrease in the synthesis of the disaccharidase. The cells proliferate continuously at the permissive temperature (33 degrees C), but proliferation ceases at the nonpermissive temperature (39.5 degrees C). To our knowledge, this is the first description of the establishment of epithelial cell lines from both small intestine and colon of the same mouse strain. The success reported here indicates that this transgenic mouse will be a useful source of tissue for the study of the mechanisms that control the proliferation and eventual differentiation and senescence of the cells of the intestinal mucosa. These mice will also be a useful source of cells for attempts to culture cells from other tissues that have proved difficult to culture in vitro.

Surfactant-immobilized fibronectin enhances bioactivity and regulates sensory neurite outgrowth.

A PEO-containing surface coating was investigated as a means to control neurite outgrowth in the presence of serum. Various ratios of end-group-activated tri-block copolymer Pluronic F108 were used to immobilize the extracellular matrix protein fibronectin (FN). Primary cultures of dorsal root ganglion neurons were cultured on F108-immobilized FN or, as a control, on FN adsorbed from solution directly to polystyrene. Although FN surface concentration could be controlled in a dose-dependent manner by either technique, dose-dependent control of neuronal behaviors was best achieved on F108-immobilized FN. This effect was similar regardless of the presence of serum in the culture medium. F108-immobilized FN supported twofold greater maximal neurite outgrowth than did directly adsorbed FN. Furthermore, at similar surface concentrations, F108-FN was significantly more active in promoting neurite outgrowth. Polypropylene filament bundles treated with F108-immobilized FN supported robust outgrowth from explants of dorsal root ganglia, demonstrating the utility of the surface coating on clinically relevant materials with more complex shapes. The ability to control neuronal behaviors in a serum-resistant manner, coupled with enhanced biologic activity, demonstrates the potential for surfactant-based immobilization as a method for generating biointeractive materials for tissue engineering.

Antigenic expression by cells derived from human gliomas does not correlate with morphological classification.

We have found that cell populations derived from human gliomas can be divided into antigenic classes which are not predictable on the basis of standard morphological analysis and which, most frequently, do not support the lineage assignations of various tumours as determined by traditional neuropathological methods. For example, only 6/60 cultures derived from astrocytomas expressed glial fibrillary acidic protein (GFAP), an astrocyte specific marker, and all six of these cultures were derived from morphological categories which more frequently gave rise to populations which did not express GFAP. None of the seven oligodendrogliomas or four oligo-astrocytomas examined expressed antigens specifically expressed by oligodendrocytes. Most tumour-derived populations, from all classes of tumour and all grades of malignancy, expressed cell-surface fibronectin, an extracellular matrix protein only rarely found on the surfaces of CNS macroglia; such cells did not express glial-specific antigens (e.g., GFAP) in vitro. Investigation of antigen expression in tumour biopsies indicated that some tumours also consisted largely or wholly of cells which expressed fibronectin in situ. Fibronectin-expressing cells were aneuploid and were not contact inhibited in their growth, indicating that they were transformed cells. We have also identified two previously unknown antigenic phenotypes among the gliomas.

Vascular smooth muscle cells of H-2Kb-tsA58 transgenic mice. Characterization of cell lines with distinct properties.

BACKGROUND: The vascular wall is composed of at least two different populations of smooth muscle cells that are distinct in their structure and protein composition. According to the developmental stage of tissue taken for culture, the ratio between cells of epithelioid phenotype and spindle-shaped cells is variable. In particular, the epithelioid cells display characteristic features associated with immaturity. Because their increased appearance can be observed in endothelial denudation, the represent a dedifferentiated, proliferative smooth muscle cell type with a repair function in vascular injury. METHODS AND RESULTS: To investigate this cellular heterogeneity, we established vascular smooth muscle cell lines from H-2Kb-tsA58 transgenic mice. Due to temperature-sensitive expression of the SV 40 large T-antigen in cells derived from this mouse strain, our smooth muscle lines were conditionally immortalized from the onset of their life in culture. Thus, we were able to clone cell lines representing the two different phenotypes described so far. Epithelioid cells derived from newborn animals are characterized by their expression of cytokeratins and the development of tight junctional complexes. Spindle-shaped cells, which could be isolated from newborn or adult animals, corresponded in phenotype and protein expression to smooth muscle cell lines established previously. CONCLUSIONS: The special properties of vascular smooth muscle cells of the epithelioid phenotype suggest an endothelial replacement function in the course of injury to the vascular wall.

Regional and developmental variations in metabolite concentration in the rat brain and eye: a study using 1H NMR spectroscopy and high performance liquid chromatography.

Regional and developmental changes in metabolite concentrations were measured by 1H NMR spectroscopy and HPLC of perchloric acid extracts from rat brain and eye. The highest concentrations of N-acetylaspartate were found in grey matter as opposed to white matter with concentration increasing with age from neonate to adult, while the related compound N-acetylaspartylglutamate was highest in adult optic nerve. Creatine and choline-containing compounds were present in all regions throughout development, with higher levels of creatine found in grey matter compared to other regions. Choline-containing compounds were present at the highest concentrations in the eye at all ages examined, and tended to decrease in concentration to minimum values in adulthood in all regions. The presence of hypotaurine in corpus callosum and optic nerve was consistent with the metabolic profiles of O-2A progenitor cells and oligodendrocytes, which are cells composing these tissues. The neurotransmitters glutamate and GABA reached their highest concentrations in the olfactory bulb (higher than in adult cortex).