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R Mirsky

Publications and source records attributed to R Mirsky.

At least 73 records · Page 4Linked to original sources
PubMed1991

The effects of cAMP on differentiation of cultured Schwann cells: progression from an early phenotype (04+) to a myelin phenotype (P0+, GFAP-, N-CAM-, NGF-receptor-) depends on growth inhibition.

The present experiments were designed to clarify the relationship between cAMP elevation, proliferation and differentiation in Schwann cells. They were carried out on short-term cultures of cells obtained from neonatal rat sciatic nerves. It was found that the myelin-related phenotype was expressed in response to agents that elevate or mimic intracellular cAMP (forskolin, cholera toxin, cAMP analogues), provided cell division was absent. This phenotype included upregulation of the major myelin protein P0 and downregulation of GFAP, N-CAM, A5E3, and NGF receptor. In contrast, when cells were cultured in conditions where cell division occurred, elevation of intracellular cAMP produced an alternative response, characterized by DNA synthesis and absence of myelin-related differentiation. The cAMP mediated induction of an early Schwann cell antigen, 04, followed a different pattern since it was induced equally in dividing and nondividing cells. These observations are consistent with the proposal that during development of the rat sciatic nerve: (a) cAMP elevation, possibly induced by axon-associated factors, is a primary signal responsible for the induction of 04 expression in proliferating Schwann cells during the premyelination period; (b) subsequent withdrawal of cells associated with the larger axons from the cell cycle acts as a permissive secondary signal for induction of myelination, since in quiescent cells the ongoing cAMP elevation will trigger myelination.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Spontaneous immortalisation of Schwann cells in culture: short-term cultured Schwann cells secrete growth inhibitory activity.

In the developing peripheral nerve, Schwann cells proliferate rapidly and then become quiescent, an essential step in control of Schwann cell differentiation. Cell proliferation is controlled by growth factors that can exert positive or inhibitory influences on DNA synthesis. It has been well established that neonatal Schwann cells divide very slowly in culture when separated from neurons but here we show that when culture was continued for several months some cells began to proliferate rapidly and non-clonal lines of immortalised Schwann cells were established which could be passaged for over two years. These cells had a similar molecular phenotype to short-term cultured Schwann cells, except that they expressed intracellular and cell surface fibronectin. The difference in proliferation rates between short- and long-term cultured Schwann cells appeared to be due in part to the secretion by short-term cultured Schwann cells of growth inhibitory activity since DNA synthesis of long-term, immortalised Schwann cells was inhibited by conditioned medium from short-term cultures. This conditioned medium also inhibited DNA synthesis in short-term Schwann cells stimulated to divide by glial growth factor or elevation of intracellular cAMP. The growth inhibitory activity was not detected in the medium of long-term immortalised Schwann cells, epineurial fibroblasts, a Schwannoma (33B), astrocytes or a fibroblast-like cell-line (3T3) and it did not inhibit serum-induced DNA synthesis in epineurial fibroblasts, 33B cells or 3T3 cells. The activity was apparently distinct from transforming growth factor-beta, activin, IL6, epidermal growth factor, atrial natriuretic peptide and gamma-interferon and was heat and acid stable, resistant to collagenase and destroyed by trypsin treatment. We raise the possibility that loss of an inhibitory autocrine loop may contribute to the rapid proliferation of long-term cultured Schwann cells and that an autocrine growth inhibitor may have a role in the cessation of Schwann cell division that precedes differentiation in peripheral nerve development.

Animals↗
PubMed1990

04 and A007-sulfatide antibodies bind to embryonic Schwann cells prior to the appearance of galactocerebroside; regulation of the antigen by axon-Schwann cell signals and cyclic AMP.

In the rat sciatic nerve, the relationship between Schwann cells, axons, the extracellular matrix and perineurial sheath cells undergoes extensive modification between embryo day 15 and the onset of myelination during the first postnatal day. Little is known about molecular changes in Schwann cells in this important prenatal period. In the present paper, we use immunofluorescence to study the prenatal development and postnatal regulation of the antigen(s) recognized by the 04 monoclonal antibody and a well-characterized rat monoclonal antibody to sulfatide, A007. We show that, in a series of immunochemical tests, the 04 antibody recognizes only sulfatide in neonatal and adult rat nerves. Both antibodies first bind to Schwann cells in the sciatic nerve at embryo day 16-17, and all Schwann cells bind both antibodies at birth. In the adult nerve, both nonmyelin-forming and myelin-forming cells are labelled with the antibodies. Schwann cells dissociated from embryo day 15 nerves and cultured in the absence of axons develop neither 04 nor A007 binding on schedule, and 04-positive and A007-positive Schwann cells from postnatal nerves lose the ability to bind these antibodies during the first few days in culture. Schwann cells in the distal stump of transected nerves also sharply down-regulate cell surface binding of 04. High numbers of 04-positive or A007-positive Schwann cells reappear in cultures treated with agents that mimic or elevate intracellular cAMP. We conclude that two anti-sulfatide antibodies 04 and A007, recognize an antigen, probably sulfatide, that appears very early in Schwann cell development (one to two days prior to galactocerebroside) but is nevertheless subject to upregulation by axonal contact or elevation of intracellular cAMP.

Animals↗
PubMed1990

Three markers of adult non-myelin-forming Schwann cells, 217c(Ran-1), A5E3 and GFAP: development and regulation by neuron-Schwann cell interactions.

Immunohistochemical methods are used to investigate in detail the development and regulation of three proteins (217c(Ran-1), A5E3 and GFAP) specifically associated with adult non-myelin-forming Schwann cells in the rat sciatic nerve, from embryo day 15 to maturity. 217c(Ran-1), which is probably the NGF-receptor, and A5E3 are expressed by the majority of cells in the nerve at embryo day 15 and by essentially all cells at embryo day 18. GFAP first appears at embryo day 18; this is an intrinsically programmed developmental event which occurs in cultured Schwann cells even in the absence of serum. Postnatally, the expression of 217c(Ran-1), A5E3 and GFAP is suppressed in cells that form myelin but retained in non-myelin-forming Schwann cells. Mature myelin-forming cells nevertheless maintain the potential to express all three proteins but will only do so if removed from contact with myelinated axons. In neuron-free cultures Schwann cells express all three proteins. This work, together with our previous observations on N-CAM, shows that removal of a diverse set of surface proteins and a change in intermediate filament expression is one of the major consequences of axon to Schwann cell signalling during myelination in the rat sciatic nerve. Unlike myelin-forming cells, adult non-myelin-forming Schwann cells remain very similar to embryonic and newborn cells with respect to expression of surface proteins, in contrast to the previously established developmental changes that occur in their surface lipids.

Animals↗
PubMed1989

Schwann cells co-cultured with stimulated T cells and antigen express major histocompatibility complex (MHC) class II determinants without interferon-gamma pretreatment: synergistic effects of interferon-gamma and tumor necrosis factor on MHC class II induction.

Schwann cells (SC) do not express major histocompatibility complex (MHC) class II antigens under normal culture conditions. SC can, however, be induced in vitro to express MHC class II molecules by exposure to high concentrations of interferon-gamma (IFN-gamma) and can present antigens to antigen-specific T cell lines. In the present study immunohistochemical labeling showed that most SC (greater than 90%) prepared from rat neonatal sciatic nerves expressed MHC class II molecules when cultured together with mycobacterial antigen and T cells, and as a consequence were able to function as antigen-presenting cells in lymphoproliferation assays, without requiring pretreatment with IFN-gamma. Antigen or T cells alone were ineffective in stimulating MHC class II expression and induction of class II molecules was MHC restricted, requiring the presence of syngeneic T cells. Addition of monoclonal antibody DB1, directed against IFN-gamma to co-cultures of SC and T lymphocytes stimulated with antigen, prevented the induction of MHC class II antigen on SC. When SC were incubated with recombinant (r)IFN-gamma alone, up to 50% of SC showed positive labeling for MHC class II antigen. This level of expression was enhanced to greater than 80% when recombinant tumor necrosis factor (rTNF) was also added. rTNF alone had no effect, and addition of DBI antibody inhibited the synergistic effects of rTNF on MHC class II expression. The effects of rIL 4 were also investigated but neither rIL 4 alone nor rIL 4 in combination with rIFN-gamma induced MHC class II expression by SC. These results show that in the presence of sensitized T lymphocytes and antigen, SC do not require pretreatment with exogenous rIFN-gamma to express MHC class II antigens and function as antigen-presenting cells. T cell-derived TNF and IFN-gamma appear to act as mediators of the T cell-induced expression of MHC class II by SC.

Animals↗
PubMed1989

Transforming growth factor-beta and gamma-interferon have dual effects on growth of peripheral glia.

The influence of transforming growth factor-beta (TGF-beta) and gamma-interferon on DNA synthesis in Schwann cells and enteric glia in culture has been studied. TGF-beta stimulated the DNA synthesis of short-term (less than 2 weeks in culture) Schwann cells, whereas gamma-interferon was ineffective. The stimulatory effect of TGF-beta was additive to the stimulation of DNA synthesis due to axonal membrane fragments. In contrast to their effect on short-term Schwann cells, both TGF-beta and gamma-interferon inhibited DNA synthesis in enteric glial cells and in long-term (over 3 months in culture) Schwann cells. When short-term Schwann cells were stimulated to divide by axolemma or glial growth factor, gamma-interferon did not inhibit this enhanced DNA synthesis although it suppressed DNA synthesis induced by cAMP analogues. These results raise the possibility that TGF-beta and gamma-interferon might have a role in controlling glial proliferation during development and/or regeneration of the peripheral nervous system.

Animals↗
PubMed1989

Non-myelin-forming Schwann cells proliferate rapidly during Wallerian degeneration in the rat sciatic nerve.

Transection of a mixed peripheral nerve results in the degeneration of axons and breakdown of myelin in the distal stump. These events are accompanied by a sharp but transient Schwann cell proliferation. The present study seeks to determine whether both myelin-forming and non-myelin-forming Schwann cells enter a proliferative phase under these conditions, or whether the dividing cells are chiefly recruited from one or other of the Schwann cell populations. The macrophage recruitment into the transected distal stumps has also been timed and quantitated, since it has been suggested that macrophages are an important source of Schwann cell mitogens in degenerating peripheral nerves. Incorporation of [3H]-thymidine and autoradiography was used as a measure of cell proliferation, and cell type markers and immunohistochemistry were used to identify myelin-forming and non-myelin-forming Schwann cells. The cells were removed from the distal stump of the rat sciatic nerve and sympathetic trunk at various times after transection and proliferation measured during the first 24 h in culture. It was found that in the sciatic nerve, which contains a mixture of myelinated and unmyelinated fibres, both myelin-forming cells, identified by presence of the myelin protein Po, and non-myelin-forming cells (Po- cells) showed a substantial elevation in [3H]-thymidine labelling index at day 2 postoperatively, which was similar in magnitude for the two categories of cell. The proliferation rate of both Po+ and Po- cells remained elevated for up to 8 days after transection.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Type I collagen preparations inhibit DNA synthesis in glial cells of the peripheral nervous system.

The mechanisms underlying cessation of glial proliferation in the developing peripheral nervous system are obscure. One possibility, as yet little explored, is that mitotic inhibitory signals play a part in regulating glial cell numbers. In this study we demonstrate that type I collagen preparations from several different sources can inhibit the rate of DNA synthesis in purified populations of enteric glia and both short-term and long-term secondary Schwann cells in dissociated cell cultures. When these cells are grown on gelled or dried type I collagen substrata, they proliferate at substantially lower rates than on polylysine substrata. In contrast, type III or V collagen preparations do not inhibit glial DNA synthesis and laminin, fibronectin, type IV collagen, and secreted matrix from bovine corneal endothelial cells all stimulate thymidine incorporation. The inhibitory effect is not observed with heat denatured type I collagen preparations, but is seen equally in serum-containing medium, in medium containing fibronectin-free serum, or in serum-free medium, suggesting that the interaction of collagen with the cells requires structurally intact collagen molecules and does not occur via intermediary linkage to fibronectin. The inhibition on collagen is accompanied by a shape change from a more flattened morphology to a narrow spindle form. The labeling index of a rat Schwannoma cell line, 33B, is not inhibited on type I collagen substrata. These results demonstrate that type I collagen preparations inhibit the DNA synthesis levels of early postnatal peripheral glial cells in vitro. It remains to be determined whether this effect occurs via direct collagen-cell membrane interactions or whether it depends on accessory molecules, perhaps present in the collagen preparations themselves, since these are not purified to absolute homogeneity.

Animals↗
PubMed1989

Control of peripheral glial cell proliferation: enteric neurons exert an inhibitory influence on Schwann cell and enteric glial cell DNA synthesis in culture.

Neuronal membranes from rat dorsal root ganglia provide a mitogenic signal to cultured Schwann cells and it has been suggested this is an important factor in regulating Schwann cell numbers during development. In this study, the influence of enteric neurons on the DNA synthesis of both Schwann cells and enteric glia has been investigated as well as the effect of axonal membrane fractions (axolemma) on enteric glia. The proliferation rate of rat Schwann cells and enteric glia was assessed in culture using [3H]thymidine uptake and autoradiography in combination with immunolabelling to identify cell types. When purified rat Schwann cells were co-cultured with guinea pig enteric neurons, their DNA synthesis rate was reduced compared with control cultures of pure Schwann cells or Schwann cells not close to neurites or neuronal cell bodies. Nevertheless, in accordance with previous findings that sensory neurons stimulate Schwann cell division, these Schwann cells increased their DNA synthesis rate when in contact with neurites from purified guinea pig or adult rat dorsal root ganglion neurons and on exposure to bovine axolemmal fractions. The enteric neurons also suppressed the DNA synthesis of enteric glia in co-cultures of purified enteric neurons and enteric glia, while bovine axolemma stimulated their DNA synthesis. These results indicate that a mitotic inhibitory signal is associated with enteric neurons and can exert its effect on both Schwann cells and enteric glia, and that enteric glia, like Schwann cells, are stimulated to divide by axolemmal fractions. It thus seems possible that during development glial cell numbers in the peripheral nervous system may be controlled by both positive and negative regulators of cell growth.

Animals↗
PubMed1988

Antigenic markers and laminin expression in cultured enteric neural cells.

In this study, polyclonal and monoclonal antibodies have been used in conjunction with standard immunohistochemical methods to define markers which can be used to identify and study the main cell types present in the outgrowth area of explant cultures of myenteric plexus from newborn guinea pig. We show that all of the neurons binds antibodies to the glycoprotein Thy-1 and the antibody A2B5 which recognizes gangliosides. All enteric glial cells bind antibodies to the calcium-binding protein S100, and the A2B5 monoclonal antibody and ca. 95% of glia are labeled by the antibody LB1, which recognizes the GD3 ganglioside. Most fibroblasts are labelled by antibodies to Thy-1 and the matrix glycoprotein fibronectin. Thus enteric neurons can be defined serologically as Thy-1+/A2B5+/S100- cells; enteric glia as S100+ cells and fibroblasts as A2B5-/S100-cells. The markers have been used to demonstrate that laminin is made by both enteric glia and fibroblasts. They have also been used to show that ca. 5% of neurons and less than 5% of enteric glia bind the antibody Leu 7 (HNK-1, L2), thus revealing the subpopulations of neurons and glia show differential cell surface expression of the carbohydrate epitope recognized by the antibody. In the accompanying paper, we demonstrate that two of the antibodies (LB1 and Thy-1) can be used to generate purified populations of neurons and glia.

Animals↗
PubMed1988

Establishment and properties of separate cultures of enteric neurons and enteric glia.

In this paper methods are described for the preparation of two types of culture derived from myenteric explants: (a) highly enriched neuronal cell cultures, and (b) purified glial cells (greater than 98%). Both procedures combine the technique of antibody complement-mediated cytolysis with the use of an antimitotic agent. Immunohistochemical methods were used to compare the purified cells to their counterparts in mixed cultures (see accompanying paper). Antibodies to the glycoprotein Thy-1 and the monoclonal antibody A2B5 which recognizes gangliosides, labelled the cell surface of all enteric neurons in enriched cultures while subpopulations of the neurons expressed the Leu 7 carbohydrate epitope, the neurotransmitter 5-hydroxytryptamine and the neuropeptides substance P, methionine-enkephalin and vasoactive intestinal polypeptide. Autoradiographic experiments show that a subpopulation of enriched neurons exhibit high-affinity uptake sites for gamma-[3H]aminobutyric acid (GABA). All purified enteric glia continue to express the calcium binding protein S100, the basement membrane glycoprotein laminin and the antigens recognized by the A2B5 antibody, and subpopulations of glia are labelled by the monoclonal antibodies LB1 which binds to GD3 gangliosides, and Leu 7. Thus enteric neurons and glia can survive independently of each other and express molecular properties which are present in cultures normally containing both cell types.

Animals↗
PubMed1988

Macrophage-like cells in muscularis externa of mouse small intestine: immunohistochemical localization of F4/80, M1/70, and Ia-antigen.

Macrophage-like cells (MLC) in mouse small intestine are situated in the muscularis externa in the subserosal layer at the level of Auerbach's plexus, and at the level of the deep muscular plexus. By combined labelling with FITC-dextran and immunohistochemical techniques, the MLC were shown to express the macrophage markers F4/80 and M1/70.15. The MLC appeared to be constitutively IE-antigen-positive, but did not contain lysozyme. It is suggested that MLC, like Langerhans cells, belong to a specialized class of cells in the mononuclear phagocyte system.

Animals↗
PubMed1988

Immunohistochemical localisation and electrophysiological actions of GABA in prevertebral ganglia in guinea-pig.

Immunohistochemical techniques were used to detect the presence of a GABA-like material in prevertebral sympathetic ganglia in guinea-pigs. Varicose, immunolabelled nerve fibres were observed in close proximity to sympathetic neurones in inferior mesenteric ganglia and coeliac ganglia. Non-varicose, immunolabelled nerve fibres were observed in lumbar colonic nerves and superior coeliac nerves, i.e. in nerve bundles peripheral to prevertebral ganglia. Immunolabelling was also present in neurones in the myenteric plexus and in nerve fibres in the circular muscle of the colon, as shown previously (Hills et al., Neuroscience, 22 (1987) 301-312). However, GABA-like immunoreactivity was not observed in the cell bodies of prevertebral ganglia nor in splanchnic nerves central to prevertebral ganglia. It was concluded from these results that prevertebral ganglia in guinea-pig receive a GABAergic innervation from neurones peripheral to the ganglia, possibly from GABA-containing neurones in the myenteric plexus of the gastrointestinal tract. Intracellular recordings were made from sympathetic neurones in the inferior mesenteric ganglion (IMG). Application of GABA onto the IMG caused a slow depolarisation of sympathetic neurones, during which there was a marked decrease in the input resistance of IMG cells. Application of GABA also depressed excitatory postsynaptic potentials (EPSPs) and action potentials in sympathetic neurones excited by cholinergic nerve fibres in the lumbar colonic nerves. The reversal potential of the GABA-induced slow depolarisation was -37 mV, a value close to the chloride equilibrium potential for sympathetic neurones. The actions of GABA were reversibly reduced by the GABAA antagonist, bicuculline, and were modulated in a predictable manner by substituting chloride ions with methane-sulphonate ions. These results indicated that GABA, and presumably GABAergic nerves in prevertebral ganglia, modulate the excitability of sympathetic neurones by acting on GABAA receptors linked to a chloride ionophore.

Animals↗
PubMed1988

Reversal of nerve damage in streptozotocin-diabetic rats by acute application of insulin in vitro.

1. Immunohistochemical, immunoblotting and release experiments were performed on ileum from control rats, from 8-week streptozotocin-diabetic rats and from diabetic rats after acute application of insulin in vitro. 2. There was an increase in vasoactive-intestinal-polypeptide-like and a decrease in calcitonin-gene-related-peptide-like immunoreactivity in the myenteric plexus of the diabetic rat ileum, although electrically evoked release of both peptides from enteric nerves was defective. Acute application of insulin in vitro reversed the defective release and changes in immunoreactivity of vasoactive intestinal polypeptide and calcitonin-gene-related peptide seen in the enteric nerves of streptozotocin-diabetic rat ileum. 3. In addition, using a monoclonal neurofilament antibody RT 97 that recognizes a phosphorylated neurofilament epitope present in normal enteric nerves, it was shown that this phosphorylated neurofilament epitope was absent in diabetic nerves, even though a polyclonal neurofilament antibody revealed that neurofilaments were present in both axons and cell bodies of the myenteric plexus of diabetic rat ileum. After only 2 h of insulin incubation in vitro, the phosphorylated neurofilament epitope was again present in the nerves. 4. It is suggested that the abnormal distribution of phosphorylated neurofilaments and defective storage and release of vasoactive intestinal polypeptide and calcitonin-gene-related peptide in the present study may be a more general feature of diabetes. The restoration of these abnormalities by continuous acute insulin application in vitro shown here suggests that the availability of a steady level of insulin might prevent some of the changes which occur in early stages of diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Myelinated, but not unmyelinated axons, reversibly down-regulate N-CAM in Schwann cells.

There is evidence from chicks and mice that N-CAM expression in Schwann cells is subject to significant regulation during development and following injury. In the present work, rat sciatic nerve and immunohistochemical methods have been used to study developmental and injury-related modulation of N-CAM in Schwann cells, using cell type specific markers to identify different Schwann cell populations, and cell counting to quantify their size. The study has sought to determine unambiguously whether immature Schwann cells in developing nerves and denervated Schwann cells in injured adult nerves express surface N-CAM, and has investigated the temporal relationship between the gradual loss of surface N-CAM and the differentiation of myelin-forming Schwann cells, monitored by the sequential appearance of the glycolipid galactocerebroside and the myelin-specific protein P0. Further points examined are whether this down-regulation of N-CAM is rapidly reversible following loss of axonal contact, and whether N-CAM reappearance in Schwann cells depends on protein synthesis. In nerves from 17- to 18-day embryos, 90% of the Schwann cells, identified with Ran-1 antibodies, expressed surface N-CAM. In nerves from newborn rats many cells are in the early stage of myelin synthesis and therefore express galactocerebroside, although they have not yet acquired P0. Suspension staining of dissociated cells from this nerve showed that 92% of the galactocerebroside-positive cells were also N-CAM positive. In suspension staining of nerves from 5-day, 10-day and adult rats, P0-positive cells were essentially N-CAM negative.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Gamma interferon, but not Mycobacterium leprae, induces major histocompatibility class II antigens on cultured rat Schwann cells.

In order to investigate the possible role of Schwann cells in immune reactions, and in particular their involvement in the response to infection with Mycobacterium leprae, it was determined under what conditions Schwann cells express major histocompatibility complex class II (MHC class II) antigens, since these molecules are thought to have a key role in antigen presentation during cellular immune responses. In situ and in vitro preparations from newborn and adult rat sciatic nerves were used as a model system to examine this question. Schwann cells in dissociated cell cultures did not express immunohistochemically detectable amounts of MHC class II antigens. Teased nerve preparations from the sciatic nerves of healthy adult rats showed no detectable immunolabelling of either myelin-forming or non-myelin-forming Schwann cells. When dissociated Schwann cell cultures derived from the sciatic nerves of either neonatal or adult rats were treated with 10, 50 or 100 units of gamma interferon, MHC class II antigens were detectable on the surface of some Schwann cells 48 h after addition of the interferon. By 72 h, 32.29 +/- 3.9% of Schwann cells in the cultures from neonatal rats and 53.32 +/- 5.4% of Schwann cells in cultures from adult rats, identified by the presence of intracellular S-100, were clearly MHC class II-positive, especially at doses of 50 and 100 units per ml of gamma interferon. Some, but not all, of the fibroblastic cells were very weakly MHC class II-positive. Infection of the cultures with Mycobacterium leprae did not induce MHC class II antigen expression in either Schwann cells or fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗