Migration Stimulating Factor (MSF): Its Role in the Tumour Microenvironment.

Migration Stimulating Factor (MSF) is a 70 kDa truncated isoform of fibronectin (FN); its mRNA is generated from the FN gene by an unusual two-stage processing. Unlike full-length FN, MSF is not a matrix molecule but a soluble protein which displays cytokine-like activities not displayed by any other FN isoform due to steric hindrance. There are two isoforms of MSF; these are referred to as MSF+aa and MSF-aa, while the term MSF is used to include both.MSF was first identified as a motogen secreted by foetal and cancer-associated fibroblasts in tissue culture. It is also produced by sprouting (angiogenic) endothelial cells, tumour cells and activated macrophages. Keratinocytes and resting endothelial cells secrete inhibitors of MSF that have been identified as NGAL and IGFBP-7, respectively. MSF+aa and MSF-aa show distinct functionality in that only MSF+aa is inhibited by NGAL.MSF is present in 70-80% of all tumours examined, expressed by the tumour cells as well as by fibroblasts, endothelial cells and macrophages in the tumour microenvironment (TME). High MSF expression is associated with tumour progression and poor prognosis in all tumours examined, including breast carcinomas, non-small cell lung cancer (NSCLC), salivary gland tumours (SGT) and oral squamous cell carcinomas (OSCC). Epithelial and stromal MSF carry independent prognostic value. MSF is also expressed systemically in cancer patients, being detected in serum and produced by fibroblast from distal uninvolved skin. MSF-aa is the main isoform associated with cancer, whereas MSF+aa may be expressed by both normal and malignant tissues.The expression of MSF is not invariant; it may be switched on and off in a reversible manner, which requires precise interactions between soluble factors present in the TME and the extracellular matrix in contact with the cells. MSF expression in fibroblasts may be switched on by a transient exposure to several molecules, including TGFß1 and MSF itself, indicating an auto-inductive capacity.Acting by both paracrine and autocrine mechanisms, MSF stimulates cell migration/invasion, induces angiogenesis and cell differentiation and alters the matrix and cellular composition of the TME. MSF is also a survival factor for sprouting endothelial cells. IGD tri- and tetra-peptides mimic the motogenic and angiogenic activities of MSF, with both molecules inhibiting AKT activity and requiring αvß3 functionality. MSF is active at unprecedently low concentrations in a manner which is target cell specific. Thus, different bioactive motifs and extracellular matrix requirements apply to fibroblasts, endothelial cells and tumour cells. Unlike other motogenic and angiogenic factors, MSF does not affect cell proliferation but it stimulates tumour growth through its angiogenic effect and downstream mechanisms.The epithelial-stromal pattern of expression and range of bioactivities displayed puts MSF in the unique position of potentially promoting tumour progression from both the "seed" and the "soil" perspectives.

Effects of 4-aminopyridine on demyelinated axons, synapses and muscle tension.

Several clinical trials have demonstrated that 4-amino-pyridine (4-AP), a potassium channel-blocking agent, improves symptoms in some patients with multiple sclerosis. The beneficial effects have typically been attributed to the restoration of conduction to demyelinated axons, since this effect was previously demonstrated experimentally. However, the clinical dose is approximately 250-1000 times lower than that used experimentally, potentially making extrapolation of the experimental findings unreliable. To examine the action(s) of 4-AP in demyelinating disorders, the drug was administered at clinical doses, both in vivo and in vitro, to rat dorsal column axons which had been experimentally demyelinated by the intraspinal injection of ethidium bromide. 4-AP had no consistent effect in restoring conduction to demyelinated axons, even to axons which were held just on the verge of conducting by adjusting the lesion temperature. However, 4-AP had prominent effects that did not involve demyelinated axons, including the potentiation of synaptic transmission and an increase in skeletal muscle twitch tension. We propose that these latter effects may be largely responsible for the beneficial action of 4-AP in multiple sclerosis patients. If so, the dominant effects of 4-AP in multiple sclerosis patients are independent of demyelination, and it follows that 4-AP may be beneficial in other neurological disorders in which function is diminished.

Demyelination: the role of reactive oxygen and nitrogen species.

This review summarises the role that reactive oxygen and nitrogen species play in demyelination, such as that occurring in the inflammatory demyelinating disorders multiple sclerosis and Guillain-Barré syndrome. The concentrations of reactive oxygen and nitrogen species (e.g. superoxide, nitric oxide and peroxynitrite) can increase dramatically under conditions such as inflammation, and this can overwhelm the inherent antioxidant defences within lesions. Such oxidative and/or nitrative stress can damage the lipids, proteins and nucleic acids of cells and mitochondria, potentially causing cell death. Oligodendrocytes are more sensitive to oxidative and nitrative stress in vitro than are astrocytes and microglia, seemingly due to a diminished capacity for antioxidant defence, and the presence of raised risk factors, including a high iron content. Oxidative and nitrative stress might therefore result in vivo in selective oligodendrocyte death, and thereby demyelination. The reactive species may also damage the myelin sheath, promoting its attack by macrophages. Damage can occur directly by lipid peroxidation, and indirectly by the activation of proteases and phospholipase A2. Evidence for the existence of oxidative and nitrative stress within inflammatory demyelinating lesions includes the presence of both lipid and protein peroxides, and nitrotyrosine (a marker for peroxynitrite formation). The neurological deficit resulting from experimental autoimmune demyelinating disease has generally been reduced by trial therapies intended to diminish the concentration of reactive oxygen species. However, therapies aimed at diminishing reactive nitrogen species have had a more variable outcome, sometimes exacerbating disease.

NaG: a sodium channel-like mRNA shared by Schwann cells and other neural crest derivatives.

NaG, a member of subfamily 2, was originally characterized as a "glial" sodium channel, and is expressed at high levels in Schwann cells in vivo. However, NaG has also been shown by in situ hybridization to be highly expressed in rat dorsal root ganglion (DRG) neurons, which, like Schwann cells, are neural crest derivatives. In the present study, we used non-isotopic in situ hybridization with a riboprobe for NaG, in conjunction with RT-PCR, to determine whether NaG is expressed in tissues related to the DRG either by neural crest origin or sensory function. We found that the expression of significant levels of NaG mRNA was restricted to derivatives of the neural crest (neurons of DRG, trigeminal ganglion and mesencephalic nucleus of the trigeminal nerve, and Schwann and satellite cells); the absence of NaG from superior cervical ganglion neurons and adrenal medulla chromaffin cells indicates that not all neural crest derived neural elements express NaG. NaG was not observed in sensory neurons (retina, vestibular ganglion, spiral ganglion, olfactory epithelium) that are not of neural crest origin. Our results indicate that NaG mRNA is expressed not only in Schwann cells, but also in a spectrum of neuronal cell types with afferent function of neural crest origin. NaG thus appears to represent a transcript encoding a sodium channel or sodium channel-like protein that is uniquely expressed by both Schwann cells and afferent neurons of neural crest origin.

Conduction in segmentally demyelinated mammalian central axons.

The prominent symptoms associated with central demyelinating diseases such as multiple sclerosis (MS) are primarily caused by conduction deficits in affected axons. The symptoms may go into remission, but the mechanisms underlying remissions are uncertain. One factor that could be important is the restoration of conduction to affected axons, but it is not known whether demyelinated central axons resemble their peripheral counterparts in being able to conduct in the absence of repair by remyelination. In the present study we have made intra-axonal recordings from central axons affected by a demyelinating lesion, and then the axons have been labeled ionophoretically to permit their subsequent identification. Ultrastructural examination of 23 labeled preparations has established that some segmentally demyelinated central axons can conduct, and that they can do so over continuous lengths of demyelination exceeding several internodes (2500 micron). Such segmentally demyelinated central axons were found to conduct with the anticipated reduction in velocity and a refractory period of transmission (RPT) as much as 34 times the value obtained from the nondemyelinated portion of the same axon; the RPT was typically prolonged to 2-5 times the normal value. We conclude that some segmentally demyelinated central axons can conduct, and we propose that the restoration of conduction to such axons is likely to contribute to the remissions commonly observed in diseases such as MS.

Sodium channel alpha-subunit mRNAs I, II, III, NaG, Na6 and hNE (PN1): different expression patterns in developing rat nervous system.

The expression of sodium channel alpha-subunit mRNAs I, II, III, NaG, Na6 and hNE (PN1) was examined in developing (E17-P30) hippocampus, cerebellum, spinal cord and dorsal root ganglia using non-isotopic in situ hybridization cytochemistry. The results showed distinct patterns of expression for each of the sodium channel mRNAs with maturation of the nervous system. In the hippocampus, sodium channel mRNA I was not detected at any developmental time, while mRNA II showed increasing hybridization signal between E17 and P30. Sodium channel mRNA III was more prevalent at late embryonic and early postnatal times, and was barely detectable at P30. The transcript for NaG showed transient expression between P2 and P15, being expressed at low levels at E17 and not being detectable at P30. Sodium channel mRNA Na6 exhibited a high level of expression between E17 and P15 in the hippocampal formation, with an attenuation of the signal by P30. hNE (PN1) mRNA was not detected in the hippocampus at any time examined. In the cerebellum, sodium channel mRNA I was not detected at E17 or P2, but became detectable in Purkinje cells at P15 and continued to show a low level of expression in these cells at P30. mRNA I was not detected at any time examined in granule cells of the cerebellum. Sodium channel mRNA II exhibited increasing expression in the developing cerebellum, and showed increasing signal in Purkinge cells beginning on P2 and granule cells on P15. Sodium channel mRNA III was down-regulated with development in the cerebellum, although mRNA III was readily detected at E17, it was not detected in any layers of the cerebellum by P15. NaG mRNA showed a peak of expression at P2, and was present at low levels at E17 and P15 and not detectable at P30. Na6 mRNA was highly expressed in the E17 cerebellum; this mRNA was present at high levels in Purkinje cells throughout development, although in granule cells the signal was attenuated at P15-P30. Sodium channel hNE (PN1) mRNA was not detected in the cerebellum at any time in development. In the spinal cord, sodium channel mRNA I showed increasing expression beginning at P2 and was highly expressed, particularly in ventral motor neurons, by P30. Sodium channel II mRNA was detected at all stages of development in the spinal cord; in contrast, mRNA III was detected at E17 and P2, but showed very low levels of expression by P30. NaG mRNA exhibited a transient expression in spinal cord at P2, but was not detectable at E17 and P30. Na6 mRNA was detectable at very low levels at E17 and became highly expressed at P2, prior to a reduction of the signal at P15 and P30. hNE (PN1) mRNA was not detected in the spinal cord at any time in development. In the dorsal root ganglia, sodium channel I mRNA hybridization signal was detected in DRG neurons at P2, with slightly increased levels at P15 and P30. Sodium channel II mRNA exhibited a relatively constant, moderate level of expression at all developmental ages. Sodium channel III mRNA was highly expressed in DRG neurons at E17 but was down-regulated with further development so that it was not detectable by P30. NaG mRNA was strongly expressed by some DRG neurons at all stages of development from E17 to P30; in general the level of NaG labelling was greater in larger neurons than in smaller neurons. Na6 mRNA showed increasing expression with development in DRG neurons; at E17, low levels of Na6 mRNA were detected and by P15 to P30 high levels of expression were present in some neurons. hNE (PN1) mRNA was present in DRG neurons at P2, and was up-regulated with further development so that by P30 hNE (PN1) was expressed in all DRG neurons sizes. These results demonstrate that sodium channel alpha-subunit mRNAs I, II, III, NaG, Na6 and hNE (PN1) exhibit distinct spatial and temporal patterns of expression in nervous tissue, and suggest that the expression of the sodium channel alpha-subunits is differentially regulated. (ABSTRACT TRUNCATED)

Blood-brain barrier permeability in astrocyte-free regions of the central nervous system remyelinated by Schwann cells.

The patency of the blood-brain barrier was examined during the development and repair of focal demyelinating lesions induced in the dorsal columns of rats by the intraspinal injection of ethidium bromide, with or without concomitant irradiation. Blood-brain barrier integrity was determined by the intravenous injection of horseradish peroxidase or by the immunofluorescent localization of endogenous albumin. Following repair, the central area of the lesions was remyelinated by Schwann cells and lacked astrocytes. In unirradiated lesions, demyelination was established at one week and the lesion was largely repaired by remyelination by 12 weeks. Horseradish peroxidase extravasation was absent at one day after injection, but was present at three days and throughout the period of repair. With one exception, all animals which exhibited regions of demyelination also exhibited horseradish peroxidase extravasation. No horseradish peroxidase was seen in lesions where all the demyelinated axons had been repaired by remyelination, and strong albumin immunofluorescence was also absent from such lesions. Albumin immunoreactivity was also absent from normal spinal cords, although it was prominent in normal sciatic nerves and dorsal roots. Irradiation of lesions resulted in a delay in the repair by remyelination, and repair of the blood-brain barrier was similarly delayed. Promotion of Schwann cell remyelination has been suggested as a potential therapy for central demyelinating disorders such as multiple sclerosis; however, central regions remyelinated by Schwann cells lack astrocytes, cells which have been implicated in the induction and maintenance of the blood-brain barrier. Since blood-brain barrier opening may be an early step in the production of new lesions, a defective barrier could allow such remyelinated regions to act as foci for further lesion development. We conclude, however, that the remyelination of central demyelinating lesions by Schwann cells is accompanied by recovery of properties of an intact blood-brain barrier, despite the lack of astrocytes. The present findings support the idea that promotion of remyelination by Schwann cells may form an effective therapy for central demyelinating diseases.

Orphan nuclear receptor ROR alpha gene: isoform-specific spatiotemporal expression during postnatal development of brain.

We analyzed expression of mouse orphan nuclear receptor ROR alpha during postnatal development of rodent brain. Using a riboprobe corresponding to the 3'-end of mROR alpha cDNA a peak of ROR alpha expression was observed at postnatal 16 day (P16) in the Purkinje cells of cerebellum, neurons of the thalamus and the olfactory bulb. The hippocampus was also shown to express ROR alpha with an earlier peak at P7. Expression in cell types other than the Purkinje cells appeared transient. On the other hand, when a probe to the 5'-end of mROR alpha cDNA was used, we observed patterns of ROR alpha expression that are different from those observed with the 3'-probe. No specific transcripts of ROR alpha were detected with the 5'-probe in the Purkinje cells until P16. Additionally, the relative level of the hybridization signals with the 5'-probe and the 3'-probe were different among the various brain regions. Together with the previous findings that ROR alpha comprises at least four isoforms which differ from one another in their N-terminal regions, these observations suggest that the spatiotemporal expression of ROR alpha is under isoform-specific regulation. The timing of its expression suggests that ROR alpha may be involved in regulation of postnatal maturation of specific classes of neurons.

Restoration of normal conduction properties in demyelinated spinal cord axons in the adult rat by transplantation of exogenous Schwann cells.

Although remyelination of demyelinated CNS axons is known to occur after transplantation of exogenous glial cells, previous studies have not determined whether cell transplantation can restore the conduction properties of demyelinated axons in the adult CNS. To examine this issue, the dorsal columns of the adult rat spinal cord were demyelinated by x-irradiation and intraspinal injections of ethidium bromide. Cell suspensions of cultured astrocytes and Schwann cells derived from neonatal rats transfected with the (beta-galactosidase) reporter gene were injected into the glial-free lesion site. After 3-4 weeks nearly all of the demyelinated axons were remyelinated by the transplanted Schwann cells. The dorsal columns were removed and maintained in an in vitro recording chamber; conduction properties were studied using field potential and intra-axonal recording techniques. The demyelinated axons exhibited conduction slowing and block, and a reduction in their ability to follow high-frequency stimulation. Axons remyelinated by transplantation of cultured Schwann cells exhibited restoration of conduction through the lesion, with reestablishment of normal conduction velocity. The axons remyelinated after transplantation showed enhanced impulse recovery to paired-pulse stimulation and greater frequency-following capability as compared with both demyelinated and control axons. These results demonstrate the functional repair of demyelinated axons in the adult CNS by transplantation of cultured myelin-forming cells from the peripheral nervous system in combination with astrocytes.

Changes in the distribution of a calcium-dependent ATPase during demyelination and remyelination in the central nervous system.

A calcium-adenosine triphosphatase (Ca(2+)-ATPase) activity expressed by CNS nerve fibres has been examined during demyelination and remyelination in rats, 21-26 days after an intraspinal injection of ethidium bromide. The Ca(2+)-ATPase distribution was determined cytochemically, using a technique believed primarily to reflect the presence of ecto-ATPases. We confirm that in normal nerve fibres Ca(2+)-ATPase activity was present on the external surface of the myelin sheath, and on the axolemma at the nodes of Ranvier. Labelling of the internodal axolemma was restricted to small, scattered, punctate regions. However, following demyelination the Ca(2+)-ATPase activity was expressed continuously along both the exposed, previously internodal axolemma of entirely naked axons, and it was particularly prominent at sites of contact between axons and glial-cell processes. During remyelination (which in this lesion is accomplished predominantly by Schwann cells) the proportion of the axonal surface exhibiting Ca(2+)-ATPase activity decreased in concert with the progressive thickening of the new myelin sheath. The non-myelin forming plasmalemma of Schwann cells was positive for the Ca(2+)-ATPase activity, but activity was abruptly lost at the site of compaction between the inner and outer leaflets of the forming myelin sheath. Ecto-ATPase activity is a property of some cell adhesion molecules, and it follows that the changes observed in the distribution of ATPase activity in this study may reflect changes in the axolemma which are important for the successful repair of the lesion by remyelination. The ATPase activity may, for example, reflect the changing distribution of molecules important in aiding axo-glial recognition and the establishment of axo-glial contacts.

A mechanism for ectopic firing in central demyelinated axons.

Intracellular recordings of ectopic activity were made from demyelinated axons in rat dorsal columns using in vitro techniques. On-going bursts of discharges were observed in some axons, and these were sometimes superimposed upon slow depolarizing potentials. These intracellularly recorded, depolarizing potentials were strongly reminiscent of the slow negative potentials previously recorded extracellularly from the periaxonal region of normal myelinated fibres following potassium loading of this space. Also, in previously silent demyelinated axons, ectopic activity could sometimes be induced by brief periods of high frequency stimulation applied remotely from the lesion. The generation of the ectopic activity can be explained in terms of an artificially increased periaxonal concentration of potassium which may lead to the opening of internodal potassium channels and an inward potassium current. We suggest that a raised periaxonal potassium concentration may occur within compartments in demyelinating lesions, and that it can result in the generation of ectopic impulses.

Expression of sodium channel alpha- and beta-subunits in the nervous system of the myelin-deficient rat.

Using subtype-specific riboprobes and a non-isotope in situ hybridization technique, the pattern of expression of the mRNAs for voltage dependent sodium channel alpha-subunits I, II, III and NaG, and the beta 1-subunit were compared in myelin-deficient rats and unaffected male littermates. Tissues examined included the hippocampus, cerebellum, spinal cord and dorsal root ganglia. Previous studies have demonstrated that the expression of sodium channel alpha- and beta 1-subunits follows a distinct temporal and spatial pattern during development, characterized in part by greater expression of alpha-subunit III and its mRNA during development than in the adult. We examined animals of 20-22 days of age, a time when, according to earlier reports, the unaffected animals should nearly have reached an adult expression pattern. Normal male littermates were indeed found to express a sodium channel subunit mRNA pattern generally consistent with previous reports on adult rats. Myelin-deficient animals exhibited an expression pattern identical to the unaffected littermates, indicating that myelination is not required for the progression from the embryonic to the adult expression pattern of sodium channel subunits.

Internodal potassium currents can generate ectopic impulses in mammalian myelinated axons.

Positive clinical symptoms can arise from ectopic action potentials in several disorders of myelinated axons. Here we report that an elevated K+ concentration in the periaxonal, internodal space can cause internodal K+ currents to become excitatory, resulting in slow potential oscillations and associated bursts of ectopic spikes. Ectopic firing may therefore be favoured by conditions in which periaxonal K+ buffering is compromised.

Conduction properties of central nerve fibers remyelinated by Schwann cells.

Demyelination of central axons arises from a number of conditions, including multiple sclerosis and spinal cord compression. The demyelination disrupts conduction and leads directly to the production of symptoms. Repair of the demyelination by peripheral myelinating cells could potentially relieve the symptoms, but the conduction properties of central axons remyelinated by Schwann cells have yet to be studied in detail. This paper examined the conduction properties of such axons. Large focal demyelinating and remyelinating lesions were induced in the dorsal columns of rats by the intraspinal injection of ethidium bromide. Recordings of compound action potentials conducted through these lesions were then made at various recovery times. Thus the changing conduction properties of the affected fibers could be correlated with the different stages of lesion development. During the early stages of demyelination there was widespread conduction block, with no evidence of appreciable conduction occurring with prolonged latency or refractory period of transmission (RPT). However, with the onset of remyelination by Schwann cells, conduction was restored in many axons, and most, if not all, of the affected axons eventually showed successful conduction through the lesion. Initially the conduction was characterized by very prolonged latency, long RPT, and an inability to conduct fast trains of impulses. These deficits became less prominent as remyelination progressed. In chronically remyelinated axons the RPT was restored to within normal limits, although some deficit in both conduction velocity and the ability to conduct trains of impulses persisted. Since these deficits were not severe we conclude that remyelination of central demyelinated axons by Schwann cells should be effective in promoting the restoration of normal function.

End-tidal carbon dioxide (CO2) monitoring in small animals.

A simple modification of a commercially available capnometer (Traverse Medical Monitors model 2000) is described which suits it for use with small animals such as rats. The modification involves the omission of the fluids separator from the gas pathway, while maintaining a connection to the separator to permit the continued function of the "automatic zero" feature, which corrects for any zero-drift. The capnometer has several functions which recommend its use even when artificial ventilation is not required.