Glycolysis inhibition improves photodynamic therapy response rates for equine sarcoids.

Photodynamic therapy (PDT) holds great promise in treating veterinary and human dermatological neoplasms, including equine sarcoids, but is currently hindered by the amount of photosensitiser and light that can be delivered to lesions thicker than around 2 mm, and by the intrinsic antioxidant defences of tumour cells. We have developed a new PDT technique that combines an efficient transdermal penetration enhancer solution, for topical delivery of 5-aminolevulinic acid (ALA) photosensitiser, with acute topical post-PDT application of the glycolysis inhibitor lonidamine. We show that the new PDT combination treatment selectively kills sarcoid cells in vitro, with repeated rounds of treatment increasing sarcoid sensitisation to PDT. In vivo, ALA PDT followed by 600 µM lonidamine substantially improves treatment outcomes for occult, verrucous, nodular and fibroblastic sarcoids after 1 month (93% treatment response in 27 sarcoids), compared with PDT using only ALA (14% treatment response in 7 sarcoids).

Targeting tumour energy metabolism potentiates the cytotoxicity of 5-aminolevulinic acid photodynamic therapy.

BACKGROUND: Cancerous cells usually exhibit increased aerobic glycolysis, compared with normal tissue (the Warburg effect), making this pathway an attractive therapeutic target. METHODS: Cell viability, cell number, clonogenic assay, reactive oxygen (ROS), ATP, and apoptosis were assayed in MCF-7 tumour cells and corresponding primary human mammary epithelial cells (HMEC). RESULTS: Combining the glycolysis inhibitors 2-deoxyglucose (2DG; 180 mM) or lonidamine (300 µM) with 10 J cm(-2) 5-aminolevulinic acid (ALA) photodynamic therapy (PDT) increases MCF-7 cytotoxicity (by 3.5-fold to 70% death after 24 h, and by 10-fold in 9-day clonogenic assays). However, glycolysis inhibition only slightly increases HMEC PDT cytotoxicity (between two-fold and three-fold to a maximum of 9% death after 24 h). The potentiation of PDT cytotoxicity only occurred if the glycolysis inhibitors were added after ALA incubation, as they inhibited intracellular accumulation of photosensitiser if coincubated with ALA. CONCLUSION: As 2DG and lonidamine are already used as cancer chemotherapeutic agents, our results are directly translatable to combination therapies with existing topical PDT.

Virolysis of feline calicivirus and human GII.4 norovirus following chlorine exposure under standardized light soil disinfection conditions.

The relationship between the infectivity of the feline calicivirus (FCV) vaccine strain F-9 and capsid destruction (virolysis) in response to available chlorine was investigated under standardized light soil disinfection conditions. Virolysis was measured using RNase pretreatment (in order to destroy exposed RNA following chlorine treatment) and quantitative reverse transcription PCR. A comparison between the results of plaque assays and virolysis following exposure of FCV F-9 grown in tissue culture to different concentrations of available chlorine showed a similar log-linear relationship, with >4-log reductions occurring at 48 and 66 ppm, respectively. Three non-epidemiologically linked human GII.4 noroviruses (NoVs) present in dilute clinical samples showed behavior similar to each other and were 10 times more resistant to virolysis than cultured FCV F-9. FCV F-9 when present in dilute human GII.4 samples acquired increased resistance to virolysis approaching that of human NoVs. This study represents a direct comparison between the virolysis of a surrogate virus (FCV F-9) and that of human GII.4 NoVs within the same matrix in response to available chlorine. The results support the view that matrix effects have a significant effect on virus survival.

A two-dimensional gel electrophoretic study of proteins synthesized and released by degenerating adult mouse sciatic nerve.

Previous two-dimensional (2-D) gel electrophoretic studies of proteins secreted by degenerating mammalian peripheral nerves (Ignatius et al., 1986, Proc. Natl. Acad. Sci. USA 83: 1125-1129; Muller et al., 1986, J. Cell Biol. 102: 393-402) detected the up-regulation of two proteins of 67-70 and 34-37 kDa, although they failed to resolve proteins smaller than about 15 kDa or with isoelectric points greater than 8. In the present study, we have used 2-D gels that can resolve proteins in the molecular mass range 3.6-200 kDa and isoelectric point range 2.4-10.6. This revealed that the incorporation of radiolabel by three diffusible proteins with apparent molecular mass/isoelectric point values of 38/5-6, 27-31/4-5, and 8/>10 was increased in the distal stumps of sciatic nerves 4 days after lesion, while the radiolabel incorporation by a further two proteins (15/5.3 and 12.5-17.5/6.8-7.5) was increased in the distal nerve stump 15 days after lesion. The possible cellular sources of these proteins were assessed by comparing protein secretion from unoperated nerves with nerve segments maintained in culture for 4 days (in which the contribution from recruited macrophages would be expected to be minimal) and segments of nerve that had been frozen and then replaced in situ for 4 days (in which the contribution from nerve sheath cells would be expected to be minimal). This revealed that three of the proteins up-regulated in lesioned nerves (27-31/4-5, 15/5.3, and 12.5-17.5/6.8-7.5) are probably sheath cell products, while the other two (38/5-6 and 8/>10) may be secreted mainly by macrophages (or other cells) that infiltrate the frozen nerve segments. The identity of these proteins and their possible involvement in axonal regeneration remain to be determined.

Defects in pathfinding by cranial neural crest cells in mice lacking the neuregulin receptor ErbB4.

Mouse embryos with a loss-of-function mutation in the gene encoding the receptor tyrosine kinase ErbB4 exhibit misprojections of cranial sensory ganglion afferent axons. Here we analyse ErbB4-deficient mice, and find that morphological differences between wild-type and mutant cranial ganglia correlate with aberrant migration of a subpopulation of hindbrain-derived cranial neural crest cells within the paraxial mesenchyme environment. In transplantation experiments using new grafting techniques in cultured mouse embryos, we determine that this phenotype is non-cell-autonomous: wild-type and mutant neural crest cells both migrate in a pattern consistent with the host environment, deviating from their normal pathway only when transplanted into mutant embryos. ErbB4 signalling events within the hindbrain therefore provide patterning information to cranial paraxial mesenchyme that is essential for the proper migration of neural crest cells.

Chondroitin sulphate-binding molecules may pattern central projections of sensory axons within the cranial mesenchyme of the developing mouse.

During mammalian hindbrain development, sensory axons grow along highly stereotyped routes within the cranial mesenchyme to reach their appropriate entry points into the neuroepithelium. Thus, trigeminal ganglion axons always project to rhombomere (r)2, whilst facial/acoustic ganglia axons always project to r4. Axons are never observed to enter the mesenchyme adjacent to r3, raising the possibility that r3 mesenchyme contains an axon growth-inhibitory activity. Conversely, in mice which lack the erbB4 receptor (normally expressed in r3), trigeminal and facial/acoustic ganglia axons misproject into r3 mesenchyme, suggesting that the putative axon barrier is absent. To investigate this hypothesis, we have developed an in vitro model in which dissociated wild-type embryonic trigeminal ganglion neurons are cultured on longitudinal cryosections of embryonic mouse head. We observed that on wild-type embryonic day 10 (E10) cryosections, neurites generally failed to grow into r3 mesenchyme from the adjacent r2 or r4 mesenchyme. This barrier was removed if cryosections were pretreated with chondroitinase or were washed with excess chondroitin 6-sulphate or hypertonic saline. By contrast, when trigeminal neurons were seeded onto cryosections of E10 erbB4 -/- embryo heads their neurites readily entered mutant r3 mesenchyme. Immunohistochemical analysis demonstrated chondroitin-sulphated proteoglycans throughout the cranial mesenchyme in both wild-type and erbB4 -/- embryos. We propose that trigeminal axons are excluded from wild-type r3 mesenchyme by a growth-inhibitory activity which associates with chondroitin-sulphated proteoglycans and that the synthesis of this activity may rely on signals transduced by erbB receptors.

Behaviour of DRG sensory neurites at the intact and injured adult rat dorsal root entry zone: postnatal neurites become paralysed, whilst injury improves the growth of embryonic neurites.

The dorsal root entry zone is a PNS-CNS junction between Schwann cells and astrocytes, defining the site where dorsal root ganglia (DRG) axons enter the adult mammalian spinal cord. Following dorsal root injury (rhizotomy), DRG axons regenerate within the PNS environment of the root but stop at the DREZ and fail to re-enter the spinal cord. We have used an in vitro model to compare how neurites growing from embryonic (E13) and postnatal (P0 and adult) DRG neurons behave at the uninjured and rhizotomized adult rat DREZ. We find that both freshly dissected and conditioned-lesioned postnatal DRG neurons seldom grow neurites across cryosections of the uninjured or rhizotomized DREZ. However, embryonic DRG neurons more readily grow neurites across cryosections of the uninjured and 7-day post-lesion (dpl) DREZ and are dramatically better able to cross the 21 dpl DREZ. This enhanced growth was abolished by co-incubation with a function-blocking antiserum to beta1-integrin receptors, whilst immunoreactivity for some beta1-integrin ligands (tenascin-C and fibronectin) increased at the DREZ by 21 dpl, suggesting that beta1-integrin ligands may stimulate the growth of embryonic neurites across the 21 dpl DREZ. Fluorescence time-lapse video-microscopy was used to record the behaviour of dye-labelled postnatal DRG neurites as they encounter the uninjured adult DREZ in vitro. Neurites rarely turned around at the DREZ, but instead became paralysed. Of a variety of chemical modifications to uninjured DREZ cryosections, only treatment with methanol, chloroform, or the protease inhibitor D-phe-pro-arg chloromethylketone hydrochloride (PPACK, 100 microM) caused any increase in the proportion of postnatal neurites which crossed the DREZ.

Effects of extracellular matrix components on axonal outgrowth from peripheral nerves of adult animals in vitro.

Relatively little is known of the growth requirements for regenerating axons of the peripheral nervous system of adult animals. In the present study, we show that extracellular matrix material secreted by the Engelbreth-Holm-Swarm tumor cell line (matrigel) supports axonal growth from explanted peripheral nerve-dorsal root ganglia (DRG) preparations of adult mice and amphibia in serum-free media, without addition of growth factors. Axonal growth in matrigel was much more profuse than that in the more commonly used gels of type 1 collagen and, after some days in culture, was accompanied by migration of Schwann cells along axons. The most abundant protein in matrigel is laminin, which has been shown in many studies to support axonal growth but, surprisingly, antisera to laminin did not inhibit axonal growth in matrigel. To determine the ability of the major components of matrigel, laminin, type IV collagen, and heparan sulfate proteoglycan (HSPG), to support axonal growth, these proteins were added to preparations of mouse peripheral nerve-DRGs in type I collagen gels. Regenerating axons were significantly longer in the presence of laminin and type IV collagen than in control cultures, while HSPG had a slight inhibitory effect. In this assay system, however, diluted matrigel solution was even more effective in stimulating axonal growth than laminin or type IV collagen, either alone or in combination. The results suggest that in addition to laminin and type IV collagen, other components within matrigel may contribute to its ability to support axonal growth.

Border controls at the mammalian spinal cord: late-surviving neural crest boundary cap cells at dorsal root entry sites may regulate sensory afferent ingrowth and entry zone morphogenesis.

Boundary caps (BCs) form when neural crest cells, migrating ventrally alongside the neural tube, arrest at sites where axons will enter and exit. However, nothing is known of their subsequent fate and functions. We have found late-surviving neural crest BC cell clusters at proximal dorsal root entry sites throughout rat spinal cord development. Sensory afferents cross BCs to enter the spinal cord, while exiting astrocyte processes, destined to form the dorsal root entry zone (DREZ) after birth, are temporarily stalled in their vicinity. To test whether contact with BC cells influences neurite outgrowth from dorsal root ganglia, neurons were cultured on embryonic dorsal root/spinal cord cryosections. Neurites that entered CNS territory preferentially extended over BC cells. Thus, BC cells could be instrumental in regulating afferent ingrowth and DREZ morphogenesis in mammalian spinal cord development.

Expression of a developmentally regulated, phosphorylated isoform of microtubule-associated protein 1B in sprouting and regenerating axons in vitro.

We have developed a novel culture system for studying axonal regeneration. Short lengths of spinal nerves with their attached dorsal root ganglia were removed from adult mice, explanted into Matrigel and maintained in serum-free medium for up to eight days. Profuse outgrowth of unfasciculated, naked axons occurred within 6 h from the cut ends of the peripheral nerve, dorsal roots and eventually from the ganglion itself, and continued to grow throughout the observation period. Some axons were entirely smooth, whilst others showed prominent varicosities. The former stained with antibody RT97, a marker for large-calibre, myelinated axons, whilst the latter stained with antibodies to calcitonin gene-related peptide, predominantly a marker for unmyelinated and small-diameter myelinated sensory axons. All axons stained with a monoclonal antibody (150) that recognizes a developmentally regulated phosphorylated isoform of the microtubule-associated protein 1B [Gordon-Weeks P. R. et al. (1993) Eur. J. Neurosci. 5, 1302-1311]. Monoclonal antibody 150 staining was observed along the entire length of all axons growing out of the explant; the proximal regions of these axons within the explant itself did not stain. The staining extended to the growth cones, which had elaborate morphologies. Other antibodies (e.g. to growth-associated protein 43) labelled axons within the nerve, as well as those growing in Matrigel. In preparations where the peripheral nerve had been crushed half-way along its length at the time of explantation, monoclonal antibody 150 staining was absent from axons in the nerve proximal to the crush, but present in axons which had regenerated within the nerve distal to the crush. The results indicate that re-expression during axonal regeneration of the phosphorylated isoform of microtubule-associated protein 1B recognized by monoclonal antibody 150 is restricted to the newly formed lengths of regenerated axons. The correlation between its expression and axonal growth during development and regeneration suggests that it may play a role in axonal extension. Our observations also demonstrate the usefulness of these explant cultures for studying axonal regeneration.

An in vitro model of the rat dorsal root entry zone reveals developmental changes in the extent of sensory axon growth into the spinal cord.

The dorsal root entry zone (DREZ) forms the junction between the dorsal roots of the peripheral nervous system and the spinal cord. In rats older than 1 week, lesioned primary sensory axons regenerate within the dorsal roots but stop at the DREZ, and are thus unable to reconnect with the spinal cord. To analyze the causes of this failure, we have developed a culture model of the interaction of sensory axon growth cones with the intact DREZ, whereby dissociated dorsal root ganglion neurons from rats of various ages are grown on longitudinal cryosections of rat spinal cord, incorporating the DREZ and attached dorsal roots, from neonatal, 1-week-old (P6), or adult animals. Neurites of all ages grew along the roots to the DREZ, where their ability to cross into the spinal cord depended on both their age and that of the spinal cord substrate. Neurites from neonatal neurons failed to cross either the P6 or adult DREZ, but a substantial proportion crossed the immature neonatal DREZ. Early embryonic neurites exhibited substantial crossing on both immature and adult DREZ. These findings strongly suggest that soon after birth, the normal mammalian DREZ acquires growth inhibitory activity that is recognized by the axons of postnatal but not early embryonic sensory neurons.

Cellular migration and axonal outgrowth from adult mammalian peripheral nerves in vitro.

It is known that following peripheral nerve transections, sheath cells proliferate and migrate to form a bridge between nerve stumps, which may facilitate axonal regeneration. In the present investigations, cellular migration and axonal outgrowth from nerves of adult mice were studied in vitro using collagen gels. During the first 3 days in culture, profuse migration of fibroblasts and macrophages occurred from the ends of sciatic nerve segments, which had been lesioned in situ a few days prior to explantation, but not from segments of normal nerves. The mechanism of cellular activation in the lesioned nerves was not determined, but migration was blocked by suramin, which inhibits the actions of several growth factors. The migrating cells, which form the bridge tissue, may promote axonal regeneration in two ways. Firstly, axonal outgrowth from isolated intercostal nerves was significantly increased in co-cultures with bridges from lesioned sciatic nerves. This stimulatory effect was inhibited by antibodies to 2.5S nerve growth factor. Secondly, the segments of bridge tissue contracted when removed from animals. It is possible that fibroblasts within the bridge exert traction that would tend to pull the lesioned stumps of peripheral nerve together, as in the healing of skin wounds. The traction may also influence deposition of extracellular matrix materials, such as collagen fibrils, which could orient the growth of the regenerating axons toward the distal nerve stump.

Protein synthesis and release by normal and lesioned axolotl peripheral nerves.

Previous studies in urodeles (Holder et al., 1982, J. Physiol. 326:371; Holder et al., 1984, Proc. R. Soc. Lond. B 222:477; Aaronson et al., 1995, Neuroscience 66:201) have shown that regenerating axons of peripheral nerves tend to grow toward distal nerve stumps, which is consistent with the hypothesis that axonal growth may be stimulated by factors released from degenerating nerves. In the present study we used two-dimensional gel electrophoresis and autoradiography to compare the incorporation of radiolabeled methionine into proteins which are synthesized and released in vitro by segments of normal and previously cut axolotl sciatic nerves, within the isoelectric point range 2.4-10.6 and molecular weight range 3.6-200 kDa. In the distal portion of nerves cut 7 days previously in vivo, the synthesis of at least six secreted proteins was significantly greater than in undamaged nerves. The possible cellular sources of these proteins was assessed by comparing protein release from normal nerves with nerve segments maintained in culture for 7 days (in which the contribution from recruited macrophages would be expected to be minimal) and segments of nerve which had been frozen and then replaced in situ for 7 days (in which the contribution from sheath cells would be expected to be minimal).(ABSTRACT TRUNCATED AT 250 WORDS)

Oriented growth of regenerating axons in axolotl forelimbs is consistent with guidance by diffusible factors from distal nerve stumps.

Previous studies have shown that when peripheral nerves in axolotl limbs are cut and surgically misdirected, regenerating axons grow back to the original pathways and innervate their correct muscles. In the present study however, we demonstrate that when given a choice between their correct nerve stump and an incorrect stump (forearm flexor nerve), regenerating extensor cranialis nerve axons grow towards both pathways. This result suggests that the directed growth of regenerating axons in the peripheral nervous system may be in response to factor(s) released from the distal nerve stumps, but that in this region of the limb, axons were unable to differentiate between correct and incorrect pathways. Growing axons appeared to be accompanied by neural sheath cells, whilst activated macrophages remained near the cut nerve stumps. Possible mechanisms by which regenerating axons may eventually innervate their correct targets are discussed.

Macrophage response during axonal regeneration in the axolotl central and peripheral nervous system.

We have used a monoclonal antibody (5F4) and Griffonia lectin to study the recruitment of macrophages after crushing axolotl central and peripheral axons. In both cases axonal regeneration begins within one to two days and, in the CNS, proceeds at a rate of about 0.05 mm per day. However, in the spinal cord, macrophage entry is restricted to the lesion site whilst in peripheral nerves macrophages rapidly enter the distal nerve stump after injury. These results suggest that the role (if any) played by macrophages during axonal regeneration may differ in these two situations.

Expression of GAP-43 in normal and regenerating nerves in the frog.

A polyclonal antiserum to chicken, growth-associated protein-43 (GAP-43), raised in rabbit, was shown to recognize a molecule with similar properties to GAP-43 in frogs. Using this antiserum, GAP-43 immunoreactivity was shown to be present throughout the brain and white matter of the spinal cord of larval frogs, but became restricted to specific regions in the adult frog central nervous system. In the peripheral nervous system, GAP-43 was present in normal tadpole and adult axons. After cutting the adult sciatic nerve, GAP-43 slowly disappeared from axons in the distal stump, but appeared in Schwann cells and other (uncharacterized) cells. The constitutive expression of GAP-43 in the adult frog sciatic nerve may be related to the phenomenon of remodelling of motor end-plates, which is known to occur throughout life in frogs.

Effects of freezing a segment of peripheral nerve on subsequent protein release and axonal regeneration in the frog.

Previous studies in frogs have shown that axons from the proximal stump of a cut nerve will grow toward the distal stump, possibly in response to diffusible trophic factors produced by cells of the nerve sheath. In the present experiments, the synthesis and release of proteins in vitro, from proximal and distal stumps of frog sciatic nerves, were studied 1, 4, and 14 days after nerve section in vivo. Using two-dimensional gel electrophoresis to separate released proteins, a marked increase in the synthesis of two lipoproteins of 37 and 67 kDa was seen, initially in both proximal and distal stumps, but by 14 days these proteins were produced exclusively by the distal stump. To see if the production of these proteins was correlated with subsequent reinnervation of the distal stump, isolated nerve segments were removed from the frogs and either replaced immediately or frozen (to kill sheath cells) and replaced. After 2 weeks, the pattern of newly synthesized proteins released by both the frozen and nonfrozen nerve segments was similar although freezing severely impaired the reinnervation of the nerve segment. These results suggest that although the 37- and 67-kDa lipoproteins may have a role in nerve regeneration, their presence per se is not sufficient to support the reinnervation of a distal stump of a cut peripheral nerve and that additional factors may therefore be required.

A study of the expression of laminin in the spinal cord of the frog during development and regeneration.

In the present experiments, we have used an affinity-purified polyclonal antibody to laminin to determine the time course of expression of laminin in the central nervous system (CNS) of Rana temporaria tadpoles during normal development and during restoration of the dorsal columns of the spinal cord. Immunoblotting analysis indicated that in the peripheral nervous system (PNS) of adult frogs the antibody recognized proteins of molecular weights 350-400 and 205-220 kDa, corresponding to the A and B chains respectively of mammalian laminin. Immunohistochemistry with our antibody suggested that laminin was absent from the tadpole spinal cord, and did not appear even in the basal lamina of the blood vessels within the spinal cord until after metamorphosis. Furthermore, there was no evidence of laminin expression in the dorsal columns after hemisection of the spinal cord. However, throughout development laminin was present in basal lamina outside the CNS, in particular in the pial membranes, and in the basal lamina of blood vessels and sheath cells in the dorsal and ventral roots. Electron microscopy showed that the blood vessels of CNS capillaries had basal laminae throughout development that was morphologically indistinguishable from that seen in peripheral vessels.