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Objective: This review examines the evidence of neural inhibition as a mechanism underlying pain relief and anesthetic effect of photobiomodulation (PBM)., Background: PBM for pain relief has also been used for more than 30 years; however, the mechanism of its effectiveness has not been well understood., Methods: We review electrophysiological studies in humans and animal models and cell culture studies to examine neural responses to PBM., Results: Evidence shows that PBM can inhibit nerve function in vivo, in situ, ex vivo, and in culture. Animal studies using noxious stimuli indicate nociceptor-specific inhibition with other studies providing direct evidence of local conduction block, leading to inhibited translation of pain centrally. Evidence of PBM-disrupted neuronal physiology affecting axonal flow, cytoskeleton organization, and decreased ATP is also presented. PBM changes are reversible with no side effects or nerve damage., Conclusions: This review provides strong evidence in neuroscience identifying inhibition of neural function as a mechanism for the clinical application of PBM in pain and anesthesia.

Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is an inflammatory neuropathy, classically characterised by a slowly progressive onset and symmetrical, sensorimotor involvement. However, there are many phenotypic variants, suggesting that CIDP may not be a discrete disease entity but rather a spectrum of related conditions. While the abiding theory of CIDP pathogenesis is that cell-mediated and humoral mechanisms act together in an aberrant immune response to cause damage to peripheral nerves, the relative contributions of T cell and autoantibody responses remain largely undefined. In animal models of spontaneous inflammatory neuropathy, T cell responses to defined myelin antigens are responsible. In other human inflammatory neuropathies, there is evidence of antibody responses to Schwann cell, compact myelin or nodal antigens. In this review, the roles of the cellular and humoral immune systems in the pathogenesis of CIDP will be discussed. In time, it is anticipated that delineation of clinical phenotypes and the underlying disease mechanisms might help guide diagnostic and individualised treatment strategies for CIDP., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

The neuroglia of the peripheral nervous system (PNS) are derived from the neural crest and are a diverse family of cells. They consist of myelinating Schwann cells, non-myelinating Schwann cells, satellite cells, and perisynaptic Schwann cells. Due to their prominent role in the formation of myelin, myelinating Schwann cells are the best recognised of these cells. However, Schwann cells and the other neuroglia of the PNS have many functions that are independent of myelination and contribute significantly to the functioning of the peripheral nerve in both health and disease. Here we discuss the contribution of PNS neuroglial cells to clinical deficit in neurodegenerative disease, peripheral neuropathy, and pain., (© 2014 Peripheral Nerve Society.)

Schwann cells are primarily discussed in the context of their ability to form myelin. However there are many subtypes of these neural crest derived cells including satellite cells of the dorsal root ganglia and autonomic ganglia, the perisynaptic Schwann cells of the neuromuscular junction and the non-myelin forming Schwann cells which ensheathe the unmyelinated fibres of the peripheral nervous system which are about 80% of peripheral nerves. This review discusses the many functions of these Schwann cell subsets including their seminal role in axonal ensheathment, perineuronal organisation, maintenance of normal neural function, synapse formation, response to damage and repair and an increasingly recognised active role in pain syndromes., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Serine/threonine protein kinase C βII isoform (PKCβII) or the pain receptor transient receptor potential vanilloid 1 (TRPV1) have been separately implicated in mediating heat hyperalgesia during inflammation or diabetic neuropathy. However, detailed information on the role of PKC βII in nociceptive signaling mediated by TRPV1 is lacking. This study presents evidence for activation and translocation of the PKC βII isoform as a signaling event in nociception mediated by activation of TRPV1 by capsaicin. We show that capsaicin induces translocation of cytosolic PKCβII isoform fused with enhanced green fluorescence protein (PKCβII-EGFP) in dorsal root ganglion (DRG) neurons. We also show capsaicin-induced translocation in Chinese Hamster Ovarian (CHO) cells co-transfected with TRPV1 and PKCβII-EGFP, but not in CHO cells expressing PKCβII-EGFP alone. By contrast, the PKC activator phorbol-12-myristate-13-acetate (PMA) induced translocation of PKCβII-EGFP which was sustained and independent of calcium or TRPV1. In addition PMA-induced sensitization of TRPV1 to capsaicin response in DRG neurons was attenuated by PKCβII blocker CGP 53353. Capsaicin response via TRPV1 in the DRG neurons was confirmed by TRPV1 antagonist AMG 9810. These results suggested a novel and potential signaling link between PKCβII and TRPV1. These cell culture models provide a platform for investigating mechanisms of painful neuropathies mediated by nociceptors expressing the pain sensing gene TRPV1, and its regulation by the PKC isoform PKCβII.

Low-level laser therapy (LLLT) has been shown in clinical trials to relieve chronic pain and the World Health Organization has added LLLT to their guidelines for treatment of chronic neck pain. The mechanisms for the pain-relieving effects of LLLT are however poorly understood. We therefore assessed the effects of laser irradiation (LI) on somatosensory-evoked potentials (SSEPs) and compound muscle action potentials (CMAPs) in a series of experiments using visible (λ = 650 nm) or infrared (λ = 808 nm) LI applied transcutaneously to points on the hind limbs of rats overlying the course of the sciatic nerve. This approximates the clinical application of LLLT. The 650-nm LI decreased SSEP amplitudes and increased latency after 20 min. CMAP proximal amplitudes and hip/ankle (H/A) ratios decreased at 10 and 20 min with increases in proximal latencies approaching significance. The 808-nm LI decreased SSEP amplitudes and increased latencies at 10 and 20 min. CMAP proximal amplitudes and H/A ratios decreased at 10 and 20 min. Latencies were not significantly increased. All LI changes for both wavelengths returned to baseline by 48 h. These results strengthen the hypothesis that a neural mechanism underlies the clinical effectiveness of LLLT for painful conditions., (© 2011 Peripheral Nerve Society.)

Early pathological studies in patients with acute and chronic inflammatory demyelinating neuropathies, and the animal model experimental autoimmune neuritis (EAN) showed similarities in the process of demyelination. These studies focused on compact myelin proteins and peptides as targets of immune attack in Guillain-Barré syndrome (GBS), chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), and EAN. However, serological studies in patients with subsets of GBS highlighted the importance of gangliosides - glycolipids enriched in non-compact Schwann cell regions and the node, paranodal, and internodal axolemma. In the acute motor axonal neuropathy (AMAN) rabbit model, antibodies to the ganglioside GM1 bind in the nodal region, impair Na channel clustering and disturb Schwann cell/axon organisation. Schwann cell neurobiological studies now highlight the importance of adhesion molecules, including neurofascins, gliomedin, contactins, and NrCAM to Schwann cell/axon integrity. Changes to nodal fine structure by immune responses against such molecules may provide a mechanism for reversible conduction failure or block. Recovery of patients with CIDP or multifocal motor neuropathy (MMN) following treatment may sometimes be better explained by reversal of conduction failure than remyelination or regeneration. This review considers the importance of the intricate molecular arrangements at the nodal and paranodal regions in inflammatory neuropathies such as CIDP. Early images of compact myelin stripping and phagocytosis, may have diverted the research focus away from these vital non-compact myelin Schwann cell areas., (© 2011 Peripheral Nerve Society.)

A variety of molecules are reported to be involved in chronic pain. This review outlines the specifics of protein kinase C (PKC), its isoforms and their role in modulating thermo-sensitive transient receptor potential (TRP) channels TRPV1-4, TRPM8, and TRPA1. Anatomically, PKC and thermo-sensitive TRPs are co-expressed in cell bodies of nociceptive dorsal root ganglion (DRG) neurons, which are used as physiological correlates of peripheral and central projections involved in pain transmission. In the past decade, modulation of painful heat-sensitive TRPV1 by PKC has received the most attention. Recently, PKC modulation of other newly discovered thermo-sensitive pain-mediating TRPs has come into focus. Such modulation may occur under conditions of chronic pain resulting from nerve damage or inflammation. Since thermo-TRPs are primary detectors of acute pain stimuli, their modulation by PKC can severely alter their function, resulting in chronic pain. Comprehensive knowledge of pain signaling involving interaction of specific isoforms of PKC with specific thermo-sensitive TRP channels is incomplete. Such information is necessary to dissect out modality specific mechanisms to better manage the complex polymodal nature of chronic pain. This review is an attempt to update the readers on current knowledge of PKC modulation of thermo-sensitive TRPs and highlight implications of such modulation for pain signaling.

The catabolic pathway of l-tryptophan (l-trp), known as the kynurenine pathway (KP), has been implicated in the pathogenesis of a wide range of brain diseases through its ability to lead to immune tolerance and neurotoxicity. As endothelial cells (ECs) and pericytes of the blood-brain-barrier (BBB) are among the first brain-associated cells that a blood-borne pathogen would encounter, we sought to determine their expression of the KP. Using RT-PCR and HPLC/GC-MS, we show that BBB ECs and pericytes constitutively express components of the KP. BBB ECs constitutively synthesized kynurenic acid, and after immune activation, kynurenine (KYN), which is secreted basolaterally. BBB pericytes produced small amounts of picolinic acid and after immune activation, KYN. These results have significant implications for the pathogenesis of inflammatory brain diseases in general, particularly human immunodeficiency virus (HIV)-related brain disease. Kynurenine pathway activation at the BBB results in local immune tolerance and neurotoxicity: the basolateral secretion of excess KYN can be further metabolized by perivascular macrophages and microglia with synthesis of quinolinic acid. The results point to a mechanism whereby a systemic inflammatory signal can be transduced across an intact BBB to cause local neurotoxicity.

Background: Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). Although the pathogenesis of MS remains unknown, it is widely regarded as an autoimmune disease mediated by T-lymphocytes directed against myelin proteins and/or other oligodendrocyte epitopes., Methods: In this study we investigated the gene expression profiles of peripheral blood cells from patients with RRMS during the relapse and the remission phases utilizing gene microarray technology. Dysregulated genes encoded in regions associated with MS susceptibility from genomic screens or previous transcriptomic studies were identified. The proximal promoter region polymorphisms of two genes were tested for association with disease and expression level., Results: Distinct sets of dysregulated genes during the relapse and remission phases were identified including genes involved in apoptosis and inflammation. Three of these dysregulated genes have been previously implicated with MS susceptibility in genomic screens: TGFbeta1, CD58 and DBC1. TGFbeta1 has one common SNP in the proximal promoter: -508 T>C (rs1800469). Genotyping two Australian trio sets (total 620 families) found a trend for over-transmission of the T allele in MS in females (p < 0.13). Upregulation of CD58 and DBC1 in remission is consistent with their putative roles in promoting regulatory T cells and reducing cell proliferation, respectively. A fourth gene, ALOX5, is consistently found over-expressed in MS. Two common genetic variants were confirmed in the ALOX5 putative promoter: -557 T>C (rs12762303) and a 6 bp tandem repeat polymorphism (GGGCGG) between position -147 and -176; but no evidence for transmission distortion found., Conclusion: The dysregulation of these genes tags their metabolic pathways for further investigation for potential therapeutic intervention.

The use of 2 monoclonal antibodies during double immunohistochemistry would enable the use of a greater variety of antibody combinations. Here, we demonstrate a simple, cost effective method of double indirect immunostaining of cultured cells using primary antibodies from the same species. This method uses microwaving of cell samples immediately after the application of the first secondary antibody, and significantly reduces the level of nonspecific staining. This technique does not elute the antibodies, nor raise the sample temperature above 37 degrees C.

We report the formation of 830 nm (cw) laser-induced, reversible axonal varicosities, using immunostaining with beta-tubulin, in small and medium diameter, TRPV-1 positive, cultured rat DRG neurons. Laser also induced a progressive and statistically significant decrease (p<0.005) in MMP in mitochondria in and between static axonal varicosities. In cell bodies of the neuron, the decrease in MMP was also statistically significant (p<0.05), but the decrease occurred more slowly. Importantly we also report for the first time that 830 nm (cw) laser blocked fast axonal flow, imaged in real time using confocal laser microscopy and JC-1 as mitotracker. Control neurons in parallel cultures remained unaffected with no varicosity formation and no change in MMP. Mitochondrial movement was continuous and measured along the axons at a rate of 0.8 microm/s (range 0.5-2 microm/s), consistent with fast axonal flow. Photoacceptors in the mitochondrial membrane absorb laser and mediate the transduction of laser energy into electrochemical changes, initiating a secondary cascade of intracellular events. In neurons, this results in a decrease in MMP with a concurrent decrease in available ATP required for nerve function, including maintenance of microtubules and molecular motors, dyneins and kinesins, responsible for fast axonal flow. Laser-induced neural blockade is a consequence of such changes and provide a mechanism for a neural basis of laser-induced pain relief. The repeated application of laser in a clinical setting modulates nociception and reduces pain. The application of laser therapy for chronic pain may provide a non-drug alternative for the management of chronic pain.

Important mechanisms that regulate inhibitory and facilitatory effects on TRPV1-mediated nociception are desensitization and phosphorylation, respectively. Using Ca2+-imaging, we have previously shown that desensitization of TRPV1 upon successive capsaicin applications was reversed by protein kinase C activation in dorsal root ganglion neurons and CHO cells. Here, using both Ca2+-imaging and patch-clamp methods, we show that PMA-induced activation of PKCepsilon is essential for increased sensitivity of desensitized TRPV1. TRPV1 has two putative substrates S502 and S800 for PKCepsilon-mediated phosphorylation. Patch-clamp analysis showed that contribution of single mutant S502A or S800A towards increased sensitivity of desensitized TRPV1 is indistinguishable from that observed in a double mutant S502A/S800A. Since S502 is a non-specific substrate for TRPV1 phosphorylation by kinases like PKC, PKA or CAMKII, evidence for a role of PKC specific substrate S800 was investigated. Evidence for in vivo phosphorylation of TRPV1 at S800 was demonstrated for the first time. We also show that the expression level of PKCepsilon paralleled the amount of phosphorylated TRPV1 protein using an antibody specific for phosphorylated TRPV1 at S800. Furthermore, the anti-phosphoTRPV1 antibody detected phosphorylation of TRPV1 in mouse and rat DRG neurons and may be useful for research regarding nociception in native tissues. This study, therefore, identifies PKCepsilon and S800 as important therapeutic targets that may help regulate inhibitory effects on TRPV1 and hence its desensitization.

Multiple sclerosis (MS) is an enigmatic disease of the central nervous system resulting in sclerotic plaques with the pathological hallmarks of demyelination and axonal damage, which can be directly or indirectly orchestrated by cells from the peripheral circulation. The majority of patients with MS follow a relapsing-remitting course in the early stages of the disease (RRMS) but most ultimately enter a secondary progressive phase (SPMS). About 10% of patients follow a primary progressive course from the onset (PPMS). We measured gene expression in whole blood of people with and without chronic progressive MS (CPMS), PPMS and SPMS, to discover genes which may be differentially expressed in peripheral blood in active disease, and so identify pathologically significant genes and pathways; and we investigated genetic differences in the promoters of dysregulated genes encoded in genomic regions associated with MS. If SPMS and PPMS were independently compared to the controls, there was little overlap in the set of most dysregulated genes. Ribosomal protein genes, whose expression is usually associated with cell proliferation and activation, were dramatically over-represented in the set of most down-regulated genes in PPMS compared to SPMS (P < 10(-4), chi(2)). The T cell proliferation gene IL7R (CD127) was also underexpressed in PPMS, but was up-regulated in SPMS compared to the controls. One interleukin 7 receptor (IL7R) promoter single nucleotide polymorphism (SNP), -504 C, was undertransmitted in PPMS trios (P = 0.05, TDT), and carriers of this allele were under-represented in PPMS cases from two independent patient cohorts (combined P = 0.006, FE). The four known IL7R promoter haplotypes were shown to have similar expression levels in healthy controls, but not in CPMS (P < 0.01, t test). These data support the hypothesis that PPMS has significant pathogenetic differences from SPMS, and that IL7R may be a useful therapeutic target in PPMS.

The mechanisms causing persistence of embryonal cells that later give rise to tumors is unknown. One tumorigenic factor in the embryonal childhood tumor neuroblastoma is the MYCN protooncogene. Here we show that normal mice developed neuroblast hyperplasia in paravertebral ganglia at birth that completely regressed by 2 weeks of age. In contrast, ganglia from MYCN transgenic (TH-MYCN) mice demonstrated a marked increase in neuroblast hyperplasia and MycN expression during week 1. Regression of neuroblast hyperplasia was then delayed and incomplete before neuroblastoma tumor formation at 6 and 13 weeks in homo- and hemizygote mice, respectively. Paravertebral neuronal cells cultured from perinatal TH-MYCN mice exhibited 3- to 10-fold resistance to nerve growth factor (NGF) withdrawal, compared with normal mice. Both low- and high-affinity NGF receptors were expressed in perinatal neuroblast hyperplasia but not in neuroblastoma tumor tissue. MYCN transgene amplification was present at low levels in perinatal neuroblast hyperplasia from both homo- and hemizygote TH-MYCN mice. However, only in hemizygous mice did tumor formation correlate with a stepwise increase in the frequency of MYCN amplification. These data suggest that inappropriate perinatal MycN expression in paravertebral ganglia cells from TH-MYCN mice initiated tumorigenesis by altering the physiologic process of neural crest cell deletion. Persisting embryonal neural crest cells underwent further changes, such as MYCN amplification and repression of NGF receptor expression, during tumor progression. Our studies provide a model for studying perinatal factors influencing embryonal tumor initiation.

Ca2+ selective ion channels of vanilloid receptor subtype-1 (TRPV1) in capsaicin-sensitive dorsal root ganglion (DRG) neurons and TRPV1 transfected Chinese hamster ovarian (CHO) cells are desensitized following calcium-dependent tachyphylaxis induced by successive applications of 100 nM capsaicin. Tachyphylaxis of TRPV1 to 100 nM capsaicin stimuli was not observed in the absence of extracellular calcium. Capsaicin sensitivity of desensitized TRPV1 ion channels recovered on application of phorbol-12-myristate-13-acetate (PMA). PMA-induced recovery of desensitized TRPV1 was primarily due to influx of extracellular calcium observed during re-application of capsaicin following desensitization. Capsazepine blocked the re-sensitization to capsaicin by PMA. Protein kinase C (PKC) inhibitory peptide PKC fragment 19-36 also inhibited re-sensitization to capsaicin by PMA. Reversal of capsaicin-induced desensitization by PMA was prevented by a mutation of TRPV1 where phosphorylation sites serine502 and serine800 were replaced with alanine. This study provides evidence for a role of PKC in reversing capsaicin-induced calcium-dependent desensitization of TRPV1 ion channels.

Glutamate excitotoxicity has been implicated in neuronal death and damage in many neurodegenerative disorders. The potential neuroprotective role of the plasma membrane calcium ATPase (PMCA) and the NMDA receptor were investigated in rat and human brain neurons after a glutamate insult. Investigation of potential mechanisms of neuronal survival revealed that surviving rat cerebellar granule cells expressed the mRNA of new PMCA isoforms 2b and 2c. There was no observable change in expression of PMCA isoforms or NMDA receptor NR2 subtypes in human cortical neurons. This study shows that subsets of rat and human neurons are resistant to glutamate-induced excitotoxicity and the mechanisms employed to enable survival differ between rat and human neurons.

Human fetal tissue is a scarce resource that has been used in Australia for biomedical research since 1980. From 1994 to 2002, it has been used for research by 19 biomedical researchers at 12 separate Australian institutions (four universities, six major teaching hospitals and two research institutes). With an average of 265 samples distributed annually, researchers have conducted experiments in biomedical research with the approval of their Human Ethics Committees, and published 74 manuscripts in peer reviewed journals over the past decade. The tissue is obtained from therapeutic termination of pregnancies at 8-20 weeks', but mostly 14-18 weeks', gestation. The average number of fetuses obtained over the past 10 years was 108 per annum. Our understanding of the pathogenesis of human diseases such as diabetes, multiple sclerosis, retinopathy of prematurity and osteoporosis has been advanced because of such experiments, and better drug treatment of disorders such as osteoarthritis has been made possible with the use of human fetal tissue. The benefits of human fetal tissue research need greater recognition.

Oxidative stress has been implicated in the pathogenesis of a number of human neurodegenerative disorders of the central nervous system (CNS), including Alzheimer's disease (AD). To better understand the pathological effects of oxidative stress on CNS neurons we used a primary human brain cell culture model of hydrogen peroxide-induced oxidative stress. Neuronal and astrocytic morphology was visualised by immunofluorescence with antibodies to the neuron-specific microtubule component beta-tubulin III and against glial fibrillary acidic protein (GFAP), respectively. After exposure to 40 mM H(2)O(2) for 60-90 min, axonal swelling was observed, which developed into axonal beading after 48 h. No beading was observed in GFAP-positive astrocytes. Despite the concentration of H(2)O(2) used, neurons remained attached to the substratum and showed no signs of apoptosis. This was attributed to the neuroprotective effect of the B-27 medium supplement, which contained antioxidants. The axonal swelling and beading was consistent with a disruption of microtubules by oxidative stress and subsequent hold-up of axonal transport.

Within the brain, quinolinic acid (QUIN) is an important neurotoxin, especially in AIDS dementia complex (ADC). Its production by monocytic lineage cells is increased in the context of inflammation. However, it is not known whether QUIN promotes inflammation. Astrocytes are important in immunoregulation within the brain and so we chose to examine the effects of QUIN on the astrocyte. Using purified primary human fetal astrocyte cultures, we determined chemokine production using ELISA assays and RT-PCR and chemokine receptor expression using immunocytochemistry and RT-PCR with QUIN in comparison to TNFalpha, IL-1beta, and IFNgamma. We found that QUIN induces astrocytes to produce large quantities of MCP-1 (CCL2) and lesser amounts of RANTES (CCL5) and IL-8 (CXCL8). QUIN also increases SDF-1alpha (CXCL12), HuMIG (CXCL9), and fractalkine (CX(3)CL1) mRNA expression. Moreover, QUIN leads to upregulation of the chemokine receptor expression of CXCR4, CCR5, and CCR3 in human fetal astrocytes. Most of these effects were comparable to those induced by TNFalpha, IL-1beta, and IFNgamma. The present work represents the first evidence that QUIN induces chemokine and chemokine receptor expression in astrocytes and is at least as potent as classical mediators such as inflammatory cytokines. These results suggest that QUIN may be critical in the amplification of brain inflammation, particularly in ADC., (Copyright 2003 Wiley-Liss, Inc.)

There is good evidence that the kynurenine pathway (KP) and one of its products, quinolinic acid (QUIN) play a role in the pathogenesis of neurological diseases. Monocytic cells are known to be the major producers of QUIN. However, macrophages have the ability to produce approximately 20 to 30-fold more QUIN than microglia. The molecular origin of this difference has not been clarified yet. Using unstimulated and IFN-gamma-stimulated cultures of human fcetal microglia and adult macrophages, we assayed mRNA expression of 8 key enzymes of the KP using RT-PCR and QUIN production using GC-MS. We found that after stimulation with IFN-gamma microglia produced de novo 20-fold less QUIN than macrophages. This quantitative difference in the ability to produce QUIN appears to be associated with a lower expression of 3 important enzymes of the KP in microglia: indoleamine 2,3-dioxygenase (IDO), kynureninase (KYNase) and kynurenine hydroxylase (KYN(OH)ase). These results suggest that activated infiltrating macrophages are the most potent QUIN producers during brain inflammatory diseases with playing a lesser role.

Little is known about the mechanisms of transport of neurotropic herpesviruses, such as herpes simplex virus (HSV), varicella-zoster virus, and pseudorabies virus, within neurons. For these viruses, which replicate in the nucleus, anterograde transport from the cell body of dorsal root ganglion (DRG) neurons to the axon terminus occurs over long distances. In the case of HSV, unenveloped nucleocapsids in human DRG neurons cocultured with autologous skin were observed by immunoelectron microscopy to colocalize with conventional ubiquitous kinesin, a microtubule-dependent motor protein, in the cell body and axon during anterograde axonal transport. Subsequently, four candidate kinesin-binding structural HSV proteins were identified (VP5, VP16, VP22, and US11) using oligohistidine-tagged human ubiquitous kinesin heavy chain (uKHC) as bait. Of these viral proteins, a direct interaction between uKHC and US11 was identified. In vitro studies identified residues 867 to 894 as the US11-binding site in uKHC located within the proposed heptad repeat cargo-binding domain of uKHC. In addition, the uKHC-binding site in US11 maps to the C-terminal RNA-binding domain. US11 is consistently cotransported with kinetics similar to those of the capsid protein VP5 into the axons of dissociated rat neurons, unlike the other tegument proteins VP16 and VP22. These observations suggest a major role for the uKHC-US11 interaction in anterograde transport of unenveloped HSV nucleocapsids in axons.

Apolipoprotein E (apoE) plays a role in the distribution of lipid within many organs and cell types in the human body, including the central nervous system (CNS). The apoE4 isoform is also an established risk factor for late-onset Alzheimer's disease (AD), however its role in the aetiology of the disease remains largely unknown. Therefore, as AD is a late-onset disease, we sort to investigate how conditions hypothesised to model ageing affect apoE metabolism, such as the transport of apoE along the secretory pathway. Two of these models include oxidative stress and calcium deregulation. Using apoE-EGFP-expressing astrocytoma cell lines we established that vesicle number and velocity are up-regulated under oxidative stress conditions, and slowed under KCl induced calcium deregulation. Although these findings apply to cells in general under these two stress conditions, the up-regulation of apoE in particular may be a response to cell injury with implications for neurodegeneration such as that found with late-onset AD., (Copyright 2002 Elsevier Science Ltd. All rights reserved.)

Apolipoprotein E (apoE), a well characterized protein, forms lipoprotein complexes with cholesterol. Such complexes formed are endocytosed via the LDL receptor family by many cell types in particular within the human central nervous system (CNS). The apoE-endocytic pathway leads to apoE degradation. However, it has recently been indirectly shown that apoE can be retained intracellularly and then re-secreted. To investigate the fate of endocytosed apoE isoforms E2 and E3 within human CNS cells in real-time, we added the CNS form of these apoE isoforms, linked to a green fluorescent protein (EGFP), to cultured human foetal brain tissue. There was bi-directional trafficking of apoE-EGFP in neuron and astrocyte processes and 'stationary' perinuclear vesicles in type-I astrocytes. Thus, active apoE recycling in cells with defined processes suggests a role for apoE in mediating signalling through receptor-mediated endocytosis.

Interferon-beta(1b) (IFN-beta(1b)) has limited efficacy in the treatment of relapsing-remitting multiple sclerosis (RRMS). The kynurenine pathway (KP) is chiefly activated by IFN-gamma and IFN-alpha, leading to the production of a variety of neurotoxins. We sought to determine whether IFN-beta(1b) induces the KP in human monocyte-derived macrophages, as one explanation for its limited efficacy. Serial dilutions of IFN-beta(1b) (at concentrations comparable to those found in the sera of IFN-beta(1b)-treated patients) were added to human macrophage cultures. Supernatants were collected at various time points and assayed for the KP end product, quinolinic acid (QUIN). The effect of IFN-beta(1b) on the KP enzymes indoleamine 2,3-dioxygenase (IDO), 3-hydroxyanthranilate dioxygenase (3HAO), and quinolinate phosphoribosyltransferase (QPRTase) mRNA expression was assessed by semiquantitative RT-PCR. IFN-beta(1b) (> or =10 IU/ml) led to increased mRNA expression of both IDO and QUIN production (7901 +/- 715 nM) after 72 h at 50 IU/ml IFN-beta(1b) (p < 0.0001). This study demonstrates that IFN-beta(1b), in pharmacologically relevant concentrations, induces KP metabolism in human macrophages and may be a limiting factor in its efficacy in the treatment of MS. Inhibitors of the KP may be able to augment the efficacy of IFN-beta in MS.

There is good evidence that the kynurenine pathway (KP) and one of its products, quinolinic acid (QUIN), play a role in the pathogenesis of neurological diseases, in particular AIDS dementia complex. Although QUIN has been shown to be produced in neurotoxic concentrations by macrophages and microglia, the role of astrocytes in QUIN production is controversial. Using cytokine-stimulated cultures of human astrocytes, we assayed key enzymes and products of the KP. We found that human astrocytes lack kynurenine hydroxylase so that large amounts of kynurenine and the QUIN antagonist kynurenic acid were produced. However, the amounts of QUIN that were synthesized were subsequently completely degraded. We then showed that kynurenine in concentrations comparable with those produced by astrocytes led to significant production of QUIN by macrophages. These results suggest that astrocytes alone are neuroprotective by minimizing QUIN production and maximizing synthesis of kynurenic acid. However, it is likely that, in the presence of macrophages and/or microglia, astrocytes become indirectly neurotoxic by the production of large concentrations of kynurenine that can be secondarily metabolized by neighbouring or infiltrating monocytic cells to form the neurotoxin QUIN.

Apolipoprotein E (apoE) plays a role in the distribution of lipid within many organs and cell types in the human body, including neurons and astrocytes of the central nervous system (CNS). The apoE4 isoform is also a genetic risk factor for late onset Alzheimer's disease (AD). However, the mechanism by which apoE is involved in AD is largely unknown. In order to understand how apoE is involved in the distribution of lipid in the CNS, we sought to investigate not only the origin of intraneuronal apoE, but the pathway by which it is processed once synthesized. We have established that human neurons can synthesize apoE in the presence of astrocytes, and that intracellular neuronal apoE is processed through the rough endoplasmic reticulum, golgi, and CD63-positive lysosomes where it may be stored before secretion. Our results also suggest that apoE synthesis is regulated by a feedback mechanism, controlled by the neuron itself. This regulatory mechanism may be essential to the maintenance of neuronal cholesterol concentrations and in turn membrane stability., (Copyright 2001 Wiley-Liss, Inc.)

The association of the E4 isoform of apolipoprotein E (apoE) as a genetic risk factor for late onset Alzheimer's disease (AD) has been well established. Central nervous system (CNS) neurons are specifically affected so that defining the mechanisms by which two of the major human apoE isoforms act within CNS neurons is important to our understanding of their effect on neuronal maintenance and function. We have developed a cell culture model using human brain tissue to characterize exogenous apoE transport. We have tracked the association of apoE3 and E4 with CD63, the GTP-binding protein rab5a and the acidic hydrolase cathepsin D, which localize lysosomes, early endosomes, and late endosomes/lysosomes, respectively. Double immunostaining and confocal laser scanning microscopy revealed by z-series that after 30 min most intraneuronal apoE colocalized with rab5a, whereas no astrocyte apoE/rab5a colocalization was detected. Conversely, apoE3 and CD63 did not colocalize in neurons, even after 1 h, but was colocalized in astrocytes. Also, there was approximately 9% apoE3 colocalization with cathepsin D in neurons, whereas up to 87% of apoE4 vesicles were colocalized. In astrocytes, the proportion of apoE3 colocalized with cathepsin D was greater than that in neurons, but still significantly different from that found with apoE4. These immunohistological data demonstrate that, in neurons, apoE can be endocytosed via a rab5a-regulated vesicle-mediated pathway and that beyond this stage there may be isoform specific differences in apoE trafficking present in both neurons and astrocytes., (Copyright 2001 Academic Press.)

Capsaicin, a pungent ingredient of hot chilli peppers, triggered Ca(2+) influx in dorsal root ganglion (DRG) neurons, which express specific vanilloid receptors of type 1, with ED(50)<100 nM. An increase in capsaicin concentration to 10 microM inhibited Ca(2+) clearance from the cytosol, but did not affect the amplitude of intracellular Ca(2+) elevation. In DRG neurons, 10 microM capsaicin also produced a significant drop in mitochondrial membrane potential (Deltapsi), as measured with the mitochondria-specific potentiometric fluorescent dye JC-1. Similar loss of mitochondrial potential upon application of capsaicin was observed in non-neuronal primary (human lymphocytes) and transformed (human myeloid leukaemia cell line, HL-60) cells. The EC(50) values for capsaicin-induced mitochondrial depolarisation were 6.9 microM (DRG neurons), 200 microM (human lymphocytes) and 150 microM (HL-60 cells). Removal of extracellular Ca(2+) or an application of the antioxidant trolox attenuated capsaicin-induced dissipation of Deltapsi in DRG neurons, but not in human lymphocytes and HL-60 cells. Rotenone, an inhibitor of complex I of the mitochondrial respiratory chain, and oligomycin, an inhibitor of F(0)F(1)-ATPase, significantly enhanced the mitochondrial depolarisation produced by capsaicin in DRG neurons. In human lymphocytes and HL-60 cells, only oligomycin potentiated the effect of capsaicin. From our results, we suggest that, in DRG neurons and non-neuronal cells, capsaicin dissipates Deltapsi, possibly due to a direct inhibition of complex I of the mitochondrial respiratory chain. The presence of vanilloid receptor-1 in DRG neurons makes their mitochondria 20-30-fold more sensitive to the depolarising effect of capsaicin compared with non-neuronal cells lacking vanilloid receptor-1. The higher sensitivity of DRG neurons to capsaicin may underlie a selective neurotoxicity of capsaicin towards sensory neurons.

This is the first study to report the presence of T and B lymphocyte markers and antigen presenting-like molecules in a marsupial bandicoot. Intra-cytoplasmic markers for CD3 and CD5, as well as surface Thy-1.1 and CD1a molecules were located in lymphocytes of T dependent regions of immuno-lymphoid tissue in the northern brown bandicoot using immunohistochemical techniques. Similarly, intra-cytoplasmic domains of CD79a, CD79b molecules and surface IgG molecules enabled characterisation of B lymphocytes and plasma cells. The phenotypic expression of these molecules parallels findings in eutherians, suggesting firstly the conservation of lineage epitopes for T and B subsets and secondly, the potential for similar functional properties of immune system cells between marsupials and eutherians. In addition, the presence of MHC class II and CD1a molecules on dendritic-like cells may indicate similar mechanisms for antigen processing and presentation as reported in eutherians. The use of such immune system cell markers will enable functional studies to characterise the marsupial immune system as well as ontogeny studies of immune competence.

The cellular mechanisms involved in axonogenesis are still unclear. In the present work we found that formation of neurites in cultured neonatal dorsal root ganglion neurons co-incided with the redistribution of highly charged mitochondria. Radially distributed in subplasmalemmal space 3 h after plating, highly charged mitochondria formed clusters in the hillocks of predominant neurites during the next 2448 h and then redistributed into the axons. These results provide evidence that accumulation of a critical mass of charged mitochondria at the site of the future axonal hillock may represent the slow initiation stage of axonogenesis, followed by a fast growth phase.

We report for the first time the rearrangement of mitochondrial arrays in developing dorsal root ganglion (DRG) neurons isolated from neonatal rats in culture. Neurons were loaded with the mitochondria-specific fluorescent dye JC-1, and three-dimensional (3D) reconstruction of mitochondrial fluorescence was performed by confocal laser sectioning in fresh neurons and neurons kept in culture up to a week. We found that after 24 hours the mitochondria become reorganised to form clusters in the axonal hillocks. Axonal extension and neuronal network formation coincided with a redistribution of the mitochondrial clusters. In the extended axons the mitochondria become spaced along the axonal length; however, they formed clusters in the branch points and growth cones. We conclude that the initial clusters of mitochondria may be storage pools of mobile mitochondria able to be mobilised to provide energy for axonal transport during neuronal regeneration and neuronal outgrowth. These findings may have relevance to the rate of axonal regeneration and axonal transport in adult DRG neurons, and neuronal polarisation and axonal outgrowth regulation in developing DRG neurons.

The role of astrocytes in the production of the neurotoxin quinolinic acid (QUIN) and other products of the kynurenine pathway (KP) is controversial. Using cytokine-stimulated human astrocytes, we assayed key enzymes and products of the KP. We found that astrocytes lack kynurenine-hydroxylase so that large amounts of kynurenine (KYN) and kynurenic acid (KYNA) were produced, while minor amounts of QUIN were synthesised that were completely degraded. We then showed that kynurenine added to macrophages led to significant production of QUIN. These results suggest that astrocytes alone are neuroprotective by minimising QUIN production and maximising synthesis of KYNA. However, it is likely that, in the presence of macrophages and/or microglia, astrocytes are neurotoxic by producing large concentrations of KYN that can be metabolised by neighbouring monocytic cells to QUIN.

This study demonstrates that rat Schwann cells can reactivate resting experimental allergic neuritis generating P(2) and P(2) peptide specific CD4(+) T cell lines. T cell proliferation was significantly greater to P(2) than to P(2) peptide (SP-26) or ovalbumin (OA). Four-level analysis of variance showed that T cell proliferation with endogenous or exogenous P(2) was not significantly different for Schwann cells plus cytokine IFN-gamma (P = 0.5071) unlike P(2) peptide or OA specific T cells (P = 0.0056 and 0.0003, respectively). Untreated Schwann cells were more effective inducers than irradiated or fixed Schwann cells. As stimulated CD4(+) P(2) T cells produce IFN-gamma and TNF-alpha, this could exacerbate blood nerve barrier breakdown that has been increasingly implicated in inflammatory demyelinating neuropathies (IDNs). This would permit entry of antibodies and complement, thereby contributing to the demyelination process. Schwann cell induced reactivation of CD4(+) T cells may therefore play a role in IDNs., (Copyright 1999 Academic Press.)

We report for the first time the development and morphological characteristics of the spleen thymus, lymph nodes and liver of the northern brown bandicoot, Isoodon macrourus. To date few marsupial species have been studied. The development and morphological characteristics of the organs shared the typical features of those few other marsupials studied as well as those of eutherians. These suggest comparative functional properties with the eutherian immune system. The thymus differentiated within the first week of pouch life and showed evidence of immunolymphopoiesis up to juvenile stages. The spleen, though seeded by lymphocytes within the first week of pouch life, was slower to mature, but differentiated and showed signs of immunocompetency by the time young left the pouch. The mature spleen displayed the same anatomical blood filtering and immunosurveillance properties as that of the eutherian spleen, with evidence of erythrocyte destruction, thrombopoietic activity, activation and differentiation of immunocompetent lymphocytes. However, the absence of sheathed capillary structures in the spleen may indicate differences in the humoral response to circulating antigens. Similarly, lymph nodes also mature by this stage with anterior nodes appearing before posterior nodes. The mature lymph nodes displayed structural features of secondary immuno-lymphoid organs consistent with production of immune responses. Finally, the liver displayed haemopoietic activity for the first four weeks of pouch life. The pattern of development in the bandicoot appears to parallel the pattern reported for other marsupials, yet the thymus matures considerably earlier than previously reported and may be of significance in the development and onset of cell-mediated immunity. Current studies to characterise cellular components, such as T/B lymphocyes and accessory cells of these organs will help to define the mechanisms of immune recognition, activation and hence outline the basis of the marsupial humoral and cellular immunity.

Proinflammatory cytokines contribute to the regulation of the disease process in inflammatory neuropathies. Cellular localisation of cytokine expression in CIDP nerve biopsies should provide further insight into the pathogenic mechanisms of the disease and the individual cells involved. In this study in situ hybridisation was used to determine the exact localisation and identity of cells that express TNF alpha, IFN gamma and IL-2 mRNA within the CIDP nerve. Paraffin embedded and frozen sural nerve biopsies from three acute phase CIDP patients were used for the study. Sections of these samples were probed with digoxigenin labelled oligoprobes for TNF alpha, IFN gamma and IL-2. The results demonstrate localisation of cytokine expression to the inner rim of the perineurium, epineurial and endoneurial blood vessels and infiltrating inflammatory cells. In addition strong staining for TNF alpha. mRNA was widespread in the endoneurium in areas consistent with/suggestive of Schwann cells. Expression of cytokines in the perineurium and endoneurial blood vessels may have pertinent implications with respect to the breakdown of the blood nerve barrier associated with CIDP. In the very least the potential for an immunomodulatory role may be ascribed to these cells.

Schwann cells are capable of producing many immunomodulatory molecules, which indicates that they may play an active role in autoimmune diseases of the peripheral nervous system. We have previously reported production of the prostanoids prostaglandin E2 and thromboxane A2, products of arachidonic acid metabolism, by Schwann cells. This study reports that Schwann cells are capable of producing leukotriene C4, also a product of arachidonic acid metabolism. Production of leukotriene C4 was in response to pre-incubation of the Schwann cells with the cytokines interferon-gamma and tumour necrosis factor-alpha followed by incubation with dimethylsulfoxide. The cytokines alone did not elicit a response nor did stimulation with calcium ionophore, phorbol ester or lipopolysaccharide.

The involvement of astrocytes in Kynurenine pathway (KP) metabolism is still poorly understood. In the present study, we investigated the ability of human fetal astrocytes in vitro to produce quinolinic and picolinic acids using mass spectrometry. In parallel, we estimated the level of expression of five major KP enzymes using RT-PCR. The results demonstrated that astrocytes express most KP enzymes, except for kynurenine-hydroxylase. This in vitro study provides novel informations regarding the ability of human fetal astrocytes to degrade L-tryptophan along the KP.

The motor protein kinesin is a heterotetramer composed of two heavy chains of approximately 120 kDa and two light chains of approximately 65 kDa protein. Kinesin motor activity is dependent on the presence of ATP and microtubules. The kinesin light chain-binding site in human kinesin heavy chain was determined by reconstituting in vitro a complex of recombinant heavy and light chains. The proteins expressed in bacteria included oligohistidine-tagged fragments of human ubiquitous kinesin heavy chain, spanning most of the stalk and all of the tail domain (amino acids 555-963); and untagged, essentially full-length human kinesin light chain (4-569) along with N-terminal (4-363) and C-terminal (364-569) light chain fragments. Heavy chain fragments were attached to Ni2+-charged beads and incubated with untagged light chain fragments. Analysis of eluted complexes by SDS-PAGE and immunoblotting mapped the light chain-binding site in heavy chain to amino acids 771-813, a region close to the C-terminal end of the heavy chain stalk domain. In addition, only the full-length and N-terminal kinesin light chain fragments bound to this heavy chain region. Within this heavy chain region are four highly conserved contiguous heptad repeats (775-802) which are predicted to form a tight alpha-helical coiled-coil interaction with the heptad repeat-containing N-terminus of the light chain, in particular region 106-152 of human light chain. This predicted hydrophobic, alpha-helical coiled-coil interaction is supported by both circular dichroism spectroscopy of the recombinant kinesin heavy chain fragment 771-963, which displays an alpha-helical content of 70%, and the resistance of the heavy/light chain interaction to high salt (0.5 M).

Apolipoprotein E (apoE) has been confirmed as a risk factor for late-onset Alzheimer's disease (AD) and is associated with neurofibrillary tangles and senile plaques, the microscopic pathological characteristics of AD. There has been no direct evidence that human central nervous system neurons can take up and internalize exogenous apoE, which may be important in order for apoE to be involved in the development of the disease. This paper demonstrates by immunohistochemistry and confocal microscopy that cultured human brain neurons can take up and internalize exogenous recombinant human apoE3. We confirm that neurons express the low-density lipoprotein receptor-related protein (LRP) but do not express the low-density lipoprotein receptor. We also demonstrate that the LRP mediates the neuronal uptake of apoE.

Objective: Concentrations of quinolinic acid, an N-methyl-D-aspartate agonist, are often elevated for long periods of time in the cerebrospinal fluid (CSF) and brain tissue of patients with AIDS dementia complex (ADC). This study was designed to test the hypothesis that chronic exposure of human neurons to quinolinic acid levels equivalent to those in the CSF of ADC patients is neurotoxic., Design and Methods: Human fetal brain 14-16 weeks post-menses was cultured in medium with no detectable levels of quinolinic acid. After 4 weeks, 350 or 1200 nmol/l quinolinic acid was added to the feeding medium for a further 5 weeks. Neurotoxicity was evaluated using immunohistochemistry, transmission and scanning electron microscopy, and image analysis., Results: A total of 1200 nmol/l quinolinic acid caused altered cell associations, a decrease in cell density and decreased microtubule-associated protein (MAP)-2 immunoreactivity compared with cultures exposed to 350 nmol/l quinolinic acid or controls. Image analysis of neurons in randomly selected fields revealed significantly swollen cells (P < 0.0001) compared with those treated with 350 nmol/l quinolinic acid or controls. Dendritic varicosities and discontinuous microtubular arrays were present in neurons exposed to both quinolinic acid concentrations, but not in control cultures., Conclusions: This study is the first to assess quinolinic acid levels in the experimental medium, and demonstrates that chronic exposure of human neurons to concentrations of quinolinic acid equivalent to those in the CSF of patients with ADC leads to alterations in dendritic ultrastructure and MAP-2 immunoreactivity, which is consistent with ADC pathology.

This paper described several developmental stages of the male urogenital system in the koala Phascolarctos cinereus, employing both light and scanning electron microscopy. There are few studies of the development of the urogenital system in male marsupials. Findings by White and Timms (1994) that male koalas can be infected with Chlamydia psittaci emphasise the importance of studies on male animals and in particular their reproductive system. Specimens in our study ranged in age from 15 days postnatal to adults. Due to the rarity of such specimens, details of each specimen are linked to the changes of the structures at each available stage. Light microscopy revealed that differentiation of the gonads had commenced by 15 days postnatal and that the cytological arrangements of the urogenital system are essentially the same as those of other mammals. Scanning electron microscopy revealed stereocilia and microvilli along the lumen of each ductus epididymis and cilia and microvilli along the lumina of the vasa deferentia and urethra. The development of these structures coincided with the onset of sexual maturation, sperm production and differentiation at about three years of age.

The AIDS dementia complex (ADC) is a consequence of excessive immune activation driven at least in part by systemic HIV infection and probably brain infection. Quinolinic acid (QUIN) is a neurotoxic tryptophan metabolite produced by macrophages in response to stimulation with cytokines or infection with HIV-1. Consequently it has been implicated in ADC pathogenesis. However, macrophages infected with HIV-1 synthesize numerous neurotoxic substances. Therefore we conducted experiments using human fetal brain tissue to determine the relative importance of QUIN as a neurotoxin in ADC. Human macrophages were infected with HIV-1 in vitro using a viral isolate from a demented patient. 6-Chloro-D-tryptophan, an inhibitor of QUIN biosynthesis, was added to half the macrophage cultures to block formation of QUIN. Supernatants containing QUIN (SQpos) or in which QUIN biosynthesis had been inhibited (SQneg) were then added to human fetal brain aggregate cultures. Toxicity was evaluated using lactate dehydrogenase efflux, trypan blue exclusion, immunohistochemistry, image analysis, and electron microscopy. Each technique showed a reduction of toxicity in SQneg-treated cultures. These studies confirm the significance of QUIN as a neurotoxin in ADC and suggest that neuroprotective strategies may have a place in the treatment of this disease.

Schwann cells, the myelin forming glial cells of peripheral nerves, have been implicated as having an immunoregulatory role in inflammatory demyelinating neuropathies (IDNs) such as Guillain Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP). We employed rat IFN-gamma, a cytokine released by macrophages and CD4+ T-cells during inflammatory demyelination of the peripheral nervous system, to examine the distribution and abundance of MHC class I, MHC class II and ICAM-1 on Lewis rat Schwann cells and fibroblasts in vitro. MHC class I, class II and ICAM-1 molecules were immunolabelled with 30 nm colloidal gold and observed by scanning electron microscopy. Incubation with IFN-gamma for 24 and 72 h, resulted in the clustering of MHC class I and ICAM-1 on Schwann cells and fibroblasts with MHC class II randomly distributed as single particles. MHC class I and ICAM-1 were upregulated after 24 h incubation in the presence of IFN-gamma, whereas MHC class II was upregulated after 72 h. The difference in the rate of upregulation may indicate differences in the recycling and/or synthesis of these molecules. Changes in distribution such as clustering, in conjunction with the upregulation of these molecules, suggest a role for Schwann cells in the restimulation of specifically primed CD4+ T-cells in IDNs.

Apolipoprotein E has been shown to be a risk factor for late-onset Alzheimer's disease, with the apolipoprotein epsilon 4 allele conferring the risk. Apolipoprotein E is found in neurofibrillary tangles and senile plaques, the pathological characteristics of Alzheimer's disease. To date there is no direct evidence that human neurons can take up exogenous apolipoprotein E, which is necessary if apolipoprotein E is involved in the formation of neurofibrillary tangles. To examine apolipoprotein E uptake we employed the human NTera2/D1 cell line, which can be induced by retinoic acid to differentiate into postmitotic NTera2-N neurons, which have the characteristics and morphology of human central nervous system neurons. We defined the cell line as genotype apolipoprotein epsilon 3/3 and demonstrated that the cells do not synthesize apolipoprotein E but can take up and internalize exogenous recombinant apolipoprotein E3. We also confirmed the expression of the low-density lipoprotein receptor-related protein, a known receptor for apolipoprotein E. The NTera2/D1 cell line therefore provides a useful human cell model for examining the effects of other apolipoprotein E isoforms with a view to defining intraneuronal interactions of apolipoprotein E.

The interaction of autologous human fetal neurons with epidermal cells was studied by culturing fetal dorsal root ganglia (DRG) in the center of a dual chamber system with epidermal explants in the outer chamber. The two chambers were separated by two concentric stainless steel annular rings adherent to the substratum by silicon grease and agarose. Axons from the DRG penetrated the agarose barrier, growing into the exterior chamber by 10 d in vitro (DIV) and extended past sparse peripheral fibroblasts to interact specifically with epidermal cells by 12 to 16 DIV. Scanning electron microscopy (SEM) showed single or multiple neuronal fascicles terminating on epidermal cells with spatular, veillike or bulbous axon termini. Transmission electron microscopy (TEM) showed fine axonal termini between epidermal cells, separated by an intercellular gap. The specificity of axonal targeting for epidermal cells rather than fibroblasts was also demonstrated by infecting the DRG with Herpes simplex virus type-1 (HSV-1). Specific anterograde transport of HSV-1 along axons to keratin-expressing epidermal cells was demonstrated by immunofluorescence and immunoperoxidase staining using monoclonal antibodies to viral glycoprotein D. This model allows the study of the mechanism of the specific interactions between neurons and epidermal cells analogous to those in fetal development and after cutaneous nerve regeneration.

The Schwann cell or its myelin membrane appears to be the focus of autoimmune attack in several peripheral neuropathies. Potential Schwann cell antigens have therefore been extensively studied. Of the known Schwann cell proteins, MBP, P2, MAG, PLP, PO, PMP-22 and Connexin 32, gene defects of the latter three have recently been shown to be responsible for some forms of hereditary sensory and motor neuropathies. The L2/HNK-1 epitope, common to MAG, Po and PMP-22, is the target of autoantibody damage in neuropathies associated with certain IgM paraproteinaemias. P2, the inciting antigen in rat EAN, has no demonstrated role in human neuropathies. The same appears true for the glycolipid galactocerebroside, a neuritogen in rabbits. Gangliosides are currently under intense study, but the antigen for the common inflammatory neuropathies remains undefined. Evidence for the potential role of Schwann cells in immune modulation is provided; Schwann cells produced MHC molecules, the adhesion molecules I-CAM1, L1, L2-HNK-1, Ng-CAM, N-cadherisn, the cytokines IL-1, IL-6 and TNF-alpha and the inflammatory prostanoids, PGE-2 AND TxA2.