Experimental Allergic Neuritis Research Articles

Decitabine induces regulatory T cells, inhibits the production of IFN-gamma and IL-17 and exerts preventive and therapeutic efficacy in rodent experimental autoimmune neuritis

Guillain-Barré syndrome (GBS) is an immune-mediated acute disorder of the peripheral nervous system. Despite treatment, there is an associated mortality and severe disability in 9 to 17% of the cases. Decitabine (DAC) is a hypomethylating drug used in myelodisplastic syndrome, that has been shown to exert immunomodulatory effects. We have evaluated the effects of DAC in two rodent models of GBS, the Experimental Allergic Neuritis (EAN). Both prophylactic and therapeutic treatment with DAC ameliorated the clinical course of EAN, increasing the numbers of thymic regulatory T cells and reducing the production of proinflammmatory cytokines. Our data suggest the possible use of decitabine for the treatment of GBS.

Evidence from Human and Animal Studies: Pathological Roles of CD8+ T Cells in Autoimmune Peripheral Neuropathies

Autoimmune peripheral neuropathies such as Guillain-Barre Syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) affect millions of people worldwide. Despite significant advances in understanding the pathology, the molecular and cellular mechanisms of immune-mediated neuropathies remain elusive. T lymphocytes definitely play an important role in disease pathogenesis and CD4+ T cells have been the main area of research for decades. This is partly due to the fact that the most frequent animal model to study autoimmune peripheral neuropathy is experimental allergic neuritis (EAN). As it is induced commonly by immunization with peripheral nerve proteins, EAN is driven mainly by CD4+ T cells. However, similarly to what has been reported for patients suffering from multiple sclerosis, a significant body of evidence indicates that CD8+ T cells may play a pathogenic role in GBS and CIDP disease development and/or progression. Here, we summarize clinical studies pertaining to the presence and potential role of CD8+ T cells in autoimmune peripheral neuropathies. We also discuss the findings from our most recent studies using a transgenic mouse line (L31 mice) in which the T cell co-stimulator molecule B7.2 (CD86) is constitutively expressed in antigen presenting cells of the nervous tissues. L31 mice spontaneously develop peripheral neuropathy, and CD8+ T cells are found accumulating in peripheral nerves of symptomatic animals. Interestingly, depletion of CD4+ T cells accelerates disease onset and increases disease prevalence. Finally, we point out some unanswered questions for future research to dissect the critical roles of CD8+ T cells in autoimmune peripheral neuropathies.

Pathophysiology of autoimmune polyneuropathies

The most common autoimmune neuropathies include the acute inflammatory polyneuropathy [the Guillain-Barré Syndrome(s)]; chronic inflammatory demyelinating polyneuropathy (CIDP), multifocal motor neuropathy (MMN) and IgM anti-MAG-antibody mediated paraproteinemic neuropathy. These neuropathies occur when immunologic tolerance to peripheral nerve components (myelin, Schwann cell, axon, and motor or ganglionic neurons) is lost. Based on the immunopathologic similarities with experimental allergic neuritis induced after immunization with nerve proteins, disease transfer experiments with the patients' serum or with intraneural injections, and immunocytochemical studies on the patients' nerves, it appears that both cellular and humoral factors, either independently or in concert with each other, play a role in the cause of these neuropathies. Although in some of them there is direct evidence for autoimmune reactivity mediated by specific antibodies or autoreactive T lymphocytes, in others the underlying immune-mediated mechanisms have not been fully elucidated, in spite of good response to immunotherapies. The review highlights the factors associated with breaking the T-cell tolerance, the T-cell activation and costimulatory molecules, the immunoregulatory T-cells and relevant cytokines and the antibodies against peripheral nerve glycolipids or glycoproteins that seem to be of pathogenic relevance. Antigens in the nodal, paranodal and juxtaparanodal regions are discussed as potentially critical targets in explaining conduction failure and rapid recovery. Based on the immunopathologic network believed to play a fundamental role in the pathogenesis of these neuropathies, future therapeutic directions are highlighted using new biological agents against T-cells, cytokines, B-cells, transmigration and transduction molecules.

Antibodies to Gliomedin Cause Peripheral Demyelinating Neuropathy and the Dismantling of the Nodes of Ranvier

Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) are conditions that affect peripheral nerves. The mechanisms that underlie demyelination in these neuropathies are unknown. Recently, we demonstrated that the node of Ranvier is the primary site of the immune attack in patients with GBS and CIDP. In particular, GBS patients have antibodies against gliomedin and neurofascin, two adhesion molecules that play a crucial role in the formation of nodes of Ranvier. We demonstrate that immunity toward gliomedin, but not neurofascin, induced a progressive neuropathy in Lewis rats characterized by conduction defects and demyelination in spinal nerves. The clinical symptoms closely followed the titers of anti-gliomedin IgG and were associated with an important deposition of IgG at nodes. Furthermore, passive transfer of antigliomedin IgG induced a severe demyelinating condition and conduction loss. In both active and passive models, the immune attack at nodes occasioned the loss of the nodal clusters for gliomedin, neurofascin-186, and voltage-gated sodium channels. These results indicate that primary immune reaction against gliomedin, a peripheral nervous system adhesion molecule, can be responsible for the initiation or progression of the demyelinating form of GBS. Furthermore, these autoantibodies affect saltatory propagation by dismantling nodal organization and sodium channel clusters. Antibodies reactive against nodal adhesion molecules thus likely participate in the pathologic process of GBS and CIDP.

Homeostatic regulation of the endoneurial microenvironment during development, aging and in response to trauma, disease and toxic insult

The endoneurial microenvironment, delimited by the endothelium of endoneurial vessels and a multi-layered ensheathing perineurium, is a specialized milieu intérieur within which axons, associated Schwann cells and other resident cells of peripheral nerves function. The endothelium and perineurium restricts as well as regulates exchange of material between the endoneurial microenvironment and the surrounding extracellular space and thus is more appropriately described as a blood–nerve interface (BNI) rather than a blood–nerve barrier (BNB). Input to and output from the endoneurial microenvironment occurs via blood–nerve exchange and convective endoneurial fluid flow driven by a proximo-distal hydrostatic pressure gradient. The independent regulation of the endothelial and perineurial components of the BNI during development, aging and in response to trauma is consistent with homeostatic regulation of the endoneurial microenvironment. Pathophysiological alterations of the endoneurium in experimental allergic neuritis (EAN), and diabetic and lead neuropathy are considered to be perturbations of endoneurial homeostasis. The interactions of Schwann cells, axons, macrophages, and mast cells via cell–cell and cell–matrix signaling regulate the permeability of this interface. A greater knowledge of the dynamic nature of tight junctions and the factors that induce and/or modulate these key elements of the BNI will increase our understanding of peripheral nerve disorders as well as stimulate the development of therapeutic strategies to treat these disorders.

Membrane attack complex of complement is not essential for immune mediated demyelination in experimental autoimmune neuritis

Antibody deposition and complement activation, especially membrane attack complex (MAC) formation are considered central for immune mediated demyelination. To examine the role of MAC in immune mediated demyelination, we studied experimental allergic neuritis (EAN) in Lewis rats deficient in complement component 6 (C6) that cannot form MAC. A C6 deficient Lewis (Lewis/C6−) strain of rats was bred by backcrossing the defective C6 gene, from PVG/C6− rats, onto the Lewis background. Lewis/C6− rats had the same C6 gene deletion as PVG/C6− rats and their sera did not support immune mediated haemolysis unless C6 was added. Active EAN was induced in Lewis and Lewis/C6− rats by immunization with bovine peripheral nerve myelin in complete Freund's adjuvant (CFA), and Lewis/C6− rats had delayed clinical EAN compared to the Lewis rats. Peripheral nerve demyelination in Lewis/C6− was also delayed but was similar in extent at the peak of disease. Compared to Lewis, Lewis/C6− nerves had no MAC deposition, reduced macrophage infiltrate and IL-17A, but similar T cell infiltrate and Th1 cytokine mRNA expression. ICAM-1 and P-selectin mRNA expression and immunostaining on vascular endothelium were delayed in Lewis C6− compared to Lewis rats' nerves. This study found that MAC was not required for immune mediated demyelination; but that MAC enhanced early symptoms and early demyelination in EAN, either by direct lysis or by sub-lytic induction of vascular endothelial expression of ICAM-1 and P-selectin.

Dynamic change OX40/OX40L mRNA in experimental allergic neuritis

To examine the expression of mRNA of Oxford 40(OX40) and Oxford 40 ligand(OX40L) in the sciatic nerve, spleen, peripheral blood mononuclear cells and lymph nodes of experimental allegic neuritis (EAN). Thirty-six Lewis rats were randomly assigned into an EAN group and a CFA group. The rats were sacrificed on 9th, 17th, and 26th day after immunization. OX40 and OX40L mRNA was detected by reverse transcription polymerase chain reaction in the spleen, sciatic nerves, peripheral blood mononuclear cells and lymphonodes. The peak of clinical course came on 17th day after the immunization in EAN. The mRNA expression of OX40/OX40L was higher on 8th day and 17th day than that on 26th day after the immunization (P<0.05). There was significant difference between the EAN group and the CFA group at the 3 time points (P<0.05); rats in the CFA group didn't have any clinical manifestations. The mRNA expression of OX40 and OX40L in the EAN group raised in the sciatic nerves and lymph nodes at the above 3 time points (P<0.05). Weak expression was seen in the peripheral blood mononuclear cells. OX40 and OX40L may play a role in the pathogenesis of experimental allegic neuritis.

Ultrastructural study of experimental allergic neuritis in the chicken. I. Cell migration, granuloma formation and demyelination.

Summary Experimental allergic neuritis (EAN) was produced in order to compare its features with the demyelinating changes of peripheral nerves in chickens with Marek's disease (MD). Under the light microscope, mononuclear cells accumulated around the venular areas. Electron microscopically, mononuclear cells in the venular lumen developed slender, cytoplasmic processes, which were inserted into the cytoplasm of the venular endothelium. Subsequently, intracytoplasmic migration of the mononuclear cells was consistently observed at the venular wall. Furthermore, we suggest that mononuclear cells can also penetrate the nucleus of the endothelial cells. Granuloma formation was frequently observed within the perivenular areas where the mononuclear cells infiltrated and accumulated. Primary demyelination was coincidentally seen within the areas of cellular infiltration. The pattern of primary demyelination was divided into two types, i. e., external and internal demyelination. It is considered that the sequence of demyelinating changes in EAN is analogous to that in the peripheral nerve fibres in chickens with MD. Zusammenfassung Ultrahistologische Untersuchung der experimentellen allergischen Neuritis des Kükens I. Zellmigration, Granulombildung und Demyelinisierung Eine experimentelle allergische Neuritis (EAN) wurde induziert, um sie bei der Demyelinisierung der peripheren Nervenfasern zu vergleichen, die bei Küken mit Marekscher Erkrankung beobachtet werden. Lichtmikroskopisch stellte sich eine Anhäufung von mononukleären Zellen um die Venülen dar. Ultrahistologisch zeigten die mononukleären Zellen des Venenlumens flache Zytoplasmafortsätze, die ins Zytoplasma der venösen Endothelzellen eingefügt waren. In der Folge wurde eine konstante Wanderung der mononukleären Zellen in das Zytoplasma der venösen Endothelzellen beobachtet. Daneben gibt es Hinweise dafür, daß die mononukleären Zellen auch den Endothelzellkern durchwandern können. Häufig wurde eine Granulombildung in den perivenösen Arealen gesehen mit starker Anhäufung mononukleärer Zellen. Eine primäre Demyelinisierung wurde gleichzeitig in diesen Arealen beobachtet. Zwei Demyelinisierungsmuster werden unterteilt: eine „äußere” und eine „innere” Demyelinisierung. Es wird postuliert, daß der Ablauf der Entmarkungserscheinungen bei der EAN analog zu den Veränderungen der peripheren Nervenfasern von Küken verläuft, die an Marekscher Lähmung erkrankt sind. Résumé Recherches ultrahistologiques sur la neurite allergique expérimentale du poulet I. Migration cellulaire, formation granulomateuse et démyélinisation Une neurite allergique expérimentale (EAN) a été induite chez des poulets pour la comparer à celle accompagnée d'une démyélinisation des fibres nerveuses périphériques que l'on observe dans la maladie de Marek. Une concentration de cellules mononucléées autour des veines a été observée au microscope optique. Ultrahistologiequement, les cellules mononucléées du lumen veineux ont montré des appendices cytoplasmiques plats imbriques dans le cytoplasme des cellules endothéliales veineuses. On a observé par la suite une migration constante des cellules mononucléées dans le cytoplasme des cellules de l'endothélium veineux. Des signes ont montré que les cellules mononucléées pouvaient également traverser le noyau de la cellule endothéliale. Une formation granulomateuse a été fréquemment observée dans l'espace périveineux avec un fort rassemblement de cellules mononucléées. Une démyélinisation primaire a été observée simultanément dans ces espaces. Deux sortes de démyélinisation ont été définies: une démyélinisation «externe» et «interne». On suppose que l'évolution de la démyélinisation lors de la neurite allergique expérimentale conduit aux mêmes lésions des fibres nerveuses périphériques que celles des poulets atteints par la maladie de Marek. Resumen Estudios ultrahistológicos sobre la neuritis alérgica experimental en el pollito I. Migración celular, formación granulomatosa y desmielinización Se indujo una neuritis alérgica experimental (NAE) en pollitos para poderla comparar con la desmielinización de la fibras nerviosas periféricas que se observa en la enfermedad de Marek (EM). Al fotomicroscopio se apreció una concentración de células mononucleadas alrededor de las vénulas. Ultra-histológicamente mostraban las células mononucleadas de la luz venosa unos apéndices citoplásmicos planos imbricados en el citoplasma de las células endoteliales venosas. Subsecuentemente, se observó una migración constante de las células mononucleadas hacia el citoplasma de las células del endotelio venoso. Al lado de esto, hay indicios de que las células mononucleadas pueden atravesar también el núcleo de la célula endotelial. Frecuentemente se observó una formación granulomatosa en los espacios perivenosos con acumulación fuerte de células mononucleadas. Al mismo tiempo se apreció una desmielinización primaria en estos espacios. Los modelos de desmielinización primaria se dividieron en dos tipos: una desmielinización «externa» y otra «interna». Se considera que el curso de los fenómenos de desmielinización en la NAE es análogo al de las fibras nerviosas periféricas en los pollitos con la EM.

Th1-like cell responses to peripheral nerve myelin components over the course of experimental allergic neuritis in Lewis rats.

Experimental allergic neuritis (EAN) is a T cell-mediated animal model of Guillain-Barré syndrome characterized by inflammation and demyelination of peripheral nerves. EAN can be induced by immunization of rats with bovine peripheral nerve myelin (BPM) or the myelin proteins P2 or P0, but the extent of T cell responses over the course of EAN is incompletely defined. We studied the T cell responses to these proteins and the glycolipid GM1 by enumerating T helper type 1 (Th1)-like cells secreting interferon-gamma (IFN-gamma) after short-term culture of mononuclear cells (MNC) in presence of antigen. Already 7 days post immunization (p.i.) with BPM and before onset of clinical EAN, lymph nodes contained elevated levels of P2 responsive T cells. At the height of EAN on day 14 p.i. and during recovery, T cell levels responding to BPM, P0 and GM1 were also elevated. The same temporal profiles and specificities were registered for antigen reactive spleen MNC. The results implicate that Th1-like cells with multiple specificities including the glycolipid GM1 occur at increased levels in lymphoid organs in EAN rats, and that IFN-gamma may be an important effector molecule in the induction of nerve damage.

Autonomic dysfunction in experimental allergic neuritis.

Beat-to-beat variation (R-R variation) in the electrocardiogram was studied in experimental allergic neuritis in the Sprague-Dawley rat. Reduced R-R variations were found in 2 of 10 animals, probably as a sign of autonomic dysfunction. The vagal nerves from these two animals, studied in vitro, showed disturbed conduction. In one animal prolonged conduction latencies to supramaximal electrical stimuli were found. Vagal nerves from controls and from animals without clinical symptoms showed normal conduction. Histologically, the vagal nerves from affected animals showed a slight inflammatory cell infiltration and signs of demyelination but there was no evidence of involvement of the brainstem vasomotor nuclei. Thus, we suggest that the autonomic dysfunction in experimental allergic neuritis, measured as reduced R-R variations, is caused by a peripheral vagal neuropathy.

Oestrogen treatment reduces duration of experimental allergic neuritis in rats and suppresses T cell responses to myelin

The effects of oestrogen and pregnancy on the disease course of experimental allergic neuritis (EAN) in rats were investigated. Pregnant rats were totally protected from EAN and long term 17-beta-oestradiol treatment significantly shortened the disease duration. Lymph node cells from oestrogen treated rats had a suppressed proliferative response when stimulated with myelin or PPD, both when the response was measured immediately after cell preparation and after 72 h cell culture in vitro. Serum levels of IgG antibodies against myelin, P2 or PPD did not differ between treated and non-treated rats although oestrogen treated rats had significantly higher levels of total IgG. Immunohistochemical stainings of nerve roots showed less intensive invasion of T lymphocytes in the oestrogen treated group while immunoreactivity to both class I and II major histocompatibility complex antigens did not differ in between the groups. These findings show that oestrogen ameliorates EAN and suggest that this effect is mediated by suppression of T cell dependent immunity. Factors in addition to oestrogen may be operative during pregnancy when a total protection from EAN is obtained.

Experimental allergic neuritis: Dynamics of Ia-expressing cells, T lymphocytes of different subsets in peripheral nerve tissue and serum IgG response

Experimental allergic neuritis: Dynamics of Ia-expressing cells, T lymphocytes of different subsets in peripheral nerve tissue and serum IgG response

Changes in immune response, demyelination and axonal damage in experimental allergic neuritis

Changes in immune response, demyelination and axonal damage in experimental allergic neuritis

Disruption of neurofascin and gliomedin at nodes of Ranvier precedes demyelination in experimental allergic neuritis

High densities of voltage-gated sodium (Nav) channels at nodes of Ranvier enable the rapid regeneration and propagation of the action potentials along myelinated axons. In demyelinating pathologies, myelin alterations lead to conduction slowing and even to conduction block. In order to unravel the mechanisms of conduction failure in inflammatory demyelinating diseases, we have examined two models of Guillain-Barré syndrome: the experimental allergic neuritis induced in the Lewis rat by immunization against peripheral myelin (EAN-PM) and against a neuritogenic P2 peptide (EAN-P2). We found that Nav channel clusters were disrupted at EAN-PM nodes. Neurofascin and gliomedin, two cell adhesion molecules involved with aggregating Nav channels at nodes, were selectively affected prior to demyelination in EAN-PM, indicating that degradation of the axo-glial unit initiated node alteration. This was associated with autoantibodies to neurofascin and gliomedin. Node disruption was, however, independent from complement deposition at nodes, and deposits of the terminal complement complex (C5b-9) were found on the external surface of Schwann cells in EAN-PM. In these animals, the paranodal junctions were also affected and Kv1 channels, which are normally juxtaparanodal, were found dispersed at nodes and paranodes. Altogether, these alterations were associated with conduction deficits in EAN-PM ventral spinal roots. EAN-P2 animals also exhibited inflammatory demyelination, but did not show alteration in nodal clusters or autoantibodies. Our results highlighted the complex mechanisms underlying conduction abnormalities in demyelinating disorders, and unraveled neurofascin and gliomedin as two novel immune targets in experimental allergic neuritis.

Study on inhibitive action of bone marrow stromal cells transplantation on immunologic reaction of experimental allergic neuritis

The study was designed to establish animal experimental groundworks for a clinical practicable therapeutic route of stem cell repair for Guillain-Barre syndrome from eradicating its imunopathology angle. We separated, cultivated, identified bone marrow derived stroma cells of healthy Lewis rat to obtain activatory specific T lymphocyte by P0 180-199 compositus immunoemulsion induction in post foot pad of Lewis rat, thereby to set up P0 180-199 specific T cell line in vitro. CFA injection was used as contrast control. We set up experimental allergic neuritis model by P0180-199 specific T lymphocytes adoptive immunity andset up allogeneil graft group by BMSCs injection from vena caudalis. PBS injection group was used as contrast control. Both groups were grade by EAN clinical score on 16 days after immune. All experimental animals were killed on 6 days after immune to observe lymphocyte infiltration status in sciatic nerve. T lymphocyte proliferation test in vitro was done by separating, cultivating the obtained allergic T lymphocytes from both groups and stimulating them by P0180-199 or ConA, detecting their proliferation ability by -counter. None antigen stimulation group was used as contrast control. At last we detected serum IFN-, TNF- and IL-4 level of BMSC transplantation group and control group on 6 days after immune by ELISA method. The results shown BMSCs grown well when primary cultivated in vitro, shown stable function and strong cloning ability in serial subcultivation. BMSCs shown CD14 negative expression and CD44 positive expression, the cell purity was above 85%. The sensitized process was slow and clinic symptoms were severe after immune from metapodium pad, obvious symptoms were observed on 10 days. Specific T cell line could be obtained by this method. Symptoms of adoptive immune animals were slight and appeared rapidly which were easy for experimental observation. There was significant difference in BMSCs transplantation and PBS control group by P<0.001 on 46 days after adoptive immune; The infiltration cells was found much more in control group than in BMSC group by P<0.01. Specific P0 antigen or ConA stimulation test shown: Lymphocytes shown lower proliferation ability in BMSCs transplantation group than in PBS control group by P<0.01, the difference was significant; There are no significant difference in both group without any antigen stimulation. Serum IFN- level was higher in control group than in BMSC group by P<0.01; TNF- level was obviously higher in control group than in BMSC group by P<0.001; IL-4 level was lower in control group than in BMSC group by P<0.01. Conclusion: BMSCs are easily to be obtained and cultivated in vitro with strong proliferation ability. Effective EAN model can be established by adoptive immune method. BMSC transplantation could effectively inhibit pathologic condition progression of EAN. BMSC transplantation has inhibitory action on inflammatory cells infiltration in target organ-ischiadic nerve of EAN. BMSC transplantation can effectively inhibit proliferation reaction ability of specific T lymphocytes. BMSC transplantation downregulates serum IFN-, TNF- level, upregulates protective cytokine IL-4 level.

Cellular mRNA expression of interferon-gamma, IL-4 and transforming growth factor-beta (TGF-beta) by rat mononuclear cells stimulated with peripheral nerve myelin antigens in experimental allergic neuritis.

Experimental allergic neuritis (EAN) serves as a useful model for inflammation in the peripheral nervous system. To study the potential role of important immunoregulatory and effector cytokines in EAN, we examined the expression of mRNA for interferon-gamma (IFN-gamma), IL-4 and TGF-beta by in situ hybridization in lymph node and splenic cells cultured with bovine peripheral nerve myelin (BPM), P2 and P0 during the course of EAN in Lewis rats. Levels of IFN-gamma mRNA-expressing mononuclear cells (MNC) from lymph nodes and spleens roughly correlated with clinical status, consistent with a disease-promoting role for IFN-gamma. BPM, P0 and P2-reactive IFN-gamma mRNA-expressing T cells appeared in lymph nodes and spleen before onset of the disease, whereas a significant TGF-beta response to BPM, P2 and P0 was observed at lower levels than the IFN-gamma response and at onset of recovery, consistent with a disease down-regulating role of TGF-beta. IL-4 mRNA-expressing cells were found at levels similar to TGF-beta mRNA-expressing cells, and with the latest peak of the three cytokines examined. This result suggests that IL-4 may also suppress IFN-gamma expression at late recovery phase of EAN.

Preventive and curative effects of cyclophosphamide in an animal model of Guillain Barrè syndrome

The immunosuppressive agent cyclophosphamide (CY) was tested in rat experimental allergic neuritis (EAN), a preclinical model of Guillain Barrè syndrome (GBS). CY prophylaxis (day 0 and 14 post-immunization [p.i.]) effectively prevents clinical and histological signs of EAN and also reduces the cytokine and the NF-κB p65 expression in the nervous tissue. When administered therapeutically (day 14th p.i.) to rats with established disease CY only affects the clinical symptoms. Both the prophylactic and therapeutic treatment with CY reduced ex vivo antigen-specific T cell proliferative responses. These results warrant studies with CY in those cases of GBS resistant to conventional therapies.

Expression of TLR4 and TLR9 mRNA in Lewis Rats with Experimental Allergic Neuritis

Objective: Experimental autoimmune neuritis (EAN) is a T cell-mediated animal model of the Guillain-Barré syndrome characterized by inflammation and demyelination of the peripheral nervous system (PNS). The key pathogenesis of EAN is the imbalance between Th1- and Th2-type immune response. Toll-like receptor (TLR) is a receptor of the innate immune system. It can recognize the antigen by pathogen-associated molecular patterns, and activate the antigen-presenting cells, producing costimulating molecules to activate T cells. In this study, we observed the expression of mRNA of TLR4 and TLR9 in EAN rats. Methods: Male Lewis rats were immunized with the component of PNS myelin sheath protein P0 180–199 (100 µg) and Freund’s complete adjuvant (CFA). The clinical signs of rats and the pathological changes in the sciatic nerves were observed. The rats in the immunized group and the control group were sacrificed on day 7, 16, 24 and 33 after immunization. To observe the histopathological changes, sciatic nerve was embedded in paraffin, sectioned and stained with HE and Weil’s stain. The mRNA expression of TLR4 and TLR9 in spleen, sciatic nerve, peripheral blood monocytes and lymph nodes was detected by reverse transcriptase PCR dynamically. Results: The EAN group reached the peak of the clinical score at day 16 after the immunization, and the clinical manifestation obviously improved at day 33 after the immunization. The mRNA expression of TLR4 peaked at day 16, then decreased gradually, but at day 33, it was still higher than that of the CFA control group (p < 0.05); there were significant differences among the four time points (p < 0.05). The mRNA expression of TLR9 was upregulated during the entire course of EAN compared to the CFA and the control group (p < 0.05). Conclusions: TLR4 and TLR9 may play a role in the pathogenesis of EAN by taking part in the activation phage and effector phage.

Targeting Inflammatory Demyelinating Lesions to Sites of Wallerian Degeneration

In Theiler’s murine encephalomyelitis virus (TMEV) infection, an animal model for multiple sclerosis (MS), axonal injury precedes inflammatory demyelinating lesions, and the distribution of axonal damage present during the early phase of infection corresponds to regions where subsequent demyelination occurs during the chronic phase. We hypothesized that axonal damage recruits inflammatory cells to sites of Wallerian degeneration, leading to demyelination. Three weeks after TMEV infection, axonal degeneration was induced in the posterior funiculus of mice by injecting the toxic lectin Ricinus communis agglutinin (RCA) I into the sciatic nerve. Neuropathology was examined 1 week after lectin injection. Control mice, infected with TMEV but receiving no RCA I, had inflammatory demyelinating lesions in the anterior/lateral funiculi. Other control mice that received RCA I alone did not develop inflammatory lesions. In contrast, RCA I injection into TMEV-infected mice induced lesions in the posterior funiculus in addition to the anterior/lateral funiculi. We found no differences in lymphoproliferative responses or antibody titers against TMEV among the groups. This suggests that axonal degeneration contributes to the recruitment of inflammatory cells into the central nervous system by altering the local microenvironment. In this scenario, lesions develop from the axon (inside) to the myelin (outside) (Inside-Out model).

647: CD4 + CD25 + T cells prevent induction of demyelination in experimental allergic neuritis (EAN)

Immune tolerance is induced and maintained by T cells, which express CD4 and CD25s the IlI-2 receptor alpha chain. We examine the role of these cells in Lewis rats that had recovered from EAN and were resistant to further disease induction. The CD4+CD25+ and CD4+CD25- T cell subsets were prepared form Lewis rats that had recovered from EAN and from normal Lewis rats and their function compared. First we transferred 5x106 CD4+CD25+ T cells on the day of immunization with bovine peripheral nerve myelin (n=12), these cells suppressed severity of disease to peak at a mean score of 1, compared to controls (n=10) peaking at 2.5 and those given 5×106 CD4+CD25– T cells which peaked at 3 (n=5).