Bovine Peripheral Nerve Myelin Research Articles

Isolation, Purification and Verification of Peripheral Nerve Myelin Derived from Bovine Cauda Equina

Background: Animal models of peripheral neuroinflammation are essential to understanding the pathogenesis of, and deducing new treatments for demyelinating polyneuropathies. This paper updates and describes adapted methods for the isolation, purification and verification of bovine peripheral nerve myelin used to reliably induce peripheral neuritis in susceptible mice. Methods: Myelin was isolated via discontinuous sucrose and continuous cesium chloride density gradient ultracentrifugation from frozen adult bovine cauda equina. Negatively stained electron microscopy was employed to confirm myelin structure. Phase contrast and routine light microscopy, gravimetric analyses, gel electrophoresis and lens culinaris fluorescent western blotting, analytical colorimetric assays and indirect immunohistochemistry were performed to analyze whole myelin or its protein and lipid subcomponents. Myelin isolates were used to induce experimental autoimmune neuritis in 8-12 Weeks old female Swedish Jim Lambert/ Jackson mice. Results: Negatively-stained electron microscopy demonstrated the two-dimensional repetitive lamella structure of myelin and a lack of organelle contamination. Lipids accounted for ~72% of the dry mass of bovine peripheral nerve myelin, while ~25% was protein/ proteolipid and <4% insoluble residue based on gravimetric analyses. Glycoproteins accounted for the majority of isolated myelin proteins, with myelin protein zero (~28 KDa) being the most prevalent protein. Phospholipids accounted for ~60% of the total dry weight of bovine peripheral nerve myelin. Surface expression of galactocerebroside and sulfatide, two major lipid subcomponents present in myelin, was detected by indirect fluorescent immunohistochemistry. Bovine peripheral nerve myelin reliably induced a severe macrophagepredominant demyelinating polyneuropathy with associated axonal loss in female mice, consistent with experimental autoimmune neuritis. Conclusions: These updated and adapted methods using readily available resources can be applied to rapidly isolate, purify and verify peripheral nerve myelin from animal sources, and determine its protein and lipid composition prior to use in peripheral neuritis animal models.

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.

Selective expression and cellular localization of pro-inflammatory chemokine ligand/receptor pairs in the sciatic nerves of a severe murine experimental autoimmune neuritis model of Guillain-Barré syndrome

To determine if specific pro-inflammatory chemokine ligand/receptor pairs expressed in the peripheral nerves of Guillain-Barré syndrome patients are expressed in a severe murine experimental autoimmune neuritis (sm-EAN) model and to determine their cellular localization as a prerequisite to designing potentially therapeutic interventions in vivo. Sm-EAN was induced in 8-12-week-old female SJL/J mice using bovine peripheral nerve myelin emulsified in complete Freund adjuvant with pertussis toxin and recombinant mouse interleukin-12 acting as co-adjuvants, with appropriate controls. Mice were evaluated for neuromuscular weakness and weighed daily. Dorsal caudal tail and sciatic nerve motor electrophysiological studies were performed at expected maximal severity. Sciatic nerves were harvested and specific chemokine ligand/receptor expression was determined using real-time quantitative reverse transcriptase polymerase chain reaction and indirect fluorescent immunohistochemistry. CCL2/CCR2, CXCL10/CXCR3 and CCL5/CCR1, CCR5 expression was significantly increased in the sciatic nerves of sm-EAN mice compared with controls. CCL2 was expressed on Schwann cells with CCR2 expressed on F4/80+ macrophages and CD3+ T cells. CXCL10 was expressed on endoneurial endothelial cells and within the endoneurial interstitium, with CXCR3 expressed on CD3+ T-lymphocytes. CCL5 co-localized to axons, with CCR1 and CCR5 expression on F4/80+ macrophages and rare CD3+ T cells. This study suggests that CCL2 expressed by Schwann cells and CXCL10 expressed by endoneurial endothelial cells may induce F4/80+ macrophage and CD3+ T cell-mediated inflammation and demyelination in sm-EAN. CCL2-CCR2 and CXCL10-CXCR3 signalling pathways are potential targets for therapeutic intervention in peripheral nerve inflammation.

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.

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.

Amino acid copolymer-specific IL-10-secreting regulatory T cells that ameliorate autoimmune diseases in mice.

IL-10-secreting regulatory T cell lines specific to glatiramer acetate [poly(Y,E,A,K)n] or poly(Y,F,A,K)n have been established from the enlarged spleen and lymph nodes that result from copolymer treatment of SJL mice in which experimental autoimmune encephalomyelitis was induced by PLP139-151. These CD4+CD25+T cell lines secrete high levels of IL-10 and IL-13 but only small amounts of IL-4 and virtually no TGF-beta, IL-17, IL-6, IFN-gamma, or TNF-alpha. Their phenotypes are particularly characterized by the absence of Foxp3 and the presence of two TNFR family members, CD30 and GITR. The lines proliferated specifically to the immunizing copolymers but were autoantigen-nonspecific, in that the same T cell line could suppress autoimmunity induced by three different autoantigens in SJL mice, i.e., PLP139-151(EAE), MBP85-99 (EAE), and bovine peripheral nerve myelin (experimental autoimmune neuritis), indicating they function by bystander suppression.

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).

Effective treatment of experimental autoimmune neuritis with Fc fragment of human immunoglobulin

IV immunoglobulin (IVIg) and its Fc fragment proved effective in preventing further progression of experimental autoimmune neuritis (EAN) in the rat induced by whole bovine peripheral nerve myelin and shortening disease duration. This effectiveness was associated with significant differences in electrophysiological parameters including less prolongation of somatosensory evoked potential (S wave) latencies, better maintained S wave amplitudes, less reduction of distal motor nerve conduction velocity, and better maintained amplitudes of compound muscle action potentials of dorsal foot muscles after stimulation at ankle and hip. Moreover, treatment with IVIg and Fc fragments resulted in less extensive inflammation and demyelination in nerve roots evidenced by significantly lower histological grades. The current study provides direct evidence for the first time that Fc fraction of IVIg is the effective component in the treatment of rat EAN.

B cells play a cooperative role via CD40L–CD40 interaction in T cell-mediated experimental autoimmune neuritis in Lewis rats

The expression of co-stimulatory molecules CD40 and CD40L was examined over the course of experimental autoimmune neuritis (EAN) induced in Lewis rats by immunization with bovine peripheral nerve myelin. In draining lymph nodes, highest level of CD40L expression was seen on day 7 post immunization (p.i.), i.e. before onset of clinical signs of EAN, while CD40 expression was increased on day 14 p.i., i.e. at peak of clinical disease. In contrast, both CD40 and CD40L expressing cells in sciatic nerves, a target organ of EAN, peaked on day 14 p.i., large numbers of both expressing cells were mainly detected on day 14-21 p.i. After co-culture with EAN rat B cells bearing CD40, P0 peptide 180-199-specific T cell line cells exhibited a rapid down-regulation of CD40L expression. Furthermore, EAN rats had enhanced P0 peptide 180-199-specific antibody responses on day 14 p.i., which might have contributed to their aggravated EAN and further demonstrated the role of antibodies in EAN. The results indicate that CD40L-CD40 interactions are involved in the initiation of the antigen-specific T cell responses associated with the generation and development of EAN, and may mediate autoantibody production in EAN. Evidently, B cells play a cooperative role via CD40L-CD40 interaction in T cell-mediated EAN of Lewis rats.

Lipooligosaccharide of Campylobacter jejuni prevents myelin-specific enteral tolerance to autoimmune neuritis—a potential mechanism in Guillain-Barré syndrome?

Campylobacter jejuni-induced enteritis is the most common infection preceding Guillain-Barré syndrome (GBS), an immune-mediated polyradiculoneuritis. The acute autoimmune attack is thought to be based on C. jejuni antigens which may mimick antigens of the peripheral nervous system. Additional pathomechanisms, like disturbance of natural T cell immunoregulation by C. jejuni, have not been evaluated so far. In experimental autoimmune neuritis (EAN), a T lymphocyte-mediated animal model of human GBS, tolerance to myelin-derived autoantigens can be induced by oral feeding of the respective antigen. Here we investigated whether the lipooligosaccharide (LOS) fraction of C. jejuni may directly alter immunologic tolerance through gastrointestinal pathways. While EAN, actively induced by immunization with bovine peripheral nerve myelin could be ameliorated by precedent feeding of myelin, feeding of C. jejuni LOS along with the myelin antigen not only prevented the tolerizing effects of oral myelin but even accelerated the onset of overt EAN and augmented the myelin-specific B cell response. These findings provide evidence that LOS of C. jejuni, as produced in the gut during C. jejuni-induced enteritis, can disturb natural tolerance to definite proteins which may be or may mimic peripheral nerve antigens. In human patients this may be one of the potential mechanisms to explain why C. jejuni enteritis is a common trigger of GBS.

Biphasic form of experimental autoimmune neuritis in dark agouti rats and its oral therapy by antigen‐specific tolerization

A new and biphasic form of experimental autoimmune neuritis (EAN) is described in dark agouti rats (DA rats) and is inducible by a single immunization with bovine peripheral nerve myelin (BPM) in complete Freund's adjuvant (DA-EAN). Animals develop a mild episode of disease; after recovery, 66-100% of the rats suffer from a more severe bout of EAN with paraparesis 25-30 days after immunization. By histology, DA-EAN is an inflammatory and demyelinating polyradiculoneuropathy virtually without axonal damage. Demyelination affects mainly spinal roots. This is also reflected by markedly increased F-wave latencies in nerve conduction studies of sciatic nerves. In sciatic nerves, inflammation and demyelination are found only focally and may be the histopathologic basis for conduction failure in some fibers. Immunologic investigations revealed stronger proliferative responses of DA than of Lewis rat lymph node cells to BPM and various peptides derived from the P2 protein. Proliferative and Th1-cytokine responses were particularly pronounced in spleen during the late phase of DA-EAN as compared to the monophasic EAN of Lewis rats. The data suggest that persistent lymphocyte proliferation with secretion of interferon (IFN)-gamma may be relevant for the relapsing course of DA-EAN whereas epitope spreading may explain the increased severity of the second bout of disease. The extended Th1 response in DA rats did not go along with a lack of downregulatory mechanisms, because the second DA-EAN attack was self-limited and splenocytes from DA rats produced considerable amounts of interleukin (IL)-10 and transforming growth factor (TGF)-beta. To substantiate further a functional immunoregulation in DA rats, we modulated DA-EAN by antigen-specific oral tolerization, which is known to involve active suppressor mechanisms. Preventive feeding of BPM in combination with cholera toxin (CT) induced a long-lasting resistance to DA-EAN. Even therapeutic administration of BPM or BPM/CT after onset of signs of disease significantly mitigated the further course of disease and prevented development of paraparesis. Because DA-EAN is easily inducible and leads consistently to relapses in most rats, it can be used for studies of immune factors that determine a relapsing course of autoimmunity. Furthermore, DA-EAN may serve as a model for relapsing inflammatory demyelinating polyneuropathies such as chronic inflammatory demyelinating polyneuropathy (CIDP) and for treatment studies. Our findings on effective prevention and therapy of DA-EAN by oral application of myelin/CT corroborate this form of immunomodulation as a treatment strategy for cell-mediated processes in chronic inflammatory neuropathies.

Role Of Campylobacter Jejuni In Experimental Allergic Neuritis: A Morphological And Biochemical Study

Objective: The aim of the study was to evaluate if Campylobacter jejuni (C.j.) when used as an adjuvant would be able to produce a different form of Experimental Allergic Neuritis (EAN). We present here some preliminary results. Background: EAN is considered the in vivo model of Guillain‐Barrè Syndrome (GBS), which is often preceded by c.j. infection. EAN can be induced in Lewis rats by immunization with bovine peripheral nerve myelin in complete Freund's adjuvant (CFA), an emulsion formed by oil‐in‐water and dead mycobacteria. An adjuvant is usually necessary for the induction of EAN because it enhances the immunogenicity of the antigen. Clinically EAN is characterized by an acute monophasic course and progressive tail and limb weakness. The pathological finding is represented by marked demyelination affecting the roots and the sciatic nerve. Methods: 4 Lewis rats were immunized with an emulsion containing 2 mg of bovine peripheral myelin and C.j. strain Penner 0:41 in incomplete Freund's adjuvant (IFA). They were compared to 4 controls immunized with the same amount of peripheral myelin in CFA. The clinical course of the disease and the histological pattern of the roots and the sciatic nerve were examined. Anti‐peripheral myelin, anti‐C.jejuni and anti‐GM1 antibodies' reactivity was detected by an ELISA assay. A biochemical study was performed to test the role of cell‐ and humoral‐mediated responses. Results: The Lewis rats immunized with the C.j. as an adjuvant showed a delayed onset and a milder course of disease. Pathology in the roots was characterized by predominant demyelination, whereas the sciatic nerve presented very little signs of damage. Conclusion: This serotype of C.j. appears to be a less effective adjuvant in inducing EAN rather than Mycobacteria. Further studies are necessary to elucidate the pathogenetic mechanisms involved in GBS.

EXPERIMENTAL ALLERGIC NEURITIS IN THE SJL/J MOUSE: INDUCTION OF SEVERE AND REPRODUCIBLE DISEASE WITH BOVINE PERIPHERAL NERVE MYELIN AND PERTUSSIS TOXIN WITH OR WITHOUT INTERLEUKIN‐12

We report a reproducible model of experimental allergic neuritis (EAN) with severe clinical signs and consistent pathological features in mice. Pertussis toxin (PT) in the presence or absence of murine recombinant interleukin‐12 (mrIL‐12) was used as an adjuvant with bovine peripheral nerve myelin (BPNM) to induce clinical EAN in SJL/J mice. After immunization with a combination of BPNM in complete Freund's adjuvant (CFA) and PT, mice developed severe consistent signs of EAN. The additional treatment of immunized mice with mrIL‐12 prolonged the course of EAN characterized by earlier clinical signs of the disease and delayed the recovery stage. Mice injected with BPNM and CFA without PT developed mild clinical signs. Histological examination of the caudae equinae and the sciatic nerves taken from mice with clinical signs of EAN during the recovery stage revealed severe demyelination, remyelination and remnants of mononuclear cell infiltration. Moderate to severe EAN can be induced in SJL/J mice by the injection of a combination of BPNM in CFA and PT. This model can provide a better understanding of mechanism of demyelination in infiltrating peripheral neuropathy.

Structure of a major oligosaccharide of PASII/PMP22 glycoprotein in bovine peripheral nerve myelin.

The amino acid sequence of the glycopeptide obtained from bovine PASII/PMP22 protein in the PNS myelin was determined to be Gln-Asn-Cys-Ser-Thr, where the asparagine was glycosylated. To eliminate all the contaminated P(o) glycopeptides from the PASII/PMP22 glycopeptide preparation, we used a fluorescent probe, N-[2-(2-pyridylamino)ethyl]maleimide, which reacts with the cysteine of the PASII/PMP22 glycopeptides. The labeled PASII/PMP22 glycopeptides were isolated by HPLC and were digested further with glycopeptidase A. The resultant oligosaccharides were conjugated with 2-aminopyridine (PA) as a fluorescent tag. One major PA-oligosaccharide, OPPE1, was purified by HPLC. The structure of OPPE1 was elucidated by fast atom bombardment mass spectrometry and (1)H-NMR studies and comparing the derivatives of PA-OPPE1 and PA-oligosaccharides of gamma-globulin on HPLC. The structure, SO(4)-3GlcAbeta1-3Galbeta1-4GlcNAcbeta1-2Manalpha1+ ++-6(GlcNAcbeta1-4) (GlcNAcbeta1-2Manalpha1-3)Manbeta1-4GlcNAcbeta1- 4(Fucalpha1-6)GlcNAc- PA, was identical to the pyridylaminated form of the major oligosaccharide D8 of bovine P(o) previously reported.

Experimental allergic neuritis in the SJL/J mouse: induction of severe and reproducible disease with bovine peripheral nerve myelin and pertussis toxin with or without interleukin-12

We report a reproducible model of experimental allergic neuritis (EAN) with severe clinical signs and consistent pathological features in mice. Pertussis toxin (PT) in the presence or absence of murine recombinant interleukin-12 (mrIL-12) was used as an adjuvant with bovine peripheral nerve myelin (BPNM) to induce clinical EAN in SJL/J mice. After immunization with a combination of BPNM in complete Freund’s adjuvant (CFA) and PT, mice developed severe consistent signs of EAN. The additional treatment of immunized mice with mrIL-12 prolonged the course of EAN characterized by earlier clinical signs of the disease and delayed the recovery stage. Mice injected with BPNM and CFA without PT developed mild clinical signs. Histological examination of the caudae equinae and the sciatic nerves taken from mice with clinical signs of EAN during the recovery stage revealed severe demyelination, remyelination and remnants of mononuclear cell infiltration. Moderate to severe EAN can be induced in SJL/J mice by the injection of a combination of BPNM in CFA and PT. This model can provide a better understanding of mechanism of demyelination in infiltrating peripheral neuropathy.

Induction of experimental autoimmune neuritis in CD4 −8 − C57BL/6J mice

The C57BL/6J mice strain is known to be reputedly resistant to induction of experimental autoimmune neuritis (EAN), an animal model of Guillain–Barré syndrome in humans. Here we describe the induction of EAN in mice of the C57BL/6J background by transfer into naive syngeneic recipients bovine peripheral nerve myelin (BPM)-primed donor lymph node cells that had been stimulated in vitro with the bovine peripheral nervous system (PNS) myelin P2 protein peptide 57–81 followed by challenge with BPM, Freund's complete adjuvant and pertussis toxin. EAN was more severe, both clinically and histologically, and accompanied by extensive infiltration of inflammatory cells and demyelination in peripheral nerves when examined on day 30 after transfer of primed T cells from CD4 −8 − mice into identical naive hosts than after transfer of cells from primed wild type, CD4 −/ − or CD8 −/ − mice to corresponding recipient animals. EAN in CD4 −8 − mice was also associated with elevated numbers of P2 peptide-reactive interferon-γ (IFN-γ) secreting cells and αβ T cells were present in lymph nodes and spleens. The data suggest that PNS myelin activated T cells from an EAN-resistant mice strain are capable of homing to the PNS. The expanded CD4 −8 − αβ T cells may have helper and effector functions, related to initiation of EAN in the CD4 −8 − mice. Lack of CD4 + and CD8 + expressing cells does not prevent the initiation of an autoimmune disease.

Thalidomide prolongs experimental autoimmune neuritis in Lewis rats.

Thalidomide is reported to have immunomodulatory and anti-inflammatory effects, which have led to its use in the treatment of a number of immune-mediated disorders, including leprosy, discoid lupus and Behcet's disease, and to prevent immunological rejection phenomena following skin and bone marrow grafts. Experimental autoimmune neuritis (EAN) is a CD4+ T-cell-mediated demyelinating autoimmune disease, which represents an animal model for the study of the immunopathogenesis and immunotherapy of Guillain-Barré syndrome (GBS) in humans. We examined the effect of thalidomide in Lewis rats with EAN, which was induced by immunization with bovine peripheral nerve myelin (BPM) and complete Freund's adjuvant (CFA). Thalidomide prolonged clinical EAN when given at a dose of 200 mg/kg/day by gavage. This clinical effect was associated with increased numbers of inflammatory cells in sciatic nerve sections and elevated numbers of interferon-gamma (IFN-gamma) mRNA-expressing cells among lymph node mononuclear cells from thalidomide-treated EAN rats on day 17 postimmunization, i.e. at the peak of clinical EAN. The finding that thalidomide prolongs clinical EAN is in agreement with the clinical polyneuropathy reported in patients receiving treatment with thalidomide and limits its clinical usefulness.

Clomipramine and Imipramine Suppress Clinical Signs and T and B Cell Response to Myelin Proteins in Experimental Autoimmune Neuritis in Lewis Rats

5-Hydroxytryptamine (5-HT) reuptake inhibitors of the zimeldine-type have induced polyneuropathies similar to Guillain-Barré syndrome (GBS) in patients with endogenous depression. Some monoamine neurotransmitters have been shown to affect immune reactionsin vivoandin vitroin a concentration-dependent manner. We therefore studied the effect of the monoamine reuptake inhibitory anti-depressants, clomipramine and imipramine on specific immune response and the clinical course of experimental autoimmune neuritis (EAN), the animal model of GBS in humans. Clomipramine and imipramine both suppressed clinical signs of EAN induced by immunization with bovine peripheral nerve myelin (BPM), when given at a dose of 20mg/kg/day intraperitoneally, via osmotic pumps. Clomipramine and imipramine reduced the numbers of Th1 cells secreting IFN-γ in response to the neuritogenic myelin proteins BPM, P0 and P2 among lymph node mononuclear cells (MNC) from rats with EAN. The levels of cells secreting IgG antibodies to BPM, P2 and GM1 in lymph nodes were reduced at the height of EAN in clomipramine and imipramine treated animals. The action of clomipramine and imipramine on induced IFN-γ and anti-myelin antibodies suggests that the mechanism for the suppressive effect of those substances on EAN symptoms may be due to an action on myelin T and B cell autoreactivity. Considering that the main common pharmacological principle of clomipramine and imipramine is to increase the functional activity of the nor-adrenaline (NA) and serotonin (5-HT) of the monoamines, it seems justified to postulate that the actions of clomipramine and imipramine demonstrated in this study to some extent involve NA and/or 5-HT. The immunomodulatory effects of clomipramine and imipramine call for further research on the potential role of drugs acting on the monoamine system in the treatment of autoimmune diseases, and for further studies of immunological mechanisms in the pathogenesis of depressive disorders.

Nasal administration of multiple antigens suppresses experimental autoimmune myasthenia gravis, encephalomyelitis and neuritis

Oral tolerization with acetylcholine receptor (AChR) and myelin basic protein (MBP) prior to immunization with AChR+MBP+complete Freund's adjuvant (CFA) alleviated clinical signs of experimental autoimmune myasthenia gravis (EAMG)+experimental allergic encephalomyelitis (EAE) and AChR- or MBP-specific T and B cell responses. Tolerance induced via the nasal route needs much less tolerogen and may still be as effective as oral tolerance induction. We now immunized Lewis rats with AChR+MBP+bovine peripheral nerve myelin (BPM)+CFA, which resulted in a multiphasic clinical picture with a combination of clinical signs of the EAMG+EAE+experimental allergic neuritis (EAN), accompanied by massive macrophage infiltrations in sections of muscle, spinal cord and sciatic nerve, and strong T and B cell responses to AChR, MBP and BPM in lymphoid organs. Nasal administration of μg doses of AChR+MBP+BPM prior to immunization with a mixture of these antigens+CFA effectively suppressed the incidence and severity of clinical disease, reduced macrophage infiltrations in sections of muscle, spinal cord and sciatic nerve, and down-regulated autoreactive T cell responses to the three antigens in lymphoid organs. Numbers of AChR-, MBP-, BPM-reactive Th1 type of cytokine interferon (IFN)-γ, tumor necrosis factor (TNF)-α mRNA expression in lymph node cells were markedly suppressed, while transforming growth factor-β (TGF-β) mRNA expression was upregulated from nasally tolerized rats, suggesting an active suppression mechanism may act partly in the induction of tolerance. The results implicate the possibility to establish multiple autoantigen-based vaccination for the prevention of autoimmune diseases in humans.

Modulation of experimental autoimmune neuritis in Lewis rats by oral application of myelin antigens

Experimental autoimmune neuritis (EAN) in Lewis rats is a T cell-mediated disease and serves as an animal model of human inflammatory demyelinating neuropathies. EAN can be induced by immunization with complete bovine peripheral nerve myelin (BPM), the myelin protein P2 or its neuritogenic peptide, each emulsified in complete Freund's adjuvant (CFA). The present study evaluates the effect of oral tolerization with BPM or P2 protein on the development of actively induced EAN. Oral administration of BPM strongly suppressed clinical and histological signs of EAN subsequently induced by BPM/CFA, but feeding of P2 protein alone did not affect its course. In contrast, feeding of BPM did not mitigate the course of EAN subsequently induced by immunization with neuritogenic P2 peptide/CFA. Oral therapy with BPM after onset of myelin-induced EAN only slightly ameliorated the further course of disease, but significantly reduced lethality of this severe form of disease. The findings suggest that immunogenicity of the antigens fed determine strength of tolerance, that downregulation of EAN occurs at the site of immunization and not in the nerve, and that active suppression rather than specific anergization is operative in mediating resistance to EAN. However, only partial tolerance to myelin-induced EAN was achieved in naive animals by transfer of spleen/LN cells from rats orally tolerized with BPM. Although methodic factors may have limited the effect of the cells, the result is suggestive of some contribution of anergy to oral tolerance in the present model. Cholera toxin and LPS were identified as oral adjuvants for BPM and prolonged the state of tolerance. However, LPS exhibited proinflammatory properties if EAN was induced early after BPM/LPS-feeding. Thus, oral application of a mixture of myelin components in combination with cholera toxin may be a useful treatment for chronic inflammatory neuropathies considered autoimmune in nature.