Sandra Amor

Myelin/oligodendrocyte glycoprotein–deficient (MOG-deficient) mice reveal lack of immune tolerance to MOG in wild-type mice

We studied the immunological basis for the very potent encephalitogenicity of myelin/oligodendrocyte glycoprotein (MOG), a minor component of myelin in the CNS that is widely used to induce experimental autoimmune encephalomyelitis (EAE). For this purpose, we generated a mutant mouse lacking a functional mog gene. This MOG-deficient mouse presents no clinical or histological abnormalities, permitting us to directly assess the role of MOG as a target autoantigen in EAE. In contrast to WT mice, which developed severe EAE following immunization with whole myelin, MOG-deficient mice had a mild phenotype, demonstrating that the anti-MOG response is a major pathogenic component of the autoimmune response directed against myelin. Moreover, while MOG transcripts are expressed in lymphoid organs in minute amounts, both MOG-deficient and WT mice show similar T and B cell responses against the extracellular domain of MOG, including the immunodominant MOG 35-55 T cell epitope. Furthermore, no differences in the fine specificity of the T cell responses to overlapping peptides covering the complete mouse MOG sequence were observed between MOG+/+ and MOG-/- mice. In addition, upon adoptive transfer, MOG-specific T cells from WT mice and those from MOG-deficient mice are equally pathogenic. This total lack of immune tolerance to MOG in WT C57BL/6 mice may be responsible for the high pathogenicity of the anti-MOG immune response as well as the high susceptibility of most animal strains to MOG-induced EAE.

Role of immune responses in protection and pathogenesis during Semliki Forest virus encephalitis

The course of Semliki Forest virus (SFV) A7(74) infection in immunocompetent BALB/c, athymic nu/nu and severe combined immunodeficient (SCID) mice was compared. BALB/c mice remained healthy and exhibited transient viraemia and infectious virus in the brain from days 2 to 7. Antibodies were detectable by day 5. In comparison, SCID mice displayed a high incidence of paralysis and died: the average day of death was day 23. From infection until death, virus was present in blood and brain. No antibodies were detectable. Athymic mice were intermediate with a transient viraemia and a persistent (> 210 days) sub-clinical central nervous system (CNS) infection. These mice produced anti-viral IgM but not IgG. The pattern of infection in BALB/c or nu/nu mice could be recreated in infected SCID mice by transfer of immune serum from BALB/c or nu/nu mice with the important exception that although BALB/c immune serum could abolish infectivity titres in the CNS, scattered cells positive for viral RNA remained. Transfer of serum decreased mortality and delayed the onset of paralysis. Transfer to infected SCID mice of a non-neutralizing IgG anti-E2 monoclonal antibody did not affect the viraemia but could also reduce brain virus titres. Irrespective of specific immune responses, virus replication in CNS cells was restricted, was generally non-cytopathic and in the absence of specific immune responses could persist. From day 14 lesions of inflammatory, primary demyelination were observed throughout the CNS of BALB/c mice. In contrast, despite prolonged brain virus titres, no demyelinating lesions were observed in infected nu/nu or SCID mice. Lesions could be initiated in the latter by transfer of spleen cells but not antibody. In summary, the focal restricted infection in the CNS of adult mice infected with SFV A7(74) is independent of specific immune responses. IgM antibodies clear the viraemia. IgG antibodies including non-neutralizing antibodies reduce and clear infectious virus but cells positive for viral RNA remain. These may normally be cleared by T cell responses which are damaging and give rise to lesions of demyelination.

Characterization of the cellular and cytokine response in the central nervous system following Semliki Forest virus infection

Cytokines are important mediators in the pathogenesis of central nervous system (CNS) inflammatory diseases including multiple sclerosis (MS), experimental allergic encephalomyelitis (EAE), viral encephalitis and virus induced demyelinating diseases. We have used immunohistochemical techniques to characterize the mononuclear cell infiltrate and cytokine profiles in the CNS following infection of mice with the demyelinating A7(74) strain of Semliki Forest virus (SFV), an important viral model of MS. Mononuclear cell infiltrates in the CNS, first observed at 3 days and maximal during clearance of infectious virus, were comprised predominantly of CD8+ lymphocytes. F4/80+ macrophage/microglia and CD45/B220+ B lymphocytes were most numerous during the subsequent phase of demyelination. CD4+ T-lymphocytes were observed at low levels throughout infection. By immunostaining MHC class I, IL-1beta , IL-3 and TGF beta1 were constitutively expressed in normal mice and were upregulated following infection. MHC class II, IL-1alpha, IL-2, IL-2R, TNF-alpha and IL-6 were strongly upregulated in the CNS of SFV-infected mice and mice with chronic relapsing EAE. The spatial and temporal distribution of these cytokines during the course of disease was analysed. Whereas IL-1alpha, IL-1beta, IL-10, and TGF beta1 were observed on day 3 following infection GMCSF, IL-2 and TNF alpha were first apparent at day 7 when the cellular infiltration in the CNS was most intense. In contrast IFN gamma and IL-6 were first observed on day 10 prior to the demyelination phase of disease. Cytokines in the lesions of demyelination suggest a role in the pathogeneisis of myelin damage. Based on cytokine profiles no clear bias of either a Th1 or Th2 response was observed in the CNS during infection.

Non-human primate models of multiple sclerosis.

Summary: The phylogenetic proximity between non‐human primate species and humans is reflected by a high degree of immunological similarity. Non‐human primates therefore provide important experimental models for disorders in the human population that are caused by the immune system, such as autoimmune diseases. In this paper we describe non‐human primate models of multiple sclerosis, a chronic inflammatory and demyelinating disease of the human central nervous system. While reviewing data from the literature and our own research we will discuss the unique role of such models in the research of basic disease mechanisms and the development of new therapies.

The protective effects of omega-6 fatty acids in experimental autoimmune encephalomyelitis (EAE) in relation to transforming growth factor-beta 1 (TGF-β1) up-regulation and increased prostaglandin E2 (PGE2) production

Polyunsaturated fatty acids are known to affect the immune response and administration of the omega‐6 fatty acid linoleic acid has been reported to be beneficial in multiple sclerosis (MS) and EAE. In this study we have investigated the effects of oral feeding of plant lipid rich in the omega‐6 fatty acid gamma‐linolenic acid from Borago officinalis on acute and relapse disease and the immune response in EAE using SJL mice. EAE was induced by an encephalitogenic peptide (92–106) of myelin oligodendrocyte glycoprotein (MOG), and mice were fed the plant lipid daily from 7 days after EAE induction to assess the effects on acute disease and from day 25 to assess the effects on disease relapse. The clinical incidence and histological manifestations of acute EAE, and the clinical relapse phase of chronic relapsing EAE (CREAE) were markedly inhibited by omega‐6 fatty acid feeding. A significant increase in the production of TGF‐β1 in response to concanavalin A (Con A) at day 13 and a significant increase in TGF‐β1 and PGE2 to Con A, PPD and MOG peptide (92–106) at day 21 were detected in spleen mononuclear cells from fatty acid‐fed mice. There was no difference in interferon‐gamma, IL‐4 and IL‐2 production between the fatty acid‐fed and control groups. Significantly higher TGF‐β mRNA expression was found in the spleens of omega‐6 fatty acid‐fed mice at day 21. There were no differences in spleen cell proliferative response to Con A, PPD and MOG peptide (92–106). Biochemical analysis of spleen cell membrane fatty acids revealed significant increases in the eicosanoid precursor fatty acids dihomo‐γ‐linolenic acid and arachidonic acid in response to gamma‐linolenic acid feeding, indicating rapid metabolism to longer chain omega‐6 fatty acids. These results show that oral feeding of gamma‐linolenic acid‐rich plant lipid markedly affects the disease course of acute EAE and CREAE and is associated with an increase in cell membrane long chain omega‐6 fatty acids, production of PGE2 and gene transcription and, on activation, secretion of TGF‐β1.

Cell Biology of Autoimmune Diseases

Autoimmune diseases such as insulin-dependent diabetes mellitus, rheumatoid arthritis, and multiple sclerosis are common in the western world and are often devastating diseases which pose serious health problems. The key feature of such diseases is the development and persistence of inflammatory processes in the apparent absence of pathogens, leading to chronic breakdown of selected tissues. To date, no comprehensive explanation can be given for the onset or persistence of autoimmunity. As a rule, the chronic activation of helper T lymphocytes reactive against self proteins appears to be crucial for fueling the destructive autoimmune process, but why this occurs remains to be established. In this review, we present an overview on the rules that govern activation of T lymphocytes and on the factors that control it. The contribution of both genetic and environmental factors are discussed, clarifying that most autoimmune disease are of multifactorial origin. Special emphasis is given to the contribution of infectious events and the role of stress proteins in the process. In attempts to dissect the mechanisms involved in autoimmunity and to develop ways of blocking disease, experimental animal models are widely employed. We describe the various experimental models that exist for the study of multiple sclerosis, diabetes, and other autoimmune diseases and on the experience that has been gained in such models with experimental therapies to block the activation of self-reactive T lymphocytes. The lessons that can be drawn from these studies provide hope that continued efforts will lead to the successful development of antigen-specific strategies which block the development of autoimmunity also in humans.

High incidence of spontaneous disease in an HLA-DR15 and TCR transgenic multiple sclerosis model

Multiple sclerosis (MS) is thought to involve CD4 T cell recognition of self myelin, many studies focusing on a pathogenic role for anti-myelin, HLA-DR15-restricted T cells. In experimental allergic encephalomyelitis, it is known which epitopes trigger disease and that disease is associated with determinant spread of T cell reactivity. Characterization of these events in human MS is critical for the development of peptide immunotherapies, but it has been difficult to define the role of determinant spread or define which epitopes might be involved. In this study, we report humanized transgenic mice, strongly expressing HLA-DR15 with an MS-derived TCR; even on a RAG-2 wild-type background, mice spontaneously develop paralysis. Disease, involving demyelination and axonal degeneration, correlates with inter- and intramolecular spread of the T cell response to HLA-DR15-restricted epitopes of myelin basic protein, myelin oligodendrocyte glycoprotein, and αB-crystallin. Spread is reproducible and progressive, with two of the epitopes commonly described in responses of HLA-DR15 patients. The fact that this pattern is reiterated as a consequence of CNS tissue damage in mice demonstrates the value of the transgenic model in supplying an in vivo disease context for the human responses. This model, encompassing pathologically relevant, spontaneous disease with the presentation of myelin epitopes in the context of HLA-DR15, should offer new insights and predictions about T cell responses during MS as well as a more stringent test bed for immunotherapies.

Immunization With Neurofilament Light Protein Induces Spastic Paresis and Axonal Degeneration in Biozzi ABH Mice

Axonal damage is the major cause of irreversible neurologic disability in patients with multiple sclerosis. Although axonal damage correlates with antibodies against neurofilament light (NF-L) protein, a major component of the axonal cytoskeleton, the possible pathogenic role of autoimmunity to axonal antigens such as NF-L has so far been ignored. Here we show that Biozzi ABH mice immunized with NF-L protein develop neurologic disease characterized by spastic paresis and paralysis concomitant with axonal degeneration and inflammation primarily in the dorsal column of the spinal cord. The inflammatory central nervous system lesions were dominated by F4/80+ macrophages/microglia and relatively low numbers of CD4+ and CD8+ T-cells. In splenocyte cultures, proliferation to NF-L was observed in CD4+ T-cells accompanied by the production of the proinflammatory cytokine interferon-&ggr;. Elevated levels of circulating antibodies recognizing recombinant mouse NF-L were present in the serum, and immunoglobulin deposits were observed within axons in spinal cord lesions of mice exhibiting clinical disease. These data provide evidence that autoimmunity to NF-L protein induces axonal degeneration and clinical neurologic disease in mice, indicating that autoimmunity to axonal antigens, as described in multiple sclerosis, may be pathogenic rather than acting merely as a surrogate marker for axonal degeneration.

Evaluating the validity of animal models for research into therapies for immune-based disorders

The last few decades of the 20th century have shown an intensified search for safer and more effective medications against chronic diseases that burden ageing societies of the western world. The impressive development of biotechnological production techniques has greatly facilitated the pharmaceutical development of relatively non-toxic biological molecules. However, despite the huge investments, only a few effective therapies for immune-based diseases have reached the clinic. In this article we use examples from monoclonal antibody trials to discuss the validity and predictive strength of the animal models currently used for the development of effective therapies.

Native myelin oligodendrocyte glycoprotein promotes severe chronic neurological disease and demyelination in Biozzi ABH mice

Myelin oligodendrocyte glycoprotein (MOG) is a powerful encephalitogen for experimental autoimmune demyelination. However, the use of MOG peptides or recombinant proteins representing part of the protein fails to fully address the possible pathogenic role of the full‐length myelin‐derived protein expressing post‐translational modifications. Immunization of mice with central nervous system tissues from wild‐type (WT) and MOG‐deficient (MOG–/–) mice demonstrates that MOG in myelin is necessary for the development of chronic demyelinating experimental autoimmune encephalomyelitis (EAE) in mice. While immunization with WT spinal cord homogenate (SCH) resulted in a progressive EAE phenotype, MOG–/– SCH induced a mild self‐limiting acute disease. Following acute EAE with MOG–/– SCH, mice developed T cell responses to recombinant mouse MOG (rmMOG), indicating that MOG released from myelin is antigenic; however, the lack of chronic disease indicates that such responses were not pathogenic. Chronic demyelinating EAE was observed when MOG–/– SCH was reconstituted with a dose of rmMOG comparable to MOG in myelin (2.5% of total white matter‐derived protein). These data reveal that while immunization with the full‐length post‐translational modified form of MOG in myelin promotes the development of a more chronic autoimmune demyelinating neurological disease, MOG (and/or other myelin proteins) released from myelin during ongoing disease do not induce destructive autoimmunity.