Mauro C. Dal Canto

Neuropathological changes in two lines of mice carrying a transgene for mutant human Cu,Zn SOD, and in mice overexpressing wild type human SOD: a model of familial amyotrophic lateral sclerosis (FALS)

Two different lines of mice, G1 and G20, carrying a transgene for a mutant form of Cu,Zn SOD, found in a family with familial amyotrophic lateral sclerosis (FALS), develop clinical and pathological changes which are, in their late stages, strikingly similar to those in human disease. We have analyzed the distribution and characteristics of lesions in the central and peripheral nervous systems of such mice. The most affected structure was the spinal cord, followed by the medulla, pons and midbrain. The early stages of the disease were characterized by vascular degeneration of anterior horn neurons and their processes, while, in the late stages, the main changes consisted of neuronal loss and atrophy of the anterior horns and the deposition in these areas of multiple filamentous inclusions resembling Lewy bodies. In the late stages of the disease, the white matter of the spinal cord was also involved, particularly in the anterior and lateral columns. Posterior columns were also involved, but to a much lesser degree. The brainstem structures also showed vacuolar degeneration of several motor nuclei and of several groups neurons in the reticular formation. Anterior roots and peripheral nerves showed the classical features of Wallerian degeneration. The dorsal root ganglia, with rare exceptions, were unremarkable. The posterior roots showed mild changes in the most severely affected mice. Changes in these two affected lines were compared to changes in mice overexpressing wild type, rather than mutant human Cu,Zn SOD. These mice never developed clinical disease, although, pathologically, they developed very mild vacuolar changes in the anterior horns of the spinal cord and in motor axons. This study shows that although simple overexpression of SOD may be injurious to motor neurons, albeit very mildly, the mutant form is necessary to produce both clinical disease and severe pathological changes which, in the chronic stage of the disease, have striking similarities to human familial ALS. A dominant gain of function, therefore, is the most likely pathogenesis of tissue injury induced by mutations in Cu,Zn SOD.

Blockade of CD28/B7-1 Interaction Prevents Epitope Spreading and Clinical Relapses of Murine EAE

Relapsing experimental autoimmune encephalomyelitis (R-EAE) induced with the immunodominant epitope from proteolipid protein, PLP139-151, is characterized by the development of recurrent relapses with recruitment of T cells reactive to additional myelin peptides, including PLP178-191 (epitope spreading). In this study, we have determined that the CD28/B7 costimulatory pathway is involved in this process. We found preferential up-regulation of B7-1 during the course of R-EAE and a selective increase in its functional costimulatory activity, relative to B7-2. Anti B7-1 F(ab) fragment therapy, but not anti B7-2 MAb therapy, blocked clinical relapses, ameliorated CNS pathology, and blocked epitope spreading. These results suggest that the maintenance of autoimmune reactivity in EAE depends on CD28/B7-1-dependent costimulation of newly recruited T cells responsible for epitope spreading. These studies have important implications for the role of epitope spreading in disease progression and the clinical application of costimulatory antagonists in autoimmune diseases.

Age-Dependent Penetrance of Disease in a Transgenic Mouse Model of Familial Amyotrophic Lateral Sclerosis

The mutation gly93-->ala of Cu,Zn superoxide dismutase (SOD) is found in patients with familial amyotrophic lateral sclerosis and causes motor neuron disease when expressed in transgenic mice. The progression of clinical and pathological disease was studied in a line of mice designated G1H. Clinical disease started at 91 +/- 14 days of age with fine shaking of the limbs, followed by paralysis and death by 136 +/- 7 days of age. Pathological changes begin by 37 days of age with vacuoles derived from swollen mitochondria accumulating in motor neurons. At the onset of clinical disease (90 days), significant death of somatic motor neurons innervating limb muscles has occurred; mice at end-stage disease (136 days) show up to 50% loss of cervical and lumbar motor neurons. However, neither thoracic nor cranial motor neurons show appreciable loss despite vacuolar changes. Autonomic motor neurons also are not affected. Mice that express wild-type human Cu,Zn SOD remain free of disease, indicating that mutations cause neuron loss by a gain-of-function. Thus, the age-dependent penetrance of motor neuron disease in this transgenic model is due to the gradual accumulation of pathological damage in select populations of cholinergic neurons.

Mechanisms of immunotherapeutic intervention by anti-CD40L (CD154) antibody in an animal model of multiple sclerosis

Relapsing experimental autoimmune encephalomyelitis (R-EAE) in the SJL mouse is a Th1-mediated autoimmune demyelinating disease model for human multiple sclerosis and is characterized by infiltration of the central nervous system (CNS) by Th1 cells and macrophages. Disease relapses are mediated by T cells specific for endogenous myelin epitopes released during acute disease, reflecting a critical role for epitope spreading in the perpetuation of chronic central CNS pathology. We asked whether blockade of the CD40-CD154 (CD40L) costimulatory pathway could suppress relapses in mice with established R-EAE. Anti-CD154 antibody treatment at either the peak of acute disease or during remission effectively blocked clinical disease progression and CNS inflammation. This treatment blocked Th1 differentiation and effector function rather than expansion of myelin-specific T cells. Although T-cell proliferation and production of interleukin (IL)-2, IL-4, IL-5, and IL-10 were normal, antibody treatment severely inhibited interferon-gamma production, myelin peptide-specific delayed-type hypersensitivity responses, and induction of encephalitogenic effector cells. Anti-CD154 antibody treatment also impaired the expression of clinical disease in adoptive recipients of encephalitogenic T cells, suggesting that CD40-CD154 interactions may be involved in directing the CNS migration of these cells and/or in their effector ability to activate CNS macrophages/microglia. Thus, blockade of CD154-CD40 interactions is a promising immunotherapeutic strategy for treatment of ongoing T cell-mediated autoimmune diseases.

Induction of active and adoptive relapsing experimental autoimmune encephalomyelitis (EAE) using an encephalitogenic epitope of proteolipid protein

Proteolipid protein (PLP) is a major component of the central nervous system (CNS) myelin membrane and has been shown to induce acute experimental autoimmune encephalomyelitis (EAE) in genetically susceptible animals. Here we describe conditions by which a relapsing-remitting form of EAE can be reliably induced in SJL/J mice either actively immunized with the major encephalitogenic PLP peptide, PLP13-151(S), or following adoptive transfer of PLP139-151(S)-specific T cells. The disease follows a reliable relapsing-remitting course with acute clinical signs first appearing 6-20 days after priming or transfer and relapses first appearing at 30-45 days. The initial onset of disease correlates with delayed-type hypersensitivity (DTH) reactivity specific for PLP139-151(S), in the apparent absence of T cell reactivity to the major myelin basic protein (MBP) peptide. Histologically, both the active and adoptive forms of the disease are characterized by extensive mononuclear cell infiltration and severe demyelination of the CNS. These results suggest that T cell responses specific for PLP139-151(S) are sufficient to induce clinical and histological R-EAE in SJL/J mice. This model should prove useful for examination of the cellular and molecular events involved in clinical relapses and perhaps in determining the role of PLP-specific T cell responses in multiple sclerosis (MS).

A virus-induced molecular mimicry model of multiple sclerosis

Molecular mimicry is the process by which virus infection activates T cells that are cross-reactive with self antigens. Infection of SJL/J mice with the neurotropic picornavirus Theilers murine encephalomyelitis virus (TMEV) leads to a progressive CD4(+) T cell-mediated demyelinating disease similar to multiple sclerosis. To study the potential of virus-induced molecular mimicry to initiate autoimmune demyelination, a nonpathogenic TMEV variant was engineered to encode a 30-mer peptide encompassing the immunodominant encephalitogenic myelin proteolipid protein (PLP139-151) epitope. Infection with the PLP139-151-encoding TMEV led within 10-14 days to a rapid-onset paralytic demyelinating disease characterized by PLP139-151-specific CD4(+) Th1 responses; insertion of a non-self ovalbumin sequence led to restoration of the normal late-onset disease. Early-onset disease was also observed in mice infected with a TMEV encoding PLP139-151 with an amino acid substitution at the secondary T cell receptor (TCR) contact residue (H147A), but not in mice infected with TMEV encoding a PLP139-151 substitution at the primary TCR contact (W144A). Most significantly, mice infected with TMEV encoding a Haemophilus influenzae mimic peptide, sharing only 6 of 13 amino acids with PLP139-151, displayed rapid-onset disease and developed cross-reactive PLP139-151-specific CD4(+) Th1 responses. To our knowledge, this is the first study showing that a naturally infectious virus encoding a myelin epitope mimic can directly initiate organ-specific T cell-mediated autoimmunity.

Monocytes/macrophages isolated from the mouse central nervous system contain infectious Theiler's murine encephalomyelitis virus (TMEV)

Knowledge of the cells in which Theilers murine encephalomyelitis virus (TMEV) persists is crucial to understanding the pathogenesis of TMEV-induced demyelinating disease; however, it is still uncertain whether oligodendrocytes or macrophages are the primary target for persistence. In this study, mononuclear cells (MNC) isolated directly from central nervous system (CNS) inflammatory infiltrates of TMEV-infected mice on discontinuous Percoll gradients were found to contain infectious TMEV. Macrophages appeared to be the principal MNC infected as determined by two-color immunofluorescence. Infectious center assay and double immunostaining together indicated the presence and possible synthesis of TMEV in approximately 1 in 225 to 1 in 1000 CNS macrophages, with 1 to 7 PFU produced per macrophage. On the basis of these findings, limited replication in macrophages is consistent with the total CNS virus content detected at any time during the persistent phase of the infection as well as the slow pace of the infection.

De Novo Central Nervous System Processing of Myelin Antigen Is Required for the Initiation of Experimental Autoimmune Encephalomyelitis

We demonstrate the absolute requirement for a functioning class II-restricted Ag processing pathway in the CNS for the initiation of experimental autoimmune encephalomyelitis (EAE). C57BL/6 (B6) mice deficient for the class II transactivator, which have defects in MHC class II, invariant chain (Ii), and H-2M (DM) expression, are resistant to initiation of myelin oligodendrocyte protein (MOG) peptide, MOG35–55-specific EAE by both priming and adoptive transfer of encephalitogenic T cells. However, class II transactivator-deficient mice can prime a suboptimal myelin-specific CD4+ Th1 response. Further, B6 mice individually deficient for Ii and DM are also resistant to initiation of both active and adoptive EAE. Although both Ii-deficient and DM-deficient APCs can present MOG peptide to CD4+ T cells, neither is capable of processing and presenting the encephalitogenic peptide of intact MOG protein. This phenotype is not Ag-specific, as DM- and Ii-deficient mice are also resistant to initiation of EAE by proteolipid protein peptide PLP178–191. Remarkably, DM-deficient mice can prime a potent peripheral Th1 response to MOG35–55, comparable to the response seen in wild-type mice, yet maintain resistance to EAE initiation. Most striking is the demonstration that T cells from MOG35–55-primed DM knockout mice can adoptively transfer EAE to wild-type, but not DM-deficient, mice. Together, these data demonstrate that the inability to process antigenic peptide from intact myelin protein results in resistance to EAE and that de novo processing and presentation of myelin Ags in the CNS is absolutely required for the initiation of autoimmune demyelinating disease.

Experimental models of virus‐induced demyelination of the central nervous system

One of the arguments in favor of a viral pathogenesis for multiple sclerosis is the existence of several experimental and natural animal models of virus‐induced primary demyelination. This review deals comprehensively with such models. Well‐known examples of demyelinating viral infections in their natural host are JHM, Theiler, visna, and canine distemper encephalomyelitides. Recent reports of experimental murine infections with pathogens such as vesicular stomatitis, Chandipura, herpes simplex, Venezuelan equine encephalomyelitis, and Semliki Forest viruses are also discussed. The thrust of the review is to include viral models suspected of producing primary demyelination on an immunopathological basis.

A Critical Role for B7/CD28 Costimulation in Experimental Autoimmune Encephalomyelitis: A Comparative Study Using Costimulatory Molecule-Deficient Mice and Monoclonal Antibody Blockade

The B7/CD28 pathway provides critical costimulatory signals required for complete T cell activation and has served as a potential target for immunotherapeutic strategies designed to regulate autoimmune diseases. This study was designed to examine the roles of CD28 and its individual ligands, B7-1 and B7-2, in experimental autoimmune encephalomyelitis (EAE), a Th1-mediated inflammatory disease of the CNS. EAE induction in CD28- or B7-deficient nonobese diabetic (NOD) mice was compared with the effects of B7/CD28 blockade using Abs in wild-type NOD mice. Disease severity was significantly reduced in CD28-deficient as well as anti-B7-1/B7-2-treated NOD mice. B7-2 appeared to play the more dominant role as there was a moderate decrease in disease incidence and severity in B7-2-deficient animals. EAE resistance was not due to the lack of effective priming of the myelin peptide-specific T cells in vivo. T cells isolated from CD28-deficient animals produced equivalent amounts of IFN-γ and TNF-α in response to the immunogen, proteolipid protein 56–70. In fact, IFN-γ and TNF-α production by Ag-specific T cells was enhanced in both the B7-1 and B7-2-deficient NOD mice. In contrast, peptide-specific delayed-type hypersensitivity responses in these animals were significantly decreased, suggesting a critical role for CD28 costimulation in in vivo trafficking and systemic immunity. Collectively, these results support a critical role for CD28 costimulation in EAE induction.