Michael C. Levin

Autoimmunity due to molecular mimicry as a cause of neurological disease

One hypothesis that couples infection with autoimmune disease is molecular mimicry. Molecular mimicry is characterized by an immune response to an environmental agent that cross-reacts with a host antigen, resulting in disease. This hypothesis has been implicated in the pathogenesis of diabetes, lupus and multiple sclerosis (MS). There is limited direct evidence linking causative agents with pathogenic immune reactions in these diseases. Our study establishes a clear link between viral infection, autoimmunity and neurological disease in humans. As a model for molecular mimicry, we studied patients with human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a disease that can be indistinguishable from MS (refs. 5,6,7). HAM/TSP patients develop antibodies to neurons. We hypothesized these antibodies would identify a central nervous system (CNS) autoantigen. Immunoglobulin G isolated from HAM/TSP patients identified heterogeneous nuclear ribonuclear protein-A1 (hnRNP-A1) as the autoantigen. Antibodies to hnRNP-A1 cross-reacted with HTLV-1-tax, the immune response to which is associated with HAM/TSP (refs. 5,9). Immunoglobulin G specifically stained human Betz cells, whose axons are preferentially damaged. Infusion of autoantibodies in brain sections inhibited neuronal firing, indicative of their pathogenic nature. These data demonstrate the importance of molecular mimicry between an infecting agent and hnRNP-A1 in autoimmune disease of the CNS.

Cross-Reactivity between Immunodominant Human T Lymphotropic Virus Type I tax and Neurons: Implications for Molecular Mimicry

Human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is associated with immunoreactivity to HTLV-I tax. Antibodies isolated from patients with HAM/TSP and monoclonal antibodies (MAbs) to HTLV-I tax stained neurons. In neuronal extracts, HAM/TSP immunoglobulin G identified heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) as the autoantigen. Importantly, tax MAbs reacted with hnRNP A1. To identify the epitope recognized by the tax MAbs, the fine epitope specificity of the antibodies was determined using overlapping peptides. This analysis identified an epitope at the C-terminus (tax(346-353)), which overlaps a human immunodominant domain. Preincubation of this peptide with tax MAbs inhibited antibody binding to tax, hnRNP A1, and neurons. This indicates that a cross-reactive immune response between HTLV-I tax and neuronal hnRNP A1 is contained within the human immunodominant epitope of tax and suggests that molecular mimicry plays a role in the pathogenesis of HAM/TSP.

Molecular mimicry: Cross‐reactive antibodies from patients with immune‐mediated neurologic disease inhibit neuronal firing

Recent data indicate that molecular mimicry may play a role in the pathogenesis of human‐T‐lymphotropic virus type‐1 (HTLV‐1)‐associated myelopathy/tropical spastic paraparesis (HAM/TSP), an immune‐mediated disease of the central nervous system (CNS). Specifically, HAM/TSP patients developed antibodies that cross‐react with heterogeneous nuclear ribonuclear protein A1 (hnRNP A1), an antigen highly expressed in neurons. Antibodies to HTLV‐1‐tax cross‐reacted with hnRNP A1, suggesting molecular mimicry between the two proteins. In support of this hypothesis, HAM/TSP IgG and antibodies to hnRNP A1 and HTLV‐1‐tax inhibited neuronal firing, suggesting that these antibodies can be pathogenic. We extended these observations by carrying out studies on over 20 different neurons. We also tested IgG isolated from six different HAM/TSP patients and two HTLV‐1 seronegative controls and added experiments that control for antibody isotype, antibody target, and neuron viability. In these studies, IgG was infused into the extracellular space during whole‐cell current clamp recordings of neurons. Our results confirm that in contrast to normal IgG, IgG from all HAM/TSP patients completely inhibited neuronal firing. Affinity‐purified antibodies specific for hnRNP A1 and a monoclonal antibody to HTLV‐1‐tax (which reacted with hnRNP A1 and whose epitope overlaps the human immunodominant epitope of tax) also inhibited neuronal firing. Monoclonal antibodies to neurofilament did not change neuronal firing. These data indicate that antibodies to neurons can be pathogenic, that biologic activity can be affected by a cross‐reactive epitope between HTLV‐1‐tax and hnRNP A1, and that molecular mimicry may play a role in the pathogenesis of HAM/TSP.

Autoantibodies that recognize functional domains of hnRNPA1 implicate molecular mimicry in the pathogenesis of neurological disease

As a model for molecular mimicry in neurological disease, we study people infected with human T-lymphotropic virus type 1 (HTLV-1) who develop HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP), an immune-mediated disease of the central nervous system (CNS). In HAM/TSP, data suggests molecular mimicry is the result of cross-reactive antibodies between HTLV-1-tax and heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), a protein over-expressed in human CNS neurons. The hnRNP A1 epitope recognized by autoantibodies was unknown. In this study, we hypothesized that antibodies purified from HAM/TSP patients would react with functionally significant domains of hnRNP A1. Western blotting of functionally significant deletion mutants and overlapping fusion proteins using HAM/TSP IgG revealed two core epitopes within the C-terminal region of hnRNP A1. The first (aminoacids 191-SSQRGRSGSGNF-202), overlapped the RGG domain and the second (aminoacids 293-GQYFAKPRNQGG-304), with the M9 shuttling sequence, two functionally important regions of hnRNP A1. Monoclonal antibodies to HTLV-1-tax also reacted with the epitopes. These data fulfill an important criterion of molecular mimicry, namely that mimicking epitopes are not random, but include biologically significant regions of target proteins. This suggests an important role for the cross-reactive immune response between HTLV-1 and hnRNP A1 in the pathogenesis of immune-mediated neurological diseases via molecular mimicry.

IgG in brain correlates with clinicopathological damage in HTLV-1 associated neurologic disease

Objective: To test the authors’ hypothesis that antibody deposition in autopsy specimens from patients with human T-lymphotropic virus type 1–associated myelopathy/tropical spastic paraparesis (HAM/TSP) would correlate with CNS damage. Methods: Endogenous immunoglobulin G (IgG) was detected using antihuman IgG in autopsy tissues from HAM/TSP and control patients. IgG was isolated from the CSF, CNS, and sera of patients with HAM/TSP and tested for reactivity to heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), an autoantigen recently associated with molecular mimicry in HAM/TSP. Results: In situ IgG localized to elements of the corticospinal system including neurons of the frontal cortex and precentral gyrus, as well as throughout axons in subcortical white matter, periventricular white matter, posterior limb of the internal capsule, midbrain, pons, and medulla. Similarly, there was IgG deposition within the posterior-column/medial lemniscal sensory system, including the arcuate fibers of the cranial-cervical junction, the nucleus cuneatus, and throughout the course of the medial lemniscus in the medulla, pons, and midbrain. IgG from brain, CSF, and serum of the patients with HAM/TSP showed immunoreactivity with hnRNP A1. Conclusion: Patients with HAM/TSP develop antibodies specific for neurons and axons that are preferentially damaged in the CNS.

HTLV-1 Induced Molecular Mimicry in Neurological Disease

As a model for molecular mimicry, we study patients infected with human T-lymphotropic virus type 1 (HTLV-1) who develop a neurological disease called HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a disease with important biological similarities to multiple sclerosis (MS) (Khan et al. 2001; Levin et al. 1998, 2002a; Levin and Jacobson 1997). The study of HAM/TSP, a disease associated with a known environmental agent (HTLV-1), allows for the direct comparison of the infecting agent with host antigens. Neurological disease in HAM/TSP patients is associated with immune responses to HTLV-1-tax (a regulatory and immunodominant protein) and human histocompatibility leukocyte antigen (HLA) DRB1*0101 (Bangham 2000; Jacobson et al. 1990; Jeffery et al. 1999; Lal 1996). Recently, we showed that HAM/TSP patients make antibodies to heterogeneous nuclear ribonuclear protein A1 (hnRNP A1), a neuron-specific autoantigen (Levin et al. 2002a). Monoclonal antibodies to tax cross-reacted with hnRNP A1, indicating molecular mimicry between the two proteins. Infusion of cross-reactive antibodies with an ex vivo system completely inhibited neuronal firing indicative of their pathogenic nature (Kalume et al. 2004; Levin et al. 2002a). These data demonstrate a clear link between chronic viral infection and autoimmune disease of the central nervous system (CNS) in humans and, we believe, in turn will give insight into the pathogenesis of MS.

A role for hypertrophic astrocytes and astrocyte precursors in a case of rapidly progressive multiple sclerosis

The purpose of this study was to examine the roles played by astrocytes in a case of rapidly progressive multiple sclerosis (MS). Within early-active and active lesions, hypertrophic astrocytes played an important role in lesion patho logy through the phagocytosis of myelin and axonal debris and through the internalization of other glial cells, including astrocytes. In addition to this critical role, hypertrophic astrocytes, in areas that lack significant inflammation (within the adjacent normal appearing white matter and within late remyelinating lesions) were found to be active in myelin and axonal debris phagocytosis with no evidence of cellular internalization. Hypertrophic astrocytes therefore not only play an important role in the patho genesis of MS lesions but also exert a continued deleterio us effect upon tissue in the absence of significant inflammation. In addition, we found evidence for a significant population of vimentin-positive, glial fibrillary acidic protein (GFA P)-negative, bipolar, astrocyte precursors within the late remyelinating lesions. Their significance is not known but a possible role may include their participatio n in the successful remyelination of the lesion.

Proteomic analysis of phosphotyrosyl proteins in the rat brain: Effect of butorphanol dependence

Butorphanol (17‐cyclobutylmethyl‐3,14‐dihydroxymorphinan) tartrate (Stadol) is a mixed agonist‐antagonist opioid analgesic agent that is about five to seven times as potent as morphine in analgesic effects. The chronic use of butorphanol produces physical dependence in humans and animals. Phosphorylation plays a very important role in developing butorphanol dependence; however, global phosphorylation events induced by chronic butorphanol administration have not been reported. The aim of this study is to determine the alteration of tyrosine phosphorylation of brain frontal cortical proteins in butorphanol‐dependent rats using a proteomic approach. Dependence was produced by continuous intracerebroventricular (i.c.v.) infusion of butorphanol (26 nmol/μl/hr) for 72 hr via osmotic minipump in rats. Similar patterns of protein expression were detected by two‐dimensional electrophoresis (2‐DE) in brain frontal cortex of butorphanol‐dependent and saline‐treated control rats. All 65 phosphotyrosyl (p‐Tyr) protein spots detected in pH 3–10 phosphotyrosine 2‐DE of control rat brains were detected in butorphanol‐dependent rat brains. The densities of most p‐Tyr protein spots were increased in butorphanol‐dependent rat brains compared to saline‐treated control samples. Eighteen additional p‐Tyr protein spots were detected in pH 3–10 2‐DE images of butorphanol‐dependent rat brains. Immobilized pH strips with three different narrow pH ranges were examined to improve the resolution of p‐Tyr proteins in 2‐DE gels. Fifty‐three p‐Tyr protein spots were identified as known proteins involved in cell cytoskeleton, cell metabolism, and cell signaling. This proteomic approach can provide useful information for understanding the complex mechanism of butorphanol dependence in vivo.

Localization of retrovirus in the central nervous system of a patient co-infected with HTLV-1 and HIV with HAM/TSP and HIV-associated dementia

Persons co-infected with HTLV-1 and HIV are at increased risk for neurologic disease. These patients may develop HAM/TSP and/or HIV-associated dementia. In this study, we localized cells infected with retrovirus in the central nervous system (CNS) of a patient with both HAM/TSP and HIV-associated dementia. HTLV-1 was localized to astrocytes and HIV to macrophage/microglia. There was no co-infection of a single cell phenotype in this patient. These data suggest that mechanisms other than co-infection of the same CNS cell may play a role in the development of neurologic disease in patients dual infected with HTLV-1 and HIV.