Stephen L. Hauser - US grants

Several in vivo and in vitro studies suggest that muscle-derived factors influence the development and the maintenance of synaptic contacts at the neuromuscular junction. Using a chick system, this project will focus on the characterization of such "motoneuron growth factors" in order to help clarify the relationship between the neuron and its muscle target, and perhaps to provide new insights into the pathogenesis and treatment of human diseases in which motoneurons are selectively lost, such as Amyotrophic Lateral Sclerosis (ALS). Monoclonal antibodies will be produced against a neurite-promoting factor for spinal neurons which is present in chick neonatal muscle extracts. In preliminary work, a 10-4 fold purification of neonatal factor has been achieved, and this material will be used as the starting immunogen. These monoclonal antibodies will then be used to purify the factor to homogeneity, to characterize the active molecule, and to study its function and tissue distribution in normal and in disease states. A second approach is based upon the observation that repeated immunizations with chick neonatal muscle extracts partially purified for neurite-promoting activity can result, in rabbits, in a progressive paralytic disease with prominent atrophy; pathologic examination has revealed a pattern of abnormalities consistent with distal denervation. The immunopathologic mechanisms and the active substances responsible for this syndrome will be studied. Finally the functional effect of circulating antibodies from patients with motor system disease on in vitro neurite extension by dissociated chick spinal cord cells, and the binding of these antibodies to muscle or to nervous system structures will be studied. The purification of neurite-promoting or of disease-inducing substances from muscle will permit direct testing of antibody reactivity against these substances in human disease states.

[unreadable] DESCRIPTION (provided by applicant): In this proposal, we will study the immune response responsible for a unique form of experimental allergic encephalomyelitis (EAE) in a nonhuman primate. Following immunization with whole white matter in adjuvant, Callithrix jacchus (C. jacchus) marmosets develop a relapsing-remitting disease and prominent acute and chronic demyelination closely resembling human multiple sclerosis (MS). In the initial funding period, we have found that the MS-like lesion appears to result from a complex immune response requiring both encephalitogenic T-cells and pathogenic antibody. T-cells reactive against myelin basic protein (MBP) are capable of mediating the inflammatory component of marmoset EAE. Demyelination, on the other hand, is antibody mediated. We have also found that the quantitatively minor myelin protein, myelin oligodendrocyte glycoprotein (MOG), is an important antigen in this system, as immunization against MOG or passive transfer of anti-MOG antibody reproduces the core features of the disease. We propose to utilize a number of state-of-the-art technologies to fully characterize the cellular and humoral immune response against MOG in C. jacchus, to study the role of determinant spreading in chronic disease, and to identify the antigen targets of demyelinating antibody and the mechanisms of antibody mediated demyelination. These studies will provide a foundation for immunotherapy based upon an understanding of the antigens and the immune mechanisms responsible for a complex autoimmune disease. [unreadable] [unreadable] MS affects approximately 350,000 Americans and is thus, excluding trauma, the most important acquired neurologic disease arising in early- to mid-adulthood. Because MS affects women more often than men, it is a particularly significant women's health problem. It is essential that useful model systems for this disabling disease be developed and studied. Advantages of the C. jacchus model for the study of MS include its clinical and pathologic similarity to MS, the natural chimerism of marmosets that permits adoptive transfer of T-cells, and the extensive conservation of immune and nervous system genes and proteins between human and nonhuman primate species. Equally important, work to date in C. jacchus has found that a diverse immune response to more than one antigen appears to be responsible for the MS-like lesion, a finding that parallels emerging concepts of the pathogenesis of human MS. [unreadable] [unreadable]

DESCRIPTION (Adapted from the Investigator's Abstract): The overall goals of this study are: (1) to define the natural history of quantitative MRI and brainwide MTR and HMRSI in patients with primary progressive (PP)MS and healthy controls and, (2)determine the treatment effect of mitoxantrone on those imaging measures. In contrast to conventional T2 weighted imaging which does not distinguish between edema, inflammation, demyelination, and axonal loss, MTR and HMRSI provide more specific imaging markers of demyelination and axonal loss. As there are no longitudinal studies of the combination these complementary imaging measures in PPMS, this study will further our understanding of the natural history of this clinical pattern of MS which is characterized predominantly by diffuse rather than focal abnormalities in brain white matter. The goals will be accomplished by adding annual MT and HMRSI to conventional MRI scans being obtained in patients participating in an ongoing placebo-controlled trial of mitoxantrone in PPMS. Scanning will be continued in 27 placebo recipients for 4 years after completing the 1 year treatment phase of that study and comparable scanning will be obtained from 27 healthy controls. Specific Aims: 1) To compare changes in brain wide histograms of MT ratios, T2 relaxation times and HMRSI spectral intensities of N-acetyl aspartate (NAA), creatine/phosphocreatine (Cr) and choline (Chol) in patients randomly assigned to treatment with MTXN 12 mg/m2 or placebo administered IV every third month for up to 12 months. 2) To compare cross-sectional and annual brain-wide changes in quantitative parameters derived from MT, T2 and HMRSI data for healthy controls and untreated patients with PPMS over a period of 5 years and to investigate how these data correlate with conventional imaging endnotes such as changes in brain volume, ventricular volume, T1 lesion load and T2 lesion load. 3) To determine whether annual changes in quantitative parameters derived from MT, T2 and HMRSI data from patients with PPMS correlate with annual sustained changes in clinical measures of disability and conventional imaging endpoints such as changes in brain volume, ventricular volume, Tl lesion load and T2 lesion load.

DESCRIPTION (provided by applicant): The goal of this application is to identify and characterize the complete repertoire of genes encoded in the MHC region that predispose and/or modulate the expression of autoimmune disease. Following the recent NINDS-sponsored workshop on MHC Genetics in Autoimmune Diseases and the subsequent announcement of an RFA to extend interdisciplinary science in this area, we created a Consortium, named The International MHC and Autoimmunity Genetics Network (IMAGEN) to tackle this problem in a meaningful and decisive manner. The IMAGEN investigators represent a large, diverse, and broad-based collaborative team of scientists at eight academic centers with synergistic skills; demonstrated expertise in MHC genetics and biology; clinical expertise in identifying endophenotypes; history of mutual productive collaborations; and experience in large scale genotyping and state-of-the-art analytical approaches. The basic structure of this collaborative project proposes a common base screen with a panel of more than1500 highly informative SNPs and replication for all diseases. Biologically relevant clinical endpoints will be incorporated into the analysis to assess the role of HLA variants in progression. Specific aim 3 for each project will address disease-specific questions. The primary screen will allow us to: 1) map the association signal(s) across the entire MHC to identify regions of the maximal signal; 2) identify extended MHC haplotypes carrying the strongest association signals; 3) identify recombinant chromosomes that maximally delimit the association; 4) make testable hypotheses as to whether different autoimmune diseases are influenced by a single association with a particular locus, or a single association with an extended haplotype or multiple, independent associations across the MHC. The focus is on Multiple Sclerosis, Rheumatoid Arthritis, IgA Deficiency, Common Variable Immunodeficiency, Myasthenia Gravis, Systemic Lupus Erythematosus and Ulcerative Colitis. We believe that the clinical dataset assembled for this project is unmatched anywhere in the world. An Administrative Core at UCSF will coordinate activities and interactions for the overall project. A second Core at the Broad Institute will be responsible for generation of genotypes, data QC, storage, and interaction with BISC.

DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is a common and severe disorder of the central nervous system characterized by chronic inflammation, myelin loss, gliosis, varying degrees of axonal and oligodendrocyte pathology, and progressive neurological dysfunction. MS pathogenesis includes a complex genetic component. In spite of intensive long-standing efforts, the knowledge of MS genetics remains incomplete. Our overall objective is to characterize the repertoire of genes that predispose to MS and modulate its presentation. Their identification is now possible as a result of rapid progress in defining the landscape of genetic organization and cataloging variation across the human genome. This proposal builds on the availability of second generation, high-quality genome-wide association results and comprehensive phenotypic data in a large MS cohort. We propose four main research goals: In Specific Aim 1 high-coverage genome-wide genetic data from a total of approximately 17,000 subjects affected with MS will be pooled and analyzed using a multi-stage and multi- analytical approach to map unambiguous association signals from sequence (SNPs) and copy number (CNVs) polymorphisms and identify novel disease candidate genes, leading to robust and testable hypotheses as to which are the specific common allelic variants conferring susceptibility. Using the meta-analysis-derived genotypes, together with open databases and novel algorithms, we will develop a composite global map of the effects of epistatic interactions and biochemical networks of genomic variation underlying MS pathogenesis. Specific Aim 2 takes advantage of the wealth of phenotypic data available for the different datasets to assess disease course variables and correlations to genotype. Important clinical metrics such as age and site of disease onset, disability at entry of study and progression, and changes in lesion distribution and burden will be incorporated into the analysis of genetic data. This aim directly addresses the question of clinical heterogeneity in MS and the correlation between different phenotypes and genotypes. In Specific Aim 3 we intend to generate high-coverage sequence information for the regulatory regions, exons and flanking regions of genes with unequivocal evidence of association for the discovery of rare variants associated with MS. To efficiently resequence DNA in a large dataset (3,000 patients / 3,000 controls), we will create 120 pools of 50 DNA samples each. Approximately 1,000 low-frequency variants will be genotyped in a validation cohort consisting of 10,000 MS subjects and 10,000 control subjects. As technologies advance and costs retreat, whole exome re-sequencing will be pursued in the second half of the funding cycle. Finally, in Specific Aim 4 we will integrated all the generated data and build an array, the "MS Fine-Mapping Gene-chip," consisting of a comprehensive compendium of common and rare variants covering and flanking confirmed associations with susceptibility and disease expression. All relevant variants will be tested in an independent dataset (10,000 cases / 10,000 controls) for association individually and by combining multiple alleles within a single gene and across multiple genes to assess of causative cumulative effects. From this dataset, we expect to generate a minimal set of DNA variants that individually or in combination can aid in prediction of disease risk and/or progression. The availability of a large and well-characterized cohort as described here, coupled with the aid of high-powered laboratory technologies, provides an outstanding opportunity to identify and characterize MS-related genes. This information may translate into clinically useful genetic biomarkers and reveal novel targets for therapy. PUBLIC HEALTH RELEVANCE: Public Health Relevance Statement Multiple sclerosis (MS) is a common cause of severe neurological disability resulting from the interruption of myelinated tracts in the central nervous system. MS is second only to trauma as a cause of neurologic disability in young adults, affecting approximately 2 million people worldwide and more than 400,000 individuals in the US. Remarkably, the incidence of MS seems to have increased considerably over the last century, and this increase may have occurred primarily in women. The socioeconomic consequences of this long-lasting disease are staggering as 75- 85% of patients are eventually unemployed and at high risk for social isolation. Conservative estimates indicate that this chronic illness results in healthcare costs exceeding $200 billion annually in the United States alone. Thus, MS is the second most costly neurological disorder after Alzheimer's disease. Despite important advances in therapeutics, none of the currently available disease-modifying drugs convincingly alter the long-term prognosis of the disease. Clinical manifestations are extremely diverse, but very little is known about the underlying cause of this variability. It can vary from a benign illness to a rapidly evolving and incapacitating disease. Onset may be abrupt or insidious, and early symptoms may be severe or seem so trivial that a patient may not seek medical attention for months or years. Most patients ultimately experience progressive disability and twenty-five years after onset approximately 80% of affected individuals will require assistance with ambulation. Thus over the long-term, MS is most often a severe disease requiring profound lifestyle adjustments to the affected and their families. We aim to identify the genes and the gene-specific variants that code for products involved in MS susceptibility. We anticipate that there may be several genes involved in MS risk. These genes may work independently or together, and affect susceptibility in concert with environmental factors. Particular combinations of inherited genetic variants may also determine when symptoms develop, or how the disease progresses. Their identification will help to define the basic etiology of MS, improve risk assessment, and influence therapeutics.

? DESCRIPTION (provided by applicant): Monoclonal antibodies against CD20 are a highly effective therapy for multiple sclerosis (MS) currently in phase III clinical development. CD20 is commonly viewed as an archetypical B cell marker. However, a subset of human T lymphocytes (T cells) also expresses CD20. Presently, not much is known about the functional or pathological relevance of CD20-expressing T cells, but a possible involvement of this T cell subpopulation in autoimmune disorders has been suggested. CD20+ T cells can assume a pro-inflammatory Th17 phenotype in rheumatoid arthritis (RA) and MS, and increased numbers of CD3+CD20dim T cells can be found in peripheral blood (PB) of MS patients. Like CD20+ B cells, CD3+CD20dim T cells are effectively depleted from PB of MS and RA patients by the anti-CD20 antibody rituximab, which may, in part, be responsible for the effectiveness of anti-CD20 therapeutic strategies. Unpublished preliminary experiments suggest that CD3+CD20dim T cells in PB may be increased during MS relapses; CD20-expressing T cells are also present in CSF but an association with disease-activity has yet to be studied. Furthermore, next-generation deep T cell receptor ß-chain variable region (TCR-Vß) immune-repertoire sequencing suggests that identical CD20dim T cell clonotypes in peripheral blood and CSF may be involved in MS disease-activity. To our knowledge, no murine equivalent to human CD3+CD20dim T cells has been identified. However, treatment of mice with an antibody specific for MS4aB1, a murine CD20 homolog expressed on T cells, was found to ameliorate disease severity of experimental autoimmune encephalomyelitis (EAE), theoretically mimicking the therapeutic effect of rituximab-mediated CD3+CD20dim T cell depletion, in the absence of B cell depletion, in humans. The objective of this research program is to delineate the potential pathological involvement of CD3+CD20dim T cells in the immune pathology of MS. Methods: We will perform extensive phenotypic, transcriptional, and functional characterizations of CD20+ T cells in PB (Aim 1), to examine whether CD20+ T cells and/or other T cell subsets can provide an antigen-specific, immunologically active, and sustained connection between the periphery and CNS compartments (Aim 2), and to determine their prevalence in MS CSF during different stages of the disease (Aim 2) and compared to other neurological diseases (Aim 3).

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system affecting nearly one million individuals in the United States. The disease is characterized by acute and chronic inflammation, myelin loss; oligodendrocyte, neuronal and axonal pathology; and progressive neurological dysfunction. A number of breakthrough translational discoveries, and especially the highly beneficial effects of B cell depleting drugs, have set the stage for a growing, yet still imperfect, therapeutic pipeline. Research will fall short of its potential to improve patient outcomes until the trigger(s) of disease onset and modifiers of progression are identified. Central to this project is the hypothesis that B cells presenting in the cerebrospinal fluid (CSF) and peripheral blood during early MS play key roles in triggering MS and in mediating ongoing progressive disease activity. We propose to interrogate unique patient cohorts, including an incident cohort, with novel enabling technologies to identify triggers of MS and modifiers of the clinical course. Well characterized clinical populations, high-field serial magnetic resonance imaging (MRI), genetic information organized as functional operational networks, and a focus on B cell biology are the central elements of this initiative. A primary goal will be to characterize the molecular diversity of B cells and their receptors at various stages of disease to identify pathogenic populations and their antigenic targets. A multi-layered experimental strategy includes single cell B cell transcriptomics, together with comprehensive phage-displayed synthetic human, viral and microbial peptidomes for the screening of antibody fingerprints against external drivers in the serum, CSF, and recombinant antibody libraries. The integrative analysis will contextualize the data using clinical, MRI and genetic determinants, striving to apply rigorous statistical principles, including independent replication.