autoantigens not previously implicated in disease. Reactiv-ities were conserved in humanized T-cell receptor (TCR)/HLA DR2 transgenic mice sharing this TCR with appropriateantigen presentation and T-cell activation in vivo followingpeptide administration. In vivo studies are ongoing both inTCR/HLA DR2 and HLA DR2 transgenic mice to determine thepathological relevance of these cross reactivities and theirrelationship to antigenic spreading in vivo. Together, thesestudies emphasize the highly degenerate antigen recogni-tion of autoreactive T-cells implicated in MS, and suggestantigen specific approaches may target cells with reactiv-ities beyond those originally considered.

doi:10.1016/j.clim.2006.04.237

Sa.6. An Estrogen Receptor A Ligand IsNeuroprotective in Experimental AutoimmuneEncephalomyelitis.Laurie Beth Morales, Kyi Kyi Loo, Hong-biao Liu,Cory Peterson, Seema Tiwari-Woodruff, Rhonda Voskuhl.Neurology, University of California Los Angeles, LosAngeles, CA.

Multiple Sclerosis is an inflammatory, neurodegenerativedisease for which experimental autoimmune encephalomy-elitis (EAE) is a model. Treatments with estrogens have beenshown to decrease the severity of EAE through anti-inflam-matory mechanisms. Here we investigated whether treat-ment with an estrogen receptor alpha (ERa) ligand is bothnecessary and sufficient to recapitulate the estrogen medi-ated protection in EAE. EAE was induced in wild type, ERa orERh deficient mice, each treated with the highly selectiveERa agonist, propyl pyrazole triol (PPT), to determine theeffect on clinical outcomes as well as inflammatory andneurodegenerative changes. ERa ligand treatment amelio-rated clinical disease in both wild type and ERh knock outmice, but not in ERa knock outmice. It also induced favorablechanges in autoantigen specific cytokine production in theperipheral immune system and decreased CNS white matterinflammation and demyelination. Interestingly, neuronalabnormalities accompanied by monocyte/microglial activa-tion were observed in gray matter of spinal cords of EAE miceat the earliest stage of clinical disease, and treatment witheither estradiol or the ERa ligand significantly reduced thisgray matter pathology. Treatment with this ERa ligand ishighly selective in vivo, mediating both anti-inflammatoryand neuroprotective effects in EAE. Supported by NIH grantNS45433 and NMSS grant RD3407, both to R.R.V.

doi:10.1016/j.clim.2006.04.238

Sa.7. Distinct Roles for PD-L1 and PD-L2 inRegulating Host Immunity in EAE.Loise Francisco, Keturah Brown, Mary Keir, Arlene Sharpe.Pathology, Brigham & Women’s Hospital, Harvard MedicalSchool, Boston, MA.

Experimental autoimmune encephalomyelitis (EAE) is ananimal model of Multiple Sclerosis (MS) dependent on auto-

reactive T-cells. Negative costimulatory molecules, such asthe programmed death-1 ligands, PD-L1 and PD-L2, areessential for the maintenance of peripheral tolerance andfor controlling the development and reactivity of disease-inducing self-reactive T-cells. PD-L1 is expressed on restingand activated T, B, myeloid and dendritic cells, as well asnonlymphoid tissues. In contrast, expression of PD-L2 islimited to professional antigen presenting cells (APCs). Wehave found that PD-L1 and PD-L2 have distinct roles inregulating the pathogenesis of EAE. To dissect their individualcontributions in modulating the immune response, wegenerated various PD-Ligand chimeric mice and comparedthese animals to wild-type (WT), PD-L1, PD-L2, and PD-L1/L2deficient mice for susceptibility to EAE. After immunizationwith myelin oligodendrocyte glycoprotein (MOG35-55), wefound that PD-Ligand�/� animals developed disease morerapidly than WT mice. PD-L1/L2�/�mice developed clinicaldisease sooner and more severe than WT, PD-L1�/� and PD-L2�/� mice. Studies of chimeric mice revealed a delay indisease onset in mice expressing only PD-L2 in the peripherybut lack both PD-Ligands in the central nervous system (CNS).Conversely, mice expressing neither of the PD-Ligands in theperiphery and only PD-L2 in the CNS had early disease onsetand more severe EAE than other chimeric and WT animals.Moreover, mice only expressing PD-L1 in the CNS and lack PD-Ligand expression in the periphery have delayed diseaseonset and less disease severity. These results suggest that PD-L2 expressed on APCs in peripheral lymph nodes may be thecritical to regulating disease induction and that PD-L1expression in the target organ is essential for host protectionduring the effector phase of EAE.

doi:10.1016/j.clim.2006.04.239

Sa.8. Comprehensive Phenotyping of MultipleSclerosis: Changes in Monocytes with Ifn-Beta-1a(AvonexRRRR) Treatment.Aaron Kantor,1 Jun Deng,1 Christopher Becker,1 Hua Lin,1

Emmanuelle Waubant,4 Joesph Lacy,3 Donald Bennett,2

Susan Goelz.2 1Biomarker Discovery Sciences, PPD, MenloPark, CA; 2Reasearch, Biogen Idec, Cambridge, MA;3Neurology, Palo Alto Medical Foundatin, Palo Alto, CA;4Neurology, UCSF MS Center, San Francisco, CA.

Purpose: This was a cross-sectional pharmacodynamicPhase IV molecular phenotyping study to identify potentialbiomarkers and better understand the therapeutic andimmunomodulatory effects of AvonexR in Multiple Sclerosis(MS) subjects. Methods: The study was multi-center, open-label, and prospective with cross-sectional and within grouppharmacodynamic comparisons. The cohorts were: 1) Healthy(n = 35), 2) Naı̈ve (n = 35)-MS patients with no previousinterferon therapy, 3) Stable (n = 35)-MS patients on AVONEXRtreatmentwhowere clinically stable, and 4) Breakthrough (n =16)-MS patients on AVONEXR treatment with a history ofclinical relapses or disease progression. Broad-based molecu-lar phenotyping was used to generate new hypotheses.Thousands of analytical variables were tracked with Surro-ScanTM laser scanning cytometry, immunoassays for specificsoluble proteins, and mass-spectrometry based differential

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proteomic and metabolomic profiling. Results: A strong withindrug group pharmacodynamic effect was observed 1.5 vs. 6days post weekly drug injection. Hundreds of differences wereobserved at a p-value b0.001. Most major cell populationsdecreased short-term after treatment. The striking exceptionis monocytes, which increased both in absolute counts (20—40%) and as a fraction of total leukocytes (50—75%). Changes inmultiple monocyte-associated molecules were also observedshort-term post injection, including: HLA class II, CCR5,CD11b, CD38, CD40, CD54, CD64, CD69, CD 86 and TLR2 andTLR4. Monocyte related soluble factors, such as MCP2 andinterferon-inducible gene products such as MxA and IP-10 alsoshowed short-term changes. Differences between cohortswere subtler and less common. MS and Healthy groupdifferences included B-cells, sCD14, and sVCAM. Stable andBreakthrough Avonex group differences included interferon-inducible gene products and sVCAM.

doi:10.1016/j.clim.2006.04.240

Sa.9. Examination of the B-Cell Repertoire in theCNS of Patients with MS.Kevin O’Connor,1 Shannon McArdel,2 Elizabeth Bradshaw,1

David Anderson,1 Nicole Moore,2 Moira Brady,2

David Hafler.1 1Neurology, Harvard Medical School, Boston,MA; 2Neurology, Brigham andWomen’s Hospital, Boston, MA.

B-cells isolated from the CNS lesions and CSF of patientswith MS are clonally expanded, show a restricted Ig genevariable (V) region expression repertoire and have accumulat-ed somatic mutations in these V region genes. These char-acteristics are, collectively, suggestive of a T-cell mediated,antigen-driven immune response. However, the degree towhich B-cell populations in the CSF represent those in MSlesions has not yet been described. Here we examined therepertoire of heavy and light Ig V region genes of B-cells fromthe peripheral blood (PB), CSF, and multiple white-matterlesions of a single MS patient in order to assess peripheralversus local activation and proliferation. Ig V region genesequences from individual CD19+, CD138+ or CD19+/CD138+cellswerealigned to theirmost homologous germline sequenceand analyzed for nucleotide mutations, CDR3 sequenceidentity, isotype and V region gene family. Examination of over150 sequences revealed that CSF and lesion populations displaydistinctive profiles of B-cells. CSF contained IgM and IgGisotypes at a nearly 1:1 ratio; although the average numberof mutations was higher in CSF than in peripheral blood,suggesting antigen experience, we did not detect any clonallyrelated or identical cells within CSF. Variable gene usageemulated that found inPB. Sequencesobserved in CSFwerenotobserved in lesions or peripheral blood. In contrast, sequencesfrom lesions contained exclusively IgG isotypes, identicalclones in multiple libraries as well as clonal variants. Variablegene usage in lesions was restricted. These results suggest thatthe B-cell response occurring within lesion tissue is primarilyself-contained, potentially due to germinal center formationproximal to individual CNS lesions.

doi:10.1016/j.clim.2006.04.241

Sa.10. Protective Effects of Testosterone Treatmentin Multiple Sclerosis.Stefan Gold,1 Barbara Giesser,1,2 Ann Drain,1

Vinitia Tandon,3 Ronald Swerdloff,3 Nancy Sicotte,1

Rhonda Voskuhl.1 1Neurology, UCLA, Los Angeles, CA;2Cousins Center, UCLA, Los Angeles, CA; 3Endocrinology,Habor-UCLA Medical Center, Torrance, CA.

Background: There is a distinct female preponderance ofautoimmune diseases including Multiple Sclerosis (MS),suggesting a role for sex hormones and/or chromosomes indisease susceptibility. In vitro and experimental data indicatethat testosterone may exert beneficial effects in inflamma-tory autoimmune disease by shifting immune responses froma Th1-like pattern towards Th2. Furthermore several lines ofevidence indicate that testosterone may have neuroprotec-tive properties. Indeed, testosterone treatment has beenfound to ameliorate animal models of MS and other Th1-likeautoimmune diseases. Methods: Ten male patients withrelapsing-remitting MS (RRMS) were treated with 100mg(transdermal) testosterone (T) daily in an open label studywith a six-month observational period (months �6 to 0)followed by twelve months of treatment with T (months 1—12). Clinical examinations and blood draws were conductedevery three months. We further obtained monthly brain MRIscans. Results: There was a significant improvement incognitive function on PASAT testing that was accompaniedby a slowing in annualized brain atrophy rate on MRI. Delayedtype hypersensitivity skin tests to the recall antigen tetanusshowed significantly decreased responses during treatmentas compared to pre-treatment. Supernatants from PBMCstimulated cultures showed a significant decrease in IL-2production, while brain-derived neurotrophic factor (BDNF)and platelet-derived growth factor (PDGF) production byPBMCs were each increased significantly during treatment ascompared to pretreatment. Conclusion: These data suggest aneuroprotective effect of T treatment in male patients withRRMS. This research was supported by grants from theNational Multiple Sclerosis Society (CA1028 and RG3239) toRRV.

doi:10.1016/j.clim.2006.04.242

Sa.11. The XX Sex Chromosome Complement, AsCompared to the XY, Confers Greater Susceptibilityto Experimental Autoimmune Encephalomyelitis.Deborah Smith,1 Sienmi Du,1 Arthur Arnold,2

Rhonda Voskuhl.1 1Neurology, UCLA, Los Angeles, CA;2Physiological Science, UCLA, Los Angeles, CA.

Multiple Sclerosis (MS) is one of numerous autoimmunediseases that affects females more frequently than males. Todetermine the contribution of sex chromosomes to thisgender difference in susceptibility, we used an animal modelof MS with a known female predominance, experimentalautoimmune encephalomyelitis (EAE) in SJLmice. Mice whichhave Sry, the gene that encodes for testicular development,deleted from the Y chromosome have been backcrossed ontothe SJL strain, resulting in EAE susceptible mice which differin their complement of sex chromosomes, while having the

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