Novel oxazolo-oxazole derivatives of FTY720 reduce endothelial cell permeability, immune cell chemotaxis and symptoms of experimental autoimmune encephalomyelitis in mice

The immunomodulatory FTY720 (fingolimod) is presently approved for the treatment of relapsing-remitting multiple sclerosis. It is a prodrug that acts by modulating sphingosine 1-phosphate (S1P) receptor signaling. In this study, we have developed and characterized two novel oxazolo-oxazole derivatives of FTY720, ST-968 and the oxy analog ST-1071, which require no preceding activating phosphorylation, and proved to be active in intact cells and triggered S1P1 and S1P3, but not S1P2, receptor internalization as a result of receptor activation. Functionally, ST-968 and ST-1071 acted similar to FTY720 to abrogate S1P-triggered chemotaxis of mouse splenocytes, mouse T cells and human U937 cells, and reduced TNFa- and LPS-stimulated endothelial cell permeability. The compounds also reduced TNFα-induced ICAM-1 and VCAM-1 mRNA expression, but restored TNFα-mediated downregulation of PECAM-1 mRNA expression. In an in vivo setting, the application of ST-968 or ST-1071 to mice resulted in a reduction of blood lymphocytes and significantly reduced the clinical symptoms of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice comparable to FTY720 either by prophylactic or therapeutic treatment. In parallel to the reduced clinical symptoms, infiltration of immune cells in the brain was strongly reduced, and in isolated tissues of brain and spinal cord, the mRNA and protein expressions of ICAM-1 and VCAM-1, as well as of matrix metalloproteinase-9 were reduced by all compounds, whereas PECAM-1 and tissue inhibitor of metalloproteinase TIMP-1 were upregulated. In summary, the data suggest that these novel butterfly derivatives of FTY720 could have considerable implication for future therapies of multiple sclerosis and other autoimmune diseases.

Investigation of leptomeningeal enhancement in MS: A postcontrast FLAIR MRI study

OBJECTIVE To investigate possible leptomeningeal contrast enhancement using postcontrast fluid-attenuated inversion recovery (FLAIR) MRI as an additional marker of inflammation in patients with multiple sclerosis (MS). METHODS A cohort of 112 patients (73 women) with clinically definitive MS or a clinically isolated syndrome suggestive of CNS demyelination were included. A pathologic control group of 5 stroke patients was also examined. MRI was performed on a 3T system including FLAIR, T2-weighted, T1-weighted-contrast injection, followed by T1-weighted and FLAIR. RESULTS Of the 112 patients, 39 had an acute relapse at the time of MRI. In total, 96 contrast-enhancing lesions were identified on postcontrast T1-weighted images. The pathologic control group demonstrated the sensitivity of postcontrast FLAIR images demonstrating leptomeningeal enhancement in all cases. In contrast, only 1 out of 112 examined patients with MS showed a single area of abnormal leptomeningeal contrast enhancement. CONCLUSIONS In contrast to intraparenchymal blood-brain barrier (BBB) dysfunction that is frequently seen in patients with MS, BBB dysfunction of leptomeningeal vessels is usually not detectable in patients with early MS.

Cell surface levels of endothelial ICAM-1 influence the transcellular or paracellular T-cell diapedesis across the blood-brain barrier

The extravasation of CD4(+) effector/memory T cells (TEM cells) across the blood-brain barrier (BBB) is a crucial step in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) or multiple sclerosis (MS). Endothelial ICAM-1 and ICAM-2 are essential for CD4(+) TEM cell crawling on the BBB prior to diapedesis. Here, we investigated the influence of cell surface levels of endothelial ICAM-1 in determining the cellular route of CD4(+) TEM -cell diapedesis across cytokine treated primary mouse BBB endothelial cells under physiological flow. Inflammatory conditions, inducing high levels of endothelial ICAM-1, promoted rapid initiation of transcellular diapedesis of CD4(+) T cells across the BBB, while intermediate levels of endothelial ICAM-1 favored paracellular CD4(+) T-cell diapedesis. Importantly, the route of T-cell diapedesis across the BBB was independent of loss of BBB barrier properties. Unexpectedly, a low number of CD4(+) TEM cells was found to cross the inflamed BBB in the absence of endothelial ICAM-1 and ICAM-2 via an obviously alternatively regulated transcellular pathway. In vivo, this translated to the development of ameliorated EAE in ICAM-1(null) //ICAM-2(-/-) C57BL/6J mice. Taken together, our study demonstrates that cell surface levels of endothelial ICAM-1 rather than the inflammatory stimulus or BBB integrity influence the pathway of T-cell diapedesis across the BBB.

Neutrophils mediate blood-spinal cord barrier disruption in demyelinating neuroinflammatory diseases

Disruption of the blood-brain and blood-spinal cord barriers (BBB and BSCB, respectively) and immune cell infiltration are early pathophysiological hallmarks of multiple sclerosis (MS), its animal model experimental autoimmune encephalomyelitis (EAE), and neuromyelitis optica (NMO). However, their contribution to disease initiation and development remains unclear. In this study, we induced EAE in lys-eGFP-ki mice and performed single, nonterminal intravital imaging to investigate BSCB permeability simultaneously with the kinetics of GFP(+) myeloid cell infiltration. We observed a loss in BSCB integrity within a day of disease onset, which paralleled the infiltration of GFP(+) cells into the CNS and lasted for ∼4 d. Neutrophils accounted for a significant proportion of the circulating and CNS-infiltrating myeloid cells during the preclinical phase of EAE, and their depletion delayed the onset and reduced the severity of EAE while maintaining BSCB integrity. We also show that neutrophils collected from the blood or bone marrow of EAE mice transmigrate more efficiently than do neutrophils of naive animals in a BBB cell culture model. Moreover, using intravital videomicroscopy, we demonstrate that the IL-1R type 1 governs the firm adhesion of neutrophils to the inflamed spinal cord vasculature. Finally, immunostaining of postmortem CNS material obtained from an acutely ill multiple sclerosis patient and two neuromyelitis optica patients revealed instances of infiltrated neutrophils associated with regions of BBB or BSCB leakage. Taken together, our data provide evidence that neutrophils are involved in the initial events that take place during EAE and that they are intimately linked with the status of the BBB/BSCB.

Novel insights into the development and maintenance of the blood-brain barrier

The blood-brain barrier (BBB) is essential for maintaining homeostasis within the central nervous system (CNS) and is a prerequisite for proper neuronal function. The BBB is localized to microvascular endothelial cells that strictly control the passage of metabolites into and out of the CNS. Complex and continuous tight junctions and lack of fenestrae combined with low pinocytotic activity make the BBB endothelium a tight barrier for water soluble moleucles. In combination with its expression of specific enzymes and transport molecules, the BBB endothelium is unique and distinguishable from all other endothelial cells in the body. During embryonic development, the CNS is vascularized by angiogenic sprouting from vascular networks originating outside of the CNS in a precise spatio-temporal manner. The particular barrier characteristics of BBB endothelial cells are induced during CNS angiogenesis by cross-talk with cellular and acellular elements within the developing CNS. In this review, we summarize the currently known cellular and molecular mechanisms mediating brain angiogenesis and introduce more recently discovered CNS-specific pathways (Wnt/β-catenin, Norrin/Frizzled4 and hedgehog) and molecules (GPR124) that are crucial in BBB differentiation and maturation. Finally, based on observations that BBB dysfunction is associated with many human diseases such as multiple sclerosis, stroke and brain tumors, we discuss recent insights into the molecular mechanisms involved in maintaining barrier characteristics in the mature BBB endothelium.

PreImplantation factor promotes neuroprotection by targeting microRNA let-7

Dysfunction and loss of neurons are the major characteristics of CNS disorders that include stroke, multiple sclerosis, and Alzheimer's disease. Activation of the Toll-like receptor 7 by extracellular microRNA let-7, a highly expressed microRNA in the CNS, induces neuronal cell death. Let-7 released from injured neurons and immune cells acts on neighboring cells, exacerbating CNS damage. Here we show that a synthetic peptide analogous to the mammalian PreImplantation factor (PIF) secreted by developing embryos and which is present in the maternal circulation during pregnancy inhibits the biogenesis of let-7 in both neuronal and immune cells of the mouse. The synthetic peptide, sPIF, destabilizes KH-type splicing regulatory protein (KSRP), a key microRNA-processing protein, in a Toll-like receptor 4 (TLR4)-dependent manner, leading to decreased production of let-7. Furthermore, s.c. administration of sPIF into neonatal rats following hypoxic-ischemic brain injury robustly rescued cortical volume and number of neurons and decreased the detrimental glial response, as is consistent with diminished levels of KSRP and let-7 in sPIF-treated brains. Our results reveal a previously unexpected mechanism of action of PIF and underscore the potential clinical utility of sPIF in treating hypoxic-ischemic brain damage. The newly identified PIF/TLR4/KSRP/let-7 regulatory axis also may operate during embryo implantation and development.

Recurrent Nicolau syndrome associated with subcutaneous glatiramer acetate injection--a case report.

BACKGROUND Glatiramer acetate is worldwide used as first line treatment in relapsing remitting multiple sclerosis. Local skin reactions associated with glatiramer acetate are common, however, only isolated cases of severe local injection site reactions known as Nicolau Syndrome have been reported so far. CASE PRESENTATION We describe the case of a recurrent Nicolau Syndrome occurred during longstanding glatiramer acetate treatment in a woman with multiple sclerosis. The haemorrhagic patch necrotized and was treated locally as a deep second degree burn with excision of dead skin tissue and was healed. Treatment with glatiramer acetate was definitely suspended. CONCLUSIONS GA injections can be complicated by isolated or recurrent Nicolau Syndrome, a potentially life-threatening condition of which neurologists should be aware.

Subcellular distribution of FTY720 and FTY720-phosphate in immune cells - another aspect of Fingolimod action relevant for therapeutic application

FTY720 (Fingolimod; Gilenya®) is an immune-modulatory prodrug which, after intracellular phosphorylation by sphingosine kinase 2 (SphK2) and export, mimics effects of the endogenous lipid mediator sphingosine-1-phosphate. Fingolimod has been introduced to treat relapsing-remitting multiple sclerosis. However, little has been published about the immune cell membrane penetration and subcellular distribution of FTY720 and FTY720-P. Thus, we applied a newly established LC-MS/MS method to analyze the subcellular distribution of FTY720 and FTY720-P in subcellular compartments of spleen cells of wild type, SphK1- and SphK2-deficient mice. These studies demonstrated that, when normalized to the original cell volume and calculated on molar basis, FTY720 and FTY720-P dramatically accumulated several hundredfold within immune cells reaching micromolar concentrations. The amount and distribution of FTY720 was differentially affected by SphK1- and SphK2-deficiency. On the background of recently described relevant intracellular FTY720 effects in the nanomolar range and the prolonged application in multiple sclerosis, this data showing a substantial intracellular accumulation of FTY720, has to be considered for benefit/risk ratio estimates.

Sphingosine kinase 2 deficient mice exhibit reduced experimental autoimmune encephalomyelitis: Resistance to FTY720 but not ST-968 treatments

The immunomodulatory drug FTY720 is presently approved for the treatment of relapsing-remitting multiple sclerosis. It is a prodrug that requires activation by sphingosine kinase 2 (SK-2) to induce T cell homing to secondary lymphoid tissue. In this study, we have investigated the role of SK-2 in experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. We show that SK-2 deficiency reduced clinical symptoms of EAE. Furthermore, in SK-2-deficient mice, the protective effect of FTY720 on EAE was abolished, while the non-prodrug FTY720-derivative ST-968 was still fully active. Protection was paralleled by reduced numbers of T-lymphocytes in blood and a reduced blood-brain-barrier leakage. This correlated with reduced mRNA expression of ICAM-1, VCAM-1, but enhanced expression of PECAM-1. A similar regulation of permeability and of PECAM-1 was seen in primary cultures of isolated mouse brain vascular endothelial cells and in a human immortalized cell line upon SK-2 knockdown. In summary, these data demonstrated that deletion of SK-2 exerts a protective effect on the pathogenesis of EAE in C57BL/6 mice and that SK-2 is essential for the protective effect of FTY720 but not of ST-968. Thus, ST-968 is a promising novel immunomodulatory compound that may be a valuable alternative to FTY720 under conditions where SK-2 activity is limited.

Inflammation and Optic Nerve Regeneration

Neuroinflammation has long been studied for its connection to the development and progression of Multiple Sclerosis. In recent years, the field has expanded to look at the role of inflammatory processes in a wide range of neurological conditions and cognitive disorders including stroke, amyotrophic lateral sclerosis, and autism. Researchers have also started to note the beneficial impacts of neuroinflammation in certain diseases. Neuroinflammation: New Insights into Beneficial and Detrimental Functions provides a comprehensive view of both the detriments and benefits of neuroinflammation in human health. Neuroinflammation: New Insights into Beneficial and Detrimental Functions opens with two chapters that look at some fundamental aspects of neuroinflammation in humans and rodents. The remainder of the book is divided into two sections which examine both the detrimental and beneficial aspects of inflammation on the brain, spinal cord and peripheral nerves, on various disease states, and in normal aging. These sections provide a broad picture of the role neuroinflammation plays in the physiology and pathology of various neurological disorders. Providing cross-disciplinary coverage, Neuroinflammation: New Insights into Beneficial and Detrimental Functions will be an essential volume for neuroimmunologists, neurobiologists, neurologists, and others interested in the field.

Treatment of optic neuritis with erythropoietin (TONE): a randomised, double-blind, placebo-controlled trial-study protocol.

INTRODUCTION Optic neuritis leads to degeneration of retinal ganglion cells whose axons form the optic nerve. The standard treatment is a methylprednisolone pulse therapy. This treatment slightly shortens the time of recovery but does not prevent neurodegeneration and persistent visual impairment. In a phase II trial performed in preparation of this study, we have shown that erythropoietin protects global retinal nerve fibre layer thickness (RNFLT-G) in acute optic neuritis; however, the preparatory trial was not powered to show effects on visual function. METHODS AND ANALYSIS Treatment of Optic Neuritis with Erythropoietin (TONE) is a national, randomised, double-blind, placebo-controlled, multicentre trial with two parallel arms. The primary objective is to determine the efficacy of erythropoietin compared to placebo given add-on to methylprednisolone as assessed by measurements of RNFLT-G and low-contrast visual acuity in the affected eye 6 months after randomisation. Inclusion criteria are a first episode of optic neuritis with decreased visual acuity to ≤0.5 (decimal system) and an onset of symptoms within 10 days prior to inclusion. The most important exclusion criteria are history of optic neuritis or multiple sclerosis or any ocular disease (affected or non-affected eye), significant hyperopia, myopia or astigmatism, elevated blood pressure, thrombotic events or malignancy. After randomisation, patients either receive 33 000 international units human recombinant erythropoietin intravenously for 3 consecutive days or placebo (0.9% saline) administered intravenously. With an estimated power of 80%, the calculated sample size is 100 patients. The trial started in September 2014 with a planned recruitment period of 30 months. ETHICS AND DISSEMINATION TONE has been approved by the Central Ethics Commission in Freiburg (194/14) and the German Federal Institute for Drugs and Medical Devices (61-3910-4039831). It complies with the Declaration of Helsinki, local laws and ICH-GCP. TRIAL REGISTRATION NUMBER NCT01962571.

Comparison of Routine Brain Imaging at 3 T and 7 T.

OBJECTIVE The aim of this study was to compare quantitative and semiquantitative parameters (signal-to-noise ratio [SNR], contrast-to-noise ratio [CNR], image quality, diagnostic confidence) from a standard brain magnetic resonance imaging examination encompassing common neurological disorders such as demyelinating disease, gliomas, cerebrovascular disease, and epilepsy, with comparable sequence protocols and acquisition times at 3 T and at 7 T. MATERIALS AND METHODS Ten healthy volunteers and 4 subgroups of 40 patients in total underwent comparable magnetic resonance protocols with standard diffusion-weighted imaging, 2D and 3D turbo spin echo, 2D and 3D gradient echo and susceptibility-weighted imaging of the brain (10 sequences) at 3 T and 7 T. The subgroups comprised patients with either lesional (n = 5) or nonlesional (n = 4) epilepsy, intracerebral tumors (n = 11), demyelinating disease (n = 11) (relapsing-remitting multiple sclerosis [MS, n = 9], secondary progressive MS [n = 1], demyelinating disease not further specified [n = 1]), or chronic cerebrovascular disorders [n = 9]). For quantitative analysis, SNR and CNR were determined. For a semiquantitative assessment of the diagnostic confidence, a 10-point scale diagnostic confidence score (DCS) was applied. Two experienced radiologists with additional qualification in neuroradiology independently assessed, blinded to the field strength, 3 pathology-specific imaging criteria in each of the 4 disease groups and rated their diagnostic confidence. The overall image quality was semiquantitatively assessed using a 4-point scale taking into account whether diagnostic decision making was hampered by artifacts or not. RESULTS Without correction for spatial resolution, SNR was higher at 3 T except in the T2 SPACE 3D, DWI single shot, and DIR SPACE 3D sequences. The SNR corrected by the ratio of 3 T/7 T voxel sizes was higher at 7 T than at 3 T in 10 of 11 sequences (all except for T1 MP2RAGE 3D).In CNR, there was a wide variation between sequences and patient cohorts, but average CNR values were broadly similar at 3 T and 7 T.DCS values for all 4 pathologic entities were higher at 7 T than at 3 T. The DCS was significantly higher at 7 T for diagnosis and exclusion of cortical lesions in vascular disease. A tendency to higher DCS at 7 T for cortical lesions in MS was observed, and for the depiction of a central vein and iron deposits within MS lesions. Despite motion artifacts, DCS values were higher at 7 T for the diagnosis and exclusion of hippocampal sclerosis in mesial temporal lobe epilepsy (improved detection of the hippocampal subunits). Interrater agreement was 69.7% at 3 T and 93.3% at 7 T. There was no significant difference in the overall image quality score between 3 T and 7 T taking into account whether diagnostic decision making was hampered by artifacts or not. CONCLUSIONS Ultra-high-field magnetic resonance imaging at 7 T compared with 3 T yielded an improved diagnostic confidence in the most frequently encountered neurologic disorders. Higher spatial resolution and contrast were identified as the main contributory factors.

The genetic basis of thoracic aortic aneurysms and dissections: Genetic heterogeneity and mapping of TAAD1 and TAAD2 loci

Thoracic aortic aneurysms leading to aortic dissections (TAAD) are a major cause of morbidity and mortality in the United States. TAAD is a complication of some known genetic disorders, such as Marfan syndrome and Turner syndrome, but the majority of familial cases are not due to a known genetic syndrome. Previous studies by our group have established that nonsyndromic, familial TAAD is inherited in an autosomal dominant manner with decreased penetrance and variable expression. Using one large family with multiple members with TAAD for the genome wide scan, a major locus for familial TAAD was mapped to 5q13–14 (TAAD1). Nine out of 15 families studied were linked to this locus, establishing that TAAD1 was a major locus, and that there was genetic heterogeneity for the condition. Mapping of TAAD2 locus was accomplished using a single large family with multiple members with TAAD not linked to known loci of aneurysm formation. This established a second novel locus for familial TAAD on 3p24–25 (LOD score of 4.3), termed the TAAD2 locus. Two putative loci with suggestive LOD scores were mapped on 4q and 12q through a genome scan carried out using three families. TAAD phenotype in 12 families did not segregate with known loci, indicating further genetic heterogeneity. An STS-tagged BAC based contig was constructed for 7.8Mb and 25Mb critical interval of TAAD1 and TAAD2 respectively and characterized to identify the defective gene. The hypothesis that the defective genes responsible for the TAAD1 and TAAD2 encoded extracellular matrix (ECM) proteins, the major components of the elastic fiber system in the aortic media was tested. Four genes encoding ECM proteins, versican, thrombospondin-3, CRTL1, on TAAD1 and FBLN2 at TAAD2 were sequenced, but no disease-causing mutations were identified. Studies to identify the defective gene are initiated through the positional candidate gene approach using combination of bioinformatics and expression studies. The identification of the TAAD susceptibility genes will allow for presymptomatic diagnosis of individuals at risk for this life threatening disease. The identification of the molecular defects that contribute to TAAD will also further our understanding of the proteins that provide structural integrity to the aortic wall. ^

In-vivo diffusion tensor imaging of rat spinal cord with a phased array coil at 7T

Magnetic resonance imaging (MRI) is a non-invasive technique that offers excellent soft tissue contrast for characterizing soft tissue pathologies. Diffusion tensor imaging (DTI) is an MRI technique that has shown to have the sensitivity to detect subtle pathology that is not evident on conventional MRI. ^ Rats are commonly used as animal models in characterizing the spinal cord pathologies including spinal cord injury (SCI), cancer, multiple sclerosis, etc. These pathologies could affect both thoracic and cervical regions and complete characterization of these pathologies using MRI requires DTI characterization in both the thoracic and cervical regions. Prior to the application of DTI for investigating the pathologic changes in the spinal cord, it is essential to establish DTI metrics in normal animals. ^ To date, in-vivo DTI studies of rat spinal cord have used implantable coils for high signal-to-noise ratio (SNR) and spin-echo pulse sequences for reduced geometric distortions. Implantable coils have several disadvantages including: (1) the invasive nature of implantation, (2) loss of SNR due to frequency shift with time in the longitudinal studies, and (3) difficulty in imaging the cervical region. While echo planar imaging (EPI) offers much shorter acquisition times compared to spin-echo imaging, EPI is very sensitive to static magnetic field inhomogeneities and the existing shimming techniques implemented on the MRI scanner do not perform well on spinal cord because of its geometry. ^ In this work, an integrated approach has been implemented for in-vivo DTI characterization of rat spinal cord in the thoracic and cervical regions. A three element phased array coil was developed for improved SNR and extended spatial coverage. A field-map shimming technique was developed for minimizing the geometric distortions in EPI images. Using these techniques, EPI based DWI images were acquired with optimized diffusion encoding scheme from 6 normal rats and the DTI-derived metrics were quantified. ^ The phantom studies indicated higher SNR and smaller bias in the estimated DTI metrics than the previous studies in the cervical region. In-vivo results indicated no statistical difference in the DTI characteristics of either gray matter or white matter between the thoracic and cervical regions. ^

Characterization of anti -sulfatide autoantibodies in demyelinating diseases

Multiple sclerosis (MS) is the most common autoimmune disease of the central nerve system and Guillain Barré Syndrome (GBS) is an inflammatory neuropathy involving the peripheral nerves. Anti-myelin immunoglobins may play a role in the demyelination processes of the both diseases. Sulfatide is an abundant glycolipid on myelin and is a candidate target antigen for disease related autoantibodies. The objective of this study was to characterize anti-sulfatide antibodies and compare antibodies from GBS and MS patients with fetal antibodies. Our hypothesis is that some B cells producing disease-associated autoantibodies are derived from or related to B cells of the fetal repertoire. Here we report that reactivity of plasma IgM against sulfatide was elevated in twelve MS patients compared with twelve normal subjects. This result implies that anti-sulfatide antibodies are disease-related. A total of sixteen human B lymphocyte clones producing anti-sulfatide autoantibodies were isolated from MS patients, GBS patients and a human fetus. Seven of the clones were from three MS patients, four of the clones were from three GBS patients and five were from the spleen of a twenty-week human fetus. Sequences have been obtained for the heavy and light chain variable regions (VDJ and VJ regions) of all of the anti-sulfatide immunoglobulins. Seven of the sixteen antibodies used VH3 for the variable region gene of the heavy chain consistent with the rate of VH3 usage in randomly selected B cells. Somatic mutations were significantly more frequent in the patient antibodies than in the fetus and somatic mutations in CDR's (Complementarity Determining Region) were significantly more frequent than in framework regions. No significant difference was found between patients and fetus in length of VH CDRIII. However, it is reported that antibodies from randomly selected normal adult B cells have longer CDRIII lengths than those of the fetus (Sanz I, 1991 Journal of Immunology Sep 1;147(5):1720-9). Our results are consistent with derivation of the precursors of B cells producing these autoantibodies from B cells related to those of the fetal repertoire. These findings are consistent with a model in which quiescent B cells from clones produced early in development undergo proliferation in dysregulated disease states, accumulating somatic mutations and increasing in reactivity toward self-antigens. ^ Epitope mapping and molecular modeling were done to elucidate the relationships between antibody structure and binding characteristics. The autoantibodies were tested for binding activity to three different antigens: sulfatide, galactoceramide and ceramide. Molecular modeling suggests that antibodies with positive charge surrounded by or adjacent to hydrophobic groups in the binding pocket bind to the head of sulfatide via the sulfate group through electrostatic interactions. However, the antibodies with hydrophobic groups separated from positive charges appear to bind to the hydrophobic tail of sulfatide. This observation was supported by a study of the effect of NaCl concentration on antigen binding. The result suggested that electrostatic interactions played a major role in sulfate group binding and that hydrophobic interactions were of greater importance for binding to the ceramide group. Our three-dimensional structure data indicated that epitope specificity of these antibodies is more predictable at the level of tertiary than primary structure and suggested positive selection based on structure occurred in the. formation of those autoantibodies. ^