Single-nucleotide polymorphisms in HLA- and non-HLA genes associated with the development of antibodies to interferon-ß therapy in multiple sclerosis patients.

Interferons-ß (IFN-ß) are the most widely used immunomodulatory drugs for treatment of multiple sclerosis (MS). The development of neutralizing antibodies (NABs) against IFN-ß is one of the main reasons for treatment failure. While formulation of the drug has a proven impact on the development of NABs, the genetic predisposition to develop antibodies is poorly understood. We performed genome-wide single-nucleotide polymorphism (SNP) genotyping in 362 MS patients of whom 178 had developed and 184 had not developed antibodies on IFN-ß therapy. Four candidate SNPs were validated in an independent cohort of 350 antibody-positive and 468 antibody-negative MS patients. One SNP within the human leucocyte antigen (HLA) region (rs9272105, P-value: 3.56 × 10⁻¹°) and one SNP in an intergenic region on chromosome 8q24.3 (rs4961252, P-value: 2.92 × 10⁻8 showed a genome-wide significant association with the anti-IFN-ß antibody titers. We found no interaction between the genome-wide significant SNPs (rs9272105 and rs4961252) in our study and the previously described HLA-DR*0401 or *0408 alleles, indicating an additive effect of SNPs and HLA alleles. Testing for these SNPs and the HLA-DR*0401 or *0408 alleles allows to identify patients at risk to develop antibodies to IFN-ß and may provide helpful information for individual treatment decisions.

More CLEC16A gene variants associated with multiple sclerosis.

OBJECTIVES: Recently, associations of several single-nucleotide polymorphisms (SNPs) within the CLEC16A gene with multiple sclerosis (MS), type-I diabetes, and primary adrenal insufficiency were reported. METHODS: We performed linkage disequilibrium (LD) fine mapping with 31 SNPs from this gene, searching for the region of highest association with MS in a German sample consisting of 603 patients and 825 controls. RESULTS: Four SNPs located in intron 19 of the CLEC16A gene were found associated. We could replicate the finding for SNP rs725613 and were able to show for the first time the association of rs2041670, rs2080272 and rs998592 with MS. CONCLUSION: All described base polymorphisms are mapping to one LD block of approximately 50 kb within intron 19 of the CLEC16A gene, suggesting a pivotal role of this region for susceptibility of MS and possibly also for other autoimmune diseases.

CCL19 is constitutively expressed in the CNS, up-regulated in neuroinflammation, active and also inactive multiple sclerosis lesions.

CCL19 and CCL21 bind to CCR7, which is crucial for both inducing an immune response and establishing immunological tolerance. We report that in the normal human brain CCL19, but not CCL21, is transcribed, and detectable as a protein in tissue lysates and in cerebrospinal fluid. In both active and inactive multiple sclerosis (MS) lesions CCL19 transcripts were elevated. In cerebrospinal fluid from MS and OIND patients CCL19 protein was increased. In relapsing-remitting and secondary progressive MS patients CCL19 correlated with intrathecal IgG production. This study suggests that CCL19 plays a role in both the physiological immunosurveillance of the healthy CNS and the pathological maintenance of immune cells in the CNS of MS patients.

New approaches to dissect degeneracy and specificity in T cell antigen recognition.

The acquired immune system is a complex and very effective defense against invading pathogens such as bacteria and viruses. T cells are central to the acquired immune system by controlling B and T cell activation and induction of T cell effector functions. The key event for T cell activation is the recognition of a specific antigen by the T cell receptor. During the past decade antigen recognition of T cells has been investigated intensively leading to new insights into the molecular mechanisms of T cell activation. In addition to the resolution of the molecular structure of the trimolecular complex (T cell receptor, peptide, major histocompatibility complex) functional studies have demonstrated the flexibility of the T cell receptor interaction with its ligand. These observations have had strong implications for the understanding of T cell selection, maturation, and repertoire maintenance. In addition, the flexibility of the T cell receptor has provided the basis for novel methods to dissect antigen recognition and define the repertoire of ligands for a given receptor. Here, we summarize recent progress on T cell recognition and method innovations with respect to future studies in autoimmune diseases.

NS-257, a novel competitive AMPA receptor antagonist, interacts with kainate and NMDA receptors.

In this study, we examined the effects of a novel, water-soluble, putative competitive AMPA receptor antagonist, 1,2,3,6,7, 8-hexahydro-3-(hydroxyimino)-N,N,7-trimethyl-2-oxobenzo[2,1- b:3, 4-c']dipyrrole-5-sulfonamide (NS-257) on AMPA, kainate and NMDA receptors using the two-electrode voltage-clamp technique in Xenopus oocytes. All glutamate receptor subtypes were inhibited by NS-257 in a voltage-independent way. When kainate was applied to oocytes injected with total mouse brain mRNA, mainly AMPA receptors were activated. The antagonistic effects of NS-257 on these kainate-induced currents were concentration-dependent and competitive. In the same way, NS-257 blocked kainate-induced currents recorded from oocytes expressing homomeric GluR-1 receptors. In our experiments higher concentrations (>1 microM) of NS-257 also produced inhibitory effects on kainate and to a lesser extent on NMDA receptor function as indicated by recordings from GluR-6 or NR-1b/2A cRNA injected oocytes. While NMDA receptor function was inhibited in a competitive fashion, kainate responses recorded from homomeric GluR-6 receptors were blocked in a mixed competitive-noncompetitive manner. This mixed antagonistic action of NS-257 might have been caused by preincubating oocytes with concanavalin A, which blocks desensitization of kainate receptors. Although NS-257 appeared to be a less potent AMPA receptor antagonist then other known antagonists like NBQX, its main advantage over all other reported compounds so far is its higher aqueous solubility which still represents the major weakness of the other AMPA receptor antagonists, especially for clinical use.

[New approaches in research of therapy of multiple sclerosis]. / Neue Forschungsansätze zur Therapie der multiplen Sklerose.

BACKGROUND: Multiple sclerosis is a chronic inflammatory demyelinating disease of the central nervous system. With a prevalence of 0.1-0.15% in Germany multiple sclerosis is the most common cause of severe disability in young adults. PATHOGENESIS: Epidemiological and family studies demonstrate the role of environmental and genetic factors in the pathogenesis of multiple sclerosis. Based on those observations and findings in experimental animal models, it is believed that multiple sclerosis is caused by an autoimmune process. However, target antigens and mechanisms leading to tissue destruction are largely unknown. THERAPY: Since the efficacy of current immunomodulatory and immunosuppressive therapies (beta-interferons, glatiramer acetate, mitoxantrone) is limited, it is necessary to develop new strategies for the treatment of multiple sclerosis. To reach this goal, a much better understanding of disease pathogenesis is necessary which takes into account the clinical, paraclincial and histopathological heterogeneity of the disease. CONCLUSION: Only further intensive research activity on basic mechanisms of disease pathogenesis and a consequent development of resulting therapeutic strategies--from animal models to phase III studies--will result in significant improvement of the long-term course of multiple sclerosis.

Differential effects of fingolimod (FTY720) on immune cells in the CSF and blood of patients with MS.

BACKGROUND: The oral immunomodulator fingolimod (FTY720) has recently been shown to be highly effective in relapsing-remitting multiple sclerosis (MS). Fingolimod is a functional antagonist of the sphingosine-1-phosphate receptor 1 and thereby inhibits sphingosine-1-phosphate-dependent lymphocyte egress from secondary lymphoid tissues, resulting in a pronounced lymphopenia in the peripheral blood. The effects of fingolimod treatment on the CSF of patients with MS have not been studied so far. METHODS: We analyzed the leukocyte count, albumin quotient, immunoglobulin G (IgG) index, and oligoclonal bands in the CSF of fingolimod-treated patients with MS. Moreover, we performed immunophenotyping of CSF and peripheral blood leukocytes by flow cytometry. The results were compared to those from treatment-naive or natalizumab-treated patients with MS and patients with other inflammatory and noninflammatory neurologic diseases. RESULTS: Fingolimod therapy significantly decreased CSF leukocyte counts, but had little impact on the extent of intrathecal IgG synthesis and presence of oligoclonal bands in the CSF. Fingolimod decreased the proportion of CSF CD4+ T cells but to a lesser extent than in the peripheral blood. While fingolimod strongly reduced B cells in the periphery, it had little impact on B cells in the CSF. The percentage of CSF CD8+ T cells, NK cells, and monocytes increased compared to treatment-naive patients. The CD4+/CD8+ T-cell ratio in CSF reversed in most of the patients. CONCLUSION: Fingolimod treatment has a profound impact on CSF, which to some extent differs from the peripheral effects of the drug.

Antibody responses to EBV and native MOG in pediatric inflammatory demyelinating CNS diseases.

BACKGROUND: Epstein-Barr virus (EBV) has been discussed as a possible causative agent in inflammatory demyelinating diseases of the CNS. Cross-reactivity between EBV and myelin proteins has been proposed as a potential mechanism by which EBV could elicit an autoimmune response targeting the CNS. Recently, high antibody titers to native myelin oligodendrocyte glycoprotein (nMOG) were found in children affected by the first inflammatory demyelinating event. The relation between antibody responses to EBV and nMOG has not been addressed in children so far. METHODS: We investigated the occurrence of antibodies to nMOG, EBV nuclear antigen 1 (EBNA-1), and early antigen (EA) in a case-control study including children with acute disseminated encephalomyelitis (ADEM, n = 19), children with clinically isolated syndrome (CIS, n = 25), children with other neurologic diseases (n = 28), and healthy children (n = 30). Immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies against the extracellular part of nMOG were assessed by a cell-based assay, and EBV-specific IgG antibodies to EBNA-1 and IgM antibodies to EA were assessed by ELISA. RESULTS: Serum IgG antibodies to EBNA-1 were present in 43% of controls (25/58), 42% of children with ADEM (8/19), and 64% of children with CIS (16/25), whereas IgM antibodies to EA were detected in only 16% of children with ADEM (3/19). High antibody titers to nMOG were only found in children with ADEM and CIS but were not related to the seropositivity to EBV. Moreover, in EBV-seropositive children, we did not observe any correlation between anti-EBNA-1 and anti-nMOG IgG antibody titers. CONCLUSION: High serum immunoglobulin G titers to native myelin oligodendrocyte glycoprotein are found in a significant number of children affected by clinically isolated syndrome or acute disseminated encephalomyelitis. These antibodies are not related to the antibody response to Epstein-Barr virus.

Immunologic, clinical, and radiologic status 14 months after cessation of natalizumab therapy.

OBJECTIVE: Natalizumab is a humanized recombinant monoclonal antibody against very late activation antigen-4 approved for the treatment of patients with multiple sclerosis (MS). A phase II study failed to demonstrate a difference between natalizumab treatment groups and the placebo group with regard to gadolinium enhancing lesions on MRI 3 months after discontinuation of therapy. The objective of this study was to assess clinical MS disease activity, surrogate disease markers on MRI, immunologic parameters in peripheral blood and CSF, as well as safety in patients with MS after discontinuation of natalizumab therapy. METHODS: This study is a longitudinal and serial cross-sectional assessment, in which 23 patients who were treated with natalizumab in the context of two phase III clinical trials were originally enrolled. A subgroup of patients was followed over 14 months. The annual relapse rate, neurologic disease progression assessed by the Expanded Disability Status Scale, disease surrogate markers on MRI, cellular and humoral immune markers in peripheral blood and CSF, and adverse events of the drug were monitored. RESULTS: With regard to clinical disease activity, neuroimaging, and immune responses, the majority of patients in our cohort were stable. Decreased lymphocyte cell numbers and altered cell ratios returned to normal 14 months after cessation of natalizumab. No infectious complications were observed. CONCLUSION: This is the first long-term follow-up of patients who discontinued natalizumab. We did not observe a clinical, radiographic, or immunologic rebound phenomenon after discontinuation of natalizumab therapy.

Sex-specific usage patterns of sleeping sites in grey mouse lemurs (Microcebus murinus) in northwestern Madagascar.

Sleeping sites are a potentially important resource for grey mouse lemurs since they are confronted with high daily temperature fluctuations and a high predation pressure. In order to determine the existence and degree of resource competition, sleeping site characteristics, locations, and usage patterns as well as sleeping group compositions were investigated in a 3 month field study in a dry deciduous forest of northwestern Madagascar. The daily sleeping sites of females were on average better insulated and safer than those of males. Males used more sleeping sites and changed the site more often than females. During the whole study, males slept alone, whereas the females formed stable sleeping groups in on average 83.7% of the days. Sex-specific differences in usage patterns might be explained by intersexual resource competition and female dominance and could possibly be related to differential parental investment of the sexes. The underlying study indicates that sleeping sites may be a restricted and defendable resource for grey mouse lemurs. The investigation gives new insights into the distribution patterns and social organization of this species.

Digger wasp versus cricket: mechanisms underlying the total paralysis caused by the predator's venom.

The data presented here describe neurophysiological experiments addressing the question of cellular mechanisms underlying the total paralysis of locomotor behavior in crickets occurring after being stung by females of the digger wasp species Liris niger. The Liris venom effects have been studied by both in vivo recordings from identified neurons of the well-described giant fiber pathway and in vitro recordings from cultured neurons isolated from the terminal ganglion of crickets. The total paralysis of the prey is characterized by a general block of action potential generation as well as by a block of synaptic transmission. Intracellular recordings from neurons in intact ganglia under single electrode voltage-clamp conditions, as well as whole-cell patch-clamp recordings from cultured cricket neurons consistently show that the block of action potential generation by the Liris venom is due to a block of voltage-gated sodium inward currents in neurons of the stung ganglia. Furthermore, our data provide evidence that the Liris venom also blocks calcium currents in identified neurosecretory neurons. On the other hand, outward currents are not affected by the Liris venom. The in vitro recordings suggest that the Liris venom contains active venom components, which, at least for the observed block of inward currents, do not require a metabolic modification. Because venom application does not affect the ACh-induced EPSPs in giant interneurons, the Liris venom does not seem to influence the postsynaptic ACh receptors. The possible pre- and postsynaptic sites of venom action and the functional consequences on synaptic transmission within the giant fiber system are discussed.

Patterns of cerebrospinal fluid pathology correlate with disease progression in multiple sclerosis.

Multiple sclerosis is a chronic inflammatory and demyelinating disease of the CNS with, as yet, an unknown aetiology. Temporal profile, intensity and treatment responses are highly variable in multiple sclerosis suggesting pathogenetic heterogeneity. This hypothesis has been supported by histopathological studies disclosing at least four different subtypes of acute demyelinating lesions. Although stratification of multiple sclerosis patients into these categories would be extremely helpful for clinical studies, this approach is impractical as it requires brain biopsy. In this study we investigated CSF cytology from 60 multiple sclerosis patients by flow cytometry. We identified different patterns of CSF cytology, which were independent of immunological parameters in the peripheral blood. The most variable CSF parameter was the B cell to monocyte ratio, which remained stable during different phases of disease in selected patients. The ratio correlated with disease progression but not with disability or disease duration in a retrospective, consecutive analysis. A high ratio (predominance of B cells) was associated with more rapid disease progression, whereas a low ratio (predominance of monocytes) was found in patients with slower progression. Our study demonstrates the existence and potential clinical relevance of different CSF cytology patterns. We hypothesize that CSF cytology patterns may reflect the heterogeneity in the pathogenesis of multiple sclerosis.