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Factor H autoantibodies in neuromyelitis optica
Abstracts
556 Analysis of auto-antibodies in schizophrenia Carolin Hoffmanna, Matthew J. Lindemannb, Vincent C. Ramspergerb, Lakshmanan Sureshb, Jo Stevensa, Mario Losena, Peter Molenaara, Marc H. De Baetsa, Marc De Hertc, Jim Van Osa, Bart Bp Ruttena, Pilar Martinez-martineza a
Department of Psychiatry and Neuropsychology, Maastricht University, Maastricht, Netherlands; bIMMCO Diagnostics, Inc., New York, United States; cUniversity Psychiatric Centre Catholic University Leuven, Catholic University Leuven, Leuven, Belgium Objectives: Schizophrenia is a neuropsychiatric disorder with a complex etiology and pathogenesis. While symptomatic treatment is available, there are no treatments that target the biological disease mechanisms. Recently, autoantibodies to neuronal cell surface antigens have been identified in patients with schizophrenia. Autoimmunity is affecting nearly 10% of the general population and its clinical spectrum varies from endocrine, musculoskeletal to neuropsychiatric syndromes. Since autoimmune patients frequently suffer from more than one autoimmune disease, we analyzed common autoantibodies as disease markers (or risk factors) to test the hypothesis that schizophrenia is correlated to autoimmunity. Methods: We tested sera from 232 schizophrenia, bipolar disorder and myasthenia gravis patients and healthy controls for the presence of autoantibodies by ELISA and immunofluorescence. The assays included: anti-nuclear antibodies (ANA), double-stranded DNA (dsDNA), and ribosomal P protein (RPP). ANA IFA screening was performed on ImmunGlo HEp-2 slides and the acquisition of images and interpretation of patterns was performed on the Immco i-Sight automated imaging system. Results: Prevalence of anti-dsDNA autoantibodies was found to be overall relatively high and to significantly differ between disease and control groups. Schizoaffective patients had highest titers of anti-dsDNA autoantibodies. The autoantibody titer anti-RPP differs significantly between the groups. Paranoid and disorganized patients had highest titers of anti-RPP autoantibodies. No significant difference was detected in the presence of ANA autoantibodies in the different study groups. Conclusions: We confirmed that a subgroup of schizophrenia patients had increased levels of autoantibodies. These findings are the first step to improve diagnosis and possibly treatment of schizophrenic patients with autoimmune etiology. Currently, control sera are being tested to match the sample size and the age and sex distribution of the disease group.
101
Créteil, Créteil, France; cUniversity Psychiatric Centre Catholic University Leuven, Catholic University Leuven, Campus Kortenberg, Leuven, Belgium; d Department of Psychiatry, Erasmus University Medical Center, Rotterdam, Netherlands Introduction: The etiology of schizophrenia is multifactorial and highly diverse. Recent research suggests the involvement of autoimmunity in a subset of patients with schizophrenia and antibodies have been identified against neurotransmitter receptors. Up to date, only few reports have documented autoantibodies in psychiatric disease. In encephalitis, anti-N-methyl-D-aspartate glutamate receptor antibodies (NMDAr-AB) were shown to be causative for major psychotic symptoms of the disease. Antibodies directed against the same antigen were also found to be more common (6%– 10%) in patients with schizophrenia than in healthy controls, but subsequent studies were not consistent. Additional studies with larger sample size and relevant control groups are needed to determine whether NMDAr-AB are relevant to the etiology of schizophrenia. Objective: We aimed to identify the frequency of NMDAr-autoantibodies in schizophrenia and healthy controls. Methods: Our study compared the presence of NMDAr-AB of the immunoglobulin isotype G (IgG) in sera of 200 patients with schizophrenia and 200 healthy controls. Sera were screened using an established cell-based assay with antigen transfected HEK cells expressing the NMDAr subunit 1 alone or in combination with NMDAr subunit 2B. Sera identified as positive on cells were also tested for reactivity on rat brain tissue. Results: 3 patients were identified as anti-NMDAR-AB positive of which one also reacts on rat brain tissue. No control sera contained autoantibodies against the tested antigen. Conclusion: The results indicate that there might be a subgroup of patients with an underlying autoimmune reaction and warrant further research. Characterization of autoimmune schizophrenia is especially important because ultimately, the antibody positive patient group will be eligible for conventional treatment for antibody mediated diseases.
doi:10.1016/j.jneuroim.2014.08.271
304 Factor H autoantibodies in neuromyelitis optica Zsolt Illesa, Barbara Uzonyib, Mihaly Jozsib a
doi:10.1016/j.jneuroim.2014.08.270
Institute of Clinical Research and Department of Neurology, University of Southern Denmark, Odense, Denmark; bMTA-ELTE Immunology Research Group, Department of Immunology, Eötvös Loránd University, Budapest, Hungary
543 Analysis of pathogenic autoantibodies against the N-methyl-D-aspartate glutamate receptor in schizophrenia Carolin Hoffmanna, Gisela Nogales-gadeaa, Jo Stevensa, Mario Losena, Andrei Szokeb, Marion Leboyerb, Marc De Hertc, Nico J.M. Van Beverend, Peter Molenaara, Wim A. Buurmana, Marc H. De Baetsa, Bart B.F. Ruttena, Jim Van Osa, Pilar Martinez-Martineza a
Department of Psychiatry and Neuropsychology, Maastricht University, Maastricht, Netherlands; bPôle de Psychiatrie, Hôpital Henri Mondor-Albert Chenevier-Assistance Publique-Hôpitaux de Paris, Université Paris-Est
Neuromyelitis optica (NMO) is an inflammatory disease characterized by pathogenic complement-activating autoantibodies against aquaporin 4 (AQP4), the main water channel of the central nervous system. NMO is frequently associated with other autoantibodies and antibody-mediated diseases. Since autoantibodies against complement regulating factors may contribute to the activation of the complement system, here we examined the presence of antibodies against the complement regulator protein factor H in the serum of patients with NMO. The study cohort included 45 patients, who were all seropositive for AQP4 autoantibodies. Serum samples were screened by ELISA for autoantibodies using purified factor H as target antigen and human
102
Abstracts
serum albumin as control antigen. Specific IgG binding to factor H was detected in 4 samples (~9%), while no IgG binding to factor H was detected from 25 control sera. We could also detect factor H–IgG complexes using a monoclonal antibody to factor H as a catch antibody in ELISA. The binding sites of the factor H autoantibodies were mapped using recombinant constructs representing various factor H domains. All four autoantibodies bound to factor H domains 19–20, and also recognized the homologous protein CFHR1. This binding pattern was similar to factor H autoantibodies associated with atypical hemolytic uremic syndrome (HUS), used as positive controls in the assays. In conclusion, our results demonstrate that factor H autoantibodies are not uncommon in NMO. Our data also suggest that generation of antibodies against complement regulating factors among other autoantibodies may contribute to the complement-mediated damage.
ANA and anti-nucleosoma antibodies showed similar temporal pattern to AQP4 antibodies, and were also increased during NMO relapses. Abnormal elevation of both anti-nucleosoma and ANA was more common in anti-AQP4 seropositive samples. SLE was always active during NMO relapse, and the first clinical sign of NMO occurred in the active phase of SLE. Our data indicate that AQP4 antibodies in the sera are of complement-activating IgG1 subtypes years before the first attack of NMO, but during attacks of NMO, IgM antibodies also appear or are present. In our NMO patients with SLE, the very first relapse was associated with the activity of SLE and elevation of SLE-specific autoantibodies. This may indicate that (i) either a general B cell activation contributes to the manifestation of NMO, or (ii) in patients with SLE other autoantibodies may precipitate NMO relapses. doi:10.1016/j.jneuroim.2014.08.273
doi:10.1016/j.jneuroim.2014.08.272
306 Longitudinal serological and clinical data indicate that SLE activity may contribute to NMO relapses
435 Sulfatides autoreactivity in multiple sclerosis Maria Ivanovaa, Amir Tukhvatulinb, Alina Dzharullaevab, Denis Logunovb, Maria Zakharovaa a
Zsolt Illesa, Katalin T. Kovacsb, Sudhakar R. Kalluric, Tunde Csepanyd, Yvonne Schaeferc, Magdolna Simoe, Laszlo Rokuszf, Timea Berkig, Bernhard Hemmerc a
Institute of Clinical Research and Department of Neurology, University of Southern Denmark, Odense, Denmark; bDepartment of Rheumatology and Immunology, University of Pecs, Pecs, Hungary; cClinic of Neurology, Technische Universität München, Munich, Germany; dDepartment of Neurology, University of Debrecen, Debrecen, Hungary; eDepartment of Neurology, Semmelweis University, Budapest, Hungary; f1st Department of Internal Medicine, Military Hospital — State Health Centre, Budapest, Hungary; gDepartment of Immunology and Biotechnology, University of Pecs, Pecs, Hungary Neuromyelitis optica (NMO) mediated by autoantibodies against aquaporin 4 (AQP4) is frequently associated with other antibodymediated autoimmune diseases, but the relationship between serological and clinical activities of the associated diseases is not well established. In 6 patients with NMO associated with SLE, (i) we examined longitudinal changes of AQP4 antibodies of IgM and IgG classes and IgG subtypes, and related them to the clinical activity of NMO; (ii) we examined longitudinal changes of ANA, anti-nucleosoma and antidsDNA, and related them to the clinical activity of NMO; (iii) we investigated both NMO- and SLE-specific autoantibodies before and after the first clinical attack of NMO; and (iv) we compared the longitudinal relationship between anti-AQP4 and SLE-related autoantibodies. Total IgG, subtypes and IgM of AQP4 antibodies were measured by a cell-based flow cytometry assay in 3 samples obtained during NMO relapses and 17 samples collected during remissions; the human glioblastoma cell line LN18 was stably transduced using a lentiviral vector to over express human AQP4-M23. Anti-nucleosoma, ANA and anti-dsDNA were determined by ELISA. In 3 samples obtained 2–5 years before the first NMO relapse in 2 patients, anti-AQP4 antibodies were already present and had an IgG1 subtype. During NMO relapses, the titer of anti-IgG1 antibodies increased or was elevated, and became undetectable in some of the samples collected during remission. IgM antibodies also appeared or were present during relapses, and decreased thereafter. Anti-dsDNA,
Research Center of Neurology, Russian Academy of Medical Sciences, Moscow, Russian Federation; bGamaleya Research Institute of Epidemiology and Microbiology, Ministry of Health of the Russian Federation, Moscow, Russian Federation Objective: Sulfatides are among the most abundant myelin lipids. Multiple sclerosis (MS) is characterized by CNS inflammation and demyelination. Alteration of glycolipid levels (i.e. sulfatides) in MS patients' brain has been shown. Lipid composition alterations lead to induction of demyelination in animal models. Evidence of lipid autoreactivity has been shown in vitro, in animal models and in MS patients, but their mechanisms, pattern and role remain to be elucidated. The aim of this study was to assess innate and adaptive immune response to sulfatide. Methods: Sulfatide IgG antibodies were studied in serum of 51 MS patients (36 relapsing-remitting (RRMS) and 15 secondary progressive (SPMS)) and 30 healthy controls by ELISA (enzyme-linked immunosorbent assay). Obtained data were compared with patients' clinical characteristics. Innate immunity response was studied in vitro. THP-1 cells were incubated with sulfatides 100 μg/ml–1 μg/ml; and TLR ligands: Pam3CSK4 (TLR2 ligand) — 10 ng/ml–10 pg/ml; LPS Escherichia coli (TLR4 ligand) — 10 μg/ml–10 ng/ml; ODN2009 (TLR9 ligand) — 100 μg/ml–1 μg/ml; and combinations of sulfatides with mentioned TLR ligands. TLR-ligand interactions were analyzed by measurement of reporter gene expression (secreted alkaline phosphatase — SEAP) controlled by NF-kB-dependent promoter. Results: Anti-sulfatide antibodies were detected in 24/51 (47%) MS patients and 8/30 (27%) of healthy controls. There were no difference between sulfatide seropositivity in RRMS (17/36 — 47%) and SPMS (7/15 — 47%). No correlation of clinical characteristics, such as age, sex, EDSS, relapse rate and disease duration with sulfatide seropositivity was observed. Seropositive SPMS patients switched to progressive course faster than seronegative (mean disease duration before switching to progressive course 40 months vs. 115 months, respectively), although not sufficient, the patients' quantity hampered the evaluation of statistical significance between groups. Incubation of THP-1 with sulfatides did not lead to NF-kB activation irrespective of concentrations. 100 μg/ml of sulfatides potentiated NF-kB activation by Pam3CSK4 but such effect was not observed, when TLR4 or TLR9 ligands were used.
556 Analysis of auto-antibodies in schizophrenia Carolin Hoffmanna, Matthew J. Lindemannb, Vincent C. Ramspergerb, Lakshmanan Sureshb, Jo Stevensa, Mario Losena, Peter Molenaara, Marc H. De Baetsa, Marc De Hertc, Jim Van Osa, Bart Bp Ruttena, Pilar Martinez-martineza a
Department of Psychiatry and Neuropsychology, Maastricht University, Maastricht, Netherlands; bIMMCO Diagnostics, Inc., New York, United States; cUniversity Psychiatric Centre Catholic University Leuven, Catholic University Leuven, Leuven, Belgium Objectives: Schizophrenia is a neuropsychiatric disorder with a complex etiology and pathogenesis. While symptomatic treatment is available, there are no treatments that target the biological disease mechanisms. Recently, autoantibodies to neuronal cell surface antigens have been identified in patients with schizophrenia. Autoimmunity is affecting nearly 10% of the general population and its clinical spectrum varies from endocrine, musculoskeletal to neuropsychiatric syndromes. Since autoimmune patients frequently suffer from more than one autoimmune disease, we analyzed common autoantibodies as disease markers (or risk factors) to test the hypothesis that schizophrenia is correlated to autoimmunity. Methods: We tested sera from 232 schizophrenia, bipolar disorder and myasthenia gravis patients and healthy controls for the presence of autoantibodies by ELISA and immunofluorescence. The assays included: anti-nuclear antibodies (ANA), double-stranded DNA (dsDNA), and ribosomal P protein (RPP). ANA IFA screening was performed on ImmunGlo HEp-2 slides and the acquisition of images and interpretation of patterns was performed on the Immco i-Sight automated imaging system. Results: Prevalence of anti-dsDNA autoantibodies was found to be overall relatively high and to significantly differ between disease and control groups. Schizoaffective patients had highest titers of anti-dsDNA autoantibodies. The autoantibody titer anti-RPP differs significantly between the groups. Paranoid and disorganized patients had highest titers of anti-RPP autoantibodies. No significant difference was detected in the presence of ANA autoantibodies in the different study groups. Conclusions: We confirmed that a subgroup of schizophrenia patients had increased levels of autoantibodies. These findings are the first step to improve diagnosis and possibly treatment of schizophrenic patients with autoimmune etiology. Currently, control sera are being tested to match the sample size and the age and sex distribution of the disease group.
101
Créteil, Créteil, France; cUniversity Psychiatric Centre Catholic University Leuven, Catholic University Leuven, Campus Kortenberg, Leuven, Belgium; d Department of Psychiatry, Erasmus University Medical Center, Rotterdam, Netherlands Introduction: The etiology of schizophrenia is multifactorial and highly diverse. Recent research suggests the involvement of autoimmunity in a subset of patients with schizophrenia and antibodies have been identified against neurotransmitter receptors. Up to date, only few reports have documented autoantibodies in psychiatric disease. In encephalitis, anti-N-methyl-D-aspartate glutamate receptor antibodies (NMDAr-AB) were shown to be causative for major psychotic symptoms of the disease. Antibodies directed against the same antigen were also found to be more common (6%– 10%) in patients with schizophrenia than in healthy controls, but subsequent studies were not consistent. Additional studies with larger sample size and relevant control groups are needed to determine whether NMDAr-AB are relevant to the etiology of schizophrenia. Objective: We aimed to identify the frequency of NMDAr-autoantibodies in schizophrenia and healthy controls. Methods: Our study compared the presence of NMDAr-AB of the immunoglobulin isotype G (IgG) in sera of 200 patients with schizophrenia and 200 healthy controls. Sera were screened using an established cell-based assay with antigen transfected HEK cells expressing the NMDAr subunit 1 alone or in combination with NMDAr subunit 2B. Sera identified as positive on cells were also tested for reactivity on rat brain tissue. Results: 3 patients were identified as anti-NMDAR-AB positive of which one also reacts on rat brain tissue. No control sera contained autoantibodies against the tested antigen. Conclusion: The results indicate that there might be a subgroup of patients with an underlying autoimmune reaction and warrant further research. Characterization of autoimmune schizophrenia is especially important because ultimately, the antibody positive patient group will be eligible for conventional treatment for antibody mediated diseases.
doi:10.1016/j.jneuroim.2014.08.271
304 Factor H autoantibodies in neuromyelitis optica Zsolt Illesa, Barbara Uzonyib, Mihaly Jozsib a
doi:10.1016/j.jneuroim.2014.08.270
Institute of Clinical Research and Department of Neurology, University of Southern Denmark, Odense, Denmark; bMTA-ELTE Immunology Research Group, Department of Immunology, Eötvös Loránd University, Budapest, Hungary
543 Analysis of pathogenic autoantibodies against the N-methyl-D-aspartate glutamate receptor in schizophrenia Carolin Hoffmanna, Gisela Nogales-gadeaa, Jo Stevensa, Mario Losena, Andrei Szokeb, Marion Leboyerb, Marc De Hertc, Nico J.M. Van Beverend, Peter Molenaara, Wim A. Buurmana, Marc H. De Baetsa, Bart B.F. Ruttena, Jim Van Osa, Pilar Martinez-Martineza a
Department of Psychiatry and Neuropsychology, Maastricht University, Maastricht, Netherlands; bPôle de Psychiatrie, Hôpital Henri Mondor-Albert Chenevier-Assistance Publique-Hôpitaux de Paris, Université Paris-Est
Neuromyelitis optica (NMO) is an inflammatory disease characterized by pathogenic complement-activating autoantibodies against aquaporin 4 (AQP4), the main water channel of the central nervous system. NMO is frequently associated with other autoantibodies and antibody-mediated diseases. Since autoantibodies against complement regulating factors may contribute to the activation of the complement system, here we examined the presence of antibodies against the complement regulator protein factor H in the serum of patients with NMO. The study cohort included 45 patients, who were all seropositive for AQP4 autoantibodies. Serum samples were screened by ELISA for autoantibodies using purified factor H as target antigen and human
102
Abstracts
serum albumin as control antigen. Specific IgG binding to factor H was detected in 4 samples (~9%), while no IgG binding to factor H was detected from 25 control sera. We could also detect factor H–IgG complexes using a monoclonal antibody to factor H as a catch antibody in ELISA. The binding sites of the factor H autoantibodies were mapped using recombinant constructs representing various factor H domains. All four autoantibodies bound to factor H domains 19–20, and also recognized the homologous protein CFHR1. This binding pattern was similar to factor H autoantibodies associated with atypical hemolytic uremic syndrome (HUS), used as positive controls in the assays. In conclusion, our results demonstrate that factor H autoantibodies are not uncommon in NMO. Our data also suggest that generation of antibodies against complement regulating factors among other autoantibodies may contribute to the complement-mediated damage.
ANA and anti-nucleosoma antibodies showed similar temporal pattern to AQP4 antibodies, and were also increased during NMO relapses. Abnormal elevation of both anti-nucleosoma and ANA was more common in anti-AQP4 seropositive samples. SLE was always active during NMO relapse, and the first clinical sign of NMO occurred in the active phase of SLE. Our data indicate that AQP4 antibodies in the sera are of complement-activating IgG1 subtypes years before the first attack of NMO, but during attacks of NMO, IgM antibodies also appear or are present. In our NMO patients with SLE, the very first relapse was associated with the activity of SLE and elevation of SLE-specific autoantibodies. This may indicate that (i) either a general B cell activation contributes to the manifestation of NMO, or (ii) in patients with SLE other autoantibodies may precipitate NMO relapses. doi:10.1016/j.jneuroim.2014.08.273
doi:10.1016/j.jneuroim.2014.08.272
306 Longitudinal serological and clinical data indicate that SLE activity may contribute to NMO relapses
435 Sulfatides autoreactivity in multiple sclerosis Maria Ivanovaa, Amir Tukhvatulinb, Alina Dzharullaevab, Denis Logunovb, Maria Zakharovaa a
Zsolt Illesa, Katalin T. Kovacsb, Sudhakar R. Kalluric, Tunde Csepanyd, Yvonne Schaeferc, Magdolna Simoe, Laszlo Rokuszf, Timea Berkig, Bernhard Hemmerc a
Institute of Clinical Research and Department of Neurology, University of Southern Denmark, Odense, Denmark; bDepartment of Rheumatology and Immunology, University of Pecs, Pecs, Hungary; cClinic of Neurology, Technische Universität München, Munich, Germany; dDepartment of Neurology, University of Debrecen, Debrecen, Hungary; eDepartment of Neurology, Semmelweis University, Budapest, Hungary; f1st Department of Internal Medicine, Military Hospital — State Health Centre, Budapest, Hungary; gDepartment of Immunology and Biotechnology, University of Pecs, Pecs, Hungary Neuromyelitis optica (NMO) mediated by autoantibodies against aquaporin 4 (AQP4) is frequently associated with other antibodymediated autoimmune diseases, but the relationship between serological and clinical activities of the associated diseases is not well established. In 6 patients with NMO associated with SLE, (i) we examined longitudinal changes of AQP4 antibodies of IgM and IgG classes and IgG subtypes, and related them to the clinical activity of NMO; (ii) we examined longitudinal changes of ANA, anti-nucleosoma and antidsDNA, and related them to the clinical activity of NMO; (iii) we investigated both NMO- and SLE-specific autoantibodies before and after the first clinical attack of NMO; and (iv) we compared the longitudinal relationship between anti-AQP4 and SLE-related autoantibodies. Total IgG, subtypes and IgM of AQP4 antibodies were measured by a cell-based flow cytometry assay in 3 samples obtained during NMO relapses and 17 samples collected during remissions; the human glioblastoma cell line LN18 was stably transduced using a lentiviral vector to over express human AQP4-M23. Anti-nucleosoma, ANA and anti-dsDNA were determined by ELISA. In 3 samples obtained 2–5 years before the first NMO relapse in 2 patients, anti-AQP4 antibodies were already present and had an IgG1 subtype. During NMO relapses, the titer of anti-IgG1 antibodies increased or was elevated, and became undetectable in some of the samples collected during remission. IgM antibodies also appeared or were present during relapses, and decreased thereafter. Anti-dsDNA,
Research Center of Neurology, Russian Academy of Medical Sciences, Moscow, Russian Federation; bGamaleya Research Institute of Epidemiology and Microbiology, Ministry of Health of the Russian Federation, Moscow, Russian Federation Objective: Sulfatides are among the most abundant myelin lipids. Multiple sclerosis (MS) is characterized by CNS inflammation and demyelination. Alteration of glycolipid levels (i.e. sulfatides) in MS patients' brain has been shown. Lipid composition alterations lead to induction of demyelination in animal models. Evidence of lipid autoreactivity has been shown in vitro, in animal models and in MS patients, but their mechanisms, pattern and role remain to be elucidated. The aim of this study was to assess innate and adaptive immune response to sulfatide. Methods: Sulfatide IgG antibodies were studied in serum of 51 MS patients (36 relapsing-remitting (RRMS) and 15 secondary progressive (SPMS)) and 30 healthy controls by ELISA (enzyme-linked immunosorbent assay). Obtained data were compared with patients' clinical characteristics. Innate immunity response was studied in vitro. THP-1 cells were incubated with sulfatides 100 μg/ml–1 μg/ml; and TLR ligands: Pam3CSK4 (TLR2 ligand) — 10 ng/ml–10 pg/ml; LPS Escherichia coli (TLR4 ligand) — 10 μg/ml–10 ng/ml; ODN2009 (TLR9 ligand) — 100 μg/ml–1 μg/ml; and combinations of sulfatides with mentioned TLR ligands. TLR-ligand interactions were analyzed by measurement of reporter gene expression (secreted alkaline phosphatase — SEAP) controlled by NF-kB-dependent promoter. Results: Anti-sulfatide antibodies were detected in 24/51 (47%) MS patients and 8/30 (27%) of healthy controls. There were no difference between sulfatide seropositivity in RRMS (17/36 — 47%) and SPMS (7/15 — 47%). No correlation of clinical characteristics, such as age, sex, EDSS, relapse rate and disease duration with sulfatide seropositivity was observed. Seropositive SPMS patients switched to progressive course faster than seronegative (mean disease duration before switching to progressive course 40 months vs. 115 months, respectively), although not sufficient, the patients' quantity hampered the evaluation of statistical significance between groups. Incubation of THP-1 with sulfatides did not lead to NF-kB activation irrespective of concentrations. 100 μg/ml of sulfatides potentiated NF-kB activation by Pam3CSK4 but such effect was not observed, when TLR4 or TLR9 ligands were used.