Andreas Weishaupt

Publications of Andreas Weishaupt

• Human IgG directed against amphiphysin induces anxiety behavior in a rat model after intrathecal passive transfer.

  Authors: Christian Geis, Benedikt Grünewald, Andreas Weishaupt, Thomas Wultsch, Klaus V Toyka, Andreas Reif, Claudia Sommer

  Journal of neural transmission (Vienna, Austria : 1996). 02/2012;

  Stiff person syndrome with auto-antibodies against amphiphysin is characterized by muscular stiffness, spasms, and anxiety which is a less appreciated core symptom. Here, we report that intrathecalStiff person syndrome with auto-antibodies against amphiphysin is characterized by muscular stiffness, spasms, and anxiety which is a less appreciated core symptom. Here, we report that intrathecal application of purified immunoglobulin G-antibodies against amphiphysin from one patient induce anxiety behavior in rats. Immunostaining demonstrated binding of anti-amphiphysin antibodies to brain structures which are associated with anxiety disorders, such as the amygdala. We propose that antibody-mediated amphiphysin deficiency may account for anxiety behavior in stiff person syndrome via presynaptic dysregulation of GABAergic pathways.

• Human stiff-person syndrome IgG induces anxious behavior in rats.

  Authors: Christian Geis, Andreas Weishaupt, Benedikt Grünewald, Thomas Wultsch, Andreas Reif, Manfred Gerlach, Ron Dirkx, Michele Solimena, Daniela Perani, Manfred Heckmann, Klaus V Toyka, Franco Folli, Claudia Sommer

  PloS one. 01/2011; 6(2):e16775.

  Anxiety is a heterogeneous behavioral domain playing a role in a variety of neuropsychiatric diseases. While anxiety is the cardinal symptom in disorders such as panic disorder, co-morbid anxiousAnxiety is a heterogeneous behavioral domain playing a role in a variety of neuropsychiatric diseases. While anxiety is the cardinal symptom in disorders such as panic disorder, co-morbid anxious behavior can occur in a variety of diseases. Stiff person syndrome (SPS) is a CNS disorder characterized by increased muscle tone and prominent agoraphobia and anxiety. Most patients have high-titer antibodies against glutamate decarboxylase (GAD) 65. The pathogenic role of these autoantibodies is unclear. We re-investigated a 53 year old woman with SPS and profound anxiety for GABA-A receptor binding in the amygdala with (11)C-flumazenil PET scan and studied the potential pathogenic role of purified IgG from her plasma filtrates containing high-titer antibodies against GAD 65. We passively transferred the IgG fraction intrathecally into rats and analyzed the effects using behavioral and in vivo electrophysiological methods. In cell culture, we measured the effect of patient IgG on GABA release from hippocampal neurons. Repetitive intrathecal application of purified patient IgG in rats resulted in an anxious phenotype resembling the core symptoms of the patient. Patient IgG selectively bound to rat amygdala, hippocampus, and frontal cortical areas. In cultured rat hippocampal neurons, patient IgG inhibited GABA release. In line with these experimental results, the GABA-A receptor binding potential was reduced in the patient's amygdala/hippocampus complex. No motor abnormalities were found in recipient rats. The observations in rats after passive transfer lead us to propose that anxiety-like behavior can be induced in rats by passive transfer of IgG from a SPS patient positive for anti-GAD 65 antibodies. Anxiety, in this case, thus may be an antibody-mediated phenomenon with consecutive disturbance of GABAergic signaling in the amygdala region.

• Glatiramer acetate attenuates pro-inflammatory T cell responses but does not directly protect neurons from inflammatory cell death.

  Authors: Alexander M Herrmann, Kerstin Göbel, Ole J Simon, Nico Melzer, Michael K Schuhmann, Max-Philipp Stenner, Andreas Weishaupt, Christoph Kleinschnitz, Stefan Bittner, Patrick Meuth, Olaf Stuve, Thomas Budde, Bernd C Kieseier, Heinz Wiendl, Sven G Meuth

  The American journal of pathology. 10/2010; 177(6):3051-60.

  Glatiramer acetate (GA) is a synthetic, random, basic copolymer capable of modulating adaptive T cell responses. In animal models of various inflammatory and degenerative central nervous systemGlatiramer acetate (GA) is a synthetic, random, basic copolymer capable of modulating adaptive T cell responses. In animal models of various inflammatory and degenerative central nervous system disorders, GA-induced T cells cross the blood-brain barrier, secrete high levels of anti-inflammatory cytokines and neurotrophins, and thus both reduce neuronal damage and promote neurogenesis. Recently, it has been suggested that GA itself may permeate the (impaired) blood-brain-barrier and directly protect neurons under conditions of inflammation-mediated neurodegeneration. To test this hypothesis, we examined the direct effects of GA on neuronal functionality and T cell-mediated neuronal apoptosis in culture, acute brain slices, and focal experimental autoimmune encephalomyelitis. GA caused a depolarization of the resting membrane potential and led to an immediate impairment of action potential generation in neurons. Moreover, GA-incubated neurons underwent dose-dependent apoptosis. Apoptosis of ovalbumin peptide-loaded major histocompatibility complex class I-expressing neurons induced by ovalbumin-specific effector T cells could be reduced by pre-incubation of T cells, but not neurons with GA. Similar results could be found using acute brain slices. In focal experimental autoimmune encephalomyelitis, lesion size and neuronal apoptosis could be limited by pretreating rats with GA, whereas intracerebral GA application into the inflammatory lesion had no effect on neuronal survival. Our data suggest that GA attenuates adaptive pro-inflammatory T cell responses, but does not exert direct neuroprotective effects.

• Stiff person syndrome-associated autoantibodies to amphiphysin mediate reduced GABAergic inhibition.

  Authors: Christian Geis, Andreas Weishaupt, Stefan Hallermann, Benedikt Grünewald, Carsten Wessig, Thomas Wultsch, Andreas Reif, Nadiya Byts, Marcus Beck, Sibylle Jablonka, Michael K Boettger, Nurcan Üçeyler, Wernher Fouquet, Manfred Gerlach, Hans-Michael Meinck, Anna-Leena Sirén, Stephan J Sigrist, Klaus V Toyka, Manfred Heckmann, Claudia Sommer

  Brain : a journal of neurology. 09/2010; 133(11):3166-80.

  Synaptic inhibition is a central factor in the fine tuning of neuronal activity in the central nervous system. Symptoms consistent with reduced inhibition such as stiffness, spasms and anxiety occurSynaptic inhibition is a central factor in the fine tuning of neuronal activity in the central nervous system. Symptoms consistent with reduced inhibition such as stiffness, spasms and anxiety occur in paraneoplastic stiff person syndrome with autoantibodies against the intracellular synaptic protein amphiphysin. Here we show that intrathecal application of purified anti-amphiphysin immunoglobulin G antibodies induces stiff person syndrome-like symptoms in rats, including stiffness and muscle spasms. Using in vivo recordings of Hoffmann reflexes and dorsal root potentials, we identified reduced presynaptic GABAergic inhibition as an underlying mechanism. Anti-amphiphysin immunoglobulin G was internalized into neurons by an epitope-specific mechanism and colocalized in vivo with presynaptic vesicular proteins, as shown by stimulation emission depletion microscopy. Neurons from amphiphysin deficient mice that did not internalize the immunoglobulin provided additional evidence of the specificity in antibody uptake. GABAergic synapses appeared more vulnerable than glutamatergic synapses to defective endocytosis induced by anti-amphiphysin immunoglobulin G, as shown by increased clustering of the endocytic protein AP180 and by defective loading of FM 1-43, a styryl dye used to label cell membranes. Incubation of cultured neurons with anti-amphiphysin immunoglobulin G reduced basal and stimulated release of γ-aminobutyric acid substantially more than that of glutamate. By whole-cell patch-clamp analysis of GABAergic inhibitory transmission in hippocampus granule cells we showed a faster, activity-dependent decrease of the amplitude of evoked inhibitory postsynaptic currents in brain slices treated with antibodies against amphiphysin. We suggest that these findings may explain the pathophysiology of the core signs of stiff person syndrome at the molecular level and show that autoantibodies can alter the function of inhibitory synapses in vivo upon binding to an intraneuronal key protein by disturbing vesicular endocytosis.

• Mild experimental autoimmune encephalitis as a tool to induce blood-brain barrier dysfunction.

  Authors: Michael Boettger, Andreas Weishaupt, Christian Geis, Klaus Toyka, Claudia Sommer

  Journal of neural transmission (Vienna, Austria : 1996). 11/2009;

  The blood-brain barrier (BBB) serves as a border limiting access of immunoglobulins from the circulation into the brain. This becomes relevant when studying the pathogenesis of antibody-mediatedThe blood-brain barrier (BBB) serves as a border limiting access of immunoglobulins from the circulation into the brain. This becomes relevant when studying the pathogenesis of antibody-mediated autoimmune CNS disorders. Here, we characterized the BBB dysfunction in a model of mild experimental adoptive transfer autoimmune encephalomyelitis (AT-EAE). We show that large molecules can readily penetrate the BBB between days 3 and 7 after EAE-induction. This model may be valuable for studying putative pathogenic effects of immunoglobulins in the central nervous system.

• Stable silencing of the glucocorticoid receptor in myelin-specific T effector cells by retroviral delivery of shRNA: Insight into neuroinflammatory disease.

  Authors: Denise Tischner, Jens van den Brandt, Andreas Weishaupt, Fred Lühder, Marco J Herold, Holger M Reichardt

  European journal of immunology. 09/2009;

  Autoimmune responses in the CNS can be induced by adoptive transfer of CD4(+) T effector cells after antigen-restimulation and expansion of clonal cell lines in vitro. However, pathogenic factorsAutoimmune responses in the CNS can be induced by adoptive transfer of CD4(+) T effector cells after antigen-restimulation and expansion of clonal cell lines in vitro. However, pathogenic factors remain partially elusive due to the lack of appropriate methods to achieve gene inactivation. Here we describe a protocol for stable gene silencing in differentiated rat T cells by retroviral transfer of small hairpin RNAs. Through the combination of an expression cassette containing the green fluorescent protein with a puromycin selection cassette this allows for the generation of pure knockdown cell lines suitable for tracking in animals. Exemplified for the glucocorticoid receptor, we demonstrate that gene silencing renders T effector cells unresponsive to ligand-induced apoptosis and gene regulation without affecting their ability to induce EAE in rats. Interestingly, glucocorticoid administration remains effective in the treatment of EAE despite strongly diminished glucocorticoid receptor expression in antigen-specific T cells. This highlights an important role of other cell types and bystander T cells as targets of glucocorticoid therapy. Collectively, our approach provides a simple tool for stable and efficient gene silencing in T effector cells, which should help to better understand brain autoimmune pathophysiology.

• TASK1 modulates inflammation and neurodegeneration in autoimmune inflammation of the central nervous system.

  Authors: Stefan Bittner, Sven G Meuth, Kerstin Göbel, Nico Melzer, Alexander M Herrmann, Ole J Simon, Andreas Weishaupt, Thomas Budde, Douglas A Bayliss, Martin Bendszus, Heinz Wiendl

  Brain : a journal of neurology. 08/2009;

  We provide evidence that TWIK-related acid-sensitive potassium channel 1 (TASK1), a member of the family of two-pore domain potassium channels relevant for setting the resting membrane potential andWe provide evidence that TWIK-related acid-sensitive potassium channel 1 (TASK1), a member of the family of two-pore domain potassium channels relevant for setting the resting membrane potential and balancing neuronal excitability that is expressed on T cells and neurons, is a key modulator of T cell immunity and neurodegeneration in autoimmune central nervous system inflammation. After induction of experimental autoimmune encephalomyelitis, an experimental model mimicking multiple sclerosis, TASK1(-/-) mice showed a significantly reduced clinical severity and markedly reduced axonal degeneration compared with wild-type controls. T cells from TASK1(-/-) mice displayed impaired T cell proliferation and cytokine production, while the immune repertoire is otherwise normal. In addition to these effects on systemic T cell responses, TASK1 exhibits an independent neuroprotective effect which was demonstrated using both a model of acutely prepared brain slices cocultured with activated T cells as well as in vitro cultivation experiments with isolated optic nerves. Anandamide, an endogenous cannabinoid and inhibitor of TASK channels, reduced outward currents and inhibited effector functions of T cells (IFN-gamma production and proliferation); an effect completely abrogated in TASK1(-/-) mice. Accordingly, preventive blockade of TASK1 significantly ameliorated experimental autoimmune encephalomyelitis after immunization. Therapeutic application of anandamide significantly reduced disease severity and was capable of lowering progressive loss of brain parenchymal volume as assessed by magnetic resonance imaging. These data support the identification and characterization of TASK1 as potential molecular target for the therapy of inflammatory and degenerative central nervous system disorders.

• Increase of MCP-1 (CCL2) in myelin mutant Schwann cells is mediated by MEK-ERK signaling pathway.

  Authors: Stefan Fischer, Andreas Weishaupt, Jakob Troppmair, Rudolf Martini

  Glia. 07/2008; 56(8):836-43.

  Macrophages are critically involved in the pathogenesis of genetically caused demyelination, as it occurs in inherited demyelinating neuropathies. On the basis of the observation that upregulation ofMacrophages are critically involved in the pathogenesis of genetically caused demyelination, as it occurs in inherited demyelinating neuropathies. On the basis of the observation that upregulation of the Schwann cell-derived chemokine MCP-1 (CCL2) is a pathologically relevant mechanism for macrophage activation in mice heterozygously deficient for the myelin component P0 (P0+/-), we posed the question of the intracellular signaling cascade involved. By using western blot analysis of peripheral nerve lysates the MAP-kinases extracellular signal-regulated kinase 1/2 (ERK1/2) and MAP kinase/ERK kinase 1/2 (MEK1/2) showed an early and constantly increasing activation in P0 mutants. Furthermore, in nerve fibers from the P0+/- mutants, Schwann cell nuclei were much more often positive for phosphorylated ERK1/2 than in nerve fibers from wild type mice. In vitro experiments using the MEK1/2-inhibitor CI-1040 decreased ERK1/2-phosphorylation and MCP-1 expression in a Schwann cell-derived cell line. Finally, systemic application of CI-1040 lead to a decreased ERK1/2-phosphorylation and substantially reduced MCP-1-production in peripheral nerves of P0+/- mutant mice. Our study identifies MEK1/2-ERK1/2 signaling as an important intracellular pathway that connects the Schwann cell mutation with the activation of pathogenetically relevant macrophages in the peripheral nerves. These findings may have important implications for the treatment of inherited peripheral neuropathies in humans.

• The two-pore domain potassium channel TASK3 functionally impacts glioma cell death.

  Authors: Sven G Meuth, Alexander M Herrmann, Chi W Ip, Tatyana Kanyshkova, Stefan Bittner, Andreas Weishaupt, Thomas Budde, Heinz Wiendl

  Journal of neuro-oncology. 06/2008; 87(3):263-70.

  Two-pore domain K(+) channels, a recently discovered family of ion channels with a unique membrane topology, have been shown to be critically involved in cell death. We here address the functionalTwo-pore domain K(+) channels, a recently discovered family of ion channels with a unique membrane topology, have been shown to be critically involved in cell death. We here address the functional role of TASK3 (TWIK-related acid-sensitive K(+) channel, KCNK9) in human glioblastoma in vitro and in vivo. Human glioma cell lines (n = 5) as well as glioma specimens (n = 5) constitutively express TASK3 mRNA and protein. The functional impact of the potassium channel on cell survival was investigated using a medium with high (25 mM) extracellular potassium over 7 days. Using flow cytometric assessment, we show that under these culture conditions 97 +/- 0.76% of all glioma cells survived. Application of the TASK channel opener isoflurane (1 vol%) resulted in a 30 +/- 4% reduction of cell survival in different glioma cell lines. Simultaneous application of isoflurane and the TASK channel blockers bupivacaine (20 microM) and spermine (500 microM) completely reversed this effect. Our results demonstrate the expression of TASK3 in glioma cells in vitro and in vivo and provide a direct link between the TASK3 channel function and glioma cell survival. This implies that TASK3 channels may possibly represent a novel molecular target for the treatment of this type of cancer.

• Atorvastatin partially prevents an inflammatory barrier breakdown of cultured human brain endothelial cells at a pharmacologically relevant concentration.

  Authors: Mathias Buttmann, Alexander Lorenz, Andreas Weishaupt, Peter Rieckmann

  Journal of neurochemistry. 09/2007; 102(4):1001-8.

  3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (i.e. statins) are currently under clinical investigation as a prophylactic immunomodulatory treatment for neurological diseases where an3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (i.e. statins) are currently under clinical investigation as a prophylactic immunomodulatory treatment for neurological diseases where an inflammatory disruption of the blood-brain barrier plays a pathogenic role. Here, we investigated whether atorvastatin pre-treatment modulates inflammatory-induced barrier dysfunction of cultured human brain microvascular endothelial cells (HBMEC). Pre-treatment of immortalized HBMEC with atorvastatin (50 nmol/L to 1 micromol/L) dose-dependently prevented an inflammatory up-regulation of monocyte chemoattractant protein-1/CCL2 but not of interleukin-8/CXCL8 and intercellular adhesion molecule-1 expression by tumor necrosis factor-alpha or interleukin-1beta. It antagonized an inflammatory up-regulation of claudin-3 expression while zonula occludens-1 and occludin protein levels remained unaltered. Like immortalized HBMEC, primary HBMEC also showed a reduction of claudin-3 and of inducible CCL2 expression following atorvastatin pre-treatment. On a functional level, atorvastatin pre-treatment of HBMEC strongly and dose-dependently reduced adhesion of activated T lymphocytes to pre-activated primary endothelium. Atorvastatin effects could partially be abolished by parallel mevalonate treatment. These anti-inflammatory effects of atorvastatin were observed already at a pharmacologically relevant concentration of 50 nmol/L. Our results obtained with human brain endothelial cells demonstrate how statins may partially prevent an inflammatory-mediated blood-brain barrier breakdown in humans.

• Antigen therapy of experimental autoimmune encephalomyelitis selectively induces apoptosis of pathogenic T cells.

  Authors: Denise Tischner, Andreas Weishaupt, Jens van den Brandt, Chi Wang Ip, Thomas Kerkau, Ralf Gold, Holger M Reichardt

  Journal of neuroimmunology. 03/2007; 183(1-2):146-50.

  Administration of high-dose myelin antigen induces massive T cell apoptosis in experimental autoimmune encephalomyelitis (EAE) but the nature of the target cells remains elusive. Here we have used aAdministration of high-dose myelin antigen induces massive T cell apoptosis in experimental autoimmune encephalomyelitis (EAE) but the nature of the target cells remains elusive. Here we have used a cell line established in eGFP-transgenic Lewis rats to distinguish between pathogenic and bystander T cells in adoptive transfer EAE. Intravenous application of gpMBP strongly reduced the amount of encephalitogenic cells in spinal cord and spleen while the number of the other T cells remained constant. This could be attributed to their differential sensitivity to apoptosis. Thus, antigen therapy selectively targets pathogenic T cells and should therefore limit potential adverse effects.

• Polyclonal expansion of regulatory T cells interferes with effector cell migration in a model of multiple sclerosis.

  Authors: Denise Tischner, Andreas Weishaupt, Jens van den Brandt, Nora Müller, Niklas Beyersdorf, Chi Wang Ip, Klaus V Toyka, Thomas Hünig, Ralf Gold, Thomas Kerkau, Holger M Reichardt

  Brain : a journal of neurology. 11/2006; 129(Pt 10):2635-47.

  Recruitment of naturally occurring CD4+ CD25+ regulatory T (T(reg)) cells is a highly promising approach for the treatment of experimental autoimmune encephalomyelitis (EAE), a widely used model ofRecruitment of naturally occurring CD4+ CD25+ regulatory T (T(reg)) cells is a highly promising approach for the treatment of experimental autoimmune encephalomyelitis (EAE), a widely used model of multiple sclerosis. Here, we studied the in vivo interaction of T(reg) cells, induced by the monoclonal anti-CD28 antibody JJ316, with encephalitogenic T cell lines established from eGFP-transgenic rats. By tracking these fluorescent cells using flow cytometry and confocal microscopy, we found that the activation and expansion of T(reg) cells inhibited infiltration of the CNS by pathogenic T cells. Interference with effector cell migration occured within the secondary lymphoid organs, since the early therapeutic effects were achieved despite the absence of T(reg) cells in the spinal cord. However, the delayed homing to the CNS seen after prophylactic JJ316 administration indicates that T(reg) cells may play an additional role within the target tissue. In addition, the blood-brain barrier remained largely intact after JJ316 treatment, the secretion of T(H)2 cytokines was augmented and the encephalitogenic T cells exhibited a reduced secretion of IFN-gamma. This in turn resulted in a reduced expression of the chemokine receptor CXCR-3 on effector T cells which may interfere with their capacity to infiltrate the CNS. Importantly, these effects were not achieved by direct action of JJ316 on the encephalitogenic cells. Our data rather suggest that polyclonal activation of T(reg) cells in the secondary lymphoid organs is instrumental in preventing the pathological transmigration of encephalitogenic T cells into the CNS. We anticipate that these results may help to better understand the role of T(reg) cells in controlling autoimmunity in the CNS.

• Therapeutic efficacy of IL-17 neutralization in murine experimental autoimmune encephalomyelitis.

  Authors: Harald H Hofstetter, Saleh M Ibrahim, Dirk Koczan, Niels Kruse, Andreas Weishaupt, Klaus V Toyka, Ralf Gold

  Cellular immunology. 11/2005; 237(2):123-30.

  Experimental autoimmune encephalomyelitis (EAE) is widely regarded as an animal model of the human disease multiple sclerosis. A multitude of studies has investigated the neuroantigen-specific T-cellExperimental autoimmune encephalomyelitis (EAE) is widely regarded as an animal model of the human disease multiple sclerosis. A multitude of studies has investigated the neuroantigen-specific T-cell mediated cytokine pattern present in animals with EAE. In particular, the role of the so-called Th1- and Th2-cytokines has been addressed. In a recent study, it has been demonstrated that IL-23 rather than IL-12 is critical for modulating the character of the developing immune response towards a proinflammatory response and leading to EAE. IL-17 is a crucial effector cytokine, whose production is specifically triggered by IL-23, and it has been shown to be an essential inflammatory mediator in other autoimmune diseases and inflammatory conditions. This led us to investigate the role of IL-17 in EAE. Strong antigen-specific production of IL-17 was demonstrated both in peripheral immune organs and in the CNS in acute and chronic EAE, as demonstrated by ELISPOT and RT-PCR analysis. Therapeutic neutralization of IL-17 with IL-17-receptor-Fc-protein in acute EAE ameliorated clinical symptoms. Neutralization of IL-17 with a monoclonal antibody also ameliorated the disease course. We conclude that IL-17 is crucially involved in the cytokine network as an effector cytokine in EAE.

• Presynaptic effects of immunoglobulin G from patients with Lambert-Eaton myasthenic syndrome: their neutralization by intravenous immunoglobulins.

  Authors: Brigitte Buchwald, Raheleh Ahangari, Andreas Weishaupt, Klaus V Toyka

  Muscle & nerve. 05/2005; 31(4):487-94.

  Intravenous immunoglobulin (IVIg) treatment improves muscle strength in Lambert-Eaton myasthenic syndrome (LEMS), but its specific mode of action is unknown. We have delineated its mode of action onIntravenous immunoglobulin (IVIg) treatment improves muscle strength in Lambert-Eaton myasthenic syndrome (LEMS), but its specific mode of action is unknown. We have delineated its mode of action on neuromuscular blocking properties of LEMS IgG. The effect of sera and purified IgG from six patients with LEMS on evoked quantal release was investigated after direct application to the motor nerve terminal by the perfused macro-patch-clamp electrode in mouse hemidiaphragms. The effect of LEMS IgG was analyzed alone and after coincubation with different concentrations of IVIg or its Fab fragments. All LEMS sera and purified LEMS IgG fractions taken before IVIg treatment inhibited evoked quantal release in a dose-dependent manner. When LEMS IgG was coincubated with a therapeutic IVIg preparation, presynaptic inhibitory activity of LEMS IgG was diminished in a dose-dependent fashion. Monovalent Fab fragments were as effective in neutralizing the activity of LEMS IgG as whole IVIg. These direct neutralizing effects of IVIg may explain its therapeutic efficacy.

• Modulation of experimental autoimmune encephalomyelitis by administration of cells expressing antigenic peptide covalently linked to MHC class II.

  Authors: Andreas Weishaupt, Matthias Kreiss, Ralf Gold, Thomas Herrmann

  Journal of neuroimmunology. 08/2004; 152(1-2):11-9.

  The MHC class II molecule RT1Bl covalently linked with gpMBP-71-90 was expressed in P80 cells (mouse mastocytoma P815 expressing rat-CD80) and i.v. injection ameleriorated active and adoptiveThe MHC class II molecule RT1Bl covalently linked with gpMBP-71-90 was expressed in P80 cells (mouse mastocytoma P815 expressing rat-CD80) and i.v. injection ameleriorated active and adoptive transfer (AT) experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Spinal cord of animals with AT-EAE showed significant increase of apoptotic T-cells at maximum of disease after injection of P80-RT1Bl-MBP-71-90 but not of P80RT1Bl or P80 cells. The data demonstrate a possible therapeutic effect on EAE by provision of T-cell receptor (TCR) and costimulatory signals by genetically engineered antigen presenting cells (APC) and suggest induction of T-cell apoptosis as important mechanism of action.

• Tolerance induction by intrathymic expression of P0.

  Authors: Lucian Visan, Ioana A Visan, Andreas Weishaupt, Harald H Hofstetter, Klaus V Toyka, Thomas Hünig, Ralf Gold

  Journal of immunology (Baltimore, Md. : 1950). 03/2004; 172(3):1364-70.

  Genetic deficiency or instability of myelin protein zero (P0) results in hereditary motor sensory neuropathy. In view of recent advances in gene therapy, substitution of the molecular defect mayGenetic deficiency or instability of myelin protein zero (P0) results in hereditary motor sensory neuropathy. In view of recent advances in gene therapy, substitution of the molecular defect may become realistic in the near future. Here we investigate the impact of genetic deficiency of P0 on selection of the autoreactive T cell repertoire in the corresponding mouse model. We show that P0 mRNA transcripts are expressed in thymic stroma, similar to other myelin proteins and that expression of intact P0 protein can be detected by Western blot. Using a library of overlapping 20mer peptides spanning the entire length of P0 and applying the ELISPOT technique, we detected a strong immune response toward P0 extracellular domain peptide aa 41-60 in P0(-/-) knockout mice, but not in heterozygous P0(+/-) or wild-type (wt) mice. In addition, one cryptic epitope and two subdominant epitopes of P0 were identified. Using P0(-/-) into wt bone marrow (BM) chimeras we found that P0 expression in the host suffices for full tolerance induction, which is in line with its presence in thymic stroma. However, repopulation of P0(-/-) mice with wt BM led to partial induction of tolerance, suggesting that BM derived cells can also express this protein. Our findings may have implications for secondary autoimmunity developing after gene therapy in hereditary neuropathies and other diseases with genetically determined protein deficiency, because the repaired protein will then represent a foreign, nontolerized Ag.

• Effects of intravenous immunoglobulins on T cell and oligodendrocyte apoptosis in high-dose antigen therapy in experimental autoimmune encephalomyelitis.

  Authors: Andreas Weishaupt, Tanja Kuhlmann, Lisa M Schönrock, Klaus V Toyka, Wolfgang Brück, Ralf Gold

  Acta neuropathologica. 11/2002; 104(4):385-90.

  Intravenous immunoglobulins (IVIg) are purified preparations of immunoglobulins from plasma of healthy human donors containing polyclonal IgG and various immunomodulatory contaminants. IVIg may exertIntravenous immunoglobulins (IVIg) are purified preparations of immunoglobulins from plasma of healthy human donors containing polyclonal IgG and various immunomodulatory contaminants. IVIg may exert its therapeutic effect at several levels of the immune network. Antigen-specific therapy of adoptive transfer-(AT)-EAE in Lewis rats leads to elevated serum levels of tumor necrosis factor-alpha (TNF-alpha). TNF-alpha release at sites of inflammation increased apoptosis of autoaggressive T cells in spinal cord in situ and oligodendrocyte apoptosis. In addition, autoinflammatory T cells in liver were destroyed and caused liver damage by TNF-alpha release. To explore a possible neutralizing effect of IVIg on TNF-alpha secreted by antigen-specific T cells, we analyzed T cell and oligodendrocyte apoptosis as well as liver damage in rats that had been injected with myelin basic protein (MBP) and co-treated intravenously with human IVIg. As in our earlier studies, we found that TNF-alpha serum levels were raised by antigen therapy and decreased with concomitant IVIg administration. Using IVIg treatment, antigen-induced T cell apoptosis in inflamed spinal cord and liver of MBP/IVIg-treated animals was significantly reduced compared to control rats treated with MBP/albumin. T cell apoptosis decreased to levels observed in EAE rats receiving albumin only. In addition, serum levels of liver enzymes were raised after MBP/albumin administration and decreased by co-treatment with IVIg, indicating protection of hepatocytes by the neutralization of TNF-alpha. In contrast, oligodendrocyte apoptosis in animals receiving MBP/IVIg was significantly higher than in EAE controls. This indicates that IVIg may have different tissue-specific effects. Besides neutralization of TNF-alpha-mediated cell death, IVIg may also interfere with the network of local immune cells, thus modulating survival of glial cells.

• Truncation of the neuritogenic peptide bP2(60-70) results in the generation of altered peptide ligands with the potential to interfere with T cell activation.

  Authors: Martin Offenhäusser, Alexandra S Herr, Jörg Hartkamp, Marca Wauben, Tim Magnus, Oliver Grauer, Silvia Seubert, Andreas Weishaupt, Klaus V Toyka, Ralf Gold, Jakob Troppmair

  Journal of neuroimmunology. 09/2002; 129(1-2):97-105.

  Due to the central role of T cells in the pathogenesis of inflammatory diseases of the peripheral nervous system like the Guillain-Barré syndrome, specific immunotherapies aim at modifying T cellDue to the central role of T cells in the pathogenesis of inflammatory diseases of the peripheral nervous system like the Guillain-Barré syndrome, specific immunotherapies aim at modifying T cell responses. Use of truncated mutants of the neuritogenic peptide of myelin basic protein (MBP) has been shown to anergize autoreactive T cells and to reverse experimental autoimmune encephalitis (EAE). To establish a rationale basis for the use of altered peptide ligands (APLs) in the treatment of autoimmune diseases we designed a set of N- and C-terminally truncated mutants of the minimal experimental autoimmune neuritis (EAN) inducing bovine P2 (bP2) (60-70) peptide and compared them for the ability to induce immune responses and T cell receptor (TCR) cell signaling. Truncated peptides bound to MHC class II molecules and induced TCR internalization and expression of interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) with decreasing potency. None of the shortened mutants elicited a proliferative response in P2-specific T cells. Stimulation of these antigen-specific T cells with peptide bP2(62-69) using antigen presenting cells (APCs) prepulsed with bP2(60-70) resulted in a significant decrease of the proliferative response. In agreement with the observed effects on T cell activation, analysis of TCR signaling demonstrated a lack of CD3 epsilon phosphorylation and MAPK activation. Moreover, repeated injection of bP2(62-69) significantly slowed progression of adoptive transfer EAN (AT-EAN). Taken together, these findings strongly suggest that peptide bP2(62-69) can favorably modulate the antigen-induced response of neuritogenic T cells.

• Intravenous immunoglobulins neutralize blocking antibodies in Guillain-Barré syndrome.

  Authors: Brigitte Buchwald, Raheleh Ahangari, Andreas Weishaupt, Klaus V Toyka

  Annals of neurology. 07/2002; 51(6):673-80.

  Intravenous immunoglobulin (IVIg) treatment ameliorates the course of Guillain-Barré syndrome (GBS), but its specific mode of action is unknown. We attempted to delineate the effect of IVIg onIntravenous immunoglobulin (IVIg) treatment ameliorates the course of Guillain-Barré syndrome (GBS), but its specific mode of action is unknown. We attempted to delineate the effect of IVIg on neuromuscular blocking antibodies in GBS. A total of seven GBS serum samples were examined for blocking antibodies and the effect of IVIg with a macro-patch-clamp technique in mouse hemidiaphragms. First, serum was tested before and after treatment with IVIg. Second, we investigated with coincubation experiments whether the IVIg was capable of neutralizing neuromuscular blocking antibodies in GBS serum or affinity-purified immunoglobulin G (IgG) fractions. Finally, the mechanism of the neutralizing effect was studied by the coincubation of active blocking GBS IgG with Fab and Fc fragments prepared from IVIg. All GBS sera (two adults and two children) and GBS IgG fractions (three adults) taken before treatment with IVIg blocked evoked quantal release by approximately 90%. Blocking activity was markedly reduced in sera obtained after treatment with IVIg. Coincubation of the pretreatment blocking serum with the posttreatment serum, or with the IVIg preparation used for treatment, reduced the blocking activity of the pretreatment GBS serum. When GBS IgG was coincubated with IVIg, the blocking activity of GBS IgG was diminished dose-dependently. Monovalent and divalent Fab fragments prepared from the IVIg were as effective as whole IVIg, but Fc fragments were ineffective. Therapeutic IVIg is capable of neutralizing neuromuscular blocking antibodies in GBS by a dose-dependent, antibody-mediated mechanism. This may, in part, explain its therapeutic efficacy.

• Analysis of maturation states of rat bone marrow-derived dendritic cells using an improved culture technique.

  Authors: Oliver Grauer, Gisela Wohlleben, Silvia Seubert, Andreas Weishaupt, Eckhart Kämpgen, Ralf Gold

  Histochemistry and cell biology. 05/2002; 117(4):351-62.

  In this study, we examined in more detail the development of rat bone marrow-derived dendritic cells (BMDC). A two-stage culture system was used to propagate BMDC from rat bone marrow precursors.In this study, we examined in more detail the development of rat bone marrow-derived dendritic cells (BMDC). A two-stage culture system was used to propagate BMDC from rat bone marrow precursors. BMDC developed within clusters of proliferating cells after repetitive addition of rat granulocyte/macrophage colony-stimulating factor and rat interleukin (IL)-4 at a concentration of 5 ng/ml to the cultures. Fluorescence-activated cell sorter analysis performed at an early stage of development (day 6) revealed an immature phenotype with intermediate levels of major histocompatibility complex (MHC) class II expression and low levels of the costimulator molecules CD80 and CD86. Upon further culture, a strong upregulation of MHC class II, costimulatory and adhesion molecules could be observed, whereas macrophage marker antigens were downregulated. Late-stage BMDC (day 10) showed a high expression of MHC class I and II, ICAM-1, Ox62 and CD11c, and revealed a split pattern of B7-1 and B7-2. The cell yield was about 40% of the initially plated bone marrow cells with 80% MHC class II-high and less than 20% MHC class II-low positive cells. Full maturation of rat BMDC (day 12) with an almost uniform expression of B7 was achieved by subsequent subculture and further stimulation with rat tumour necrosis factor alpha (TNF-alpha), lipopolysaccharide (LPS) or soluble CD40 ligand (CD40L). Analysis of the cell supernatant revealed a strong IL-12 production after LPS or CD40L, and to a lesser extent after TNF-alpha stimulation. Additionally, LPS-treated, but not CD40L-treated BMDC secreted TNF-alpha into the supernatant. Early-stage BMDC sufficiently triggered a T cell receptor (TCR) downregulation, but did not stimulate naive T cells in an allogeneic mixed leukocyte reaction (MLR) and revealed a low stimulatory capacity in an antigen-specific T cell assay. In contrast, late-stage BMDC and especially fully mature BMDC strongly induced TCR internalisation, elicited high T cell responses in the allogeneic MLR similar to those obtained by mature rat spleen dendritic cells and efficiently activated antigen-specific T cells. In conclusion, this protocol allows easy access to large numbers of rat BMDC at defined maturation stages and selective studies for the manipulation of immune responses in rat models.