Zugl.: Konstanz, Univ., Diss., 2005

Universiẗat, Diss., 2004--Würzburg.

Die EAE ist eine Autoimmunerkrankung im Modell der Ratte. Sie ist eine inflammatorische , hauptsächlich durch CD4+ T-Zellen vermittelte Erkrankung des ZNS. In der Arbeit wurde die Interaktion von MHC, Peptid und TCR, welche typisch für diese Erkrankung sind, näher charakterisiert. / The EAE is an autoimmun desease in the model of the rat. It is characterized by inflammation of the CNS, mediated mainly by CD4+ T-cells. This work characterizes further the relevant interaction of MHC, peptid and TCR, typical found in the EAE.

Universiẗat, Diss., 2007--Giessen.

Universiẗat, Diss., 2007--Giessen.

Die Anzahl neurologischer Erkrankungen bei denen Autoantikörper gegen zentralnervöse An-tigene bekannt sind, hat in den letzten Jahren deutlich zugenommen. Allerdings gibt es nur für wenige dieser Erkrankungen hinreichende experimentelle Belege für eine pathogene Wir-kung der Autoantikörper. Zwei dieser Erkrankungen wurden im Rahmen dieser Arbeit näher untersucht: die Juvenile Neuronale Zeroid-Lipofuszinose (JNCL) mit Autoantikörpern gegen die 65 kD Isoform der Glutamatdecarboxylase und das Stiff Person Syndrom (SPS) mit Auto-antikörpern gegen Amphiphysin. Die phänotypische Charakterisierung der cln3 knockout-Maus, einem Mausmodell für die JNCL, zeigte eine progressive Verschlechterung der motorischen und koordinativen Fä-higkeiten, eingeschränktes reizbedingtes Lernen und gesteigertes angstähnliches Verhalten. Diese Symptome ähneln denen der humanen Erkrankung. Elektrophysiologisch konnte eine Antikörper-induzierte zerebelläre Dysfunktion identifiziert werden, die einer verminderten lokalen GABAergen Hemmung zugeordnet wird. Eine Reduktion der Antiköperproduktion im Tiermodell durch eine Depletion der Plasmazellen durch den Proteseinhibitor Bortezomib hatte einen positiven Effekt auf die Krankheitsentwicklung. Im zweiten experimentellen Teil der Arbeit wurde der Einfluss von Autoantikörpern gegen Amphiphysin von Patienten mit SPS auf die synaptische Transmission untersucht. Es zeigte sich hierbei in Patch-Clamp Experimenten eine Störung der GABAergen Übertragung v.a. bei hochfrequenter Stimulation, was im Einklang mit dem vermuteten Antikörper-induzierten Endozytosedefekt steht. Passiver Transfer von humanen Autoantikörpern gegen Amphiphysin induzierte angst-ähnliches Verhalten in Ratten, einem weiteren Kernsymptom des SPS. Aktive Immunisierung gegen Amphiphysin und anschließende Öffnung der Blut-Hirn-Schranke in Mäusen führte zu einer subklinischen Veränderung der Reflexverarbeitung von Ia Afferenzen auf Motoneurone im Rückenmark der Mäuse. Insgesamt konnten in zwei Erkrankungen des ZNS autoimmune Mechanismen identifi-ziert werden, die zu einer Antikörper-induzierten Fehlregulation der zentralen synaptischen Transmission führen. Diese Ergebnisse können wegweisend sein auch für die Erforschung der Pathophysiologie anderer Antikörper-assoziierte Erkrankungen des ZNS. / A growing number of neurological disorders are associated with autoantibodies targeting an-tigens within the central nervous system. Only in few cases experimental evidence corrobo-rates a pathogenic role of the autoantibodies. Two autoantibody associated diseases were investigated in detail in this work: the Juvenile neuronal ceroid lipofuscinoses (JNCL) with au-toantibodies against the 65kD isoform of the glutamate decarboxylase and the Stiff Person Syndrome (SPS) with autoantibodies against amphiphysin. The analysis of cln3 knockout mice, an animal model of the JNCL, revealed a phenotype resembling the human disorder, including progressive motor decline, limited cued learning and an increase in anxiety-related behavior. Electrophysiological analysis revealed an autoan-tibody mediated cerebellar dysfunction, which is best explained by diminished local GABAer-gic inhibition. A reduction of autoantibody production in the cln3 knockout mice by depletion of plasma cells after treatment with Bortezomib had a positive effect on the disease out-come. In the second experimental part, the effect of autoantibodies to amphiphysin from SPS patients on synaptic transmission was analysed. In patch-clamp experiments the GABAergic synaptic transmission was found to be disturbed primarily during high-frequent stimulation. This is in line with the hypothesized defect of synaptic vesicle endocytosis induced by autoan-tibodies. Passive transfer of human autoantibodies to rats induced anxiety-related behavior, a key symptom of SPS. The active immunization of mice against amphiphysin and subsequent opening of the blood brain barrier led to subclinical disturbances of the Ia afferent-motor neuron reflex pathway within the spinal cord. In conclusion, for two CNS disorders autoimmune mechanisms were identified leading to antibody-induced deregulations of central synaptic transmission. These findings may have implication for the research on pathomechanisms of other putative antibody mediated dis-orders.

Primary immune-mediated hemolytic anemia (IMHA) is an important cause of serious morbidity and mortality in dogs. Despite numerous studies examining the demographics, treatment options, and prognostic indicators of disease, the mechanisms that underlie immune dysregulation remain poorly understood. The purpose of this study was to directly identify unique erythrocyte membrane antigens in dogs diagnosed with primary IMHA. Blood samples were obtained from dogs presented to the Ontario Veterinary College Teaching Hospital with primary IMHA prior to treatment, and also from control dogs (healthy dogs and dogs with non-immunologic anemia). Antibodies bound to erythrocyte membranes were eluted using xylene. Immunoblots using patient eluates reacted against pooled canine erythrocyte lysates, and autologous patient plasma reacted against xylene eluates, were performed. These results were compared to results of similar experiments using samples from control dogs. Bands appearing in patient but not control samples were considered potential autoantigens, and were submitted for identification by liquid chromatography followed by tandem mass spectrometry. Samples from 13 dogs with primary IMHA, 4 dogs with non-immunologic anemia, and 2 healthy dogs, were analyzed. Immunoblotting confirmed the presence of immunoglobulin in eluates from all dogs. Semi-quantitatively, eluates from IMHA patients contained more immunoglobulin than those of control dogs. Mass spectrometry identified complement C3 in patient but not in control dog samples. Additional peptides identified by mass spectrometry in patient but not control dog samples included peroxiredoxin 2 and calpain. The former comprises a cytosolic hydrogen peroxide scavenger, and has been associated with erythrocyte membranes under oxidative stress conditions inducing spherocytosis. Calpain is a calcium-dependent protease that may become activated with oxidative stress and induce erythrocyte apoptosis. These findings suggest that oxidative stress and apoptosis contribute to the pathogenesis of canine IMHA. / OVC Pet Trust Fund, American Kennel Club Canine Health Foundation

Tierärztl. Hochsch., Diss., 2004--Hannover.

Immune-mediated polyneuropathies like chronic inflammatory demyelinating polyradiculoneuropathy or Guillain-Barré syndrome are rare diseases of the peripheral nervous system. A subgroup of patients harbors autoantibodies against nodal or paranodal antigens, associated with a distinct phenotype and treatment response. In a part of patients with pathologic paranodal or nodal immunoreactivity the autoantigens remain difficult or impossible to determine owing to limitations of the used detection approach - usually ELISAs (enzyme-linked-immunosorbent-assays) - and incomplete knowledge of the possible autoantigens. Due to their high-throughput, low sample consumption and high sensitivity as well as the possibility to display many putative nodal and paranodal autoantigens simultaneously, peptide microarray-based approaches are prime candidates for the discovery of novel autoantigens, point-of-care diagnostics and, in addition, monitoring of pathologic autoimmune response. Current applications of peptide microarrays are however limited by high false-positive rates and the associated need for detailed follow-up studies and validation. Here, robust peptide microarray-based detection of antibodies and the efficient validation of binding signals by on-chip neutralization is demonstrated. First, autoantigens were displayed as overlapping peptide libraries in microarray format. Copies of the biochips were used for the fine mapping of antibody epitopes. Next, binding signals were validated by antibody neutralization in solution. Since neutralizing peptides are obtained in the process of microarray fabrications, neither throughput nor costs are significantly altered. Similar in-situ validation approaches could contribute to future autoantibody characterization and detection methods as well as to therapeutic research. Areas of application could be expanded to any autoimmune-mediated neurological disease as a long-term vision. / Immunvermittelte Polyneuropathien wie die chronisch-inflammatorische demyelinisierende Polyradikuloneuropathie oder das Guillain-Barré-Syndrom sind seltene Erkrankungen des peripheren Nervensystems. Bei einem Teil dieser Patienten lassen sich Autoantikörper gegen nodale oder paranodale Antigene nachweisen, was mit einem bestimmten Phänotyp und Therapienansprechen assoziiert ist. Aufgrund der Einschränkungen verwendeter Detektionsansätze – üblicherweise ELISAs (Enzyme-linked Immunosorbent Assays) – sowie der unvollständigen Kenntnis potenzieller Autoantigene bleibt es bisher zum Teil schwierig bis unmöglich bei nachgewiesener pathologischer paranodaler bzw. nodaler Immunreaktivität die entsprechenden Autoantigene zu identifizieren. Die hohe Durchsatzleistung, der geringe Verbrauch an Probenmaterial, die hohe Sensitivität sowie die Möglichkeit zahlreiche mutmaßliche nodale und paranodale Autoantigene zeitgleich darzustellen machen Peptid-Microarray-basierte Ansätze zu wesentlichen Kandidaten für die Entdeckung neuer Autoantigene, für Point-of-Care-Diagnostik und darüber hinaus für das Monitoring pathologischer Autoimmunantworten. Durch die hohe Rate falsch positiver Ergebnisse sowie die damit verbundene Notwendigkeit detaillierter Folgestudien und Validierungen sind die gegenwärtigen Anwendungen von Peptid-Microarrays jedoch limitiert. In dieser Arbeit wird eine robuste, Peptid-Microarray-basierte Detektion von Antikörpern sowie eine effiziente Validierung der Bindungssignale mittels On-chip Neutralisation demonstriert. Zuerst wurden die Autoantigene als überlappende Peptidbüchereien im Microarray-Format dargestellt. Kopien der Biochips wurden für die Feinkartierung der Antikörper-Epitope verwendet. Mittels Antikörperneutralisation in Lösung wurden die Bindungssignale anschließend validiert. Da die neutralisierenden Peptide im Microarray- Herstellungsprozess gewonnen werden, ergeben sich weder beim Durchsatz noch bei den Kosten signifikante Änderungen. Vergleichbare In-situ-Validierungsansätze könnten zu künftigen Autoantikörper Charakterisierungen, Detektionsmethoden sowie zu therapeutischen Forschungsansätzen beitragen. Als langfristige Vision könnten die Anwendungsgebiete auf jede beliebige autoimmun-vermittelte neurologische Krankheit ausgeweitet werden.

Autoimmunity develops over an extended period of time as the result of an amalgamation of genetic, environmental, and immunologic events. Though the precise etiological factors leading to most autoimmune disease are awaiting consensus, a common thread of the autoimmune paradigm is the inappropriate activation of tissue-specific immune cells by one or more autoantigen, which begins the destruction of the tissue. To prohibit immunopathology and fine-tune the immune responses in healthy individuals, the stimulatory activities of effector/memory T (Teffs) cells must be counteracted by the suppressive mechanisms of regulatory T cells (Tregs). Thus, the potential to modulate the ratio between Teff and Tregs in autoimmune patients has been widely investigated with high hopes to permanently cure certain autoimmune diseases such as type 1 diabetes militus (T1D). Autoantigen therapies, which attempt to induce Tregs to suppress pathogenic effector cells in an autoantigen-specific manner, have shown efficacy in preventing T1D in mice, but have largely failed in clinical trials. One approach to improve the effectiveness of islet autoantigen vaccinations is to combine them with an additional modulator of the T cell response which favors a regulatory phenotype. In the work presented here, we asked whether the addition of anti-interleukin-7 receptor-alpha (anti-IL-7Rα) monoclonal antibodies (mAbs) to islet autoantigen immunizations would modulate the T cell response and prevent T1D in non-obese diabetic (NOD) mice. It was found that anti-IL-7Rα mAbs reduced the absolute numbers of islet antigen-specific T cells when immunized with islet peptide in conjunction with the commonly used vaccine adjuvant alum. Such treatments were also observed to increase nonspecific IL-2, IFN-𝛾, and IL-10 cytokine production, resulting in no improvement of T1D onset prevention. In another approach, we generated a conjugate vaccine by conjugating islet autoantigens to the immunogenic carrier protein, Keyhole Limpet Hemocyanin (KLH). We found that islet antigen-KLH (Ag-KLH) vaccination resulted in significant expansion of the desirable antigen-specific Tregs. Further, Ag-KLH immunization successfully delayed, and in some cases entirely prevented, T1D onset in NOD mice. Indicating that KLH-conjugated vaccine may represent a promising approach for future autoantigen therapies against autoimmunity. Interestingly, administration of anti-IL-7Rα mAbs did not improve these outcomes. To the contrary, we again observed excessive nonspecific cytokine production induced by IL-7Rα blockade that inhibited the beneficial effects of Ag-KLH vaccination. Taken together, we concluded that the addition of anti-IL-7Rα mAbs did not improve the efficacy of autoantigen vaccinations to prevent T1D. Significant work still remains to better characterize and isolate the beneficial effects of anti-IL-7Rα mAbs to treat autoimmunity.