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1
AZZONI, ELISABETTA. "IDENTIFICAZIONE DI AUTOANTIGENI IN PATOLOGIE NEUROLOGICHE AUTOIMMUNI." Doctoral thesis, Università degli studi di Trieste, 2005. http://hdl.handle.net/10077/14657.
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RUSSO, LUCIA. "Ricerca di nuovi autoantigeni nel diabete di tipo 1:immunoproteomica delle isole pancreatiche umane." Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2010. http://hdl.handle.net/2108/209587.
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Introduction: Type 1 Diabetes is the autoimmune form of diabetes mellitus and accounts for about 5-10%
of all cases of diabetes. It is one of the most common severe chronic childhood illnesses characterized by
insulin deficiency as a result of the progressive T-cell mediated destruction of pancreatic Langerhans islet -
cells. The factors initiating the destructive process are largely unknown but genetic and nongenetic factors
are involved. It manifests a biphasic progression with a preclinical phase “insulitis” and a clinical phase
“diabetes onset”. The preclinical phase is also characterized by the presence of circulating autoAbs targeted
to a different, yet limited, series of molecules that are expressed specifically and unspecifically in the
pancreatic islet -cells. To date, 5 are the known autoAbs: ICA (Islets Cell Ab), GADA (Glutammic Acid
Decarboxylase 65kDa Ab), IA-2A (Tyrosin phosphatase-like insulinoma Ag 2 Ab), IAA (Insulin Ab),
ZnT8A (Cation efflux Zinc Transporter 8 Ab). Search for T1D-autoAbs in patients with recent diagnosis of
diabetes is utilized for diagnostic purposes, and in first-degree relatives as predictive markers of disease.
The first type of autoAb, islet-cell autoantibody, “ICA” was described >35 years ago; however, the
entire panel of T1D-autoAgs is not complete at the moment. For example, it has been recently (2007)
identified a new major autoAg, ZnT8, that was found in 26% of T1D subjects previously classified as
autoAbs-negative. Because up to 5% of children with the clinical features/diagnosis of T1D test negative for
known autoAbs it is likely that other specific autoAbs/Ags remain to be identified.
Aim: We set up a search for new autoantigens involved in T1D using a method that has to be considered
innovative in its application to this organ-specific autoimmune disease, that we named immunoproteomic
approach; in addition we developed a radioimmunobinding assay to the goal of detecting serum reactivity on
a large-scale as validation of two new potential autoantigen molecules identified by immunoproteomic.
Materials and Methods: We studied the autoantibody repertoire of subject with diabetes of early onset (1-
10 years of age), divided in different groups of sera: A1) Subjects with diabetes negative to all autoAbs
(ICA, GADA, IA-2A, IAA, ZnT8A) and to the search for mutations in neonatal diabetes genes -INS,
KCNJ11, ABCC8 and GCK- (5 sera); B) T1D subjects positive only to IAA (4 sera); C) T1D subjects
positive only to ZnT8A (4sera) and D) T1D subjects positive only to GADA and IA-2A (8 sera). Sera from
patients with diabetes due to insulin mutations were used as “negative” control (group E1). The reactivity of
these sera was tested against cytoplasmic/membrane-enriched protein fraction from human pancreatic islets
or from exocrine pancreas sera in order to exclude any source of contamination of pancreatic islets from this
tissue. After bidimensional electrophoresis and classical Western Blot, images were acquired and spot
detection/matching performed by Progenesis software. Spots revealed by control sera (group E1) were
subtracted as “noise” in each categories of T1D-sera. For matching the reactivity between groups of sera we
detected specific spots of each group and common spots to two or more groups of the type 1 diabetic
patients. These spots were identified on the corresponding stain gel by Mass Spectrometry.
Among identified protein spots of islets two molecules (IOH-X1 and IOH-X2) were chosen to be
validated as new potential autoAgs with a “radioimmunobinding assay” in subjects with T1D developed
early (≤10 years of age). We studied IOH-X1 in five constructs (ORF: aa 1-445; N-terminal: aa 1-125;
Domain 2: aa 120-225; Central Fragment: aa 215-337 and C-terminal: aa 338-445), whereas IOH-X2 in
three constructs (ORF: aa 1-165; N-terminal: aa1-80 and C-terminal: aa 75-165).
ORF and selected constructs were amplified by PCR from corresponding human islet cDNA using
specific coupled of primers. They were cloned in an eucariotic vector and then expressed in an in vitrocoupled transcription/translation reticulocyte lysate reaction in presence of a radioactive aminoacid
(MetS35). The products purified on a sephadex column were used with human serum samples of diabetic and
control patients in an immunoprecipitation assay and finally the radioactivity was valued by an appropriate
instrument.
Results: Matching revealed that among protein spots detected in pancreatic islet cytoplasmic/membraneenriched protein fraction, 10 were in common between negative to 5 autoAbs sera (group A1) and those
IAA positive (group B), 24 were in common between group A1 and those ZnT8A positive sera (group C)
and 14 spots were common to all three groups. We then proceeded to identifying these protein spots by
MALDI MS/MS. Our initial analysis revealed the IOH-X1 protein among spots detected by all groups of
T1D-sera and the IOH-X2 molecule among protein spots detected only by the negative to 5 autoAb sera
group. Among potential autoantigens we also identified tubulins, a common spot by all three groups of sera,
and the Protein disulfide isomerase/PDIA3, as a specific spot of ZnT8A positive sera group. Tubulins and
PDI are known as uncommon antigens in T1D, already identified by others.
To confirm protein identity of spots we performed a preliminary validation by Western Blot. We
recognized with protein-specific primary antibodies the corresponding spots that were picked and identified
on stain-gel by Maldi MS/MS as IOH-X1 and IOH-X2. At the same time, to confirm the robustness of our
method, we set up the detection of a known autoAg spot by using GADA positive sera and subsequently a
polyclonal anti-GAD65 antibody on the same nitrocellulose.
In our preliminary study we tested only the ORF and the C-terminal construct of IOH-X1 protein
whereas the others remain to be analysed. In the C-terminus assay, applied on 100 patients with type 1
diabetes, 100 controls (obese) and 100 children with coeliac disease, when the 99° percentile cut-off was
utilized, we found that 24% patients with T1D and 9% patients with coeliac disease were positive to the
assay.
Conclusions: These results seem to indicate that our “immunoproteomic” method can detect known T1D
autoantigens as well as it’s feasible for novel autoantigens identification. Further analysis remain to be
performed to assess if these potential new biomarkers may be useful to improving the accuracy of T1D
diagnosis in high risk patients and general population, and to provide new targets for tolerance induction
strategies
3
Blöcker, Inga-Madeleine. "Epitopmapping des epidermalen Autoantigens BP230." Lübeck Zentrale Hochschulbibliothek Lübeck, 2010. http://d-nb.info/1003311334/34.
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Schumann, Frank. "Autoantigene bei der rheumatoiden Arthritis." [S.l.] : [s.n.], 2004. http://www.diss.fu-berlin.de/2004/199/index.html.
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Raith, Albert Johann. "Charakterisierung des potenziellen Autoantigens cRALBP." Diss., lmu, 2005. http://nbn-resolving.de/urn:nbn:de:bvb:19-47172.
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Frazer, Hilary Elizabeth. "Autoantigens in connective tissue disease." Thesis, Queen's University Belfast, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.328058.
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Tengnér, Pia. "Immune responses to the Ro and La autoantigens /." Stockholm, 1999. http://diss.kib.ki.se/1999/91-628-3590-4/.
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Mewar, Devesh. "Studies on autoantigens in rheumatoid arthritis." Thesis, University of Sheffield, 2003. http://etheses.whiterose.ac.uk/3455/.
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This thesis describes the use of phage display for the isolation of autoantigens in rheumatoid arthritis. The potential of the technology is demonstrated by the isolation of an autoantigen, eukaryotic translation elongation factor 1a1 (eEF 1 (x 1)) from a fibroblast cDNA library using rounds of selective enrichment with IgG from RA patients. Subsequently in order to isolate joint-specific antigens a phage-displayed cDNA library from rheumatoid pannus was generated and screened with analogous procedures. From the clones isolated, putative candidate autoantigens were identified. The presence of anti- eEF 1a1 autoantibodies in approximately 20% of patients with RA was confirmed and extended in larger panels of sera, and the finding of anti-eEF la1 shown to be relatively specific for RA. In contrast autoantibodies to the activation-induced negative regulator of T cells, CTLA-4 were not found in contrast to a previous report. The relevance of these findings for the use of antibodies in the diagnosis and prediction of disease characteristics in RA are discussed.
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Iwobi, Mabel Uzoamaka. "Salivary autoantigens in human rheumatic diseases." Thesis, University of Sussex, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.260048.
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Ciudad, García María Teresa. "Autoantigen Processing. How immunodominant thyroglobulin peptides are generated and presented by HLA-DR molecules." Doctoral thesis, Universitat Autònoma de Barcelona, 2015. http://hdl.handle.net/10803/325144.
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Las células dendríticas (DC) son las APCs más capaces, expresan toda la maquinaria necesaria y tienen una gran capacidad de presentación eficaz. Hemos establecido que el peptidoma de APCs profesionales puede ser estudiado sin tener que recurrir a líneas celulares, usando un pequeño número de DCs derivadas de monocitos que tienden a seleccionar péptidos de muy alta afinidad que forman familias de péptidos (nested sets) como norma. Sin embargo, también presentan péptidos no convencionales. 20% de los péptidos presentados por todos los alelos eran péptidos N o C terminales. La mayoría de los C terminales se encuentran justo en el extremo de la proteína, proceden de proteínas citosólicas y no se agrupan en nested sets. También muestran residuos preferentes de corte enzimático, en comparación con los péptidos internos.
Hemos utilizado moDCs y un modelo in vitro de digestión en un sistema "libre de células" (CFS) para analizar el procesamiento y la presentación de un autoantígeno tiroideo, la tiroglobulina. Tiroglobulina nativa o desnaturalizada capturada por MoDC generó muchos péptidos, con nested sets dominantes y ningún péptido derivado de los N o C terminales de la proteína. Se obtuvo un patrón similar cuando las DC se pulsaron con extractos de tejido tiroideo. Sin embargo, si la tiroglobulina se digería por las catepsinas coloidales (B, L y S) a pH 7,4 antes de pulsar las DCs, la presentación de péptidos de tiroglobulina quedó casi completamente eliminada. La gran cantidad de tiroglobulina intacta en el extracto de tejido podría ser la causa de estos datos y que la pre-digestión debe haber destruido todos los epítopos de tiroglobulina capaces de ser presentados. Sin embargo, cuando hicimos el mismo experimento utilizando el CFS, la tiroglobulina predigerida se presentó tan eficientemente como la proteína purificada. Por lo tanto, la pre-digestión no destruye los epítopos, pero en las DC los fragmentos pueden haber sido degradados antes de llegar al MIIC. Por lo tanto, el estado del antígeno es extremadamente relevante para su presentación por DCs pero el método CFS puede ayudar a identificar etapas del procesamiento que se pueden perder en el análisis de péptidos presentados por DC.
Dos nested sets abundantes, de alta afinidad afinidad y dominantes de la tiroglobulina fueron identificados. Uno, asociado a HLA-DR3 con el core VVVDPSIRH y uno asociado a HLA-DR15 con el core IMQYFSHFI. El grupo VVVDPSIRH contiene el péptido Tg2098, definido como inmunodominante en un modelo in vivo de tiroiditis en ratones transgénicos HLA-DR3. El mismo péptido se identificó en el peptidoma de tiroides de pacientes de Graves' HLA-DR3+. HLA-DR15 no se asocia negativamente a tiroiditis pero en un modelo similar, ratones transgénicos HLA-DR15 no desarrollaron la enfermedad usando las mismas condiciones. Curiosamente, en DCs HLA-DR15/DR3, la mayoría de los péptidos de tiroglobulina fueron presentados por HLA-DR15 pero péptidos con este core no se encontraron en muestras de tiroides HLA-DR15+ afectados. También se encontró un segundo nested set asociado a HLA-DR3, independiente de la fuente de antígeno o el método de procesamiento. Este segundo nested set, alrededor del core VIFDANAPV, no era tan abundante como el VVVDPSIRH y contenía un péptido (Tg1574), conocido por no generar respuestas de células T en el mismo modelo que el péptido Tg2098.
La diferencia funcional entre estos dos péptidos correlacionaron con dos características que son importantes en la definición de inmunodominancia, es decir, la sensibilidad a las catepsinas y a HLA-DM. Tg1574 era resistente a catepsinas mientras que Tg2098 era parcialmente sensible. Tras la digestión con varias combinaciones de catepsinas, Tg1574 no generó variantes intermedias. Sin embargo, Tg2098 fue recortado en los extremos, generando una serie de variantes, su core se mantuvo resistente a la proteolisis. Además, Tg1574 era mucho más sensible a HLA-DM que Tg2098. La interpretación de estos datos y la identificación de cuál de los dos péptidos de tiroglobulina se presenta preferentemente en el timo, serán necesarios para demostrar plenamente nuestra hipótesis.DCs are the most capable APCs, express all the necessary machinery and have a high capacity of efficient presentation. We have established that the peptidome from unpulsed professional APC can be studied without having to resort to cell lines, using small numbers of monocyte-derived DCs that tend to select very high-affinity peptides forming nested sets as a norm. However, they also presented unconventional peptides. 20% of the peptides presented by all alleles were N-terminal or C-terminal peptides. Most C-terminal peptides were located at the very extreme of the protein, pertained to cytosolic proteins and did not cluster in nested sets. They also show preferent cleavage residues, compared to internal peptides. We have used MoDCs and an in vitro digestion model set up as a cell-free system (CFS) to analyze the processing and presentation of an AITD autoantigen, thyroglobulin. Native or denatured purified thyroglobulin captured by MoDC generated many peptides, with dominant nested sets and no peptide derived from the N- or C-terminus of the protein. Very similar pattern was obtained wen MoDC were pulsed with colloid-enriched thyroid tissue extracts. Yet, if thyroglobulin was digested by the colloid cathepsins (B, L and S) at pH 7.4 prior to pulsing, MoDCs presentation of thyroglobulin peptides was almost completely abrogated. The large amount of intact thyroglobulin in the tissue extract could be accountable for these data and that the pre-digestion must have destroyed any thyroglobulin epitope that could be presented. However, when we did the same experiment using the CFS, predigested thyroglobulin was as efficiently presented as purified protein. Therefore, pre-digestion did not destroy epitopes, the fragments may have been degraded before reaching the MIIC in MoDCs. Thus, the state of the antigen is extremely relevant for its presentation by MoDCs but, the CFS method may help identifying steps of the processing events that may be lost when analyzing DC-presented peptides. Two abundant and high affinity dominant nested sets were identified from thyroglobulin. One, associated to HLA-DR3 with the VVVDPSIRH core and one associated to HLA-DR15 with the core IMQYFSHFI. The VVVDPSIRH set contains peptide Tg2098, defined as immunodominant in an in vivo model of thyroiditis in HLA-DR3 transgenic mice. The same peptide was identified in the HLA-DR3+ peptidome from GD patients' thyroids. HLA-DR15 is not negatively associated to AITD but in a similar model, HLA-DR15 transgenic mice did not develop the disease using the same conditions. Interestingly, in HLA-DR15/DR3 MoDCs, most thyroglobulin peptides were presented by HLA-DR15 but peptides with this core were not identified in HLA-DR15+ thyroid samples affected by GD. A second nested set associated to HLA-DR3 was also found, independent of the source of antigen or the processing method. This second HLA-DR3 nested set, around the VIFDANAPV core, was not as abundant as the VVVDPSIRH and contained a peptide (Tg1574), known not to generate T cell responses in the same EAT model as the Tg2098 peptide. The functional difference between these two peptides correlated with two characteristics that are important in the definition of immunodominance, i.e. sensitivity to cathepsins and to HLA-DM. Tg1574 was cathepsin-resistant whereas Tg2098 was partially sensitive. Upon digestion with several combinations of cathepsins, Tg1574 did not generate any intermediate variants and between 45 and 100% remained intact. In contrast, Tg2098 was trimmed at the peptide ends generating a number of variants, its core was maintained resistant to cleavage and only between 6 and 26% remained intact. In addition, Tg1574 was much more sensitive to HLA-DM than Tg2098. Interpreting these data after identifying what of the two peptides is preferentially presented in thymus will be necessary to fully demonstrate our hypothesis.
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Welin, Henriksson Elisabet. "Autoantigenic properties of the U1-70K protein /." Stockholm, 1998. http://diss.kib.ki.se/1998/91-628-2818-5/.
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Lundberg, Karin. "Arthritogenic and immunogenic properties of modified autoantigens /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-303-5/.
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Mulcahy, Anthony Francis. "The molecular cloning and characterisation of autoantigens." Thesis, University of Newcastle Upon Tyne, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.242453.
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Riemekasten, Gabriela. "Die Rolle eines SmD1Peptids bei der Entstehung von pathogenetisch bedeutsamen Autoantikörpern beim systemischen Lupus erythematodes." Doctoral thesis, Humboldt-Universität zu Berlin, Medizinische Fakultät - Universitätsklinikum Charité, 2003. http://dx.doi.org/10.18452/13858.
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Morgan, James. "Analysis of candidate retinal autoantigens in autoimmune uveitis." Thesis, University of Nottingham, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.415718.
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Gramaglia, Irene. "MHC mimicry with autoantigens : possible role in autoimmunity." Thesis, King's College London (University of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.244691.
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Neophytou, Pavlos Ioanni. "Approaches to the cloning of beta-cell autoantigens." Thesis, University of Cambridge, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.339471.
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Nandedkar, Neha Dhananjay. "Autoantigens and Insulin Receptor in Type 1 Diabetes." University of Toledo Health Science Campus / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=mco1556215572248676.
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Blöcker, Inga-Madeleine [Verfasser]. "Epitopmapping des epidermalen Autoantigens BP230 / Inga-Madeleine Blöcker." Lübeck : Zentrale Hochschulbibliothek Lübeck, 2010. http://d-nb.info/1003311334/34.
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Hu, Hong-gang. "Das Protein DEK im Chromatin menschlicher Zellen." Aachen Shaker, 2005. http://d-nb.info/988919354/04.
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McKee, Hayley Jane. "Aggrecan as a candidate autoantigen in rheumatoid arthritis." Thesis, University of Newcastle Upon Tyne, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.323445.
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Schubert, David. "Arthritisinduktion durch Immunität gegen ein systemisch exprimiertes Autoantigen." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2005. http://dx.doi.org/10.18452/15271.
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Ungefähr 1% der Bevölkerung der westlichen Welt leidet an rheumatoider Arthritis (RA). In einem T-Zellrezeptor transgenen Mausmodell, dem K/BxN Modell, wird die ubiquitär exprimierte Glukose-6-phosphat Isomerase (G6PI) von autoreaktiven T- und B-Zellen erkannt. Diese Mäuse entwickeln spontan eine antikörpervermittelte Arthritis, die viele Gemeinsamkeiten mit der humanen RA aufweist. In dieser Arbeit wurde untersucht, ob die Immunisierung mit G6PI eine Arthritis auch in nicht-transgenen Mäuse induzieren kann. Die Immunisierung mit heterologer humaner G6PI führte zur Entwicklung einer peripheren symmetrischen Polyarthritis in über 95% der DBA/1 Mäuse. Damit konnte zum ersten Mal gezeigt werden, dass eine Immunreaktion gegen ein systemisches exprimiertes Antigen zur einer organspezifischen Erkrankung in normalen nicht-transgenen Mäusen führt. Die Tiere entwickeln nach 9 Tagen eine Arthritis, die bis Tag 15 ihr Maximum erreicht hat und dann langsam abnimmt. Histologisch ist die Arthritis durch eine frühe Synovitis charakterisiert, gefolgt von massiven Erosionen des Knorpel und Knochens und anschließenden Reparaturprozessen, inklusive Fibrose. Obwohl die Tiere hohe Antikörpertiter entwickeln, kann die Arthritis nicht durch aufgereinigte Antikörper kranker Mäuse transferiert werden. Trotzdem spielen Antikörper eine große Rolle, da FcR-gamma-Kette defiziente Mäuse eine Arthritis mit geringer Inzidenz und mildem Verlauf entwickeln. Die Depletion der CD4 positiven Zellen verhindert die Entwicklung der Arthritis völlig, und eine Depletion während der Erkrankung führt zur schnellen Heilung. Daneben ist für die Entwicklung der Arthritis auch das Komplementsystem und TNF-alpha entscheidend, was durch Depletion von C5 bzw. durch Blockade von TNF-alpha gezeigt wurde. Zusätzlich wurde die Rolle der G6PI bei der Pathogenese der RA im Menschen untersucht. RA-Patienten zeigten keine erhöhte Frequenz von CD4 positiven T-Zellen, die nach Restimulation mit G6PI TNF-alpha oder IFN-gamma produzierten. Außerdem konnten keine erhöhten anti-G6PI Titer in Patienten mit RA oder anderen rheumatischen Erkrankungen detektiert werden.About 1% of the of the population of the western world suffers from rheumatoid arthritis (RA). In a T-cell receptor transgenic mouse model, the K/BxN model, the ubiquitously expressed glucose-6-phosphate isomerase (G6PI) is recognized by autoreactive T- and B-cells. These mice do develop an antibody dependent arthritis which show a lot of features of human RA. In this study it was examined whether arthritis could be induced in normal non-transgenic mice by immunization with G6PI. Immunization with heterologous human G6PI induces a symmetric polyarthritis in over 95% of DBA/1 mice. Therewith showing for the first time that an immune reaction against an systemic expressed antigen will lead to the development of an organ specific disease in normal non-transgenic mice. The mice develop arthritis 9d after immunization, reach their maximum at d15 and then arthritis slowly resolve. Histologically, the disease is characterized by early synovitis followed by massive cartilage destruction and erosions of the bones and later repair processes including fibrosis. Although antibody titers in the mice are high, transfer of purified anti-G6PI antibodies of sick mice alone do not transfer disease. Anyway, antibodies seem to play a major role since FcR-gamma-chain deficient mice develop disease with a much lower frequency and reduced severity. Depletion of CD4 positive T cells completely prevents disease and depletion during disease leads to an rapid resolution of arthritis. Aside this, complement and TNF-alpha is critical for the development of arthritis, which could shown by depletion of C5 and blockade of TNF-alpha. In addition, the role of G6PI in the pathogenesis of RA in humans was examined. RA patients do not show a higher frequency of CD4 positive T-cells which produce TNF-alpha and IFN-gamma after restimulation with G6PI. Furthermore, no elevated anti-G6PI titers could be detected in RA patients and in patients with other rheumatic diseases.
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Berger, Sara Maria Luginbühl Sarah. "Strukturelle und funktionelle Gemeinsamkeiten von Autoantigenen /." [S.l.] : [s.n.], 2008. http://opac.nebis.ch/cgi-bin/showAbstract.pl?sys=000279107.
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Ardesjö, Brita. "Autoantigens in Inflammatory Bowel Disease and Primary Sclerosing Cholangitis." Doctoral thesis, Uppsala universitet, Institutionen för medicinska vetenskaper, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8677.
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Inflammatory bowel disease (IBD) comprises diseases that are characterized by chronic or relapsing inflammation of the gastrointestinal tract. Primary sclerosing cholangitis (PSC) is an extraintestinal manifestation in IBD. Immunoreactivity against an autoantigen that is expressed both in the gastrointestinal tract and the biliary tract could be the link between these diseases. A possible source of such an antigen is goblet cells. Immunostainings of normal human tissues using IBD patient sera showed goblet cell immunoreactivity against goblet cells in all parts of the gastrointestinal tract. The most frequent immunostaining was found against goblet cells in the appendix against which 84% (42/50) of IBD patients compared to 8% (4/50) of healthy blood donors showed immunoreactivity. To identify the corresponding antigen we used three different approaches, investigation of immunoreactivity to different candidate proteins compared to IBD sera, immunoscreening of an appendiceal cDNA library, and immunoprecipitation of protein lysates from mucin producing cells followed by SDS-PAGE and 2D gel electrophoresis. These approaches led to the identification of several candidate autoantigens of which complement C3 is the most promising. A novel staining pattern with strong immunoreactivity to granules and the apical membrane of biliary epithelial cells was identified with 35% (12/34) of PSC sera compared to none of healthy controls (n=28). Screening of a cDNA library from normal human choledochus identified PDZ domain containing 1 (Pdzk1) and Glutathion S transferase theta 1 (GSTT1) as potential candidates. Pdzk1 is an interesting candidate which is expressed in the intestinal tract and bile ducts. GSTT1 antibodies were not specific for PSC and are thought to develop as an alloimmune response in patients with the GSTT1-null genotype. In conclusion, we have identified specific immunoreactivity to goblet cells and biliary epithelial cells using sera from patients with IBD and PSC respectively. We have also identified several potential autoantigens.
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Zhu, Jianhui. "Induction of the cellular expression of human Ro autoantigens." Thesis, McGill University, 1995. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=39900.
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Ro autoantigens are intracellular ribonucleoproteins of unknown function. Autoantibodies to these antigens are detected frequently in patients with systemic lupus erythematosus (SLE) and involved in the pathogenesis of lupus skin lesions. Although the mechanisms responsible for the induction of these autoantibodies and immunologic tissue damage are unclear, one possibility is that Ro autoantigens are expressed on the cell surface and induce an immune response. Cell surface expression of Ro antigens has been reported previously following ultraviolet B (UVB) irradiation or estrogen stimulation of human keratinocytes. In this thesis, the effect of human cytomegalovirus (CMV) infection on the surface expression of Ro antigens and calreticulin on human fibroblasts and keratinocytes was investigated using a fixed cell enzyme-linked immunoassay (ELISA), immunofluorescence, flow cytometry (FACS) analysis and immunoblotting. CMV infection of cultured human fibroblast cells was found to increase the cell surface expression of calreticulin, but not 60kD/Ro antigen. However, CMV infection, in combination with UVB irradiation, synergistically induced the expression of 52kD/Ro antigen, but not 60kD/Ro or calreticulin, on the surface of these cells. This enhanced expression of 52kD/Ro autoantigen on CMV and UVB treated cells was significant and specific, compared with untreated cells, cells infected with CMV or irradiated with UVB only, and cells subjected to other treatments including low pH. These studies were then extended to human keratinocytes, which are relevant to the skin disease associated with the presence of anti-Ro antibodies in SLE. Human CMV was demonstrated to be capable of infecting keratinocytes in vitro and induced the surface expression of 60kD/Ro antigen, but not 52kD/Ro and calreticulin, on human keratinocytes. As there was no increase in total cellular expression of 60kD/Ro antigen after viral infection, 60kD/Ro antigen appears to be redistributed from the
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Schirmer, Jan Henrik [Verfasser], and Friedrich [Akademischer Betreuer] Haag. "Charakterisierung putativer humaner Autoantigene / Jan Henrik Schirmer. Betreuer: Friedrich Haag." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2012. http://d-nb.info/1025150910/34.
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Härkönen, Taina. "Cross-reactive immune responses between enteroviruses and islet cell autoantigens." Helsinki : University of Helsinki, 2002. http://ethesis.helsinki.fi/julkaisut/mat/bioti/vk/harkonen/.
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Duan, Fei. "Immune rejection of mouse tumors expressing mutated self /." Access full-text from WCMC:, 2007. http://proquest.umi.com/pqdweb?did=1432805051&sid=8&Fmt=2&clientId=8424&RQT=309&VName=PQD.
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Schmalzl, Thomas Benedikt. "Charakterisierung des Autoantigens "mitochondriale Malat-Dehydrogenase" bei der equinen rezidivierenden Uveitis." Diss., lmu, 2005. http://nbn-resolving.de/urn:nbn:de:bvb:19-36151.
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Ekwall, Olov. "Pteridine dependent hydroxylases as autoantigens in autoimmune polyendocrine syndrome type 1." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2001. http://publications.uu.se/theses/91-554-4941-7/.
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Arif, Sefina. "Autoantibodies and autoantigens in type 1 diabetes and premature ovarian failure." Thesis, King's College London (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.285177.
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Chung, Chen-Yen. "CD4+ T cell responses to myelin autoantigens : activation, memory and tolerance." Thesis, University of Edinburgh, 2009. http://hdl.handle.net/1842/4013.
Full textAbstract:
Experimental autoimmune encephalomyelitis (EAE) is a CD4+ T cell mediated autoimmune disease of the central nervous system and shares many characteristics with multiple sclerosis (MS). Induction of EAE is mediated by myelin reactive CD4+ T helper (Th) cells, particularly Th1 and Th17 cells, which can be provoked by the immunization with myelin derived protein (or peptide) and Toll-like receptor (TLR) stimulus (eg, complete Freund¡s adjuvant, CFA). If given an injection of soluble peptide before immunization, mice do not develop EAE (they are tolerant). This approach has been widely applied, evoking tolerance in primary responses (i.e., in naive T cells). Therefore the first hypothesis of this thesis is that peptide induced protection from EAE is a result from T cell deletion or / and anergy. As MS patients have ongoing disease and over 85% of MS patients develop a relapsing-remitting course, memory T cells are key targets when considering peptide-induced tolerance as a therapeutic strategy. Thus, a model for ¡memory EAE¡ was established to test a second hypothesis that the myelin reactive memory T cells can be controlled by the administration of soluble peptide. Here, adoptive transfer of T cells from T cell receptor transgenic mice (2D2) recognizing myelin oligodendrocyte glycoprotein 35-55 (pMOG) was used to investigate the pMOG-reactive memory responses. Soluble pMOG administration could induce a transient expansion of 2D2 T cells followed by their loss through apoptosis. A model using double immunization was established by immunizing mice first with pMOG together with unmethylated CpG oligonucleotide (CpG) as an adjuvant, and subsequently immunizing with pMOG in CFA. This produced EAE with early onset and high incidence compared to mice which received pMOG/CFA only. Cells from mice that received the double immunization protocol produced high levels of IFN-γ, suggesting that memory T cell responses have been triggered in the mice. Administration of soluble peptide before secondary immunization could ameliorate EAE, indicating that memory T cells are susceptible to tolerance induction. pMOG-reactive memory T cells were further assessed by isolating CD4+ CD25- CD44high CD62Llow cells from pMOG-experienced 2D2 mice. These cells showed early and high production of IFN-γ, and early but transient production of IL-2, compared with naive population. These data provide basic information relevant to translating peptide-induced T cell tolerance from mice to humans.
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Bourquin, Carole. "Vaccination with DNA encoding a myelin autoantigen exacerbates experimental autoimmune encephalitis." Diss., lmu, 2000. http://nbn-resolving.de/urn:nbn:de:bvb:19-106.
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Ottosson, Lars. "Molecular characterization of the Ro52 autoantigen and its disease related epitopes /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-185-7/.
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Davies, Marie Louise. "Autoantigen specific T cell responses in relation to systemic lupus erythematosus." Thesis, University of Birmingham, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.394161.
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Macdonald, Pamela. "The role of apoptosis in autoantigen presentation during primary biliary cirrhosis." Thesis, University of Newcastle Upon Tyne, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.405349.
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Whitehead, Clark Merrill. "The identification and characterization of two human autoantigens, HsEg5 and ASE-1." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape10/PQDD_0027/NQ49554.pdf.
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Al-bukhari, Talat Abdullah. "Investigation of the epitope specificities of antibodies to islet β-cell autoantigens." Thesis, University of Nottingham, 2001. http://eprints.nottingham.ac.uk/13868/.
Full textAbstract:
Glutamic acid decarboxylase-65 (GAD-65) and the tyrosine phosphatase-like protein IA-2 are major targets of autoimmunity in type I diabetes mellitus (type I DM), stiff-man syndrome (SMS) and autoimmune polyendocrine syndrome (APS). In this study, the precise epitopes in GAD-65 of three different mouse monoclonal antibodies (MoAbs) (N-terminal MoAb within amino acid residues 4-17. C-terminal MoAb within amino acid residues 572-585 and GAD-6). human monoclonal antibody (b96.11 huAb) and polyclonal antibodies (SMS patients' sera) were investigated. The precise epitopes in IA-2 of two different MoAbs (76B and 76F) were also investigated. These precise epitope investigations were performed using two different phage-displayed random peptide libraries with different characteristics (T7 phage library gene X C9C and linear 9-mers, and M13 filamentous phage library gene III C7C and linear 12-mers and gene VIII 5C4C4). Sequencing of N-terminal and C-terminal MoAb reactive peptides which were obtained from the successful biopanning using M13 gene III linear 12-mers phage library and were selected by high affinity binding in immnuo-blotting assay using nitro-cellulose membranes and in capture ELISA revealed that the most significant motif recognised was P-G-x-x-x-W-S-F and F-L-I-x-E-I/V/L-D-x-L respectively which showed conservative substitutions and may correspond to the position 4-10 amino acids (aa) of GAD-65 (P-G-S-G-F-W-S-F) and to the position 573-581 aa of GAD-65 (F-L-I-E-E-I-E-R-L), respectively. To further define the N-terminal MoAb epitope, sequencing of N-terminal MoAbs reactive peptides which were obtained from the successful biopanning using M13 gene VIII 5C4C4 phage Iibrary and were selected by high affinity binding in immnuo-blotting assay using nitro-cellulose membrane and in capture ELISA. revealed a motif of S-T-P which does not correspond to 4-17 aa of GAD-65 and does not overlap with the previous motif of the N-terminal MoAb. i.c. P-G-S-G-F-W-S-F (4-10 aa) of GAD-65. Therefore, the M13 pVIlI 5C4C4 worked with N-terminal MoAb by expressing a relevant sequence for the N-terminal MoAb but which is unlike its epitope in GAD-65. To further define the N-terminal MoAb epitope sequencing of N-terminal MoAb reactive peptides, which were obtained from the successful biopanning using T7 gene X linear 9-mers phage library and were selected by high affinity binding in immnuo-blotting assay using nitro-cellulose membrane revealed a motif of P-X-X-G which may correspond to 4-7 aa of GAD-65 (P-G-S-G) which overlaps with the previous motif P-G-S-G-F-W-S-F (4-10aa). Sequencing of GAD-6 MoAb reactive peptides which were obtained from the successful biopanning using T7 gene X C9C phage library and were selected by high affinity binding in immnuo-blotting assay using nitro-cellulose membrane and in ELISA, revealed two different motifs of R/K-L/ A/I-x-K and M-x-x-A which showed conservative substitutions and may correspond to the position 525-52X and of GAD-65 (R-L-S-K) and to the position 523-526 aa of GAD-65 (M-S-R-L) respectively, which overlap with each other. To further define the GAD-6 epitope sequencing of GAD-6 reactive peptides which were obtained from the successful biopanning using T7 gene X linear 9-mers phage library and were selected by high affinity binding in immnuo-blotting assay using nitro-cellulose membrane revealed a motif of R-x-x-K, which may correspond to 525-528 aa of GAD-65 (R-L-S-K) and overlaps with the previous motif of the GAD-6 selected from T7 gene X C9C phage library. To further define the GAD-6 epitope sequencing of GAD-6 reactive peptides which were obtained from the successful biopanning using M13 gene VIII 5C4C4 phage library and were selected by high affinity binding in immnuo-blotting assay using nitro-cellulose membrane and in capture ELISA revealed a motif or M-x-x-A which may correspond to 523-526 aa of GAD-65 (M-S-R-L), and overlaps with the previous motif selected from T7 gene X C9C phage library. Thus, the overall motif or GAD-6 may correspond to 523-528 aa of GAD-65 (M-S-R-L-S-K). To further define the GAD-6 epitope, sequencing or GAD-6 reactive peptides which were obtained from the successful biopanning using MI3 gene III C7C and linear 12-mers phage libraries, did not show a clear motif and did not show reactivity with GAD-6 by capture ELISA. A possible explanation for this is that the peptides which are specific to GAD-6 are not present in these M13 pIII phage libraries. Sequencing of b96.11 huAb reactive peptides, which were obtained from the successful biopanning using M13 gene III linear 12-mers phage library and were selected by moderate affinity binding in immnuo-blotting assay using nitro-cellulose membrane revealed two different motifs of IV-T/S-A/G/L-T/S-A\L and S-T/S-G/A/L/I which showed conservative substitutions and may correspond to the position 332-336 aa of GAD-65 (V-S-A-T-A) and to the position 338-340 aa of GAD-65 (T-T-V). respectively. Thus the overall motif of b96.11 might correspond to 332-340 aa of GAD-65. In a capture ELISA system for the detection of GAD-65 specific antibodies, b78 huAb bound slightly better with GAD-6 (rather than N-terminal MoAb ) as the capture MoAb but b96.11 bound much better with GAD-6 as the capture MoAb. This might suggest that GAD-6 does interfere with b78 huAb binding to GAD-65 more than it does with b96.11 huAb. However it must also suggest that the GAD-6 and b78 huAb epitopes are not directly overlapping. Sequencing of SMS sera reactive peptides which were obtained from the successful biopanning using M13 gene III linear 12-mers phage library and were selected by moderate affinity binding in immnuo-blotting assay using nitro-cellulose membrane revealed two different motifs of L/A-A-x-T/S-R/H/K and of T/S-T-V-I/L-F-E-L/G/I/V/A-H/K-L/G-x-K/R which showed conservative substitutions and may correspond to the position 371-375 aa of GAD-65 (L-L-M-S-R) and to the position 463-472 aa of GAD-65 (T-T-G-F-E-A-H-V-D-K). respectively. as public epitopes of SMS patients' sera. Sequencing of 76B and 76F MoAbs reactive peptides, which were obtained from the successful biopanning using T7 gene X C9C and M 13 gene III linear 12-mers phage libraries and were selected by high affinity binding in immnuo-blotting assay using nitro-cellulose membrane and in ELISA, revealed that the most significant motif recognised was D-x-K-P-L-S and F-x-Y-Q, respectively which may correspond to the position 477-482 aa of IA-2 (D-Q-K-P-L-S) and to the position 626-629 aa of IA-2 (F-E-Y-Q) respectively. The studies described in this thesis have shown that the epitope mapping of different antibodies on GAD-65 and IA-2 may help to understand the relationship between antigenicity and structure in these autoantigens which are targets in type I DM and related disorders (e.g. SMS and APS).
39
Dromey, James Anthony. "Lymphocytes and the autoimmune response to islet autoantigens in Type 1 diabetes." Thesis, King's College London (University of London), 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.400496.
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Tremble, Jennifer Margaret. "Cellular and humoral responses to islet cell autoantigens in insulin dependent diabetes." Thesis, King's College London (University of London), 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.286687.
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PAILLARD-COURTIN, SOPHIE. "Etude du mecanisme d'interaction de la proteine ku, autoantigene nucleaire, avec l'adn." Paris 7, 1992. http://www.theses.fr/1992PA077146.
Full textAbstract:
Nous avons etudie en detail les modalites d'interaction de la proteine ku avec l'adn. La proteine se deplace en glissant le long de la molecule d'adn apres avoir reconnu les extremites de l'adn. Les extremites sont egalement indispensables pour la dissociation du complexe adn/proteine. La proteine ku est donc fixee sur l'adn a la maniere d'une perle enfilee sur le fil d'un collier. D'autre part, la proteine une fois fixee sur l'adn, est la cible d'un clivage proteolytique specifique
42
Franke, Claudia. "Das Protein La/SS-B: Vom Autoantigen zur Zielstruktur für die Immuntherapie." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2011. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-27655.
Full textAbstract:
Das La-Protein wurde als Autoantigen bei Autoimmunpatienten, die an SLE oder Sjögren-Syndrom erkrankt sind, entdeckt. Es kommt in phosphorylierter Form im Zellkern aller Eukaryonten vor und nimmt Aufgaben bei der Faltung, Prozessierung und nukleären Retention von RNA-Polymerase III-Transkripten wahr. Unter normalen zellulären Bedingungen ist das La-Protein außerdem in der Lage, zwischen Zellkern und Zytoplasma zu pendeln. Bei Zellstress, der nach UV-Exposition oder während einer viralen Infektion entsteht, wird das Protein verstärkt im Zytoplasma beobachtet, wo es an der Cap-unabhängigen Translation zellulärer und ggf. viraler Proteine beteiligt ist. Wird in der Zelle daraufhin Apoptose induziert, so ist das La-Protein auf der Zellmembran bzw. in apoptotischen Körperchen nachweisbar.
Ein wesentlicher Bestandteil dieser Arbeit war die Untersuchung verschiedener monoklonaler anti-La-Antikörper. Einige wenige konnten durch wiederholte Immunisierung von Mäusen mit rhLa-Protein generiert werden. Im Gegensatz dazu resultierte die einmalige Übertragung von gegen das hLa-Protein aktivierten CD4+ T-Zellen auf eine hLaTg-Maus in der Gewinnung mehrerer La-spezifischer Antikörper. Die Sequenzanalyse der Gene, die für die variablen Antikörperdomänen codieren, bestätigte, dass es sich um individuell rekombinierte und hypermutierte Immunglobuline handelt. Die Antikörper zeichneten sich außerdem durch unterschiedliche Eigenschaften bei der Bindung von humanem und murinem La-Protein in der Immunfluoreszenz, im Immunoblot oder während der Immunpräzipitation aus. Für die IgG-Antikörper konnten die Epitopbereiche innerhalb des La-Proteins eingegrenzt werden. Auffällig waren die kurzen linearen Peptidepitope, die von den auf konventionelle Art erzeugten Antikörpern gebunden wurden. Hingegen erkannten alle Antikörper, die aus dem adoptiven T-Zell-Transfer hervorgegangen waren, Konformationsepitope. Darüber hinaus wurde gezeigt, dass einige mAks aber auch anti-La-Patientenseren die reduzierte von der oxidierten Form des La-Proteins unterscheiden können. Unerwartet ist die Erkenntnis, dass sich offensichtlich zahlreiche B-Zellen mit anti-La-Spezifität von wenigen variablen Ketten ableiten und dass diese bei einer herkömmlichen Immunisierung entweder nicht aktiviert werden (und deshalb nicht in der Milz zu finden sind) oder sogar eliminiert werden.
Der Import des La-Proteins in den Zellkern wird durch die klassischen Transportmoleküle Karyopherin-α und Karyopherin–β vermittelt. Für den Shuttlingprozess muss das Protein auch wieder aus dem Kern exportiert werden. Da es kontroverse Daten bezüglich eines Crm1-abhängigen Kernexports gab, wurde das Shuttlingverhalten von GFP-La-Fusionsproteinen in dieser Arbeit genauer analysiert. Mit Hilfe von Heterokaryonexperimenten konnte bestätigt werden, dass sowohl das hLa- als auch das mLa-Protein zwischen humanen und murinen Zellkernen pendeln kann und dass der Export unabhängig von Crm1 stattfindet. Aufgrund der kurzen Verweildauer im Zytoplasma schienen die Proteine quantitativ im Zellkern vorzuliegen, doch ein Teil konnte stets in den im Heterokaryon enthaltenen Nachbarzellkernen detektiert werden. Die Verwendung von N-terminal deletierten La-Fragmenten, die alle über das C-terminale NLS verfügten, gab Aufschluss über die Regulation des Shuttlings. Es zeigte sich, dass die Menge des exportierten Proteins von einem nukleären Retentionspartner festgelegt wird, der das La-Protein bindet und dadurch im Zellkern festhält. Wird diese Assoziation aufgehoben, gelangt das La-Protein in das Zytoplasma. Dort ist es allerdings nicht detektierbar, da das NLS einen umgehenden Import zurück in den Zellkern hervorruft. Zusätzlich wurde die Auswirkung von zellulärem Stress (z. B. durch ROS) auf die intrazelluläre Lokalisation des Proteins untersucht. Unter oxidativen zellulären Bedingungen wird einerseits die Wechselwirkung mit dem nukleären Retentionspartner aufgehoben und andererseits findet kein Kernimport über Karyopherin-α mehr statt. Aus diesem Grund reichert sich das La-Protein nun verstärkt im Zytoplasma an.
Darüber hinaus wurde nachgewiesen, dass das La-Protein von apoptotischen Zellen freigesetzt wird und daraufhin auf die Membran von Nachbarzellen binden kann. Die Bindungs-eigenschaften wurden mit rhLa-Protein genauer untersucht. Das La-Protein war auf Endothel- und Epithelzellen nachweisbar und die Bindung fand sowohl bei Inkubation auf Eis als auch bei 37 °C statt. Da das La-Protein auch über DNA-Bindungseigenschaften verfügt, war es in der Lage, DNA auf der Zelloberfläche zu immobilisieren. Innerhalb von PBMCs wurde es selektiv auf Antigen-präsentierenden Zellen nachgewiesen. Diese Eigenschaften lassen eine Beteiligung des Proteins bei der Induktion von anti-dsDNA-Antikörpern in Autoimmunpatienten vermuten.
Es ist bekannt, dass die Bedingungen (Virusinfektion, UV-Exposition), die zur Translokation des La-Proteins auf die Zelloberfläche führen, bei SLE-Patienten Krankheitsschübe auslösen können. Bisher wurden anti-La-Autoantikörper aber eher nicht als pathophysiologisch erachtet, da sie bei der Bindung an bereits apoptotische Zellen keine weiteren Schäden verursachen können. Jedoch wurde in dieser Arbeit gezeigt, dass das La-Protein apoptotischer Zellen auf der Oberfläche von lebenden Zellen in der Umgebung nachgewiesen werden kann. Daran könnten anti-La-Autoantikörper binden und eine Komplement- oder NK-Zell-vermittelte Zerstörung der Nachbarzellen hervorrufen. Dadurch entstehen zusätzliche Gewebeschäden. Im Chromfreisetzungstest waren NK-Zellen tatsächlich in der Lage, La-dekorierte Zielzellen Antikörper-abhängig zu lysieren, sofern zusätzliche in vitro Stimuli präsent waren, die z. B. eine virale Infektion simulierten.
Die Immuntherapie von Tumoren ist auf bestimmte Zielstrukturen auf den Tumorzellen angewiesen, über welche die Wirkstoffe spezifisch zu den maligne transformierten Zellen gebracht werden. Die Therapeutika, die sich oft von mAks gegen diese Zielstrukturen ableiten, müssen für verschiedene Tumorarten individuell entwickelt werden. Da das La-Protein von apoptotischen Zellen freigesetzt wird und auf die Membran benachbarter (bestrahlungsresistenter) Zellen binden kann, ist es in Kombination mit einer vorangegangenen Bestrahlung als universelle Zielstruktur für die Immuntherapie nutzbar. Aus diesem Grund wurde unter Verwendung eines ausführlich in dieser Arbeit charakterisierten anti-La-Antikörpers ein rekombinantes bispezifisches Antikörperderivat entwickelt. Es ist in der Lage, das La-Protein auf der Oberfläche von Tumorzellen zu binden und auf diesen zytotoxische T-Lymphozyten zu immobilisieren. Durch die Quervernetzung werden die T-Lymphozyten aktiviert und induzieren in den Zielzellen Apoptose. Das neue Antikörperderivat verspricht eine vielseitige Anwendung in Kombination mit Strahlentherapie oder auch mit rekombinanten Antikörpermolekülen, die gegen spezifische Zielstrukturen auf den Tumorzellen gerichtet sind.
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Locke, James. "The role of joint-associated autoantigen-specific immune responses in rheumatoid arthritis." Thesis, University of Newcastle Upon Tyne, 2012. http://hdl.handle.net/10443/1773.
Full textAbstract:
Rheumatoid arthritis (RA) is an autoimmune disease characterised by chronic inflammation at multiple joints. Because of the association of the condition with certain alleles of the DRB1 gene which encodes the β-chains of human leukocyte antigen (HLA)-DR molecules, it has been postulated that auto-reactive CD4+ T-cells - specific for joint-associated autoantigens presented in the context of these RA-associated HLA-DR molecules – are involved in the initiation or perpetuation of the disease process. In addition, citrullination of autoantigen-derived peptides may enhance their binding affinity to RA-associated HLA-DR molecules, implicating citrullinated autoantigens in RA pathogenesis. Since RA primarily affects the joints, proteins of articular origin and those expressed in the joints such as human cartilage glycoprotein 39 (HCgp39), type II collagen (CII), aggrecan, α-enolase, fibrinogen and vimentin are candidate autoantigens in RA. However, it has been difficult to consistently detect T-cell responses to these candidate RA autoantigens in RA patients. In this project, I hypothesised that T-cell responses to candidate autoantigens are heightened in RA patients compared to healthy subjects and that the T-cell responses of RA patients are pro-inflammatory while those of healthy subjects are not. I first optimised an experimental system for detecting autoantigen-specific T-cell responses and applied the protocol to measure proliferative and cytokine production responses of peripheral blood mononuclear cells (PBMC) from RA patients, healthy subjects and disease controls to unmodified and citrullinated whole protein and peptide forms of the candidate RA autoantigens mentioned above. Responses to the candidate autoantigens were readily detectable in RA patients, healthy subjects and disease controls. Furthermore, I found no evidence of increased immunogenicity of citrullinated peptides versus their unmodified counterparts. Thus the study failed to provide evidence that T-cell responses to either unmodified or citrullinated autoantigens are important in RA pathogenesis.
44
Falconer, Jane. "T cell recognition of proteoglycan aggrecan : a candidate autoantigen in inflammatory arthritis." Thesis, University of Newcastle Upon Tyne, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.525062.
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Franke, Claudia. "Das Protein La/SS-B: Vom Autoantigen zur Zielstruktur für die Immuntherapie." Doctoral thesis, Technische Universität Dresden, 2009. https://tud.qucosa.de/id/qucosa%3A25267.
Full textAbstract:
Das La-Protein wurde als Autoantigen bei Autoimmunpatienten, die an SLE oder Sjögren-Syndrom erkrankt sind, entdeckt. Es kommt in phosphorylierter Form im Zellkern aller Eukaryonten vor und nimmt Aufgaben bei der Faltung, Prozessierung und nukleären Retention von RNA-Polymerase III-Transkripten wahr. Unter normalen zellulären Bedingungen ist das La-Protein außerdem in der Lage, zwischen Zellkern und Zytoplasma zu pendeln. Bei Zellstress, der nach UV-Exposition oder während einer viralen Infektion entsteht, wird das Protein verstärkt im Zytoplasma beobachtet, wo es an der Cap-unabhängigen Translation zellulärer und ggf. viraler Proteine beteiligt ist. Wird in der Zelle daraufhin Apoptose induziert, so ist das La-Protein auf der Zellmembran bzw. in apoptotischen Körperchen nachweisbar.
Ein wesentlicher Bestandteil dieser Arbeit war die Untersuchung verschiedener monoklonaler anti-La-Antikörper. Einige wenige konnten durch wiederholte Immunisierung von Mäusen mit rhLa-Protein generiert werden. Im Gegensatz dazu resultierte die einmalige Übertragung von gegen das hLa-Protein aktivierten CD4+ T-Zellen auf eine hLaTg-Maus in der Gewinnung mehrerer La-spezifischer Antikörper. Die Sequenzanalyse der Gene, die für die variablen Antikörperdomänen codieren, bestätigte, dass es sich um individuell rekombinierte und hypermutierte Immunglobuline handelt. Die Antikörper zeichneten sich außerdem durch unterschiedliche Eigenschaften bei der Bindung von humanem und murinem La-Protein in der Immunfluoreszenz, im Immunoblot oder während der Immunpräzipitation aus. Für die IgG-Antikörper konnten die Epitopbereiche innerhalb des La-Proteins eingegrenzt werden. Auffällig waren die kurzen linearen Peptidepitope, die von den auf konventionelle Art erzeugten Antikörpern gebunden wurden. Hingegen erkannten alle Antikörper, die aus dem adoptiven T-Zell-Transfer hervorgegangen waren, Konformationsepitope. Darüber hinaus wurde gezeigt, dass einige mAks aber auch anti-La-Patientenseren die reduzierte von der oxidierten Form des La-Proteins unterscheiden können. Unerwartet ist die Erkenntnis, dass sich offensichtlich zahlreiche B-Zellen mit anti-La-Spezifität von wenigen variablen Ketten ableiten und dass diese bei einer herkömmlichen Immunisierung entweder nicht aktiviert werden (und deshalb nicht in der Milz zu finden sind) oder sogar eliminiert werden.
Der Import des La-Proteins in den Zellkern wird durch die klassischen Transportmoleküle Karyopherin-α und Karyopherin–β vermittelt. Für den Shuttlingprozess muss das Protein auch wieder aus dem Kern exportiert werden. Da es kontroverse Daten bezüglich eines Crm1-abhängigen Kernexports gab, wurde das Shuttlingverhalten von GFP-La-Fusionsproteinen in dieser Arbeit genauer analysiert. Mit Hilfe von Heterokaryonexperimenten konnte bestätigt werden, dass sowohl das hLa- als auch das mLa-Protein zwischen humanen und murinen Zellkernen pendeln kann und dass der Export unabhängig von Crm1 stattfindet. Aufgrund der kurzen Verweildauer im Zytoplasma schienen die Proteine quantitativ im Zellkern vorzuliegen, doch ein Teil konnte stets in den im Heterokaryon enthaltenen Nachbarzellkernen detektiert werden. Die Verwendung von N-terminal deletierten La-Fragmenten, die alle über das C-terminale NLS verfügten, gab Aufschluss über die Regulation des Shuttlings. Es zeigte sich, dass die Menge des exportierten Proteins von einem nukleären Retentionspartner festgelegt wird, der das La-Protein bindet und dadurch im Zellkern festhält. Wird diese Assoziation aufgehoben, gelangt das La-Protein in das Zytoplasma. Dort ist es allerdings nicht detektierbar, da das NLS einen umgehenden Import zurück in den Zellkern hervorruft. Zusätzlich wurde die Auswirkung von zellulärem Stress (z. B. durch ROS) auf die intrazelluläre Lokalisation des Proteins untersucht. Unter oxidativen zellulären Bedingungen wird einerseits die Wechselwirkung mit dem nukleären Retentionspartner aufgehoben und andererseits findet kein Kernimport über Karyopherin-α mehr statt. Aus diesem Grund reichert sich das La-Protein nun verstärkt im Zytoplasma an.
Darüber hinaus wurde nachgewiesen, dass das La-Protein von apoptotischen Zellen freigesetzt wird und daraufhin auf die Membran von Nachbarzellen binden kann. Die Bindungs-eigenschaften wurden mit rhLa-Protein genauer untersucht. Das La-Protein war auf Endothel- und Epithelzellen nachweisbar und die Bindung fand sowohl bei Inkubation auf Eis als auch bei 37 °C statt. Da das La-Protein auch über DNA-Bindungseigenschaften verfügt, war es in der Lage, DNA auf der Zelloberfläche zu immobilisieren. Innerhalb von PBMCs wurde es selektiv auf Antigen-präsentierenden Zellen nachgewiesen. Diese Eigenschaften lassen eine Beteiligung des Proteins bei der Induktion von anti-dsDNA-Antikörpern in Autoimmunpatienten vermuten.
Es ist bekannt, dass die Bedingungen (Virusinfektion, UV-Exposition), die zur Translokation des La-Proteins auf die Zelloberfläche führen, bei SLE-Patienten Krankheitsschübe auslösen können. Bisher wurden anti-La-Autoantikörper aber eher nicht als pathophysiologisch erachtet, da sie bei der Bindung an bereits apoptotische Zellen keine weiteren Schäden verursachen können. Jedoch wurde in dieser Arbeit gezeigt, dass das La-Protein apoptotischer Zellen auf der Oberfläche von lebenden Zellen in der Umgebung nachgewiesen werden kann. Daran könnten anti-La-Autoantikörper binden und eine Komplement- oder NK-Zell-vermittelte Zerstörung der Nachbarzellen hervorrufen. Dadurch entstehen zusätzliche Gewebeschäden. Im Chromfreisetzungstest waren NK-Zellen tatsächlich in der Lage, La-dekorierte Zielzellen Antikörper-abhängig zu lysieren, sofern zusätzliche in vitro Stimuli präsent waren, die z. B. eine virale Infektion simulierten.
Die Immuntherapie von Tumoren ist auf bestimmte Zielstrukturen auf den Tumorzellen angewiesen, über welche die Wirkstoffe spezifisch zu den maligne transformierten Zellen gebracht werden. Die Therapeutika, die sich oft von mAks gegen diese Zielstrukturen ableiten, müssen für verschiedene Tumorarten individuell entwickelt werden. Da das La-Protein von apoptotischen Zellen freigesetzt wird und auf die Membran benachbarter (bestrahlungsresistenter) Zellen binden kann, ist es in Kombination mit einer vorangegangenen Bestrahlung als universelle Zielstruktur für die Immuntherapie nutzbar. Aus diesem Grund wurde unter Verwendung eines ausführlich in dieser Arbeit charakterisierten anti-La-Antikörpers ein rekombinantes bispezifisches Antikörperderivat entwickelt. Es ist in der Lage, das La-Protein auf der Oberfläche von Tumorzellen zu binden und auf diesen zytotoxische T-Lymphozyten zu immobilisieren. Durch die Quervernetzung werden die T-Lymphozyten aktiviert und induzieren in den Zielzellen Apoptose. Das neue Antikörperderivat verspricht eine vielseitige Anwendung in Kombination mit Strahlentherapie oder auch mit rekombinanten Antikörpermolekülen, die gegen spezifische Zielstrukturen auf den Tumorzellen gerichtet sind.
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Sanati, Mohammad Hossein. "Cloning and characterisation of a novel mitochondrial autoantigen associated with multiple sclerosis." Thesis, Sanati, Mohammad Hossein (1996) Cloning and characterisation of a novel mitochondrial autoantigen associated with multiple sclerosis. PhD thesis, Murdoch University, 1996. https://researchrepository.murdoch.edu.au/id/eprint/52268/.
Full textAbstract:
Multiple Sclerosis (MS) is a chronic neurological disease of the central nervous system, characterised by a cellular immune response in the early stages followed by demyelination. Although the cause of MS is unknown, there is much evidence that points to MS as an autoimmune disease. To test the hypothesis that an autoantigen is involved in MS, we screened a Xgtl 1 human foetal spinal cord cDNA library using IgG 6 from patients with MS. From screening 2x10 recombinant phage, 6 positive clones were identified. Proteins expressed by these phage on bacterial lysate plates appeared to react specifically with pooled MS IgG but not with pooled IgG from normal human sera. The positive clones were amplified by the polymerase chain reaction and sequenced. After searching GenBank, analysis of the sequences showed a high percent similarity between three clones and the mitochondrial gene encoding the ND4 component of human NADH:ubiquinone reductase (Complex I).
Because the region cloned was associated with an active site in Complex I, submitochondrial particles were purified from different sources and the inhibition of Complex I was tested in presence of MS IgG. The pooled MS IgG strongly inhibited Complex I activity in sub-mitochondrial particles with decylubiquinone as a substrate. A peptide CLANSNYERTHSR, which is part of the ND4 protein in Complex I, was conjugated with a maleimido-thiol bond to diphtheria toxoid and used as an autoantigen and as an immunogen in a rabbit. To remove any IgG which bound to diphtheria toxoid and the bovine serum albumin blocking agent in the ELISA, sera were pre-adsorbed before being incubated with the peptide conjugate. About 20 % of patients with MS had antibody to the peptide and when present, the level was found to fluctuate. This is the first time that a mitochondrially encoded protein has been shown to be an autoantigen. Autoantibodies to the ND4 peptide were found in patients with Leber’s Hereditary Optic Neuropathy (LHON), which is of great interest as the commonest mutation site in this mitochondrial disease is adjacent to the epitope. In preliminary experiments the autoantibodies were also found in association with other autoantibodies in diseases where there is known to be extensive damage to cells.
There is a clear molecular mimicry between the ND4 epitope and a predicted epitope in a plasmid which encodes a surface protein in spirochaetes. Cross reaction between the spirochaete protein and the ND4 peptide was demonstrated by using the rabbit antibody to ND4.
While the most likely explanation for the presence of the autoantibody to Complex I is that it is a marker of damage to mitochondria in MS, it may have a secondary role in the pathogenesis if a spirochaete triggers an autoimmune response to cells presenting peptides from damaged mitochondrial proteins on their surface.
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Buse, Barbara Christina. "Identifikation von sechs potenziellen Autoantigenen bei Hunden mit dilatativer Kardiomyopathie." Diss., lmu, 2007. http://nbn-resolving.de/urn:nbn:de:bvb:19-72257.
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Lapointe, Elvy. "Caractérisation du système autoantigène/autoanticorps Sa dans la polyarthrite rhumatoïde." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape4/PQDD_0021/MQ56925.pdf.
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Lapointe, Elvy. "Caractérisation du système autoantigène/autoanticorps Sa dans la polyarthrite rhumatoïde." Sherbrooke : Université de Sherbrooke, 2000.
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Nguyen, Thi Bang Tam. "Regulation der Genexpression des Diabetes-assoziierten Autoantigens IA-2 in INS-1 Betazellen." [S.l.] : [s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=966456009.
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