Genetik und Immunologie der Antikörper gegen citrullinierte Peptidantigene bei der rheumatoiden Arthritis

Für die Diagnose der rheumatoiden Arthritis (RA) haben sich in den letzten 10 Jahren die Autoantikörper gegen citrullinierte Peptidantigene (ACPA) etabliert, doch hinsichtlich ihrer Entstehung und ihrer Beteiligung an der Pathogenese der RA ist bisher sehr wenig bekannt. Die vorliegende Arbeit untersucht die Immunglobulin G (IgG) Subklassenantwort der ACPA hinsichtlich ihrer immunologischen und genetischen Charakteristika.For the last 10 years the antibodies against citrullinated peptide antigens (ACPA) have been established for the diagnosis of rheumatoid arthritis (RA). Whereas a good tool for rheumatologists, only a little is known about their development and their involvement in RA pathogenesis. The present study investigates the ACPA immunoglobulin G (IgG) subclass response in respect of its immunological and genetic features

Einsendeaufgabe: „Projektmanagement - Methoden und Instrumente“ ; Beispiel: bio-medizinische Grundlagenforschung (Mausmodell der rheumatoiden Arthritis)

Diese Arbeit stellt die Prüfungsleistung zu dem Kurs „Projektmanagement: Methoden und Instrumente“ dar. Herr Dipl.-Ing. (FH) Thomas Sadewasser leitete den Kurs. In dieser Arbeit soll ein kurzer Überblick über einige Methoden des Projektmanagements anhand eines Projekts der bio-medizinischen Grundlagenforschung gegeben werden. Die vorliegende Arbeit wurde ausschließlich mit Open-Source-Software erstellt und bietet daher zusätzlich Softwarevorschläge - als Alternative für die zumeist teuren proprietären Lösungen - mit denen jedermann eigene Projekte im Betriebssystem seiner Wahl planen kann

Integrative modeling of transcriptional regulation in response to antirheumatic therapy

<p>Abstract</p> <p>Background</p> <p>The investigation of gene regulatory networks is an important issue in molecular systems biology and significant progress has been made by combining different types of biological data. The purpose of this study was to characterize the transcriptional program induced by etanercept therapy in patients with rheumatoid arthritis (RA). Etanercept is known to reduce disease symptoms and progression in RA, but the underlying molecular mechanisms have not been fully elucidated.</p> <p>Results</p> <p>Using a DNA microarray dataset providing genome-wide expression profiles of 19 RA patients within the first week of therapy we identified significant transcriptional changes in 83 genes. Most of these genes are known to control the human body's immune response. A novel algorithm called TILAR was then applied to construct a linear network model of the genes' regulatory interactions. The inference method derives a model from the data based on the Least Angle Regression while incorporating DNA-binding site information. As a result we obtained a scale-free network that exhibits a self-regulating and highly parallel architecture, and reflects the pleiotropic immunological role of the therapeutic target TNF-alpha. Moreover, we could show that our integrative modeling strategy performs much better than algorithms using gene expression data alone.</p> <p>Conclusion</p> <p>We present TILAR, a method to deduce gene regulatory interactions from gene expression data by integrating information on transcription factor binding sites. The inferred network uncovers gene regulatory effects in response to etanercept and thus provides useful hypotheses about the drug's mechanisms of action.</p

Expert-independent classification of mature B-cell neoplasms using standardized flow cytometry: a multicentric study

Reproducible expert-independent flow-cytometric criteria for the differential diagnoses between mature B-cell neoplasms are lacking. We developed an algorithm-driven classification for these lymphomas by flow cytometry and compared it to the WHO gold standard diagnosis. Overall, 662 samples from 662 patients representing 9 disease categories were analyzed at 9 laboratories using the previously published EuroFlow 5-tube-8-color B-cell chronic lymphoproliferative disease antibody panel. Expression levels of all 26 markers from the panel were plotted by B-cell entity to construct a univariate, fully standardized diagnostic reference library. For multivariate data analysis, we subsequently used canonical correlation analysis of 176 training cases to project the multidimensional space of all 26 immunophenotypic parameters into 36 2-dimensional plots for each possible pairwise differential diagnosis. Diagnostic boundaries were fitted according to the distribution of the immunophenotypes of a given differential diagnosis. A diagnostic algorithm based on these projections was developed and subsequently validated using 486 independent cases. Negative predictive values exceeding 92.1% were observed for all disease categories except for follicular lymphoma. Particularly high positive predictive values were returned in chronic lymphocytic leukemia (99.1%), hairy cell leukemia (97.2%), follicular lymphoma (97.2%), and mantle cell lymphoma (95.4%). Burkitt and CD101 diffuse large B-cell lymphomas were difficult to distinguish by the algorithm. A similar ambiguity was observed between marginal zone, lymphoplasmacytic, and CD102 diffuse large B-cell lymphomas. The specificity of the approach exceeded 98% for all entities. The univariate immunophenotypic library and the multivariate expert-independent diagnostic algorithm might contribute to increased reproducibility of future diagnostics in mature B-cell neoplasms

The Prerequisites for Central Tolerance Induction against Citrullinated Proteins in the Mouse.

To assess the prerequisites for negative selection of peptidylcitrulline-specific T cells in the thymus. In detail, we here analyzed murine medullary thymic epithelial cells for the expression of peptidylarginine deiminases (PAD) and subsequent citrullination.Medullary thymic epithelial cells were sorted, their mRNA was isolated and the expression of Pad genes was analyzed by quantitative PCR. Citrullination was detected by Western Blot in lysates of sorted medullary thymic epithelial cells and histologically by immunofluorescence of thymic thin sections.Pad2 and Pad4 are the main Pad isoforms expressed in mature medullary thymic epithelial cells of the mouse and their levels of expression are comparable to that of insulin (Ins2), another highly and promiscuously expressed protein in the thymus. Citrullination was detected in medullary thymic epithelial cells as shown by Western Blot and immunofluorescence.Even though we here show that the murine thymus harbors the prerequisites for central tolerance to PAD and citrullinated peptides, it remains an open question whether the emergence of peptidylcitrulline-specific T cells and of autoantibodies recognizing citrullinated epitopes is caused by a failure of central or peripheral tolerance mechanisms

Mycophenolate mofetil prevents the delayed T cell response after pilocarpine-induced status epilepticus in mice.

Growing clinical and laboratory evidence corroborates a role for the immune system in the pathophysiology of epilepsy. In order to delineate the immune response following pilocarpine-induced status epilepticus (SE) in the mouse, we monitored the kinetics of leukocyte presence in the hippocampus over the period of four weeks. SE was induced following a ramping protocol of pilocarpine injection into 4-5 weeks old C57BL/6 mice. Brains were removed at days 1-4, 14 or 28 after SE, and the hippocampi were analyzed via flow cytometry, via quantitative reverse transcriptase PCR (qRT-PCR) and via immunohistochemistry. Epileptogenesis was confirmed by Timm staining of mossy fiber sprouting in the inner molecular layer of the dentate gyrus. The flow cytometry data revealed a biphasic immune response following pilocarpine-induced SE with a transient increase in activated CD11b+ and F4/80+ macrophages within the first four days replaced by an increase in CD3+ T-lymphocytes around day 28. This delayed T cell response was confirmed via qRT-PCR and via immunohistochemistry. In addition, qRT-PCR data could show that the delayed T cell response was associated with an increased CD8/CD4 ratio indicating a cytotoxic T cell response after SE. Intriguingly, early intervention with mycophenolate mofetil administration on days 0-3 after SE prevented this delayed T cell response. These results show an orchestrated immunological sequela and provide evidence that the delayed T cell response is sensitive to early immunomodulatory intervention

The Anti-mutated Citrullinated Vimentin Response Classifies Patients with Rheumatoid Arthritis into Broad and Narrow Responders

ABSTRACT. Objective. Autoantibodies against citrullinated peptide antigens (ACPA) are routinely determined to diagnose rheumatoid arthritis (RA) and are predictive of a more severe course of the disease. We here set out to address an involvement of ACPA in the pathogenesis of RA and investigated the recognition pattern of antibodies against 2 citrullinated antigens in more detail. Methods. The sera of 77 patients fulfilling the American College of Rheumatology criteria for RA were analyzed for subclass titers of anti-mutated citrullinated vimentin (MCV) and anticyclic citrullinated peptide (CCP) antibodies by combining subclass specific detection antibodies with commercially available CCP and MCV ELISA plates. Cross-reactivities between anti-MCV and anti-CCP antibodies were detected using a sequential ELISA system. Results. IgG1, IgG3, and IgG4 titers among anti-MCV and anti-CCP antibodies correlated significantly. Cross-reactivity of MCV-specific antibodies against CCP could be detected in 8 of 16 patients&apos; sera; however, cross-binding of MCV-specific IgG4 was weaker compared to total IgG. Conclusion. The inherent capacity of IgG4 to exchange F(ab) arms provides insight into the anti-MCV antibody diversity and suggests a classification of ACPA positive patients into broad and narrow responders

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ABSTRACT. Objective. Autoantibodies against citrullinated peptide antigens (ACPA) are routinely determined to diagnose rheumatoid arthritis (RA) and are predictive of a more severe course of the disease. We here set out to address an involvement of ACPA in the pathogenesis of RA and investigated the recognition pattern of antibodies against 2 citrullinated antigens in more detail. Methods. The sera of 77 patients fulfilling the American College of Rheumatology criteria for RA were analyzed for subclass titers of anti-mutated citrullinated vimentin (MCV) and anticyclic citrullinated peptide (CCP) antibodies by combining subclass specific detection antibodies with commercially available CCP and MCV ELISA plates. Cross-reactivities between anti-MCV and anti-CCP antibodies were detected using a sequential ELISA system. Results. IgG1, IgG3, and IgG4 titers among anti-MCV and anti-CCP antibodies correlated significantly. Cross-reactivity of MCV-specific antibodies against CCP could be detected in 8 of 16 patients&apos; sera; however, cross-binding of MCV-specific IgG4 was weaker compared to total IgG. Conclusion. The inherent capacity of IgG4 to exchange F(ab) arms provides insight into the anti-MCV antibody diversity and suggests a classification of ACPA positive patients into broad and narrow responders

Interplay between P. gingivalis, F. nucleatum and A. actinomycetemcomitans in murine alveolar bone loss, arthritis onset and progression

Abstract Increasing evidence supports the association of periodontitis with rheumatoid arthritis. Even though a prominent role has been postulated for Porphyromonas gingivalis, many bacterial species contribute to the pathogenesis of periodontal disease. We therefore investigated the impact of Porphyromonas gingivalis as well as other major pathobionts on the development of both, periodontitis and arthritis in the mouse. Pathobionts used - either alone or in combination - were Porphyromonas gingivalis, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomintans. Periodontitis was induced via oral gavage in SKG, DBA/1 and F1 (DBA/1 × B10.Q) mice and collagen-induced arthritis was provoked via immunization and boost with bovine collagen type II. Alveolar bone loss was quantified via micro computed tomography, arthritis was evaluated macroscopically and histologically and serum antibodies were assessed. Among the strains tested, only F1 mice were susceptible to P. gingivalis induced periodontitis and showed significant alveolar bone loss. Bone loss was paralleled by antibody titers against P. gingivalis. Of note, mice inoculated with the mix of all three pathobionts showed less alveolar bone loss than mice inoculated with P. gingivalis alone. However, oral inoculation with either F. nucleatum or A. actinomycetemcomintans alone accelerated subsequent arthritis onset and progression. This is the first report of a triple oral inoculation of pathobionts combined with collagen-induced arthritis in the mouse. In this interplay and this particular genetic setting, F. nucleatum and A. actinomycetemcomitans exerted a protective impact on P. gingivalis induced alveolar bone loss. By themselves they did not induce periodontitis yet accelerated arthritis onset and progression

Citrullination is present in isolated mTEC and within medullary areas of the thymus.

<p>(A) Western blot to detect citrullination (clone F95) and the reference protein (GAPDH) in lysates from FACS sorted mTECs, muscle (positive control) and liver (negative control). The arrow indicates the band present in all samples that was used for a relative expression analysis with respect to GAPDH (arrowhead): muscle: 21-fold, mTEC: 28-fold and liver: 0.75-fold. Numbers within the band of the protein marker lane indicate protein sizes in kDa. (B) Identification of mTEC in thin sections of murine thymi by dual immunofluorescence staining with anti-Aire (green) and anti-cytokeratin 5 (red) or UEA-1 (red). (C) Detection of citrullination in mTEC areas of thymic thin sections. mTECs were stained with UEA-1 (red) and RA-specific epitopes are labeled with an ACPA-high serum mixture consisting of three RA patient's sera (green) (upper right panel). A mix of two age-matched healthy control sera was used as negative control (upper left panel). Staining with a rabbit anti-citrulline serum (green) shows citrullination within mTEC areas (lower right panel). Rabbit normal serum was used as a negative control (lower left panel). All results shown were derived from NMRI thymi.</p