Untersuchungen zur Verwendungder Next Generation Sequenziertechnologiefür die Analyse von Antikörpergenenbei Gesunden und Autoimmunpatienten.

Antikörper sind Glykoproteine der adaptiven Immunantwort. Ihre Funktion ist die Abwehr von Fremdkörpern, z.B. Krankheitserregern, jedoch führen Störungen des Immunsystems zu Autoimmunerkrankungen. Die Next Generation Sequenziertechnologie ist geeignet, einen schnellen Vergleich zwischen Gesunden und an Rheumatoider Arthritis (RA) leidenden Patienten auf Antikörpergenebene zu ermöglichen. Davor müssen jedoch die experimentellen sowie bioinformatischen Methoden für den Einsatz der Technologie etabliert werden. Im experimentellen Teil dieser Arbeit wurde mittels quantitativer Real-Time Polymerase-Kettenreaktion (qRT-PCR) und Polymerase-Kettenreaktion (PCR) die Basis für den erfolgreichen Testlauf mittels Genome Sequencer FLX Instrument geschaffen. Im bioinformatischen Teil wurde mit Hilfe von Perl-Skripts und einer speziell für die Next Generation Sequenzierdaten programmierten PostgreSQL-Datenbank eine schnelle Analyse von großen Mengen an Next Generation Sequenzierdaten ermöglicht. Somit konnten signifikante Differenzen in der Expression von IgG und IgD mittels qRT-PCR festgestellt werden. Außerdem wurden anhand der Next Generation Sequenzierdaten Unterschiede bezüglich der Antikörperentstehung, -reifung sowie -diversität zwischen Gesunden und RA-Patienten im Hinblick auf die Gennutzung von VH4(DP63) bei IgG und IgD festgestellt. Weitere Untersuchungen sind auf biologischer Ebene notwendig, um die bioinformatisch ermittelten Unterschiede in der Gennutzung zu erforschen und möglicherweise für die therapeutische Anwendung bei RA geeignete Antikörper zu identifizieren.Abkürzungsverzeichnis .............................................................................................................3 I. Abkürzungen .............................................................................................................3 II. Nukleotidbezeichnungen ....................................................................................................6 1. EINLEITUNG ..............................................................................................................7 1.1. Antikörper: Bildung, Struktur, Funktion ...................................................................7 1.2. Antikörper und ihre Rolle bei Autoimmunerkrankungen ......................................11 1.3. Next Generation Sequenzierung ................................................................................12 1.4. Bioinformatische Analyse von Antikörpergensequenzen ........................................14 1.5. Zielsetzung ...................................................................................................................16 2. MATERIAL .............................................................................................................................17 2.1. Experimenteller Teil .....................................................................................................17 2.1.1. Laborausstattung ....................................................................................................17 2.1.2. Verbrauchsmaterial .................................................................................................17 2.1.3. Chemikalien, Enzyme, Kit-Systeme, Lösungen, Puffer .........................................17 2.1.4. Primer .....................................................................................................................19 2.1.5. RNA- und cDNA-Proben .......................................................................................20 2.1.6. DNA-Längenstandards ...........................................................................................21 2.2. Bioinformatischer Teil .................................................................................................22 2.2.1. Hardware ................................................................................................................22 2.2.2. Software .................................................................................................................22 2.2.3. Internetquellen: Anwendungen und Datenbanken .................................................23 2.2.4. Antikörper-Sequenzen ............................................................................................23 2.2.5. Primer .....................................................................................................................24 3. METHODEN ...........................................................................................................................25 3.1. Experimenteller Teil .....................................................................................................25 3.1.1. DNA-Verdau und Reverse Transkription ...............................................................25 3.1.2. qRT-PCR ................................................................................................................26 3.1.3. Sequenzierungsvorbereitung ..................................................................................31 3.1.4. Polymerase-Kettenreaktion ....................................................................................35 3.1.5. Aufreinigung von PCR-Produkten .........................................................................37 3.1.6. DNA-Gelelektrophorese ........................................................................................38 3.1.7. Überblick der durchgeführten Experimente ...........................................................38 3.2. Bioinformatischer Teil .................................................................................................40 3.2.1. Unterschiedliche Vortests mittels Perl-Programme ...............................................40 3.2.2. Primer-Analysen .....................................................................................................41 3.2.3. VBASE2 „Statistic Analysis“ .................................................................................42 3.2.4. nextIGbase Datenbank ...........................................................................................42 3.2.5. Statistische Auswertung .........................................................................................42 4. ERGEBNISSE .........................................................................................................................43 4.1. Experimentelle Untersuchungen ...............................................................................43 4.1.1. Effizienz-Test und Kreuzreaktivitätstest der qRT-PCR-Primer ............................43 4.1.2. Mengenverhältnis zwischen IgG und IgD bei Gesunden und Patienten ................45 4.1.3. Einsatz von cDNA Pools ........................................................................................46 4.1.4. Ermittlung der cDNA Stoffmenge in den Proben ..................................................47 4.1.5. Vorbereitung der Next Generation Amplicon Sequenzierung ................................48 4.2. Bioinformatische Untersuchungen .............................................................................54 4.2.1. Genauigkeit der Identifizierung von V Genen in gekürzten Sequenzen und Ermittlung der minimal erforderlichen Read-Länge ..............................................54 4.2.2. V-Gen-Analyse auf Nukleotid-Homopolymeren ...................................................57 4.2.3. Bestimmung der Cut Off Werte in den scFv-Testsequenzen ..................................59 4.2.4. Anzahl der durch Fw-Primer verursachten Mutationen in den scFv-Testsequenzen .....................................................................................60 4.2.5. V-Gen-Coverage ....................................................................................................61 4.3. Auswertung der Next Generation Sequenzierdaten .................................................66 4.3.1. VBASE2 „Statistic Analysis“ und nextIGbase .......................................................66 4.3.2. Vergleich der Antikörpersequenzen zwischen Gesunden und Autoimmunpatienten ......................................................................................67 5. DISKUSSION ..........................................................................................................................78 6. ZUSAMMENFASSUNG ............................................................................................................95 7. DANKSAGUNG .......................................................................................................................96 8. LITERATURVERZEICHNIS ....................................................................................................97 9. ANHANG ..............................................................................................................................10

Suppression of p75 Neurotrophin Receptor Surface Expression with Intrabodies Influences Bcl-xL mRNA Expression and Neurite Outgrowth in PC12 Cells

Background: Although p75 neurotrophin receptor (p75NTR) is the first neurotrophin receptor isolated, its diverse physiological functions and signaling have remained elusive for many years. Loss-of-function phenotypic analyses for p75NTR were mainly focused at the genetic level; however these approaches were impacted by off-target effect, insufficient stability, unspecific stress response or alternative active splicing products. In this study, p75NTR surface expression was suppressed for the first time at the protein level by endoplasmic reticulum (ER) retained intrabodies. Results: Three monoclonal recombinant antibody fragments (scFv) with affinities in the low nanomolar range to murine p75NTR were isolated by antibody phage display. To suppress p75NTR cell surface expression, the encoding genes of these scFvs extended by the ER retention peptide KDEL were transiently transfected into the neuron-like rat pheochromocytoma cell line PC12 and the mouse neuroblastoma x mouse spinal cord hybrid cell line NSC19. The ER retained intrabody construct, SH325-G7-KDEL, mediated a downregulation of p75NTR cell surface expression as shown by flow cytometry. This effect was maintained over a period of at least eight days without activating an unfolded protein response (UPR). Moreover, the ER retention of p75NTR resulted in downregulation of mRNA levels of the anti-apoptotic protein Bcl-xL as well as in strong inhibition of NGF-induced neurite outgrowth in PC12 cells. Conclusion: The ER retained intrabody SH325-G7-KDEL not only induces phenotypic knockdown of this p75NTR but als

Targeted Inactivation of Cerberus Like-2 Leads to Left Ventricular Cardiac Hyperplasia and Systolic Dysfunction in the Mouse

Previous analysis of the Cerberus like 2 knockout (Cerl2(-/-)) mouse revealed a significant mortality during the first day after birth, mostly due to cardiac defects apparently associated with randomization of the left-right axis. We have however, identified Cerl2-associated cardiac defects, particularly a large increase in the left ventricular myocardial wall in neonates that cannot be explained by laterality abnormalities. Therefore, in order to access the endogenous role of Cerl2 in cardiogenesis, we analyzed the embryonic and neonatal hearts of Cerl2 null mutants that did not display a laterality phenotype. Neonatal mutants obtained from the compound mouse line Cer2(-/-)Fundacao para a Ciencia e Tecnologia (FCT); IBB/CBME [PEst-OE/EQB/LA0023/2011]; FCT [SFRH/BD/62081/2009]info:eu-repo/semantics/publishedVersio

Rise and Fall of an Anti-MUC1 Specific Antibody

So far, human antibodies with good affinity and specificity for MUC1, a transmembrane protein overexpressed on breast cancers and ovarian carcinomas, and thus a promising target for therapy, were very difficult to generate.A human scFv antibody was isolated from an immune library derived from breast cancer patients immunised with MUC1. The anti-MUC1 scFv reacted with tumour cells in more than 80% of 228 tissue sections of mamma carcinoma samples, while showing very low reactivity with a large panel of non-tumour tissues. By mutagenesis and phage display, affinity of scFvs was increased up to 500fold to 5,7×10(-10) M. Half-life in serum was improved from below 1 day to more than 4 weeks and was correlated with the dimerisation tendency of the individual scFvs. The scFv bound to T47D and MCF-7 mammalian cancer cell lines were recloned into the scFv-Fc and IgG format resulting in decrease of affinity of one binder. The IgG variants with the highest affinity were tested in mouse xenograft models using MCF-7 and OVCAR tumour cells. However, the experiments showed no significant decrease in tumour growth or increase in the survival rates. To study the reasons for the failure of the xenograft experiments, ADCC was analysed in vitro using MCF-7 and OVCAR3 target cells, revealing a low ADCC, possibly due to internalisation, as detected for MCF-7 cells.Antibody phage display starting with immune libraries and followed by affinity maturation is a powerful strategy to generate high affinity human antibodies to difficult targets, in this case shown by the creation of a highly specific antibody with subnanomolar affinity to a very small epitope consisting of four amino acids. Despite these "best in class" binding parameters, the therapeutic success of this antibody was prevented by the target biology

Isolation and Characterisation of a Human-Like Antibody Fragment (scFv) That Inactivates VEEV In Vitro and In Vivo

Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus genus and several species of this family are pathogenic to humans. The viruses are classified as potential agents of biological warfare and terrorism and sensitive detection as well as effective prophylaxis and antiviral therapies are required

Multipotency and cardiomyogenic potential of human adipose-derived stem cells from epicardium, pericardium, and omentum

BACKGROUND: Acute myocardial infarction (MI) leads to an irreversible loss of proper cardiac function. Application of stem cell therapy is an attractive option for MI treatment. Adipose tissue has proven to serve as a rich source of stem cells (ADSCs). Taking into account the different morphogenesis, anatomy, and physiology of adipose tissue, we hypothesized that ADSCs from different adipose tissue depots may exert a diverse multipotency and cardiogenic potential. METHODS: The omental, pericardial, and epicardial adipose tissue samples were obtained from organ donors and patients undergoing heart transplantation at our institution. Human foreskin fibroblasts were used as the control group. Isolated ADSCs were analyzed for adipogenic and osteogenic differentiation capacity and proliferation potential. The immunophenotype and constitutive gene expression of alkaline phosphatase (ALP), GATA4, Nanog, and OCT4 were analyzed. DNA methylation inhibitor 5-azacytidine was exposed to the cells to stimulate the cardiogenesis. Finally, reprogramming towards cardiomyocytes was initiated with exogenous overexpression of seven transcription factors (ESRRG, GATA4, MEF2C, MESP1, MYOCD, TBX5, ZFPM2) previously applied successfully for fibroblast transdifferentiation toward cardiomyocytes. Expression of cardiac troponin T (cTNT) and alpha-actinin (Actn2) was analyzed 3 weeks after initiation of the cardiac differentiation. RESULTS: The multipotent properties of isolated plastic adherent cells were confirmed with expression of CD29, CD44, CD90, and CD105, as well as successful differentiation toward adipocytes and osteocytes; with the highest osteogenic and adipogenic potential for the epicardial and omental ADSCs, respectively. Epicardial ADSCs demonstrated a lower doubling time as compared with the pericardium and omentum-derived cells. Furthermore, epicardial ADSCs revealed higher constitutive expression of ALP and GATA4. Increased Actn2 and cTNT expression was observed after the transduction of seven reprogramming factors, with the highest expression in the epicardial ADSCs, as compared with the other ADSC subtypes and fibroblasts. CONCLUSIONS: Human epicardial ADSCs revealed a higher cardiomyogenic potential as compared with the pericardial and omental ADSC subtypes as well as the fibroblast counterparts. Epicardial ADSCs may thus serve as the valuable subject for further studies on more effective methods of adult stem cell differentiation toward cardiomyocytes

The bear in Eurasian plant names: Motivations and models

Ethnolinguistic studies are important for understanding an ethnic group's ideas on the world, expressed in its language. Comparing corresponding aspects of such knowledge might help clarify problems of origin for certain concepts and words, e.g. whether they form common heritage, have an independent origin, are borrowings, or calques. The current study was conducted on the material in Slavonic, Baltic, Germanic, Romance, Finno-Ugrian, Turkic and Albanian languages. The bear was chosen as being a large, dangerous animal, important in traditional culture, whose name is widely reflected in folk plant names. The phytonyms for comparison were mostly obtained from dictionaries and other publications, and supplemented with data from databases, the co-authors' field data, and archival sources (dialect and folklore materials). More than 1200 phytonym use records (combinations of a local name and a meaning) for 364 plant and fungal taxa were recorded to help find out the reasoning behind bear-nomination in various languages, as well as differences and similarities between the patterns among them. Among the most common taxa with bear-related phytonyms were Arctostaphylos uva-ursi (L.) Spreng., Heracleum sphondylium L., Acanthus mollis L., and Allium ursinum L., with Latin loan translation contributing a high proportion of the phytonyms. Some plants have many and various bear-related phytonyms, while others have only one or two bear names. Features like form and/or surface generated the richest pool of names, while such features as colour seemed to provoke rather few associations with bears. The unevenness of bear phytonyms in the chosen languages was not related to the size of the language nor the present occurence of the Brown Bear in the region. However, this may, at least to certain extent, be related to the amount of the historical ethnolinguistic research done on the selected languages

Controlled Rounding in Low Noise Digital Filter Structures

Several IIR digital filter structures are known which exhibit freedom of limit cycles with magnitude truncation (MT) as rounding mechanism. Other well-known structures provide low noise properties with respect to quantization. Often, nonlinear stability and quantization noise reduction are incompatible. Integrator-based structures, for example, which are common for narrowband lowpass filter applications, show an excellent low noise behaviour but they are not free from limit cycles. With controlled rounding (CR), the direction of quantization is made dependent on some suitably chosen control signal within the structure. Such a quantization mechanism has been shown to stabilize the direct form structure [1] and is easy to implement: simply subtract the control signal from the signal to be quantized, truncate the difference (MT), and subsequently add the control signal. The more difficult part is to find a suitable control signal. This contribution describes how controlled rounding can be..

N Biotechnol

For studying human antibody variable (V)-gene usage in any group of individuals or for the generation of recombinant human antibody libraries for phage display, quality and yield of the amplified V-gene repertoire is of utmost importance. Key parameters affecting the amplification of full antibody repertoires are V-gene specific primer design, complementary DNA (cDNA) synthesis from total RNA extracts of peripheral blood mononuclear cells (PBMCs) and ultimately the polymerase chain reaction (PCR). In this work we analysed all these factors; we performed a detailed bioinformatic analysis of V-gene specific primers based on VBASE2 and evaluated the influence of different commercially available reverse transcriptases on cDNA synthesis and polymerases on PCR efficiency. The primers presented cover near to 100% of all functional and putatively functional V-genes in VBASE2 and the final protocol presents an optimised combination of commercial enzymes and reaction additives for cDNA synthesis and PCR conditions for V-gene amplification. Finally, applying this protocol in combination with different immunoglobulin (Ig) chain specific reverse primers we were able to amplify rearranged antibody genes of different isotypes under investigation