Background: Factors predicting survival in men with lymph node-positive prostate cancer are still poorly defined. Patients and Methods: 193 prostate cancer patients with histopathologically proven lymph node involvement with a median follow-up of 7.3 years were studied. 94% of patients received immediate hormonal therapy. Kaplan-Meier curves were calculated to evaluate overall survival rates and compared with the log-rank test. Cumulative disease-specific and competing mortality rates were calculated by competing risk analysis and compared with the Pepe-Mori test. Cox proportional hazard models were used to determine the independent significance of predictors of all-cause mortality. Results: Age (70 years or older vs. younger), Gleason score (8–10 vs. 7 or lower) and the number of involved nodes (3 or more vs. 1–2) were identified as independent predictors of all-cause mortality. When patients with 0–1 of these risk factors were compared with those with 2–3 risk factors, all-cause (rates after 10 years 21% vs. 71%, p < 0.0001), disease-specific (12 vs. 37%, p = 0.009) and competing mortality (9 vs. 33%, p = 0.02) differed significantly. Conclusions: Some of the excess mortality in patients with poor-risk lymph node-positive prostate cancer may be attributed to increased competing mortality, possibly caused by an interaction between comorbid diseases and hormonally treated persistent or progressive prostate cancer. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Einleitung/ Zielstellung Zusätzlich zur Gammastrahlung emittiert 99mTc ca. 5 niederenergetische Auger-Elektronen mit Reichweiten von wenigen Nanometern im Gewebe. Diese haben für die nuklear-medizinische Diagnostik keine Bedeutung. Es wird jedoch über eine therapeutische Nutzung diskutiert, wofür eine Anreicherung der Auger-Elektronen-Emitter in einem strahlensensitiven Zellkompartiment erforderlich ist. Ziel der Arbeit war es, verschiedene [99mTc]Tc-Radiopharmaka hinsichtlich ihres Uptakeverhaltens, der subzellulärer Verteilung und des Retentionsverhaltens in vitro zu untersuchen, sowie die mutmaßlich durch den Auger-Effekt hervorgerufene Radiotoxizität der [99mTc]Tc-markierten Radiopharmaka zu vergleichen und die gewonnenen Ergebnisse in Hinblick auf potentielle extranukleäre strahlensensitive Targets zu interpretieren. Material und Methode Durchgeführt wurden die Versuche im ersten Abschnitt der Arbeit an Natrium-Iodid-Symporter (NIS)-positiven FRTL-5-Schilddrüsenzellen. Von [99mTc] Pertechnetat ([99mTc]TcO4-), [99mTc]TcO4- nach Vorinkubation von Perchlorat ([99mTc]TcO4-/ ClO4-), [99mTc]Tc-Hexakis-2-Methoxyisobutylisonitril ([99mTc]Tc-MIBI), [99mTc]Tc-Hexamethyl-Propylenaminoxim ([99mTc]Tc-HMPAO) und [99mTc]TcO4- nach Vorinkubation von Zinn-Pyrophosphat (Sn- PYP/ [99mTc]TcO4-) wurden die intrazelluläre Radio¬nuklid¬aufnahme und die subzelluläre Verteilung untersucht. Basierend auf den Ergebnissen dieser Versuche wurde die mittlere absorbierte Zellkerndosis kalkuliert. Zur Beurteilung der strahlenbiologischen Wirkung wurde das klonogene Zellüberleben mit der Anzahl residualer gH2AX-Foci (DNA-Schaden) verglichen und die Wirkung der [99mTc]Tc Tracer auf den Zellzyklus von FRTL-5-Zellen untersucht. Im zweiten Abschnitt der Arbeit wurde an EGFR-positiven A431-Zellen die radiotoxische Wirkung in Abhängigkeit von der intra¬zellulären Lokalisation von [99mTc]Alexa(488)-C225-Cyclooctin-Dpa-Tc(CO)3 ([99mTc]Tc-C225), [99mTc]Tc-HMPAO und [99mTc]TcO4- auf das klonogene Zellüberleben untersucht. Ergebnisse und Diskussion Aufgrund verschiedener Uptakemechanismen zeigte jedes der untersuchten [99mTc]Tc-Radiopharmaka Unterschiede im zeitlichen Verlauf der Uptakekinetik. Durch Blockierung des NIS durch ClO4- konnte eine intrazelluläre Aufnahme von [99mTc]TcO4- verhindert werden, wogegen durch Vorinkubation mit Sn-PYP die zelluläre Aufnahme von [99mTc]TcO4- um das 22-fache gesteigert wurde. [99mTc]Tc-MIBI und [99mTc]Tc-HMPAO wurden aufgrund ihrer lipophilen Eigenschaften unabhängig vom NIS passiv in die Zelle transportiert. Die Untersuchung der intrazellulären Verteilung ergab für [99mTc]Tc-HMPAO und Sn-PYP/ [99mTc]TcO4- eine vergleichbar hohe Anreicherung in der Membran/Organellen-Fraktion sowie in der Zellkernfraktion. Von [99mTc]TcO4- und [99mTc]Tc-MIBI wurde die Hauptaktivität in der Zytosol-Fraktion und nur geringe Anteile in der Membran/Organellen-Fraktion sowie in der Zellkernfraktion nachgewiesen. In guter Übereinstimmung zur subzellulären Verteilung zeigten Sn-PYP/ [99mTc]TcO4- und [99mTc]Tc-HMPAO eine fast vollständige, hingegen [99mTc]Tc-MIBI und [99mTc]TcO4- nur eine geringe Retention. Aufgrund der genannten Unterschiede wurde bei gleicher inkubierter Aktivitätskonzentration folgende Reihenfolge der resultierenden Zellkerndosis ermittelt: [99mTc]TcO4- < [99mTc]Tc-MIBI < [99mTc]Tc-HMPAO < Sn-PYP/ [99mTc]TcO4-. [99mTc]TcO4- und [99mTc]Tc-HMPAO zeigten eine ähnliche Wirkung auf das klonogene Zellüberleben und auf den Zellzyklus. Jedoch bewirken sie eine wesentlich stärkere Reduzierung des Überlebens und einen stärkeren G2-Arrest als [99mTc]Tc-MIBI und Sn-PYP/ [99mTc]TcO4-, wobei [99mTc]Tc-MIBI bei allen drei untersuchten biologischen Endpunkten die geringste Wirkung zeigte. Bei einer vergleichbaren Reduktion des Zellüberlebens von [99mTc]TcO4- und [99mTc]Tc-HMPAO induzierte [99mTc]Tc-HMPAO lediglich die Hälfte der gH2AX-Foci im Vergleich zu [99mTc]TcO4-. Die trotz geringerem DNA-Schaden vergleichbare radiotoxische Wirkung zeigte, dass das klonogene Zellüberleben nicht allein vom DNA-Schaden abhängt. Daraus folgt, dass es außer der Kern-DNA noch weitere strahlensensitive Kompartimente gibt, die durch [99mTc]Tc-HMPAO stärker geschädigt wurden als von den anderen untersuchten [99mTc]Tc Tracern. Ein mögliches extranukleäres strahlensensitives Target ist die Zellmembran, so dass im zweiten Teil der Arbeit zur Überprüfung der Radiosensitivität der Zellmembran die radiotoxische Wirkung von [99mTc]Tc-C225 an EGFR-positiven A431-Zellen untersucht wurde. [99mTc]Tc-C225 wurde über den EGFR und [99mTc]Tc-HMPAO aufgrund seiner Lipophilie durch Diffusion intrazellulär aufgenommen. [99mTc]TcO4- dagegen zeigte keine intrazelluläre Aufnahme in die NIS-negativen Zellen und wurde als Referenz für eine extrazelluläre Bestrahlung verwendet. [99mTc]Tc-C225 wies nach einstündiger Inkubation eine Membranbindung von lediglich 10 % auf, die im Laufe von 24 h auf 1,9 % absank. Dies zeigte, dass [99mTc]Tc-C225 rasch in den A431-Zellen internalisiert wurde und dass nur bei sehr kurzen Inkubationszeiten von einer spezifischen Zellmembranmarkierung gesprochen werden kann. [99mTc]Tc-HMPAO ging keine Bindung an die Zellmembran ein. Weiterhin wurde bei der Inkubation steigender Aktivitäts- und Antikörperkonzentrationen von [99mTc]Tc C225 eine Sättigung des EGFR beobachtet, woraus eine wesentlich geringere Zellkerndosis als bei Inkubation von [99mTc]Tc-HMPAO resultierte. Im Vergleich des klonogenen Zellüberlebens zeigten [99mTc]Tc-C225 und [99mTc]Tc-HMPAO bei gleicher Zellkerndosis keine Unterschiede in der radiobiologischen Wirkung. Somit konnte lediglich eine Verstärkung der radiotoxischen Effekte von [99mTc]Tc-C225 an A431-Zellen im Vergleich zur ausschließlich extrazellulären Verteilung von [99mTc]TcO4- gezeigt werden. Schlussfolgerung Die Untersuchung der radiotoxischen Wirkung von [99mTc]Tc-C225 ermöglichte bei den angewendeten Versuchsbedingungen keine Rückschlüsse auf die Strahlensensitivität der Zellmembran. Weiterführende Arbeiten zur Entwicklung eines 99mTc-markierbaren spezifischen Membranmarkers wären notwendig, um klären zu können, ob die Zellmembran ein ähnlich strahlensensitives Target wie die nukleäre DNA ist. Dosimetrische Betrachtungen an den als Modellsystemen dienenden FRTL-5- und A431-Zellen deuten darauf hin, dass aufgrund ungenügender Anreicherung eine therapeutische Wirkung der Auger-Elektronen im Tumorgewebe eher unrealistisch ist. Damit sollte aus gegenwärtiger Sicht die klinische Anwendung von 99mTc auf den diagnostischen Einsatz beschränkt bleiben. Jedoch könnte 99mTc als Auger-Elektronen-Emitter bei spezifischer Anreicherung in definierten Zellkompartimenten als Nano-Tool zur Erforschung der Strahlensensitivität einzelner Zellbestandteile eingesetzt werden. / Introduction In addition to gamma radiation, 99mTc emits approximately 5 low energy Auger and internal conversion electrons per decay, resulting in high ionization density proximal to the radionuclide’s decay position. Low-energy Auger electrons with path lengths of only nanometers cannot be utilized for diagnostic procedures; however, they have frequently been discussed for therapeutic applications. To achieve a radiobiological effect, an intracellular accumulation and distribution in relevant cell compartments of the Auger electron emitter is required. Aim The aim of the thesis was the comparison of different [99mTc]Tc-labeled compounds concerning their intracellular uptake, subcellular distribution and retention in vitro. Furthermore the radiotoxicity caused by the Auger effect has to be investigated. Material and Methods The intracellular radionuclide uptake, subcellular distribution (ProteoExtract®-Kit) and retention of [99mTc] pertechnetate ([99mTc]TcO4-), [99mTc]TcO4- after pre-incubation of perchlorate ([99mTc]TcO4-/ClO4-), [99mTc]TcO4- after pre-incubation of stannous pyrophosphate ([99mTc]TcO4-/Sn-PYP), [99mTc]Tc-hexamethyl-propylene-aminoxime ([99mTc]Tc-HMPAO) and [99mTc]Tc-hexakis-2-methoxyisobutylisonitrile ([99mTc]Tc-MIBI) were quantified in sodium-iodide symporter (NIS)-positive rat thyroid FRTL-5 cells. Basing on these results the mean absorbed nucleus dose was calculated. Radiotoxicity was investigated using phosphorylated histone H2AX (gH2AX foci), clonogenic cell survival and cell cycle analyzes. Additionally the radiotoxicity of [99mTc]Alexa(488)-C225-Cyclooctin-Dpa-Tc(CO)3 ([99mTc]Tc-C225) was compared with the one of [99mTc]TcO4- and [99mTc]Tc -HMPAO depending on the subcellular distribution in EGFR-positive A431 cells. Results and Discussion For the analyzed [99mTc]Tc-labeled compounds we detected differences in the time courses of the uptake kinetics caused by different uptake mechanisms into the FRTL-5 cells. The radionuclide uptake of [99mTc]TcO4- was blocked in the presence of perchlorate and increased by a factor of approximately 22 after pre-incubation of Sn-PYP. The lipophilic complexes [99mTc]Tc-MIBI and [99mTc]Tc-HMPAO crossed the cell membrane through passive transport via diffusion. The compartmental analysis indicated that [99mTc]Tc-HMPAO and [99mTc]TcO4-/Sn-PYP revealed a comparable high uptake in the nucleus and in the membrane/organelle fraction. [99mTc]TcO4- and [99mTc]Tc-MIBI were preferentially distributed in the cytosol, with lower amounts of the accumulated activity in both the membranes/organelles and the nucleus compared with the other compounds. In good agreement with the subcellular distribution [99mTc]Tc-HMPAO, [99mTc]TcO4-/Sn-PYP showed a nearly complete retention and [99mTc]TcO4-, [99mTc]Tc-MIBI a low retention. Due to the differences mentioned above the following sequence of the calculated mean nucleus dose for identical activity concentrations was determined: [99mTc]TcO4- < [99mTc]Tc-MIBI < [99mTc]Tc-HMPAO < Sn PYP/ [99mTc]TcO4-. [99mTc]TcO4- and [99mTc]Tc-HMPAO caused a similar reduction of the cell survival and a dose dependent G2-arrest. [99mTc]Tc-MIBI and Sn-PYP/ [99mTc]TcO4- are both less radiotoxic in terms of the estimated nucleus dose compared with [99mTc]TcO4- and [99mTc]Tc-HMPAO. Despite the similar effect on the cell survival [99mTc]Tc-HMPAO induced only half of the residual gH2AX foci than [99mTc]TcO4-. These findings reveal that clonogenic cellular survival is not solely determined by the DNA-DSB response, which may suggest the involvement of extra-nuclear radiosensitive targets in cell inactivation. A possible extra-nuclear radiosensitive target is the cell membrane. That’s why the aim of the second part of the thesis is the investigation of the radiosensitivity of the cell membrane. Therefore the radiotoxic influence of [99mTc]Tc-C225 was analyzed at EGFR-positive A431 cells. [99mTc]Tc-C225 was taken up over the EGFR and the lipophilic [99mTc]Tc-HMPAO was transported via diffusion over the cell membrane. In contrast, [99mTc]TcO4- did not show any intracellular uptake into the NIS-negative cells and therefore was used as extracellular reference. An incubation of [99mTc]Tc-C225 for one hour resulted to a membrane binding of only 10 %, which was reduced to 1.9 % after 24 hours. This demonstrated a fast internalization into A431-cell. Therefore only in the case of a very short incubation time [99mTc]Tc-C225 leads to a specific targeting of the cell membrane. [99mTc]Tc-HMPAO did not bind to the cell membrane. Furthermore the incubation of increasing concentrations of activity and antibody resulted in a saturation of the EGFR, leading to a significant lower nucleus dose in comparison to the incubation of [99mTc]Tc-HMPAO. Concerning the clonogenic cell survival no differences in the radiotoxicity of [99mTc]Tc-C225 and [99mTc]Tc-HMPAO were observed for equal nucleus dose. Thus only an amplification of the radiotoxic effects of [99mTc]Tc-C225 in comparison to the extracellular distribution in A431 cells of 99mTc-pertechnetate was observed. Conclusion The investigation of the radiotoxic effect of [99mTc]Tc-C225 did not allow any conclusions about the radiosensitivity of the cell membrane under the given experimental conditions. For clarifying if the radiosensitivity of the cell membrane is comparable to the one of the nucleus DNA further experiments for the development of a [99mTc]Tc-labeled specific target for the cell membrane are necessary. On the basis of the dosimetric considerations of the FRTL-5 cells and A431 cells used as model systems it can be concluded that because of an insufficient accumulation a therapeutic radiotoxic effect of the Auger electrons is not realistic. Therefore the clinical use of 99mTc should be limited to the diagnostics. Nevertheless specific accumulated Auger electrons of 99mTc could be applied in the field of investigation as nano-tools for the subcellular analysis of radiotoxicity.

Proteins and their interactions are fundamental to cellular life. Disruption of protein-protein, protein-RNA, or protein-DNA interactions can lead to disease, by affecting the function of protein complexes or by affecting gene regulation. A better understanding of these interactions on the molecular level gives rise to new methods to predict protein interaction, and is critical for the rational design of new therapeutic agents that disrupt disease-causing interactions. This thesis consists of three parts that focus on various aspects of protein interactions and their prediction in the context of disease. In the first part of this thesis, we classify interfaces of protein-protein interactions. We do so by systematically computing all binding sites between protein domains in protein complex structures solved by X-ray crystallography. The result is SCOPPI, the Structural Classification of Protein Protein Interfaces. Clustering and classification of geometrically similar interfaces reveals interesting examples comprising viral mimicry of human interface binding sites, gene fusion events, conservation of interface residues, and diversity of interface localisations. We then develop a novel method to predict protein interactions which is based on these structural interface templates from SCOPPI. The method is applied in three use cases covering osteoclast differentiation, which is relevant for osteoporosis, the microtubule-associated network in meiosis, and proteins found deregulated in pancreatic cancer. As a result, we are able to reconstruct many interactions known to the expert molecular biologist, and predict novel high confidence interactions backed up by structural or experimental evidence. These predictions can facilitate the generation of hypotheses, and provide knowledge on binding sites of promising disease-relevant candidates for targeted drug development. In the second part, we present a novel algorithm to search for protein binding sites in RNA sequences. The algorithm combines RNA structure prediction with sequence motif scanning and evolutionary conservation to identify binding sites on candidate messenger RNAs. It is used to search for binding sites of the PTBP1 protein, an important regulator of glucose secretion in the pancreatic beta cell. First, applied to a benchmark set of mRNAs known to be regulated by PTBP1, the algorithm successfully finds significant binding sites in all benchmark mRNAs. Second, collaborators carried out a screen to identify changes in the proteome of beta cells upon glucose stimulation while inhibiting gene expression. Analysing this set of post-transcriptionally controlled candidate mRNAs for PTBP1 binding, the algorithm produced a ranked list of 11 high confident potential PTBP1 binding sites. Experimental validation of predicted targets is ongoing. Overall, identifying targets of PTBP1 and hence regulators of insulin secretion may contribute to the treatment of diabetes by providing novel protein drug targets or by aiding in the design of novel RNA-binding therapeutics. The third part of this thesis deals with gene regulation in disease. One of the great challenges in medicine is to correlate genotypic data, such as gene expression measurements, and other covariates, such as age or gender, to a variety of phenotypic data from the patient. Here, we address the problem of survival prediction based on microarray data in cancer patients. To this end, a computational approach was devised to find genes in human cancer tissue samples whose expression is predictive for the survival outcome of the patient. The central idea of the approach is the incorporation of background knowledge information in form of a network, and the use of an algorithm similar to Google s PageRank. Applied to pancreas cancer, it identifies a set of eight genes that allows to predict whether a patient has a poor or good prognosis. The approach shows an accuracy comparable to studies that were performed in breast cancer or lymphatic malignancies. Yet, no such study was done for pancreatic cancer. Regulatory networks contain information of transcription factors that bind to DNA in order to regulate genes. We find that including background knowledge in form of such regulatory networks gives highest improvement on prediction accuracy compared to including protein interaction or co-expression networks. Currently, our collaborators test the eight identified genes for their predictive power for survival in an independent group of 150 patients. Under a therapeutic perspective, reliable survival prediction greatly improves the correct choice of therapy. Whereas the live expectancy of some patients might benefit from extensive therapy such as surgery and chemotherapy, for other patients this may only be a burden. Instead, for this group, a less aggressive or different treatment could result in better quality of the remaining lifetime. Conclusively, this thesis contributes novel analytical tools that provide insight into disease-relevant interactions of proteins. Furthermore, this thesis work contributes a novel algorithm to deal with noisy microarray measurements, which allows to considerably improve prediction of survival of cancer patients from gene expression data.

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Objective: In many countries the access to high quality medical service depends on socioeconomic factors. Therefore, these factors are associated with the treatment and prognosis of many diseases. In Germany health care is claimed to be independent from such factors due to obligatory health insurance and a well-developed medical infrastructure. Thus, socioeconomically caused health disparities should be absent. The aim of this study was to analyze the association between socioeconomic factors and the survival of oral cavity cancer in Germany. Patients and methods: In this descriptive cohort study socioeconomic status related factors as well as demographic, tumor-specific, and comorbidity factors of 500 patients treated for oral cavity cancer were obtained in the university hospital of Dresden. Pearson correlation was used to describe associations between continuous variables. Associations between categorical variables were assessed using the chi-square test. Overall and recurrence-free survival were studied using the Kaplan-Meier method. Log-rank test was carried out to test between-group differences. Cox proportional hazard models were used to estimate the risk of death and the risk of recurrence. Results: Significant differences in overall survival were found between the different educational levels and sex. Seventy-nine percent of the patients did not have a university degree or master craftsman/craftswoman. Less discrepancy was observed according to the marital status (49.4% married/engaged vs. 47.8% single, divorced, or widowed). In the multivariable analysis only sex, age at diagnosis, the Charlson score, the number of positive lymph nodes, and the nodal status were identified as independent predictors for overall survival whereas sex and the age at diagnosis were identified as independent predictors for recurrence-free survival. Conclusion: Despite the equitable health system in Germany, significant associations between overall survival of oral cavity cancer and different socioeconomic factors could be found. For elimination of these disparities, health education programs should be established in socially deprived areas. Furthermore, clinicians should keep these factors in mind when determining recall periods for dental check-ups.

Gedächtnis-B-Zellen und Plasmazellen sind essentielle Komponenten der protektiven Immunität. Die Mechanismen ihrer Induktion, ihres Überlebens und der Gedächtnis-B-Zellreaktivierung sind allerding bisher nur unvollständig verstanden. Um unser Wissen diesbezüglich zu erweitern, wurden in der vorliegenden Arbeit zum einen Charakteristika von Primär- und Sekundärimmunantworten nach Immunisierung mit Keyhole Limpet Hemocyanin (KLH) untersucht und zum anderen das Vorkommen sowie der Phänotyp humaner Gedächtnis-B-Zellen in verschiedenen lymphatischen Geweben analysiert. Die primäre parenterale KLH Immunisierung führte auf serologischer und zellulärer Ebene zu einer Reihe unerwarteter Ergebnisse, welche u. a. das Auftreten von IgA Antikörpern und die gleichzeitige Präsenz von hoch- und wenig mutierten primären Plasmablasten beinhalteten. Die Untersuchung der Gedächtnis-B-Zellverteilung in verschiedenen lymphatischen Geweben ergab den größten Gedächtnis-B-Zellpool in der Milz. Blut-, Tonsillen-, Knochenmarks- und Milz-Gedächtnis-B-Zellen wiesen nur wenige phänotypische Unterschiede auf. Einer davon war die CD69 Expression auf tonsillären ruhenden Gedächtnis-B-Zellen, was darauf hindeutet, dass tonsilläre Gedächtnis-B-Zellen tatsächlich sessil sein könnten. Die hier erhaltenen Ergebnisse stellen neue Erkenntnisse über bisher unbeschriebene Mechanismen bei parenteralen Immunantworten wie zum Beispiel die Induktion von IgA Antikörpern sowie die potentielle Rekrutierung kreuzreaktiver Gedächtnis-B-Zellen dar. Es bleibt zu klären, wie die Reaktivierung solcher Gedächtnis-B-Zellen reguliert ist. Derartiges Wissen wäre insbesondere für Therapien von Erkrankungen des Immunsystems, wie Autoimmunität, von Bedeutung. Das potentiell patrouillierende Verhalten der Gedächtnis-B-Zellen ist ein markanter Unterschied zu den nischenabhängigen sessilen Plasmazellen und deutet weitestgehend darauf hin, dass Gedächtnis-B-Zellen nicht auf derartige Nischen angewiesen sind. / Memory B cells (mBC) and antibodies are major mediators of protective immune responses yet the mechanisms of their induction, maintenance and mBC reactivation are poorly understood. Therefore, to enhance knowledge in this regard this study comprehensively characterized a human primary and secondary B cell immune response to Keyhole Limpet Hemocyanin (KLH). Secondly, mBC maintenance was investigated by a systematic analysis of mBC presence, frequency and phenotype within different lymphoid organs. Parenteral primary KLH immunization yielded unexpected results on the serological and B cellular level, including KLH-specific IgA antibody induction, the simultaneous presence of low and highly mutated circulating KLH-specific primary plasmablasts and only little clonal overlap between the primary, memory and secondary KLH-specific B cell repertoires. With respect to the organ distribution of human mBC, the spleen was identified as a major mBC reservoir. Splenic, tonsillar, bone marrow and blood mBC pools exhibited a largely comparable phenotype. Yet, we found tonsillar mBC to express CD69. Due to their resting state tonsillar mBC could therefore constitute a tissue resident cell population. The observations described allow insights into hitherto unknown potential mechanisms behind primary immune responses, i.e. prominent IgA induction by parenteral challenge and inclusion of cross-reactive mBC. The so far unclear regulatory players involved deserve future investigation, as such knowledge may be crucial for therapeutic interventions in immune system disorders. Furthermore, strikingly different to the resident plasma cells in the bone marrow, mBC appear to distribute between lymphoid organs and continuously recirculate in peripheral blood indicative of their potential permanent screening activities, suggesting that human mBC do not require one dedicated niche for their principle survival.

Plasmazellen gehören zu den Effektorzellen des adaptiven Immunsystems. Langlebige Plasmazellen tragen durch kontinuierliche Sekretion protektiver Antikörper wesentlich zum humoralen Gedächtnis bei und überleben hauptsächlich in spezialisierten Nischen des Knochenmarks. Bislang ist jedoch kein Marker bekannt, mit dessen Hilfe langlebige Plasmazellen eindeutig identifiziert werden können. Die vorliegende Arbeit befasst sich mit der molekularbiologischen, phänotypischen und funktionellen Charakterisierung von reifen Plasmazellen im gesunden humanen Knochenmark, die sich durch die differentielle Expression von CD19 unterscheiden. Dabei konnte festgestellt werden, dass CD19negative Plasmazellen durch eine vergleichsweise geringere Expression von CD45 und HLADR einen höheren Reifegrad aufweisen als CD19positive Plasmazellen. Zudem lässt die vermehrte Expression von CD28, Mcl1, Bcl2 sowie die schwächere Expression u.a. von CD95 darauf schließen, dass CD19negative Plasmazellen im Knochenmark eine bessere Überlebenskapazität besitzen als CD19positive Plasmazellen. Da beide Plasmazellpopulationen ähnliche Antigen-Spezifitäten aufweisen, Plasmazellen im Knochenmark von Säuglingen ausschließlich CD19 exprimieren und nach sekundärer Vakzinierung im Blut detektierbare Plasmablasten und Plasmazellen ebenfalls CD19 auf ihrer Oberfläche exprimieren, weist die Gesamtheit der Daten darauf hin, dass sich CD19negative Plasmazellen im Kindesalter in situ aus reifen CD19positiven Plasmazellen im Knochenmark entwickeln. Die CD19negative Plasmazellpopulation leistet durch hohe Halbwertszeit und Stabilität einen konstanten Beitrag zur Aufrechterhaltung des humoralen Gedächtnisses. Die CD19positive Plasmazellpopulation stellt hingegen eine flexible Komponente dar, die eine Anpassung der humoralen Immunität und des humoralen Gedächtnisses an aktuelle Herausforderungen auch im Erwachsenenalter ermöglicht. / Plasma cells are effector cells of the adaptive immune system. Humoral memory is sustained by long-lived plasma cells that continuously secrete protective antibodies and mostly reside in specialized niches in the bone marrow. So far, no marker is known that could distinguish long-lived plasma cells from short-lived ones. The present work addresses the biomolecular, phenotypical and functional characterization of mature plasma cells in healthy human bone marrow that differ in their expression of the surface marker CD19. CD19negative plasma cells showed higher maturity than CD19positive plasma cells as they expressed lesser amounts of CD45 and HLADR. Moreover, higher expression of CD28, Mcl1 and Bcl2 and lesser expression of CD95 argues for a better survival capacity of CD19negative plasma cells. Both plasma cell populations showed similar antigen specificities. All plasmablasts and plasma cells detectable in blood after secondary vaccination expressed CD19, as well as all plasma cells isolated from infant bone marrow. These results indicate that CD19negative plasma cells mainly develop during childhood by further differentiation of mature CD19positive plasma cells in situ in the bone marrow. CD19negative plasma cells represent a long-lived and stable component of the adaptive immune system and humoral memory, whereas the CD19positive plasma cell population displays a flexible element allowing for adaption of humoral immunity to new challenges throughout a lifetime.

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