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- Center for Interdisciplinary Clinical Research, Würzburg University, Würzburg, Germany (1)
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„Black esophagus“ oder „akute Ösophagusnekrose“ (AÖN) ist eine seltene Erkrankung, die sich makroskopisch durch eine zirkumferente Schwarzverfärbung der Ösophagusmukosa mit abruptem Ende am gastroösophagealen Übergang auszeichnet. Die genaue Pathogenese ist unbekannt; es werden multifaktorielle Einflüsse wie z. B. Säurereflux, Ischämie und verringerte Schutzmechanismen der Mukosa als mögliche Ursachen diskutiert.
Vorgestellt werden 2 Obduktionsfälle, die typische Befunde einer AÖN aufwiesen. Zusätzlich hatten Fall 1 eine Candida-Infektion und Fall 2 eine Appendizitis, sodass eine infektiöse Genese in beiden Fällen eine Rolle gespielt haben könnte.
Über die Rolle der Neuroinflammation bei Entstehung und Progression der Demenz vom Alzheimer-Typ
(2022)
Die vorliegende Studie bringt neue Erkenntnisse bezüglich der Rolle und Ver-teilung der Mikroglia und der eingewanderten Monozyten im Verlauf der Alz-heimer Erkrankung in postmortem Gehirnen. Im Gegensatz zu Studien an Tiermodellen konnten wir in unserer Kohorte eine nur sehr geringe Beteili-gung myeloischer Monozyten an der AD Pathologie beobachten, so dass man annehmen kann, dass bei Menschen die Immunantwort des Gehirns haupt-sächlich von den hirneigenen Mikrogliazellen getragen wird. Dies wurde an humanem postmortem Hirngewebe bis zu diesem Zeitpunkt noch nicht unter-sucht.
Zudem konnte gezeigt werden, dass die vulnerablen, früh von Tangles und Plaques betroffenen Hirnregionen auch eine frühe Mikrogliareaktion aufwei-sen und insbesondere von proinflammatorischen Zellen besiedelt werden und dass die Reaktion in manchen Regionen im Verlauf zunimmt, während in an-deren eine Abflachung oder sogar Abnahme beobachtet wird.
Zielsetzung dieser Arbeit war die Untersuchung von Gehirngewebe in der asymptomatischen Phase der SIV-Infektion zur Erfassung immunologischer Vorgänge bei der Entstehung einer SIV-Enzephalitis am Beispiel der gewebeständigen Lymphozyten und Makrophagen. Wir benutzten das etablierte SIV-AIDS Modell mit Makakenaffen. Untersucht wurden 24 Versuchstiere im Zeitraum von bis zu 20 Wochen, die zuvor mit einem der beiden Virusstämme (SIVmac251/ 32H oder SIVmac251/ MPBMC) infiziert worden waren, vier der Versuchstiere entwickelten eine SIV-Enzephalitis, eines dieser Tiere war mit Selegilin behandelt worden. Fünf weitere infektfreie Makaken dienten als Kontrollen. Nach definierten Zeiträumen wurden die Versuchstiere getötet, die Gehirne präpariert und mittels Immunhistochemie Lymphozyten und Makrophagen angefärbt. Im weiteren führten wir eine lichtmikroskopische Morphometrie, getrennt nach einzelnen Kompartimenten des Gehirns, wie Cortex, subcorticalem weißem Mark und perivaskulärem Raum, durch. Die Ergebnisse unserer Arbeit — die infizierte, asymptomatische Tiere mit den Kontrolltieren vergleicht — zeigen eine ausgeprägte lymphozytäre Reaktion speziell im Cortex, zum Teil auch im subcorticalen Mark. Die Makrophagen zeigen eine Zunahme der Zelldichte perivaskulär und im Cortex, welche quantitativ deutlich unter der der Lymphoyzten bleibt. Die Tiere mit Enzephalitis zeigen allesamt eine corticale, wie auch subcorticale Zunahme der lymphozytären Zelldichte, bei den Makrophagen zeigt sich eine überwiegend generelle Stimulierung in allen drei Kompartimenten. Die Ergebnisse unserer Arbeit — die sich durch die Untersuchung asymptomatischer Versuchstiere sehr gut an die Verhältnisse bei der HIV-Infektion annähert — lassen vermuten, daß es dem Immunsystem mit Hilfe der gewebeständigen Lymphozyten im Cortex und subcorticalen Mark für eine ganze Weile gelingt, den Ausbruch einer Enzephalitis und damit der AIDS Erkrankung zu verzögern.
Background
Wilms’ tumor 1-associating protein (WTAP) is a nuclear protein, which is ubiquitously expressed in many tissues. Furthermore, in various types of malignancies WTAP is overexpressed and plays a role as an oncogene. The function of WTAP in diffuse large B-cell lymphoma (DLBCL), however, remains unclear.
Methods
Immunohistochemistry was applied to evaluate the levels of WTAP expression in DLBCL tissues and normal lymphoid tissues. Overexpression and knock-down of WTAP in DLBCL cell lines, verified on mRNA and protein level served to analyze cell proliferation and apoptosis in DLBCL cell lines by flow cytometry. Finally, co-immunoprecipitation (Co-IP), IP, and GST-pull down assessed the interaction of WTAP with Heat shock protein 90 (Hsp90) and B-cell lymphoma 6 (BCL6) as well as determined the extend of its ubiquitinylation.
Results
WTAP protein levels were consistently upregulated in DLBCL tissues. WTAP promoted DLBCL cell proliferation and improved the ability to confront apoptosis, while knockdown of WTAP in DLBCL cell lines allowed a significant higher apoptosis rate after treatment with Etoposide, an anti-tumor drug. The stable expression of WTAP was depended on Hsp90. In line, we demonstrated that WTAP could form a complex with BCL6 via Hsp90 in vivo and in vitro.
Conclusion
WTAP is highly expressed in DLBCL, promoting growth and anti-apoptosis in DLBCL cell lines. WTAP is a client protein of Hsp90 and can appear in a complex with BCL6 and Hsp90 in DLBCL. Down-regulation of WTAP could improve the chemotherapeutic treatments in DLBCL.
Despite improved survival in the Rituximab (R) era, a considerable number of patients with diffuse large B-cell lymphoma (DLBCL) ultimately die from the disease. Functional imaging using [18F]fluorodeoxyglucose-PET is suggested for assessment of residual viable tumor very early during treatment but is compromised by non-specific tracer retention in inflammatory lesions. The PET tracer [18F]fluorodeoxythymidine (FLT) as surrogate marker of tumor proliferation may overcome this limitation. We present results of a prospective clinical study testing FLT-PET as superior and early predictor of response to chemotherapy and outcome in DLBCL. 54 patients underwent FLT-PET prior to and one week after the start of R-CHOP chemotherapy. Repetitive FLT-PET imaging was readily implemented into the diagnostic work-up. Our data demonstrate that the reduction of FLT standard uptake valuemean (SUVmean) and SUVmax one week after chemotherapy was significantly higher in patients achieving complete response (CR, n=48; non-CR, n=6; p<0.006). Martingale-residual and Cox proportional hazard analyses showed a significant monotonous decrease of mortality risk with increasing change in SUV. Consistent with these results, early FLT-PET response showed relevant discriminative ability in predicting CR. In conclusion, very early FLT-PET in the course of R-CHOP chemotherapy is feasible and enables identification of patients at risk for treatment failure.
Background:
During the last years, (19)F-MRI and perfluorocarbon nanoemulsion (PFC) emerged as a powerful contrast agent methodology to track cells and to visualize inflammation. We applied this new modality to visualize deep tissue abscesses during acute and chronic phase of inflammation caused by Staphylococcus aureus infection.
Methodology and Principal Findings:
In this study, a murine thigh infection model was used to induce abscess formation and PFC or CLIO (cross linked ironoxides) was administered during acute or chronic phase of inflammation. 24 h after inoculation, the contrast agent accumulation was imaged at the site of infection by MRI. Measurements revealed a strong accumulation of PFC at the abscess rim at acute and chronic phase of infection. The pattern was similar to CLIO accumulation at chronic phase and formed a hollow sphere around the edema area. Histology revealed strong influx of neutrophils at the site of infection and to a smaller extend macrophages during acute phase and strong influx of macrophages at chronic phase of inflammation.
Conclusion and Significance:
We introduce (19)F-MRI in combination with PFC nanoemulsions as a new platform to visualize abscess formation in a murine thigh infection model of S. aureus. The possibility to track immune cells in vivo by this modality offers new opportunities to investigate host immune response, the efficacy of antibacterial therapies and the influence of virulence factors for pathogenesis.
Personalized oncology is a rapidly evolving area and offers cancer patients therapy options that are more specific than ever. However, there is still a lack of understanding regarding transcriptomic similarities or differences of metastases and corresponding primary sites. Applying two unsupervised dimension reduction methods (t-Distributed Stochastic Neighbor Embedding (t-SNE) and Uniform Manifold Approximation and Projection (UMAP)) on three datasets of metastases (n = 682 samples) with three different data transformations (unprocessed, log10 as well as log10 + 1 transformed values), we visualized potential underlying clusters. Additionally, we analyzed two datasets (n = 616 samples) containing metastases and primary tumors of one entity, to point out potential familiarities. Using these methods, no tight link between the site of resection and cluster formation outcome could be demonstrated, or for datasets consisting of solely metastasis or mixed datasets. Instead, dimension reduction methods and data transformation significantly impacted visual clustering results. Our findings strongly suggest data transformation to be considered as another key element in the interpretation of visual clustering approaches along with initialization and different parameters. Furthermore, the results highlight the need for a more thorough examination of parameters used in the analysis of clusters.
Follikuläre Lymphome (FL) machen etwa 25-40% der Non-Hodgkin-Lymphome aus und sind in der Regel bereits bei Diagnosestellung nicht mehr auf den Lymphknoten beschränkt, sondern systemische Erkrankungen. In jüngeren Untersuchungen konnte gezeigt werden, dass die selten diagnostizierten limitierten Stadien (Ann Arbor I und II) der Erkrankung häufig einen nur partiellen Befall der betroffenen Lymphknoten durch das Lymphom zeigen. In diesen frühen Stadien kolonisieren follikuläre Lymphome präexistente Follikel (in situ- Lymphom) und breiten sich dann offenbar auf die übrigen Follikel des Lymphknotens aus, bevor ein systemischer Befall des gesamten Organismus feststellbar ist. Ziel der vorliegenden Arbeit war es zunächst zu untersuchen, auf welchem Weg die Zellen eines Tumorklons im follikulären Lymphom die Keimzentren eines Lymphknotens kolonisieren. Dazu wurde die genetische Verwandtschaft der einzelnen Tumorsubklone untereinander anhand ihrer individuellen Mutationsmuster bestimmt. Mit Hilfe von daraus berechneten phylogenetischen Stammbäumen konnte die Ausbreitung der Subklone auf die vorbestehenden Keimzentren nachvollzogen werden. Zweitens sollte in dieser Studie der Frage nachgegangen werden, ob die Tumorsubklone auch unter dem Einfluss der Keimzentrumsumgebung stehen, die in der physiologischen B-Zell-Reifung für die enorme Vielfalt der Antikörperspezifität sorgt (Hypermutation). Anhaltende Mutationen (ongoing mutations) innerhalb eines Tumorklons würden auf einen solchen erhaltenen Einfluss der Hypermutationsmaschinerie hinweisen. Schließlich sollte untersucht werden, ob es auch in follikulären Lymphomen eine antigenabhängige B-Zell-Reifung gibt, wie sie bei der physiologischen „Optimierung“ von Antikörpern auf die korrespondierenden Antigene zu finden ist. Material und Methode: Sieben Fälle von follikulären Lymphomen von vier Patienten (davon einer mit einem und einer mit zwei Rezidiven ihrer Lymphomerkrankung) wurden morphologisch und immunhistochemisch reevaluiert. Pro Fall wurden bis zu zehn Follikel mikrodisseziert und pro Follikel die VH-Gene von bis zu zehn Subklonen sequenziert. Computerunterstützt wurden sowohl die genetische Verwandtschaft der Tumorsubklone untereinander und ihre Verteilung auf die einzelnen Follikel, als auch das Verhältnis von R- zu S- Mutationen in den verschiedenen Abschnitten des BCR-Gens und damit ein möglicher Antigen-Einfluss auf die Hypermutation analysiert. Ergebnisse: Ein FL Grad I zeigte ein deutliches Clustering von genetisch miteinander verwandten Tumorsubklonen im selben Follikel. Dennoch fand sich ein moderater interfollikulärer Austausch der Subklone. Bei morphologisch höhergradigen FL (Grad II und IIIa) nahm das Clustering deutlich ab und der interfollikuläre Austausch zu, bis im zweiten Rezidiv eines Patienten ein weitgehend diffuses Wachstum resultierte. Als Ausdruck des erhaltenen Einflusses des Keimzentrums zeigten alle Primärtumoren (FL Grad I und II) noch ongoing mutations, während bei FL in Progression keine ongoing mutations mehr feststellbar waren. Eine Häufung von R-Mutationen in den antigenbindenden Domänen des B-Zell-Rezeptors (CDR) und S-Mutationen in den strukturellen Domänen (FR) als Hinweis auf eine antigen-gesteuerte Hypermutation in den Tumorsubklonen fand sich nur in einem FL Grad I. Aus den genetischen Analysen ergaben sich aber Hinweise auf eine erhaltene Funktionalität des B-Zell-Rezeptors in allen sieben Fällen.
In der vorliegenden Arbeit wurden die Expressionshäufigkeiten von MAGE-A-Antigenen, E -Cadherin, Laminin-5-gamma-2, MMP2 und MMP9 in Plattenepithelkarzinomen im Kopf- und Halsbereich untersuchen. Hierbei findet der Vergleich zwischen Primärtumoren und korrespondierenden Lymphknotenmetastasen besondere Beachtung. Um die Hypothese zu verifizieren, dass die o.g. Parameter einen signifikanten Einfluss auf die Progression und Metastasierung haben, wird der Zusammenhang zwischen den in der vorliegenden Arbeit gewonnenen Ergebnissen und diversen klinischen Parametern mittels Korrelationsanalyse untersucht.
Zur Entscheidungshilfe in der Therapiefindung des Mammakarzinoms haben sich bezüglich der Indikation zur Chemotherapie neben den klinischen und histopathologischen Kriterien in den letzten Jahren vorrangig Multigentests etabliert. In der vorliegenden Arbeit wurden Zusammenhänge zwischen dem Oncotyp DX® und 18 immunhistochemischen Markern aus der Tumorbiologie für 78 Fälle hormonrezeptorpositiver, Her2/neu negativer Mammafrühkarzinome mit niedrigem Lymphknotenstatus untersucht. Es erfolgten immunhistochemische Färbungen an Microtissue-Arrays der Tumorproben. Für die Marker AMACR, Cyclin D1, p53, MDM2 und PDL1 ergab sich eine klare statistisch signifikante Korrelation zum Recurrence-Score®des Oncotyp DX® und mit Einschränkungen auch für CDK4. Die Marker p27, Bcl2 und Glut 1 erreichten ein etwas niedrigeres Signifikanzniveau in der statistischen Analyse. Der immunhistochemische Routinemarker Ki67% zeigte eine hochsignifikante Korrelation mit dem Recurrence-Score®. Hierdurch ergeben sich neue Perspektiven zur Risikostratifizierung des Mammakarzinoms, wie beispielsweise die konsekutive Entwicklung eines immunhistochemischen Scores mit prädiktivem Wert für den Recurrence-Score® mit klinischer Anwendung als Prätest oder als eigenständiges Stratifizierungstool bei Brustkrebs.
Die histologische Technik der Tissue-Microarrays ist eine sehr effiziente Methode, um eine große Anzahl auch heterogener Lymphome wie des Hodgkin-Lymphoms bei hohem Durchsatz unter homogenen Färbebedingungen zu untersuchen. Die vorliegende Arbeit konnte zeigen, dass ein Probeschnitt zur vorherigen Auswahl stanzwürdigen Tumorareals nicht nötig ist. Die so genannte Blindstanzung trug weniger als einen Prozentpunkt (0,9%) zum Verlust der auswertbaren Fälle bei. Dennoch war bei einzelnen Parametern (LMP1 und EBER) ein hoher Gewebeverlust zu beobachten. In einer Stichprobe von 2696 Stanzen waren es 24% (631 Stanzen) bedingt durch die Färbetechnik, aber auch durch unterschiedliche Vorbehandlungen und Originalfixationen des Probematerials. In dieser Studie wurden 1212 Fälle zur Immuntypisierung von Tumorzellen des klassischen und nodulär lymphozyten-prädominanten Hodgkin-Lymphoms untersucht. Die Fälle von c-HL wiesen eine häufige Expression von CD30- und CD15-Oberflächenmarkern und kaum B-Zell-Marker auf, während im NLP-HL die Expression in umgekehrter Häufigkeit vorlag. Diese bisher größte Untersuchung von T-Zell-Markern an H-/RS-Zellen, erstmalig auch an NLP-HL, ergab eine bis zu 6-fach höhere Frequenz in der NLP-HL bei häufigerer B-Zell-Marker-Expression. Die in der Literatur beschriebene Rangordnung der Expressionshäufigkeit von Oberflächenantigenen im c-HL (CD2 > CD4 > CD3 > CD5 > CD8) wurde bestätigt und wich nur in den Markern CD3 und CD5 ab: Perforin >> CD4 > CD5 > CD3 > CD8 > GranzymB > TIA-1 > CD7. Tzankov et al. [45] fanden in ihrer Untersuchung mit 259 c-HL-Fällen eine Häufigkeit von 5% T-Zell-Marker-Expression. Die vorliegende Arbeit mit 1147 untersuchten c-HL-Fällen kam zum Ergebnis einer deutlich höheren T-Zell-Marker-Expressionshäufigkeit von 20,1%. Der pathophysiologische Mechanismus der T-Zell-Marker-Expression ist bis heute noch unklar, könnte aber als eine alternative Signalkaskade zur Aufrechterhaltung des Zellzyklus unter geändertem Zellmilieu gedeutet werden. Ein weiterer Fokus dieser Arbeit betraf die Gruppe der Studienteilnehmer 60 Jahre und älter, um Hinweisen auf das „age-related“ EBV-associated Lymphom und der Rolle der Mikrosatelliten-Instabilität nachzugehen. So fand sich eine signifikante Häufung von Markern für eine EBV-Infektion (EBER, LMP1) in der Gruppe der über 60-Jährigen. Die Expression von DNA-Reparaturenzymen, deren Ausbleiben auf Mikrosatelliten-Instabilität gedeutet hätte, unterschied sich zwischen jüngeren und älteren Studienteilnehmern nicht.
The mitotic spindle assembly checkpoint (SAC) maintains genome stability and marks an important target for antineoplastic therapies. However, it has remained unclear how cells execute cell fate decisions under conditions of SAC‐induced mitotic arrest. Here, we identify USP9X as the mitotic deubiquitinase of the X‐linked inhibitor of apoptosis protein (XIAP) and demonstrate that deubiquitylation and stabilization of XIAP by USP9X lead to increased resistance toward mitotic spindle poisons. We find that primary human aggressive B‐cell lymphoma samples exhibit high USP9X expression that correlate with XIAP overexpression. We show that high USP9X/XIAP expression is associated with shorter event‐free survival in patients treated with spindle poison‐containing chemotherapy. Accordingly, aggressive B‐cell lymphoma lines with USP9X and associated XIAP overexpression exhibit increased chemoresistance, reversed by specific inhibition of either USP9X or XIAP. Moreover, knockdown of USP9X or XIAP significantly delays lymphoma development and increases sensitivity to spindle poisons in a murine Eμ‐Myc lymphoma model. Together, we specify the USP9X–XIAP axis as a regulator of the mitotic cell fate decision and propose that USP9X and XIAP are potential prognostic biomarkers and therapeutic targets in aggressive B‐cell lymphoma.
Oncogenic transformation of lung epithelial cells is a multistep process, frequently starting with the inactivation of tumour suppressors and subsequent development of activating mutations in proto-oncogenes, such as members of the PI3K or MAPK families. Cells undergoing transformation have to adjust to changes, including altered metabolic requirements. This is achieved, in part, by modulating the protein abundance of transcription factors. Here, we report that the ubiquitin carboxyl-terminal hydrolase 28 (USP28) enables oncogenic reprogramming by regulating the protein abundance of proto-oncogenes such as c-JUN, c-MYC, NOTCH and ∆NP63 at early stages of malignant transformation. USP28 levels are increased in cancer compared with in normal cells due to a feed-forward loop, driven by increased amounts of oncogenic transcription factors such as c-MYC and c-JUN. Irrespective of oncogenic driver, interference with USP28 abundance or activity suppresses growth and survival of transformed lung cells. Furthermore, inhibition of USP28 via a small-molecule inhibitor resets the proteome of transformed cells towards a ‘premalignant’ state, and its inhibition synergizes with clinically established compounds used to target EGFR\(^{L858R}\)-, BRAF\(^{V600E}\)- or PI3K\(^{H1047R}\)-driven tumour cells. Targeting USP28 protein abundance at an early stage via inhibition of its activity is therefore a feasible strategy for the treatment of early-stage lung tumours, and the observed synergism with current standard-of-care inhibitors holds the potential for improved targeting of established tumours.
Untersuchungen zur Expression der Rezeptortyrosinkinase c-kit in Thymuskarzinomen und Thymomen
(2005)
Thymome und Thymuskarzinome sind seltene mediastinale Neoplasien. In der vorliegenden Arbeit wurde die Expression und der Aktivierungsstatus der Rezeptortyrosinkinase c-kit in Thymustumoren untersucht und deren diagnostische und klinische Relevanz aufgezeigt. Das c-kit-Gen wurde auf Mutationen untersucht sowie der Aktivierungsstatus im c-kit-Transduktionspfad nachgeschalteter Proteine festgestellt.
Follikuläre Lymphome (FL) zählen zu den Non-Hodgkin-Lymphomen und stellen die
größte Untergruppe der B-Zell-Lymphome dar. Bedingt durch ihren meist indolenten
Verlauf werden sie oft erst in einem fortgeschrittenen klinischen Stadium III/IV
diagnostiziert und stellen dann eine systemische Erkrankung dar.
Gelegentlich wird in der histopathologischen Untersuchung eines befallenen
Lymphknotens eine nur partielle Infiltration beobachtet, die häufig auch in den
angeschlossenen Stagingmaßnahmen mit einer nur lokalen Tumorausbreitung (klinisches
Stadium I/II) assoziiert ist. Ein solches lokal begrenztes Stadium kann gemäß der
Standard-Behandlungsprotokolle mit einer alleinigen Strahlentherapie ausreichend
kontrolliert werden.
Ziel der vorliegenden Arbeit war es zum einen, eine mögliche Assoziation einer nur
partiellen Lymphknoteninfiltration beim FL mit einem lokal begrenzten klinischen Stadium
zu untersuchen. Zum anderen sollte die Inzidenz einer nur partiellen Lymphknoten-
Infiltration beim FL bestimmt werden.
Der Vergleich der Studienkohorte mit einer nur partiellen Lymphknoteninfiltration, definiert
als zumindest ein vollständig erhaltener Lymphfollikel, mit der Kontrollkohorte zeigte
einen hochsignifikanten Unterschied: In der Studienkohorte befanden sich 38 von 40 Fälle
(95%) in einem lokalen Stadium, wohingegen die Kontrollkohorte mit vollständiger
Lymphknoteninfiltration nur bei 10 von 49 Patienten (20%) ein lokales Krankheitsstadium
(p<0.001) aufwies.
Um die erhaltenen Ergebnisse zu validieren, wurden alle FL Grad 1-3A aus dem
exemplarischen Jahr 2001 untersucht. Hier zeigte sich in 34 Fällen (11 %) eine nur
partielle Infiltration. In allen 18 Fällen mit mindestens einem vollständig erhaltenen
reaktiven Keimzentrum lag in Übereinstimmung mit der initialen Studienkohorte ein lokales
Krankheitsstadium I/II vor (p<0.001).
Die erhaltenen Ergebnisse zeigen eindrücklich, dass follikuläre Lymphome mit einem nur
partiellen Befall der Lymphknoten häufig mit einem (noch) lokalen klinischen Stadium
assoziiert sind. In diesen Fällen käme eine alleinige Bestrahlung als Therapieoption in
Betracht.
Marginalzonen-Lymphome (MZL) gehören zur Gruppe der indolenten Non-Hodgkin-Lymphome der B-Zell-Reihe, zu denen nach der aktuellen WHO-Klassifikation auch die primär kutane Marginalzonen-Lymphome (PCMZL) zählen. Eine klonale Leicht- und Schwerkettenexpression kann immunhistochemisch speziell in MZL mit sekretorischer/plasmozytoider Differenzierung (unabhängig von ihrer Primärlokalisation) nachgewiesen werden. In Voruntersuchungen war aufgefallen, dass von primär kutanen MZL ungewöhnlich häufig IgG bzw. IgG4 exprimiert wird, während extrakutane MZL auch nach Literaturangaben eine präferentielle IgM-Expression aufweisen. In der hier vorgelegten Arbeit wurde die Prävalenz einer IgG4-Expression an einer großen Kohorte von sekretorisch/plasmazellulär differenzierten MZL untersucht. Hierzu wurde die Immunglobulinschwerkettenexpression an 169 MZL unterschiedlicher Primärlokalisationen immunhistochemisch analysiert. Es konnte gezeigt werden, dass PCMZL überzufällig häufig IgG exprimieren (78 %, 35/49), wobei der Anteil IgG4-positiver PCMZL mit 54 % (19 von 35) sogar über dem der anderen drei IgG-Subklassen lag (46 %, 16/35). Unter den 120 anderen, nicht kutanen MZL war lediglich ein okuläres MZL positiv für die Schwerkette IgG4.
Ferner wurde an dem in dieser Arbeit näher charakterisierten Kollektiv der PCMZL molekularbiologische Untersuchungen zur Frage einer MyD88 (L265P)-Mutation durchgeführt, die letztendlich in keinem der diesbezüglich auswertbaren 45 PCMZL nachgewiesen werden konnte.
In der vorliegenden Arbeit wurde das Vorkommen neu erworbener N-Glykosylierungsmotive in t(14;18)-positiven und -negativen FL der lokalisierten (FL I/II) und fortgeschrittenen Stadien (FL III/IV), sowie zum Zeitpunkt der Primärdiagnose und des Rezidivs untersucht. Dabei wurde der jeweilige Haupttumorklon mit Hilfe von „Next Generation Sequencing“ und unter Verwendung des „LymphoTrack® Assays“ in einer Serie von 68 kryoasservierten FL identifiziert 36 t(14;18)-negative und 32 t(14;18)-positive FL. Die Frequenz neu erworbener N-Glykosylierungsmotive unterschied sich signifikant zwischen t(14;18)-positiven und -negativen PD/R-FL III/IV, während man zwischen t(14;18)-positiven und -negativen PD/R-FL I/II keinen Unterschied beobachten konnte. Des Weiteren zeigten t(14;18)-negative PD/R-FL I-IV im Vergleich zu t(14;18)-positiven PD/R-FL I-IV signifikant häufiger einen Zugewinn neuer N-Glykosylierungsmotive in der FR3 Region des BCL2 Gens, sowie eine vermehrte Nutzung des IGHV4-34 Keimbahngens. Interessanterweise beschränkte sich die Nutzung des IGHV4-34 Gens auf PD-FL und konnte in R-FL nicht nachgewiesen werden. Da sowohl das Vorkommen neu erworbener N-Glykosylierungsmotive in FR3 als auch die Nutzung von IGHV4-34 im Zusammenhang mit Autoimmunerkrankungen beschrieben wurden, deuten unsere Ergebnisse darauf hin, dass die Subgruppe der t(14;18)-negativen FL im pathologischen Prozess der Onkogenese mehr auf die Stimulation durch (Auto)-Antigene als durch die Stimulation des B-Zell Rezeptors mit Lektinen (DC-SIGN) angewiesen sein könnte.
Bei vielen Karzinomen spielt EGFR und das KRAS-Onkogen eine wichtige Rolle in der Tumorentstehung. Da bei den seltenen Karzinomen an Kopfspeicheldrüsen sehr wenig über molekulare Mechanismen der Tumorgenese bekannt ist, war es das Ziel der Arbeit den EGFR-Signalweg zu untersuchen. Es wurden Paraffinschnitte von 43 Speicheldrüsenkarzinomen von den Typen ACC, MEC und Adeno-Ca NOS mit dem phosphorylierten EGFR-Antikörper gefärbt und mit klinisch-pathologischen Daten korreliert. Weiterhin wurde eine Mutationsanalyse der kras-Gensequenz durchgeführt. In allen Fällen war das kras-Gen vom Wildtyp. Bei der Expressionsanalyse von EGFR stellte sich heraus, dass 79% der Proben einen aktivierten EGF-Rezeptor besitzen. Statistisch signifikante Korrelationen gab es zwischen der EGFR-Expression und dem Patientenalter, dem zervikalen Lymphknotenbefall und der Tumorgröße. Der EGF-Signaltransduktionsweg ist bei den untersuchten Karzinomen der Kopfspeicheldrüsen im überwiegenden Masse aktiviert, ohne dass eine autonome Aktivierung beim KRAS-Onkogen vorliegt.
In der vorliegenden Studie haben wir mit Hilfe von SHARP-Screening, einer 16S-rRNA-basierten Heterogenitäts- und phylogenetischen Analyse, die mikrobielle Diversität in entzündlichen Lymphadenitiden ohne vorherige Kenntnis der jeweiligen Erreger an einer Serie von 15 Lymphknoten untersucht. Die Methode wurde erstmals auf diese Fragestellung angewandt. Sie konnte für die Verwendung von paraffineingebettetem Gewebe adaptiert werden, so dass auch Gewebeproben analysiert werden konnten, von denen kein Gefriermaterial zur Verfügung stand und die in Routineverfahren eingebettet und nach Standardmethoden gefärbt wurden. SHARP-Screening beinhaltet zwei komplementäre Schritte: Zuerst erfolgte die Erstellung einer Genbank aller bakteriellen Gene aus der gesamten extrahierten DNA des analysierten Gewebes durch gezielte Amplifikation des 16S-rRNA-Gens mittels universeller eubakterieller Primer. Als zweiter Schritt wurde nach der Transformation mittels Analyse des Restriktionsfragmentlängenpolymorphismus die Selektion der jeweiligen unterschiedlichen Phylotypen der enthaltenen 16S-rRNA-Gene durchgeführt (insgesamt 400). Nach der Sequenzierung wurden die 16S-rRNA-Gene durch den Vergleich mit bekannten bakteriellen Sequenzen mit Hilfe des „Basic-Local-Alignment-Search-Tool“ (BLAST) identifiziert. SHARP-Screening hat sich als geeignete Methode zur Analyse der gesamten, in einer Gewebeprobe enthaltenen bakteriellen Flora erwiesen. Dabei wurden zum Teil andere Erreger gefunden, als aus dem histologischen Bild vermutet wurden. So konnte zum Beispiel mit dem Nachweis von Gluconacetobacter sacchari, als potentieller Erreger einer septischen Granulomatose, eine alternative Differentialdiagnose zur histologisch vermuteten Katzenkratzkrankheit aufgezeigt werden. Darüber hinaus konnten auch gleichartige histologische Bilder bei dem gleichen identifizierten Erreger beobachtet werden. Zum Beispiel konnten im Zusammenhang mit dem Auftreten von Ödemen, Nekrosen, granulomatös eitrigen Veränderungen und einer ausgeprägten Sinus-histiozytose im Lymphknotengewebe immer wieder Comamonadaceae bzw. Janthinobacterium nachgewiesen werden. Oft zeigte sich nicht ein einzelner Erreger der Lymphadenitis, sondern ein ganzes Spektrum, wobei aus dem Vorhandensein der 16S-rRNA nicht auf das Vorhandensein vitaler Erreger geschlossen werden kann. Dennoch erlaubt die Häufigkeit der entsprechenden Klone eine semiquantitative Abschätzung der Bedeutung des jeweiligen Erregers. So wies SHARP-Screening auch Homologien zu Paracoccus yeeii nach. Eine Spezies, die mit klassischen Methoden häufig übersehen wird, die in Lymphknoten jedoch eine pathogene Rolle spielen kann. Im Zusammenhang mit dem histologischen Verdacht auf ein Malignom wurden in einigen Fällen Streptomyces, Roseomonas gilardii rosea und Stenotrophomonas maltophila nachgewiesen, die auch in der Literatur häufig bei immunsupprimierten Patienten vorkommen. Bei dem Verdacht auf ein Lymphgranuloma venerum wurde eine Cyanobacterium-Spezies detektiert, die es nach Literaturangaben Chlamydia trachomatis erst möglich macht, den eigenen Aminosäurestoffwechsel zu betreiben. Insgesamt dürften vom SHARP-Screening noch weitere tiefgreifende Erkenntnisse der bakteriellen Diversität und kausaler Erregerassoziationen in Erkrankungen des lymphatischen Systems zu erwarten sein.
Knochensialoprotein (BSP) ist ein Protein der extrazellulären Matrix im Knochen und mineralisierten Geweben, wird aber auch von verschiedenen Tumorzellen exprimiert (Bellahcene et al., 1994, 1997, 1998). Dies ist assoziiert mit einer schlechten Prognose und einem erhöhten Risiko für eine spätere Entwicklung von Knochenmetastasen. Diel et al. (1999) konnte zeigen, dass ein erhöhter Serum-BSP-Wert bei Patientinnen mit Mammakarzinom zu einem gehäuften Auftreten von Knochenmetastasen im Laufe der Erkrankung führt. BSP scheint ein Marker für die Entstehung von Knochenmetastasen zu sein. In der Literatur ist ein Antikörper beschrieben, der ein Epitop des BSP erkennt, welches im BSP aus Tumorzellen nicht glykosyliert ist, im BSP aus mineralisiertem Gewebe allerdings schon (Armbruster et al., 2009). Im Tiermodell konnte gezeigt werden, dass Knochenmetastasen verhindert werden können bei gleichzeitiger Gabe von Tumorzellen und Antikörpern gegen BSP beziehungsweise, dass bei vorhandenen Knochenmetastasen eine Behandlung der Tiere mit einem Anti-BSP-Antikörper die Metastasen zurückbildet (Bäuerle et al., 2005, 2006). In der aktuellen Arbeit wird die Expression von BSP an menschlichem Gewebe von Knochenmetastasen mit unterschiedlichen Primärtumoren mittels Immunhistochemie untersucht. Insgesamt wurden 35 Fälle von Knochenmetastasen mit Primärtumor eines Mammakarzinoms untersucht, wobei 22,9% eine BSP Expression aufweisen, davon 5,7% eine starke. Knochenmetastasen mit dem Primärtumor Prostatakarzinom sind mit 8 Fällen repräsentiert, wobei 75% positiv für BSP sind, davon 25% stark positiv. Die einzelnen Fälle zeigen eine starke BSP Expression im Stroma und eine schwache BSP Expression der Tumorzellen. Diese Ergebnisse des Antikörpers gegen normal glykosyliertes BSP wurden verglichen mit dem Antikörper gegen nicht glykosyliertes BSP. Der Nachweis von BSP in Tumorzellen zeigt dasselbe Ergebnis, BSP im Stroma wird durch den Antikörper gegen nicht- glykosyliertes BSP intensiver dargestellt. Daraus lässt sich folgern, dass der Antikörper gegen nicht- glykosyliertes BSP nicht spezifisch für die Isoform des BSP aus Tumorzellen ist, sondern gleichermaßen in der Routinediagnostik von BSP eingesetzt werden kann. Die Untersuchung könnte sogar darauf hinweisen, dass dieser Antikörper die nicht- glykosylierte Isoform im Stroma erkennt und damit bei Untersuchung des Stromas die bessere Alternative darstellt.
The paracaspase Malt1 is a central regulator of antigen receptor signaling that is frequently mutated in human lymphoma. As a scaffold, it assembles protein complexes for NF-kappa B activation, and its proteolytic domain cleaves negative NF-kappa B regulators for signal enforcement. Still, the physiological functions of Malt1-protease are unknown. We demonstrate that targeted Malt1-paracaspase inactivation induces a lethal inflammatory syndrome with lymphocyte-dependent neurodegeneration in vivo. Paracaspase activity is essential for regulatory T cell (Treg) and innate-like B cell development, but it is largely dispensable for overcoming Malt1-dependent thresholds for lymphocyte activation. In addition to NF-kappa B inhibitors, Malt1 cleaves an entire set of mRNA stability regulators, including Roquin-1, Roquin-2, and Regnase-1, and paracaspase inactivation results in excessive interferon gamma (IFN gamma) production by effector lymphocytes that drive pathology. Together, our results reveal distinct threshold and modulatory functions of Malt1 that differentially control lymphocyte differentiation and activation pathways and demonstrate that selective paracaspase blockage skews systemic immunity toward destructive autoinflammation.
Zusammenfassung Die Langzeitresultate von aortocoronaren Venenbypässen unter Verwendung von Vena saphena magna Interponaten hängen neben vielen anderen Faktoren maßgeblich von der Integrität des Gefäßendothels ab. Ein intaktes Endothel spielt für die Offenheit des Grafts eine entscheidende Rolle, da Endothelverletzungen die Entwicklung vorzeitiger thrombotischer Graftverschlüsse triggern und auch an den späten Graftverschlüssen durch Intimahyperplasie und Einsprossung glatter Muskelzellen beteiligt sind. So spielt die Vermeidung intraoperativer Endothelschädigungen der Venengrafts durch die Lagerungsmedien eine entscheidende Rolle. Diese Arbeit hatte zum Ziel, das Endothel von Venengrafts nach Inkubation mit verschiedenen Lagerungslösungen mit direkten Nachweismethoden wie Rasterelektronen- und Transelektronenmikroskopie zu untersuchen. Untersucht wurden sieben cm lange Venensegmente von sechs Patienten, die sich einer ACVB-Operation unterzogen. Die Präparation der Venen fand unter standardisierten Bedingungen statt. Anschließend erfolgte die Inkubation jeweils eines Drittels der entnommenen Segmente für 45 Minuten in einer der folgenden Lagerungsmedien, physiologische Kochsalzlösung, Medium 199 + 20mM HEPES + 5% bovines Serumalbumin und Medium 199 + 20mM HEPES + 20% humanes Serumalbumin. Die Auswertung des Endothelzellschadens erfolgte mittels raster- und transelektronenmikroskopischer Untersuchungen sowie histopathologischer Aufarbeitung. Venensegmente nach Lagerung in physiologischer Kochsalzlösung zeigen signifikante Schädigungen der Endothelzelloberfläche. Bereits nach 45-minütiger Lagerung findet sich in den rasterelektronenmikroskopischen Aufnahmen eine 56,5%ige Ablösung der Endothelzellschicht, transelektronenmikroskopisch kann man Zellschädigungen im Sinne von Zellhydrops und Karyolyse nachweisen. Dagegen findet man nach Lagerung in Medium 199 mit 20%igem Albuminanteil bei Betrachtung mit dem Rasterelektronenmikroskop deutlich geringere Zellschädigungen. Das Endothel von Venen nach Inkubation mit Nährmedium mit 5%igem Albuminanteil stellt sich nahezu intakt, ohne wesentliche Zerstörungen der Zelloberfläche dar. Unsere Arbeit konnte belegen, dass die Lagerungsmethode einen deutlichen Einfluss auf das Gefäßendothel ausübt. Um möglichst große Anteile intakten Endothels zu gewährleisten, bedarf es einer Modifizierung der bisherigen Handhabung der Venenlagerung während einer aortocoronaren Venenbypass-Operation. Eine Möglichkeit dazu könnte in der Lagerung in Zellkulturmedium mit einem 5%igen Albuminanteil gesehen werden.
Intrakranielle Blutungen sind im Säuglingsalter seltene, aber lebensbedrohende Ereignisse. Neben Gefäßmissbildungen, Stoffwechseldefekten sowie Störungen der Blutgerinnung kommen v. a. nichtakzidentielle Traumata, Schütteltrauma in Betracht. Die klinische Diagnostik umfasst hinsichtlich der Blutungsgenese neben Sonographie und MRT als apparatives Verfahren auch eine Fundoskopie sowie laborchemische Analysen, insbesondere der Gerinnungsparameter. Für die Blutgerinnung ist das fettlösliche Vitamin K essenziell: Frühe, klassische und späte Vitamin-K-Mangel-Blutungen werden dabei unterschieden. Um ein gehäuftes Wiederauftreten von Vitamin-K-Mangel-Blutungen bei Neugeborenen und jungen Säuglingen zu verhindern, bedarf es einer hinreichenden Aufklärung der Eltern. Eine Verweigerung der Prophylaxe scheint Folge einer weltanschaulich begründeten Ablehnung der Schulmedizin und ein zunehmendes Phänomen in wohlhabenden Industrieländern zu sein.
Humane oder humanisierte monoklonale Antikörper haben sich in den letzten zehn Jahren als Arzneimittel etabliert. Sie sind hochspezifisch und zeigen in ihrer Anwendung im Vergleich zu konventionellen Therapeutika viel weniger Nebenwirkungen. In den 80er Jahren gelang es am Pathologischen Institut der Universität Würzburg eine Reihe von humanen Antikörpern aus Patienten zu isolieren, die hochspezifisch mit malignen Zellen reagieren und diese sowohl in vitro als auch im experimentellen Tiermodel selektiv durch Induktion von Apoptose töten. Um die Wirkungsweise von monoklonalen Antikörpern in der Krebstherapie zu erhöhen, werden die meisten in Kombination mit herkömmlichen Methoden, wie Chemotherapie, eingesetzt. Die ideale Therapieform sind hinsichtlich der Nebenwirkungen sog. Cocktails aus verschiedenen monoklonalen Antikörpern. Allerdings sind die Studien hierzu noch wenig fortgeschritten. Das Ziel dieser Arbeit war es, in präklinischen Versuchsreihen den Einsatz verschiedener tumorspezifischer humaner monoklonaler Antikörper als Cocktail und in Kombination mit Chemotherapie zu evaluieren. Hierzu wurden neun Antikörper in 32 verschiedenen Antikörperkombinationen hinsichtlich ihrer Auswirkungen auf die in vitro Proliferation einer Pankreaskarzinom-Zellinie untersucht. In Immunfluoreszenz-Aufnahmen ließ sich zeigen, dass kombinierte Antikörper an unterschiedlichen Stellen an der Zelle binden, was eindeutig auf verschiedene Zielstrukturen hinweist. Einige werden dabei endozytiert, während andere auf der Zellmembran bleiben. Interessanterweise ließen sich Kombinationen identifizieren, deren antiproliferative Wirkung sowohl additiv als auch synergistisch ist, das heißt größer als die Summe ihrer Einzelaktivitäten. Wurden Antikörper mit Zytostatika (5-Flurouracil) kombiniert, so ließen sich ebenfalls synergistische Effekte beobachten. In FACS-Analysen zeigt sich ein gesteigertes Bindungsverhalten der Antikörper, wenn die Zellen mit 5-FU vorinkubiert wurden. Zusammenfassend bestätigen die Ergebnisse dieser Arbeit die Beobachtung, dass die Wirkung humaner monoklonaler Antikörper in Kombination mit Chemotherapie erhöht werden kann. Für die Zukunft humaner Antikörper als Therapiemittel gegen maligne Erkrankungen mag allerdings noch wichtiger sein, dass Antiköper in Cocktails tatsächlich synergistische Wirkung zeigen können.
Die Entstehung maligner Zellen durch irreversible genetische Veränderungen ist ein allgegenwärtiger Prozess im menschlichen Organismus. Allein die spontane Mutationsrate genügt um in einem Organismus permanent transformierte Zellen entstehen zu lassen, welche den Körper in kürzester Zeit überschwemmen würden. Auch wenn bestimmte genetische Schäden frühzeitig durch Reparaturmechanismen beseitigt werden und sich nicht jede transformierte Zelle in einem Tumor manifestiert, so ist die eigentliche Frage nicht, warum Krebs entsteht, sondern warum er bei der hohen Mutationsrate so selten auftritt. Verantwortlich für die frühe Erkennung und Beseitigung transformierter Zellen ist das körpereigene Immunsystem, das in der Lage ist die meisten aberranten Zellen zu entfernen, sodass der manifeste Tumor die Ausnahme und nicht die Regel ist. Der menschliche Organismus verfügt über ein angeborenes und ein erworbenes Immunsystem. Bis heute ist nicht eindeutig geklärt, ob maligne Zellen mit ihren veränderten Oberflächenstrukturen erst eine Immunantwort induzieren müssen oder ob, wie bei der Abwehr infektiöser Partikel, die angeborene Immunität für die Beseitigung von Tumorzellen verantwortlich ist. Die in dieser Arbeit verwendete humane Hybridoma Technologie (Immortalisierung menschlicher Lymphozyten und Isolierung monoklonaler Antikörper) bietet die einzigartige Möglichkeit, sowohl aus an Krebs erkrankten Patienten als auch aus gesunden Probanden tumorspezifische Antikörper zu isolieren und durch deren genauere Charakterisierung Einblicke in die humorale Immunität gegen maligne Zellen zu erhalten. In der vorliegenden Arbeit werden fünf humane monoklonale Antikörper beschrieben, die aus verschiedenen Tumorpatienten gewonnen wurden, sowie zwei Antikörper, die aus gesunden Probanden isoliert werden konnten. In allen Fällen erwiesen sich die Antikörper als tumorspezifisch, d.h. sie reagieren nicht mit gesundem Gewebe und sind demnach keine Autoantikörper. Es handelt sich weiterhin in allen Fällen um Antikörper des IgM-Isotyps; es konnten keinen Antikörper anderer Ig-Klassen isoliert werden. Genetische Analysen ergaben, dass alle isolierten Antikörper gering oder gar nicht mutiert waren, was bedeutet, dass sie nicht durch Stimulation affinitätsgereift sind. Zudem konnte demonstriert werden, dass alle Antikörper Apoptose von Tumorzellen induzieren und dass sie an eine Zuckerkette ihrer Antigene binden oder solche Carbohydrate zumindest entscheidend in die Bindung involviert sind. Die Eigenschaften der in dieser Arbeit beschriebenen Antikörper wurden mit anderen bereits etablierten IgM-Antikörpern verglichen. Hierbei stellte sich heraus, dass alle Antikörper, welche sich als tumorspezifisch erwiesen, ähnliche Eigenschaften zeigen. Interessant ist zudem die Beobachtung, dass die Keuzreaktion der Antikörper, also ihre Reaktion mit anderen Tumorgeweben, reziprok mit dem Mutations-grad korreliert ist. Je mehr Mutationen ein Antikörper aufweist, desto eingeschränkter und spezifischer sind demnach seine Reaktionen mit anderen Tumoren. Dies deutet darauf hin, dass auch innerhalb der Keimbahn-kodierten Antikörper durch vereinzelte Mutationen eine höhere Variabilität erzeugt werden kann. Ähnlich wie bei der Affinitätsreifung der erworbenen Immunität scheint sich auch hier die Spezifität mit der Anzahl der Mutationen zu erhöhen. Zusammenfassend weisen die erhaltenen Ergebnisse darauf hin, dass zumindest die humorale Immunität gegen maligne Zellen das Resultat der angeborenen Immunität ist und nicht von Tumorzellen induziert wird. Dies bedeutet zudem, dass Moleküle wie natürliche Antikörper in der Immunität eine viel größere Rolle spielen als bisher angenommen. Ähnliche Ergebnisse wurden bereits bei der Untersuchung der Immunität gegen bakterielle Antigene erzielt, sodass hier vermutet werden kann, dass die gleichen Mechanismen zugrunde liegen wie bei der Abwehr transformierter Zellen. Darüber hinaus wird die Frage beantwortet, warum ein manifester Tumor eine Ausnahme bleibt. Die angeborene, primäre Immunität verfügt über ein existierendes Repertoire an Rezeptoren, welche eine ausreichende Variabilität aufweisen, und muss daher nicht erst über ein komplexes System von Erkennung und Stimulation, wie die adaptierte Immunität, induziert werden. Dieser logistische Vorsprung der natürlichen Immunität garantiert eine permanente Überwachung und eine schnelle Reaktion gegenüber veränderten Zellen und fremden Partikeln.
The transcription factor NFATc1 has been shown to regulate the activation and differentiation of T-cells and B-cells, of DCs and megakaryocytes. Dysregulation of NFAT signaling was shown to be associated with the generation of autoimmune diseases, malignant transformation and the development of cancer [71]. The primary goal of this work was to gain insights on Nfatc1 induction and regulation in lymphocytes and to find new direct NFATc1 target genes. Three new BAC -transgenic reporter mouse strains (tgNfatc1/Egfp, tgNfatc1/DE1 and tgNfatc1/DE2) were applied to analyze Nfatc1 induction and regulation in primary murine B- and T-cells. As a result, we were able to show the persistent requirement of immunoreceptor-signaling for constant Nfatc1 induction, particularly, for NFATc1/αA expression. Furthermore, we showed that NF-κB inducing agents, such as LPS, CpG or CD40 receptor engagement, in combination with primary receptor-signals, positively contributed to Nfact1 induction in B-cells [137]. We sought to establish a new system which could help to identify direct NFATc1 target genes by means of ChIP and NGS in genom-wide approaches. We were able to successfully generate a new BAC-transgene encoding a biotinylatable short isoform of NFATc1, which is currently injected into mice oocyte at the TFM in Mainz. In addition, in vivo biotinylatable NFATc1–isoforms were cloned and stably expressed in the murine B-cell lymphoma line WEHI-231. The successful use of these cells stably overexpressing either the short NFATc1/αA or the long NFATc1/βC isoform along with the bacterial BirA biotin ligase was confirmed by intracellular stainings, FACS analysis, confocal microscopy and protein IP. By NGS, we detected 2185 genes which are specifically controlled by NFATc1/αA, and 1306 genes which are exclusively controlled by NFATc1/βC. This shows that the Nfatc1 locus encodes “two genes” which exhibit alternate, in part opposite functions. Studies on the induction of apoptosis and cell-death revealed opposed roles for the highly inducible short isoform NFATc1/αA and the constantly expressed long isoform NFATc1/βC. These findings were confirmed by whole transcriptome-sequencing performed with cells overexpressing NFATc1/αA and NFATc1/βC. Several thousand genes were found to be significantly altered in their expression profile, preferentially genes involved in apoptosis and PCD for NFATc1/βC or genes involved in transcriptional regulation and cell-cycle processes for NFATc1/αA. In addition we were able to perform ChIP-seq for NFATc1/αA and NFATc1/βC in an ab-independent approach. We found potential new target-sites, but further studies will have to address this ambitious goal in the future. In individual ChIP assays, we showed direct binding of NFATc1/αA and NFATc1/βC to the Prdm1 and Aicda promoter regions which are individually controlled by the NFATc1 isoforms.
The Nuclear Factors of Activated T cells (NFATs) are critical transcription factors that direct gene expression in immune and non-immune cells. Interaction of T cells with Ag-presenting cells results in the clustering of T-cell antigen receptor (TCR), co-receptors and integrins. Subsequent signal transduction resulting in NFAT activation leads to cytokine gene expression. Among the NFATs expressed in T cells, NFATc1 shows a unique induction property, which is essential for T cell differentiation and activation. It was revealed before that 3 major isoforms of NFATc1 are generated in activated T cells – the inducible short NFATc1/A, and the longer isoforms NFATc1/B and C. However, due to alternative splicing events and the existence of two different promoters and two alternative polyadenylation, we show here that 6 isoforms are synthesized in T cells which differ in their N-terminal and C-terminal peptides. In these experiments, we have identified these 6 isoforms by semi-quantitative long distance RT-PCR in several T cells subsets, and the inducible properties of 6 isoforms were investigated in those cells. The short NFATc1/A which is under control of the P1 promoter and the proximal pA1 polyadenylation site was the most prominent and inducible isoform in T effector cells. The transcription of the longer NFATc1/B and C isoforms is constitutive and even reduced in activated T lymphocytes. In addition to NFATc1 autoregulation, we tried to understand the NFATc1 gene regulation under the control of PKC pathways by microarray analysis. Compared to treatment of T cells with ionomycin alone (which enhances Ca++ flux), treatment of cells with the phorbolester TPA (leading to PKC activation) enhanced the induction of NFATc1. Microarray analysis revealed that PKC activation increased the transcription of NF-B1, Fos and JunB, which are important transcription factors binding to the regulatory regions of the NFATc1 gene. Besides the promoting effect of these transcription factors, we provided evidence that p53 and its targeting gene, Gadd45, exerted a negative effect on NFATc1 gene transcription. Summarizing all these results, we drew novel conclusions on NFATc1 expression, which provide a more detailed view on the regulatory mechanisms of NFATc1 transcription. Considering the high transcription and strong expression of NFATc1 in various human lymphomas, we propose that similar to NF-B, NFATc1/A plays a pivotal role in lymphomagenesis.
TP53 mutations have been associated with anaplasia in Wilms tumour, which conveys a high risk for relapse and fatal outcome. Nevertheless, TP53 alterations have been reported in no more than 60% of anaplastic tumours, and recent data have suggested their presence in tumours that do not fulfil the criteria for anaplasia, questioning the clinical utility of TP53 analysis. Therefore, we characterized the TP53 status in 84 fatal cases of Wilms tumour, irrespective of histological subtype. We identified TP53 alterations in at least 90% of fatal cases of anaplastic Wilms tumour, and even more when diffuse anaplasia was present, indicating a very strong if not absolute coupling between anaplasia and deregulation of p53 function. Unfortunately, TP53 mutations do not provide additional predictive value in anaplastic tumours since the same mutation rate was found in a cohort of non-fatal anaplastic tumours. When classified according to tumour stage, patients with stage I diffuse anaplastic tumours still had a high chance of survival (87%), but this rate dropped to 26% for stages II–IV. Thus, volume of anaplasia or possible spread may turn out to be critical parameters. Importantly, among non-anaplastic fatal tumours, 26% had TP53 alterations, indicating that TP53 screening may identify additional cases at risk. Several of these non-anaplastic tumours fulfilled some criteria for anaplasia, for example nuclear unrest, suggesting that such partial phenotypes should be under special scrutiny to enhance detection of high-risk tumours via TP53 screening. A major drawback is that these alterations are secondary changes that occur only later in tumour development, leading to striking intratumour heterogeneity that requires multiple biopsies and analysis guided by histological criteria. In conclusion, we found a very close correlation between histological signs of anaplasia and TP53 alterations. The latter may precede development of anaplasia and thereby provide diagnostic value pointing towards aggressive disease.
The aim of the present study was a refined analysis of neuroinflammation including TMEM119 as a useful microglia-specific marker in forensic assessments of traumatic causes of death, e.g., traumatic brain injury (TBI). Human brain tissue samples were obtained from autopsies and divided into cases with lethal TBI (n = 25) and subdivided into three groups according to their trauma survival time and compared with an age-, gender-, and postmortem interval-matched cohort of sudden cardiovascular fatalities as controls (n = 23). Brain tissue samples next to cortex contusions and surrounding white matter as well as samples of the ipsilateral uninjured brain stem and cerebellum were collected and stained immunohistochemically with antibodies against TMEM119, CD206, and CCR2. We could document the highest number of TMEM119-positive cells in acute TBI death with highly significant differences to the control numbers. CCR2-positive monocytes showed a significantly higher cell count in the cortex samples of TBI cases than in the controls with an increasing number of immunopositive cells over time. The number of CD206-positive M2 microglial cells increased survival time-dependent. After 3 days of survival, the cell number increased significantly in all four regions investigated compared with controls. In sum, we validate a specific and robustly expressed as well as fast reacting microglia marker, TMEM119, which distinguishes microglia from resident and infiltrating macrophages and thus offers a great potential for the estimation of the minimum survival time after TBI.
Thymic hyperplasia (TH) with lymphoepithelial sialadenitis (LESA)-like features (LESA-like TH) has been described as a tumor-like, benign proliferation of thymic epithelial cells and lymphoid follicles. We aimed to determine the frequency of lymphoma and autoimmunity in LESA-like TH and performed retrospective analysis of cases with LESA-like TH and/or thymic MALT-lymphoma. Among 36 patients (21 males) with LESA-like TH (age 52 years, 32–80; lesion diameter 7.0 cm, 1–14.5; median, range), five (14%) showed associated lymphomas, including four (11%) thymic MALT lymphomas and one (3%) diffuse large B-cell lymphoma. One additional case showed a clonal B-cell-receptor rearrangement without evidence of lymphoma. Twelve (33%) patients (7 women) suffered from partially overlapping autoimmune diseases: systemic lupus erythematosus (n = 4, 11%), rheumatoid arthritis (n = 3, 8%), myasthenia gravis (n = 2, 6%), asthma (n = 2, 6%), scleroderma, Sjögren syndrome, pure red cell aplasia, Grave’s disease and anti-IgLON5 syndrome (each n = 1, 3%). Among 11 primary thymic MALT lymphomas, remnants of LESA-like TH were found in two cases (18%). In summary, LESA-like TH shows a striking association with autoimmunity and predisposes to lymphomas. Thus, a hematologic and rheumatologic workup should become standard in patients diagnosed with LESA-like TH. Radiologists and clinicians should be aware of LESA-like TH as a differential diagnosis for mediastinal mass lesions in patients with autoimmune diseases.
The cystine/glutamate antiporter xCT is an important source of cysteine for cancer cells. Once taken up, cystine is reduced to cysteine and serves as a building block for the synthesis of glutathione, which efficiently protects cells from oxidative damage and prevents ferroptosis. As melanomas are particularly exposed to several sources of oxidative stress, we investigated the biological role of cysteine and glutathione supply by xCT in melanoma. xCT activity was abolished by genetic depletion in the Tyr::CreER; Braf\(^{CA}\); Pten\(^{lox/+}\) melanoma model and by acute cystine withdrawal in melanoma cell lines. Both interventions profoundly impacted melanoma glutathione levels, but they were surprisingly well tolerated by murine melanomas in vivo and by most human melanoma cell lines in vitro. RNA sequencing of human melanoma cells revealed a strong adaptive upregulation of NRF2 and ATF4 pathways, which orchestrated the compensatory upregulation of genes involved in antioxidant defence and de novo cysteine biosynthesis. In addition, the joint activation of ATF4 and NRF2 triggered a phenotypic switch characterized by a reduction of differentiation genes and induction of pro-invasive features, which was also observed after erastin treatment or the inhibition of glutathione synthesis. NRF2 alone was capable of inducing the phenotypic switch in a transient manner. Together, our data show that cystine or glutathione levels regulate the phenotypic plasticity of melanoma cells by elevating ATF4 and NRF2.
The transcriptional coactivator Bob1 promotes the development of follicular T helper cells via Bcl6
(2016)
Follicular T helper (Tfh) cells are key regulators of the germinal center reaction and long-term humoral immunity. Tfh cell differentiation requires the sustained expression of the transcriptional repressor Bcl6; however, its regulation in CD4\(^+\) T cells is incompletely understood. Here, we report that the transcriptional coactivator Bob1, encoded by the Pou2af1 gene, promotes Bcl6 expression and Tfh cell development. We found that Bob1 together with the octamer transcription factors Oct1/Oct2 can directly bind to and transactivate the Bcl6 and Btla promoters. Mixed bone marrow chimeras revealed that Bob1 is required for the expression of normal levels of Bcl6 and BTLA, thereby controlling the pool size and composition of the Tfh compartment in a T cell-intrinsic manner. Our data indicate that T cell-expressed Bob1 is directly involved in Tfh cell differentiation and required for mounting normal T cell-dependent B-cell responses.
While numerous experiments on NFAT were already performed with CD4+ T cells showing defective cytokine release and a reduced T helper cell development, no detailed studies existed for CD8+ T cells. From this point, we wanted to examine the impact of NFATc1 and c2 on the physiological functions of CD8+ T cells in vitro and in vivo. Therefore, we used a murine infection model with the bacteria Listeria monocytogenes and mice in which NFATc1 was specifically depleted in the T cell compartment.
Our first in vitro studies showed a typical NFATc1 and c2 nuclear translocation and changes on mRNA levels upon T cell activation similarly in CD4+ as well as in CD8+ T cells extracted from wild type mice. NFAT nuclear translocation is important for target gene activation and generation of effector functions. Stimulated T cell populations lacking NFATc1 and/or NFATc2 showed a markedly decreased expression of Th1/Tc1 cytokines, as e.g. IL 2 and IFNγ being important for the clearance of intracellular pathogens. From our in vitro model for the generation of allogenically reactive cytotoxic CD8+ T cells, we revealed a decreased killing and lytic granule-release capacity in Nfatc1 inactivated CD8+ T cells whereas NFATc2-/- cytotoxic T cells did not show an altered cytotoxic response compared to wild type cells.
Interestingly, we found lytic granules accumulated and mitochondria not getting translocated to the immunological synapse upon re-stimulation in NFATc1-deficient CD8+ T cells. Together with results showing the CsA insensitivity of the CTL killing/degranulation capacities, we assume that some major cellular processes are affected by NFATc1 which are not directly linked to the TCR-induced signal transduction cascade.
We also showed the importance of NFATc1 in T cells during intracellular infections with the bacteria Listeria monocytogenes in an in vivo mouse model. After five days, only few bacteria were detected in wt mice whereas high amounts of Listeria particles were extracted from livers of Nfatc1fl/fl x Cd4 cre mice. Although the reactivity towards the pathogen was similar in both groups, a decreased cytokine expression in NFATc1-/- CD8+ T cells was observed together with an altered memory cell generation.
Our results show the importance of NFATc1 in CD8+ T cells and give some clue for a possible connection to other basal cellular functions, as e.g. the formation of an immunological synapse.
Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) can present with different histopathological growth patterns. The impact of these histopathological growth patterns on relapse characteristics is unknown. We therefore analyzed paired biopsies obtained at initial diagnosis and relapse from 33 NLPHL patients who had received first‐line treatment within German Hodgkin Study Group (GHSG) trial protocols, and from a second cohort of 41 relapsed NLPHL patients who had been treated outside GHSG studies. Among the 33 GHSG patients, 21 patients presented with a typical growth pattern at initial diagnosis, whereas 12 patients had a variant histology. The histopathological growth patterns at initial diagnosis and at relapse were consistent in 67% of cases. A variant histology at initial diagnosis was associated with a shorter median time to lymphoma recurrence (2.8 vs 5.2 years; P = .0219). A similar tendency towards a shorter median time to lymphoma recurrence was observed for patients presenting with a variant histology at relapse, irrespective of the growth pattern at initial diagnosis. Results obtained from the 41 NLPHL patients who had been treated outside GHSG studies were comparable (median time to lymphoma recurrence for variant histology vs typical growth pattern at initial diagnosis: 1.5 vs 7.0 years). In conclusion, the histopathological growth pattern remains consistent at relapse in the majority of NLPHL cases, and has major impact on the time of relapse.
The defense against invading pathogens is, amongst other things, mediated via the action of antibodies. Class-switched antibodies and antibodies of high affinity are produced by plasma cells descending from germinal center B (GCB) cells. GCB cells develop in the germinal center (GC), a specialized microstructure found in the B-cell follicle of secondary lymphoid organs. GCB-cell maturation and proliferation are supported by follicular T- helper (Tfh) cells. On the other hand, follicular regulatory T (Tfr) cells control this process in quantity and quality preventing, for instance, the formation of autoantibodies directed against endogenous structures. The development of GCB, Tfh and Tfr cells essentially depends on the migration into the GC, which is mediated via the expression of the chemokine receptor CXCR5.
One transcription factor highly expressed in follicular T cells, comprising Tfh and Tfr cells, is NFATc1. Tfr cells additionally express the transcriptional repressor Blimp-1, which is not expressed in Tfh cells. We found that NFATc1 is transactivating Cxcr5 via response elements in the promoter and enhancer in vitro. Blimp-1 binds to the same elements, transactivating Cxcr5 expression in cooperation with NFATc1, whilst mediating Cxcr5- repression on its own. In Tfr cells Blimp-1 suppresses CXCR5 expression in the absence of NFATc1. Blimp-1 itself is necessary to restrict Tfr-cell frequencies and to mediate Tfr- cell function as in mice with Blimp-1-ablated Tregs high frequencies of Tfr cells do not reduce GCB- or Tfh cell frequencies. NFATc1 and Blimp-1 double deficient Tfr cells show additional loss of function, which becomes visible in clearly expanded antibody titers.
To evaluate the function of NFATc1 in Tfr cells, we not only deleted it, but also overexpressed a constitutive active form of NFATc1/aA (caNFATc1/aA) in regulatory T cells (Tregs). The latter is leading to an upregulation of CXCR5 per cell, without changing Tfh or Tfr-cell frequencies. However, the high density of surface CXCR5 enhances the migration of Tfr cells deep into the GC, which results in a tighter control of the antigen- specific humoral immune response. Additionally, caNFATc1/aA increases the expression of genes coding for Tfr effector molecules like Il1rn, Il10, Tigit and Ctla4. Interestingly, this part of the transcriptional change is dependent on the presence of Blimp-1. Furthermore, Blimp-1 regulates the expression of multiple chemokine receptor genes on the background of caNFATc1/aA.
In contrast, when caNFATc1/aA is overexpressed in all T cells, the frequencies of Tfh- and GCB cells are dominantly reduced. This effect seems to stem from the conventional T- cell (Tcon) side, most probably originating from increased secretion of interleukin-2 (IL- 2) via the caNFATc1/aA overexpressing Tcons. IL-2 is known to hinder the germinal center reaction (GCR) and it might in its abundance not be neutralizable by Tfr cells.
Taken together, NFATc1 and Blimp-1 cooperate to control the migration of Tfr cells into the GC. Tfr cells in the GC depend on NFATc1 and Blimp-1 to perform their proper function. Overexpression of caNFATc1 in Tregs strengthens Tfr function in a Blimp-1-dependent manner, whilst overexpression of caNFATc1 in all T cells dominantly diminishes the GCR.
SUMMARY GABP is a heterodymeric member of Ets-family transcription factors. It consists of two subunits – GABPa which contains DNA binding domain and GABPb, which provides transcriptional activation domain and nuclear localization signal. GABPa/b complex is essential for transcriptional activation of multiple lineage-restricted and housekeeping genes, several viral genes, and in some cases might function as transcriptional repressor. Large variety of data indicates involvement of GABP in the complex regulation of cell growth, specified by quiescence, stimulation/proliferation, apoptosis and senescence. Expression level of GABPa subunit is rapidly increased when resting cells enter S-phase, and GABPa/b complex is critical to promote the continuity of the cell cycle. Conditional inactivation of GABPa expression in mouse embryonic fibroblasts results in a complete block of proliferation and acquisition of senescence-like phenotype. However, the influence of GABP on the other cell growth determinant – the apoptosis – remains largely obscure. Therefore we aimed to investigate the influence of GABPa/b expression level on the cell growth in vitro. Using siRNA approach we achieved efficient but only transient down-regulation of GABPa expression which precluded further cell growth studies. Persistent increase of the expression of GABPb subunit only resulted in a positive effect on the cell growth speed. Simultaneous conditional overexpression of both GABPa and GABPb subunits though, strongly reduced the growth of the affected cell cultures in reversible and in expression level dependent manner. Interestingly, GABPa/b overexpressing cells did show neither cell cycle arrest nor massive induction of apoptosis. However, more detailed analyses revealed that dampened apoptotic processes were taking place in GABPa/b−overexpressing cells, starting with a prominent activation of caspase-12. Interestingly, activation of downstream effector caspases was rather suppressed explaining a weak increase of apoptotic cells in GABPa/b overexpressing cultures. This effect suggests that the activation of caspase-12 by elevated amounts of exogenous GABPa/b reflects the normal physiological mechanism of caspase-12 regulation.
In this thesis we have investigated the effect of NFAT (Nuclear Factor of Activated T Cell) transcription factors on the expression of Rag-(Recombination Activating Genes) genes in murine thymus. The protein products of Rag genes, RAG1 and RAG2, are critical for the recombination and generation of the TCR (T Cell Receptor) repertoire during thymocyte development, and their expression can be suppressed by the activity of NFAT factors. In thymus, the expression of Rag1 and Rag2 genes is induced at the double-negative (DN, CD4-8-) 3 stage, down-regulated at the DN4 stage, re-induced at the double-positive (DP, CD4+8+) stage, and suppressed again at the single-positive (SP, CD4+8- or CD4-8+) stage. Although it is known that TCR signaling suppresses the expression of Rag1 and Rag2 at the SP stage, the signals that mediate the Rag gene down-reulation remain elusive. Here we report that both the calcineurin-NFAT-signaling and MAPKinase signaling pathways, which are activated by TCR signaling during positive selection, mediate the Rag gene down-regulation in DP thymocytes. The calcineurin-NFAT pathway suppresses both the Rag1 and the Rag2 gene expression. This pathway has a stronger suppressive effect on the Rag1 than the Rag2 gene. A synergistic activity between the two NFAT factors NFATc2 and NFATc3 is essential for calcineurin-NFAT signaling to efficiently suppress the Rag gene expression in DP thymocytes. It is likely that the calcineurin-NFAT signaling down-regulates Rag gene expression by suppressing both the Rag anti-silencer element (ASE) activity and the Rag promoter activity. Similarly, MEK-ERK signaling of MAPK signaling pathway mediates the Rag gene suppression in DP thymocytes although the mechanism through which MEK-ERK mediates the Rag gene down-regulation has to be elucidated. In DN thymocytes, it appears that neither the calcineurin-NFAT signaling nor MAPK signaling is involved in the Rag gene down-regulation. However, a role for these two signaling pathways in the Rag gene up-regulation in DN thymocytes is not excluded. In DN thymocytes, pre-TCR signaling stimulates the expression both Nfatc1 and Nfatc2 genes but has no effect on Nfatc3 gene expression. In DN thymocytes, pre-TCR signaling activates Nfatc1α expression but not Nfatc1ß expression, i.e. the two promoters controling Nfatc1 gene xpression are differently controled by pre-TCR signals. Nfatc1α gene expression in DN thymocytes is mainly regulated by the MAPK signaling pathway because activation of Nfatc1α is mediated by MEK-ERK signaling but opposed by JNK signaling. Calcineuirn-NFAT and p38 signaling pathways are not involved in Nfatc1α promoter regulation in DN thymocytes. In DP thymocytes, TCR signaling up-regulates Nfatc1 and Nfatc2 expression but down-regulates Nfatc3 expression. In DP thymocytes, TCR signaling activates Nfatc1α expression. The activation of Nfatc1α in DP thymocytes is mediated by NFATc1, but not or to a less degree by NFATc2 and NFATc3. MEK-ERK, JNK, and p38 signaling pathways are involved in Nfatc1α gene activation in DP thymocytes, probably by activating NFAT trans-activation activity. All these findings illustrate that in thymocytes the expression of NFAT transcription factors – which are essential for thymic development - is controled at multiple levels.
Objectives: The aim of this study was to evaluate the efficiency of cetuximab-based anti-EGFR treatment and Aurora kinase A / B knockdown as a function of Aurora kinase polymorphism in HNSCC cell lines.
Materials and methods: First, protein expression of Aurora kinase A / B and EGFR and Aurora kinase A polymorphism were studied in tumour samples.
The survival and proliferation of Aurora kinase A homo- (Cal27) and heterozygous (HN) HNSCC cell lines was evaluated using a colony formation assay and a flow cytometric assay. Also, aneuploidy was determined. EGFR signalling pathway were visualised by western blotting.
Results: Immunohistochemistry revealed the overexpression of Aurora kinase A / B in HNSCC. The knockdown of each kinase caused a significant decrease in clonogenic survival, independent of Aurora kinase A polymorphism. In contrast, cetuximab treatment impaired clonogenic survival only in the Aurora kinase A-homozygous cell line (Cal27).
Conclusion: This study provides in vitro evidence for the predictive value of Aurora kinase A polymorphism in the efficiency of cetuximab treatment. Resistance to cetuximab treatment can be overcome by simultaneous Aurora kinase A/B knockdown.
The cellular microenvironment in follicular lymphoma is of biological and clinical importance. Studies on the clinical significance of non-malignant cell populations have generated conflicting results, which may partly be influenced by poor reproducibility in immunohistochemical marker quantification. In this study, the reproducibility of manual scoring and automated microscopy based on a tissue microarray of 25 follicular lymphomas as compared to flow cytometry is evaluated. The agreement between manual scoring and flow cytometry was moderate for CD3, low for CD4, and moderate to high for CD8, with some laboratories scoring closer to the flow cytometry results. Agreement in manual quantification across the 7 laboratories was low to moderate for CD3, CD4, CD8 and FOXP3 frequencies, moderate for CD21, low for MIB1 and CD68, and high for CD10. Manual scoring of the architectural distribution resulted in moderate agreement for CD3, CD4 and CD8, and low agreement for FOXP3 and CD68. Comparing manual scoring to automated microscopy demonstrated that manual scoring increased the variability in the low and high frequency interval with some laboratories showing a better agreement with automated scores. Manual scoring reliably identified rare architectural patterns of T-cell infiltrates. Automated microscopy analyses for T-cell markers by two different instruments were highly reproducible and provided acceptable agreement with flow cytometry. These validation results provide explanations for the heterogeneous findings on the prognostic value of the microenvironment in follicular lymphoma. We recommend a more objective measurement, such as computer-assisted scoring, in future studies of the prognostic impact of microenvironment in follicular lymphoma patients.
The proteome profiles of the olfactory bulb of juvenile, adult and aged rats - an ontogenetic study
(2015)
Background:
In this study, we searched for proteins that change their expression in the olfactory bulb (oB) of rats during ontogenesis. Up to now, protein expression differences in the developing animal are not fully understood. Our investigation focused on the question whether specific proteins exist which are only expressed during different development stages. This might lead to a better characterization of the microenvironment and to a better determination of factors and candidates that influence the differentiation of neuronal progenitor cells.
Results:
After analyzing the samples by two-dimensional polyacrylamide gel electrophoresis (2DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), it could be shown that the number of expressed proteins differs depending on the developmental stages. Especially members of the functional classes, like proteins of biosynthesis, regulatory proteins and structural proteins, show the highest differential expression in the stages of development analyzed.
Conclusion:
In this study, quantitative changes in the expression of proteins in the oB at different developmental stages (postnatal days (P) 7, 90 and 637) could be observed. Furthermore, the expression of many proteins was found at specific developmental stages. It was possible to identify these proteins which are involved in processes like support of cell migration and differentiation.
The Proteome Profiles of the Cerebellum of Juvenile, Adult and Aged Rats-An Ontogenetic Study
(2015)
In this study, we searched for proteins that change their expression in the cerebellum (Ce) of rats during ontogenesis. This study focuses on the question of whether specific proteins exist which are differentially expressed with regard to postnatal stages of development. A better characterization of the microenvironment and its development may result from these study findings. A differential two-dimensional polyacrylamide gel electrophoresis (2DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of the samples revealed that the number of proteins of the functional classes differed depending on the developmental stages. Especially members of the functional classes of biosynthesis, regulatory proteins, chaperones and structural proteins show the highest differential expression within the analyzed stages of development. Therefore, members of these functional protein groups seem to be involved in the development and differentiation of the Ce within the analyzed development stages. In this study, changes in the expression of proteins in the Ce at different postnatal developmental stages (postnatal days (P) 7, 90, and 637) could be observed. At the same time, an identification of proteins which are involved in cell migration and differentiation was possible. Especially proteins involved in processes of the biosynthesis and regulation, the dynamic organization of the cytoskeleton as well as chaperones showed a high amount of differentially expressed proteins between the analyzed dates.
The treatment of Parkinson's disease by transplantation of dopaminergic (DA) neurons from human embryonic mesencephalic tissue is a promising approach. However, the origin of these cells causes major problems: availability and standardization of the graft. Therefore, the generation of unlimited numbers of DA neurons from various types of stem or progenitor cells has been brought into focus. A source for DA neurons might be conditionally immortalized progenitor cells. The temperature-sensitive immortalized cell line CSM14.1 derived from the mesencephalon of an embryonic rat has been used successfully for transplantation experiments. This cell line was analyzed by unbiased stereology of cell type specific marker proteins and 2D-gel electrophoresis followed by mass spectrometry to characterize the differentially expressed proteome. Undifferentiated CSM14.1 cells only expressed the stem cell marker nestin, whereas differentiated cells expressed GFAP or NeuN and tyrosine hydroxylase. An increase of the latter cells during differentiation could be shown. By using proteomics an explanation on the protein level was found for the observed changes in cell morphology during differentiation, when CSM14.1 cells possessed the morphology of multipolar neurons. The results obtained in this study confirm the suitability of CSM14.1 cells as an in vitro model for the study of neuronal and dopaminergic differentiation in rats.
Familial gastrointestinal stromal tumors (GIST) are dominant genetic disorders that are caused by germline mutations of the type III receptor tyrosine kinase KIT. While sporadic mutations are frequently found in mastocytosis and GISTs, germline mutations of KIT have only been described in 39 families until now. We detected a novel germline mutation of KIT in exon 11 (p.Lys-558-Asn; K558N) in a patient from a kindred with several GISTs harboring different secondary somatic KIT mutations. Structural analysis suggests that the primary germline mutation alone is not sufficient to release the autoinhibitory region of KIT located in the transmembrane domain. Instead, the KIT kinase module becomes constitutively activated when K558N combines with different secondary somatic mutations. The identical germline mutation in combination with an additional somatic KIT mutation was detected in a second patient of the kindred with seminoma while a third patient within the family had a cutaneous mastocytosis. These findings suggest that the K558N mutation interferes with the juxtamembranous part of KIT, since seminoma and mastocystosis are usually not associated with exon 11 mutations.
In contrast to other haematological malignancies, targeted immunotherapy has not entered standard treatment regimens for de novo or relapsed multiple myeloma (MM) yet. While a number of IgG-formatted monoclonal antibodies are currently being evaluated in clinical trials in MM, our study aimed to investigate whether the fully human IgM monoclonal antibody PAT-SM6 that targets a tumour-specific variant of the heat shock protein GRP78 might be an attractive candidate for future immunotherapeutic approaches. We here show that GRP78 is stably and consistently expressed on the surface on tumour cells from patients with de novo, but also relapsed MM and that binding of PAT-SM6 to MM cells can specifically exert cytotoxic effects on malignant plasma cells, whereas non-malignant cells are not targeted. We demonstrate that the induction of apoptosis and, to a lesser extent, complement dependent cytotoxicity is the main mode of action of PAT-SM6, whereas antibody dependent cellular cytotoxicity does not appear to contribute to the cytotoxic properties of this antibody. Given the favourable safety profile of PAT-SM6 in monkeys, but also in a recent phase I trial in patients with malignant melanoma, our results form the basis for a planned phase I study in patients with relapsed MM.
YAP and TAZ, downstream effectors of the Hippo pathway, are important regulators of proliferation. Here, we show that the ability of YAP to activate mitotic gene expression is dependent on the Myb-MuvB (MMB) complex, a master regulator of genes expressed in the G2/M phase of the cell cycle. By carrying out genome-wide expression and binding analyses, we found that YAP promotes binding of the MMB subunit B-MYB to the promoters of mitotic target genes. YAP binds to B-MYB and stimulates B-MYB chromatin association through distal enhancer elements that interact with MMB-regulated promoters through chromatin looping. The cooperation between YAP and B-MYB is critical for YAP-mediated entry into mitosis. Furthermore, the expression of genes coactivated by YAP and B-MYB is associated with poor survival of cancer patients. Our findings provide a molecular mechanism by which YAP and MMB regulate mitotic gene expression and suggest a link between two cancer-relevant signaling pathways.
Highlights
• The integrated stress response leads to a general ATF4-dependent activation of NRF2
• ATF4 causes a CHAC1-dependent GSH depletion, resulting in NRF2 stabilization
• An elevation of NRF2 transcript levels fosters this effect
• NRF2 supports the ISR/ATF4 pathway by improving cystine and antioxidant supply
Summary
The redox regulator NRF2 becomes activated upon oxidative and electrophilic stress and orchestrates a response program associated with redox regulation, metabolism, tumor therapy resistance, and immune suppression. Here, we describe an unrecognized link between the integrated stress response (ISR) and NRF2 mediated by the ISR effector ATF4. The ISR is commonly activated after starvation or ER stress and plays a central role in tissue homeostasis and cancer plasticity. ATF4 increases NRF2 transcription and induces the glutathione-degrading enzyme CHAC1, which we now show to be critically important for maintaining NRF2 activation. In-depth analyses reveal that NRF2 supports ATF4-induced cells by increasing cystine uptake via the glutamate-cystine antiporter xCT. In addition, NRF2 upregulates genes mediating thioredoxin usage and regeneration, thus balancing the glutathione decrease. In conclusion, we demonstrate that the NRF2 response serves as second layer of the ISR, an observation highly relevant for the understanding of cellular resilience in health and disease.
Due to the wide variety of benign and malignant salivary gland tumors, classification and malignant behavior determination based on histomorphological criteria can be difficult and sometimes impossible. Spectroscopical procedures can acquire molecular biological information without destroying the tissue within the measurement processes. Since several tissue preparation procedures exist, our study investigated the impact of these preparations on the chemical composition of healthy and tumorous salivary gland tissue by Fourier-transform infrared (FTIR) microspectroscopy. Sequential tissue cross-sections were prepared from native, formalin-fixed and formalin-fixed paraffin-embedded (FFPE) tissue and analyzed. The FFPE cross-sections were dewaxed and remeasured. By using principal component analysis (PCA) combined with a discriminant analysis (DA), robust models for the distinction of sample preparations were built individually for each parotid tissue type. As a result, the PCA-DA model evaluation showed a high similarity between native and formalin-fixed tissues based on their chemical composition. Thus, formalin-fixed tissues are highly representative of the native samples and facilitate a transfer from scientific laboratory analysis into the clinical routine due to their robust nature. Furthermore, the dewaxing of the cross-sections entails the loss of molecular information. Our study successfully demonstrated how FTIR microspectroscopy can be used as a powerful tool within existing clinical workflows.
Dysregulation of the apoptotic pathway is widely recognized as a key step in lymphomagenesis. Notably, LITAF was initially identified as a p53-inducible gene, subsequently implicated as a tumor suppressor. Our previous study also showed LITAF to be methylated in 89.5% B-NHL samples. Conversely, deregulated expression of BCL6 is a pathogenic event in many lymphomas. Interestingly, our study found an oppositional expression of LITAF and BCL6 in B-NHL. In addition, LITAF was recently identified as a novel target gene of BCL6. Therefore, we sought to explore the feedback loop between LITAF and BCL6 in B-NHL. Here, our data for the first time show that LITAF can repress expression of BCL6 by binding to Region A (−87 to +65) containing a putative LITAF-binding motif (CTCCC) within the BCL6 promoter. Furthermore, the regulation of BCL6 targets (PRDM1 or c-Myc) by LITAF may be associated with B-cell differentiation. Results also demonstrate that ectopic expression of LITAF induces cell apoptosis, activated by releasing cytochrome c, cleaving PARP and caspase 3 in B-NHL cells whereas knockdown of LITAF robustly protected cells from apoptosis. Interestingly, BCL6, in turn, could reverse cell apoptosis mediated by LITAF. Collectively, our findings provide a novel apoptotic regulatory pathway in which LITAF, as a transcription factor, inhibits the expression of BCL6, which leads to activation of the intrinsic mitochondrial pathway and tumor apoptosis. Our study is expected to provide a possible biomarker as well as a target for clinical therapies to promote tumor cell apoptosis.
Background The levels and clinical relevance of Th17 cells and other interleukin-17-producing cells have not been analyzed in chronic lymphocytic leukemia. The objective of this study was to quantify blood and tissue levels of Th17 and other interleukin-17-producing cells in patients with this disease and correlate blood levels with clinical outcome.
Design and Methods: Intracellular interleukin-17A was assessed in blood and splenic mononuclear cells from patients with chronic lymphocytic leukemia and healthy subjects using flow cytometry. Interleukin-17A-producing cells were analyzed in formalin-fixed, paraffin-embedded spleen and lymph node sections using immunohistochemistry and immunofluorescence.
Results: The absolute numbers of Th17 cells in peripheral blood mononuclear cells and the percentages of Th17 cells in spleen cell suspensions were higher in patients with chronic lymphocytic leukemia than in healthy subjects; in six out of eight paired chronic lymphocytic leukemia blood and spleen sample comparisons, Th17 cells were enriched in spleen suspensions. Circulating Th17 levels correlated with better prognostic markers and longer overall survival of the patients. Two "non-Th17" interleukin-17-expressing cells were identified in chronic lymphocytic leukemia spleens: proliferating cells of the granulocytic lineage and mature mast cells. Granulocytes and mast cells in normal spleens did not express interleukin-17. Conversely, both chronic lymphocytic leukemia and healthy lymph nodes contained similar numbers of interleukin-17+ mast cells as well as Th17 cells.
Conclusions: Th17 cells are elevated in chronic lymphocytic leukemia patients with better prognostic markers and correlate with longer survival. Furthermore, non-Th17 interleukin-17A-expressing cells exist in chronic lymphocytic leukemia spleens as maturing granulocytes and mature mast cells, suggesting that the microenvironmental milieu in leukemic spleens promotes the recruitment and/or expansion of Th17 and other IL-17-expressing cells. The pathophysiology of Th17 and non-Th17-interleukin-producing cells in chronic lymphocytic leukemia and their distributions and roles in this disease merit further study.
Purpose
Multiple myeloma is a hematologic malignancy originating from clonal plasma cells. Despite effective therapies, outcomes are highly variable suggesting marked disease heterogeneity. The role of functional imaging for therapeutic management of myeloma, such as positron emission tomography with 2-deoxy-2-[18F]fluoro-D-glucose (18F-FDG-PET), remains to be determined. Although some studies already suggested a prognostic value of 18F-FDG-PET, more specific tracers addressing hallmarks of myeloma biology, e.g. paraprotein biosynthesis, are needed. This study evaluated the amino acid tracers L-methyl-[11C]-methionine (11C-MET) and [18F]-fluoroethyl-L-tyrosine (18F-Fet) for their potential to image myeloma and to characterize tumor heterogeneity.
Experimental Design
To study the utility of 11C-MET, 18F-Fet and 18F-FDG for myeloma imaging, time activity curves were compared in various human myeloma cell lines (INA-6, MM1.S, OPM-2) and correlated to cell-biological characteristics, such as marker gene expression and immunoglobulin levels. Likewise, patient-derived CD138+ plasma cells were characterized regarding uptake and biomedical features.
Results
Using myeloma cell lines and patient-derived CD138+ plasma cells, we found that the relative uptake of 11C-MET exceeds that of 18F-FDG 1.5- to 5-fold and that of 18F-Fet 7- to 20-fold. Importantly, 11C-MET uptake significantly differed between cell types associated with worse prognosis (e.g. t(4;14) in OPM-2 cells) and indolent ones and correlated with intracellular immunoglobulin light chain and cell surface CD138 and CXCR4 levels. Direct comparison of radiotracer uptake in primary samples further validated the superiority of 11C-MET.
Conclusion
These data suggest that 11C-MET might be a versatile biomarker for myeloma superior to routine functional imaging with 18F-FDG regarding diagnosis, risk stratification, prognosis and discrimination of tumor subtypes.
Purpose
While [\(^{18}\)F]-fluorodeoxyglucose ([\(^{18}\)F]FDG) is the standard for positron emission tomography/computed tomography (PET/CT) imaging of oral squamous cell carcinoma (OSCC), diagnostic specificity is hampered by uptake in inflammatory cells such as neutrophils or macrophages. Recently, molecular imaging probes targeting fibroblast activation protein α (FAP), which is overexpressed in a variety of cancer-associated fibroblasts, have become available and might constitute a feasible alternative to FDG PET/CT.
Methods
Ten consecutive, treatment-naïve patients (8 males, 2 females; mean age, 62 ± 9 years) with biopsy-proven OSCC underwent both whole-body [\(^{18}\)F]FDG and [\(^{68}\)Ga]FAPI-04 (FAP-directed) PET/CT for primary staging prior to tumor resection and cervical lymph node dissection. Detection of the primary tumor, as well as the presence and number of lymph node and distant metastases was analysed. Intensity of tracer accumulation was assessed by means of maximum (SUV\(_{max}\)) and peak (SUV\(_{peak}\) standardized uptake values. Histological work-up including immunohistochemical staining for FAP served as standard of reference.
Results
[\(^{18}\)F]FDG and FAP-directed PET/CT detected all primary tumors with a SUVmax of 25.5 ± 13.2 (FDG) and 20.5 ± 6.4 (FAP-directed) and a SUVpeak of 16.1 ± 10.3 ([\(^{18}\)F]FDG) and 13.8 ± 3.9 (FAP-directed), respectively. Regarding cervical lymph node metastases, FAP-directed PET/CT demonstrated comparable sensitivity (81.3% vs. 87.5%; P = 0.32) and specificity (93.3% vs. 81.3%; P = 0.16) to [\(^{18}\)F]FDG PET/CT. FAP expression on the cell surface of cancer-associated fibroblasts in both primary lesions as well as lymph nodes metastases was confirmed in all samples.
Conclusion
FAP-directed PET/CT in OSCC seems feasible. Future research to investigate its potential to improve patient staging is highly warranted.
C-X-C motif chemokine receptor 4 (CXCR4) is a key factor for tumor growth and metastasis in several types of human cancer. This study investigated the feasibility of CXCR4-directed imaging with positron emission tomography/computed tomography (PET/CT) using [\(^{68}\)Ga]Pentixafor in malignant pleural mesothelioma.
Six patients with pleural mesothelioma underwent [\(^{68}\)Ga]Pentixafor-PET/CT. 2′-[\(^{18}\)F]fluoro-2′-deoxy-D-glucose ([\(^{18}\)F]FDG)-PET/CT (4/6 patients) and immunohistochemistry obtained from biopsy or surgery (all) served as standards of reference. Additionally, 9 surgical mesothelioma samples were available for histological work-up.
Whereas [\(^{18}\)F]FDG-PET depicted active lesions in all patients, [\(^{68}\)Ga]Pentixafor-PET/CT recorded physiologic tracer distribution and none of the 6 patients presented [\(^{68}\)Ga]Pentixafor-positive lesions. This finding paralleled results of immunohistochemistry which also could not identify relevant CXCR4 surface expression in the samples analyzed.
In contrast to past reports, our data suggest widely absence of CXCR4 expression in pleural mesothelioma. Hence, robust cell surface expression should be confirmed prior to targeting this chemokine receptor for diagnosis and/or therapy.
Diagnosing any of the more than 30 types of T-cell lymphomas is considered a challenging task for many pathologists and currently requires morphological expertise as well as the integration of clinical data, immunophenotype, flow cytometry and clonality analyses. Even considering all available information, some margin of doubt might remain using the current diagnostic procedures. In recent times, the genetic landscape of most T-cell lymphomas has been elucidated, showing a number of diagnostically relevant mutations. In addition, recent data indicate that some of these genetic alterations might bear prognostic and predictive value. Extensive genetic analyses, such as whole exome or large panel sequencing are still expensive and time consuming, therefore limiting their application in routine diagnostic. We therefore devoted our effort to develop a lean approach for genetic analysis of T-cell lymphomas, focusing on maximum efficiency rather than exhaustively covering all possible targets. Here we report the results generated with our small amplicon-based panel that could be used routinely on paraffin-embedded and even decalcified samples, on a single sample basis in parallel with other NGS-panels used in our routine diagnostic lab, in a relatively short time and with limited costs. We tested 128 available samples from two German reference centers as part of our routine work up (among which 116 T-cell lymphomas), which is the largest routine diagnostic series reported to date. Our results showed that this assay had a very high rate of technical success (97%) and could detect mutations in the majority (79%) of tested T-cell lymphoma samples.
Introduction: Large-cell transformation (LCT) of mycosis fungoides (MF) has been associated with a higher risk of relapse and progression and, consequently, restricted prognosis. Its molecular pathogenesis has not been elucidated yet. Materials and Methods: In order to address molecular mechanisms of LCT, we performed hybrid capture panel-based sequencing of skin biopsies from 10 patients suffering from MF with LCT versus 17 patients without LCT including follow-up biopsies during clinical course, respectively (51 samples in total). The analyzed patients were attributed to three different groups based on the presence of LCT and clinical behavior. Results: While indolent MF cases without LCT did not show pathogenic driver mutations, a high rate of oncogenic alterations was detected in patients with LCT and aggressive clinical courses. Various genes of different oncogenic signaling pathways, including the MAPK and JAK-STAT signaling pathways, as well as epigenetic modifiers were affected. A high inter-individual and distinctive intra-individual mutation diversity was observed. Oncogenic RAS mutations were exclusively detected in patients with LCT. Conclusion: Our data demonstrate that LCT transition of MF is associated with increased frequency of somatic mutations in cancer-associated genes. In particular, the activation of RAS signaling — together with epigenetic dysregulation — may crucially contribute to the molecular pathogenesis of the LCT phenotype, thus conveying its adverse clinical behavior.
Objective Refractory coeliac disease (RCD) is a potentially hazardous complication of coeliac disease (CD). In contrast to RCD type I, RCD type II is a precursor entity of enteropathy-associated T-cell lymphoma (EATL), which is associated with clonally expanding T-cells that are also found in the sequentially developing EATL. Using high-throughput sequencing (HTS), we aimed to establish the small-intestinal T-cell repertoire (TCR) in CD and RCD to unravel the role of distinct T-cell clonotypes in RCD pathogenesis. Design DNA extracted from duodenal mucosa specimens of controls (n=9), active coeliacs (n=10), coeliacs on a gluten-free diet (n=9), RCD type I (n= 8), RCD type II (n= 8) and unclassified Marsh I cases (n= 3) collected from 2002 to 2013 was examined by TCR beta-complementarity- determining regions 3 (CDR3) multiplex PCR followed by HTS of the amplicons. Results On average, 106 sequence reads per sample were generated consisting of up to 900 individual TCR beta rearrangements. In RCD type II, the most frequent clonotypes (ie, sequence reads with identical CDR3) represent in average 42.6% of all TCR beta rearrangements, which was significantly higher than in controls (6.8%; p<0.01) or RCD type I (6.7%; p<0.01). Repeat endoscopies in individual patients revealed stability of clonotypes for up to several years without clinical symptoms of EATL. Dominant clonotypes identified in individual patients with RCD type II were unique and not related between patients. CD-associated, gliad-independent CDR3 motifs were only detectable at low frequencies. Conclusions TCR beta-HTS analysis unravels the TCR in CD and allows detailed analysis of individual TCR beta rearrangements. Dominant TCR beta sequences identified in patients with RCD type II are unique and not homologous to known gliadin-specific TCR sequences, supporting the assumption that these clonal T-cells expand independent of gluten stimulation.
The massive infiltration of lymphocytes into the skin is a hallmark of numerous human skin disorders. By co-culturing murine keratinocytes with splenic T cells we demonstrate here that T cells affect and control the synthesis and secretion of chemokines by keratinocytes. While pre-activated CD8\(^+\)T cells induce the synthesis of CXCL9 and CXCL10 in keratinocytes and keep in check the synthesis of CXCL1, CXCL5, and CCL20, keratinocytes dampen the synthesis of CCL3 and CCL4 in pre-activated CD8\(^+\)T cells. One key molecule is IFN-γ that is synthesized by CD8\(^+\)T cells under the control of NFATc1 and NFATc2. CD8\(^+\)T cells deficient for both NFAT factors are unable to induce CXCL9 and CXCL10 expression. In addition, CD8\(^+\)T cells induced numerous type I IFN-inducible “defense genes” in keratinocytes encoding the PD1 and CD40 ligands, TNF-α and caspase-1. The enhanced expression of type I IFN-inducible genes resembles the gene expression pattern at the dermal/epidermal interface in lichen planus, an inflammatory T lymphocyte-driven skin disease, in which we detected the expression of CXCL10 in keratinocytes in close vicinity to the infiltration front of T cells. These data reflect the multifaceted interplay of lymphocytes with keratinocytes at the molecular level.
Background
Kaposi sarcoma (KS) is a malignant disease most commonly diagnosed in the setting of a human immunodeficiency virus (HIV) infection and in patients receiving immunosuppressive treatment. Pulmonary KS has never been reported in association with endogenous Cushing’s syndrome (CS).
Case presentation
A 60-year-old woman presented with symptoms and signs of CS. Adrenal CS was confirmed by standard biochemical evaluation. Imaging revealed a right adrenal lesion (diameter 3.5 cm) and multiple pulmonary nodules, suggesting a cortisol-secreting adrenal carcinoma with pulmonary metastases. The patient underwent right adrenalectomy with a pathohistological diagnosis of an adrenal adenoma. Subsequent thoracoscopic wedge resection of one lung lesion revealed pulmonary KS with positive immunostaining for human herpes virus 8 (HHV-8). HIV-serology was negative. Hydrocortisone replacement was initiated for secondary adrenal insufficiency after surgery. Post-operative follow up imaging showed complete remission of all KS-related pulmonary nodules solely after resolution of hypercortisolism.
Conclusion
KS may occur in the setting of endogenous CS and may go into remission after cure of hypercortisolism without further specific treatment.
Sumoylation of transcription factors modulate their activity (either upregulating or downregulating) by altering protein-protein interactions as well as subcelluar/subnuclear localization. The transcription factor family of NFAT (Nuclear Factor of Activated T cells) plays an important role in cytokine gene regulation in T cells. Due to alternative usage of two promoters (P1 & P2), two polyadenylation sites (pA1 and pA2) and alternative splicing events, NFATc1 is expressed in six isoforms which are NFATc1/alphaA, betaA, alphaB, betaB, alphaC and betaC, where alpha and beta refer to two different 1st exons and A, B, C to the differentially spliced and extended C-termini. The short isoforms of NFATc1 (NF-ATc1/A) contain a relatively short C terminus whereas, the longer isoforms, B and C, span the extra C-terminal peptides of 128 and 246 aa, respectively. To analyze the specific biological effects of NFATc1 isoform, a yeast two hybrid screening of a human spleen cDNA library with extra C-terminal peptide of NFATc1 as a bait, was performed. At the end of the assay, the proteins involved in the sumoylation pathway such as Ubc9, PIAS1 were detected with highest frequencies and subsequently were were able to demonstrate that NFATc1 is sumoylated. The extent of sumoylation is isoform specific. While NFATc1/A, harboring only one sumoylation site, shows very weak sumoylation, the two additional sites within NFATc1/C lead to efficient sumoylation. This modification directs NFATc1/C into SUMO-1 bodies, which in turn colocalize with PML-nbs. Furthermore, sumoylated NFATc1/C recruits the transcriptional co-repressors HDAC (both class I as well as class II HDACs) which results in a significant decrease of the level of histone acetylation on the IL-2 promoter, an important NFATc1 target gene. As a consequence of this, a decrease of IL-2 production was observed, while NFATc1/C, which can no longer be sumoylated due to mutating the target lysines, exhibited dramatic elevated transcriptional potential on the IL2 promoter. This supports our finding from IL-2 promoter-driven reporter gene assay, which shows downregulation of NFATc1/C transactivation upon sumoylation. Hence, sumoylation exerts a negative effect on NFATc1 transcriptioanl activity. Immunofluorescence studies showed SUMO modification to relocate NFATc1/C also into transcriptionally inactive heterochromatin regions, demonstrated by H3K9 m3 (tri-methylated histone lysine 9) colocalization studies. Interestingly, in the absence of sumoylation, NFATc1 was partially colocalized with transcriptional hotspots in the nucleus, which might contribute to the higher transcription potentiality of the non-sumoylated NFATc1. It is important to note that, the transcriptional activity of other NFATc1 target genes (IL-13, IFN-gamma etc.) was positively upregulated upon sumoylation of NFATc1, suggesting a non-universal effect of sumoylation on NFATc1/C function. In conclusion, sumoylation directs NFATc1 into nuclear bodies where it interacts with transcriptional co-repressors and relocalize itself with heterochromatin, leading to repression of NFATc1/C-mediated transcription. Most importantly, the effect of NFATc1/C sumoylation is promoter specific. Taken together, SUMO modification alters the function of NFATc1 from an activator to a site-specific transcriptional repressor. This study unraveled a novel regulatory mechanism, which controls isoform specific NFATc1 function.
Background: Renal cell carcinoma (RCC) is divided into three major histopathologic groups—clear cell (ccRCC), papillary (pRCC) and chromophobe RCC (chRCC). We performed a comprehensive re-analysis of publicly available RCC datasets from the TCGA (The Cancer Genome Atlas) database, thereby combining samples from all three subgroups, for an exploratory transcriptome profiling of RCC subgroups.
Materials and Methods: We used FPKM (fragments per kilobase per million) files derived from the ccRCC, pRCC and chRCC cohorts of the TCGA database, representing transcriptomic data of 891 patients. Using principal component analysis, we visualized datasets as t-SNE plot for cluster detection. Clusters were characterized by machine learning, resulting gene signatures were validated by correlation analyses in the TCGA dataset and three external datasets (ICGC RECA-EU, CPTAC-3-Kidney, and GSE157256).
Results: Many RCC samples co-clustered according to histopathology. However, a substantial number of samples clustered independently from histopathologic origin (mixed subgroup)—demonstrating divergence between histopathology and transcriptomic data. Further analyses of mixed subgroup via machine learning revealed a predominant mitochondrial gene signature—a trait previously known for chRCC—across all histopathologic subgroups. Additionally, ccRCC samples from mixed subgroup presented an inverse correlation of mitochondrial and angiogenesis-related genes in the TCGA and in three external validation cohorts. Moreover, mixed subgroup affiliation was associated with a highly significant shorter overall survival for patients with ccRCC—and a highly significant longer overall survival for chRCC patients.
Conclusions: Pan-RCC clustering according to RNA-sequencing data revealed a distinct histology-independent subgroup characterized by strengthened mitochondrial and weakened angiogenesis-related gene signatures. Moreover, affiliation to mixed subgroup went along with a significantly shorter overall survival for ccRCC and a longer overall survival for chRCC patients. Further research could offer a therapy stratification by specifically addressing the mitochondrial metabolism of such tumors and its microenvironment.
Das Somatostatin-Analogon Octreotid ist ein in der Diagnose von Thymomen und Thymuskarzinomen wertvolles Hilfsmittel, da praktisch 100 % der Fälle eine kräftige Anreicherung in der Octreotid-Szintigraphie aufweisen, während entzündlich veränderte und nicht-neoplastische Thymi meist nicht anreichern. Auch therapeutisch zeigten 5 der 8 hier untersuchten Thymome eine ausgeprägte, objektivierbare Tumorregression unter einer Kombinationstherapie von Octreotid und Prednison, allerdings zählte die Mehrzahl der Fälle mit gutem Ansprechen zu der Gruppe der klinisch gutartigen Typ A und AB Thymome, während alle Fälle ohne Ansprechen ungünstige Typ B3 Thymome und Thymuskarzinome waren. Das unterschiedliche Anreicherungsverhalten von Normalthymi und Thymomen war nicht durch Unterschiede im immunhistochemischen Expressionmuster von SSTR-Isoformen erklärbar. Die für die Bindung von Octreotid hauptverantwortlichen Somatostatinrezeptor (SSTR) Subtypen 2A, 3 und 5 zeigten in Normalthymi und nahezu allen untersuchten Thymomen und Thymuskarzinomen eine kräftige zytoplasmatische Expression, während membranständige SSTR in Normalthymi nicht, in Thymomen und Thymuskarzinomen nur in etwa 5 % der Fälle beobachtet wurden. Auch das unterschiedliche Ansprechen der einzelnen Thymome bzw. Thymuskarzinome korrelierte nicht mit einem erkennbar unterschiedlichen Expressionsprofil der einzelnen SSTR. Die Befunde sind in erster Linie durch unterschiedliche Kinetiken bei der Internalisierung der SSTR, möglicherweise auch durch Heterokomplexbildung mit anderen Rezeptortypen erklärbar. Eine weitere Erklärungsmöglichkeit wäre eine unterschiedliche Kompetition von Octreotid mit intrinsischem Somatostatin. Unsere Ergebnisse sprechen gegen eine gängige Auffassung, nach der ausschließlich membranständige SSTR durch eine Octreotid-Therapie „attackiert“ werden können. Die Aussagekraft der in der Bildgebung objektivierten Tumorregression und der histologischen Befunde vor und nach Octreotidtherapie ist allerdings mit Vorbehalt zu bewerten, da in allen Fällen eine Kombinationstherapie mit Steroiden durchgeführt wurde und mittlerweile bekannt ist, dass auch Steroide allein bei einem Teil der Fälle zu einer Tumorremission führen können.
Targeting molecular alterations as an effective treatment for isocitrate dehydrogenase-wildtype glioblastoma (GBM) patients has not yet been established. Sterol-O-Acyl Transferase 1 (SOAT1), a key enzyme in the conversion of endoplasmic reticulum cholesterol to esters for storage in lipid droplets (LD), serves as a target for the orphan drug mitotane to treat adrenocortical carcinoma. Inhibition of SOAT1 also suppresses GBM growth. Here, we refined SOAT1-expression in GBM and IDH-mutant astrocytoma, CNS WHO grade 4 (HGA), and assessed the distribution of LD in these tumors. Twenty-seven GBM and three HGA specimens were evaluated by multiple GFAP, Iba1, IDH1 R132H, and SOAT1 immunofluorescence labeling as well as Oil Red O staining. To a small extent SOAT1 was expressed by tumor cells in both tumor entities. In contrast, strong expression was observed in glioma-associated macrophages. Triple immunofluorescence labeling revealed, for the first time, evidence for SOAT1 colocalization with Iba1 and IDH1 R132H, respectively. Furthermore, a notable difference in the amount of LD between GBM and HGA was observed. Therefore, SOAT1 suppression might be a therapeutic option to target GBM and HGA growth and invasiveness. In addition, the high expression in cells related to neuroinflammation could be beneficial for a concomitant suppression of protumoral microglia/macrophages.
The genetic mechanisms underlying adrenocortical tumor development are still largely unknown. We used high-resolution single nucleotide polymorphism microarrays (Affymetrix SNP 6.0) to detect copy number alterations (CNAs) and copy-neutral losses of heterozygosity (cnLOH) in 15 cortisol-secreting adrenocortical adenomas with matched blood samples. We focused on microalterations aiming to discover new candidate genes involved in early tumorigenesis and/or autonomous cortisol secretion. We identified 962 CNAs with a median of 18 CNAs per sample. Half of them involved noncoding regions, 89% were less than 100 kb, and 28% were found in at least two samples. The most frequently gained regions were 5p15.33, 6q16.1, 7p22.3-22.2, 8q24.3, 9q34.2-34.3, 11p15.5, 11q11, 12q12, 16q24.3, 20p11.1-20q21.11, and Xq28 (>= 20% of cases), most of them being identified in the same three adenomas. These regions contained among others genes like NOTCH1, CYP11B2, HRAS, and IGF2. Recurrent losses were less common and smaller than gains, being mostly localized at 1p, 6q, and 11q. Pathway analysis revealed that Notch signaling was the most frequently altered. We identified 46 recurrent CNAs that each affected a single gene (31 gains and 15 losses), including genes involved in steroidogenesis (CYP11B1) or tumorigenesis (CTNNB1, EPHA7, SGK1, STIL, FHIT). Finally, 20 small cnLOH in four cases affecting 15 known genes were found. Our findings provide the first high-resolution genome-wide view of chromosomal changes in cortisol-secreting adenomas and identify novel candidate genes, such as HRAS, EPHA7, and SGK1. Furthermore, they implicate that the Notch1 signaling pathway might be involved in the molecular pathogenesis of adrenocortical tumors.
Adrenocortical tumors consist of benign adenomas and highly malignant carcinomas with a still incompletely understood pathogenesis. A total of 46 adrenocortical tumors (24 adenomas and 22 carcinomas) were investigated aiming to identify novel genes involved in adrenocortical tumorigenesis. High-resolution single nucleotide polymorphism arrays (Affymetrix) were used to detect copy number alterations (CNAs) and copy neutral losses of heterozygosity (cnLOH). Genomic clustering showed good separation between adenomas and carcinomas, with best partition including only chromosome 5, which was highly amplified in 17/22 malignant tumors. The malignant tumors had more relevant genomic aberrations than benign tumors, such as a higher median number of recurrent CNA (2631 vs 94), CNAs >100 Kb (62.5 vs 7) and CN losses (72.5 vs 5.5), and a higher percentage of samples with cnLOH (91% vs 29%). Within the carcinoma cohort, a precise genetic pattern (i.e. large gains at chr 5, 7, 12, and 19, and losses at chr 1, 2, 13, 17, and 22) was associated with a better prognosis (overall survival: 72.2 vs 35.4 months, P=0.063). Interestingly, >70% of gains frequent in beningn were also present in malignant tumors. Notch signaling was the most frequently involved pathway in both tumor entities. Finally, a CN gain at imprinted “IGF2” locus chr 11p15.5 appeared to be an early alteration in a multi-step tumor progression, followed by the loss of one or two alleles, associated with increased IGF2 expression, only in carcinomas. Our study serves as database for the identification of genes and pathways, such as Notch signaling, which could be involved in the pathogenesis of adrenocortical tumors. Using these data, we postulate an adenoma-carcinoma sequence for these tumors.
Die Ursachen des Rotatorenmanschettensyndroms sind vielfältig, ausgehend von der Hypothese, dass die vorbestehenden Muskelatrophie die postoperative Refunktionalisierung beeinflusst, wurde in der vorliegenden Arbeit eine semiquantitative Bestimmung der Expression des fetalen Acetylcholinrezeptottyps untersucht. bei neurogenen Schädigung der Skelettmuskulatur kommt es zu einer Überexpression des fetalen Acetylcholinrezeptors. Ein RT-PCR-basiertes Verfahren zur Bestimmung der Expression des fetalen Acetylcholinrezeptors war methodischer Ausgangspunkt dieser Arbeit. Es konnte in 74% der untersuchten Patienten eine Überexpression des fetalen Rezeptortyps bei Rotatorenmanschettensyndrom nachgewiesen werden.
Tuberous sclerosis complex (TSC), caused by dominant mutations in either TSC1 or TSC2 tumour suppressor genes is characterized by the presence of brain malformations, the cortical tubers that are thought to contribute to the generation of pharmacoresistant epilepsy. Here we report that tuberless heterozygote \(Tsc1^{+/-}\) mice show functional upregulation of cortical GluN2C-containing N-methyl-D-aspartate receptors (NMDARs) in an mTOR-dependent manner and exhibit recurrent, unprovoked seizures during early postnatal life (<P19). Seizures are generated intracortically in the granular layer of the neocortex. Slow kinetics of aberrant GluN2C-mediated currents in spiny stellate cells promotes excessive temporal integration of persistent NMDAR-mediated recurrent excitation and seizure generation. Accordingly, specific GluN2C/D antagonists block seizures in \(Tsc1^{+/-}\) mice in vivo and in vitro. Likewise, GluN2C expression is upregulated in TSC human surgical resections, and a GluN2C/D antagonist reduces paroxysmal hyperexcitability. Thus, GluN2C receptor constitutes a promising molecular target to treat epilepsy in TSC patients.
Allogenic hematopoietic stem cell transplantation (allo-HCT) is a curative therapy for the treatment of malignant and non-malignant bone marrow diseases. The major complication of this treatment is a highly inflammatory reaction known as Graft-versus-Host Disease (GvHD). Cyclosporin A (CsA) and tacrolimus are used to treat GvHD which limits inflammation but also interferes with the anticipated Graft-versus-Leukemia (GvL) effect. These drugs repress conventional T cells (Tcon) along with regulatory T cells (Treg), which are important for both limiting GvHD and supporting GvL. Both of these drugs inhibit calcineurin (CN), which dephosphorylates and activates the nuclear factor of activated T-cells (NFAT) family of transcription factors. Here, we make use of our Cd4cre.Cas9+ mice and developed a highly efficient non-viral CRISPR/Cas9 gene editing method by gRNA-only nucleofection. Utilizing this technique, we demonstrated that unstimulated mouse T cells upon NFATc1 or NFATc2 ablation ameliorated GvHD in a major mismatch mouse model. However, in vitro pre-stimulated mouse T cells could not achieve long-term protection from GvHD upon NFAT single-deficiency. This highlights the necessity of gene editing and transferring unstimulated human T cells during allo-HCT. Indeed, we established a highly efficient ribonucleoprotein (RNP)-mediated CRISPR/Cas9 gene editing for NFATC1 and/or NFATC2 in pre-stimulated as well as unstimulated primary human T cells. In contrast to mouse T cells, not NFATC1 but NFATC2 deficiency in human T cells predominantly affected proinflammatory cytokine production. However, either NFAT single-knockout kept cytotoxicity of human CD3+ T cells untouched against tumor cells in vitro. Furthermore, mouse and human Treg were unaffected upon the loss of a single NFAT member. Lastly, NFATC1 or NFATC2-deficient anti-CD19 CAR T cells, generated with our non-viral ‘one-step nucleofection’ method validated our observations in mouse and human T cells. Proinflammatory cytokine production was majorly dependent on NFATC2 expression, whereas, in vitro cytotoxicity against CD19+ tumor cells was undisturbed in the absence of either of the NFAT members. Our findings emphasize that NFAT single-deficiency in donor T cells is superior to CN-inhibitors as therapy during allo-HCT to prevent GvHD while preserving GvL in patients.
The subclassification of diffuse large B-cell lymphoma (DLBCL) into germinal center B-cell-like (GCB) and activated B-cell-like (ABC) subtypes has become mandatory in the 2017 update of the WHO classification of lymphoid neoplasms and will continue to be used in the WHO 5\(^{th}\) edition. The RNA-based Lymph2Cx assay has been validated as a reliable surrogate of high-throughput gene expression profiling assays for distinguishing between GCB and ABC DLBCL and provides reliable results from formalin-fixed, paraffin-embedded (FFPE) material. This test has been previously used in clinical trials, but experience from real-world routine application is rare. We routinely applied the Lymph2Cx assay to day-to-day diagnostics on a series of 147 aggressive B-cell lymphoma cases and correlated our results with the immunohistochemical subclassification using the Hans algorithm and fluorescence in situ hybridization findings using break-apart probes for MYC, BCL2, and BCL6. The routine use of the Lymph2Cx assay had a high technical success rate (94.6%) with a low rate of failure due to poor material and/or RNA quality. The Lymph2Cx assay was discordant with the Hans algorithm in 18% (23 of 128 cases). Discordant cases were mainly classified as GCB by the Hans algorithm and as ABC by Lymph2Cx (n = 11, 8.6%). Only 5 cases (3.9%) were classified as non-GCB by the Hans algorithm and as GCB by Lymph2Cx. Additionally, 5.5% of cases (n = 7) were left unclassified by Lymph2Cx, whereas they were defined as GCB (n = 4) or non-GCB (n = 3) by the Hans algorithm. Our data support the routine applicability of the Lymph2Cx assay.
The Nuclear Factors of Activated T cells (NFATs) are critical transcription factors playing major roles in the control of the cell cycle, apoptosis and, probably, also cancerogenesis. Of all the four genuine NFATc family members, NFATc1 has the unique induction property which appears to be essential for T and B cell development, along with its considerable role in cytokine gene expression and function in non-lymphoid tissues and during organ development (such as in the development of muscle and heart cells). A number of studies have proved the potential role of NFATc1 protein in development of lymphomas and leukemias and provided evidence of differential expression of the same gene in different tumours (Suppression in classical Hodgkin lymphomas but overexpression in T-ALLs). Although the most commonly accepted pathway is the dephosphorylation of NFAT by calcineurin upon a rise in intracellular Ca++ leading to nuclear translocation followed by transcription of Il2 gene and related cytokines, it is quite possible that signaling mechanisms other than (or in addition to) calcineurin activation lead to NFATc1 induction as well. One of the major isoforms of NFATc1, NFATc1/αA, is the short inducible factor, produced upon full T and B cell activation. Here we used two different conditional knock-out mice as our study model. Inactivation of the murine Nfatc1 gene in bone marrow (of Cd79a/mb-1-cre x Nfatc1flx/flx mice) and spleen (of Cd23-cre x Nfatc1flx/flx mice) resulted in complete ablation of NFATc1 expression in splenic B cells. Although no severe developmental defects were found for the generation of ‘conventional’ B2 cells, NFATc1 inactivation in bone marrow B-cells led to a strong decrease in the peritoneal B1a cell population. In-vitro studies showed a clear-cut decrease in proliferation and an increase in Activation Induced Cell Death (AICD) of NFATc1-/- splenic B cells upon BCR stimulation. While NFATc1 appears to control directly the AICD of peripheral B cells, further studies revealed an effect of NFATc1 on proliferation by a sustained differentiation program controlling Ca++ flux and calcineurin activity which are needed to maintain transcription and proliferation of primary B cells. Re-expression of NFATc1 at a low dose could protect cells against AICD, whereas at a higher dose it initiated AICD. These data suggest an important dual role of NFATc1 in controlling proliferation and apoptosis of peripheral B lymphocytes. NFATc1 ablation also impaired the Ig class switch to IgG3 by T cell-independent (TI) type II antigens and impaired IgG3+ plasmablast formation when studied in-vivo by NP-Ficoll immunization or in-vitro using an in-vitro class-switch model. Contrary to the immunizations with TI-type II antigen, no significant differences were documented in Ig class switch upon immunization with NP-KLH, a T-cell dependent (TD) antigen. Taken together, the data indicate NFATc1/αA as a crucial player in the activation and function of splenic B cells upon BCR stimulation. Missing or incomplete NFATc1/αA induction appears to be one reason for the generation of B cell unresponsiveness, whereas uncontrolled NFATc1/αA expression could lead to unbalanced immune reactions and autoimmune diseases.
T lymphocytes (T cells) represent one of the major cell populations of the immune system. Named by the place of their development, the thymus, several types can be distinguished as the αβ T cells, the γδ T cells, the mucosa-associated invariant T cells (MAIT), and the natural killer T (NKT) cells. The αβ lineages of CD4+ THelper and the CD8+ T cytotoxic cells with the T cell receptor (TCR) composed of α- and β-chain are major players of the adaptive immune system. In the thymus, CD4+ and CD8+ single positive (SP) αβ cells represent the ultimate result of positive and negative selection of CD4+CD8+ double positive (DP) thymocytes. The DP population derives from the double negative (DN) thymocytes that develop from bone marrow-derived progenitors through different stages (DN1-DN4) that are characterized by CD25 and CD44 surface expression.
NFATc1, a member of the Nuclear Factor of Activated T cells (NFAT) transcription factors family, is critically involved in the differentiation and function of T cells. During thymocyte development, the nuclear expression of NFATc1 reaches the highest level at the DN3 (CD44-CD25+) stage. The hematopoietic cell-specific ablation of NFATc1 activity results in an arrest of thymocyte differentiation at the DN1 (CD44+CD25-) stage. On the other hand, over-expression of a constitutively active version of NFATc1 results in an impaired transition of DN3 cells to the DN4 (CD44-CD25-) stage, suggesting that a certain threshold level of NFATc1 activity is critical at this point.
ChIP-seq and RNA-seq analysis allowed us the identification of NFATc1/A target genes involved in lineage development as the Tcra and Tcrb gene loci. Furthermore, we identified multiple NFATc1-regulated genes that are involved in γδ T cell development. In the mouse models, Rag1Cre-Nfatc1fl/fl and Rag1Cre-E2fl/fl, in which the activity of NFATc1 or inducible NFATc1 in the latter is impaired during the early stages of thymocyte development, we observed increased numbers of γδ T cells. These γδ T cells showed an unusual overexpression of CD4, a lack of CD24 expression, and overexpression of the anti-apoptotic gene Bcl2a1a.
We hypothesize that during the DN stages NFATc1 plays an important role in regulating crucial steps of αβ thymocyte development and when NFATc1 activity is missing this may disturb αβ development resulting in alternative cell fates like γδ T cells.
Adrenocortical carcinoma (ACC) is a rare endocrine malignancy and treatment of advanced disease is challenging. Clinical trials with multi-tyrosine kinase inhibitors in the past have yielded disappointing results. Here, we investigated fibroblast growth factor (FGF) receptors and their pathways in adrenocortical tumors as potential treatment targets. We performed real-time RT-PCR of 93 FGF pathway related genes in a cohort of 39 fresh frozen benign and malignant adrenocortical, 9 non-adrenal tissues and 4 cell lines. The expression of FGF receptors was validated in 166 formalin-fixed paraffin embedded (FFPE) tissues using RNA in situ hybridization (RNAscope) and correlated with clinical data. In malignant compared to benign adrenal tumors, we found significant differences in the expression of 16/94 FGF receptor pathway related genes. Genes involved in tissue differentiation and metastatic spread through epithelial to mesechymal transition were most strongly altered. The therapeutically targetable FGF receptors 1 and 4 were upregulated 4.6- and 6-fold, respectively, in malignant compared to benign adrenocortical tumors, which was confirmed by RNAscope in FFPE samples. High expression of FGFR1 and 4 was significantly associated with worse patient prognosis in univariate analysis. After multivariate adjustment for the known prognostic factors Ki-67 and ENSAT tumor stage, FGFR1 remained significantly associated with recurrence-free survival (HR=6.10, 95%CI: 1.78 – 20.86, p=0.004) and FGFR4 with overall survival (HR=3.23, 95%CI: 1.52 – 6.88, p=0.002). Collectively, our study supports a role of FGF pathways in malignant adrenocortical tumors. Quantification of FGF receptors may enable a stratification of ACC for the use of FGFR inhibitors in future clinical trials.
Ribosomal biogenesis and protein synthesis are deregulated in most cancers, suggesting that interfering with translation machinery may hold significant therapeutic potential. Here, we show that loss of the tumor suppressor adenomatous polyposis coli (APC), which constitutes the initiating event in the adenoma carcinoma sequence for colorectal cancer (CRC), induces the expression of RNA polymerase I (RNAPOL1) transcription machinery, and subsequently upregulates ribosomal DNA (rDNA) transcription. Targeting RNAPOL1 with a specific inhibitor, CX5461, disrupts nucleolar integrity, and induces a disbalance of ribosomal proteins. Surprisingly, CX5461-induced growth arrest is irreversible and exhibits features of senescence and terminal differentiation. Mechanistically, CX5461 promotes differentiation in an MYC-interacting zinc-finger protein 1 (MIZ1)- and retinoblastoma protein (Rb)-dependent manner. In addition, the inhibition of RNAPOL1 renders CRC cells vulnerable towards senolytic agents. We validated this therapeutic effect of CX5461 in murine- and patient-derived organoids, and in a xenograft mouse model. These results show that targeting ribosomal biogenesis together with targeting the consecutive, senescent phenotype using approved drugs is a new therapeutic approach, which can rapidly be transferred from bench to bedside.
Risikostratifikation grosszelliger B-Zell Non-Hodgkin Lymphome anhand immunhistochemischer Parameter
(2007)
Die diffusen großzelligen B-Zell-Lymphome (DLBCL) stellen den häufigsten Typ aller Non-Hodgkin-Lymphome dar, sind aber morphologisch, immunologisch, genetisch und klinisch eine sehr heterogene Gruppe. Aufgrund dieser Heterogenität von DLBCL wurde in mehreren Studien untersucht, ob eine molekulare Heterogenität der Tumoren vorläge, bzw. versucht, eine molekulare Reklassifikation zu erreichen. Resultat dieser Bemühungen war eine Unterscheidung bzw. Definition einer Keimzentrums- ähnlichen (GCB-cell-like) Gruppe und einer aktivierten B-Zellen-ähnlichen (ABC-like) Gruppe, die sich in ihrem Ansprechen auf übliche Therapieschemata, mit einer deutlich besseren Prognose für die GCB-like-Gruppe, unterschieden. Die hierbei angewendete Microarray-Technologie hat den entscheidenden Nachteil, dass hierfür qualitativ hochwertige RNA zur Verfügung stehen muss. Neu ist der Ansatz, unterschiedliche Proteinexpressionsmuster am Paraffinmaterial zur Unterscheidung prognostisch relevanter Gruppen heranzuziehen. Die hierbei erzielten Daten sind allerdings in Ihren Aussagen hinsichtlich der prognostischen Wertigkeiten widersprüchlich. In der vorliegenden Arbeit wurden zunächst verschiedene biologische Parameter am Paraffinmaterial hinsichtlich ihrer prognostischen Wertigkeit in der Risikostratifikation von DLBCL untersucht. In einem ersten Schritt wurden klinische Daten von 99 de novo entstandenen großzelligen B-Zell-Lymphomen erhoben, bei denen es sich um 84 DLBCL und um elf DLBCL mit einer weiteren Komponente eines follikulären Lymphoms Grad 3B bzw. auch um vier Fälle mit ausschließlich follikulärem Wachstumsmuster handelte. Die Klassifikation der Fälle nach dem Internationalen Prognostischen Index (IPI) sowie der einzelnen klinischen Parameter des IPI zeigte eine deutliche prognostische Relevanz. In einem zweiten Schritt wurden immunhistochemische Färbungen mit verschiedenen Antikörpern durchgeführt und auf ihre prognostische Bedeutung überprüft. Als negative prognostische Parameter erwiesen sich die Negativität für CD10 sowie BCL-6, also Antigene, die mit einer Keimzentrumszell-Differenzierung assoziiert werden, sowie eine Überexpression von MUM-1, das mit einer postfollikulären Differenzierung assoziiert wird. Weiterhin konnte gezeigt werden, dass einer Expression von BCL-2 und einem Ki67-Index von unter 80 % eine negative prognostische Bedeutung zukommt. Die Stratifikation der Fälle in einen GCB- und einen ABC- Typ anhand des Hans-Klassifikators zeigte nur eine schwache Korrelation zur Überlebenswahrscheinlichkeit. In einem dritten Schritt wurde gezeigt, dass die kombinierte Analyse jeweils zweier Parameter eine relative Abhängigkeit ihrer Expression von der Expression weiterer Marker erkennen ließ. Aus diesem Grunde wurde ein Modell einer sequentiellen Addition negativer prognostischer Indikatoren entwickelt, in der bei Anwesenheit einer negativen Variable (CD10-Negativität, BCL-6 < 20%, BCL-2 positiv, MUM-1≥ 50% und Ki67 < 80%) ein negativer Faktor gewertet und die Summe dieser als Risiko-Score angegeben wurde. Die Stratifikation der Patienten anhand dieses „kombinierten immunhistochemischen Risiko-Scores“ zeigte drei prognostisch deutlich unterschiedliche Gruppen: In der Gruppe von Patienten ohne Risikofaktoren verstarb lediglich eine Patientin (die eine Behandlung abgelehnt hatte); in der Hochrisikogruppe (Score 5) verstarben alle Patienten innerhalb eines Jahres. Die multivariate Analyse des Scores ergab dabei eine Unabhängigkeit von den Parametern des IPI. In der intermediären Gruppe mit einem Risiko-Score von 1-4 zeigten sich der IPI sowie eine LDH-Erhöhung und das Vorhandensein einer B-Symptomatik als geeignete Parameter, um hier eine weitere Stratifizierung durchzuführen. Die vorliegende Arbeit stellt somit eine Erweiterung der publizierten Ansätze einer Erfassung prognostischer Indikatoren in kombinierten Algorithmen dar. Eine Verifizierung der gezeigten Ergebnisse in einer homogen behandelten Patientengruppe innerhalb einer klinischen Studie muss Ziel weiterer Untersuchungen sein.
Despite its significant overexpression in several malignant neoplasms, the expression of RPS27 in the central nervous system (CNS) is widely unknown. We identified the cell types expressing RPS27 in the CNS under normal and disease conditions. We acquired specimens of healthy brain (NB), adult pilocytic astrocytoma (PA) World Health Organization (WHO) grade I, anaplastic PA WHO grade III, gliomas WHO grade II/III with or without isocitrate dehydrogenase (IDH) mutation, and glioblastoma multiforme (GBM). RPS27 protein expression was examined by immunohistochemistry and double-fluorescence staining and its mRNA expression quantified by RT-PCR. Patients’ clinical and tumor characteristics were collected retrospectively. RPS27 protein was specifically expressed in tumor cells and neurons, but not in healthy astrocytes. In tumor tissue, most macrophages were positive, while this was rarely the case in inflamed tissue. Compared to NB, RPS27 mRNA was in mean 6.2- and 8.8-fold enhanced in gliomas WHO grade II/III with (p < 0.01) and without IDH mutation (p = 0.01), respectively. GBM displayed a 4.6-fold increased mean expression (p = 0.02). Although RPS27 expression levels did not affect the patients’ survival, their association with tumor cells and tumor-associated macrophages provides a rationale for a future investigation of a potential function during gliomagenesis and tumor immune response.
Bei der postoperativen Therapieplanung des Mammakarzinoms treten immer wieder Entscheidungsgrenzfälle auf, bei denen keine sicheren Argumente für oder gegen eine adjuvante Chemotherapie gefunden werden können. Bei 50 Hormonrezeptor-positiven, Her2/neu-negativen Mammakarzinomen ohne oder mit nur geringer nodaler Metastasierung (max. pT1a) wurde zusätzlich zu den konventionellen klinisch-pathologischen Risikofaktoren der OncotypeDX®-Multigentest veranlasst. In der Tumorkonferenz wurde bereits vor Eingang des Testergebnisses ein Votum für oder gegen eine Chemotherapie auf Basis konventioneller Parameter protokolliert; die definitive Therapieempfehlung erfolgte nach Vorliegen des Multigentest-Ergebnisses.
32 Mammakarzinome (64 %) zeigten einen niedrigen, 26 (32 %) einen mittleren und 3 (6 %) einen hohen Recurrence-Score (RS). In vielen Fällen konnte das OncotypeDX®-Ergebnis eine auf der Basis konventioneller Parameter getroffene Therapieentscheidung stützen. In fünf Fällen wurde eine zunächst favorisierte Entscheidung für eine adjuvante Therapie revidiert. In drei Fällen wurde eine zunächst nicht geplante Chemotherapie empfohlen. Allerdings führte in einigen Fällen auch eine niedrige oder intermediäre Risikokonstellation in der OncotypeDX®-Testung nicht dazu, von einer adjuvanten Chemotherapie abzuraten.
Insgesamt spricht das Ergebnis nicht dafür, einen Multigentest als Standardmethode einzusetzen. Vielmehr sollten zunächst die konventionellen, insbesondere die histopathologischen und immunhistochemischen Parameter mit großer Sorgfalt erhoben und analysiert werden. Im Zweifelsfall und nach Kosten-Nutzen-Abwägung kann ein Multigentest jedoch ein weiteres hilfreiches Argument für oder gegen eine bestimmte Therapieempfehlung liefern.
Genetische Veränderungen von Thymomen, insbesondere rekurrente Aberrationen, sind bislang unbekannt. In dieser Dissertation wurden 13 WHO Typ A Thymome, 23 WHO Typ B3 Thymome sowie 12 primäre Plattenepithelkarzinome (WHO Typ C Thymome) sowie einige seltene Primärtumoren des Thymus mittels Komparativer genomischer Hybridisierung untersucht. Mit Ausnahme eines einzigen Falles zeigten WHO Typ A Thymome keine chromosomalen Gewinne oder Verluste. Im Gegensatz dazu fanden sich bei allen Thymomen des Typs B3 genetische Aberrationen. Der Gewinn von 1q trat in 15 (65%), Gewinne von Chromosom 16 und Xq traten in jeweils 3 (13%) Fällen auf. Verluste fanden sich auf Chromosom 6 in 10 Fällen (43%) und an 13q in 6 Fällen (23%). Die häufigsten Gewinne bei primären Plattenepithelkarzinomen fanden sich auf 1q in 7 Fällen (58%), auf 17q in 4 Fällen (33%) und auf Chromosom 5 und 18 in jeweils 3 Fällen (25%). Rekurrente Verluste traten bei dieser Entität auf Chromosom 16q in 8 Fällen (67%), auf Chromosom 6 in 6 Fällen (50%), auf Chromosom 3 in 4 Fällen (33%) und auf den Chromosomen 13q sowie 17p in jeweils 3 Fällen (25%) auf. Die Dissertation zeigt, daß histologisch unterschiedliche Thymome mit unterschiedlichen, rekurrenten Aberrationen assoziiert sind. WHO Typ A Thymome weisen nur vereinzelt genetische Aberrationen auf. Thymome vom WHO Typ A und Typ B3 weisen verschiedene genetische Phänotypen auf, während Thymome vom Typ B3 sowie die primären Plattenepithelkarzinome des Thymus gemeinsame genetische Aberrationen zeigen. Neben den pathogenetischen Aspekten weist der beobachtete Verlust von Chromosom 6, auf dem sich die Genloci der HLA-Moleküle befinden, bei WHO Typ B3 Thymomen auch auf eine Rolle bei der Entstehung paraneoplastischer Autoimmunphänomene wie der Myasthenia Gravis hin.
Das murine Blimp1 (B lymphocyte-induced maturation protein) und sein humanes Homolog PRDI-BF1 (positive regulatory domain I binding factor 1) sind als terminale Differenzierungsfaktoren der myeloischen Reihe und der B-Lymphozyten (BCs) beschrieben worden. Über direkte sequenzspezifische Promotorbindung und epigenetische Modifikationen greifen sie größtenteils reprimierend in die Expression einer Vielzahl von Genen ein und werden dabei funktionell mit einer niedrigen Proliferationsrate und einem hohen Grad an Zelldifferenzierung und Apoptose in Zusammenhang gebracht. Im Zuge dieser Arbeit konnte gezeigt werden, dass Blimp1 auch in der zweiten lymphatischen Zellreihe, also in den T-Lymphozyten (TCs) und hier insbesondere in den Subtypen der T-Helfer-2-Zellen (CD4+ TH2), der CD4+ T-Gedächtniszellen (CD4+ TMem) und der regulatorischen TCs (CD4+ CD25+ TReg) exprimiert wird. Die gefundene Blimp1-Expression ist nicht konstitutiv. Vielmehr wird das Blimp1-Gen über T-Zellrezeptorsignale – über die Proteinkinase C (PKC) und den Anstieg freier intrazellulärer Ca2+-Konzentrationen – verstärkend über den Interleukin-6-Rezeptor-pathway, und über die Aktivität des Transkriptionsfaktors C/EBPβ (CCAAT/enhancer-binding protein) allein induziert. Die Transaktivierung des TATA-boxlosen Blimp1-Promotors (ca. 1 kb) durch C/EBPβ wird stärker über dessen proximale Hälfte vermittelt, an der jedoch keine direkte Bindung des Transkriptionsfaktors nachzuweisen ist, und scheint somit indirekt zu sein. Im Gegensatz dazu bindet das C/EBPβ-Protein im schwächer transaktivierenden distalen Blimp1-Promotorbereich direkt an eine kombinierte, der P1/Pu-bB-des IL-4-Promotors ähnliche NFAT/C/EBP-Bindungssequenz. Die C/EBP-Bindungsstelle liegt hierbei in direkter Nachbarschaft zum NFAT-(nuclear factor of activated T cells) Bindungsmotiv. Auch NFATc1 bindet direkt, entwickelt aber kein transaktivierendes Potential sondern reprimiert vielmehr die durch C/EBPβ-vermittelte Transaktivierung.
Die Unterscheidung zwischen körpereigenen und körperfremden Strukturen ist eine grundlegende Herausforderung der spezifischen Immunantwort. Pathologische Veränderungen dieser Abgrenzung können zu schwerwiegenden Autoimmunerkrankungen wie beispielsweise Diabetes Mellitus, Rheumatischer Arthritis oder Multipler Sklerose führen. Um unerwünschte (Auto-) Immunreaktionen zu verhindern, existieren verschiedene Formen von peripheren Toleranzmechanismen, die durch viele Transkriptionsfaktoren wie z. B. ICER (inducible cAMP early repressor), NFAT (nuclear factor of activated T cells) und Foxp3 (forkhead box protein p3) kontrolliert werden. Foxp3+ regulatorische T-Zellen (Tregs) sind spezialisierte immun-suppressive Lymphozyten, welche die Aktivierung anderer Immunzellen unterdrücken können. Einer der möglichen Mechanismen ist der Transfer zyklischen Adenosin-Monophosphats (cAMP) von Tregs in konventionelle T- und B-Lymphozyten. Die erhöhte intrazelluläre Konzentration an cAMP führt in Effektorzellen zur Induktion und Kerntranslokation von ICER. Der transkriptionelle Repressor ICER supprimiert die Expression vieler NFAT-regulierter Gene und hemmt darüber hinaus die Induktion der NFATc1/αA-Isoform selbst. Diese Isoform wird speziell in pro-inflammatorischen Effektorzellen hochreguliert und ist maßgeblich an deren spezifischem transkriptionellen Programm beteiligt. Foxp3 ist ein zentraler Faktor für die Bildung und Funktion sowohl Thymus-generierter nTregs als auch peripher (TGFβ-) induzierter iTregs. Die Kontrolle des Foxp3-Gens wird in iTregs – überraschenderweise aber nicht in nTregs – durch NFAT-Faktoren reguliert. Allerdings hemmt Foxp3 durch eine negative Rückkopplung wiederum die Induktion und Aktivität von NFATc1/αA. Dies stellt somit ein weiteres Regulativ dar, wobei Foxp3 nicht nur die Plastizität, sondern auch die Funktion von immun-suppressiven T-Zellen steuert. Zusätzlich regulieren die verschiedenen NFAT-Faktoren auch die Antigen präsentierenden dendritischen Zellen (DCs). Während NFATc1 und NFATc2 die Differenzierung und Proliferation von DCs beeinflussen, reguliert NFATc3 deren Zytokinexpression und steuert indirekt auch die nachfolgende T-Zell-Immunantwort. Die Kontrolle der Genregulation in Immunzellen durch die Transkriptionsfaktoren ICER, NFAT und Foxp3 erfüllt somit spezifische Funktionen der Immunität, reguliert aber gleichzeitig wichtige Aspekte der peripheren Toleranz, um schädliche (Auto-) Immunreaktionen zu verhindern.
Peritonitis is a common disease in man, frequently caused by fungi, such as Candida albicans; however, in seldom cases opportunistic infections with Saccharomyces cerevisiae are described. Resident peritoneal macrophages (prMΦ) are the major group of phagocytic cells in the peritoneum. They express a broad range of surface pattern recognition receptors (PRR) to recognize invaders. Yeast infections are primarily detected by the Dectin-1 receptor, which triggers activation of NFAT and NF-κB pathways.
The transcription of the Nfatc1 gene is directed by the two alternative promoters, inducible P1 and relatively constitutive P2 promoter. While the role of P1-directed NFATc1α-isoforms to promote survival and proliferation of activated lymphocytes is well-established, the relevance of constitutively generated NFATc1β-isoforms, mainly expressed in resting lymphocytes, myeloid and non-lymphoid cells, remains unclear. Moreover, former work at our department indicated different roles for NFATc1α- and NFATc1β-proteins in lymphocytes.
Our data revealed the functional role of NFATc1 in peritoneal resident macrophages. We demonstrated that the expression of NFATc1β is required for a proper immune response of prMΦ during fungal infection-induced acute peritonitis. We identified Ccl2, a major chemokine produced in response to fungal infections by prMΦ, as a novel NFATc1 target gene which is cooperatively regulated through the NFAT- and canonical NF-κB pathways. Consequently, we showed that NFATc1β deficiency in prMΦ results in a decreased infiltration of inflammatory monocytes, leading to a delayed clearance of peritoneal fungal infection.
We could further show that the expression of NFATc1β-isoforms is irrelevant for homeostasis of myeloid and adaptive immune system cells and that NFATc1α- (but not β-) isoforms are required for a normal development of peritoneal B1a cells. In contrast to the situation in myeloid cells, NFATc1β deficiency is compensated by increased expression of NFATc1α-isoforms in lymphoid cells. As a consequence, NFATc1ß is dispensable for activation of the adaptive immune system.
Taken together our results illustrate the redundancy and indispensability of NFATc1-isoforms in the adaptive and innate immune system, indicating a complex regulatory system for Nfatc1 gene expression in different compartments of the immune system and likely beyond that.
Multiple myeloma (MM) is a plasma cell disorder that is characterized by a great genetic heterogeneity. Recent next generation sequencing studies revealed an accumulation of tumor-associated mutations in receptor tyrosine kinases (RTKs) which may also contribute to the activation of survival pathways in MM. To investigate the clinical role of RTK-mutations in MM, we deep-sequenced the coding DNA-sequence of EGFR, EPHA2, ERBB3, IGF1R, NTRK1 and NTRK2 which were previously found to be mutated in MM, in 75 uniformly treated MM patients of the “Deutsche Studiengruppe Multiples Myelom”. Subsequently, we correlated the detected mutations with common cytogenetic alterations and clinical parameters. We identified 11 novel non-synonymous SNVs or rare patient-specific SNPs, not listed in the SNP databases 1000 genomes and dbSNP, in 10 primary MM cases. The mutations predominantly affected the tyrosine-kinase and ligand-binding domains and no correlation with cytogenetic parameters was found. Interestingly, however, patients with RTK-mutations, specifically those with rare patient-specific SNPs, showed a significantly lower overall, event-free and progression-free survival. This indicates that RTK SNVs and rare patient-specific RTK SNPs are of prognostic relevance and suggests that MM patients with RTK-mutations could potentially profit from treatment with RTK-inhibitors.
Rapid and Efficient Gene Editing for Direct Transplantation of Naive Murine Cas9\(^+\) T Cells
(2021)
Gene editing of primary T cells is a difficult task. However, it is important for research and especially for clinical T-cell transfers. CRISPR/Cas9 is the most powerful gene-editing technique. It has to be applied to cells by either retroviral transduction or electroporation of ribonucleoprotein complexes. Only the latter is possible with resting T cells. Here, we make use of Cas9 transgenic mice and demonstrate nucleofection of pre-stimulated and, importantly, of naive CD3\(^+\) T cells with guideRNA only. This proved to be rapid and efficient with no need of further selection. In the mixture of Cas9\(^+\)CD3\(^+\) T cells, CD4\(^+\) and CD8\(^+\) conventional as well as regulatory T cells were targeted concurrently. IL-7 supported survival and naivety in vitro, but T cells were also transplantable immediately after nucleofection and elicited their function like unprocessed T cells. Accordingly, metabolic reprogramming reached normal levels within days. In a major mismatch model of GvHD, not only ablation of NFATc1 and/or NFATc2, but also of the NFAT-target gene IRF4 in naïve primary murine Cas9\(^+\)CD3\(^+\) T cells by gRNA-only nucleofection ameliorated GvHD. However, pre-activated murine T cells could not achieve long-term protection from GvHD upon single NFATc1 or NFATc2 knockout. This emphasizes the necessity of gene-editing and transferring unstimulated human T cells during allogenic hematopoietic stem cell transplantation.
Purpose
Patients suffering from aggressive systemic peripheral lymphoma with primary central nervous system involvement (PCL) are a rare and sparsely investigated population. Recommended treatment regimens include a combination of intrathecal and systemic chemotherapy as well as whole brain radiotherapy while offering relatively poor survival.
Methods
We conducted a single-center retrospective study that analyzed safety and outcome of 4 + 4 cycles Rituximab (R)-CHOP and R-high-dose Methotrexate (HD-MTX) for newly diagnosed, transplant-eligible patients ("Ping-Pong"), followed by Cytarabine (AraC)/Thiotepa (TT), BCNU/TT, and autologous hematologic stem cell transplantation (aHSCT). We retrospectively analyzed a set of 16 patients with high-intermediate or high-risk IPI status.
Results
Overall response rate to Ping-Pong was 100% measured by CT/MRI, including 93.75% complete remissions after BCNU/TT followed by PBSCT. One patient failed to qualify for high-dose chemotherapy due to progression when receiving Cytarabine/TT. All patients experienced grade III adverse events, 3 of them a grade IV adverse event. Estimated progression-free survival is 93.75% after a 4.8-year follow-up currently.
Conclusion
Our study suggests high effectivity of R-CHOP with mid-cycle MTX with aHSCT consolidation towards acceptable OS results in this challenging patient population.
The aim of this pilot study was to investigate the diagnostic potential of TMEM119 as a useful microglia-specific marker in combination with immunostainings for phagocytic function and infiltrating capacity of monocytes in cases of lethal monosubstance intoxications by morphine (MOR), methamphetamine (METH), and of ethanol-associated death (ETH) respectively. Human brain tissue samples were obtained from forensic autopsies of cases with single substance abuse (MOR, n = 8; ETH, n = 10; METH, n = 9) and then compared to a cohort of cardiovascular fatalities as controls (n = 9). Brain tissue samples of cortex, white matter, and hippocampus were collected and stained immunohistochemically with antibodies against TMEM119, CD68KiM1P, and CCR2. We could document the lowest density of TMEM119-positive cells in MOR deaths with highly significant differences to the control densities in all three regions investigated. In ETH and METH deaths, the expression of TMEM119 was comparable to cell densities in controls. The results indicate that the immunoreaction in brain tissue is different in these groups depending on the drug type used for abuse.
Background
Despite advances in treatment of patients with non-small cell lung cancer, carriers of certain genetic alterations are prone to failure. One such factor frequently mutated, is the tumor suppressor PTEN. These tumors are supposed to be more resistant to radiation, chemo- and immunotherapy.
Results
We demonstrate that loss of PTEN led to altered expression of transcriptional programs which directly regulate therapy resistance, resulting in establishment of radiation resistance. While PTEN-deficient tumor cells were not dependent on DNA-PK for IR resistance nor activated ATR during IR, they showed a significant dependence for the DNA damage kinase ATM. Pharmacologic inhibition of ATM, via KU-60019 and AZD1390 at non-toxic doses, restored and even synergized with IR in PTEN-deficient human and murine NSCLC cells as well in a multicellular organotypic ex vivo tumor model.
Conclusion
PTEN tumors are addicted to ATM to detect and repair radiation induced DNA damage. This creates an exploitable bottleneck. At least in cellulo and ex vivo we show that low concentration of ATM inhibitor is able to synergise with IR to treat PTEN-deficient tumors in genetically well-defined IR resistant lung cancer models.
Protocadherins (PCDHs) belong to the cadherin superfamily and represent the largest subgroup of calcium-dependent adhesion molecules. In the genome, most PCDHs are arranged in three clusters, α, β, and γ on chromosome 5q31. PCDHs are highly expressed in the central nervous system (CNS). Several PCDHs have tumor suppressor functions, but their individual role in primary brain tumors has not yet been elucidated. Here, we examined the mRNA expression of PCDHGC3, a member of the PCDHγ cluster, in non-cancerous brain tissue and in gliomas of different World Health Organization (WHO) grades and correlated it with the clinical data of the patients. We generated a PCDHGC3 knockout U343 cell line and examined its growth rate and migration in a wound healing assay. We showed that PCDHGC3 mRNA and protein were significantly overexpressed in glioma tissue compared to a non-cancerous brain specimen. This could be confirmed in glioma cell lines. High PCDHGC3 mRNA expression correlated with longer progression-free survival (PFS) in glioma patients. PCDHGC3 knockout in U343 resulted in a slower growth rate but a significantly faster migration rate in the wound healing assay and decreased the expression of several genes involved in WNT signaling. PCDHGC3 expression should therefore be further investigated as a PFS-marker in gliomas. However, more studies are needed to elucidate the molecular mechanisms underlying the PCDHGC3 effects.
Elevation of intracellular cAMP in T lymphocytes, induced by agents such as IL-1α, prostaglandins or forskolin, inhibits Th1-type cytokine production but stimulates Th2-type cytokine production. The signaling pathway engaged in cAMP-mediated induction of Th2 lymphokines remains obscure and therefore my doctoral work was focused on the elucidation of cAMP pathway in primary Th lymphocytes. While forskolin treatment of EL-4 cells led both to an activation of Th2 lymphokines and inhibition of Th1 lymphokines, ectopic expression of catalytically active PKA stimulated Th2 lymphokines but failed to inhibit Th1 lymphokine expression. Thus, the PKA activity is selectively involved in the stimulation of Th2 lymphokine expression whereas other cAMP-dependent pathway(s) appears to downregulate Th1 lymphokines. By investigating different types of primary murine Th cells, it was found that active PKA enhanced IL-5 expression only in Th0 and Th2 but not in Th1 cells. This is likely due to the different levels of GATA-3 whose expression is high in Th2, moderate in Th0 and very low in Th1 cells. Ectopic expression of GATA-3 in Th1 cells induced Th2 lymphokine expression which could be further enhanced by increased cAMP levels or PKA activity. Investigations on the role of increased cAMP levels on Th2 lymphokines in D10 cells, a Th2-type cell line, led to the conclusion that elevated cAMP concentrations do not stimulate PKA but p38 activity which, through phosphorylation of GATA-3, appeared to induce IL-5 and IL-13 expression (Chen et al., 2000). While focusing on primary Th lymphocytes, it was observed that expression of the catalytic subunit α of PKA is sufficient for optimal IL-5 expression in primary Th0 cells. In addition, downregulation of IL-5 production in primary Th2 cells by the treatment with low concentrations of H-89, a PKA specific inhibitor, as well as by the ectopic expression of a negatively acting version of regulatory PKA subunit I demonstrates that active PKA plays an important role in IL-5 gene regulation. These findings using different types of primary CD4+ T lymphocytes, including Th2 cells, the one likely to represent the native IL-5 producers in vivo, demonstrates that the adenylyl cyclase/cAMP/PKA signaling pathway plays an important role in IL-5 gene expression in primary Th2 cells. Thus the importance of cAMP/PKA signaling pathway in Th2 effector function was established during this doctoral research work.
Regulatory T cells (Tregs) prevent autoimmunity but limit antitumor immunity. The canonical NF-\(\kappa\)B signaling pathway both activates immunity and promotes thymic Treg development. Here, we report that mature Tregs continue to require NF-\(\kappa\)B signaling through I\(\kappa\)B-kinase \(\beta\) (IKK\(\beta\)) after thymic egress. Mice lacking IKK\(\beta\) in mature Tregs developed scurfy-like immunopathology due to death of peripheral FoxP3\(^+\) Tregs. Also, pharmacological IKK\(\beta\) inhibition reduced Treg numbers in the circulation by ~50% and downregulated FoxP3 and CD25 expression and STAT5 phosphorylation. In contrast, activated cytotoxic T lymphocytes (CTLs) were resistant to IKK\(\beta\) inhibition because other pathways, in particular nuclear factor of activated T cells (NFATc1) signaling, sustained their survival and expansion. In a melanoma mouse model, IKK\(\beta\) inhibition after CTL cross-priming improved the antitumor response and delayed tumor growth. In conclusion, prolonged IKK\(\beta\) inhibition decimates circulating Tregs and improves CTL responses when commenced after tumor vaccination, indicating that IKK\(\beta\) represents a druggable checkpoint.
Prognostische und therapeutische Aspekte von Thymomen : eine retrospektive Studie von 582 Fällen
(2004)
Thymome sind seltene epitheliale Thymustumoren, die in der überwiegenden Zahl der Fälle die Fähigkeit zur Reifung und zum Export von T-Zellen behalten haben. Diese Fähigkeit ist als Ursache für die häufige Asoziation dieser Tumoren mit Autoimmunphänomenen (z.B Myasthenia gravis)anzunehmen. Die vorgelegte Studie zeigt die prognostische Relevanz der derzeit gültigen histologischen WHO-Klassifizierung von Thymomen. Das biologische Verhalten der einzelnen Thymomtypen korreliert dabei mit dem Ausmaß zytogenetischer Veränderungen. Wenige klinische und histologische Parameter wie der histologische Subtyp, Tumorstadium nach Masaoka sowie der Resektionsstatus reichen aus, um den Verlauf eines bestimmten Thymoms mit genügender Zuverlässigkeit prognostizieren zu können. Dies konnte in Übereinstimmung mit früheren Arbeiten in unserer Studie gezeigt werden. Somit müssen vor allem diese drei Parameter berücksichtigt werden, um eine adäquate Therapie einleiten zu können. Angaben zu Alters- und Geschlechtsverteilung können diese Befunde ergänzen, haben jedoch keine prognostische Signifikanz für die Wahl der Therapie. Die erhobenen Befunde der vorgelegten Follow-up Studie können als Grundlage prospektiver klinischer Therapiestudien dienen. Im Zentrum der Bemühungen sollte hierbei nach unseren Ergebnissen die Therapie von „high-risk“ Thymomen des Typ B und C stehen, bei denen eine primäre vollständige Resektion nicht möglich ist, oder bei denen zum Zeitpunkt der Operation bereits Metastasen bestehen. Therapieoptionen mit multimodalen Therapiestrategien müssen dafür noch weiter modifiziert und über längere Zeiträume erprobt werden. Zudem sollten klinische Studien mit Somatostatin-Analoga als neue Therapiemöglichkeit gefördert werden. Aufgrund der äußerst niedrigen Inzidenz von Thymomen und der niedrigen Frequenz von Patienten mit diesen ungünstigen Thymomverläufen werden diese Versuche nationale oder internationale Bemühungen erfordern.
Die Abschätzung des individuellen Rezidiv- und Progressrisikos eines Patienten mit einem Nierenzellkarzinom gelingt durch Bestimmung von Staging und Grading nur unzureichend. Ziel der durchgeführten Untersuchung war es neben etablierten Prognoseparametern wie Staging und Grading eines Nierentumors die VEGF-Immunreaktivität zu untersuchen und auf seine Tauglichkeit als Prognoseparameter zu überprüfen. Hierzu wurden parafin-fixierte Präparate von 200 Patienten, die an der Urologischen Universitätsklinik Würzburg zwischen 1985 und 1994 tumornephrektomiert wurden, immunhistochemisch untersucht. Zu allen Patienten ist ein postoperativer Verlauf bekannt. Die Präparate wurden zunächst nach der Avidin-Biotin-Methode gefärbt und zur Auswertung vorbereitet. Für jedes Präparat wurde nun der prozentuale Anteil der VEGF-positiven Zellen im Tumor durch zwei unabhängige Untersucher bestimmt. Als Mass der Prognose (abhängige Variable) wurden Überlebenszeit und rezidivfreie Zeit nach Tumornephrektomie gewählt. Als prognostische Parameter (unabhängige Variable) dienten die etablierten Faktoren wie Staging und Grading sowie der potentiell relevante Faktor VEGF. VEGF erwies sich, im Präparat immunhostochemisch gemessen, als statistisch signifikanter Prognosefaktor, der aber den klassischen Faktoren unterlegen ist. In einer multivariaten Prognoseanalyse und in einer Berechnung von Prognoseindizes sowie Receiver Operating Characteristics (ROC)-Kurven konnte gezeigt werden, dass eine Kombination der etablierten Prognosefaktoren mit VEGF eine statistisch signifikante Steigerung der Vorhersagegenauigkeit erreicht
Purpose
Therapeutic options for breast cancer (BC) treatment are constantly evolving. The Human Epidermal Growth Factor 2 (HER2)-low BC entity is a new subgroup, representing about 55% of all BC patients. New antibody–drug conjugates demonstrated promising results for this BC subgroup. Currently, there is limited information about the conversion of HER2 subtypes between primary tumor and recurrent disease.
Methods
This retrospective study included women with BC at the University Medical Centre Wuerzburg from 1998 to 2021. Data were retrieved from patients' records. HER2 evolution from primary diagnosis to the first relapse and the development of secondary metastases was investigated.
Results
In the HR-positive subgroup without HER2 overexpression, HER2-low expression in primary BC was 56.7 vs. 14.6% in the triple-negative subgroup (p < 0.000). In the cohort of the first relapse, HER2-low represented 64.1% of HR-positive vs. 48.2% of the triple-negative cohort (p = 0.03). In patients with secondary metastases, HER2-low was 75.6% vs. 50% in the triple negative subgroup (p = 0.10). The subgroup of HER2-positive breast cancer patients numerically increased in the course of disease; the HER2-negative overall cohort decreased. A loss of HER2 expression from primary BC to the first relapse correlated with a better OS (p = 0.018). No clinicopathological or therapeutic features could be identified as potential risk factors for HER2 conversion.
Conclusion
HER2 expression is rising during the progression of BC disease. In view of upcoming therapeutical options, the re-analysis of newly developed metastasis will become increasingly important.
Zervikale Lymphknoten stellen einen typischen Manifestationsort des klassischen Hodgkin-Lymphoms dar. Eine primäre extranodale Manifestation dieses Tumors wird in der Mehrheit der wissenschaftlichen Berichte als selten angesehen. Diese Dissertation hatte zum Ziel, möglichst viele Fälle mit dem speziellen Krankheitsbild eines klassischen Hodgkin-Lymphoms mit primär extranodaler Manifestation im Kopf-Hals-Bereich zu analysieren. Anhand der ermittelten Ergebnisse sollten Antworten auf bislang offene Fragestellungen gegeben werden. In der ersten Datenerhebungsphase wurde eine umfangreiche Analyse der bislang zu dieser Thematik veröffentlichten deutsch- und englischsprachigen Fachliteratur durchgeführt. Aus den Jahren 1924 bis 2010 konnten 103 einzeln aufgelistete Patienten ermittelt werden. Diese geeigneten Fallberichte wurden mit allen verfügbaren Einzelheiten zu histologischen, epidemiologischen und klinischen Befunden tabellarisch dokumentiert. In der zweiten Phase der Datenerhebung wurde am Lymphknotenreferenzzentrum des pathologischen Institutes der Julius-Maximilians-Universität Würzburg ein eigenes Patientenkollektiv ermittelt. Von 1999 bis zum Jahr 2008 konnten 21 Patienten analysiert werden. Auch dieses Kollektiv wurde auf histologische, epidemiologische und klinische Parameter untersucht. Die herausgearbeiteten Ergebnisse der 124 Patienten wurden in allen Einzelheiten diskutiert und die eingangs aufgestellten Fragestellungen abschließend beantwortet.
Background
Scattered extracellular deposits of amyloid within the brain parenchyma can be found in a heterogeneous group of diseases. Its condensed accumulation in the white matter without evidence for systemic amyloidosis is known as primary brain amyloidoma (PBA). Although originally considered as a tumor-like lesion by its space-occupying effect, this condition displays also common hallmarks of a neurodegenerative disorder.
Case presentation
A 50-year-old woman presented with a mild cognitive decline and seizures with a right temporal, irregular and contrast-enhancing mass on magnetic resonance imaging. Suspecting a high-grade glioma, the firm tumor was subtotally resected. Neuropathological examination showed no glioma, but distinct features of a neurodegenerative disorder. The lesion was composed of amyloid AL λ aggregating within the brain parenchyma as well as the adjacent vessels, partially obstructing the vascular lumina. Immunostaining confirmed a distinct perivascular inflammatory reaction. After removal of the PBA, mnestic impairments improved considerably, the clinical course and MRI-results are stable in the 8-year follow-up.
Conclusion
Based on our histopathological findings, we propose to regard the clinicopathological entity of PBA as an overlap between a neoplastic and neurodegenerative disorder. Since the lesions are locally restricted, they might be amenable to surgery with the prospect of a definite cure.
(1) Background: C-X-C Motif Chemokine Receptor 4 (CXCR4) and Fibroblast Activation Protein Alpha (FAP) are promising theranostic targets. However, it is unclear whether CXCR4 and FAP positivity mark distinct microenvironments, especially in solid tumors. (2) Methods: Using Random Forest (RF) analysis, we searched for entity-independent mRNA and microRNA signatures related to CXCR4 and FAP overexpression in our pan-cancer cohort from The Cancer Genome Atlas (TCGA) database — representing n = 9242 specimens from 29 tumor entities. CXCR4- and FAP-positive samples were assessed via StringDB cluster analysis, EnrichR, Metascape, and Gene Set Enrichment Analysis (GSEA). Findings were validated via correlation analyses in n = 1541 tumor samples. TIMER2.0 analyzed the association of CXCR4 / FAP expression and infiltration levels of immune-related cells. (3) Results: We identified entity-independent CXCR4 and FAP gene signatures representative for the majority of solid cancers. While CXCR4 positivity marked an immune-related microenvironment, FAP overexpression highlighted an angiogenesis-associated niche. TIMER2.0 analysis confirmed characteristic infiltration levels of CD8+ cells for CXCR4-positive tumors and endothelial cells for FAP-positive tumors. (4) Conclusions: CXCR4- and FAP-directed PET imaging could provide a non-invasive decision aid for entity-agnostic treatment of microenvironment in solid malignancies. Moreover, this machine learning workflow can easily be transferred towards other theranostic targets.
Idiopathic Parkinson’s disease (PD) is characterized by a progredient degeneration of the brain, starting at deep subcortical areas such as the dorsal motor nucleus of the glossopharyngeal and vagal nerves (DM) (stage 1), followed by the coeruleus–subcoeruleus complex; (stage 2), the substantia nigra (SN) (stage 3), the anteromedial temporal mesocortex (MC) (stage 4), high-order sensory association areas and prefrontal fields (HC) (stage 5) and finally first-order sensory association areas, premotor areas, as well as primary sensory and motor field (FC) (stage 6). Autoimmunity might play a role in PD pathogenesis. Here we analyzed whether anti-brain autoantibodies differentially recognize different human brain areas and identified autoantigens that correlate with the above-described dissemination of PD pathology in the brain. Brain tissue was obtained from deceased individuals with no history of neurological or psychiatric disease and no neuropathological abnormalities. Tissue homogenates from different brain regions (DM, SN, MC, HC, FC) were subjected to SDS-PAGE and Western blot. Blots were incubated with plasma samples from 30 PD patients and 30 control subjects and stained with anti-IgG antibodies to detect anti-brain autoantibodies. Signals were quantified. Prominent autoantigens were identified by 2D-gel-coupled mass spectrometry sequencing. Anti-brain autoantibodies are frequent and occur both in healthy controls and individuals with PD. Glial fibrillary acidic protein (GFAP) was identified as a prominent autoantigen recognized in all plasma samples. GFAP immunoreactivity was highest in DM areas and lowest in FC areas with no significant differences in anti-GFAP autoantibody titers between healthy controls and individuals with PD. The anti-GFAP autoimmunoreactivity of different brain areas correlates with the dissemination of histopathological neurodegeneration in PD. We hypothesize that GFAP autoantibodies are physiological but might be involved as a cofactor in PD pathogenesis secondary to a leakage of the blood–brain barrier.
Pilot study on the value of Raman spectroscopy in the entity assignment of salivary gland tumors
(2021)
Background
The entity assignment of salivary gland tumors (SGT) based on histomorphology can be challenging. Raman spectroscopy has been applied to analyze differences in the molecular composition of tissues. The aim of this study was to evaluate the suitability of RS for entity assignment in SGT.
Methods
Raman data were collected in deparaffinized sections of pleomorphic adenomas (PA) and adenoid cystic carcinomas (ACC). Multivariate data and chemometric analysis were completed using the Unscrambler software.
Results
The Raman spectra detected in ACC samples were mostly assigned to nucleic acids, lipids, and amides. In a principal component-based linear discriminant analysis (LDA) 18 of 20 tumor samples were classified correctly.
Conclusion
In this proof of concept study, we show that a reliable SGT diagnosis based on LDA algorithm appears possible, despite variations in the entity-specific mean spectra. However, a standardized workflow for tissue sample preparation, measurement setup, and chemometric algorithms is essential to get reliable results.
Background
Panniculitis-like T-cell lymphoma is an uncommon type of non-Hodgkin lymphoma, occurring usually in the form of nodules within the subcutaneous fat tissue of the extremities or trunk. In the literature, subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is described as a distinct type of T-cell lymphoma with a variable clinical behavior, depending on molecular phenotype of T-cell receptor (TCR) and on the presence or absence of hemophagocytic syndrome.
Case presentation
We present a bioptic and autoptic case of a 65-years old Caucasian man with panniculitic T-cell lymphoma with morphological and immunohistochemical features of SPTCL, limited to the retroperitoneal and mesenteric mass, i.e. without any cutaneous involvement, and associated with severe hemophagocytic lymphohistiocytosis.
Conclusion
A panniculitic T-cell lymphoma with morphological and molecular features of SPTCL, which is limited to mesentery, i.e. does not involve subcutaneous fat, seems to be exceedingly rare.
Background: Primary cutaneous follicular B-cell lymphoma (PCFBCL) represents an indolent subtype of Non-Hodgkin’s lymphomas, being clinically characterized by slowly growing tumors of the skin and common cutaneous relapses, while only exhibiting a low propensity for systemic dissemination or fatal outcome. Up to now, only few studies have investigated underlying molecular alterations of PCFBCL with respect to somatic mutations. Objectives: Our aim was to gain deeper insight into the pathogenesis of PCFBCL and to delineate discriminatory molecular features of this lymphoma subtype. Methods: We performed hybridization-based panel sequencing of 40 lymphoma-associated genes of 10 cases of well-characterized PCFBCL. In addition, we included two further ambiguous cases of atypical B-cell-rich lymphoid infiltrate/B-cell lymphoma of the skin for which definite subtype attribution had not been possible by routine investigations. Results: In 10 out of 12 analyzed cases, we identified genetic alterations within 15 of the selected 40 target genes. The most frequently detected alterations in PCFBCL affected the TNFRSF14, CREBBP, STAT6 and TP53 genes. Our analysis unrevealed novel mutations of the BCL2 gene in PCFBCL. All patients exhibited an indolent clinical course. Both the included arbitrary cases of atypical B-cell-rich cutaneous infiltrates showed somatic mutations within the FAS gene. As these mutations have previously been designated as subtype-specific recurrent alterations in primary cutaneous marginal zone lymphoma (PCMZL), we finally favored the diagnosis of PCMZL in these two cases based on these molecular findings. Conclusions: To conclude, our molecular data support that PCFBCL shows distinct somatic mutations which may aid to differentiate PCFBCL from pseudo-lymphoma as well as from other indolent and aggressive cutaneous B-cell lymphomas. While the detected genetic alterations of PCFBCL did not turn out to harbor any prognostic value in our cohort, our molecular data may add adjunctive discriminatory features for diagnostic purposes on a molecular level.
Four molecules of the tumor suppressor p53 assemble to cooperatively bind proapoptotic target genes. The structural basis for cooperativity consists of interactions between adjacent DNA binding domains. Mutations at the interaction interface that compromise cooperativity were identified in cancer patients, suggesting a requirement of cooperativity for tumor suppression. We report on an analysis of cooperativity mutant p53(E177R) mice. Apoptotic functions of p53 triggered by DNA damage and oncogenes were abolished in these mice, whereas functions in cell-cycle control, senescence, metabolism, and antioxidant defense were retained and were sufficient to suppress development of spontaneous T cell lymphoma. Cooperativity mutant mice are nevertheless highly cancer prone and susceptible to different oncogene-induced tumors. Our data underscore the relevance of DNA binding cooperativity for p53-dependent apoptosis and tumor suppression and highlight cooperativity mutations as a class of p53 mutations that result in a selective loss of apoptotic functions due to an altered quaternary structure of the p53 tetramer.