66 research outputs found

    Analysis of "knockout, knockin" mice that express a functional Fas Ligand molecule lacking the intracellular domain

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    Fas Ligand (FasL; CD95L; CD178; TNSF6) is a 40 kDa glycosylated type II transmembrane protein with 279 aa in mice and 281 aa in humans that belongs to the tumor necrosis factor (TNF) family. The extracellular domain (ECD) harbors a TNF homology domain, the receptor binding site, a motif for self assembly and trimerization, and several putative N-glycosylation and a metalloprotease cleavage site/s. The cytoplasmic tail of FasL is the longest of all TNFL family members and contains several conserved signaling motifs, such as a putative tandem Casein kinase I phosphorylation site, a unique proline-rich domain (PRD) and phosphorylatable tyrosine residues (Y7 in mice; Y7, Y9, Y13 in human). The FasL/Fas system is renowned for the potent induction of apoptosis in the receptor-bearing cell and is especially important for immune system functions. It is involved in the killing of target cells by natural killer (NK) and cytotoxic T cells, in the (self) elimination of effector cells following the proliferative phase of an immune response (activation-induced cell death; AICD), in the maintenance of immuneprivileged sites and in the induction and maintenance of peripheral tolerance. Owing to its potent pro-apoptotic signaling capacity and important functions, FasL expression and activity are tightly regulated at transcriptional and posttranscriptional levels and restricted to few cell types, such as immune effector cells and cells of immune-privileged sites. In contrast, Fas is expressed in a variety of tissues including lymphoid tissues, liver, heart, kidney, pancreas, brain and ovary. In addition to its pro-apoptotic function, the FasL/Fas system can also elicit nonapoptotic signals in the receptor-expressing cell. Among others, Fas-signaling exerts co-stimulatory functions in the immune system, e.g. by promoting survival, activation and proliferation of T cells. Besides the capacity to deliver a signal into receptor-bearing cells (‘forward signal’), FasL can receive and transmit signals into the ligand-expressing cell. This phenomenon has been described for several TNF family ligands and is known as ‘reverse signaling’. The first evidence for the existence of reverse signaling into FasL-bearing cells stems from two studies that demonstrated either co-stimulation of murine CD8+ T cell lines by FasL cross-linking or inhibition of activation-induced proliferation of murine CD4+ T cells. In both cases, the observed changes of proliferative behaviour critically depended on the presence of a signaling-competent FasL. Almost certainly, the FasL ICD is functionally involved in signal-transmission: (i) The ICD is highly conserved across species and harbors several signaling motifs, most notably a unique PRD. (ii) Numerous proteins have been identified which interact with the FasL PRD via their SH3 or WW domains and regulate various aspects of FasL biology, such as FasL sorting, storage, cell surface expression and the linkage of FasL to intracellular signaling pathways. (iii) Post-translational modifications of the ICD have been implicated in the sorting of FasL to vesicles and the FasL-dependent activation of Nuclear factor of activated T cells (NFAT). (iv) Proteolytic processing of FasL liberates the ICD and allows its translocation into the nucleus where it might influence gene transcription. (v) It could be shown that overexpression of the FasL ICD is sufficient to initiate reverse signaling upon concomitant T cell receptor (TCR) stimulation and ICD cross-linking. Conflicting data on the consequences of FasL reverse signaling exist, and costimulatory as well as inhibitory functions have been reported. These discrepancies probably reflect the use of artificial experimental systems. Neither the precise molecular mechanism underlying FasL reverse signaling, nor its physiological relevance have been addressed at the endogenous protein level in vivo. Therefore, a ‘knockout/knockin’ mouse model in which wildtype FasL was replaced with a deletion mutant lacking the intracellular portion (FasL Delta Intra) was established in the group of PD Dr. Martin Zörnig. In the present study, FasL Delta Intra mice were phenotypically characterized and were employed to investigate the physiological consequences of FasL reverse signaling at the molecular and cellular level. To ensure that FasL Delta Intra mice represent a suitable model to study the consequences of FasL reverse signaling, we demonstrated that activated lymphocytes from homozygous FasL Delta Intra or wildtype mice express comparable amounts of (truncated) FasL at the cell surface. The truncated protein retains the capacity to induce apoptosis in Fas receptor-positive target cells, as co-culture assays with FasL-expressing activated lymphocytes and Fas-sensitive target cells showed. Additionally, systematic screening of unchallenged mice did not reveal any phenotypic abnormalities. Notably, signs of a lymphoproliferative autoimmune disease associated with FasL-deficiency could not be detected. As several reports have implicated FasL reverse signaling in the regulation of T cell expansion and activation, proliferation of lymphocytes isolated from FasL Delta Intra and wildtype mice in response to antigen receptor stimulation was investigated. Using CFSE dilution assays it could be demonstrated that the proliferative response of CD4+ T cells, CD8+ T cells and of B cells was enhanced in the absence of the FasL ICD. Interestingly, this effect was most pronounced in B cells and could only be detected in CD4+ T cells after depletion of CD4+CD25+ regulatory T cells. To our Summary knowledge, this is the first time that FasL reverse signaling has been demonstrated in B cells. In a series of experiments, the activation of several pathways that are known to play important roles in signal-transmission initiated upon antigen receptor triggering was assessed. As a molecular correlate for the observed enhancement of activation-induced proliferation, Extracellular signal regulated kinase (ERK1/2) phosphorylation was significantly increased in FasL Delta Intra mice following antigen receptor crosslinking. Surprisingly, B cell stimulation lead to a comparable extent of activating phosphorylations on S38 in c-Raf and S218/S222 in MEK1/2 in cells isolated from wildtype and FasL Delta Intra mice, indicating that Mitogen activated protein kinases (MAPKs) upstream of ERK1/2 (Raf-1 and MEK1/2) apparently do not contribute to the differential regulation of ERK1/2. Experiments in which activation-induced Akt phosphorylation (S473) was quantified also did not suggest a participation of Phosphoinositol specific kinase 3 (PI3K)/Akt signals in this process. Instead, further characterization of the upstream pathway revealed an involvement of Phospholipase C gamma (PLC gamma) and Protein kinase C (PKC) signals in FasL-dependent ERK1/2- regulation. Previous studies in our group revealed a Notch-like processing of FasL, resulting in the transcriptional regulation of a reporter gene. Furthermore, an interaction of the FasL ICD with the transcription factor Lymphoid-enhancer binding factor-1 (Lef-1) that affected Lef-1-dependent reporter gene transcription could be demonstrated. Therefore, a molecular analysis of activated lymphocytes was performed to identify FasL reverse signaling target genes. The differential expression of promising candidates was verified by quantitative real-time PCR (qRT-PCR), which showed that the transcription of genes associated with lymphocyte proliferation and activation was increased in FasL Delta Intra mice compared to wildtype mice. Interestingly, an extensive regulation of Lef-1-dependent Wnt/beta-Catenin signalingrelated genes was found. Lef-1 mRNA (RT-PCR) and protein (intracellular FACS staining) could be detected in mature B cells, suggesting the possibility of FasL ICD-mediated inhibition of Lef-1-dependent gene expression in these cells, initiated by Notch-like processing of FasL. To investigate the consequences of FasL reverse signaling in vivo, a potential participation of the FasL ICD in the regulation of immune responses upon various challenges was analyzed. In experiments in which thymocyte proliferation or the expansion of antigen-specific T cells following a challenge with the superantigen Staphylococcus enterotoxin B (SEB), with Lymphocytic choriomeningitis virus (LCMV) or with Listeria monocytogenes were investigated, comparable results were obtained with wildtype and FasL Delta Intra mice. Likewise, the recruitment of neutrophils in a thioglycollate-induced model of peritonitis was not affected by deletion of the FasL ICD. These findings might reflect regulatory mechanisms operating in vivo, such as control exerted by regulatory T cells. Along these lines, proliferative differences in CD4+ T cells could only be detected ex vivo after depletion of CD4+CD25+ regulatory T cells. Furthermore, several in vitro studies indicate that retrograde FasL signals can be observed under conditions of suboptimal lymphocyte stimulation, but not when the TCR is optimally stimulated. Therefore, the potent initiation of antigen receptor signaling by stimuli like SEB or LCMV might have masked inhibitory FasL reverse signaling in these experiments. In agreement with the observed hyperactivation of lymphocytes in the absence of the ICD ex vivo, the increase in germinal center B cells (GCs) following immunization with the hapten 3-hydroxy 4-nitrophenylacetyl (NP) and the number of antibody-secreting PCs was significantly higher in FasL Delta Intra mice. The larger quantity of PCs correlated with increased titers of NP-binding, i.e. antigen-specific, IgM and IgG1 antibodies in the serum of FasL Delta Intra mice after immunization. These data suggest that FasL reverse signaling exerts immunmodulatory functions. Supporting this notion, a model of Ovalbumin-induced allergic airway inflammation revealed an involvement of retrograde FasL-signals in the recruitment of immune effector cells into the lung and in the activation of T cells following exposure of mice to Ovalbumin. Together, our ex vivo and in vivo findings based on endogenous FasL protein levels demonstrate that FasL ICD-mediated reverse signaling is a negative modulator of certain immune responses. It is tempting to speculate that FasL reverse signaling might be a fine-tuning mechanism to prevent autoimmune diseases, a theory which will be tested in adequate mouse models in the future.Der Fas Ligand (FasL; CD95L; CD178; TNSF6) ist ein glykosyliertes Typ II Transmembranprotein, das zur Proteinfamilie der Tumornekrosefaktor (TNF)-Liganden gehört. Das Mausprotein besteht aus 279 AminosĂ€uren, wĂ€hrend der humane FasL 281 AminosĂ€uren umfasst. Im extrazellulĂ€ren Teil des Liganden befinden sich eine TNF Homologie-DomĂ€ne, die Rezeptorbindestelle, ein Sequenzmotiv zur Selbstaggregation und Trimerisierung sowie mehrere Stellen, an denen der FasL N-glykosyliert oder von Metalloproteasen geschnitten werden kann. Die intrazellulĂ€re Region des FasL ist die lĂ€ngste aller TNFL-Familienmitglieder und enthĂ€lt verschiedene konservierte Signalmotive, z.B. ein Casein-Kinase I (CK-I) Substrat-Motiv, eine Prolin-reiche DomĂ€ne (PRD) und potentielle Tyrosin-Phosphorylierungsstellen (Maus: Y7; Mensch: Y7, Y9, Y13). Die Bindung des FasL an den zugehörigen Rezeptor Fas löst in der rezeptortragenden Zelle apoptotischen Zelltod aus. Dies spielt eine besondere Rolle fĂŒr verschiedenen Funktionen des Immunsystems, z.B. fĂŒr die Lyse von Zielzellen durch natĂŒrliche Killerzellen (NK-Zellen) und zytotoxische T-Zellen, fĂŒr die Selbsteliminierung von Effektorzellen nach der Expansionsphase einer Immunantwort (activation-induced cell death; AICD), fĂŒr die Aufrechterhaltung eines immunprivilegierten Status bestimmter Gewebe und fĂŒr die Induktion und Erhaltung der peripheren Toleranz. Im Gegensatz zu dem von vielen Zelltypen konstitutiv exprimiertem Fas-Rezeptor ist die Expression und AktivitĂ€t des FasL auf wenige Zelltypen beschrĂ€nkt (Zellen des Immunsystems und immunprivilegierte Gewebe) und wird auf transkriptioneller und post-translationaler Ebene reguliert. Ebenso wichtig wie die pro-apoptotische Funktion ist die Vermittlung nichtapoptotischer Signale durch den Fas-Rezeptor. So wurde unter anderem gezeigt, dass Fas-Aktivierung ko-stimulatorisch wirken und das Überleben, die Aktivierung und die Proliferation von T-Zellen und Tumorzellen fördern kann. ZusĂ€tzlich zu der Vermittlung des klassischen „VorwĂ€rtssignals“ in die rezeptortragende Zelle kann FasL selbst Signale empfangen und in die ligandexprimierende Zelle weiterleiten. Dieses PhĂ€nomen wird als „reverse“ oder „bidirektionale“ SignalĂŒbertragung bezeichnet und wurde ebenfalls fĂŒr andere TNFL-Familienmitglieder beschrieben. Erste Hinweise fĂŒr die Existenz einer reversen SignalĂŒbertragung in die FasL-tragende Zelle stammen aus zwei Studien, in denen gezeigt wurde, dass die Vernetzung des FasL durch Fas-Fc oder anti-FasL-Antikörper die T-Zell-Rezeptor (TZR)-vermittelte Proliferation muriner CD8+ T-Zelllinien ko-stimuliert bzw. die muriner CD4+ T-Zellen inhibiert. In beiden FĂ€llen hingen die beobachteten VerĂ€nderungen des Proliferationsverhaltens von der Anwesenheit eines funktionellen Liganden ab. Es ist anzunehmen, dass das retrograde Signal durch die intrazellulĂ€re DomĂ€ne (IZD) des FasL vermittelt wird: (i) Die IZD ist speziesĂŒbergreifend konserviert und beinhaltet mehrere Signalmotive, darunter eine innerhalb der TNFL-Familie einzigartige PRD. (ii) Eine Vielzahl von Proteinen bindet ĂŒber eine SH3 oder WW DomĂ€ne an die PRD des FasL und reguliert so verschiedene Aspekte der FasL-Biologie, wie z.B. die subzellulĂ€re Lokalisation, die Expression an der ZelloberflĂ€che und das Zusammenspiel mit intrazellulĂ€ren Signalwegen. (iii) Post-translationale Modifikationen der IZD wurden mit der Sortierung des Liganden in Vesikel und der FasL-abhĂ€ngigen Aktivierung von Nuclear factor of activated T cells (NFAT) in Verbindung gebracht. (iv) Die proteolytische Prozessierung des FasL fĂŒhrt zur Freisetzung der IZD in das Zytosol und ermöglicht die Translokation der IZD in den Zellkern, wo sie die Transkription von Genen regulieren kann. (v) Es konnte gezeigt werden, dass die Überexpression der FasL IZD ausreicht, um nach gleichzeitiger TZR-Stimulation und IZD-Vernetzung eine reverse SignalĂŒbertragung in murinen CD8+ T-Zelllinien zu initiieren. Die möglichen Konsequenzen der FasL-vermittelten reversen SignalĂŒbertragung werden kontrovers diskutiert, und sowohl ko-stimulatorische wie auch inhibitorische Funktionen sind publiziert worden. Diese sich widersprechenden Beobachtungen basieren vermutlich darauf, dass die Experimente in artifiziellen Systemen durchgefĂŒhrt wurden und weder der molekulare Mechanismus, noch die physiologische Bedeutung der reversen SignalĂŒbertragung auf der Ebene des endogenen Proteins in vivo untersucht wurden. Daher wurde in der Arbeitsgruppe von PD Dr. Martin Zörnig ein Mausmodell fĂŒr eine defekte reverse FasL SignalĂŒbertragung (FasL Delta Intra) etabliert. Diesen ‚knockout/knockin’-MĂ€usen fehlt die intrazellulĂ€re FasL DomĂ€ne, wĂ€hrend die Transmembran- und die extrazellulĂ€re DomĂ€ne weiterhin vorhanden sind. Im Verlauf der vorliegenden Arbeit wurden die FasL Delta Intra MĂ€use phĂ€notypisch charakterisiert und als Modell genutzt, um die physiologische Funktion der reversen FasL SignalĂŒbertragung auf molekularer und zellulĂ€rer Ebene aufzuklĂ€ren. Um sicherzustellen, dass die FasL Delta Intra MĂ€use ein geeignetes Modellsystem zur Analyse der reversen FasL SignalĂŒbertragung darstellen, wurden die Expression und FunktionalitĂ€t des trunkierten FasL untersucht. Die korrekte AusfĂŒhrung FasL/Fas-vermittelter Apoptose ist ein wichtiger Punkt, da natĂŒrlich vorkommende Maus-MutantenstĂ€mme, in denen die Fas-induzierte Apoptose durch Mutationen im FasL (FasLgld/gld) bzw. im Fas (Faslpr/lpr) Gen inaktiviert wird, eine schwere Autoimmunerkrankung entwickeln. Ein vergleichbarer PhĂ€notyp in FasL Delta Intra MĂ€usen könnte die Auswirkungen der defekten reversen SignalĂŒbertragung maskieren. Die Expression des FasL konnte durch RT-PCR und durchflußzytometrische FĂ€rbungen auf der OberflĂ€che aktivierter Lymphozyten in homozygoten FasL Delta Intra MĂ€usen verifiziert werden. Experimente, in denen aktivierte Lymphozyten mit Fas-sensitiven Zielzellen ko-kultiviert wurden, zeigten, dass der trunkierte FasL weiterhin Apoptose in Fas-tragenden Zellen auslösen kann. Außerdem weisen die FasL Delta Intra MĂ€use im gesunden Zustand keinerlei phĂ€notypische AuffĂ€lligkeiten auf und entwickeln keine Symptome der mit FasL-Defizienz (FasL gld/gld, FasL-/-) verbundenen lymphoproliferativen Autoimmunerkrankung. Da die meisten Studien eine Beeinflussung der T-Zell Expansion durch die reverse FasL SignalĂŒbertragung nahe legen, wurde das Proliferationsverhalten von Lymphozyten aus homozygoten FasL Delta Intra oder Wildtyp MĂ€usen nach Stimulation des jeweiligen Antigenrezeptors analysiert. Experimente zur aktivierungsinduzierten Lymphozytenexpansion belegten eine deutlich höhere ProliferationskapazitĂ€t von einzelpositiven CD4+ und CD8+ T-Zellen sowie B-Zellen ohne IZD. Dieser Effekt war in B-Zellen am stĂ€rksten ausgeprĂ€gt und konnte in CD4+ T-Zellen nur dann beobachtet werden, wenn zuvor regulatorische CD4+CD25+ T-Zellen (Tregs) depletiert wurden. Nach unserem Kenntnisstand beschreibt die vorliegende Arbeit zum ersten Mal die Auswirkungen der reverse FasL SignalĂŒbertragung in B-Zellen. Um zu verstehen, wie reverses FasL-Signaling auf aktivierungsinduzierte Lymphozytenproliferation einwirkt, wurde das Anschalten verschiedener Signalwege, die bekanntermaßen fĂŒr die vom Antigenrezeptor ausgehende Signalverarbeitung wichtig sind, untersucht. Als ein molekulares Korrelat fĂŒr die beobachtete verstĂ€rkte Proliferation wurde eine deutlich erhöhte Phosphorylierung der Extracellular signal regulated kinase (ERK1/2) nach Vernetzung des Antigerezeptors in homozygoten FasL Delta Intra MĂ€usen gefunden. Überraschenderweise schienen die ERK1/2-vorgeschalteten Mitogen-aktivierten Proteinkinasen (MAPKs) Raf-1 und MEK1/2 nicht fĂŒr die differentielle Regulation von ERK1/2 verantwortlich zu sein, da die Stimulation von Wildtyp und FasL-mutanten B-Zellen zu einem vergleichbaren Ausmaß aktivierender Phosphorylierungen in c-Raf1 (S38) und MEK1/2 (S218/S222) fĂŒhrte. Ebenso wiesen Experimente, in denen die aktivierungsinduzierte Akt-Phosphorylierung (S473) untersucht wurde, nicht auf eine Beteiligung des Phosphoinositol-spezifische Kinase 3 (PI3K)/Akt Signalwegs an der reversen FasL SignalĂŒbertragung hin. Es konnte jedoch gezeigt werden, dass durch Phospholipase C gamma (PLC gamma) und Proteinkinase C (PKC) vermittelte Signale in die FasL-abhĂ€ngige Regulation von ERK1/2 involviert sind. Unsere Gruppe konnte zeigen, dass FasL in einer Ă€hnlichen Weise wie das Signalprotein Notch prozessiert wird. Dies fĂŒhrt nach proteolytischer Freisetzung der FasL IZD in das Zytoplasma zur transkriptionellen Regulation eines Reportergens. Des Weiteren wurde in der Arbeitsgruppe von PD Dr. M. Zörnig eine Interaktion der FasL IZD mit dem Transkriptionsfaktor Lymphoid-enhancer binding factor-1 (Lef-1) aufgezeigt, die die Lef-1-abhĂ€ngige Transkription beeinflusste. Daher wurde im Rahmen dieser Doktorarbeit eine systematische Expressionsanalyse aktivierter Lymphozyten durchgefĂŒhrt. Die dabei identifizierten Zielgene der reversen FasL SignalĂŒbertragung wurden durch quantitative real-time PCR (qRT-PCR) verifiziert. Es stellte sich heraus, dass vor allem Gene, die mit der Proliferation und Aktivierung von Lymphozyten assoziiert werden, in homozygoten FasL Delta Intra MĂ€usen stĂ€rker exprimiert werden. Interessanterweise wurde ebenfalls eine extensive Regulation von Genen, die mit Lef-1-abhĂ€ngigen Wnt/beta-Catenin-Signalen im Zusammenhang stehen, beobachtet. Obwohl andere Publikationen nahe legen, dass die Expression von Lef-1 wĂ€hrend der B-Zell-Entwicklung stark herunterreguliert wird, konnten in der vorliegenden Arbeit Lef-1 mRNA (RT-PCR) und Protein (intrazellulĂ€re, durchflußzytometrische FĂ€rbung) in naĂŻven B-Zellen detektiert werden. Diese Daten weisen auf einen Zusammenhang von reverser FasL SignalĂŒbertragung und Lef-1 in reifen Lymphozyten hin. Zur AufklĂ€rung der in vivo Funktion reverser FasL SignalĂŒbertragung wurden verschiedene Infektions- und Immunisierungsmodelle angewendet, mit denen eine mögliche Beteiligung der FasL IZD an der Regulation von Immunantworten untersucht werden kann. Experimente, in denen die Thymozytenproliferation oder die Expansion Antigen-spezifischer T-Zellen in Folge einer Infektion mit dem Superantigen Staphylococcus enterotoxin B (SEB), mit dem Lymphozyten Choriomeningitis Virus (LCMV) oder dem intrazellulĂ€ren Bakterium Listeria monocytogenes evaluiert wurden, ergaben vergleichbare Ergebnisse in Wildtyp und FasL Delta Intra MĂ€usen. Auch die Rekrutierung von Neutrophilen in einem Thioglycollat-induzierten Peritonitismodell wurde nicht von der Deletion der FasL IZD beeinflußt. Möglicherweise spiegeln diese Befunde in vivo aktive, regulatorische Mechanismen wider, wie z.B. eine durch regulatorische T-Zellen ausgeĂŒbte Kontrolle. Ein Hinweis darauf ist die ex vivo Beobachtung, dass Unterschiede in der Proliferation von Wildtyp und mutanten CD4+ T-Zellen nur beobachtet werden konnten, wenn regulatorische T-Zellen vorher depletiert wurden. Außerdem zeigen verschiedene in vitro Studien, dass retrograde FasL-Signale nur bei sub-optimaler Lymphozyten-Stimulation beobachtet werden können, nicht aber bei optimaler Vernetzung des TZR. Daher könnten Stimuli wie SEB oder LCMV durch die starke Initiation von Antigenrezeptorsignalen die inhibitorische Funktion der reverse FasL Signaltransduktion maskieren. Im Einklang mit der ex vivo beobachteten Hyperaktivierung von Lymphozyten ohne FasL IZD wurden in FasL Delta

    Analysis of knockout/knockin mice that express a mutant FasL lacking the intracellular domain

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    Fas ligand (FasL; CD178; CD95L) is a type II transmembrane protein belonging to the tumour necrosis factor family; its binding to the Fas receptor (CD95; APO-1) triggers apoptosis in the receptor-bearing cell. Signalling through this pathway plays a pivotal role during the immune response and in immune system homeostasis. Similar to other TNF family members, the intracellular domain has been reported to transmit signals to the inside of the FasL-bearing cell (reverse signalling). Recently, we identified the proteases ADAM10 and SPPL2a as molecules important for the processing of FasL. Protease cleavage releases the intracellular domain, which then is able to translocate to the nucleus and to repress reporter gene activity. To study the physiological importance of FasL reverse signalling in vivo, we established knockout/knockin mice with a FasL deletion mutant that lacks the intracellular portion (FasLDeltaIntra). Co-culture experiments confirmed that the truncated FasL protein is still capable of inducing apoptosis in Fas-sensitive cells. Preliminary immune histochemistry data suggest that, in contrast to published data, the absence of the intracellular FasL domain does not alter the intracellular FasL localization in activated T cells. We are currently investigating signalling and proliferative capacities of T cells derived from homozygous FasLDeltaIntra mice to validate a co-stimulatory role of FasL reverse signalling

    11C-Methionine-PET in multiple myeloma: a combined study from two different institutions

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    11^{11}C-methionine (MET) has recently emerged as an accurate marker of tumor burden and disease activity in patients with multiple myeloma (MM). This dual-center study aimed at further corroboration of the superiority of MET as positron emission tomography (PET) tracer for staging and re-staging MM, as compared to 18^{18}F-2`-deoxy-2`-fluoro-D-glucose (FDG). 78 patients with a history of solitary plasmacytoma (n=4), smoldering MM (SMM, n=5), and symptomatic MM (n=69) underwent both MET- and FDG-PET/computed tomography (CT) at the University Centers of WĂŒrzburg, Germany and Navarra, Spain. Scans were compared on a patient and on a lesion basis. Inter-reader agreement was also evaluated. In 2 patients, tumor biopsies for verification of discordant imaging results were available. MET-PET detected focal lesions (FL) in 59/78 subjects (75.6%), whereas FDG-PET/CT showed lesions in only 47 patients (60.3%; p<0.01), accordingly disease activity would have been missed in 12 patients. Directed biopsies of discordant results confirmed MET-PET/CT results in both cases. MET depicted more FL in 44 patients (56.4%; p<0.01), whereas in two patients (2/78), FDG proved superior. In the remainder (41.0%, 32/78), both tracers yielded comparable results. Inter-reader agreement for MET was higher than for FDG (Îș = 0.82 vs Îș = 0.72). This study demonstrates higher sensitivity of MET in comparison to standard FDG to detect intra- and extramedullary MM including histologic evidence of FDG-negative, viable disease exclusively detectable by MET-PET/CT. MET holds the potential to replace FDG as functional imaging standard for staging and re-staging of MM

    FDG PET/CT depicts cutaneous plasmocytoma

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    Targeting Paraprotein Biosynthesis for Non-Invasive Characterization of Myeloma Biology

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    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

    Influence of the amount of co-infused amino acids on post-therapeutic potassium levels in peptide receptor radionuclide therapy

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    Background Peptide receptor radionuclide therapy (PRRT) is routinely used for advanced or metastasized neuroendocrine tumours (NET). To prevent nephrotoxicity, positively charged amino acids (AA) are co-infused. The aim of this study was to correlate the risk for therapy-related hyperkalaemia with the total amount of AA infused. Methods Twenty-two patients undergoing PRRT with standard activities of 177Lu-DOTATATE/-TOC were monitored during two following treatment cycles with co-infusion of 75 and 50 g of AA (L-arginine and L-lysine), respectively. Mean serum levels of potassium and other parameters (glomerular filtration rate [GFR], creatinine, blood urea nitrogen [BUN], phosphate, chloride, lactate dehydrogenase) prior to, 4 h and 24 h after AA infusion were compared. Results Self-limiting hyperkalaemia (>5.0 mmol/l) resolving after 24 h occurred in 91% (20/22) of patients in both protocols. Potassium levels, BUN, creatinine, GFR, phosphate, chloride and LDH showed a similar range at 4 h after co-infusion of 75 or 50 g of AA, respectively (p > 0.05). Only GFR and creatinine levels at 24 h varied significantly between the two co-infusion protocols (p < 0.05). Conclusions Hyperkalaemia is a frequent side effect of AA infusion in PRRT. Varying the dose of co-infused amino acids did not impact on the incidence and severity of hyperkalaemia

    18FDG-PET/CT for prognostic stratification of patients with multiple myeloma relapse after stem cell transplantation

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    The aim of this study was to investigate the prognostic value of 18F-fluoro-deoxyglucose positron emission tomography–computed tomography (18F-FDG-PET/CT) in 37 patients with a history of multiple myeloma (MM) and suspected or confirmed recurrence after stem cell transplantation (SCT). All patients had been heavily pre-treated. Time to progression (TTP) and overall survival (OS) were correlated to a number of different PET-derived as well as clinical parameters. Impact on patient management was assessed. Absence of FDG-avid MM foci was a positive prognostic factor for both TTP and OS (p10 focal lesions correlated with both TTP (p10 lesions in the appendicular skeleton proved to have the strongest association with disease progression. Intensity of glucose uptake and presence of extramedullary disease were associated with shorter TTP (p=0.037 and p=0.049, respectively). Manifestations in soft tissue structures turned out to be a strong negative predictor for both, TTP and OS (p<0.01, respectively). PET resulted in a change of management in 30% of patients. Our data underline the prognostic value of 18F-FDG-PET/CT in MM patients also in the setting of post-SCT relapse. PET/CT has a significant impact on patient management
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