610 Medizin und Gesundheit
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Schriftenreihe
Sonstige beteiligte Institutionen
- Johns Hopkins School of Medicine (18)
- IZKF Nachwuchsgruppe Geweberegeneration für muskuloskelettale Erkrankungen (7)
- Clinical Trial Center (CTC) / Zentrale für Klinische Studien Würzburg (ZKSW) (5)
- Johns Hopkins University School of Medicine (5)
- Bernhard-Heine-Centrum für Bewegungsforschung (4)
- Johns Hopkins School of Medicine, Baltimore, MD, U.S. (4)
- Klinikum Fulda (3)
- Zentraleinheit Klinische Massenspektrometrie (3)
- CHC Würzburg (Comprehensive Hearing Center) (2)
- Center for Interdisciplinary Clinical Research, Würzburg University, Würzburg, Germany (2)
ResearcherID
- D-1221-2009 (1)
Gen-Umwelt-Interaktionen haben einen wichtigen Stellenwert für das Verständnis der Entstehung psychiatrischer Erkrankungen. Für die Catechol-O-Methyltransferase (COMT)konnte kürzlich gezeigt werden, dass diese die Gehirnaktivität moduliert, während der Verarberitung negativer Stimuli. Für positive Stimuli konnte kein Effekt nachgewiesen werden. In der vorliegenden Arbeit sollte nun geprüft werden, ob Lebensereignisse, als ein Umweltfaktor, für die emotionale Verarbeitung eine Rolle spielen. Um das herauszufinden untersuchten wir 81 gesunde Probanden mittels EEG während der Darbietung positiver und negativer emotionaler Bilder. Wie erwartet moduliert COMT die EPN (early posterior negativity) für negative Bilder, aber nicht für positive. Unter Berücksichtigung der Lebensereignisse konnte der fehlende Effekt der COMT bei der positiven Bedingung aufgelöst werden. Eine hohe Lebensereignis-Last führt dabei zu einer verminderten Gehirnaktivität für positive Stimuli, was sich aber nur für den Met/Met-Genotyp zeigt. Relevant scheint das vor allem für die Entwicklung von Depressionen zu sein, da depressive Patienten häufig ihre Umwelt als weniger positiv bewerten.
In der vorliegenden Arbeit wurden die Mechanismen der Hochdosis-GC-Pulstherapie im Zusammenhang mit akuten Schüben von MS-Patienten anhand des Tiermodells der MS, der Experimentellen Autoimmunen Enzephalomyelitis (EAE), untersucht. Die EAE wurde in C57Bl/6 Mäusen und diversen GR-defizienten Mäusen durch Immunisierung mit Myelinoligodendrozytenglykoprotein (MOG35-55) induziert. Es konnte gezeigt werden, dass die Gabe von Dexamethason (Dex) den Krankheitsverlauf dosisabhängig verbessert. Die Untersuchung heterozygoter GR Knock-out Mäuse und hämatopoetischer Stammzellchimären verdeutlichte, dass der zytosolische GR (cGR) für die Vermittlung therapeutischer GC-Effekte von sehr großer Bedeutung ist. Der Einsatz zelltyp-spezifischer GR-defizienter Mäuse zeigte auf zellulärer Ebene, dass für die Vermittlung von GC-Wirkungen die Expression des GR vor allem in T-Zellen unabdingbar ist, wohingegen die GR-Expression in myeloiden Zellen in diesem Kontext keine Bedeutung hat. Durch die Analyse des molekularen Mechanismus konnte festgestellt werden, dass diese Effekte durch Apoptoseinduktion und Herunterregulieren von Adhäsionsmolekülen in peripheren, aber nicht ZNS-residenten T-Zellen erzielt wurden. Überdies wurde ersichtlich, dass Dex die T-Zellmigration in das ZNS verhinderte. Diese Beobachtung unterstützt die Hypothese, dass Dex durch Apoptoseinduktion und Immunmodulation hauptsächlich auf periphere T-Zellen wirkt und somit den ständigen Influx neuer Immunzellen in das ZNS verhindert. Ferner konnte in dieser Arbeit gezeigt werden, dass die therapeutische Gabe hochdosierten Methylprednisolons (MP) in diesem EAE-Modell ebenfalls zu einer dosisabhängigen Verbesserung der EAE führte. Diese beruhte auf einer reduzierten Lymphozyteninfiltration in das ZNS, war allerdings im Vergleich zur Dex-Therapie aufgrund geringerer Wirkpotenz weniger stark ausgeprägt. Im Gegensatz dazu führte die präventive MP-Applikation zu einem verstärkten EAE-Verlauf, der nach der Beeinflussung peripherer, hämatopoetischer Immunzellen auf eine verstärkte Proliferation autoreaktiver T-Zellen zurückzuführen ist. Im weiteren Verlauf der vorliegenden Arbeit wurde als möglicher Ersatz für die Hochdosis-GC-Pulstherapie eine nicht-steroidale, antiinflammatorische Substanz im chronischen EAE-Modell der C57Bl/6 Maus etabliert. Erste tierexperimentelle Untersuchungen mit Compound A (CpdA) offenbarten eine lediglich geringe therapeutische Breite dieser Substanz, wobei innerhalb pharmakologischer Dosierungen dennoch therapeutische Wirkungen vermittelt werden konnten. Anhand von in vitro Experimenten konnte eindeutig nachgewiesen werden, dass CpdA GR-unabhängig Apoptose induzierte, wobei Immunzellen und neuronale Zellen gegenüber CpdA besonders empfindlich reagierten. Der Einsatz T-Zell-spezifischer GR-defizienter Mäuse konnte zeigen, dass CpdA für die Vermittlung therapeutischer Wirkungen den cGR benötigt. Ferner wurde offensichtlich, dass CpdA in Abwesenheit des cGR in T-Zellen eine signifikante Verschlechterung der EAE verursachte. Durch die Anwendung physikochemischer Analysenmethoden, wie der Massenspektrometrie und 1H-NMR-Spektroskopie, konnte festgestellt werden, dass CpdA in vitro in gepufferten Medien in eine zyklische, chemisch sehr reaktive Verbindung (Aziridin) metabolisiert wird. Diese kann sehr wahrscheinlich für die Apoptose-Induktion in Zellen und die in Mäusen beobachteten neurotoxischen Ausfallerscheinungen verantwortlich gemacht werden. Durch chemische Analysen konnte in vitro in wässriger CpdA-Lösung ein weiterer Metabolit, das sympathomimetisch wirksame Synephrin, identifiziert werden. Um die Wirksamkeit adrenerger Substanzen in vivo zu testen, wurde das ß1/2-Sympathomimetikum Isoproterenol appliziert. Dieses verbesserte die EAE-Symptomatik, was sehr wahrscheinlich auf eine reduzierte Antigenpräsentation und einer damit verbundenen verminderten T-Zellinfiltration in das ZNS zurückzuführen ist.
SUMOylation, as a post-translational modification, plays a crucial role in several biological processes. Small ubiquitin-like modifier (SUMO) proteins can be reversibly linked to the lysine residues located within specific motifs on numerous target proteins, leading to the change of stability, localization, activity of target proteins, mostly by promoting or interfering with the interaction with other molecules. Consequently, it can regulate gene transcription, migration, cell cycle progression, cellular responses to stress, and tumorigenesis.
NFATc1 belongs to the Nuclear Factor of Activated T-cells (NFAT) transcription factor family, which is dephosphorylated and translocates to the nucleus upon cell stimulation, which provokes Ca2+ signalling. NFAT plays a crucial role in the development and function of the immune system. NFATc1 has three SUMOylation sites at the position of aa 349, 702, and 914. In our previous study, we demonstrated that point mutations performed on the SUMOylation sites on all three or only at the lysine residues K702 and K914 lead to enhanced expression of IL-2 in vitro. To evaluate the function of SUMOylation of NFATc1 on T cell-mediated immunity in vivo, we not only generated a transgenic mouse strain (NFATc1/ΔS+ mouse) by point mutations from Lysine to Arginine on the two SUMOylation sites within exon 10 of Nfatc1 to prevent their SUMOylation, but in combination created another mouse strain (NFATc1/ΔBC+ mouse) that is completely Nfatc1 exon 10-ablated by using the LoxP/Cre system. In NFATc1/ΔS+ T cells, we observed enhanced IL-2 production and less IL-17A and IFN-γ expression. In line with exon 10 bearing the relevant SUMO sites, NFATc1/ΔBC+ CD4+ T cells behaved similarly as NFATc1/ΔS+ ones. The mechanism is that elevated IL-2 secretion can counteract the expression of IL-17A and IFN-γ via STAT5 and Blimp-1 induction. Afterwards, Blimp-1 suppressed IL-2 itself as well as Bcl2A1. Next, we performed two disease models with our NFATc1/ΔS+ mice. In a major mismatch model for acute graft-versus-host disease, we found that the mice transplanted with NFATc1/ΔS+ CD3+ T cells developed less severe disease, and T cells proliferated less due to increased Tregs. Moreover, when transferring 2D2.NFATc1/ΔS+ Th1 plus Th17 cells to Rag1-/- mice to induce experimental autoimmune encephalitis, we also observed ameliorated disease compared to animals with transferred WT T cells as well as increased Tregs.
Taking all data together, the deficiency in SUMOylation of NFATc1 leads to an elevated IL-2 secretion in T cells and subsequent activation of STAT5, which competes with STAT3 to inhibit IL-17A production and promotes Treg expansion, as well as to an enforcement of Blimp-1 expression, which suppresses IFN-γ and IL-2 expression. Consequently and despite a short phase of enhanced IL-2 secretion, the deficiency of SUMOylation on NFATc1 can protect from autoreactive and alloreactive diseases.
Moreover, to further understand the function of SUMOylation of NFATc1 in humans, we started by establishing an in vitro 3D culture system for tonsil organoids, which was successful in the presence of feeder cells, along with IL-4 and IL-7 cytokines. To confirm that our 3D tonsil organoids can respond to real antigens, we used CMV peptides and peptides of spike proteins from Covid-19 as real antigens, and co-cultured with tonsil organoids, which indeed can generate memory cells and plasmablasts. In the end, we also compared 3D to 2D cultures. Although the total numbers of all B cell subsets were much less in 3D culture than that in 2D culture, still, it indicates that this in-vitro culture system has its limitation, while being usable to produce the similar results as 2D did. Therefore, this 3D culture system can be used as a platform to investigate NFATc1/ΔS+ or NFATc1/ΔBC+ TFH and TFR cells in the dynamic of human GC responses.
The transcriptional repressor-Blimp-1 terminates differentiation of B lymphocytes as well as myeloid cells. Our data show that Blimp-1 is highly expressed in freshly isolated murine primary T lymphocytes, particularly its minor splice variant. Ectopic expression of Blimp-1 by retroviral transduction neither dramatically altered secretion of IFN-ã or IL-4 nor did it induce the ability to suppress as regulatory T cells. However, induction of Blimp-1 resulted in not only a significant reduction in the production of IL-2 but also an inability to proliferate as well as in the reduced viability. These results demonstrate that Blimp-1 might mark end stages of lineage differentiation in T cells.
This study investigated the role of bottom-up and top-down neural mechanisms in the processing of emotional face expression during memory formation. Functional brain imaging data was acquired during incidental learning of positive ("happy"), neutral and negative ("angry" or "fearful") faces. Dynamic Causal Modeling (DCM) was applied on the functional magnetic resonance imaging (fMRI) data to characterize effective connectivity within a brain network involving face perception (inferior occipital gyrus and fusiform gyrus) and successful memory formation related areas (hippocampus, superior parietal lobule, amygdala, and orbitofrontal cortex). The bottom-up models assumed processing of emotional face expression along feed forward pathways to the orbitofrontal cortex. The top-down models assumed that the orbitofrontal cortex processed emotional valence and mediated connections to the hippocampus. A subsequent recognition memory test showed an effect of negative emotion on the response bias, but not on memory performance. Our DCM findings showed that the bottom-up model family of effective connectivity best explained the data across all subjects and specified that emotion affected most bottom-up connections to the orbitofrontal cortex, especially from the occipital visual cortex and superior parietal lobule. Of those pathways to the orbitofrontal cortex the connection from the inferior occipital gyrus correlated with memory performance independently of valence. We suggest that bottom-up neural mechanisms support effects of emotional face expression and memory formation in a parallel and partially overlapping fashion.
Despite promising clinical results in osteochondral defect repair, a recently developed bi-layered collagen/collagen-magnesium-hydroxyapatite scaffold has demonstrated less optimal subchondral bone repair. This study aimed to improve the bone repair potential of this scaffold by adsorbing bone morphogenetic protein 2 (BMP-2) and/or platelet-derived growth factor-BB (PDGF-BB) onto said scaffold. The in vitro release kinetics of BMP-2/PDGF-BB demonstrated that PDGF-BB was burst released from the collagen-only layer, whereas BMP-2 was largely retained in both layers. Cell ingrowth was enhanced by BMP-2/PDFG-BB in a bovine osteochondral defect ex vivo model. In an in vivo semi-orthotopic athymic mouse model, adding BMP-2 or PDGF-BB increased tissue repair after four weeks. After eight weeks, most defects were filled with bone tissue. To further investigate the promising effect of BMP-2, a caprine bilateral stifle osteochondral defect model was used where defects were created in weight-bearing femoral condyle and non-weight-bearing trochlear groove locations. After six months, the adsorption of BMP-2 resulted in significantly less bone repair compared with scaffold-only in the femoral condyle defects and a trend to more bone repair in the trochlear groove. Overall, the adsorption of BMP-2 onto a Col/Col-Mg-HAp scaffold reduced bone formation in weight-bearing osteochondral defects, but not in non-weight-bearing osteochondral defects.
The effect of 6-chloro-2,3,4,5-tetrahydro-3-methyi-1-H-3-benzazepine (SKF 86466), a selectlve nonimldazoline alpha-2 adrenoceptor antagonlst, on hippocampal re1ease of norepinephrine and dopamlne in conscious rats was lnvestigated by /n vlvo mlcrodialysis and high-pressure liquid chromatography. Additionally, extracellular concentrations of hippocampal dopamine (DA) and norepinephrtne (NE), durtng Infusion of selective monoamine uptake Inhibitors, were determined in freely moving rats. The basal concentration of NE in the dialysate was 4.9 ± 0.3 pg/20 pl. lntravenous admlnistratlon of 5 or 10 mgJkg of SKF 86466 was associated wlth a transierlt inc:rease (30 min) of 2-fold (12 ± 1 pg/20 ,d; p < .05) and 8-fold (39 ± 3 pg/20 pl; p < .05), respectlvely, in dlalysate NE, whereas a 1-mgfkg dose had no effect. DA was not detected in basal dlalysates, but after the adminlstratlon of 5 or 10 mgJkg of SKF 86466, 3.9 ± 0.4 and 6.4 ± 0.6 pg/20 pl, respectlvely, was present in the dialysates. The rnaxlmum increase in dialysate DA was reached 60 to 90 min after SKF 86466. The DA was not derived from plasma because plasma NE was elevated after the 5 mgJkg dose of SKF 86466 whereas no plasma DA was detected. ln order to determlne whether DA was present in noradrenergic nerve termlnals, the dopamine ß-hydroxylase Inhibitor SKF 1 02698 was administered (50 mgJkg i.p.). The Inhibitor decreased dialysate NE but DA was stin not detected in the dialysate. When SKF 86466 (5 mgJkg t.v.) was adminlstered 4 hr after SKF 102698, DA appeared in the dialysate but there was no lncrease in dialysate NE. Administration through the dialysis probe of the DA uptake Inhibitor, GBR-12909 (0.1 and 1 pM), dose-dependently lnaeased DA Ieveis to 5.7 ± 1.2 and 9.6 ± 2.8 pg/20 pl, respectively. GBR-12909 had no effect on hippocampal NE. Desipramine (5 and 10 pM) lncreased dose-dependently dialysate NE and lncreased DA concentrations to detectable Ieveis (2.7 ± 0.5 and 3.5 ± 0.7 pg/20 ,d, respectively). These results suggest that the a/pha-2 adrenoceptors modulate both NE and DA release in the rat hlppocampus and that DA detected in the hlppocampal dialysate might be released from dopaminergic neurons.
DNA damage occurs frequently during normal cellular progresses or by environmental factors. To preserve the genome integrity, DNA damage response (DDR) has evolved to repair DNA and the non-properly repaired DNA induces human diseases like immune deficiency and cancer. Since a large number of proteins involved in DDR are enzymes of ubiquitin system, it is critical to investigate how the ubiquitin system regulates cellular response to DNA damage. Hereby, we reveal a novel mechanism for DDR regulation via activation of SCF ubiquitin ligase upon DNA damage.
As an essential step for DNA damage-induced inhibition of DNA replication, Cdc25A degradation by the E3 ligase β-TrCP upon DNA damage requires the deubiquitinase Usp28. Usp28 deubiquitinates β-TrCP in response to DNA damage, thereby promotes its dimerization, which is required for its activity in substrate ubiquitination and degradation. Particularly, ubiquitination at a specific lysine on β-TrCP suppresses dimerization.
The key mediator protein of DDR, 53BP1, forms oligomers and associates with β-TrCP to inhibit its activity in unstressed cells. Upon DNA damage, 53BP1 is degraded in the nucleoplasm, which requires oligomerization and is promoted by Usp28 in a β-TrCP-dependent manner. Consequently, 53BP1 destruction releases and activates β-TrCP during DNA damage response.
Moreover, 53BP1 deletion and DNA damage promote β-TrCP dimerization and recruitment to chromatin sites that locate in the vicinity of putative replication origins. Subsequently, the chromatin-associated Cdc25A is degraded by β-TrCP at the origins. The stimulation of β-TrCP binding to the origins upon DNA damage is accompanied by unloading of Cdc45, a crucial component of pre-initiation complexes for replication. Loading of Cdc45 to origins is a key Cdk2-dependent step for DNA replication initiation, indicating that localized Cdc25A degradation by β-TrCP at origins inactivates Cdk2, thereby inhibits the initiation of DNA replication.
Collectively, this study suggests a novel mechanism for the regulation of DNA replication upon DNA damage, which involves 53BP1- and Usp28-dependent activation of the SCF(β-TrCP) ligase in Cdc25A degradation.
Das mR-Paradigma beschreibt die Fähigkeit Objekte gedanklich zu drehen und erfordert dabei komplexe neuronale Prozesse. Bisherige Studien konnten nicht klären, ob es ein spezifisches Muster der Beeinträchtigung im mR-Test bei fokalen Dystonien gibt. Die übergeordnete Fragestellung der vorliegenden Arbeit war, ob eine verlangsamte Reaktion bei der mR von körperlichen Abbildungen einen stabilen Endophänotyp fokaler Dystonien darstellt. Die Zielsetzung war die Überprüfung der Hypothesen, 1) dass bisherige Ergebnisse, die eine verlängerte Reaktionszeit von CD-Patienten bei der mR von körperlichen Abbildungen aufzeigten, reproduzierbar sind und 2) dass eine erhöhte Reaktionszeit bei der mR von körperlichen Abbildungen auch bei Patienten mit BSP vorliegt. Um dabei die mR möglichst spezifisch zu untersuchen, wurden folgende sekundäre Hypothesen formuliert: a) die kognitive Leistungsfähigkeit und b) das allgemeine Reaktionsvermögen der Teilnehmer stellen potenzielle Störfaktoren für die Reaktionszeit bei der mR-Aufgabe dar. Diese wurden neben der Händigkeit und der allgemeinen Geschicklichkeit systematisch erhoben.
23 CD-Patienten und 23 gesunde Kontrollpersonen sowie 21 BSP- und 19 HFS-Patienten wurden hinsichtlich Geschlechterverteilung, Alter und Bildungsstand verglichen. Zudem wurden Händigkeit, Fingergeschicklichkeit, allgemeine Reaktionszeit und kognitiver Status jedes Teilnehmers erhoben. Im mR-Test wurden Fotos von Körperteilen (Hand, Fuß oder Kopf) und einem nicht-körperlichen Objekt (Auto) gezeigt, die in sechs verschiedene Winkelgrade um die eigene Achse in der Bildebene rotiert waren. Die Teilnehmer wurden gebeten, die Lateralität des dargestellten Bildes per Tastendruck anzugeben. Bewertet wurden sowohl Geschwindigkeit als auch Richtigkeit der Antworten.
Im Vergleich zu gesunden Kontrollpersonen schnitten CD- und HFS-Patienten bei der mR der Hände schlechter ab, während die BSP-Patienten vergleichbare Leistungen zeigten. Es bestand ein signifikanter Zusammenhang zwischen einer verlängerten mR-Reaktionszeit und reduzierten MoCA-Scores sowie einer erhöhten mR-Reaktionszeit und verlängerter allgemeiner Reaktionszeit. Nach Ausschluss der Patienten mit MCI zeigten CD-Patienten, nicht jedoch HFS-Patienten, im Vergleich zur gesunden Kontrollgruppe weiterhin verlangsamte Reaktionszeiten der Hände.
Die vorliegende Studie konnte die Frage, ob eine verlangsamte Reaktion bei der mR von körperlichen Abbildungen einen stabilen Endophänotyp fokaler Dystonien darstellt, nicht sicher beantworten. Es stellte sich jedoch heraus, dass Kognition und allgemeine Reaktionszeit starke Einflussfaktoren bei der mR-Aufgabe sind. Dies wurde in den früheren Arbeiten nicht berücksichtigt und stellt daher ein neues und wichtiges Ergebnis dar. Die verlangsamte Reaktion bei der mR der Hände bei CD-Patienten auch nach Ausschluss von Patienten mit MCI lässt ein spezifisches Defizit der Fähigkeit der mR vermuten. Das Vorliegen einer tiefergreifenden zugrundeliegenden Netzwerkstörung, die sich auf die Leistung im mR-Test auswirkt, wäre dabei denkbar.
Neurofilament depletion improves microtubule dynamics via modulation of Stat3/stathmin signaling
(2016)
In neurons, microtubules form a dense array within axons, and the stability and function of this microtubule network is modulated by neurofilaments. Accumulation of neurofilaments has been observed in several forms of neurodegenerative diseases, but the mechanisms how elevated neurofilament levels destabilize axons are unknown so far. Here, we show that increased neurofilament expression in motor nerves of pmn mutant mice, a model of motoneuron disease, causes disturbed microtubule dynamics. The disease is caused by a point mutation in the tubulin-specific chaperone E (Tbce) gene, leading to an exchange of the most C-terminal amino acid tryptophan to glycine. As a consequence, the TBCE protein becomes instable which then results in destabilization of axonal microtubules and defects in axonal transport, in particular in motoneurons. Depletion of neurofilament increases the number and regrowth of microtubules in pmn mutant motoneurons and restores axon elongation. This effect is mediated by interaction of neurofilament with the stathmin complex. Accumulating neurofilaments associate with stathmin in axons of pmn mutant motoneurons. Depletion of neurofilament by Nefl knockout increases Stat3-stathmin interaction and stabilizes the microtubules in pmn mutant motoneurons. Consequently, counteracting enhanced neurofilament expression improves axonal maintenance and prolongs survival of pmn mutant mice. We propose that this mechanism could also be relevant for other neurodegenerative diseases in which neurofilament accumulation and loss of microtubules are prominent features.
“I tried to control my emotions”: nursing home care workers’ experiences of emotional labor in China
(2022)
Despite dramatic expansions in the Chinese nursing home sector in meeting the increasing care needs of a rapidly aging population, direct care work in China remains largely devalued and socially unrecognized. Consequently, scant attention has been given to the caregiving experiences of direct care workers (DCWs) in Chinese nursing homes. In particular, given the relational nature of care work, there is little knowledge as to how Chinese DCWs manage emotions and inner feelings through their emotional labor. This article examines the emotional labor of Chinese DCWs through ethnographic data collected with 20 DCWs in one nursing home located in an urban setting in central China. Data were analyzed using conventional content analysis and constant comparison. Participants’ accounts of sustaining a caring self, preserving professional identity, and hoping for reciprocity revealed implicit meanings about the often-conflicting nature of emotional labor and the nonreciprocal elements of care work under constrained working conditions. Importantly, the moral-cultural notion of bao (报 norm of reciprocity) was found to be central among DCWs in navigating strained resources and suggested their agency in meaning-construction. However, their constructed moral buffers may be insufficient if emotional labor continues to be made invisible by care organizations.
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.
The HECT-type ubiquitin ligase HECT, UBA and WWE Domain Containing 1, (HUWE1) regulates key cancer-related pathways, including the Myc oncogene. It affects cell proliferation, stress and immune signaling, mitochondria homeostasis, and cell death. HUWE1 is evolutionarily conserved from Caenorhabditis elegance to Drosophila melanogaster and Humans. Here, we report that the Drosophila ortholog, dHUWE1 (CG8184), is an essential gene whose loss results in embryonic lethality and whose tissue-specific disruption establishes its regulatory role in larval salivary gland development. dHUWE1 is essential for endoreplication of salivary gland cells and its knockdown results in the inability of these cells to replicate DNA. Remarkably, dHUWE1 is a survival factor that prevents premature activation of JNK signaling, thus preventing the disintegration of the salivary gland, which occurs physiologically during pupal stages. This function of dHUWE1 is general, as its inhibitory effect is observed also during eye development and at the organismal level. Epistatic studies revealed that the loss of dHUWE1 is compensated by dMyc proeitn expression or the loss of dmP53. dHUWE1 is therefore a conserved survival factor that regulates organ formation during Drosophila development.
Die vorliegende klinisch-experimentelle Arbeit beleuchtet den Zusammenhang zwischen biologischem Geschlecht, den Konzentrationen der Geschlechtshormone Testosteron, Estradiol sowie dem kardialen Protein NT-pro-BNP in vivo und der Kraftentwicklung stimulierter Herzmuskelzellen in vitro. Im Studienzeitraum wurden insgesamt 225 Patienten (35 weiblich, 190 männlich), die sich einer elektiven koronarchirurgischen Operation unter Einsatz der Herz-Lungen-Maschine unterzogen, in die Studie eingeschlossen. Im Rahmen der Operation wurden Herzmuskelproben vom linken und rechten Herzohr gewonnen. Aus diesen wurde experimentell der kontraktile Apparat isoliert. Diese Muskelfaserbündel wurden mittels Immersion in verschieden stark konzentrierten Kalziumbädern zur Kontraktion stimuliert und die resultierende Kraftentwicklung erfasst. Diese Daten wurden den im Patientenblut bestimmten Serumkonzentrationen von Estradiol, Testosteron und NT-pro-BNP gegenübergestellt. Es konnte, auch unter Berücksichtigung der Hormonkonzentrationen, weder eine Korrelation des Patientengeschlechts mit der Kraftentwicklung festgestellt werden, noch korrelierte die Konzentration von NT-pro-BNP mit der Kraftentwicklung im experimentellen Modell.
In der vorliegenden Studie wurde untersucht, ob laryngeale Konstriktionsphänomene regelhaft bei gesunden Säuglingen mit deutscher Umgebungssprache auftreten, ob es eine altersabhängige oder geschlechtsabhängige Entwicklung in der Auftrittshäufigkeit der Phänomene gibt und ob diese zusätzlich durch den Vokalisationstyp beeinflusst wird. Dazu wurden hier vier typische Vokalisationstypen im vorsprachlichen Alter definiert: spontanes Säuglingsweinen vor einer Mahlzeit in zwei Ausprägungsformen (Typ C und UC, letzterer als Typ weniger intensiven Säuglingsweinens) sowie Nichtschreivokalisationen in zwei Ausprägungsformen (UB: Übergangslaut zwischen UC und silbenartigem Vokalisieren (BB)).
In der vorliegenden Arbeit wurden solche Konstriktionsphänomene untersucht, die in der medizinischen Fachliteratur häufig mit pathologischen Zuständen der respiratorischen Regelung sowie Vokaltraktmalformationen bei Säuglingen mit Lippen-Kiefer-Gaumen-Segelspalten beschrieben werden. Es wurde hier untersucht, ob ähnliche Phänomene auch bei gesunden Säuglingen regelhaft auftreten.
Dazu wurde in einem kombinierten Längs- und Querschnittsdesign eine deskriptive Analyse von 20.406 Einzelvokalisationen von 20 Säuglingen in den ersten sieben Lebensmonaten vorgenommen. Die Vokalisationen lagen anonymisiert im Zentrum für vorsprachliche Entwicklung & Entwicklungsstörungen an der Poliklinik für Kieferorthopädie des Universitätsklinikums Würzburg vor. Es handelt sich um eine explorative, retrospektive Analyse.
Unter Verwendung von Frequenzspektren und Audiofiles wurden alle Einzelvokalisationen audio-visuell analysiert und drei Stufen mit unterschiedlicher Ausprägung der Konstriktionen einsortiert (Kategorie 1 – 3; Kategorie 0 = keine Konstriktionen in der Vokalisation). Die Kategoriendefinition wurde vom Autor der vorliegenden Arbeit in einer Voruntersuchung erarbeitet und durch weitere Kodierer getestet und als geeignet befunden.
Im Ergebnis der Arbeit konnte gezeigt werden, dass die hier untersuchten Konstriktionsphänomene regelhaft bei allen gesunden Säuglingen im Untersuchungszeitraum vorkommen. Die Auftrittshäufigkeit war dabei teilweise vom Geschlecht, vom Alter und vom Vokalisationstyp abhängig. Eine vergleichbare systematische Analyse lag bisher in der Literatur nicht vor. Die Ergebnisse werden aus physiologischer und linguistisch-phonetischer Perspektive interpretiert. Es konnte gezeigt werden, dass die im spontanen Weinen beobachteten Konstriktionsphänomene auch bei den Komfortvokalisationen (Nichtschreivokalisationen) vorkamen.
Dies stützt die Kontinuitätshypothese in der vorsprachlichen Entwicklung.
Die Arbeit hat auch widerlegt, dass alle Konstriktionsphänomene im Säuglingsweinen ein Pathologiemarker sind. Die Differenzierung zwischen physiologischen und pathologischen Konstriktionsphänomenen, die z.B. durch respiratorische Dysfunktion entstehen können (Stridor), ist eine Aufgabe für nachfolgende Arbeiten.
Für weiterführende Arbeiten mit dem Ziel der Anwendung von Stimmregisterphänomenen in der Vorsprachlichen Diagnostik sind methodisch erweiterte Ansätze bei gleichzeitig größerer Stichprobe erforderlich.
To generate infectious viral particles, viruses must specifically select their genomic RNA from milieu that contains a complex mixture of cellular or non-genomic viral RNAs. In this review, we focus on the role of viral encoded RNA structures in genome packaging. We first discuss how packaging signals are constructed from local and long-range base pairings within viral genomes, as well as inter-molecular interactions between viral and host RNAs. Then, how genome packaging is regulated by the biophysical properties of RNA. Finally, we examine the impact of RNA packaging signals on viral evolution.
Engineered vaccinia virus (VACV) strains are used extensively as vectors for the development of novel cancer vaccines and cancer therapeutics. In this study, we describe for the first time a high-throughput approach for both fluorescent rVACV generation and rapid viral titer measurement with the multi-well plate imaging system, IncuCyte\(^®\)S3. The isolation of a single, well-defined plaque is critical for the generation of novel recombinant vaccinia virus (rVACV) strains. Unfortunately, current methods of rVACV engineering via plaque isolation are time-consuming and laborious. Here, we present a modified fluorescent viral plaque screening and selection strategy that allows one to generally obtain novel fluorescent rVACV strains in six days, with a minimum of just four days. The standard plaque assay requires chemicals for fixing and staining cells. Manual plaque counting based on visual inspection of the cell culture plates is time-consuming. Here, we developed a fluorescence-based plaque assay for quantifying the vaccinia virus that does not require a cell staining step. This approach is less toxic to researchers and is reproducible; it is thus an improvement over the traditional assay. Lastly, plaque counting by virtue of a fluorescence-based image is very convenient, as it can be performed directly on the computer.
Neurodegeneration plays an essential role in Parkinson’s disease (PD). Several crucial neuronal pro-and antidegeneration markers were described to be altered in disease models accompanied by neurodegeneration. In the AAV1/2-A53T-aSyn PD rat model progressive time-dependent motor impairment and neurodegeneration in the nigrostriatal tract starting from 2 weeks after PD model induction could be found. Downregulation of Nrf2 in SN and nigrostriatal axon localization, a trend of Tau downregulation in SN and upregulation in axon localization in the AAV1/2-A53T-aSyn PD rat model were observed, indicating potential therapeutic value of these two molecular targets in PD. No alterations of SARM1 and NMNAT2 could be detected, indicating little relevance of these two molecules with our AAV1/2-A53T-aSyn rat model.
Purpose: Prostate-specific membrane antigen (PSMA)-targeted positron emission tomography (PET) imaging has become commonly utilized in patients with prostate cancer (PCa). The PSMA reporting and data system version 1.0 (PSMA-RADS version 1.0) categorizes lesions on the basis of the likelihood of PCa involvement, with PSMA-RADS-3A (soft tissue) and PSMA-RADS-3B (bone) lesions being indeterminate for the presence of disease. We retrospectively reviewed the imaging follow-up of such lesions to determine the rate at which they underwent changes suggestive of underlying PCa.
Methods: PET/CT imaging with \(^{18}\)F-DCFPyL was carried out in 110 patients with PCa and lesions were categorized according to PSMA-RADS Version 1.0. 56/110 (50.9%) patients were determined to have indeterminate PSMA-RADS-3A or PSMA-RADS-3B lesions and 22/56 (39.3%) patients had adequate follow-up to be included in the analysis. The maximum standardized uptake values (SUV\(_{max}\)) of the lesions were obtained and the ratios of SUV\(_{max}\) of the lesions to SUV\(_{mean}\) of blood pool (SUV\(_{max}\)-lesion/SUV\(_{mean}\)-bloodpool) were calculated. Pre-determined criteria were used to evaluate the PSMA-RADS-3A and PSMA-RADS-3B lesions on follow-up imaging to determine if they demonstrated evidence of underlying malignancy.
Results: A total of 46 lesions in 22 patients were considered indeterminate for PCa (i.e. PSMA-RADS-3A (32 lesions) or PSMA-RADS-3B (14 lesions)) and were evaluable on follow-up imaging. 27/46 (58.7%) lesions demonstrated changes on follow-up imaging consistent with the presence of underlying PCa at baseline. These lesions included 24/32 (75.0%) PSMA-RADS-3A lesions and 3/14 (21.4%) lesions categorized as PSMA-RADS-3B. The ranges of SUVmax and SUVmax-lesion/SUVmean-bloodpool overlapped between those lesions demonstrating changes consistent with malignancy on follow-up imaging and those lesions that remained unchanged on follow-up.
Conclusion: PSMA-RADS-3A and PSMA-RADS-3B lesions are truly indeterminate in that proportions of findings in both categories demonstrate evidence of malignancy on follow-up imaging. Overall, PSMA-RADS-3A lesions are more likely than PSMA-RADS-3B lesions to represent sites of PCa and this information should be taken into when guiding patient therapy.
The numbers of monocytes and macrophages in the walls of cerebral blood vessels were counted on perfusion-fixed frozen brain sections (16 JLffi) of spontaneously hypertensive rats (SHR), stroke-prone SHR (SHR-SP), normotensive Wistar-Kyoto (WKY) rats, and young (16-week-old) and old (2-year-old) normotensive Sprague-Dawley rats (SD-l6w and SD-2y, respectively) using monoclonal antiborlies against rat macrophages (ED2). The staining was visualized with fluoresceinlabeled second antiborlies. The ED2-specific staining in brain sections was restricted to macrophages in a perivascular location. The number of perivascular cells per square millimeter of high-power field was significantly greater in SHR-SP (8.6 ± 2.1; n = 4) and SHR (6. 7 ± 0.9; n = 6) than in normotensive WKY (4.0 ± 0.5; n = 6; p <0.01). The number of perivascular macrophages was also greater in SD-2y (7.5 ± 2.7; n = 9) than in SD-l6w (2.9 ± 1.8; n = 8; p < 0.01). No ED2 staining was found in the resident microglia or in the endothelial cells, which were identified by double staining with rhodamine-labeled anti-factor VIII-related antigen antiborlies. The results suggest that the stroke risk factors hypertension and advanced age are associated with increased subendothelial accumulation of monocytes and macrophages. This accumulation could increase the tendency for the endothelium to convert from an anticoagulant to a procoagulant surface in response to mediators released from these subendothelial cells.
The Escherichia coli effector EspJ blocks Src kinase activity via amidation and ADP ribosylation
(2014)
The hallmark of enteropathogenic Escherichia coli (EPEC) infection is the formation of actin-rich pedestal-like structures, which are generated following phosphorylation of the bacterial effector Tir by cellular Src and Abl family tyrosine kinases. This leads to recruitment of the Nck-WIP-N-WASP complex that triggers Arp2/3-dependent actin polymerization in the host cell. The same phosphorylation-mediated signalling network is also assembled downstream of the Vaccinia virus protein A36 and the phagocytic Fc-gamma receptor FcγRIIa. Here we report that the EPEC type-III secretion system effector EspJ inhibits autophosphorylation of Src and phosphorylation of the Src substrates Tir and FcγRIIa. Consistent with this, EspJ inhibits actin polymerization downstream of EPEC, Vaccinia virus and opsonized red blood cells. We identify EspJ as a unique adenosine diphosphate (ADP) ribosyltransferase that directly inhibits Src kinase by simultaneous amidation and ADP ribosylation of the conserved kinase-domain residue, Src E310, resulting in glutamine-ADP ribose.
Purpose
To achieve a highly balanced comparison of trabecular bypass stenting (IS2, iStent inject) with ab interno trabeculectomy (T, Trabectome) by exact matching.
Methods
Fifty-three IS2 eyes were matched to 3446 T eyes. Patients were matched using exact matching by baseline intraocular pressure (IOP), the number of glaucoma medications, and glaucoma type, and using nearest neighbor matching by age. Individuals without a close match were excluded. All surgeries were combined with phacoemulsification.
Results
A total of 78 eyes (39 in each group) could be matched as exact pairs with a baseline IOP of 18.3 ± 5.1 mmHg and glaucoma medications of 2.7 ± 1.2 in each. IOP in IS2 was reduced to 14.6 ± 4.2 mmHg at 3 months and in T to a minimum of 13.1 ± 3.2 mmHg at 1 month. In IS2, IOP began to rise again at 6 months, eventually exceeding baseline. At 24 months, IOP in IS2 was 18.8 ± 9.0 mmHg and in T 14.2 ± 3.5 mmHg. IS2 had a higher average IOP than T at all postoperative visits (p < 0.05 at 1, 12, 18 months). Glaucoma medications decreased to 2.0 ± 1.5 in IS2 and to 1.5 ± 1.4 in T.
Conclusion
T resulted in a larger and sustained IOP reduction compared with IS2 where a rebound occurred after 6 months to slightly above preoperative values.
The green synthesis of silver nanoparticles (SNPs) using plant extracts is an eco-friendly method. It is a single step and offers several advantages such as time reducing, cost-effective and environmental non-toxic. Silver nanoparticles are a type of Noble metal nanoparticles and it has tremendous applications in the field of diagnostics, therapeutics, antimicrobial activity, anticancer and neurodegenerative diseases. In the present work, the aqueous extracts of aerial parts of Lampranthus coccineus and Malephora lutea F. Aizoaceae were successfully used for the synthesis of silver nanoparticles. The formation of silver nanoparticles was early detected by a color change from pale yellow to reddish-brown color and was further confirmed by transmission electron microscope (TEM), UV–visible spectroscopy, Fourier transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS), X-ray diffraction (XRD), and energy-dispersive X-ray diffraction (EDX). The TEM analysis of showed spherical nanoparticles with a mean size between 12.86 nm and 28.19 nm and the UV- visible spectroscopy showed λ\(_{max}\) of 417 nm, which confirms the presence of nanoparticles. The neuroprotective potential of SNPs was evaluated by assessing the antioxidant and cholinesterase inhibitory activity. Metabolomic profiling was performed on methanolic extracts of L. coccineus and M. lutea and resulted in the identification of 12 compounds, then docking was performed to investigate the possible interaction between the identified compounds and human acetylcholinesterase, butyrylcholinesterase, and glutathione transferase receptor, which are associated with the progress of Alzheimer’s disease. Overall our SNPs highlighted its promising potential in terms of anticholinesterase and antioxidant activity as plant-based anti-Alzheimer drug and against oxidative stress.
To understand the gene regulation of an organism of interest, a comprehensive genome annotation is essential. While some features, such as coding sequences, can be computationally predicted with high accuracy based purely on the genomic sequence, others, such as promoter elements or noncoding RNAs, are harder to detect. RNA sequencing (RNA-seq) has proven to be an efficient method to identify these genomic features and to improve genome annotations. However, processing and integrating RNA-seq data in order to generate high-resolution annotations is challenging, time consuming, and requires numerous steps. We have constructed a powerful and modular tool called ANNOgesic that provides the required analyses and simplifies RNA-seq-based bacterial and archaeal genome annotation. It can integrate data from conventional RNA-seq and differential RNA-seq and predicts and annotates numerous features, including small noncoding RNAs, with high precision. The software is available under an open source license (ISCL) at https://pypi.org/project/ANNOgesic/.
Among the defense strategies developed in microbes over millions of years, the innate adaptive CRISPR-Cas immune systems have spread across most of bacteria and archaea. The flexibility, simplicity, and specificity of CRISPR-Cas systems have laid the foundation for CRISPR-based genetic tools. Yet, the efficient administration of CRISPR-based tools demands rational designs to maximize the on-target efficiency and off-target specificity. Specifically, the selection of guide RNAs (gRNAs), which play a crucial role in the target recognition of CRISPR-Cas systems, is non-trivial. Despite the fact that the emerging machine learning techniques provide a solution to aid in gRNA design with prediction algorithms, design rules for many CRISPR-Cas systems are ill-defined, hindering their broader applications.
CRISPR interference (CRISPRi), an alternative gene silencing technique using a catalytically dead Cas protein to interfere with transcription, is a leading technique in bacteria for functional interrogation, pathway manipulation, and genome-wide screens. Although the application is promising, it also is hindered by under-investigated design rules. Therefore, in this work, I develop a state-of-art predictive machine learning model for guide silencing efficiency in bacteria leveraging the advantages of feature engineering, data integration, interpretable AI, and automated machine learning. I first systematically investigate the influential factors that attribute to the extent of depletion in multiple CRISPRi genome-wide essentiality screens in Escherichia coli and demonstrate the surprising dominant contribution of gene-specific effects, such as gene expression level. These observations allowed me to segregate the confounding gene-specific effects using a mixed-effect random forest (MERF) model to provide a better estimate of guide efficiency, together with the improvement led by integrating multiple screens. The MERF model outperformed existing tools in an independent high-throughput saturating screen. I next interpret the predictive model to extract the design rules for robust gene silencing, such as the preference for cytosine and disfavoring for guanine and thymine within and around the protospacer adjacent motif (PAM) sequence. I further incorporated the MERF model in a web-based tool that is freely accessible at www.ciao.helmholtz-hiri.de.
When comparing the MERF model with existing tools, the performance of the alternative gRNA design tool optimized for CRISPRi in eukaryotes when applied to bacteria was far from satisfying, questioning the robustness of prediction algorithms across organisms. In addition, the CRISPR-Cas systems exhibit diverse mechanisms albeit with some similarities. The captured predictive patterns from one dataset thereby are at risk of poor generalization when applied across organisms and CRISPR-Cas techniques. To fill the gap, the machine learning approach I present here for CRISPRi could serve as a blueprint for the effective development of prediction algorithms for specific organisms or CRISPR-Cas systems of interest. The explicit workflow includes three principle steps: 1) accommodating the feature set for the CRISPR-Cas system or technique; 2) optimizing a machine learning model using automated machine learning; 3) explaining the model using interpretable AI. To illustrate the applicability of the workflow and diversity of results when applied across different bacteria and CRISPR-Cas systems, I have applied this workflow to analyze three distinct CRISPR-Cas genome-wide screens. From the CRISPR base editor essentiality screen in E. coli, I have determined the PAM preference and sequence context in the editing window for efficient editing, such as A at the 2nd position of PAM, A/TT/TG downstream of PAM, and TC at the 4th to 5th position of gRNAs. From the CRISPR-Cas13a screen in E. coli, in addition to the strong correlation with the guide depletion, the target expression level is the strongest predictor in the model, supporting it as a main determinant of the activation of Cas13-induced immunity and better characterizing the CRISPR-Cas13 system. From the CRISPR-Cas12a screen in Klebsiella pneumoniae, I have extracted the design rules for robust antimicrobial activity across K. pneumoniae strains and provided a predictive algorithm for gRNA design, facilitating CRISPR-Cas12a as an alternative technique to tackle antibiotic resistance.
Overall, this thesis presents an accurate prediction algorithm for CRISPRi guide efficiency in bacteria, providing insights into the determinants of efficient silencing and guide designs. The systematic exploration has led to a robust machine learning approach for effective model development in other bacteria and CRISPR-Cas systems. Applying the approach in the analysis of independent CRISPR-Cas screens not only sheds light on the design rules but also the mechanisms of the CRISPR-Cas systems. Together, I demonstrate that applied machine learning paves the way to a deeper understanding and a broader application of CRISPR-Cas systems.
Invasive fungal infections (IFIs) are difficult to diagnose and to treat and, despite several available antifungal drugs, cause high mortality rates. In the past decades, the incidence of IFIs has continuously increased. More recently, SARS-CoV-2-associated lethal IFIs have been reported worldwide in critically ill patients. Combating IFIs requires a more profound understanding of fungal pathogenicity to facilitate the development of novel antifungal strategies. Animal models are indispensable for studying fungal infections and to develop new antifungals. However, using mammalian animal models faces various hurdles including ethical issues and high costs, which makes large-scale infection experiments extremely challenging. To overcome these limitations, we optimized an invertebrate model and introduced a simple calcofluor white (CW) staining protocol to macroscopically and microscopically monitor disease progression in silkworms (Bombyx mori) infected with the human pathogenic filamentous fungi Aspergillus fumigatus and Lichtheimia corymbifera. This advanced silkworm A. fumigatus infection model could validate knockout mutants with either attenuated, strongly attenuated or unchanged virulence. Finally, CW staining allowed us to efficiently visualize antifungal treatment outcomes in infected silkworms. Conclusively, we here present a powerful animal model combined with a straightforward staining protocol to expedite large-scale in vivo research of fungal pathogenicity and to investigate novel antifungal candidates.
Regulation of effector T cells is an important mechanism to control organ-specific inflammation. Thereby regulatory T cells (Treg cells) are essential for maintaining peripheral immune tolerance and for establishing parenchyma immune homeostasis. A novel population of natural human Treg characterized by the constitutive expression of the immune-tolerogenic human HLA-G molecule has been identified. In the first part of the study, we elucidated the mechanism(s) by which CD4+ HLA-Gpos Treg modulates their cellular targets namely autologous HLA-G negative responder T cells (HLAGneg Tresp). Using a suppression system free of antigen-presenting cells (APC), we demonstrate a T-T cell interaction resulting in suppression of HLA-Gneg Tresp. We could also show that this suppression was independent of cell-cell contact. Importantly, stimulus of T cell receptor (TCR) on HLA-Gpos Treg facilitated their suppressive capacity. We also observed that removal of HLA-Gpos Treg from the established co-cultures could restore the ability of HLA-Gneg Tresp to proliferate upon TCR re-stimulation, indicating that the suppression was reversible. Further, HLA-Gpos Treg–mediated suppression was critically depending on the secretion of IL-10 but not TGF-β. Taken together, this part of the work provides an in-depth characterization of the mechanisms of how HLA-Gpos Treg suppresses T responder cells in direct T-T interactions. Understanding the suppressive mechanism used by HLA-Gpos Treg may help to develop therapeutic strategies to modulate regulatory arms of T-cell suppression. In the second part of this study, the potential role of HLA-Gpos Treg in the pathophysiological process of Multiple Sclerosis (MS), a prototypic autoimmune inflammatory central nervous system (CNS), has been investigated. We found that HLA-Gpos Treg are enriched in the cerebrospinal fluid (CSF) from MS patients, but not in non-inflammatory controls. CSFderived HLA-Gpos Treg showed predominance of central memory (CD45RA-CD27+) phenotype, exhibited markers of activation (ICOS), and had significantly higher expression of the inflammatory chemokine receptor CCR5. Importantly, these cells demonstrated as potent suppressors to autologous CD4+ T-cell proliferation. Using an in vitro model of human blood brain barrier, we showed that HLA-Gpos Treg have a strong propensity to migrate, which could be facilitated by MIP1α and RANTES (ligands of CCR5) but not MIP3β (a ligand of CCR7). The HLA-Gpos Treg migration triggered by chemokines was also associated with a gain of suppressive capacity upon cellular transmigration. In contrast to CD4+CD25+ naturally occurring FoxP3-expressing Treg, HLA-Gpos Treg from patients with MS did not exhibit impaired function, suggesting that HLA-Gpos Treg are selectively recruited to the sites of CNS inflammation in an effort to combat destructive inflammation during MS. Our results contribute to the understanding of the role and function of HLA-Gpos Treg and provide an important example of “beneficial” T-cell inflammation in CNS autoimmunity- interesting both from a patho/-physiological and a therapeutically point of view.
Aging is known to be a risk factor for structural abnormalities and functional decline in the nervous system. Characterizing age-related changes is important to identify putative pathways to overcome deleterious effects and improve life quality for the elderly. In this study, the peripheral nervous system of 24-month-old aged C57BL/6 mice has been investigated and compared to 12-month-old adult mice. Aged mice showed pathological alterations in their peripheral nerves similar to nerve biopsies from elderly human individuals, with nerve fibers showing demyelination and axonal damage. Such changes were lacking in nerves of adult 12-month-old mice and adult, non-aged humans. Moreover, neuromuscular junctions of 24-month-old mice showed increased denervation compared to adult mice. These alterations were accompanied by elevated numbers of macrophages in the peripheral nerves of aged mice. The neuroinflammatory conditions were associated with impaired myelin integrity and with a decline of nerve conduction properties and muscle strength in aged mice.
To determine the pathological impact of macrophages in the aging mice, macrophage depletion was performed in mice by oral administration of CSF-1R specific kinase (c-FMS) inhibitor PLX5622 (300 mg/kg body weight), which reduced the number of macrophages in the peripheral nerves by 70%. The treated mice showed attenuated demyelination, less muscle denervation and preserved muscle strength. This indicates that macrophage-driven inflammation in the peripheral nerves is partially responsible for the age-related neuropathy in mice.
Based on previous observations that systemic inflammation can accelerate disease progression in mouse models of neurodegenerative diseases, it was hypothesized that systemic inflammation can exacerbate the peripheral neuropathy found in aged mice. To investigate this hypothesis, aged C57BL/6 mice were intraperitoneally injected with a single dose of lipopolysaccharide (LPS; 500 μg/kg body weight) to induce systemic inflammation by mimicking bacterial infection, mostly via activation of Toll-like receptors (TLRs). Altered endoneurial macrophage activation, highlighted by Trem2 downregulation, was found in LPS injected aged mice one month after injection. This was accompanied by a so far rarely observed form of axonal perturbation, i.e., the occurrence of “dark axons” characterized by a damaged cytoskeleton and an increased overall electron density of the axoplasm. At the same time, however, LPS injection reduced demyelination and muscle denervation in aged mice. Interestingly, TREM2 deficiency in aged mice led to similar changes to LPS injection. This suggests that LPS injection likely mitigates aging-related demyelination and muscle denervation via Trem2 downregulation.
Taken together, this study reveals the role of macrophage-driven inflammation as a pathogenic mediator in age-related peripheral neuropathy, and that targeting macrophages might be an option to mitigate peripheral neuropathies in aging individuals. Furthermore, this study shows that systemic inflammation may be an ambivalent modifier of age-related nerve damage, leading to a distinct type of axonal perturbation, but in addition to functionally counteracting, dampened demyelination and muscle denervation. Translationally, it is plausible to assume that tipping the balance of macrophage polarization to one direction or the other may determine the functional outcome in the aging peripheral nervous system of the elderly.
Functional versus morphological assessment of vascular age in patients with coronary heart disease
(2021)
Communicating cardiovascular risk based on individual vascular age (VA) is a well acknowledged concept in patient education and disease prevention. VA may be derived functionally, e.g. by measurement of pulse wave velocity (PWV), or morphologically, e.g. by assessment of carotid intima-media thickness (cIMT). The purpose of this study was to investigate whether both approaches produce similar results. Within the context of the German subset of the EUROASPIRE IV survey, 501 patients with coronary heart disease underwent (a) oscillometric PWV measurement at the aortic, carotid-femoral and brachial-ankle site (PWVao, PWVcf, PWVba) and derivation of the aortic augmentation index (AIao); (b) bilateral cIMT assessment by high-resolution ultrasound at three sites (common, bulb, internal). Respective VA was calculated using published equations. According to VA derived from PWV, most patients exhibited values below chronological age indicating a counterintuitive healthier-than-anticipated vascular status: for VA(PWVao) in 68% of patients; for VA\(_{AIao}\) in 52% of patients. By contrast, VA derived from cIMT delivered opposite results: e.g. according to VA\(_{total-cIMT}\) accelerated vascular aging in 75% of patients. To strengthen the concept of VA, further efforts are needed to better standardise the current approaches to estimate VA and, thereby, to improve comparability and clinical utility.
Purpose
Growing evidence implies that transition to parenthood triggers symptoms of mental burden not only in women but likewise in men, especially in high-risk pregnancies. This is the first study that examined and compared the prevalence rates of depression, anxiety, and somatic symptom burden of expectant fathers who face different risk situations during pregnancy.
Methods
Prevalence rates of paternal depression (Edinburgh postnatal depression scale), anxiety (generalized anxiety disorder seven), and somatic symptom burden (somatic symptom scale eight) were examined in two risk samples and one control group in the third trimester of their partners’ pregnancy: risk sample I (n = 41) consist of expectant fathers whose partners were prenatally hospitalized due to medical complications; risk sample II (n = 52) are fathers whose partners were prenatally mentally distressed; and control group (n = 70) are those non-risk pregnancies.
Results
On a purely descriptive level, the data display a trend of higher symptom burden of depression, anxiety, and somatic symptoms in the two risk samples, indicating that expectant fathers, whose pregnant partners were hospitalized or suffered prenatal depression, were more prenatally distressed. Exploratory testing of group differences revealed an almost three times higher prevalence rate of anxiety in fathers whose partner was hospitalized (12.2%) compared to those non-risks (4.3%).
Conclusion
Results underline the need for screening implementations for paternal prenatal psychological distress, as well as specific prevention and treatment programs, especially for fathers in risk situations, such as their pregnant partners’ prenatal hospitalization.
The study was registered with the German clinical trials register (DRKS00020131) on 2019/12/09.
Background: Animal models of human inflammatory diseases have limited predictive quality for human clinical trials for various reasons including species specific activation mechanisms and the immunological background of the animals which markedly differs from the genetically heterogeneous and often aged patient population.
Objective: Development of an animal model allowing for testing therapeutics targeting pathways involved in the development of Atopic Dermatitis (AD) with better translatability to the patient.
Methods: NOD-scid IL2R \(\gamma^{null}\) mice engrafted with human peripheral blood mononuclear cells (hPBMC) derived from patients suffering from AD and healthy volunteers were treated with IL-4 and the antagonistic IL-4 variant R121/Y124D (Pitrakinra). Levels of human (h) IgE, amount of B-, T- and plasma-cells and ratio of CD4 : CD8 positive cells served as read out for induction and inhibition of cell proliferation and hIgE secretion. Results were compared to in vitro analysis.
Results: hIgE secretion was induced by IL-4 and inhibited by the IL-4 antagonist Pitrakinra in vivo when formulated with methylcellulose. B-cells proliferated in response to IL-4 in vivo; the effect was abrogated by Pitrakinra. IL-4 shifted CD4 : CD8 ratios in vitro and in vivo when hPBMC derived from healthy volunteers were used. Pitrakinra reversed the effect. Human PBMC derived from patients with AD remained inert and engrafted mice reflected the individual responses observed in vitro.
Conclusion: NOD-scid IL2R \(\gamma^{null}\) mice engrafted with human PBMC reflect the immunological history of the donors and provide a complementary tool to in vitro studies. Thus, studies in this model might provide data with better translatability from bench to bedside.
Objective: To evaluate the safety and effectiveness of round window (RW), oval window (OW), CliP and Bell couplers for use with an active middle ear implant. Methods: This is a multicenter, long-term, prospective trial with consecutive enrollment, involving 6 university hospitals in Germany. Bone conduction, air conduction, implant-aided warble-tone thresholds and Freiburger monosyllable word recognition scores were compared with unaided preimplantation results in 28 moderate-to-profound hearing-impaired patients after 12 months of follow-up. All patients had previously undergone failed reconstruction surgeries (up to 5 or more). In a subset of patients, additional speech tests at 12 months postoperatively were used to compare the aided with the unaided condition after implantation with the processor switched off. An established quality-of-life questionnaire for hearing aids was used to determine patient satisfaction. Results: Postoperative bone conduction remained stable. Mean functional gain for all couplers was 37 dB HL (RW = 42 dB, OW = 35 dB, Bell = 38 dB, CliP = 27 dB). The mean postoperative Freiburger monosyllable score was 71% at 65 dB SPL. The postimplantation mean SRT<sub>50</sub> (speech reception in quiet for 50% understanding of words in sentences) improved on average by 23 dB over unaided testing and signal-to-noise ratios also improved in all patients. The International Outcome Inventory for Hearing Aids (IOI-HA)quality-of-life questionnaire was scored very positively by all patients. Conclusion: A significant improvement was seen with all couplers, and patients were satisfied with the device at 12 months postoperatively. These results demonstrate that an active implant is an advantage in achieving good hearing benefit in patients with prior failed reconstruction surgery.
Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304\(\pm\)0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of \(\pm\)0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.
Many new immunotherapeutic approaches aim on the stimulatory targeting of receptors of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF) using antibodies with intrinsic or conditional agonism. There is an initial need to characterize corresponding TNFRSF receptor (TNFR)-targeting antibodies with respect to affinity, ligand binding, receptor activation and the epitope recognized. Here, we report a collection of simple and matched protocols enabling the detailed investigation of these aspects by help of Gaussia princeps luciferase (GpL) fusion proteins and analysis of interleukin-8 (IL8) production as an easily measurable readout of TNFR activation. In a first step, the antibodies and antibody variants of interest are transiently expressed in human embryonal kidney 293 cells, either in non-modified form or as fusion proteins with GpL as a reporter domain. The supernatants containing the antibody-GpL fusion proteins can then be used without further purification in cell-free and/or cellular binding studies to determine affinity. Similarly, binding studies with mutated TNFR variants enable the characterization of the antibody binding site within the TNFR ectodomain. Furthermore, in cellular binding studies with GpL fusion proteins of soluble TNFL molecules, the ability of the non-modified antibody variants to interfere with TNFL-TNFR interaction can be analyzed. Last but not least, we describe a protocol to determine the intrinsic and the Fc gamma receptor (FcγR)-dependent agonism of anti-TNFR antibodies which exploits i) the capability of TNFRs to trigger IL8 production in tumor cell lines lacking expression of FcγRs and ii) vector- and FcγR-transfected cells, which produce no or only very low amounts of human IL8. The presented protocols only require standard molecular biological equipment, eukaryotic cell culture and plate readers for the quantification of luminescent and colorimetric signals.
Antibody-based soluble and membrane-bound TWEAK mimicking agonists with FcγR-independent activity
(2023)
Fibroblast growth factor (FGF)-inducible 14 (Fn14) activates the classical and alternative NFκB (nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells) signaling pathway but also enhances tumor necrosis factor (TNF)-induced cell death. Fn14 expression is upregulated in non-hematopoietic cells during tissue injury and is also often highly expressed in solid cancers. In view of the latter, there were and are considerable preclinical efforts to target Fn14 for tumor therapy, either by exploiting Fn14 as a target for antibodies with cytotoxic activity (e.g. antibody-dependent cellular cytotoxicity (ADCC)-inducing IgG variants, antibody drug conjugates) or by blocking antibodies with the aim to interfere with protumoral Fn14 activities. Noteworthy, there are yet no attempts to target Fn14 with agonistic Fc effector function silenced antibodies to unleash the proinflammatory and cell death-enhancing activities of this receptor for tumor therapy. This is certainly not at least due to the fact that anti-Fn14 antibodies only act as effective agonists when they are presented bound to Fcγ receptors (FcγR). Thus, there are so far no antibodies that robustly and selectively engage Fn14 signaling without triggering unwanted FcγR-mediated activities. In this study, we investigated a panel of variants of the anti-Fn14 antibody 18D1 of different valencies and domain architectures with respect to their inherent FcγR-independent ability to trigger Fn14-associated signaling pathways. In contrast to conventional 18D1, the majority of 18D1 antibody variants with four or more Fn14 binding sites displayed a strong ability to trigger the alternative NFκB pathway and to enhance TNF-induced cell death and therefore resemble in their activity soluble (TNF)-like weak inducer of apoptosis (TWEAK), one form of the natural occurring ligand of Fn14. Noteworthy, activation of the classical NFκB pathway, which naturally is predominately triggered by membrane-bound TWEAK but not soluble TWEAK, was preferentially observed with a subset of constructs containing Fn14 binding sites at opposing sites of the IgG scaffold, e.g. IgG1-scFv fusion proteins. A superior ability of IgG1-scFv fusion proteins to trigger classical NFκB signaling was also observed with the anti-Fn14 antibody PDL192 suggesting that we identified generic structures for Fn14 antibody variants mimicking soluble and membrane-bound TWEAK.
Fibroblast growth factor-inducible 14 (Fn14) is a member of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF) and is activated by its ligand TNF-like weak inducer of apoptosis (TWEAK). The latter occurs as a homotrimeric molecule in a soluble and a membrane-bound form. Soluble TWEAK (sTWEAK) activates the weakly inflammatory alternative NF-κB pathway and sensitizes for TNF-induced cell death while membrane TWEAK (memTWEAK) triggers additionally robust activation of the classical NF-κB pathway and various MAP kinase cascades. Fn14 expression is limited in adult organisms but becomes strongly induced in non-hematopoietic cells by a variety of growth factors, cytokines and physical stressors (e.g., hypoxia, irradiation). Since all these Fn14-inducing factors are frequently also present in the tumor microenvironment, Fn14 is regularly found to be expressed by non-hematopoietic cells of the tumor microenvironment and most solid tumor cells. In general, there are three possibilities how the tumor-Fn14 linkage could be taken into consideration for tumor therapy. First, by exploitation of the cancer associated expression of Fn14 to direct cytotoxic activities (antibody-dependent cell-mediated cytotoxicity (ADCC), cytotoxic payloads, CAR T-cells) to the tumor, second by blockade of potential protumoral activities of the TWEAK/Fn14 system, and third, by stimulation of Fn14 which not only triggers proinflammtory activities but also sensitizes cells for apoptotic and necroptotic cell death. Based on a brief description of the biology of the TWEAK/Fn14 system and Fn14 signaling, we discuss the features of the most relevant Fn14-targeting biologicals and review the preclinical data obtained with these reagents. In particular, we address problems and limitations which became evident in the preclinical studies with Fn14-targeting biologicals and debate possibilities how they could be overcome.
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.
Background
Chronic psychological stress is associated with accelerated aging and increased risk for aging-related diseases, but the underlying molecular mechanisms are unclear.
Results
We examined the effect of lifetime stressors on a DNA methylation-based age predictor, epigenetic clock. After controlling for blood cell-type composition and lifestyle parameters, cumulative lifetime stress, but not childhood maltreatment or current stress alone, predicted accelerated epigenetic aging in an urban, African American cohort (n = 392). This effect was primarily driven by personal life stressors, was more pronounced with advancing age, and was blunted in individuals with higher childhood abuse exposure. Hypothesizing that these epigenetic effects could be mediated by glucocorticoid signaling, we found that a high number (n = 85) of epigenetic clock CpG sites were located within glucocorticoid response elements. We further examined the functional effects of glucocorticoids on epigenetic clock CpGs in an independent sample with genome-wide DNA methylation (n = 124) and gene expression data (n = 297) before and after exposure to the glucocorticoid receptor agonist dexamethasone. Dexamethasone induced dynamic changes in methylation in 31.2 % (110/353) of these CpGs and transcription in 81.7 % (139/170) of genes neighboring epigenetic clock CpGs. Disease enrichment analysis of these dexamethasone-regulated genes showed enriched association for aging-related diseases, including coronary artery disease, arteriosclerosis, and leukemias.
Conclusions
Cumulative lifetime stress may accelerate epigenetic aging, an effect that could be driven by glucocorticoid-induced epigenetic changes. These findings contribute to our understanding of mechanisms linking chronic stress with accelerated aging and heightened disease risk.
Growth factor induced signaling cascades are key regulatory elements in tissue development, maintenance and regeneration. Perturbations of these cascades have severe consequences, leading to developmental disorders and neoplastic diseases. As a major function in signal transduction, activating mutations in RAF family kinases are the cause of human tumorigenesis, where B-RAF V600E has been identified as the prevalent mutant. In order to address the oncogenic function of B-RAF V600E, we have generated transgenic mice expressing the activated oncogene specifically in lung alveolar epithelial type II cells. Constitutive expression of B-RAF V600E caused abnormalities in alveolar epithelium formation that led to airspace enlargements. These lung lesions showed signs of tissue remodeling and were often associated with chronic inflammation and low incidence of lung tumors. The inflammatory cell infiltration did not precede the formation of the lung lesions but was rather accompanied with late tumor development. These data support a model where the continuous regenerative process initiated by oncogenic B-RAF-driven alveolar disruption provides a tumor-promoting environment associated with chronic inflammation.
Polyspezifische organische Kationentransporter (OCTs) der SLC22 Familie transportieren organische Kationen entlang eines elektrochemischen Gradienten und spielen eine entscheidende Rolle bei der Ausscheidung und Gewebeverteilung von endogenen organischen Kationen und bei der Aufnahme, Ausscheidung und Verteilung von kationischen Medikamenten und Toxinen. Zu den endogenen transportierten Substanzen gehört auch der Neurotransmitter Acetylcholin (ACh), der unter anderem in der menschlichen Haut eine wichtige Rolle in der Zelldifferenzierung und –proliferation spielt. Die dermatologischen Antiinfektiva Gentianaviolett (GV) und Brillantgrün (BG) aus der Gruppe der Triphenylmethanfarbstoffe werden in der Behandlung lokaler Wundinfektionen verwendet und zeigen im klinischen Gebrauch Nebenwirkungen wie Wundheilungsstörungen. Es konnte gezeigt werden, dass die Farbstoffe die OCTs konzentrationsabhängig hemmen und es wurden die Werte der halbmaximalen Hemmkonzentration ermittelt. Dabei zeigte sich, dass GV und BG zu den Hemmstoffen mit der höchsten Affinität zu den OCTs gehören. Ein Transport der Farbstoffe durch die OCTs konnte nicht nachgewiesen werden, die toxische Wirkung auf Keratinozyten in in-vitro Versuchen mit menschlichen Zellreihen wurde bestätigt. Ein Zusammenhang zwischen den beobachteten Wundheilungsstörungen unter der Therapie mit Triphenylmethanfarbstoffen und der Hemmung des ACh-Transportes durch die OCTs konnte nicht bestätigt werden.
Background:
Sleep-related eating may occur in the context of mental illness, sleep disorders, or psychopharmacological treatment. Frequently, sleep-related eating leads to severe weight gain and, so far, there are no treatment options for the condition.
Case presentation:
We report the case of a 54-year-old white woman with depression, panic disorder, and sleep apnea under treatment with various antidepressants who developed severe sleep-related eating. Her sleep-related eating completely vanished after addition of agomelatine, it reoccurred after cessation of agomelatine, and vanished again after her re-exposure to another melatonergic drug, extended melatonin.
Conclusions:
This case suggests that melatonergic drugs lead to relief from sleep-related eating, even when the condition occurs in the context of physical and mental disorders as well as psychopharmacological treatment.
Die Nikotinamid N-Methyltransferase (NNMT) wurde als wichtiger Regulator des Energiemetabolismus in Fettzellen beschrieben. So bewahrt ein NNMT Knock-down Mäuse vor einer nahrungsinduzierten Adipositas und bei reduzierter NNMT-Expression in weißen 3T3-L1 Adipozyten zeigen diese einen erhöhten zellulären Sauerstoffverbrauch.
Für den Adipozytenstoffwechsel ist die insulinstimulierte Glukoseaufnahme wesentlich. Um den Einfluss eines NNMT-Knock-downs auf diese zu untersuchen wurde unter Nutzung der Substratspezifitäten des prokaryotischen und eukaryotischen Isoenzyms der Glukose-6-phosphat-Dehydrogenase ein enzymatisch-photometrischer Assay zur Messung der Glukoseaufnahmerate in adhärenten weißen 3T3-L1 und braunen Adipozytenkulturen entwickelt.
Mit lentiviraler Transduktion wurde in den Adipozytenkulturen ein persistenter NNMT-Knock-down induziert. Die NNMT-Aktivität wurde mit einem fluoreszenzbasierten Assay gemessen und die Glukoseaufnahmerate in deren Abhängigkeit bestimmt.
Die Reduktion der NNMT-Aktivität verminderte die Glukoseaufnahmerate der 3T3-L1 Adipozyten sowohl basal, wie auch unter Insulinstimulation. Braune Adipozyten hingegen zeigten bei verringerter NNMT-Aktivität eine erhöhte insulinstimulierte Glukoseaufnahmerate, aber keinen Unterschied der basalen Glukoseaufnahmerate.
Dieser differenzielle Einfluss auf die Glukoseaufnahme weißer und brauner Adipozyten stärkt die wichtige Rolle der NNMT, die ihr zur Regulation des Fettzellstoffwechsels zugeschrieben wird und enthüllt erstmals eine direkte Wirkung auf braune Adipozyten.
In der vorliegenden Studie wurde untersucht, ob sich bei Patienten mit Bronchialkarzinom durch eine Second-Line-Chemotherapie mit dem Tyrosinkinaseinhibitor Erlotinib ein Nutzen erzielen lässt. Dabei wurde besonderes Augenmerk auf Faktoren gelegt, die das progressionsfreie Überleben bei diesen Patienten möglicherweise beeinflussen können. Grundlage der Untersuchungen bildete ein Probanden- kollektiv von 42 Patienten, die sich mit der Diagnose nicht-kleinzelliges Bronchialkarzinom bis zum 31.12.2007 einer Second-Line-Chemotherapie mit Erlotinib unterzogen. Zu diesem Stichtag lagen für 23 Patienten Todesdaten vor. Das mediane progressionsfreie Überleben für alle untersuchten 42 Patienten betrug 3,88 Monate, wobei sich die Spanne von 0 bis 21 Monate erstreckte. Im Gesamten zeigten 59,52% der Patienten ein Ansprechen, das heißt, das progressionsfreie Überleben dauerte länger als einen Monat. Folgende allgemeine Patientenmerkmale wurden aus den Krankenblättern erhoben: Geschlecht, Alter, histologische Tumordiagnose, Tumorstadium nach Stadieneinteilung durch die TNM-Klassifikation, Metastasensorte soweit Metastasen vorlagen, Zeitpunkt der Erstdiagnose, Rauchverhalten, Allgemeinzustand nach Karnofsky-Index, Vorerkrankungen, Begleitmedikation, ausgewählte Laborwerte sowie Zeitraum und Art der First-Line-Therapie. Was die Second-Line-Therapie unter Erlotinib betrifft, so wurden hier gezielt im Verlauf die aktuelle Dosis, Änderungen der Begleittherapie, Allgemeinzustand nach Karnofsky-Index, Toxizität (besonderes Augenmerk verdienten Nebenwirkungen an der Haut und im Gastrointestinaltrakt), Auffälligkeiten von einigen Laborwerten und das Tumorverhalten untersucht. Zum Abschluss wurden das Ende der Erlotinibtherapie, Grund des Absetzens, aktueller Stand, eventuell im Anschluss an die Erlotinibtherapie durchgeführte Therapien sowie im Falle des Todes der Todeszeitpunkt erfasst. Im Gesamtüberblick konnten somit das progressionsfreie Überleben unter Erlotinib, die Einnahmedauer des Tyrosinkinaseinhibitors und das absolute Überleben seit der Erstdiagnose festgestellt werden. Hauptziel der Untersuchung war es, herauszufinden, inwiefern unsere Patienten unter Erlotinib im Sinne einer Verlängerung ihres progressionsfreien Überlebens profitieren und welche Faktoren die Ansprechrate beeinflussen. Der Einfluss ausgewählter Merkmale wurde statistisch untersucht. Im Vergleich mit Angaben in der Literatur konnte für das untersuchte Probandenkollektiv eine repräsentative Verteilung der Patientenmerkmale ermittelt und somit eine gewisse Relevanz der Ergebnisse dieser Studie angenommen werden. Statistisch signifikante Unterschiede ergaben sich für folgende Faktoren: Einnahmedauer, Krankheitsverhalten bei Therapiebeginn, Karnofsky-Index während der Behandlung, Nebenwirkungen an der Haut sowie Veränderungen in der Serumchemie. Nonresponder nahmen folglich Erlotinib wie erwartet statistisch signifikant kürzer ein, zeigten zu Beginn der Erlotinibtherapie statistisch signifikant häufiger ein fortschreitendes Krankheitsgeschehen, wiesen während der Therapie statistisch signifikant häufiger einen schlechteren Karnofsky-Index auf, erlitten statistisch signifikant weniger häufig schwere Nebenwirkungen an der Haut (Grad 3 und 4) und ihnen waren statistisch signifikant häufiger Serumwertveränderungen zuzuschreiben. Das prognostische Hauptmerkmal, das sowohl bei unserem Patientengut als auch in der Literatur signifikante Bedeutung hat, ist wohl, dass Responder statistisch signifikant häufiger schwere Nebenwirkungen an der Haut erleiden. Da Auftreten und Schweregrad von follikulären Ekzemen also mit einem positiven Ansprechen von Erlotinib korreliert sind, könnte es vielleicht nützlich sein, wenn man bereits vor Beginn der Erlotinibtherapie vorhersagen könnte, wie wahrscheinlich Patienten solche Nebenwirkungen entwickeln werden. Insgesamt kann man sagen, dass der Einsatz dieser molekular zielgerichteten Substanz zu einer Erweiterung der Therapiemöglichkeiten im palliativen Setting bei Bronchialkarzinompatienten geführt hat. Die Behandlung mit Erlotinib bedeutet ein Benefit für das Gesamtüberleben und das progressionsfreie Überleben bei Patienten mit fortgeschrittenem Bronchialkarzinom, die im Voraus eine Chemotherapie erhalten hatten. Dieses Benefit beschränkt sich nicht auf klinische Subgruppen wie weibliches Geschlecht, Adenokarzinom in der Histologie und Nichtraucher. Diese klinischen Parameter allein sind wohl folglich nicht ausreichend, um Patienten im Vornherein zu identifizieren, die wahrscheinlich von der Therapie profitieren. Vielmehr sind bessere Methoden notwendig, um möglicherweise voraussagen zu können, wer von dem Tyrosinkinaseinhibitor profitieren wird. Hoffnungen ruhen auf der Etablierung molekularer prognostischer und prädiktiver Marker in der klinischen Routine.
Hintergrund: Die Schlafbezogene Atmungsstörung (SBAS) ist eine häufige Komorbidität der systolischen Herzinsuffizienz und mit einer kürzeren Lebenserwartung assoziiert. Es steht derzeit eine Vielzahl einfacher ambulanter SBAS-Screening-Geräte zur Verfügung. Keines davon wurde jedoch bisher für Patienten mit chronischer systolischer Herzinsuffizienz validiert.
Fragestellung: Die vorliegende Untersuchung diente der Prüfung eines einfachen ambulanten SBAS-Screenings hinsichtlich diagnostischer Trennschärfe und prognostischer Relevanz für Patienten mit systolischer Herzinsuffizienz.
Methoden: Bei Patienten mit symptomatischer systolischer Herzinsuffizienz (linksventrikuläre Ejektionsfraktion (LVEF) < 45%, NYHA ≥ II) wurden nächtlicher Atemfluss, Pulsfrequenz und Sauerstoffsättigung mit dem ApneaLinkTM (Fa. ResMed) ambulant aufgezeichnet. Hieraus werden der Apnoe-/Hypopnoe-Index (AHI) sowie der Entsättigungsindex (ODI) berechnet. Die Ergebnisse wurden der Diagnose des Schlafmediziners anhand einer PSG als gültigem Goldstandard der SBAS-Diagnostik gegenübergestellt. Der Überlebensstatus wurde bei allen Patienten durch ambulante bzw. telefonische Nachuntersuchungen erfasst. Mittels ROC-Analysen wurden diagnostische und durch Cox-Regressionen prognostische Schwellenwerte des SBAS-Screenings ermittelt.
Ergebnisse: Insgesamt wurden 131 Patienten eingeschlossen: das mittlere Alter lag bei 68±13 Jahren, 110 Patienten (84%) waren männlich, 53 Patienten (41%) hatten ein NYHA-Stadium ≥3, die mittlere LVEF lag bei 34%. Bei 69 Patienten (53%) konnte eine PSG durchgeführt werden, welche bei 55 Patienten (80%) eine SBAS diagnostizierte. Bei 38 Patienten (55%) lag eine mind. mittelgradige, therapiepflichtige SBAS vor. In ROC-Analysen für ein mind. mittelgradige SBAS ergaben sich AUCs von 0.77; 0.82; 0.81; 0,79 und 0,82 für AHI; ODI; (AHI+ODI)/2; (2xAHI+ODI)/3 und (AHI+2xODI)/3. Die größtmögliche Spezifität (0,90) und Sensitivität (0,66) für die Diagnose einer Therapiepflichtigen SBAS lab bei einem cut-off von (AHI+2xODI)/3 ≥ 21/h. Die mediane Follow-up Zeit der Studie lag bei 23 (18; 27) Monaten; es starben 21 Patienten (16%). Es fand sich eine positive Korrelation zwischen (AHI+2xODI)/3 und Mortalitätsrate. Bereits für den (AHI+2xODI)/3 ≥9/h war die Mortalität signifikant erhöht (3,55 (95%CI: 1.04-12.13) p=0,04).
Schlussfolgerung: Eine therapiepflichtige SBAS lässt durch ein ambulantes Screening zuverlässig diagnostizieren. Dabei ist der ODI dem AHI überlegen. Für die bestmögliche diagnostische Trennschärfe empfehlen wir einen (AHI+2xODI)/3 von 21/h heranzuziehen, wobei bereits ein Wert von 9/h prognostisch relevant ist, so dass bei Patienten mit einem (AHI+2xODI)/3 von 9-20/h zwar auf eine schlafmedizinische Untersuchung verzichtet werden kann, eine engmaschige kardiologische Kontrolle inklusive wiederholten Screenings jedoch unabdingbar bleibt.
Bacteria lose or gain genetic material and through selection, new variants become fixed in the population. Here we provide the first, genome-wide example of a single bacterial strain’s evolution in different deliberately colonized patients and the surprising insight that hosts appear to personalize their microflora. By first obtaining the complete genome sequence of the prototype asymptomatic bacteriuria strain E. coli 83972 and then resequencing its descendants after therapeutic bladder colonization of different patients, we identified 34 mutations, which affected metabolic and virulence-related genes. Further transcriptome and proteome analysis proved that these genome changes altered bacterial gene expression resulting in unique adaptation patterns in each patient. Our results provide evidence that, in addition to stochastic events, adaptive bacterial evolution is driven by individual host environments. Ongoing loss of gene function supports the hypothesis that evolution towards commensalism rather than virulence is favored during asymptomatic bladder colonization.
Viele Studien weisen auf einen Zusammenhang zwischen einer verlängerten QTc-Zeit und dem Auftreten von Torsade-de-Pointes-Tachyarrhythmien und dem plötzlichen Herztod hin. Auch AV-Blockierungen und Schenkelblöcke
erhöhen das Risiko für kardiale Erkrankungen und Ereignisse deutlich. Eine direkte Beziehung zwischen Serumspiegeln der trizyklischen Antidepressiva
und der Verlängerung der PQ-, QRS- oder QTc-Zeit wurde bisher jedoch nicht untersucht.
Aus diesem Anlass wurde in dieser Arbeit nun versucht, eine Korrelation zwischen den Serumspiegeln der trizyklischen Antidepressiva Amitriptylin und
Doxepin bzw. ihrer Metabolite und einigen EKG-Parametern zu identifizieren und zu beschreiben. Hierfür wurden die Daten von 172 Patienten der Klinik und
Poliklinik für Psychiatrie, Psychosomatik und Psychotherapie untersucht, die eine Serumspiegelbestimmung des Talspiegels im Steady-State der TZA einen Tag vor, einen Tag nach oder am selben Tag einer EKG-Ableitung erhalten hatten und
keine pathologischen Elektrolytwerte vorwiesen.
In der Rangkorrelation zeigte sich ein signifikanter positiver Zusammenhang zwischen Nortriptylin-Spiegel und QTcB (r = 0,24; p < 0,05) sowie QTcH (r = 0,205;
p < 0,05), zwischen Nortriptylin und QTcF und QTcLC lag dieser Zusammenhang auf Trendniveau. Zwischen PQ-Zeit und Nortriptylin- (r = 0,226; p < 0,05) sowie
Summenkonzentration (r = 0,195; p < 0,05) zeigte sich ebenfalls ein signifikanter positiver Zusammenhang.
Im Mann-Whitney-U-Test waren die QTc-Zeiten der Patienten mit Summenspiegeln aus Amitriptylin und Nortriptylin jenseits der Warnschwelle
von 300 ng/ml signifikant länger als bei den Patienten mit niedrigeren Summenspiegeln (QTcB = 449 zu 432 ms; QTcF = 423 zu 410 ms; QTcH = 423 zu 410 ms; QTcLC = 421 zu 409 ms; p < 0,05) und auch die PQ-Zeit dieser Gruppe
war signifikant verlängert (PQ = 163 zu 179 ms; p < 0,05). Ähnliches galt für das Patientenkollektiv mit einem Nortriptylin-Spiegel oberhalb des Referenzbereichs von 170 ng/ml. Dieses zeigte signifikant längere
QTc-Zeiten nach allen Korrekturmethoden (QTcB = 457 zu 432 ms; QTcF = 430 zu 409 ms; QTcH = 429 zu 410 ms; QTcLC = 427 zu 409 ms; p < 0,01) und zudem
längere PQ- (164 zu 180 ms; p < 0,05) und QRS-Zeiten (98 zu 107 ms; p = 0,054).
Diese Ergebnisse machen deutlich, dass eine regelmäßige EKG-Kontrolle während der Einnahme von trizyklischen Antidepressiva notwendig ist, um kardiale Nebenwirkungen frühzeitig zu erkennen und diesen vorzubeugen.
Ebenso wichtig sind regelmäßige Serumspiegelbestimmungen, um das Risiko durch erhöhte Serumspiegel jenseits der Warnschwellen bzw. Referenzbereiche frühzeitig zu erkennen. Eine Beachtung der Tagesdosis allein reicht hier explizit nicht aus.
Im Rahmen des metabolischen Syndroms bei morbider Adipositas kommt es unter anderem zu Imbalancen im autonomen Nervensystem (ANS): ein Missverhältnis von Sympathikus und Parasympathikus (PS) führt zu einer Dysregulation von orexigenen und anorexigenen Hormonen und konsekutiv zu einer weiteren Gewichtszunahme.
Diese Arbeit untersucht die Auswirkung von bariatrischen Operationen auf das abdominale ANS anhand des parasympathisch regulierten PPP drei Monate nach Intervention. Die Veränderungen der Imbalance wurden mit Hilfe eines modifizierten Sham Feedings (MSF) ermittelt, bei dem nur die kephale Phase der Nahrungsaufnahme durchlaufen und die Mahlzeiten nicht geschluckt wird. Die PPP-Spiegel im Verlauf des MSF sind daher ausschließlich eine Darstellung des PS. Zusätzlich wurden die Verläufe der Insulin- und Glukosewerte bestimmt.
Die PPP-Spiegel der normalgewichtigen Kontrollgruppe sind signifikant höher als die der adipösen Patienten prä- und postoperativ. Die Kurvenverläufe des PPP sind aber zwischen den Gruppen vergleichbar. Somit ist die akute Reaktion des PS auf die Nahrungsaufnahme bei morbider Adipositas zwar unverändert, es zeigt sich aber keine Verbesserung der ANS-Dysfunktion kurz nach Intervention. Die Aufteilung der Patienten in Jüngere und Ältere (verglichen mit dem Durchschnittsalter der Studie) ergibt postoperativ absolut und relativ eine Zunahme der PPP-Spiegel bei den Älteren, während es bei den Jüngeren zu einem Abfall kommt. Bei halbierter Gruppegröße ist die Aussagekraft der Signifikanz jedoch eingeschränkt.
Auch ohne diabetische Stoffwechsellage gleichen sich die Insulin- und Glukosewerte der Patienten post-OP denen der Kontrollen an. Eine mögliche Ursache hierfür ist aber nicht nur eine primäre ANS-Verbesserung, denn vor allem in der ersten postoperativen Phase scheint die starke Nahrungsmittelrestriktion mehr Auswirkungen zu haben. Eine spätere Besserung der ANS-Imbalance durch z.B. Gewichtsverlust ist jedoch nicht ausgeschlossen.
In der vorliegenden Studie wurde der Zusammenhang des depressiven Syndroms mit dem Vitamin D-Spiegel an einer Stichprobe gerontopsychiatrischer Patienten (n = 140) der Neurogerontopsychiatrischen Tagesklinik Würzburg untersucht. Die Depressivität der Patienten zu Beginn und im Verlauf der Behandlung wurde zum einen mittels der ICD-10-Klassifikation, zum anderen mittels des Scores auf der GDS- und Hamilton-Skala zu Beginn und Ende des Aufenthalts in der Tagesklinik sowie bei einer poststationären Kontrolle bestimmt. Der Vitamin D-Spiegel wurde bei Behandlungsbeginn bestimmt und im Falle eines Mangels 1000 IU Vitamin D am Tag oral substituiert. Hierbei zeigte sich kein Zusammenhang zwischen der Ausprägung des depressiven Syndroms und dem Vitamin D-Spiegel zu Beginn der Behandlung. Dagegen stellte sich heraus, dass Patienten mit einem höheren Spiegel eine deutlichere Verbesserung der depressiven Symptome auf der GDS im Verlauf der Behandlung erfuhren. Außerdem bestand eine signifikante negative Korrelation zwischen BMI und Vitamin D-Spiegel sowie eine Abhängigkeit der Spiegelhöhe von der Jahreszeit. Vitamin D könnte nach den Ergebnissen dieser Studie möglicherweise eine wirkungssteigernde und nebenwirkungsarme Komedikation in der antidepressiven Therapie von älteren psychisch erkrankten Menschen darstellen. Es bedarf weiterer ausführlicher Forschung über den neurophysiologischen Zusammenhang zwischen Vitamin D und der Schwere einer depressiven Erkrankung. Besonders hinsichtlich der Verwendung von Vitamin D als Komedikation gilt es, weitere intensive Forschung in Form von gut designten, randomisierten Fall-Kontroll-Studien und prospektiven Interventionsstudien zu betreiben, um die Therapie von depressiven Patienten im höheren Lebensalter weiter zu verbessern.
Depression is a common psychiatric disorder among geriatric patients that decreases the quality of life and increases morbidity and mortality. Vitamin D as a neuro-steroid hormone might play a role in the onset and treatment of depression. In the present study, the association between depressive symptoms and vitamin D concentration in serum was evaluated. 140 patients of a psychogeriatric day-care unit were included. The geriatric depression scale (GDS) and the Hamilton depression rating scale (HDRS) were assessed at the beginning and end of treatment, GDS scores additionally 6 weeks after discharge from the day-care unit. Vitamin D levels were measured at the beginning of the treatment, routinely. Patients with levels below 30 µg/L were treated with 1000 IU vitamin D per day. There was no association between the severity of depressive symptoms and the concentration of vitamin D at the beginning of the treatment. Patients with higher vitamin D levels showed a stronger decline of depressive symptoms measured by the GDS during their stay in the day-care unit. We provide evidence that vitamin D serum levels might influence antidepressant therapy response in a geriatric population. Prospective studies are necessary to determine which patients may profit from add-on vitamin D therapy.