Regulation of T cell activation by ct-CD45

CD45 war eines der ersten signaling Moleküle die auf Leukozyten identifi-ziert worden sind und wird als Leukozyt Markermolekül verwendet. Es ist der Prototyp von Transmembranrezeptor ähnlichen Protein Tyrosin Phos-phatasen (PTPs) und es werden verschiedene Isoformen davon auf allen kernhaltigen hematopoetischen Zellen exprimiert. CD45 spielt eine essen-zielle rolle im Immunsystem indem es verschiedene Substrate dephospho-ryliert. Vor kurzem wurde, von unserer Gruppe, eine alternative Funktion für die intrazelluläre Domäne dieses Proteins (ct-CD45) gefunden. Es wurde gezeigt, dass wenn humane Monozyten und Granulozyten mit Pilz-stimuli aktiviert werden CD45 gespalten und ct-CD45 freigesetzt wird. Die-ses ct-CD45 agiert als zytokinähnlicher Faktor, der die T Zell Proliferation hemmt wenn diese mit dendritischen Zellen oder CD3 Antikörper aktiviert werden. Der zytoplasmatische Teil von CD45 kann dabei als interzellulärer Regulator zwischen dem angeborenen und dem adaptiven Immunsystem wirken. In dieser Studie wurde die direkte Wirkung von ct-CD45 auf die Aktivierung und die Funktion von T Zellen in einem APC freien System weiter untersucht. Wir haben entdeckt, dass ct-CD45 die Proliferation von peripheren T Zellen inhibiert wenn diese über CD3 oder CD3/CD63 akti-viert werden aber nicht wenn sie mit CD3/CD28 stimuliert werden. Im Ge-gensatz dazu hatte ct-CD45 keinen Einfluss auf die Proliferation von Na-belschnurblut T Zellen, die auf die gleiche Weise aktiviert worden sind. Überraschender Weise wurde dennoch die Produktion von IL-2, IL-4, IL-10, IL-13, IL-17 und IFN gamma stark reduziert, auch in den T Zellen bei denen die Proliferation nicht gehemmt worden ist. Die einzige Ausnahme war IL-4, welches eine erhöhte Produktion in Nebelschnur Blut T Zellen in der Anwesenheit von ct-CD45 zeigte. Genauso wie die Zytokine wurde auch die Expression von neu synthetisierten Aktivierungsmarkern wie CD25, CD97 und MHCII und auch die Oberflächenexpression von CD69, das zum Teil schon in intrazellulären Pools gespeichert wird und dann von dort aus an die Zelloberfläche gebracht wird, herunter reguliert. Darüber hinaus haben wir entdeckt, dass diese mit ct-CD45 behandelten T Zellen bei Restimulierung ohne ct-CD45 keine Proliferation zeigen und dass die-ser Effekt nur zum Teil durch Zugabe von IL-2 rückgängig gemacht wer-den kann. Außerdem haben wir versucht einen möglichen Rezeptorkandidaten für ct-CD45 auf aktivierten T Zellen zu finden. Es wurde gezeigt, dass dieses Protein spezifisch an aktivierte T Zellen bindet und dass diese Bindung mit mAb blockiert werden kann. Bindungsassays mit einer cDNA Bibliothek für aktivierte T Zellen haben eine Bindung zwischen ct-CD45 und einem Pro-tein (PRAT4A), das mit dem Toll-like Rezeptor 4 assoziiert ist, gezeigt. PRAT4A wurde vor kurzem entdeckt und als ER-residentes Chaperon be-schrieben, das unentbehrlich für das Trafficking der meisten TLRs ist. Obwohl dieses Protein als ER-resident beschrieben worden ist ist es uns gelungen es auch auf der Zelloberfläche von aktivierten T Zellen nachzu-weisen. In dieser Diplomarbeit haben wir gezeigt, dass PRAT4A ein potentieller Rezeptorkandidat für ct-CD45 ist und dass die Bindung dieses cytoplas-matischen Teiles von CD45 an aktivierte T Zellen zu einem Anergie ähnli-chen Stadium führt, das nur zum Teil durch Zugabe von IL-2 umgekehrt werden kann.CD45 was one of the first signaling molecules identified on leukocytes and is used as a leukocyte marker molecule. It is the prototypic member of transmembrane receptors like protein tyrosine phosphatases (PTPs) and various forms of it are expressed on all nucleated hematopoetic cells. CD45 plays an essential role in immune functions by dephosphorylating different substrates. Recently an alternative function for the intracellular domain of CD45 (ct-CD45) was discovered by our group. It was shown that CD45 is cleaved and ct-CD45 is released during activation of human monocytes and granulocytes by fungal stimuli. Furthermore ct-CD45 was found to ct-CD45 act as a cytokine like factor which inhibits T cell prolifera-tion induced by dendritic cells or CD3 antibodies. The cytoplasmatic tail of CD45 can thereby act as an intercellular regulator between the innate and the adaptive immune system. In this study the direct impact of ct-CD45 on the activation and function of T cells was further investigated in an antigen presenting cell free system. We found that ct-CD45 inhibited CD3 and CD3/CD63 but not CD3/CD28 induced proliferation in peripheral T cells and that it did not inhibit the proliferation of cord blood T cells which were stimulated the same way. Surprisingly the production of IL-2, IL-4, IL-10, IL-13, IL-17 and IFN gamma was strongly reduced even in those T cells whose proliferation was not inhibited. The only exception was IL-4 which showed an increased production in cord blood T cells upon stimulation in the presence of ct-CD45. In line with the downregulation of cytokine pro-duction the expression of de novo synthesized activation markers on the cell surface of T-cells like CD25, CD97 and MHCII and the surface ex-pression of CD69, which originate in part from preformed intracellular pools, was down regulated. Furthermore we found out that these ct-CD45 treated T cells show no proliferation upon restimulation without ct-CD45 and that this effect can be only partly reversed by addition of exogenous IL-2. In addition to that we were trying to find a possible receptor candidate for ct-CD45 on activated T cells. It was shown that this protein binds specifi-cally to activated T cells and that this binding can be blocked by mAb. Binding assays with a cDNA library for activated T cells gave evidence for interaction of ct-CD45 and a protein associated with Toll-like receptor 4 (PRAT4A). PRAT4A was recently discovered as an ER-resident chaperon which is indispensable for the trafficking of most TLRs. Even though this protein is described as ER resident binding assays showed that PRAT4A is also expressed on the cell surface of activated T cells. In this study we showed that PRAT4A is a possible receptor candidate for ct-CD45 and that the binding of this cytoplasmatic part of CD45 to acti-vated T cells lead to an anergy like hyporesponsive state that can only be partly reversed by the addition of exogenous IL-2

HOW BLOCKCHAIN FACILITATES SMART CITY APPLICATIONS– DEVELOPMENT OF A MULTI-LAYER TAXONOMY

A decade after Sathosi Nakamoto published his famous whitepaper, blockchain technology (BT) has started to become widely recognized and used beyond the cryptocurrency Bitcoin. While the financial sector is the most prominent adopter of the technology, numerous other fields of application for the ground-breaking innovation are discussed by researchers and practitioners alike. One key area in which blockchain-based applications are expected to drive radical and disruptive innovation is smart cities. BT provides unique benefits which smart cities can leverage to improve quality of life, adminis-trative processes, and environmental sustainability. However, due to the entrepreneurial dynamics and abundant fields of application for BT in smart cities, an integrated and boundary-spanning analysis is lacking. Thus, our paper aims at analysing how BT is used in different smart city business models to present a multi-layer taxonomy. For this purpose, we identified a global sample of 80 startups which offer products or services for smart cities and examined their business models. The paper explores economic and technological characteristics of blockchain based smart city applications. These unique insights will be useful for researchers, practitioners, and regulators

Retreatment with interferon-alpha and ribavirin in primary interferon-alpha non-responders with chronic hepatitis C

Background/Aims: Combination therapy with interferon-alpha (IFN-alpha) plus ribavirin is more efficacious than IFN-alpha monotherapy in previously untreated patients with chronic hepatitis C and patients with IFN-alpha relapse. Only limited data are available in IFN-alpha non-responders. In a multicenter trial we therefore evaluated the efficacy of combination therapy in IFN-alpha-resistant chronic hepatitis C. Methods: Eighty-two patients (mean age 46.8 years, 54 males, 28 females) with chronic hepatitis C were treated with IFN-alpha-2a (3 x 6 MIU/week) and ribavirin (14 mg/kg daily) for 12 weeks. Thereafter, treatment was continued only in virological responders (undetectable serum HCV RNA at week 12) with an IFN-alpha dose of 3 x 3 MIU/week and without ribavirin for a further 9 months. The primary study endpoint was an undetectable HCV RNA by RT-PCR at the end of the 24-week follow-up period. Results: After 12 weeks of combination therapy, an initial virological response was observed in 29 of 82 (35.4%) patients. Due to a high breakthrough rate after IFN-a dose reduction and ribavirin discontinuation, an end-of-treatment response was only achieved in 12 of 82 (14.6%) patients. After the follow-up period, a sustained virological response was observed in 8 of 82 (9.8%) patients. Infection with HCV genotype 3 was the only pretreatment parameter, which could predict a sustained response (HCV-1, 5%; HCV-3, 57.1%; p < 0.001). Conclusions: Despite a high initial response rate of 35.4%, sustained viral clearance was achieved only in 9.8% of the retreated primary IFN-alpha non-responders. Higher IFN-alpha induction and maintenance dose, as well as prolonged ribavirin treatment may possibly increase the virological response rates in non-responders, particularly in those infected by HCV-1

Ageism: an old concept from new perspectives.

This article is an introduction to the special issue "New horizons in ageism research: innovation in study design, methodology and applications to research, policy and practice". This special issue aims to offer a broad and innovative perspective on ageism. The first section addresses new developments in the conceptualization of ageism. This section focuses not only on the negative side of ageism, but also on benevolent ageism, which is manifested in protective attitudes and behaviors towards older persons because of their age, following the stereotype of older persons as a vulnerable group that needs protection. The second section concerns the manifestation of ageism: between traditional and underexplored arenas. The third section concerns innovative methods to explore the concept of ageism. This section relies on innovations in qualitative and quantitative methods to explore nuances in the manifestations of ageism. The next section addresses interventions to reduce or prevent ageism

New forms of ageism as a challenge for a un convention on the rights of older persons

This article contributes to the ongoing debate around the need for a new United Nations convention on the human rights of older persons. It explores new forms of ageism that have so far been overlooked in the discussion and explores the value of a new treaty from a symbolic point of view. Analyzing the instrumentalization of older persons by ageing policies, and the devaluation of older age as part of age imperialism, we argue that a new treaty can valorize older people’s social status and address them as autonomous subjects. This can be achieved by emphasizing the individual dignity of older persons and by expressing their equal legal recognition. This exploration of the conceptual challenges posed by new forms of ageism provides arguments that may prove useful for reframing the debate around a new convention.acceptedVersionPeer reviewe

Convenient diastereoselective synthesis of annulated 3-substituted-(5S*,6S*,Z)-2-(2-(2,4-dinitrophenyl)hydrazono)-5,6-diphenyl-1,3-thiazinan-4-ones

Racemic 2-(2,4-dinitrophenyl)hydrazono)-5,6-diphenyl-1,3-thiazinan-4-ones and (Z)-N '-(2,4-dinitrophenyl)-2,3-diphenylacrylohydrazide were formed during the diastereoselective reaction between 4-substituted 1-(2,4-dinitrophenyl)thiosemicarbazides and 2,3-diphenylcycloprop-2-enone under refluxing ethanol. The structures of the synthesized compounds were confirmed by single-crystal X-ray analyses. [GRAPHICS] .Peer reviewe

Familiäre Kavernome des Zentralnervensystems: Eine klinische und genetische Studie an 15 deutsche Familien

Zusammenfassung: 1928 beschrieb Hugo Friedrich Kufs erstmalig eine Familie mit zerebralen, retinalen und kutanen Kavernomen. Mittlerweile wurden über 300 weitere Familien beschrieben. Ebenfalls wurden drei Genloci 7q21-q22 (mit dem Gen CCM1), 7p15-p13 (Gen CCM2) und 3q25.2-q27 (Gen CCM3) beschrieben, in denen Mutationen zu Kavernomen führen. Das Genprodukt von CCM1 ist das Protein Krit1 (Krev Interaction Trapped 1), das über verschiedene Mechanismen mit der Angiogenese interagiert. Das neu entdeckte CCM2-Gen enkodiert ein Protein, das möglicherweise eine dem Krit1 ähnliche Funktion in der Regulation der Angiogenese hat. Das CCM3-Gen wurde noch nicht beschrieben. In dieser Arbeit werden sowohl die klinischen und genetischen Befunde bei 15 deutschen Familien beschriebe

Nanoliter Scale Parallel Liquid–Liquid Extraction for High‐Throughput Purification on a Droplet Microarray

In the current drug discovery process, the synthesis of compound libraries is separated from biological screenings both conceptually and technologically. One of the reasons is that parallel on-chip high-throughput purification of synthesized compounds is still a major challenge. Here, on-chip miniaturized high-throughput liquid–liquid extraction in volumes down to 150 nL with efficiency comparable to or better than large-scale extraction utilizing separation funnels is demonstrated. The method is based on automated and programmable merging of arrays of aqueous nanoliter droplets with organic droplets. Multi-step extraction performed simultaneously or with changing conditions as well as handling of femtomoles of compounds are demonstrated. In addition, the extraction efficiency is analyzed with a fast optical readout as well as matrix-assisted laser desorption ionization-mass spectrometry on-chip detection. The new massively parallel and miniaturized purification method adds another important tool to the chemBIOS concept combining chemical combinatorial synthesis with biological screenings on the same miniaturized droplet microarray platform, which will be essential to accelerate drug discovery

Buckled Thin-Film Transistors and circuits on soft elastomers for stretchable electronics

Although recent progress in the field of flexible electronics has allowed the realization of biocompatible and conformable electronics, systematic approaches which combine high bendability ( 3-4 %) and low complexity in the fabrication process are still missing. Here, we show a technique to induce randomly-oriented and customized wrinkles on the surface of a biocompatible elastomeric substrate, where Thin-Film Transistors (TFTs) and circuits (inverter and logic NAND gates) based on Amorphous-IGZO are fabricated. By tuning the wavelength and the amplitude of the wrinkles, the devices are fully operational while bent to 13 µm bending radii as well as while stretched up to 5%, keeping unchanged electrical properties. Moreover, a flexible rectifier is also realized, showing no degradation in the performances while flat or wrapped on an artificial human wrist. As proof of concept, transparent TFTs are also fabricated, presenting comparable electrical performances to the non-transparent ones. The extension of the buckling approach from our TFTs to circuits demonstrates the scalability of the process, prospecting applications in wireless stretchable electronics to be worn or implanted