Molecular mechanisms of human T cell anergy induced by the cytoplasmic tail of CD45

Die klassische Proteintyrosinphosphatase CD45 kann in humanen Monozyten und Granulozyten bei Aktivierung durch Zymosan oder PMA proteolytisch gespalten werden. Die Freisetzung der cytoplasmatischen Domäne von CD45 (ct-CD45) erfolgt durch aktivierungsinduzierten Zelltod. Diese Domäne bindet als löslicher Faktor an humane T-Zellen und reduziert Proliferation und Zytokinsekretion was zur Ausbildung eines anergieähnlichen Zustandes führt. Das Ziel der vorliegenden Arbeit ist es die molekularen Mechanismen, welche hinter der von ct-CD45 verursachten Anergie stehen, zu analysieren. Mit PRAT4A hat unser Labor in einer früheren Studie einen Rezeptorkandidaten für ct-CD45 identifiziert. In dieser Studie demonstrieren wir, dass die Bindung von ct-CD45 an eine PRAT4A-überexprimierende Zelllinie durch einen Antikörper gegen PRAT4A unterbunden werden kann. Es wird gezeigt, dass die Induzierung dieses Zustandes von einer verminderten Bildung von Lymphoblasten begleitet wird, was auf einen frühen Zellzyklusarrest hindeutet. Microarray-Analyse von humanen T-Zellen die in der Gegenwart von ct-CD45 aktiviert wurden, zeigte die Überexpression zweier Faktoren – Krüppel-like factor 2 (KLF2) und Schlafen family member 12 (SLFN12). Während KLF2 ein bekannter, mit der Regulation von T-Zellquieszenz assoziierter Faktor ist, weiß man wenig über SLFN12. Dieser Faktor wird nur in Primaten exprimiert und gehört einer Genfamilie an, die mit Thymozytenentwicklung und zellulärer Wachstumsregulierung in Verbindung gebracht wird. Diese Studie bestätigt die Überexpression von beiden Faktoren mittels quantitativer Realtime-PCR und identifiziert SLFN12 als möglichen Anergiefaktor. IL-2 reduzierte die Expression von SLFN12 mRNA wogegen KLF2 kaum reguliert wurde. SLFN12 wurde zudem bereits kurz nach Aktivierung in der Präsenz von ct-CD45 hochreguliert. Die Transfektion einer SLFN12 siRNA verbesserte die Proliferation von T-Zellen die in der Gegenwart von ct-CD45 aktiviert wurden. Die Expressionsmuster von Zellzyklusfaktoren unterstützen einen frühen Zellzyklusarrest durch ct-CD45, da sich die Expression von Zyklin D1, nicht aber von anderen Faktoren verringert zeigte. Zusammenfassend zeigt diese Arbeit, dass ct-CD45 über die Bindung an PRAT4A T-Zellanergie induziert und zu einer Hochregulierung von SLFN12 führt. Die spezifische Inhibierung von Zyklin D1 führt zu einem frühen Zellzyklusarrest, der sich in einer verringerten Bildung von Lymphoblasten manifestiert, welche morphologisch ruhenden Zellen ähnlich sind. Unsere Daten unterstützen daher einen neuen funktionalen Differenzierungszustand von TLymphozyten.The prototypic receptor-like protein tyrosine phosphatase CD45 is proteolytically cleaved in human monocytes and granulocytes upon activation via zymosan or PMA. The cytoplasmic tail of ct-CD45 (ct-CD45) is released via activation-induced cell death. Ct-CD45 acts as a cytokine-like factor on human T cells, thereby, potently reducing proliferation and cytokine production leading to an anergy-like state in T cells. The aim of this work is to elucidate the molecular mechanisms governing ct-CD45-induced T cell anergy. A potential receptor candidate for ct-CD45 on T cells was identified to be PRAT4A. Here, we demonstrate that binding of ct-CD45 to a murine cell line transfected with human PRAT4A can be blocked by an antibody against PRAT4A. Induction of the anergic state is accompanied by an inhibition of T cell blast formation suggesting an early cell cycle arrest. Microarray analysis of T cells activated in the presence of ct-CD45 indicated upregulation of two factors not known to be associated with anergy before - Schlafen family member 12 (SLFN12) and Krueppel-like factor 2 (KLF2). While KLF2 is a transcription factor implicated in the maintenance of T cell quiescence, little is known about SLFN12. SLFN12 is a primate-specific factor which belongs to a gene family that is involved in thymocyte development and cellular growth regulation. The expression of both factors was confirmed and analyzed via quantitative real-time PCR identifying SLFN12 as a putative anergy factor. IL-2 strongly reduced SLFN12 mRNA whereas KLF2 expression was barely changed. Furthermore only SLFN12 showed early induction in ct-CD45-treated T cells. Transfection of a SLFN12 siRNA could partially restore proliferation of anergic cells. Expression profiling of cell cycle-associated factors supported an early cell cycle arrest as cyclin D1 showed impaired induction while other factors were unaltered. Taken together, these results demonstrate that ct-CD45 induces anergy via PRAT4A subsequently leading to the induction of SLFN12. Inhibition of cyclin D1 results in an early cell cycle arrest characterized by reduced lymphoblast formation resembling cellular quiescence. Thus, our data supports a novel functional state for T cells

Next-generation sequencing of the CHO cell transcriptome

Meeting abstract from 22nd European Society for Animal Cell Technology(ESACT) Meeting on Cell Based Technologies Vienna, Austria. 15-18 May 2011(VLID)90656

HSF2BP negatively regulates homologous recombination in DNA interstrand crosslink repair

The tumor suppressor BRCA2 is essential for homologous recombination (HR), replication fork stability and DNA interstrand crosslink (ICL) repair in vertebrates. We show that ectopic production of HSF2BP, a BRCA2-interacting protein required for meiotic HR during mouse spermatogenesis, in non-germline human cells acutely sensitize them to ICL-inducing agents (mitomycin C and cisplatin) and PARP inhibitors, resulting in a phenotype characteristic of cells from Fanconi anemia (FA) patients. We biochemically recapitulate the suppression of ICL repair and establish that excess HSF2BP compromises HR by triggering the removal of BRCA2 from the ICL site and thereby preventing the loading of RAD51. This establishes ectopic expression of a wild-type meiotic protein in the absence of any other protein-coding mutations as a new mechanism that can lead to an FA-like cellular phenotype. Naturally occurring elevated production of HSF2BP in tumors may be a source of cancer-promoting genomic instability and also a targetable vulnerability

Dosimetric precision of an ion beam tracking system

<p>Abstract</p> <p>Background</p> <p>Scanned ion beam therapy of intra-fractionally moving tumors requires motion mitigation. GSI proposed beam tracking and performed several experimental studies to analyse the dosimetric precision of the system for scanned carbon beams.</p> <p>Methods</p> <p>A beam tracking system has been developed and integrated in the scanned carbon ion beam therapy unit at GSI. The system adapts pencil beam positions and beam energy according to target motion.</p> <p>Motion compensation performance of the beam tracking system was assessed by measurements with radiographic films, a range telescope, a 3D array of 24 ionization chambers, and cell samples for biological dosimetry. Measurements were performed for stationary detectors and moving detectors using the beam tracking system.</p> <p>Results</p> <p>All detector systems showed comparable data for a moving setup when using beam tracking and the corresponding stationary setup. Within the target volume the mean relative differences of ionization chamber measurements were 0.3% (1.5% standard deviation, 3.7% maximum). Film responses demonstrated preserved lateral dose gradients. Measurements with the range telescope showed agreement of Bragg peak depth under motion induced range variations. Cell survival experiments showed a mean relative difference of -5% (-3%) between measurements and calculations within the target volume for beam tracking (stationary) measurements.</p> <p>Conclusions</p> <p>The beam tracking system has been successfully integrated. Full functionality has been validated dosimetrically in experiments with several detector types including biological cell systems.</p

Standardised data collection for clinical follow-up and assessment of outcomes in differences of sex development (DSD): recommendations from the COST action DSDnet

The treatment and care of individuals who have a difference of sex development (DSD) have been revised over the past two decades and new guidelines have been published. In order to study the impact of treatments and new forms of management in these rare and heterogeneous conditions, standardised assessment procedures across centres are needed. Diagnostic work-up and detailed genital phenotyping are crucial at first assessment. DSDs may affect general health, have associated features or lead to comorbidities which may only be observed through lifelong follow-up. The impact of medical treatments and surgical (non-) interventions warrants special attention in the context of critical review of current and future care. It is equally important to explore gender development early and refer to specialised services if needed. DSDs and the medical, psychological, cultural and familial ways of dealing with it may affect self-perception, self-esteem, and psychosexual function. Therefore, psychosocial support has become one of the cornerstones in the multidisciplinary management of DSD, but its impact remains to be assessed. Careful clinical evaluation and pooled data reporting in a global DSD registry will allow linking genetic, metabolomic, phenotypic and psychological data. For this purpose, our group of clinical experts and patient and parent representatives designed a template for structured longitudinal follow-up. In this paper, we explain the rationale behind the selection of the dataset. This tool provides guidance to professionals caring for individuals with a DSD and their families. At the same time, it collects the data needed for answering unsolved questions of patients, clinicians, and researchers. Ultimately, outcomes for defined subgroups of rare DSD conditions should be studied through large collaborative endeavours using a common protocol

Observer variability of absolute and relative thrombus density measurements in patients with acute ischemic stroke

Introduction: Thrombus density may be a predictor for acute ischemic stroke treatment success. However, only limited data on observer variability for thrombus density measurements exist. This study assesses the variability and bias of four common thrombus density measurement methods by expert and non-expert observers. Methods: For 132 consecutive patients with acute ischemic stroke, three experts and two trained observers determined thrombus density by placing three standardized regions of interest (ROIs) in the thrombus and corresponding contralateral arterial segment. Subsequently, absolute and relative thrombus densities were determined using either one or three ROIs. Intraclass correlation coefficient (ICC) was determined, and Bland–Altman analysis was performed to evaluate interobserver and intermethod agreement. Accuracy of the trained observer was evaluated with a reference expert observer using the same statistical analysis. Results: The highest interobserver agreement was obtained for absolute thrombus measurements using three ROIs (ICCs ranging from 0.54 to 0.91). In general, interobserver agreement was lower for relative measurements, and for using one instead of three ROIs. Interobserver agreement of trained non-experts and experts was similar. Accuracy of the trained observer measurements was comparable to the expert interobserver agreement and was better for absolute measurements and with three ROIs. The agreement between the one ROI and three ROI methods was good. Conclusion: Absolute thrombus density measurement has superior interobserver agreement compared to relative density measurement. Interobserver variation is smaller when multiple ROIs are used. Trained non-expert observers can accurately and reproducibly assess absolute thrombus densities using three ROIs

Analysis of single nucleotide polymorphisms in the FAS and CTLA-4 genes of peripheral T-cell lymphomas

Angioimmunoblastic T-cell lymphoma (AILT) represents a subset of T-cell lymphomas but resembles an autoimmune disease in many of its clinical aspects. Despite the phenotype of effector T-cells and high expression of FAS and CTLA-4 receptor molecules, tumor cells fail to undergo apoptosis. We investigated single nucleotide polymorphisms (SNPs) of the FAS and CTLA-4 genes in 94 peripheral T-cell lymphomas. Although allelic frequencies of some FAS SNPs were enriched in AILT cases, none of these occurred at a different frequency compared to healthy individuals. Therefore, SNPs in these genes are not associated with the apoptotic defect and autoimmune phenomena in AILT

Automated entire thrombus density measurements for robust and comprehensive thrombus characterization in patients with acute ischemic stroke

Background and Purpose: In acute ischemic stroke (AIS) management, CT-based thrombus density has been associated with treatment success. However, currently used thrombus measurements are prone to inter-observer variability and oversimplify the heterogeneous thrombus composition. Our aim was first to introduce an automated method to assess the entire thrombus density and then to compare the measured entire thrombus density with respect to current standard manual measurements. Materials and Method: In 135 AIS patients, the density distribution of the entire thrombus was determined. Density distributions were described usingmedians, interquartile ranges (IQR), kurtosis, and skewedness. Differences between themedian of entire thrombusmeasurements and commonly applied manualmeasurements using 3 regions of interest were determined using linear regression. Results: Density distributions varied considerably with medians ranging from 20.0 to 62.8 HU and IQRs ranging from 9.3 to 55.8 HU. The average median of the thrombus density distributions (43.5 ± 10.2 HU) was lower than the manual assessment (49.6 ± 8.0 HU) (p<0.05). The difference between manual measurements and median density of entire thrombus decreased with increasing density (r = 0.64; p<0.05), revealing relatively higher manual measurements for low density thrombi such that manual density measurement tend overestimates the real thrombus density. Conclusions: Automatic measurements of the full thrombus expose a wide variety of thrombi density distribution, which is not grasped with currently used manual measurement. Furthermore, d

Two-year clinical follow-up of the Multicenter Randomized Clinical Trial of Endovascular Treatment for Acute Ischemic Stroke in The Netherlands (MR CLEAN): Design and statistical analysis plan of the extended follow-up study

Background: MR CLEAN was the first randomized trial to demonstrate the short-term clinical effectiveness of endovascular treatment in patients with acute ischemic stroke caused by large vessel occlusion in the anterior circulation. Several other trials confirmed that endovascular treatment improves clinical outcome at three months. However, limited data are available on long-term clinical outcome. We aimed to estimate the effect of endovascular treatment on functional outcome at two-year follow-up in patients with acute ischemic stroke. Secondly, we aimed to assess the effect of endovascular treatment on major vascular events and mortality during two years of follow-up. Methods: MR CLEAN is a multicenter clinical trial with randomized treatment allocation, open-label treatment, and blinded endpoint evaluation. Patients included were 18 years or older with acute ischemic stroke caused by a proven anterior proximal artery occlusion who could be treated within six hours after stroke onset. The intervention contrast was endovascular treatment and usual care versus no endovascular treatment and usual care. The current study extended the follow-up duration from three months to two years. The primary outcome is the score on the modified Rankin scale at two years. Secondary outcomes include all-cause mortality and the occurrence of major vascular events within two years of follow-up. Discussion: The results of our study provide information on the long-term clinical effectiveness of endovascular treatment, which may have implications for individual treatment decisions and estimates of cost-effectiveness. Trial registration:NTR1804. Registered on 7 May 2009; ISRCTN10888758. Registered on 24 July 2012 (main MR CLEAN trial); NTR5073. Registered on 26 February 2015 (extended follow-up study)