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Total numbers and percentage of hematopoietic subsets in young Pax5+/- and wild type mice.

The PDF includes legends for Supplementary Tables S1 - S5 as well as legends for Supplementary Figures S1 - S3. Supplementary Figure S1 indicates pathogens detected in mice held in the SPF and conventional animal facility. Supplementary Figure S2 represents flow cytometric, histological and gene set enrichment characterization in Pax5+/- mice. Supplementary Figure S3 refers to in vivo growth of Ba/F3 cells expressing Jak3V670A and Jak3R653H mutations, respectively.

Supplementary Table 4: Differentially expressed genes in bone marrow proB and preB cells from ETV6-RUNX1 mice compared to wild-type bone marrow proB and preB cells. (12423 genes-probesets with FDR = 0.1).

Contents: Supplementary Table 1: Characteristics of the pediatric patients enrolled in whole-genome- and whole-exome-sequencing. Supplementary Table 2: Conventional Facility Health Monitoring Report. Supplementary Table 3: Percentage of hematopoietic subsets in young ETV6-RUNX1 and wild type mice housed in conventional facility. Supplementary Table 5: Differential regulation of histonemodifying genes in ETV6-RUNX1 mice compared to human ETV6-RUNX1 pB-ALL. Supplementary Table 6: Alterations in genes related to histone modification in pB-ALL human samples Supplementary Table 7: Summary of whole genome sequencing statistics. Supplementary Table 8: Summary of exome sequencing statistics. Supplementary Table 9: Validated SNVs in the patient cohort. Supplementary Fig. 1: Distribution of genomic ETV6-RUNX1 breakpoints amongst patients. Supplementary Fig. 2: Development of Sca1-ETV6-RUNX1 mice. Supplementary Fig. 3: pB-ALL development in Sca1-ETV6-RUNX1 mice and B cell development in young Sca1-ETV6-RUNX1 mice housed in SPF facility. Supplementary Fig. 4: Tumor exome sequencing data identify genes mutated in mouse and human. Supplementary Fig. 5: Study of proB cells in young Sca1-ETV6-RUNX1 mice.

ETV6-RUNX1 is associated with the most common subtype of childhood leukemia. As few ETV6-RUNX1 carriers develop precursor B-cell acute lymphocytic leukemia (pB-ALL), the underlying genetic basis for development of full-blown leukemia remains to be identified, but the appearance of leukemia cases in time-space clusters keeps infection as a potential causal factor. Here, we present in vivo genetic evidence mechanistically connecting preleukemic ETV6-RUNX1 expression in hematopoetic stem cells/precursor cells (HSC/PC) and postnatal infections for human-like pB-ALL. In our model, ETV6-RUNX1 conferred a low risk of developing pB-ALL after exposure to common pathogens, corroborating the low incidence observed in humans. Murine preleukemic ETV6-RUNX1 pro/preB cells showed high Rag1/2 expression, known for human ETV6-RUNX1 pB-ALL. Murine and human ETV6-RUNX1 pB-ALL revealed recurrent genomic alterations, with a relevant proportion affecting genes of the lysine demethylase (KDM) family. KDM5C loss of function resulted in increased levels of H3K4me3, which coprecipitated with RAG2 in a human cell line model, laying the molecular basis for recombination activity. We conclude that alterations of KDM family members represent a disease-driving mechanism and an explanation for RAG off-target cleavage observed in humans. Our results explain the genetic basis for clonal evolution of an ETV6-RUNX1 preleukemic clone to pB-ALL after infection exposure and offer the possibility of novel therapeutic approaches. Cancer Res; 77(16); 4365–77. ©2017 AACR.

In early modern medical literature, there are increasing references to sterility and impotence in older men. This is especially true of the

Recently, a rare type of relapse was reported upon treating a B cell acute lymphoblastic leukemia (B-ALL) patient with anti-CD19 chimeric antigen receptor (CAR)-T cells caused by unintentional transduction of residual malignant B cells (CAR-B cells). We show that anti-CD19 and anti-CD20 CARs are presented on the surface of lentiviral vectors (LVs), inducing specific binding to the respective antigen. Binding of anti-CD19 CAR-encoding LVs containing supernatant was reduced by CD19-specific blocking antibodies in a dose-dependent manner, and binding was absent for unspecific LV containing supernatant. This suggests that LVs bind via displayed CAR molecules to CAR antigen-expressing cells. The relevance for CAR-T cell manufacturing was evaluated when PBMCs and B-ALL malignant B cells were mixed and transduced with anti-CD19 or anti-CD20 CAR-displaying LVs in clinically relevant doses to mimic transduction conditions of unpurified patient leukapheresis samples. Malignant B cells were transduced at higher levels with LVs displaying anti-CD19 CARs compared to LVs displaying non-binding control constructs. Stability of gene transfer was confirmed by applying a potent LV inhibitor and long-term cultures for 10 days. Our findings provide a potential explanation for the emergence of CAR-B cells pointing to safer manufacturing procedures with reduced risk of this rare type of relapse in the future., Graphical abstract, Schaser and colleagues found that lentiviral vectors encoding B cell-specific CARs display the respective CAR on the particle surface. The LV-displayed CARs retained their antigen-binding potential and specifically promoted transduction of malignant B cells. These findings provide a potential explanation for the emergence of CAR-expressing malignant cells in B-ALL patients.

Biofilms are closely packed cells held and shielded by extracellular matrix composed of structural proteins and exopolysaccharides (EPS). As matrix components are costly to produce and shared within the population, EPS-deficient cells can act as cheaters by gaining benefits from the cooperative nature of EPS producers. Remarkably, genetically programmed EPS producers can also exhibit phenotypic heterogeneity at single cell level. Previous studies have shown that spatial structure of biofilms limits the spread of cheaters, but the long-term influence of cheating on biofilm evolution is not well understood. Here, we examine the influence of EPS non-producers on evolution of matrix production within the populations of EPS producers in a model biofilm-forming bacterium, Bacillus subtilis. We discovered that general adaptation to biofilm lifestyle leads to an increase in phenotypical heterogeneity of eps expression. Apparently, prolonged exposure to EPS-deficient cheaters, may result in different adaptive strategy, where eps expression increases uniformly within the population. We propose a molecular mechanism behind such adaptive strategy and demonstrate how it can benefit the EPS-producers in the presence of cheaters. This study provides additional insights on how biofilms adapt and respond to stress caused by exploitation in long-term scenario.

The stem cell marker and RNA-binding protein Musashi-1 is overexpressed in endometriosis. Musashi-1-siRNA knockdown in Ishikawa cells altered the expression of stem cell related genes, such as OCT-4. To investigate the role of both human Musashi homologues (MSI-1 and MSI-2) in the pathogenesis of endometriosis, immortalized endometriotic 12-Z cells and primary endometriotic stroma cells were treated with Musashi-1- and Musashi-2-siRNA. Subsequently, the impact on cell proliferation, cell apoptosis, cell necrosis, spheroid formation, stem cell phenotype and the Notch signaling pathway was studied in vitro. Using the ENDOMET Turku Endometriosis database, the gene expression of stem cell markers and Notch signaling pathway constituents were analyzed according to localization of the endometriosis lesions. The database analysis demonstrated that expression of Musashi and Notch pathway-related genes are dysregulated in patients with endometriosis. Musashi-1/2-double-knockdown increased apoptosis and necrosis and reduced stem cell gene expression, cell proliferation, and the formation of spheroids. Musashi silencing increased the expression of the anti-proliferation mediator p21. Our findings suggest the therapeutic potential of targeting the Musashi–Notch axis. We conclude that the Musashi genes have an impact on Notch signaling and the pathogenesis of endometriosis through the downregulation of proliferation, stemness characteristics and the upregulation of apoptosis, necrosis and of the cell cycle regulator p21.

Many critical advances in research utilize techniques that combine high-resolution with high-content characterization at the single cell level. We introduce the MICS (MACSimaTM Imaging Cyclic Staining) technology, which enables the immunofluorescent imaging of hundreds of protein targets across a single specimen at sub-cellular resolution. MICS is based on cycles of staining, imaging, and erasure, using photobleaching of fluorescent labels of recombinant antibodies (REAfinityTM), release of antibodies (REAleaseTM) or their labels (REAdyeleaseTM). Multimarker analysis can identify potential targets for immune therapy against solid tumors. With MICS we analysed human glioblastoma, ovarian and pancreatic carcinoma, and 16 normal tissues. One potential target pair for chimeric antigen receptor (CAR) T-cell therapies identified for ovarian carcinoma is EPCAM/THY1. Using an adapter CAR T cell approach, we show selective killing of cells only in presence of both markers. MICS represents a new high content microscopy methodology to be widely used for personalized medicine.

The reduction of 4-nitrophenol (4-NP) to 4-aminophenol (4-AP) with sodium borohydride (NaBH4) in aqueous media is by far the most widely used reaction for testing the catalytic performance of a lar...

ZUSAMMENFASSUNG Ziel der Arbeit Im vorliegenden Beitrag untersuchen wir, inwiefern die Lektüre des Romans „Unendlicher Spaß“ von David Foster Wallace eine Erweiterung des psychiatrischen Wissenshorizontes in Bezug auf Abhängigkeitserkrankungen ermöglicht und wie die dargestellten gesellschaftlichen Entwicklungen zu einem umfassenderen Verständnis dieser Psychopathologie beitragen können. Ferner hinterfragen wir, ob auch Patienten von der Lektüre therapeutisch profitieren können. Methodik Der Roman „Unendlicher Spaß“ wird in Hinblick auf die Fragestellungen mit den Methoden der qualitativen Literaturanalyse interpretiert, insbesondere unter Zuhilfenahme einer Gegenüberstellung von Romanpassagen und der ICD-10-Klassifikation von Abhängigkeitserkrankungen. Ergebnisse Den ausgewählten Roman kennzeichnet eine personalisierte und hyperrealistisch-detaillierte Ausgestaltung von in psychiatrischer Fachliteratur evident dargestellten Symptomkomplexen. Der Roman eignet sich besonders zur Sensibilisierung von Psychiatern für gesellschaftliche Entwicklungen im Blick auf das Sucht- und Gesundheitsverständnis. Schlussfolgerung Psychiatrische Lektüre fiktionaler Literatur kann – unabhängig von Krankheit und Format – zu einem erweiterten Krankheitsverständnis führen und dadurch klinische Erfahrung ergänzen und reflektieren.

Background Assistance systems serving the locomotion of older people interact in many ways with the culture of a society. Since early modern times at the latest, walking aids were tantamount to human frailty; however, the cane also symbolized governmental power or reputation. Nowadays, the cane, the wheelchair, and the rollator have not only a functional significance in terms of a better mobility, they also enable people to take an active part in social life. Objective This study aimes at tracing back these provisional insights into the history of civilization and thereby analyze the roots, new forms and pictures of the handling and metaphors of these assistance systems. The goal in the context of this special issue is to decipher a central textual and pictorial symbol of old age, comparing it with more recent symbols of assistance in old age. Material and methods Methodologically, the text combines approaches of philology and history of medicine with those of the history of art. It analyzes (after a brief retrospection of ancient times and the Middle Ages) by means of textual and pictorial sources from the sixteenth to the twenty-first centuries the historical development of these aids of locomotion for older people. Additionally, it explores the cultural relevancy of these assistance systems. Results and discussion In history the medical profession paid relatively late and then only minor attention to the assistive systems analyzed here. Its semantic diversity is closely related to the age roles and stereotypes of age prevailing in certain epochs. The more the respective assistive tool is used by old people, the more suitable it is as a symbol of old age and the more biased and negative the semantic connotation seems to be. The development of a symbol of age connoting frailty, at present symbolized most clearly by the rollator, tends to refer to a pejorative image of age in a society. The cultural historical analysis suggests that a contrasting development will only be possible when the assistive systems will again fulfil a diversity of alternative functions and semantics.

The present article summarises the recommendations for the handling, histopathological workup, diagnostics and reporting in surgical pathology of biopsies and resection specimens in patients with the clinical diagnosis of endometriosis. In addition to practical aspects of pathology, the guidelines also take into account the clinical requirements for histopathology for the optimal diagnosis and therapy of the patients.Based on the definition of endometriosis of the corpus uteri (adenomyosis uteri) most commonly used in the pathological literature, this was defined in the guidelines as the detection of the endometriosis focus in the myometrium at a distance from the endomyometrial border of a medium-sized visual field (100× magnification), which in metric units corresponds to around 2.5 mm. In bowel resection specimens, the status of the resection margins had to be documented within the histopathological report.Also mentioned are the requirements for the reporting of carcinomas associated with endometriosis, including the immunohistochemical evaluation of steroid hormone receptors and mismatch repair proteins.Die vorliegende Übersicht fasst die relevanten Aspekte der S2k-Leitlinie Endometriose zusammen. Die Empfehlungen umfassen die Aufarbeitung und Befunderhebung bei Biopsien und Resektaten, die bei der klinischen Diagnose einer Endometriose entnommen wurden. Die Leitlinie berücksichtigt neben praktischen Aspekten der Pathologie ebenso die klinischen Notwendigkeiten an die Histopathologie für eine optimale Diagnostik und Therapie der Patientinnen.Basierend auf der in der pathologischen Literatur gebräuchlichsten Definition der Endometriose des Corpus uteri (Adenomyosis uteri) wurde diese in der Leitlinie definiert als der Nachweis des Endometrioseherdes im Myometrium in einem Abstand zur endomyometranen Grenze von einem mittelgroßen Gesichtsfeld (100fache Vergrößerung), was metrisch rund 2,5 mm entspricht. Bei Darmresektaten soll zum Status der Resektionsränder Stellung genommen werden.Ebenso definiert werden Anforderungen im Kontext endometrioseassoziierter Karzinome (z. B. Hormonrezeptorbestimmungen, immunhistochemische Untersuchung auf Ausfall der DNA-Mismatch-Reparaturproteine).

A major roadblock prohibiting effective cellular immunotherapy of pancreatic ductal adenocarcinoma (PDAC) is the lack of suitable tumor-specific antigens. To address this challenge, here we combine flow cytometry screenings, bioinformatic expression analyses and a cyclic immunofluorescence platform. We identify CLA, CD66c, CD318 and TSPAN8 as target candidates among 371 antigens and generate 32 CARs specific for these molecules. CAR T cell activity is evaluated in vitro based on target cell lysis, T cell activation and cytokine release. Promising constructs are evaluated in vivo. CAR T cells specific for CD66c, CD318 and TSPAN8 demonstrate efficacies ranging from stabilized disease to complete tumor eradication with CD318 followed by TSPAN8 being the most promising candidates for clinical translation based on functionality and predicted safety profiles. This study reveals potential target candidates for CAR T cell based immunotherapy of PDAC together with a functional set of CAR constructs specific for these molecules., There is an unmet clinical need to identify therapeutic options for the treatment of pancreatic cancer (PDAC). Here the authors present a systematic screening approach for the identification of potential PDAC cell surface target candidates for CAR-T cell based immunotherapy, followed by their functional validation in preclinical models.

A domain that is often neglected in the assessment of chimeric antigen receptor (CAR) functionality is the extracellular spacer module. However, several studies have elucidated that membrane proximal epitopes are best targeted through CARs comprising long spacers, while short spacer CARs exhibit highest activity on distal epitopes. This finding can be explained by the requirement to have an optimal distance between the effector T cell and target cell. Commonly used long spacer domains are the CH2-CH3 domains of IgG molecules. However, CARs containing these spacers generally show inferior in vivo efficacy in mouse models compared to their observed in vitro activity, which is linked to unspecific Fcγ-Receptor binding and can be abolished by mutating the respective regions. Here, we first assessed a CAR therapy targeting membrane proximal CD20 using such a modified long IgG1 spacer. However, despite these mutations, this construct failed to unfold its observed in vitro cytotoxic potential in an in vivo model, while a shorter but less structured CD8α spacer CAR showed complete tumor clearance. Given the shortage of well-described long spacer domains with a favorable functionality profile, we designed a novel class of CAR spacers with similar attributes to IgG spacers but without unspecific off-target binding, derived from the Sialic acid-binding immunoglobulin-type lectins (Siglecs). Of five constructs tested, a Siglec-4 derived spacer showed highest cytotoxic potential and similar performance to a CD8α spacer in a CD20 specific CAR setting. In a pancreatic ductal adenocarcinoma model, a Siglec-4 spacer CAR targeting a membrane proximal (TSPAN8) epitope was efficiently engaged in vitro, while a membrane distal (CD66c) epitope did not activate the T cell. Transfer of the TSPAN8 specific Siglec-4 spacer CAR to an in vivo setting maintained the excellent tumor killing characteristics being indistinguishable from a TSPAN8 CD8α spacer CAR while outperforming an IgG4 long spacer CAR and, at the same time, showing an advantageous central memory CAR T cell phenotype with lower release of inflammatory cytokines. In summary, we developed a novel spacer that combines cytotoxic potential with an advantageous T cell and cytokine release phenotype, which make this an interesting candidate for future clinical applications.