Micro-RNA-155 inhibits IFN-γ signaling in CD4+ T cells

Micro-RNA (miR) are increasingly recognized as critical regulators of tissue-specific patterns of gene expression. CD4+ T cells lacking miR-155, for example, exhibit bias towards Th2 differentiation, indicating that the absence of individual miR could alter CD4+ T cell differentiation. We now show that MiR-155 is induced upon T cell activation and that it promotes Th1 differentiation when over-expressed in activated CD4+ T cells. Antagonism of miR-155 leads to induction of IFN-γRα, and a functional miR-155 target site is identified within the 3′ UTR of IFN-γRα. These results identify IFN-γRα as a second miR-155 target in T cells and suggest that miR-155 contributes to Th1 differentiation in CD4+ T cells by inhibiting IFN-γ signaling

Aktivierung von RARalpha führt zur Symptombesserung in EAE durch Induktion von FoxP3

Autoimmune diseases have a high incidence and are a major cause of morbidity in the developed world. They occur when the immune system mistakes self for non-self tissue and mounts an inappropriate attack. This can be viewed as a result of decreased immunological tolerance. One autoimmune disease in particular, multiple sclerosis, has been described as the most common cause of neurological disability in young adults. Autoreactive T cells are involved in numerous autoimmune processes. In MS, T cells attack the myelin nerve sheaths around neurons. The present work examines the role of retinoids in the immune system. It is well known that retinoids can enhance immunological tolerance. However, the mechanism of how this tolerance is achieved had not been shown. The first part of our studies investigated the effects of retinoids on T cell development in vitro. In the second part, the findings were tested in an animal model of MS, i.e., EAE. We were able to show that retinoids can skew T cell differentiation towards the regulatory T cell subset, which is associated with increased immunological tolerance. This augmentation of regulatory T cell numbers occurs at the expense of the Th17 subset, which is implied in many autoimmune processes, including multiple sclerosis. Furthermore, we presented evidence that the increased regulatory T cell numbers and decreased Th17 numbers are caused by the activation of the nuclear receptor RARalpha. In vitro studies were carried out using highly specific agonists and antagonists of RARalpha as well as retroviral overexpression. Furthermore, we tested our finding of RARalpha mediated tolerance in vivo. In EAE, activation of RARalpha led to amelioration of symptoms and an increased number of regulatory T cells in the CNS. The successful amelioration of EAE by RARalpha agonists suggests that RARalpha agonists could be important drug candidates in the prevention and treatment of MS, in particular as one RARalpha activator is already used in clinical practice. In more general terms, RARalpha activators could be versatile clinical tools to modulate the immune response towards increased tolerance.Autoimmunkrankheiten haben eine hohe Inzidenz und tragen wesentlich zur Morbidität in der entwickelten Welt bei. Autoimmunerkrankungen entstehen, wenn das Immunsystem fälschlicherweise eigenes Gewebe als fremd ansieht und eine Abwehrreaktion einleitet. Dieser Prozeß kann als das Resultat einer verminderten immunologischen Toleranz gesehen werden. Multiple Sklerose ist eine Autoimmunerkrankung, die möglicherweise die häufigste Ursache neurologischer Behinderung in jungen Erwachsenen darstellt. Autoreaktive T-Zellen sind an zahlreichen autoimmunen Prozessen beteiligt. Bei der multiplen Sklerose attackieren autoreaktive T-Zellen die Myelinscheiden der Neurone. Die vorliegende Arbeit untersucht die Rolle von Retinoiden im Immunsystem. Es ist bekannt, daß Retinoide die immunologische Toleranz erhöhen können. Der Mechanismus für diese Toleranzerhöhung war jedoch bis jetzt unklar. Im ersten Teil dieser Arbeit untersuchten wir den Einfluß von Retinoiden auf die Entwicklung von T Zellen in vitro. Im zweiten Teil wurden die Ergebnisse in EAE getestet, einem Tiermodell der Multiplen Sklerose. Wir konnten demonstrieren, daß Retinoide die T Zell-Differenzierung steuern können, so dass mehr regulatorische T Zellen entstehen, die mit erhöhter immunologischer Toleranz verbunden sind. Die Erhöhung der regulatorischen T -Zell-Zahlen ging auf Kosten der Entstehung von Th17 T-Zellen, die an vielen autoimmunen Prozessen beteiligt sind, auch bei der Multiplen Sklerose. Zudem konnten wir zeigen, dass eine erhöhte Anzahl regulatorischer T-Zellen und eine verminderte Anzahl von Th17 Zellen durch die Aktivierung des nukleären Rezeptor RARalpha erzeugt wird. In vitro Studien sowohl mit hochspezifischen RARalpha Agonisten und -Antagonisten als auch mit retroviraler Überexpression wurden durchgeführt. Zusätzlich testeten wir unsere Ergebnisse in vivo. Im Tiermodell EAE führte die Aktivierung von RARalpha zu einer Symptomverbesserung und einer erhöhten regulatorischen T-Zell-Zahl im ZNS. Die erfolgreiche Symptomverbesserung bei EAE durch RARalpha-Agonisten legt nahe, daß RARalpha-Agonisten wichtige Kandidaten bei der Suche nach Medikamenten für die Multiple Sklerose sein könnten, insbesondere weil ein RARalpha-Agonist bereits eine klinische Zulassung für einen anderen Anwendungsbereich besitzt. Ganz allgemein könnten RARalpha-Agonisten ein wertvolles klinisches Werkzeug zur Modulation der Immunantwort hin zu höherer Toleranz sein

Mapping the binding site for gossypol-like inhibitors of Plasmodium falciparum lactate dehydrogenase.

Gossypol is a di-sesquiterpene natural-product in the form of a functionalised binaphthyl and is isolated from cotton plants. The compound has long been known to exhibit anti-malarial and other biological activities. Previous studies have indicated that compounds of this type target Plasmodium falciparum lactate dehydrogenase (pfLDH), an essential enzyme for energy generation within the parasite. In this study, we report that simple naphthalene-based compounds, the core of the gossypol structure, exhibit weak inhibition of the parasite lactate dehydrogenase. Crystal structures of the complexes formed by binding of these naphthalene-based compounds to their target enzyme have been used to delineate the molecular features likely to form the gossypol binding site. Two modes of binding are observed: one overlapping the pyruvate but not the co-factor site, the other bridging the binding sites for the co-factor nicontinamide group and pyruvate substrate. This latter site encompasses molecular features unique to Plasmodium forms of LDH and is likely to represent the mode of binding for gossypol derivatives that show selectivity for the parasite enzymes. We also report a substrate analogue that unexpectedly binds within the adenine pocket of the co-factor groove. Although these core pharmacophore-like molecules only exhibit low levels of inhibitory activity, these molecular snapshots provide a rational basis for renewed structure-based development of naphthalene-based compounds as anti-malarial agents

Refined sgRNA efficacy prediction improves large- and small-scale CRISPR-Cas9 applications

Genome editing with the CRISPR-Cas9 system has enabled unprecedented efficacy for reverse genetics and gene correction approaches. While off-target effects have been successfully tackled, the effort to eliminate variability in sgRNA efficacies-which affect experimental sensitivity-is in its infancy. To address this issue, studies have analyzed the molecular features of highly active sgRNAs, but independent cross-validation is lacking. Utilizing fluorescent reporter knock-out assays with verification at selected endogenous loci, we experimentally quantified the target efficacies of 430 sgRNAs. Based on this dataset we tested the predictive value of five recently-established prediction algorithms. Our analysis revealed a moderate correlation (r = 0.04 to r = 0.20) between the predicted and measured activity of the sgRNAs, and modest concordance between the different algorithms. We uncovered a strong PAM-distal GC-content-dependent activity, which enabled the exclusion of inactive sgRNAs. By deriving nine additional predictive features we generated a linear model-based discrete system for the efficient selection (r = 0.4) of effective sgRNAs (CRISPRater). We proved our algorithms' efficacy on small and large external datasets, and provide a versatile combined on-and off-target sgRNA scanning platform. Altogether, our study highlights current issues and efforts in sgRNA efficacy prediction, and provides an easily-applicable discrete system for selecting efficient sgRNAs

The non-coding RNA landscape of human hematopoiesis and leukemia

textabstractNon-coding RNAs have emerged as crucial regulators of gene expression and cell fate decisions. However, their expression patterns and regulatory functions during normal and malignant human hematopoiesis are incompletely understood. Here we present a comprehensive resource defining the non-coding RNA landscape of the human hematopoietic system. Based on highly specific non-coding RNA expression portraits per blood cell population, we identify unique fingerprint non-coding RNAs-such as LINC00173 in granulocytes-and assign these to critical regulatory circuits involved in blood homeostasis. Following the incorporation of acute myeloid leukemia samples into the landscape, we further uncover prognostically relevant non-coding RNA stem cell signatures shared between acute myeloid leukemia blasts and healthy hematopoietic stem cells. Our findings highlight the importance of the non-coding transcriptome in the formation and maintenance of the human blood hierarchy

Secreted Antiviral Entry Inhibitory (SAVE) Peptides for Gene Therapy of HIV Infection

Gene therapeutic strategies for human immunodeficiency virus type 1 (HIV-1) infection could potentially overcome the limitations of standard antiretroviral drug therapy (ART). However, in none of the clinical gene therapy trials published to date, therapeutic levels of genetic protection have been achieved in the target cell population for HIV-1. To improve systemic antiviral efficacy, C peptides, which are efficient inhibitors of HIV-1 entry, were engineered for high-level secretion by genetically modified cells. The size restrictions for efficient peptide export through the secretory pathway were overcome by expressing the C peptides as concatemers, which were processed into monomers by furin protease cleavage. These secreted antiviral entry inhibitory (SAVE) peptides mediated a substantial protective bystander effect on neighboring nonmodified cells, thus suppressing virus replication even if only a small fraction of cells was genetically modified. Accordingly, these SAVE peptides may provide a strong benefit to AIDS patients in future, and, if applied by direct in vivo gene delivery, could present an effective alternative to antiretroviral drug regimen