Crucial role of SLP-76 and ADAP for neutrophil recruitment in mouse kidney ischemia-reperfusion injury.

Neutrophils trigger inflammation-induced acute kidney injury (AKI), a frequent and potentially lethal occurrence in humans. Molecular mechanisms underlying neutrophil recruitment to sites of inflammation have proved elusive. In this study, we demonstrate that SLP-76 (SH2 domain-containing leukocyte phosphoprotein of 76 kD) and ADAP (adhesion and degranulation promoting adaptor protein) are involved in E-selectin-mediated integrin activation and slow leukocyte rolling, which promotes ischemia-reperfusion-induced AKI in mice. By using genetically engineered mice and transduced Slp76(-/-) primary leukocytes, we demonstrate that ADAP as well as two N-terminal-located tyrosines and the SH2 domain of SLP-76 are required for downstream signaling and slow leukocyte rolling. The Tec family kinase Bruton tyrosine kinase is downstream of SLP-76 and, together with ADAP, regulates PI3Kγ (phosphoinositide 3-kinase-γ)- and PLCγ2 (phospholipase Cγ2)-dependent pathways. Blocking both pathways completely abolishes integrin affinity and avidity regulation. Thus, SLP-76 and ADAP are involved in E-selectin-mediated integrin activation and neutrophil recruitment to inflamed kidneys, which may underlie the development of life-threatening ischemia-reperfusion-induced AKI in humans

Skap2 is required for β2 integrin-mediated neutrophil recruitment and functions.

Integrin activation is required for neutrophil functions. Impaired integrin activation on neutrophils is the hallmark of leukocyte adhesion deficiency (LAD) syndrome in humans, characterized by impaired leukocyte recruitment and recurrent infections. The Src kinase-associated phosphoprotein 2 (Skap2) is involved in integrin functions in different leukocyte subtypes. However, the role of Skap2 in β2 integrin activation and neutrophil recruitment is unknown. In this study, we demonstrate the crucial role of Skap2 in regulating actin polymerization and binding of talin-1 and kindlin-3 to the β2 integrin cytoplasmic domain, thereby being indispensable for β2 integrin activation and neutrophil recruitment. The direct interaction of Skap2 with the Wiskott-Aldrich syndrome protein via its SH3 domain is critical for integrin activation and neutrophil recruitment in vivo. Furthermore, Skap2 regulates integrin-mediated outside-in signaling events and neutrophil functions. Thus, Skap2 is essential to activate the β2 integrins, and loss of Skap2 function is sufficient to cause a LAD-like phenotype in mice

Directing HIV-1 for degradation by non-target cells, using bi-specific single-chain llama antibodies

While vaccination against HIV-1 has been so far unsuccessful, recently broadly neutralizing antibodies (bNAbs) against HIV-1 envelope glycoprotein were shown to induce long-term suppression in the absence of antiretroviral therapy in patients with antibody-sensitive viral reservoirs. The requirement of neutralizing antibodies indicates that the antibody mediated removal (clearance) of HIV-1 in itself is not efficient enough in these immune compromised patients. Here we present a novel, alternative approach that is independent of a functional immune system to clear HIV-1, by capturing the virus and redirecting it to non-target cells where it is internalized and degraded. We use bispecific antibodies with domains derived from small single chain Llama antibodies (VHHs). These bind with one domain to HIV-1 envelope proteins and with the other domain direct the virus to cells expressing epidermal growth factor receptor (EGFR), a receptor that is ubiquitously expressed in the body. We show that HIV envelope proteins, virus-like particles and HIV-1 viruses (representing HIV-1 subtypes A, B and C) are efficiently recruited to EGFR, internalized and degraded in the lysosomal pathway at low nM concentrations of bispecific VHHs. This directed degradation in non-target cells may provide a clearance platform for the removal of viruses and other unwanted agents from the circulation, including toxins, and may thus provide a novel method for curing

Crucial role of SLP-76 and ADAP for neutrophil recruitment in mouse kidney ischemia-reperfusion injury

Neutrophils trigger inflammation-induced acute kidney injury (AKI), a frequent and potentially lethal occurrence in humans. Molecular mechanisms underlying neutrophil recruitment to sites of inflammation have proved elusive. In this study, we demonstrate that SLP-76 (SH2 domain–containing leukocyte phosphoprotein of 76 kD) and ADAP (adhesion and degranulation promoting adaptor protein) are involved in E-selectin–mediated integrin activation and slow leukocyte rolling, which promotes ischemia-reperfusion–induced AKI in mice. By using genetically engineered mice and transduced Slp76(−/−) primary leukocytes, we demonstrate that ADAP as well as two N-terminal–located tyrosines and the SH2 domain of SLP-76 are required for downstream signaling and slow leukocyte rolling. The Tec family kinase Bruton tyrosine kinase is downstream of SLP-76 and, together with ADAP, regulates PI3Kγ (phosphoinositide 3-kinase–γ)- and PLCγ2 (phospholipase Cγ2)-dependent pathways. Blocking both pathways completely abolishes integrin affinity and avidity regulation. Thus, SLP-76 and ADAP are involved in E-selectin–mediated integrin activation and neutrophil recruitment to inflamed kidneys, which may underlie the development of life-threatening ischemia-reperfusion–induced AKI in humans

Molecular Evolution of Broadly Neutralizing Llama Antibodies to the CD4-Binding Site of HIV-1

To date, no immunization of humans or animals has elicited broadly neutralizing sera able to prevent HIV-1 transmission; however, elicitation of broad and potent heavy chain only antibodies (HCAb) has previously been reported in llamas. In this study, the anti-HIV immune responses in immunized llamas were studied via deep sequencing analysis using broadly neutralizing monoclonal HCAbs as a guides. Distinct neutralizing antibody lineages were identified in each animal, including two defined by novel antibodies (as variable regions called VHH) identified by robotic screening of over 6000 clones. The combined application of five VHH against viruses from clades A, B, C and CRF_AG resulted in neutralization as potent as any of the VHH individually and a predicted 100% coverage with a median IC50 of 0.17 µg/ml for the panel of 60 viruses tested. Molecular analysis of the VHH repertoires of two sets of immunized animals showed that each neutralizing lineage was only observed following immunization, demonstrating that they were elicited de novo. Our results show that immunization can induce potent and broadly neutralizing antibodies in llamas with features similar to human antibodies and provide a framework to analyze the effectiveness of immunization protocols

A mammalian cell based FACS-panning platform for the selection of HIV-1 envelopes for vaccine development

An increasing number of broadly neutralizing monoclonal antibodies (bnMAb) against the HIV-1 envelope (Env) protein has been discovered recently. Despite this progress, vaccination efforts with the aim to re-elicit bnMAbs that provide protective immunity have failed so far. Herein, we describe the development of a mammalian cell based FACS-panning method in which bnMAbs are used as tools to select surface-exposed envelope variants according to their binding affinity. For that purpose, an HIV-1 derived lentiviral vector was developed to infect HEK293T cells at low multiplicity of infection (MOI) in order to link Env phenotype and genotype. For proof of principle, a gp145 Env model-library was established in which the complete V3 domain was substituted by five strain specific V3 loop sequences with known binding affinities to nMAb 447-52D, respectively. Env genes were recovered from selected cells by PCR, subcloned into a lentiviral vector (i) to determine and quantify the enrichment nMAb binders and (ii) to generate a new batch of transduction competent particles. After 2 selection cycles the Env variant with highest affinity was enriched 20-fold and represented 80% of the remaining Env population. Exploiting the recently described bnMAbs, this procedure might prove useful in selecting Env proteins from large Env libraries with the potential to elicit bnMAbs when used as vaccine candidates

Use of transient and stable RNA-Interference to increase proliferation of dystrophine positive and negative myoblasts

Titelblatt, Inhaltsverzeichnis, Zusammenfassung, Danksagung, Erklärung Einleitung Material und Methoden Ergebnisse Diskussion LiteraturverzeichnisDie Duchenne Muskeldystrophie (DMD) gehört zu den häufigsten erblichen Muskeldystrophien im Kindesalter. Gegenwärtig werden die Substitution von Dystrophin oder ähnlichen Proteinen sowie die Korrektur der Mutation und damit die Wiederherstellung der Dystrophinexpression als gentherapeutische Strategien verfolgt. Jedoch führt sowohl die Herstellung der Dystrophinexpression als auch die Verwendung von viralen Transfersystemen im adulten Organismus zu Reaktionen des Immunsystems. Zudem wird eine autologe ex vivo Therapie durch eine limitierte Zahl an Myoblasten eingeschränkt. Fetale und Dystrophin-Downstream Konzepte könnten Probleme der genannten Strategien umgehen bzw. sinnvoll unterstützen. Expressionsuntersuchungen in Dystrophin negativen (Dys-) Muskelzellen zeigten Veränderungen in Genen für Zellwachstum und Differenzierung. So wiesen Biopsien von DMD-Patienten ein erhöhtes Niveau an p21 mRNA auf, einem Cyklin-abhängigen Kinase-Inhibitor, der hoch reguliert das Fortschreiten der Zellen von der G1 zur S1 Phase im Zellzyklus und somit die Zellteilung inhibiert. Ein erhöhtes Niveau von p21 schränkt somit die Zielzellen für eine autologe ex vivo oder in vivo Gen- und Zelltherapie ein. Die Reduktion von p21 würde die Möglichkeit bieten das Reservoir an Myoblasten zu erhalten oder wiederherzustellen. In der vorliegenden Arbeit erfolgte die Etablierung der p21 Inhibition in vitro durch RNA-Interferenz. Hierfür wurden optimale Transfektionsbedingungen und funktionelle siRNAs ermittelt. Die effektiven siRNAs wurden in einem lentiviralen Vektorsystem verwendet, um einen möglichst vollständigen und stabilen p21 knockdown zu erzielen. Dies geschah für primäre humane Myoblasten und für murine C2C12 Zellen, um den Effekt der p21 Inhibition auf die Proliferation zu untersuchen. Die Austestung an murinen Zellen erlaubt die Entwicklung eines Systems für einen in vivo Versuch am mdx-Mausmodell. Zwei in humanen Myoblasten wirksame siRNAs gegen p21 kamen an Dys- Zellen zum Einsatz. Die siRNA induzierte p21 Proteinreduktion betrug 72 Stunden nach Transfektion 29% bis 78%. Die p21 Proteinreduktion resultierte in einer Proliferationssteigerung von 11% bis 47% gegenüber unbehandelten Kontrollen. Die Expression von siRNA im lentiviralen Vektor führte zu einer nachhaltigen p21 Proteinreduktion von 70% fünf Wochen nach Infektion in Zellen eines DMD Patienten. Die Proliferation der infizierten Zellen stieg um 127%. Wiederholte Infektionen von Zellen eines anderen Patienten zeigten einen Proliferationsanstieg von 35% bis 63%. Die Unterschiede in der p21-Reduktion und der Proliferationssteigerung weisen auf eine hohe Varianz des siRNA Effektes hin. Hierbei können Unterschiede im Integrationsort des lentiviralen Vektors und individuelle Schwankungen in der p21 Basisexpression eine Rolle spielen. Die p21 Inhibition verhinderte eine vollständige Differenzierung. Für murine Zellen wurde ebenfalls eine siRNA identifiziert, die nach transienter Transfektion zu einer p21 Inhibition von 61% nach 48 Stunden führte. Die abgeleitete, viral exprimierte shRNA erzielte jedoch nur eine Reduktion von 30% gegenüber Kontrollen. Die Ergebnisse dieser Arbeit zeigen, dass eine stabile p21 Reduktion mittels viral vermittelter RNA- Interferenz in der Lage ist die Proliferation von Dys- Zellen zu steigen. Damit könnte der stetige Verlust von Myoblasten bei DMD Patienten eingeschränkt werden, was zum Erhalt der Zielzellen für eine Gen- und Zelltherapie führt. Weiterführende Arbeiten zur Regulation der p21 siRNA Expression müssen jedoch eine Redifferenzierung der Myoblasten sicherstellen und sich dem Infektions-bedingten Zellverlust stellen. Während eine p21 Reduktion alleine keine Ursache für eine Onkogenese zu sein scheint, müssen verbesserte retrovirale Systeme Integration-bedingte Risiken minimieren.Duchenne Muscle Dystrophy (DMD) is one of the most common heritable muscle disorders in childhood. Dystrophine substitution or gene fixing approaches to restore dystrophine expression are currently investigated as gene therapeutic strategies. But there are still obstacles to overcome like immune response to the re-established dystrophine expression as well to viral vector systems. And autologous ex vivo therapies are limited due to the small amount of available target cells. Fetal- or dystrophine-downstream concepts could bypass or assist some problems of these strategies. Comprehensive studies on expression of dystrophine negative (dys-) myoblasts showed altered expression of genes involved in cell proliferation and differentiation. Biopsies of DMD patients showed an increased level of cyclin dependent kinase inhibitor p21 mRNA compared to dystrophine positive controls. P21 regulates the G1 S1 progression within the cell cycle. An elevated level on p21 inhibits cell proliferation and limits the potential target cells for an autologous ex vivo as well as in vivo gene or cell therapy. Therefore a reduction in p21 could help to maintain or restore the reservoir of myoblasts. In the present study a p21 inhibition by RNA-Interference was established. Optimal transfection parameters were determined and potent p21 siRNAs were identified. The influences on cell proliferation were investigated. Effective siRNAs were used in a lentiviral vector system to achieve a long lasting, inducible and complete p21 knockdown. This was done in primary human myoblasts as well as in murine c2c12 cells. This parallel approach provides the opportunity to use this strategy in vivo in the mdx mouse model. Two effective siRNAs against human p21 were used on primary dystrophine negative myoblasts. P21 protein was reduced by 29% to 79% after 72 hours after transfection and resulted in an increase in proliferation by 11% to 47% compared to untreated controls. The siRNA expressed by a lentiviral vector lead to a prolonged reduction of p21 protein by 70% of myoblasts from a dys- patient five weeks after infection. The proliferation was elevated by 127%. Infection of myoblasts from a second patient resulted in a protein reduction by 35%-63%. The p21 knockdown prevented also a complete differentiation of myoblasts. Differences in p21 basis expression, or different integration sites of the lentiviral vector could be the reason for observed variances in the effect of siRNA inhibition. The p21 knockdown prevented also a complete differentiation of myoblasts. Additionally effective siRNAs for tests on murine myoblasts were identified. After transient transfection p21 protein was reduced by 61% after 48 hours. The same siRNA expressed by a lentiviral vector showed a decrease of p21 protein by only 30% compared to untreated controls. Differences in p21 basis expression, or different integration sites of the lentiviral vector could be a reason for the observed variances in the effect of siRNA inhibition. The present study shows that a stable p21 inhibition by RNA interference can lead to an increased proliferation of dys- myoblasts. This could prevent the continuous loss of myoblasts and preserve cells for an additional gene and cell therapy. Following studies which focus on regulation of siRNA expression should assure redifferentiation of treated myoblasts. Although the loss of p21 alone seemed no cause of oncogenesis, retroviral vector systems have to be improved to minimize the risk of oncogenesis by random integration

Crucial role of SLP-76 and ADAP for neutrophil recruitment in mouse kidney ischemia-reperfusion injury.

Neutrophils trigger inflammation-induced acute kidney injury (AKI), a frequent and potentially lethal occurrence in humans. Molecular mechanisms underlying neutrophil recruitment to sites of inflammation have proved elusive. In this study, we demonstrate that SLP-76 (SH2 domain-containing leukocyte phosphoprotein of 76 kD) and ADAP (adhesion and degranulation promoting adaptor protein) are involved in E-selectin-mediated integrin activation and slow leukocyte rolling, which promotes ischemia-reperfusion-induced AKI in mice. By using genetically engineered mice and transduced Slp76(-/-) primary leukocytes, we demonstrate that ADAP as well as two N-terminal-located tyrosines and the SH2 domain of SLP-76 are required for downstream signaling and slow leukocyte rolling. The Tec family kinase Bruton tyrosine kinase is downstream of SLP-76 and, together with ADAP, regulates PI3Kγ (phosphoinositide 3-kinase-γ)- and PLCγ2 (phospholipase Cγ2)-dependent pathways. Blocking both pathways completely abolishes integrin affinity and avidity regulation. Thus, SLP-76 and ADAP are involved in E-selectin-mediated integrin activation and neutrophil recruitment to inflamed kidneys, which may underlie the development of life-threatening ischemia-reperfusion-induced AKI in humans

Affinity analysis of 447-52D antibody to individual members of the Env/V3 library.

<p>HEK293T cells were separately transduced at low MOI (0.1) by one of the pQL9 derived lentiviral particles expressing the indicated chimeric Env/V3-variant, respectively. 48 h post transduction, cells were stained with MAb 447-52D and an APC-labeled secondary antibody. The mean values of two independent experiments corrected for equal GFP expression are shown. <b>A</b> A time-course with 447-52D antibody (10 µg/mL) was performed to record the incubation-time needed for equilibrium binding. FACS data are expressed as the MFI for every member of the model library at the different incubation times, respectively. <b>B</b> 447-52D antibody concentrations were serially diluted and incubated on infected cells at equilibrium incubation-time (1 h at 4°C) to obtain a concentration dependent binding profile. <b>C</b> Cell populations stained with 0.1 µg/mL 447-52D antibodies are depicted separately in order to highlight the differential binding at this concentration. <b>D</b> Dissociation constants (K_D) were calculated from the antibody-titration curves shown in (B) based on the µM concentrations derived from the 447-52D molecular weight and therefore expressed as Kd (µM). Data points were fitted by nonlinear least squares regression (One site; Fit total and nonspecific binding, Graphpad Prism 5), and the resulting K_d as well as R² values are listed (#, non-calculable).</p

FACS-panning: gating strategy.

<p>HEK293T (13×10⁶) cells were transduced at low MOI (0.1) (<b>A</b>) by lentivirus particles derived from a plasmid mixture containing equal amounts of all Env/V3 variants and (<b>B</b>) by separately lentiviral particles each encoding only one of the chimeric Env/V3-variants, respectively. Equal amounts of transduced cells were harvested 72 h post transduction and were analysed by FACS. <b>A</b> One typical sorting experiment is shown to illustrate the FACS gating and sorting strategy: 50,000 events were recorded and a series of hierarchical gates were applied to isolate single cells. GFP positive cells were gated within their main population (Gate 5) to exclude multiply infected cells (high GFP) and background noise (low GFP). The GFP main population was subsequently gated for strong 447-52D antibody binding (high APC) in relation to Env expression (coupled to the GFP signal). Therefore, a triangle shaped Gate (Gate 6) was chosen. The complete Gate hierarchy starting with all events is illustrated. <b>B</b> Evaluation of the triangle-gate strategy: HEK293T cells (13×10⁶) were transduced at low MOI (0.1) with each pQL9 Env/V3-virus variant, respectively. Cells were stained with 447-52D antibody and analyzed by FACS according to the gating strategy described in A. Cells that appeared in Gate 5 were further analyzed by calculating a linear regression curve (purple). Slope values calculated for representative variants Env/V3-MN, Env/V3-HXB2, and Env/V3-SF33 are indicated. The triangle shaped Gate 6 is shown in a log-scale dot plot. The numbers of events counted for Gate 5 and Gate 6 are shown below each plot, respectively. Decreasing amounts of cells equal decreasing affinities of Env, as detected in the sorting Gate 6, ranging from 10 counts of Env/V3-MN, 2 of Env/V3-HXB2 and 0 of Env/V3-SF33, respectively.</p