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

A Two-Step Synthesis of Avobenzone.

Avobenzone is an important agent in sunscreen that reacts with the full spectrum of UVA/UVB light. In fact, avobenzone has one of the largest UV light absorbance ranges of all sunscreen agents. The wide absorbance range of avobenzone helps the sunscreen better protect the skin. This project involved two steps to synthesize avobenzone. Starting with 4-tert-butylbenzaldehyde, 4-methoxyacetophenone, and a base, an Aldol reaction was used to create a ketol intermediate. This step involved experimentation with several bases, including sodium hydroxide and potassium-tert-butoxide, to attempt to stop the product at this intermediate ketol form. Two different mixing methods were also compared in the synthesis of this ketol intermediate. The second step involved conversion of the ketol to the diketone avobenzone product after combination with an oxidizing agent. To make this synthesis more green, a one-step synthesis relying on a Claisen reaction was also attempted

Untersuchung der Veränderungen des Zytoskeletts in humanen Lungenmakrophagen in Abhängigkeit des Aktivierungszustandes der kleinen Rho-GTPasen aufgrund der Interaktionen mit synthetischen Nanopartikel

Metalloxide sind die derzeit wichtigsten kommerziell verarbeiteten Nanopartikel (NPs). Ziel dieser Arbeit war es, synthetisch hergestellte NPs auf Silika- und Zirkoniumdioxid-Basis zu charakterisieren und Interaktionen mit vor allem humanen Alveolarmakrophagen zu untersuchen. In Makrophagen konnte via MTT-Test erst bei hohen Konzentrationen eine akute Toxizität durch ZrO2-NPs nachgewiesen werden. Mikroskopische Aufnahmen von perinukleär lokalisierten Teilchenclustern der untersuchten SiO2-NPs wurden als Grundlage zur Erforschung der intrazellulären Teilchen-Dynamik verwendet. Die Quantifizierung des für die meisten zellulären Aufnahmeprozesse essentiellen F-Aktins ergab nach Inkubation mit NPs einen Anstieg des fibrillären Aktingehalts. Als zentrale Regulatoren der Aktin-Dynamik wurden die kleinen Rho-GTPasen (RhoA, Rac1 und Cdc42) auf einen veränderten Aktivierungsgrad nach Inkubation mit SiO2-NPs untersucht. Ob und wie stark die Rho-GTPasen aktiviert wurden, war dabei abhängig von der jeweils zu betrachtenden GTPase, Oberflächenmodifikation und Konzentration der Partikel, sowie der jeweiligen Zellspezies. In humanen Interstitiellen Makrophagen konnte nach Exposition von nicht-PEGylierten SiO2-NPs via EMSA eine erhöhte Aktivierung des Transkriptionsfaktors NF-kB gezeigt werden. Die Ergebnisse dieser Arbeit sollen dazu beitragen, die Toxizität und das inflammatorische Potential der verwendeten Nanomaterialien realistisch einzuschätzen.There is an increasing exposition of humans to engineered Nanoparticles (NPs) that are present in a wide range of commercially available products, consisting of metal oxides representing actually the commercially most important NPs. Aim of this dissertation was to characterize technical silica and zirconium nanoparticles and to investigate the interactions with human lung macrophages. In macrophages, an acute toxicitiy was shown for zirconia NPs in high concentrations after an incubation time of 24 hours. Based on the microscopic images, it has been possible to develop a stochastic simulation of particle transport and particle dynamics in macrophages. Quantification of F-actin resulted in an increase after incubation with PEGylated and non-PEGylated silica NPs. The small Rho-GTPases (RhoA, Rac1 and Cdc42) play a pivotal role in regulating actin dynamics. Degree of activation depended on the selection of cell species, GTPase, surface modification and concentration of NPs. Investigations had to be carried out about how far small Rho-GTPases in macrophages are able to activate NF-kB after incubation with NPs. Results of EMSAs performed with interstitial macrophages incubated with non-PEGylated silica NPs showed an increase in NF-kB activation. Investigations in the context of this work are absolutely essential for the safe handling with nanomaterials. The findings are supposed to provide assessment of toxicity and inflammatory potential of the used nanomaterials

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

Zika virus preferentially replicates in the female reproductive tract after vaginal inoculation of rhesus macaques.

Zika virus (ZIKV) is a mosquito-transmitted virus that can cause severe defects in an infected fetus. ZIKV is also transmitted by sexual contact, although the relative importance of sexual transmission is unclear. To better understand the role of sexual transmission in ZIKV pathogenesis, a nonhuman primate (NHP) model of vaginal transmission was developed. ZIKV was readily transmitted to mature cycling female rhesus macaque (RM) by vaginal inoculation with 104-106 plaque-forming units (PFU). However, there was variability in susceptibility between the individual RM with 1->8 vaginal inoculations required to establish infection. After treatment with Depoprovera, a widely used contraceptive progestin, two RM that initially resisted 8 vaginal ZIKV inoculations became infected after one ZIKV inoculation. Thus, Depoprovera seemed to enhance susceptibility to vaginal ZIKV transmission. Unexpectedly, the kinetics of virus replication and dissemination after intravaginal ZIKV inoculation were markedly different from RM infected with ZIKV by subcutaneous (SQ) virus inoculation. Several groups have reported that after SQ ZIKV inoculation vRNA is rapidly detected in blood plasma with vRNA less common in urine and saliva and only rarely detected in female reproductive tract (FRT) secretions. In contrast, in vaginally inoculated RM, plasma vRNA is delayed for several days and ZIKV replication in, and vRNA shedding from, the FRT was found in all 6 animals. Further, after intravaginal transmission ZIKV RNA shedding from FRT secretions was detected before or simultaneously with plasma vRNA, and persisted for at least as long. Thus, ZIKV replication in the FRT was independent of, and often preceded virus replication in the tissues contributing to plasma vRNA. These results support the conclusion that ZIKV preferentially replicates in the FRT after vaginal transmission, but not after SQ transmission, and raise the possibility that there is enhanced fetal infection and pathology after vaginal ZIKV transmission compared to a mosquito transmitted ZIKV