Neutrophil Extracellular Trap-Related Extracellular Histones Cause Vascular Necrosis in Severe GN

Severe GN involves local neutrophil extracellular trap (NET) formation. We hypothesized a local cytotoxic effect of NET-related histone release in necrotizing GN. In vitro, histones from calf thymus or histones released by neutrophils undergoing NETosis killed glomerular endothelial cells, podocytes, and parietal epithelial cells in a dose-dependent manner. Histone-neutralizing agents such as antihistone IgG, activated protein C, or heparin prevented this effect. Histone toxicity on glomeruli ex vivo was Toll-like receptor 2/4 dependent, and lack of TLR2/4 attenuated histone-induced renal thrombotic microangiopathy and glomerular necrosis in mice. Anti–glomerular basement membrane GN involved NET formation and vascular necrosis, whereas blocking NET formation by peptidylarginine inhibition or preemptive anti-histone IgG injection significantly reduced all aspects of GN (i.e., vascular necrosis, podocyte loss, albuminuria, cytokine induction, recruitment or activation of glomerular leukocytes, and glomerular crescent formation). To evaluate histones as a therapeutic target, mice with established GN were treated with three different histone-neutralizing agents. Anti-histone IgG, recombinant activated protein C, and heparin were equally effective in abrogating severe GN, whereas combination therapy had no additive effects. Together, these results indicate that NET-related histone release during GN elicits cytotoxic and immunostimulatory effects. Furthermore, neutralizing extracellular histones is still therapeutic when initiated in established GN.     

The xanthine oxidase–NFAT5 pathway regulates macrophage activation and TLR‐induced inflammatory arthritis

NFAT5 (nuclear factor of activated T cells), a well‐known osmoprotective factor, can be activated by isotonic stimuli such as Toll‐like receptor (TLR) triggering. However, it is unclear how NFAT5 discriminates between isotonic and hypertonic stimuli to produce different functional and molecular outcomes. Here, we identified a novel XO–ROS–p38 MAPK–NFAT5 pathway (XO is xanthine oxidase, ROS is reactive oxygen species) that is activated in RAW 264.7 macrophages upon isotonic TLR stimulation. Unlike what is seen under hypertonic conditions, XO‐derived ROS were selectively required for the TLR‐induced NFAT5 activation and NFAT5 binding to the IL‐6 promoter in RAW 264.7 macrophages under isotonic conditions. In mouse peritoneal macrophages and human macrophages, TLR ligation also induced NFAT5 activation, which was dependent on XO and p38 kinase. The involvement of XO in NFAT5 activation by TLR was confirmed in RAW 264.7 macrophages implanted in BALB/c mice. Moreover, allopurinol, an XO inhibitor, suppressed arthritis severity and decreased the expression of NFAT5 and IL‐6 in splenic macrophages in C57BL/6 mice. Collectively, these data support a novel function of the XO–NFAT5 axis in macrophage activation and TLR‐induced arthritis, and suggest that XO inhibitor(s) could serve as a therapeutic agent for chronic inflammatory arthritis.     

Comparison of Chondroitin Sulfate and Hyaluronic Acid Doped Conductive Polypyrrole Films for Adipose Stem Cells

Polypyrrole (PPy) is a conductive polymer that has aroused interest due to its biocompatibility with several cell types and high tailorability as an electroconductive scaffold coating. This study compares the effect of hyaluronic acid (HA) and chondroitin sulfate (CS) doped PPy films on human adipose stem cells (hASCs) under electrical stimulation. The PPy films were synthetized electrochemically. The surface morphology of PPy–HA and PPy–CS was characterized by an atomic force microscope. A pulsed biphasic electric current (BEC) was applied via PPy films non-stimulated samples acting as controls. Viability, attachment, proliferation and osteogenic differentiation of hASCs were evaluated by live/dead staining, DNA content, Alkaline phosphatase activity and mineralization assays. Human ASCs grew as a homogenous cell sheet on PPy–CS surfaces, whereas on PPy–HA cells clustered into small spherical structures. PPy–CS supported hASC proliferation significantly better than PPy–HA at the 7 day time point. Both substrates equally triggered early osteogenic differentiation of hASCs, although mineralization was significantly induced on PPy–CS compared to PPy–HA under BEC. These differences may be due to different surface morphologies originating from the CS and HA dopants. Our results suggest that PPy–CS in particular is a potential osteogenic scaffold coating for bone tissue engineering.     

Inside the clinical evaluation of sleepiness: subjective and objective tools

Sleep and Breathing - To critically review the available tools for evaluating excessive daytime sleepiness (EDS) in clinical practice. Objective tests and subjective scales were divided into three...     

Local immune responses in human tuberculosis: learning from the site of infection

Host-pathogen interactions in tuberculosis should be studied at the disease site because Mycobacterium tuberculosis is predominately contained in local tissue lesions. Although M. tuberculosis infection involves different clinical forms of tuberculosis, such as pulmonary tuberculosis, pleural tuberculosis, and lymph node tuberculosis, most studies of human tuberculosis are performed using cells from the peripheral blood, which may not provide a proper reflection of the M. tuberculosis-specific immune responses induced at the local site of infection. A very low proportion of M. tuberculosis-specific effector T cells are found in the blood compared with the infected tissue, and thus there may be considerable differences in the cellular immune response and regulatory mechanisms induced in these diverse compartments. In this review, we discuss differences in the immune response at the local site of infection compared with the peripheral circulation. The cell types and immune reactions involved in granuloma formation and maintenance as well as the in situ technologies used to assess local tuberculosis pathogenesis are also described. We need to strengthen and improve the exploratory strategies used to dissect immunopathogenesis in human tuberculosis with the aim to accelerate the implementation of relevant research findings in clinical practice.     

Identification of neuronal substrates implicates Pak5 in synaptic vesicle trafficking

Synaptic transmission is mediated by a complex set of molecular events that must be coordinated in time and space. While many proteins that function at the synapse have been identified, the signaling pathways regulating these molecules are poorly understood. Pak5 (p21-activated kinase 5) is a brain-specific isoform of the group II Pak kinases whose substrates and roles within the central nervous system are largely unknown. To gain insight into the physiological roles of Pak5, we engineered a Pak5 mutant to selectively radiolabel its substrates in murine brain extract. Using this approach, we identified two novel Pak5 substrates, Pacsin1 and Synaptojanin1, proteins that directly interact with one another to regulate synaptic vesicle endocytosis and recycling. Pacsin1 and Synaptojanin1 were phosphorylated by Pak5 and the other group II Paks in vitro, and Pak5 phosphorylation promoted Pacsin1-Synaptojanin1 binding both in vitro and in vivo. These results implicate Pak5 in Pacsin1- and Synaptojanin1-mediated synaptic vesicle trafficking and may partially account for the cognitive and behavioral deficits observed in group II Pak-deficient mice.     

An Integrated Alcohol Abuse and Medical Treatment Model for Patients with Hepatitis C

Background  

Patients with chronic hepatitis C virus (HCV) infection have high rates of alcohol consumption, which is associated with progression of fibrosis and lower response rates to HCV treatment.