Poly(I:C) Induces Human Lung Endothelial Barrier Dysfunction by Disrupting Tight Junction Expression of Claudin-5.

Viral infections are often accompanied by pulmonary microvascular leakage and vascular endothelial dysfunction via mechanisms that are not completely defined. Here, we investigated the effect of the Toll-like receptor 3 (TLR3) ligand polyinosinic-polycytidylic acid [Poly(I:C)], a synthetic analog of viral double-stranded RNA (dsRNA) commonly used to simulate viral infections, on the barrier function and tight junction integrity of primary human lung microvascular endothelial cells. Poly(I:C) stimulated IL-6, IL-8, TNFα, and IFNß production in conjunction with the activation of NF-κB and IRF3 confirming the Poly(I:C)-responsiveness of these cells. Poly(I:C) increased endothelial monolayer permeability with a corresponding dose- and time-dependent decrease in the expression of claudin-5, a transmembrane tight junction protein and reduction of CLDN5 mRNA levels. Immunofluorescence experiments revealed disappearance of membrane-associated claudin-5 and co-localization of cytoplasmic claudin-5 with lysosomal-associated membrane protein 1. Chloroquine and Bay11-7082, inhibitors of TLR3 and NF-κB signaling, respectively, protected against the loss of claudin-5. Together, these findings provide new insight on how dsRNA-activated signaling pathways may disrupt vascular endothelial function and contribute to vascular leakage pathologies.

AFM Imaging Reveals Topographic Diversity of Wild Type and Z Variant Polymers of Human α1-Proteinase Inhibitor.

α1-Proteinase inhibitor (antitrypsin) is a canonical example of the serpin family member that binds and inhibits serine proteases. The natural metastability of serpins is crucial to carry out structural rearrangements necessary for biological activity. However, the enhanced metastability of the mutant Z variant of antitrypsin, in addition to folding defect, may substantially contribute to its polymerization, a process leading to incurable serpinopathy. The metastability also impedes structural studies on the polymers. There are no crystal structures of Z monomer or any kind of polymers larger than engineered wild type (WT) trimer. Our understanding of polymerization mechanisms is based on biochemical data using in vitro generated WT oligomers and molecular simulations. Here we applied atomic force microscopy (AFM) to compare topography of monomers, in vitro formed WT oligomers, and Z type polymers isolated from transgenic mouse liver. We found the AFM images of monomers closely resembled an antitrypsin outer shell modeled after the crystal structure. We confirmed that the Z variant demonstrated higher spontaneous propensity to dimerize than WT monomers. We also detected an unexpectedly broad range of different types of polymers with periodicity and topography depending on the applied method of polymerization. Short linear oligomers of unit arrangement similar to the Z polymers were especially abundant in heat-treated WT preparations. Long linear polymers were a prominent and unique component of liver extracts. However, the liver preparations contained also multiple types of oligomers of topographies undistinguishable from those found in WT samples polymerized with heat, low pH or guanidine hydrochloride treatments. In conclusion, we established that AFM is an excellent technique to assess morphological diversity of antitrypsin polymers, which is important for etiology of serpinopathies. These data also support previous, but controversial models of in vivo polymerization showing a surprising diversity of polymer topography.

Certified Nurse-Midwives' Experiences with Gestational Weight Management.

Excessive gestational weight gain is associated with fetal metabolic reprogramming and subsequent childhood obesity, as well as maternal recalcitrant obesity and its successive morbidities. We conducted a review of the literature and an explorative, descriptive study of the techniques and strategies used by a subset of certified nurse-midwives to help women achieve optimal gestational weight gain. We also identified barriers to this outcome. With more effective management approaches to gestational weight gain, adverse maternal and neonatal health outcomes could potentially be prevented. http://dx.doi.org/10.1016/j.nwh.2015.12.007.

Subvisible Particle Content, Formulation, and Dose of an Erythropoietin Peptide Mimetic Product Are Associated With Severe Adverse Postmarketing Events.

Peginesatide (Omontys(®); Affymax, Inc., Cupertino, CA) was voluntarily withdrawn from the market less than a year after the product launch. Although clinical trials had demonstrated the drug to be safe and efficacious, 49 cases of anaphylaxis, including 7 fatalities, were reported not long after market introduction. Commercialization was initiated with a multiuse vial presentation, which differs in formulation from the single-use vial presentation used in phase 3 studies. Standard physical and chemical testing did not indicate any deviation from product specifications in either formulation. However, an analysis of subvisible particulates using nanoparticle tracking analysis and flow imaging revealed a significantly higher concentration of subvisible particles in the multiuse vial presentation linked to the hypersensitivity cases. Although it is unknown whether the elevated particulate content is causally related to these serious adverse events, this report illustrates the utility of characterizing subvisible particulates not captured by conventional light obscuration.

Gestation age dependent transfer of human immunoglobulins across placenta in timed-pregnant guinea pigs.

INTRODUCTION: When administered during pregnancy, antibodies and other biologic drugs that contain the Fc part of the IgG molecule can traverse the placenta. Although it is generally accepted that the FcRn receptor mediates this process, gaps remain in our understanding of underlying details in humans and in common laboratory animal species. METHODS: We expanded our previous studies in timed-pregnant guinea pigs to both measure the transport of human (h) IgG at earlier gestation ages in vivo and evaluate FcRn function in vitro using Surface Plasmon Resonance (SPR) and Madin-Darby canine kidney cells (MDCK) that express guinea pig (gp) FcRn. RESULTS: In timed-pregnant guinea pigs both the average concentration of hIgG in the fetus and its ratio to maternal hIgG concentration increase exponentially with gestation age. Thus, hIgG fetal:maternal concentration ratios increase from an average of 1% to 3%, 17%, and 76% on GD ∼26, 35, 46, and 54, respectively. In vitro, gpFcRn immobilized on a solid surface can bind hIgG and gpIgG preparations in a similar manner. All engineered human Fc isotype-specific constructs were internalized by MDCK-gpFcRn cells at significant levels. While not significant, their recycling and hIgG transcytosis by this cell line also trend higher than background controls. DISCUSSION: Pregnant guinea pigs exhibit similarities with humans in the degree and timing of transplacental transfer as well as the ability of their FcRn to bind and internalize hIgG in vitro. Further studies are needed to guide building appropriate systems for the evaluation of FcRn mediated function of human immunoglobulin therapies.

Microtubule plus-end tracking protein CLASP2 regulates neuronal polarity and synaptic function.

Microtubule organization and dynamics are essential during axon and dendrite formation and maintenance in neurons. However, little is known about the regulation of microtubule dynamics during synaptic development and function in mammalian neurons. Here, we present evidence that the microtubule plus-end tracking protein CLASP2 (cytoplasmic linker associated protein 2) is a key regulator of axon and dendrite outgrowth that leads to functional alterations in synaptic activity and formation. We found that CLASP2 protein levels steadily increase throughout neuronal development in the mouse brain and are specifically enriched at the growth cones of extending neurites. The short-hairpin RNA-mediated knockdown of CLASP2 in primary mouse neurons decreased axon and dendritic length, whereas overexpression of human CLASP2 caused the formation of multiple axons, enhanced dendritic branching, and Golgi condensation, implicating CLASP2 in neuronal morphogenesis. In addition, the CLASP2-induced morphological changes led to significant functional alterations in synaptic transmission. CLASP2 overexpression produced a large increase in spontaneous miniature event frequency that was specific to excitatory neurotransmitter release. The changes in presynaptic activity produced by CLASP2 overexpression were accompanied by increases in presynaptic terminal circumference, total synapse number, and a selective increase in presynaptic proteins that are involved in neurotransmitter release. Also, we found a smaller increase in miniature event amplitude that was accompanied by an increase in postsynaptic surface expression of GluA1 receptor localization. Together, these results provide evidence for involvement of the microtubule plus-end tracking protein CLASP2 in cytoskeleton-related mechanisms underlying neuronal polarity and interplay between microtubule stabilization and synapse formation and activity.