Macaques infected with a CCR5-tropic simian/human immunodeficiency virus (SHIV) develop broadly reactive anti-HIV neutralizing antibodies

The development of anti-human immunodeficiency virus (anti-HIV) neutralizing antibodies and the evolution of the viral envelope glycoprotein were monitored in rhesus macaques infected with a CCR5-tropic simian/human immunodeficiency virus (SHIV), SHIVSF162P4. Homologous neutralizing antibodies developed within the first month of infection in the majority of animals, and their titers were independent of the extent and duration of viral replication during chronic infection. The appearance of homologous neutralizing antibody responses was preceded by the appearance of amino acid changes in specific variable and conserved regions of gp120. Amino acid changes first appeared in the V1, V2, C2, and V3 regions and subsequently in the C3, V4, and V5 regions. Heterologous neutralizing antibody responses developed over time only in animals with sustained plasma viremia. Within 2 years postinfection the breadth of these responses was as broad as that observed in certain patients infected with HIV type 1 (HIV-1) for over a decade. Despite the development of broad anti-HIV-1 neutralizing antibody responses, viral replication persisted in these animals due to viral escape. Our studies indicate that cross-reactive neutralizing antibodies are elicited in a subset of SHIVSF162P4 infected macaques and that their development requires continuous viral replication for extended periods of time. More importantly, their late appearance does not prevent progression to disease. The availability of an animal model where cross-reactive anti-HIV neutralizing antibodies are developed may facilitate the identification of virologic and immunologic factors conducive to the development of such antibodies.     

Field Test of the World Health Organization Multi-Professional Patient Safety Curriculum Guide

Introduction

Although the importance of training in patient safety has been acknowledged for over a decade, it remains under-utilized and under-valued in most countries. WHO developed the Multi-professional Patient Safety Curriculum Guide to provide schools with the requirements and tools for incorporating patient safety in education. It was field tested with 12 participating schools across the six WHO regions, to assess its effectiveness for teaching patient safety to undergraduate and graduate students in a global variety of settings.

Methods

The evaluation used a combined prospective/retrospective design to generate formative information on the experiences of working with the Guide and summative information on the impacts of the Guide. Using stakeholder interviews and student surveys, data were gathered from each participating school at three times: the start of the field test (baseline), soon after each school started teaching, and soon after each school finished teaching.

Results

Stakeholders interviewed were strongly positive about the Guide, noting that it emphasized universally important patient safety topics, was culturally appropriate for their countries, and gave credibility and created a focus on patient safety at their schools. Student perceptions and attitudes regarding patient safety improved substantially during the field test, and their knowledge of the topics they were taught doubled, from 10.7% to 20.8% of correct answers on the student survey.

Discussion

This evaluation documented the effectiveness of the Curriculum Guide, for both ease of use by schools and its impacts on improving the patient safety knowledge of healthcare students. WHO should be well positioned to refine the contents of the Guide and move forward in encouraging broader use of the Guide globally for teaching patient safety.     

Remodeling of atrial ATP-sensitive K⁺ channels in a model of salt-induced elevated blood pressure

Hypertension is associated with the development of atrial fibrillation; however, the electrophysiological consequences of this condition remain poorly understood. ATP-sensitive K(+) (K(ATP)) channels, which contribute to ventricular arrhythmias, are also expressed in the atria. We hypothesized that salt-induced elevated blood pressure (BP) leads to atrial K(ATP) channel activation and increased arrhythmia inducibility. Elevated BP was induced in mice with a high-salt diet (HS) for 4 wk. High-resolution optical mapping was used to measure atrial arrhythmia inducibility, effective refractory period (ERP), and action potential duration at 90% repolarization (APD(90)). Excised patch clamping was performed to quantify K(ATP) channel properties and density. K(ATP) channel protein expression was also evaluated. Atrial arrhythmia inducibility was 22% higher in HS hearts compared with control hearts. ERP and APD(90) were significantly shorter in the right atrial appendage and left atrial appendage of HS hearts compared with control hearts. Perfusion with 1 μM glibenclamide or 300 μM tolbutamide significantly decreased arrhythmia inducibility and prolonged APD(90) in HS hearts compared with untreated HS hearts. K(ATP) channel density was 156% higher in myocytes isolated from HS animals compared with control animals. Sulfonylurea receptor 1 protein expression was increased in the left atrial appendage and right atrial appendage of HS animals (415% and 372% of NS animals, respectively). In conclusion, K(ATP) channel activation provides a mechanistic link between salt-induced elevated BP and increased atrial arrhythmia inducibility. The findings of this study have important implications for the treatment and prevention of atrial arrhythmias in the setting of hypertensive heart disease and may lead to new therapeutic approaches.     

Neutrophil extracellular trap formation is associated with IL-1β and autophagy-related signaling in gout

Background

Gout is a prevalent inflammatory arthritis affecting 1–2% of adults characterized by activation of innate immune cells by monosodium urate (MSU) crystals resulting in the secretion of interleukin-1β (IL-1β). Since neutrophils play a major role in gout we sought to determine whether their activation may involve the formation of proinflammatory neutrophil extracellular traps (NETs) in relation to autophagy and IL-1β.

Methodology/Principal Findings

Synovial fluid neutrophils from six patients with gout crisis and peripheral blood neutrophils from six patients with acute gout and six control subjects were isolated. MSU crystals, as well as synovial fluid or serum obtained from patients with acute gout, were used for the treatment of control neutrophils. NET formation was assessed using immunofluorescence microscopy. MSU crystals or synovial fluid or serum from patients induced NET formation in control neutrophils. Importantly, NET production was observed in neutrophils isolated from synovial fluid or peripheral blood from patients with acute gout. NETs contained the alarmin high mobility group box 1 (HMGB1) supporting their pro-inflammatory potential. Inhibition of phosphatidylinositol 3-kinase signaling or phagolysosomal fusion prevented NET formation, implicating autophagy in this process. NET formation was driven at least in part by IL-1β as demonstrated by experiments involving IL-1β and its inhibitor anakinra.

Conclusions/Significance

These findings document for the first time that activation of neutrophils in gout is associated with the formation of proinflammatory NETs and links this process to both autophagy and IL-1β. Modulation of the autophagic machinery may represent an additional therapeutic study in crystalline arthritides.     

Phospholipase A2 subclasses in acute respiratory distress syndrome

Phospholipases A₂ (PLA₂) catalyse the cleavage of fatty acids esterified at the sn-2 position of glycerophospholipids. In acute lung injury-acute respiratory distress syndrome (ALI-ARDS) several distinct isoenzymes appear in lung cells and fluid. Some are capable to trigger molecular events leading to enhanced inflammation and lung damage and others have a role in lung surfactant recycling preserving lung function: Secreted forms (groups sPLA₂-IIA, -V, -X) can directly hydrolyze surfactant phospholipids. Cytosolic PLA₂ (cPLA₂-IVA) requiring Ca²⁺ has a preference for arachidonate, the precursor of eicosanoids which participate in the inflammatory response in the lung. Ca²⁺-independent intracellular PLA₂s (iPLA₂) take part in surfactant phospholipids turnover within alveolar cells. Acidic Ca²⁺-independent PLA₂ (aiPLA₂), of lysosomal origin, has additionally antioxidant properties, (peroxiredoxin VI activity), and participates in the formation of dipalmitoyl-phosphatidylcholine in lung surfactant. PAF-AH degrades PAF, a potent mediator of inflammation, and oxidatively fragmented phospholipids but also leads to toxic metabolites. Therefore, the regulation of PLA₂ isoforms could be a valuable approach for ARDS treatment.     

Lipase activity in Thermus thermophilus HB8: Purification and characterization of the extracellular enzyme

In this study, the lipolytic activity of Thermus thermophilus HB8 was examined. The addition of various oils increased the production of extracellular lipolytic activity, while a combination of olive oil and glucose increased both extracellular and intracellular lipolytic activity. The oxygen transfer rate had a significant influence on both biomass and production of extra- or intra-cellular lipolytic activity. The formation of white halos due to the hydrolysis of oleic acid ester (Tween 80) in agar plates containing Nile Blue and the formation of Ca²⁺-oleate indicated the secretion of lipase. When the cell-free supernatant of cells grown in basal reach medium or the corresponding intracellular extract were electrophoresed under denatured and renatured conditions, using ??-naphthyl acetate and Fast Blue RR, major bands at 56 kDa or 62 and 32 kDa were observed, respectively. The 56 kDa extracellular enzyme was partial purified and characterized. Its peak of activity occurred at 80°C and pH 7.0, while the T_1/2 was 1 h at 100°C. The K _m of the partial purified enzyme was 1 mM and the V _max was 0.044 U/mL/min when using p-nitrophenyl laurate as substrate. The presence of Ca²⁺ and Hg²⁺ stimulated lipase activity, whereas Zn²⁺, Co²⁺, or EDTA inhibited lipase activity. The highest activity was observed in the presence of coconut oil and p-nitrophenyl laurate (pNPL). Purified lipase was the most stable in the presence of various organic solvents, such as pentanol, chloroform and n-dodecane. Because of the superior thermostability and stability in the presence of organic solvents of T. thermophilus extracellular lipase, this lipase holds great promise for use in industrial applications.     

Activation of the Canonical Bone Morphogenetic Protein (BMP) Pathway during Lung Morphogenesis and Adult Lung Tissue Repair

Signaling by Bone Morphogenetic Proteins (BMP) has been implicated in early lung development, adult lung homeostasis and tissue-injury repair. However, the precise mechanism of action and the spatio-temporal pattern of BMP-signaling during these processes remains inadequately described. To address this, we have utilized a transgenic line harboring a BMP-responsive eGFP-reporter allele (BRE-eGFP) to construct the first detailed spatiotemporal map of canonical BMP-pathway activation during lung development, homeostasis and adult-lung injury repair. We demonstrate that during the pseudoglandular stage, when branching morphogenesis progresses in the developing lung, canonical BMP-pathway is active mainly in the vascular network and the sub-epithelial smooth muscle layer of the proximal airways. Activation of the BMP-pathway becomes evident in epithelial compartments only after embryonic day (E) 14.5 primarily in cells negative for epithelial-lineage markers, located in the proximal portion of the airway-tree, clusters adjacent to neuro-epithelial-bodies (NEBs) and in a substantial portion of alveolar epithelial cells. The pathway becomes activated in isolated E12.5 mesenchyme-free distal epithelial buds cultured in Matrigel suggesting that absence of reporter activity in these regions stems from a dynamic cross-talk between endoderm and mesenchyme. Epithelial cells with activated BMP-pathway are enriched in progenitors capable of forming colonies in three-dimensional Matrigel cultures.As lung morphogenesis approaches completion, eGFP-expression declines and in adult lung its expression is barely detectable. However, upon tissue-injury, either with naphthalene or bleomycin, the canonical BMP-pathways is re-activated, in bronchial or alveolar epithelial cells respectively, in a manner reminiscent to early lung development and in tissue areas where reparatory progenitor cells reside. Our studies illustrate the dynamic activation of canonical BMP-pathway during lung development and adult lung tissue-repair and highlight its involvement in two important processes, namely, the early development of the pulmonary vasculature and the management of epithelial progenitor pools both during lung development and repair of adult lung tissue-injury.     

Heterogeneity of ATP-sensitive K+ Channels in Cardiac Myocytes: ENRICHMENT AT THE INTERCALATED DISK*

Ventricular ATP-sensitive potassium (K_ATP) channels link intracellular energy metabolism to membrane excitability and contractility. Our recent proteomics experiments identified plakoglobin and plakophilin-2 (PKP2) as putative K_ATP channel-associated proteins. We investigated whether the association of K_ATP channel subunits with junctional proteins translates to heterogeneous subcellular distribution within a cardiac myocyte. Co-immunoprecipitation experiments confirmed physical interaction between K_ATP channels and PKP2 and plakoglobin in rat heart. Immunolocalization experiments demonstrated that K_ATP channel subunits (Kir6.2 and SUR2A) are expressed at a higher density at the intercalated disk in mouse and rat hearts, where they co-localized with PKP2 and plakoglobin. Super-resolution microscopy demonstrate that K_ATP channels are clustered within nanometer distances from junctional proteins. The local K_ATP channel density, recorded in excised inside-out patches, was larger at the cell end when compared with local currents recorded from the cell center. The K_ATP channel unitary conductance, block by MgATP and activation by MgADP, did not differ between these two locations. Whole cell K_ATP channel current density (activated by metabolic inhibition) was ∼40% smaller in myocytes from mice haploinsufficient for PKP2. Experiments with excised patches demonstrated that the regional heterogeneity of K_ATP channels was absent in the PKP2 deficient mice, but the K_ATP channel unitary conductance and nucleotide sensitivities remained unaltered. Our data demonstrate heterogeneity of K_ATP channel distribution within a cardiac myocyte. The higher K_ATP channel density at the intercalated disk implies a possible role at the intercellular junctions during cardiac ischemia.