Facing the urgency of therapies for progressive MS — a Progressive MS Alliance proposal

Therapies for infiltrative inflammation in multiple sclerosis (MS) have advanced greatly, but neurodegeneration and compartmentalized inflammation remain virtually untargeted as in other diseases of the nervous system. Consequently, many therapies are available for the relapsing–remitting form of MS, but the progressive forms remain essentially untreated. The objective of the International Progressive MS Alliance is to expedite the development of effective therapies for progressive MS through new initiatives that foster innovative thinking and concrete advancements. Based on these principles, the Alliance is developing a new funding programme that will focus on experimental medicine trials. Here, we discuss the reasons behind the focus on experimental medicine trials, the strengths and weaknesses of these approaches and of the programme, and why we hope to advance therapies while improving the understanding of progression in MS. We are soliciting public and academic feedback, which will help shape the programme and future strategies of the Alliance

Insulin-stimulated phosphorylation of endothelial nitric oxide synthase at serine-615 contributes to nitric oxide synthesis

Insulin stimulates endothelial NO (nitric oxide) synthesis via PKB (protein kinase B)/Akt-mediated phosphorylation and activation of eNOS (endothelial NO synthase) at Ser-1177. In previous studies, we have demonstrated that stimulation of eNOS phosphorylation at Ser-1177 may be required, yet is not sufficient for insulin-stimulated NO synthesis. We therefore investigated the role of phosphorylation of eNOS at alternative sites to Ser-1177 as candidate parallel mechanisms contributing to insulin-stimulated NO synthesis. Stimulation of human aortic endothelial cells with insulin rapidly stimulated phosphorylation of both Ser-615 and Ser-1177 on eNOS, whereas phosphorylation of Ser-114, Thr-495 and Ser-633 was unaffected. Insulin-stimulated Ser-615 phosphorylation was abrogated by incubation with the PI3K (phosphoinositide 3-kinase) inhibitor wortmannin, infection with adenoviruses expressing a dominant-negative mutant PKB/Akt or pre-incubation with TNFα (tumour necrosis factor α), but was unaffected by high culture glucose concentrations. Mutation of Ser-615 to alanine reduced insulin-stimulated NO synthesis, whereas mutation of Ser-615 to aspartic acid increased NO production by NOS in which Ser-1177 had been mutated to an aspartic acid residue. We propose that the rapid PKB-mediated stimulation of phosphorylation of Ser-615 contributes to insulin-stimulated NO synthesis

AKT activity orchestrates marginal zone B cell development in mice and humans.

The signals controlling marginal zone (MZ) and follicular (FO) B cell development remain incompletely understood. Here, we show that AKT orchestrates MZ B cell formation in mice and humans. Genetic models that increase AKT signaling in B cells or abolish its impact on FoxO transcription factors highlight the AKT-FoxO axis as an on-off switch for MZ B cell formation in mice. In humans, splenic immunoglobulin (Ig) D <sup>+</sup> CD27 <sup>+</sup> B cells, proposed as an MZ B cell equivalent, display higher AKT signaling than naive IgD <sup>+</sup> CD27 <sup>-</sup> and memory IgD <sup>-</sup> CD27 <sup>+</sup> B cells and develop in an AKT-dependent manner from their precursors in vitro, underlining the conservation of this developmental pathway. Consistently, CD148 is identified as a receptor indicative of the level of AKT signaling in B cells, expressed at a higher level in MZ B cells than FO B cells in mice as well as humans

Riboflavin/UVA Collagen Cross-Linking-Induced Changes in Normal and Keratoconus Corneal Stroma

Purpose To determine the effect of Ultraviolet-A collagen cross-linking with hypo-osmolar and iso-osmolar riboflavin solutions on stromal collagen ultrastructure in normal and keratoconus ex vivo human corneas. Methods Using small-angle X-ray scattering, measurements of collagen D-periodicity, fibril diameter and interfibrillar spacing were made at 1 mm intervals across six normal post-mortem corneas (two above physiological hydration (swollen) and four below (unswollen)) and two post-transplant keratoconus corneal buttons (one swollen; one unswollen), before and after hypo-osmolar cross-linking. The same parameters were measured in three other unswollen normal corneas before and after iso-osmolar cross-linking and in three pairs of swollen normal corneas, in which only the left was cross-linked (with iso-osmolar riboflavin). Results Hypo-osmolar cross-linking resulted in an increase in corneal hydration in all corneas. In the keratoconus corneas and unswollen normal corneas, this was accompanied by an increase in collagen interfibrillar spacing (p<0.001); an increase in fibril diameter was also seen in two out of four unswollen normal corneas and one unswollen keratoconus cornea (p<0.001). Iso-osmolar cross-linking resulted in a decrease in tissue hydration in the swollen normal corneas only. Although there was no consistent treatment-induced change in hydration in the unswollen normal samples, iso-osmolar cross-linking of these corneas did result in a compaction of collagen fibrils and a reduced fibril diameter (p<0.001); these changes were not seen in the swollen normal corneas. Collagen D-periodicity was not affected by either treatment. Conclusion The observed structural changes following Ultraviolet-A cross-linking with hypo-osmolar or iso-osmolar riboflavin solutions are more likely a consequence of treatment-induced changes in tissue hydration rather than cross-linking

Ultra-stable optical clock with two cold-atom ensembles

Atomic clocks based on optical transitions are the most stable, and therefore precise, timekeepers available. These clocks operate by alternating intervals of atomic interrogation with dead time required for quantum state preparation and readout. This non-continuous interrogation of the atom system results in the Dick effect, an aliasing of frequency noise of the laser interrogating the atomic transition. Despite recent advances in optical clock stability achieved by improving laser coherence, the Dick effect has continually limited optical clock performance. Here we implement a robust solution to overcome this limitation: a zero-dead-time optical clock based on the interleaved interrogation of two cold-atom ensembles. This clock exhibits vanishingly small Dick noise, thereby achieving an unprecedented fractional frequency instability of $6 \times 10^{-17} / \sqrt{\tau}$ for an averaging time $\tau$ in seconds. We also consider alternate dual-atom-ensemble schemes to extend laser coherence and reduce the standard quantum limit of clock stability, achieving a spectroscopy line quality factor $Q> 4 \times 10^{15}$

α1A-Adrenergic Receptor Induces Activation of Extracellular Signal-Regulated Kinase 1/2 through Endocytic Pathway

G protein-coupled receptors (GPCRs) activate mitogen-activated protein kinases through a number of distinct pathways in cells. Increasing evidence has suggested that endosomal signaling has an important role in receptor signal transduction. Here we investigated the involvement of endocytosis in α1A-adrenergic receptor (α1A-AR)-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2). Agonist-mediated endocytic traffic of α1A-AR was assessed by real-time imaging of living, stably transfected human embryonic kidney 293A cells (HEK-293A). α1A-AR was internalized dynamically in cells with agonist stimulation, and actin filaments regulated the initial trafficking of α1A-AR. α1A-AR-induced activation of ERK1/2 but not p38 MAPK was sensitive to disruption of endocytosis, as demonstrated by 4°C chilling, dynamin mutation and treatment with cytochalasin D (actin depolymerizing agent). Activation of protein kinase C (PKC) and C-Raf by α1A-AR was not affected by 4°C chilling or cytochalasin D treatment. U73122 (a phospholipase C [PLC] inhibitor) and Ro 31–8220 (a PKC inhibitor) inhibited α1B-AR- but not α1A-AR-induced ERK1/2 activation. These data suggest that the endocytic pathway is involved in α1A-AR-induced ERK1/2 activation, which is independent of Gq/PLC/PKC signaling

Pharmacological studies of the mechanism and function of interleukin-1β-induced miRNA-146a expression in primary human airway smooth muscle

<p>Abstract</p> <p>Background</p> <p>Despite the widespread induction of miR-146a during the innate immune response little is known regarding its biogenesis, function and mechanism. We have therefore examined the role of miR-146a during the interleukin (IL)-1β-stimulated IL-6 and IL-8 release and proliferation in primary human airway smooth muscle (HASM) cells.</p> <p>Methods</p> <p>HASM cells were isolated from human lung re-section, cultured to a maximum of 3 - 6 passages and then exposed to IL-1β. miR-146a expression were determined by qRT-PCR, IL-6 and IL-8 release by ELISA and proliferation using bromodeoxyuridine incorporation. The role of NF-κB and the MAP kinase pathways was assessed using pharmacological inhibitors of IKK2 (TPCA-1), JNK (SP600125), p38 MAP kinase (SB203580) and MEK-1/2 (PD98059). miR-146a function was determined following transfection of HASM with inhibitors and mimics using Amaxa electroporation.</p> <p>Results</p> <p>IL-1β induced a time-dependent and prolonged 100-fold induction in miR-146a expression, which correlated with release of IL-6 and IL-8. Exposure to IL-1β had no effect upon HASM proliferation. Pharmacological studies showed that expression of primary miR-146a was regulated at the transcriptional levels by NF-κB whilst post-transcriptional processing to mature miR-146a was regulated by MEK-1/2 and JNK-1/2. Functional studies indicated that IL-1β-induced miR-146a expression does not negatively regulate IL-6 and IL-8 release or basal proliferation. However, inhibition of IL-1β-induced IL-6 and IL-8 release was observed at the super-maximal intracellular miR-146a levels obtained by transfection with miR-146a mimics and indicates that studies using miRNA mimics can produce false positive results. Mechanistic studies showed that in the presence of super-maximal levels, the action of miR-146a mimics was mediated at a step following IL-6 and IL-8 mRNA transcription and not through down-regulation of IL-1 receptor associated kinase 1 (IRAK-1) and TNF receptor-associated factor 6 (TRAF6) protein expression, two predicted miR-146a targets involved in IL-1β signalling.</p> <p>Conclusions</p> <p>We have shown that IL-1β-induced miR-146a expression in HASM and that this was regulated at the transcriptional level by NF-κB and at the post-transcriptional level by the MEK-1/2 and JNK-1/2. Unlike previous reports, studies using miRNA inhibitors showed that miR-146a expression did not regulate IL-6 and IL-8 release or proliferation and suggest miR-146a function and mechanism is cell-type dependent.</p

Abnormalities of calcium metabolism and myocardial contractility depression in the failing heart

Heart failure (HF) is characterized by molecular and cellular defects which jointly contribute to decreased cardiac pump function. During the development of the initial cardiac damage which leads to HF, adaptive responses activate physiological countermeasures to overcome depressed cardiac function and to maintain blood supply to vital organs in demand of nutrients. However, during the chronic course of most HF syndromes, these compensatory mechanisms are sustained beyond months and contribute to progressive maladaptive remodeling of the heart which is associated with a worse outcome. Of pathophysiological significance are mechanisms which directly control cardiac contractile function including ion- and receptor-mediated intracellular signaling pathways. Importantly, signaling cascades of stress adaptation such as intracellular calcium (Ca2+) and 3′-5′-cyclic adenosine monophosphate (cAMP) become dysregulated in HF directly contributing to adverse cardiac remodeling and depression of systolic and diastolic function. Here, we provide an update about Ca2+ and cAMP dependent signaling changes in HF, how these changes affect cardiac function, and novel therapeutic strategies which directly address the signaling defects