β-Amyloid Directly Inhibits Human α4β3-Nicotinic Acetylcholine Receptors Heterologously Expressed In Human Sh-Ep1 Cells

Amyloid-β (Aβ) accumulation and aggregation are thought to contribute to the pathogenesis of Alzheimer\u27s disease (AD). In AD, there is a selective decrease in the numbers of radioligand binding sites corresponding to the most abundant nicotinic acetylcholine receptor (nAChR) subtype, which contains human α4 and β2 subunits (hα4β2-nAChR). However, the relationships between these phenomena are uncertain, and effects of Aβ on hα4β2-nAChR function have not been investigated in detail. We first confirmed expression of hα4 and hβ2 subunits as messenger RNA in transfected, human SH-EP1 cells by reverse transcription-polymerase chain reaction and mRNA fluorescence in situ, hybridization analyses. Immunoprecipitation Western analyses confirmed α4 and β2 subunit protein expression and coassembly. Whole cell current recording demonstrated heterologous expression in SH-EP1-hα4β2 cells of functional hα4β2-nAChRs with characteristic responses to nicotinic agonists or antagonists. Nicotine-induced whole cell currents were suppressed by Aβ1-42 in a dose-dependent manner. Functional inhibition was selective for Aβ1-42compared with the functionally inactive, control peptide Aβ40-1. Aβ1-42-mediated inhibition of hα4β2-nAChR function was non-competitive, voltage-independent, and use-independent. Pre-loading of cells with guanyl-5′-yl thiophosphate failed to prevent Aβ1-42- induced inhibition, suggesting that down-regulation of hα4β2-nAChR function by Aβ1-42 is not mediated by nAChR intemalization. Sensitivity to Aβj_42 antagonism at 1 nM was evident for hα4β2-nAChRs, but not for heterologously expressed human α7-nAChRs, although both nAChR subtypes were functionally inhibited by 100 nM Aβ1-42, with the magnitude of functional block being higher for 100 nM Aβ1-42 acting on hα7-nAChRs. These findings suggest that hα4β2-nAChRs are sensitive and perhaps pathophysiologically relevant targets for Aβ neurotoxicity in AD

Interferon-b Modulates Inflammatory Response in Cerebral Ischemia

BACKGROUND: Stroke is a leading cause of death in the world. In >80% of strokes, the initial acute phase of ischemic injury is due to the occlusion of a blood vessel resulting in severe focal hypoperfusion, excitotoxicity, and oxidative damage. Interferon-β (IFNβ), a cytokine with immunomodulatory properties, was approved by the US Food and Drug Administration for the treatment of relapsing-remitting multiple sclerosis for more than a decade. Its anti-inflammatory properties and well-characterized safety profile suggest that IFNβ has therapeutic potential for the treatment of ischemic stroke. METHODS AND RESULTS: We investigated the therapeutic effect of IFNβ in the mouse model of transient middle cerebral artery occlusion/reperfusion. We found that IFNβ not only reduced infarct size in ischemic brains but also lessened neurological deficits in ischemic stroke animals. Further, multiple molecular mechanisms by which IFNβ modulates ischemic brain inflammation were identified. IFNβ reduced central nervous system infiltration of monocytes/macrophages, neutrophils, CD4(+) T cells, and γδ T cells; inhibited the production of inflammatory mediators; suppressed the expression of adhesion molecules on brain endothelial cells; and repressed microglia activation in the ischemic brain. CONCLUSIONS: Our results demonstrate that IFNβ exerts a protective effect against ischemic stroke through its anti-inflammatory properties and suggest that IFNβ is a potential therapeutic agent, targeting the reperfusion damage subsequent to the treatment with tissue plasminogen activator

Prostaglandin E2 Inhibition of IL-27 Production in Murine Dendritic Cells: A Novel Mechanism That Involves IRF1

IL-27, a multifunctional cytokine produced by APCs, antagonizes inflammation by affecting conventional dendritic cells (cDC), inducing IL-10, and promoting development of regulatory Tr1 cells. Although the mechanisms involved in IL-27 induction are well studied, much less is known about the factors that negatively impact IL-27 expression. PGE2, a major immunomodulatory prostanoid, acts as a proinflammatory agent in several models of inflammatory/autoimmune disease, promoting primarily Th17 development and function. In this study, we report on a novel mechanism that promotes the proinflammatory function of PGE2 We showed previously that PGE2 inhibits IL-27 production in murine bone marrow-derived DCs. In this study, we show that, in addition to bone marrow-derived DCs, PGE2 inhibits IL-27 production in macrophages and in splenic cDC, and we identify a novel pathway consisting of signaling through EP2/EP4→induction of cAMP→downregulation of IFN regulatory factor 1 expression and binding to the p28 IFN-stimulated response element site. The inhibitory effect of PGE2 on p28 and irf1 expression does not involve endogenous IFN-β, STAT1, or STAT2, and inhibition of IL-27 does not appear to be mediated through PKA, exchange protein activated by cAMP, PI3K, or MAPKs. We observed similar inhibition of il27p28 expression in vivo in splenic DC following administration of dimethyl PGE2 in conjunction with LPS. Based on the anti-inflammatory role of IL-27 in cDC and through the generation of Tr1 cells, we propose that the PGE2-induced inhibition of IL-27 in activated cDC represents an important additional mechanism for its in vivo proinflammatory functions

Biomechanical comparisons of F.E.R.I. techniques with different type of intramedullary screws fixation for Jones fractures

Introduction: Jones fractures frequently fail to unite, and adequate fixation stability is crucial. This study aimed to elucidate the biomechanical stability of various intramedullary screw fixation constructs.Methods: Jones fracture model over the proximal 5th metatarsal of artificial bone was created in all specimens. Six groups were divided based on varied screw constructs with different screw lengths, either 30 or 40 mm, including cannulated screws—C30 and C40 groups, one high-resistance suture combined with intramedullary cannulated screws (F.E.R.I. technique)—CF30 and CF40 groups, and second-generation headless compression screws (SG-HCS) —HL30 and HL40 groups. Mechanical testing was conducted sequentially, and the maximal force (N) and stiffness (N/mm) of all constructs were recorded.Results: The maximal force (N) at 1.0 mm downward displacement in C30, C40, CF30, CF40, HL30, and HL40 groups were 0.56 ± 0.02, 0.49 ± 0.02, 0.65 ± 0.02, 0.49 ± 0.01, 0.68 ± 0.02, and 0.73 ± 0.02, respectively, and the stiffness (N/mm) in subgroups were 0.49 ± 0.01, 0.43 ± 0.01, 0.67 ± 0.01, 0.42 ± 0.01, 0.61 ± 0.01, and 0.58 ± 0.02, respectively. SG-HCS subgroups exhibited greater maximal force and stiffness than conventional cannulated screws. Screws of 30 mm in length demonstrated better stability than all 40 mm-length screws in each subgroup. In C30 fixation, the stiffness and maximum force endured increased by 1.16 and 1.12 times, respectively, compared with the C40 fixation method. There were no significant differences between CF30 and SG-HCS groups. Only the F.E.R.I technique combined with the 4.5 mm cannulated screw of 30 mm in length increased the biomechanical stability for Jones fractures.Discussion: These biomechanical findings help clinicians decide on better screw fixation options for greater stability in Jones fractures, especially when large-diameter screws are limited in use. However, this biomechanical testing of intramedullary screw fixation on Jones fracture model lacks clinical validation and no comparisons to extramedullary plate fixations. Moving forward, additional clinical and biomechanical research is necessary to validate our findings

Dithiolethione ACDT suppresses neuroinflammation and ameliorates disease severity in experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disorder characterized by the central nervous system (CNS) infiltration of myelin-specific pathogenic T cells followed by brain inflammation in association with demyelination. Similarly, experimental autoimmune encephalomyelitis (EAE), the animal model of MS, also exhibits increased CNS infiltration of pathogenic T cells, including Th1 and Th17, leading to detrimental effects of neuroinflammation and demyelination. We previously reported that 3H-1,2-dithiole-3-thione (D3T), the structurally-simplest of the sulfur-containing dithiolethiones, exerted a promising therapeutic effect in EAE. In the current study we report that 5-Amino-3-thioxo-3H-(1,2)dithiole-4-carboxylic acid ethyl ester (ACDT), a substituted derivative of D3T, exhibits anti-inflammatory properties in EAE. ACDT, administered post immunization, delayed disease onset and reduced disease severity in chronic C57BL/6 EAE, and ACDT, administered during disease remission, suppressed disease relapse in relapsing-remitting SJL/J EAE. Further analysis of the cellular and molecular mechanisms underlying the protective effects of ACDT in EAE revealed that ACDT inhibited pathogenic T cell infiltration, suppressed microglia activation, repressed neurotoxic A1 astrocyte generation, lessened blood-brain barrier disruption, and diminished MMP3/9 production in the CNS of EAE. In summary, we demonstrate that ACDT suppresses neuroinflammation and ameliorates disease severity in EAE through multiple cellular mechanisms. Our findings suggest the potential of developing ACDT as a novel therapeutic agent for the treatment of MS/EAE

Isolation of Mouse Cerebral Microvasculature for Molecular and Single-Cell Analysis

Brain microvasculature forms a specialized structure, the blood-brain barrier (BBB), to maintain homeostasis and integrity of the central nervous system (CNS). The BBB dysfunction is emerging as a critical contributor to multiple neurological disorders, including stroke, traumatic brain injury, autoimmune multiple sclerosis, and neurodegenerative diseases. The brain microvasculature exhibits highly cellular and regional heterogeneity to accommodate dynamic changes of microenvironment during homeostasis and diseases. Thus, investigating the underlying mechanisms that contribute to molecular or cellular changes of the BBB is a significant challenge. Here, we describe an optimized protocol to purify microvessels from the mouse cerebral cortex using mechanical homogenization and density-gradient centrifugation, while maintaining the structural integrity and functional activity of the BBB. We show that the isolated microvessel fragments consist of BBB cell populations, including endothelial cells, astrocyte end-feet, pericytes, as well as tight junction proteins that seal endothelial cells. Furthermore, we describe the procedures to generate single-cell suspensions from isolated microvessel fragments. We demonstrate that cells in the single-cell suspensions are highly viable and suitable for single-cell RNA-sequencing analysis. This protocol does not require transgenic mice and cell sorting equipment to isolate fluorescence-labeled endothelial cells. The optimized procedures can be applied to different disease models to generate viable cells for single-cell analysis to uncover transcriptional or epigenetic landscapes of BBB component cells

Opportunities and Challenges of Joint Edge and Fog Orchestration

Pushing contents, applications, and network functions closer to end users is necessary to cope with the huge data volume and low latency required in future 5G networks. Edge and fog frameworks have emerged recently to address this challenge. Whilst the edge framework was more infrastructure focused and more mobile operator-oriented, the fog was more pervasive and included any node (stationary or mobile), including terminal devices. This article analyzes the opportunities and challenges to integrate, federate, and jointly orchestrate the edge and fog resources into a unified framework.This work has been partially funded by the H2020 collaborative Europe/Taiwan research project 5G-CORAL (grant num. 761586

Identification of N-benzyltetrahydroisoquinolines as novel anti-neuroinflammatory agents

A series of simplified berberine analogs was designed, synthesized, and evaluated for anti-inflammatory activity. SAR studies identified N-benzyltetrahydroisoquinoline 7d as a potent berberine analog. 7d suppressed LPS-induced inflammatory cytokine levels in both BV2 cells and primary microglia. Taken together, our results suggest that simplified BB analogs have therapeutic potential as a novel class of anti-neuroinflammatory agents

Dimethyl fumarate attenuates reactive microglia and long-term memory deficits following systemic immune challenge

BACKGROUND: Systemic inflammation is associated with increased cognitive decline and risk for Alzheimer's disease. Microglia (MG) activated during systemic inflammation can cause exaggerated neuroinflammatory responses and trigger progressive neurodegeneration. Dimethyl fumarate (DMF) is a FDA-approved therapy for multiple sclerosis. The immunomodulatory and anti-oxidant properties of DMF prompted us to investigate whether DMF has translational potential for the treatment of cognitive impairment associated with systemic inflammation. METHODS: Primary murine MG cultures were stimulated with lipopolysaccharide (LPS) in the absence or presence of DMF. MG cultured from nuclear factor (erythroid-derived 2)-like 2-deficient (Nrf2 -/- ) mice were used to examine mechanisms of DMF actions. Conditioned media generated from LPS-primed MG were used to treat hippocampal neuron cultures. Adult C57BL/6 and Nrf2 -/- mice were subjected to peripheral LPS challenge. Acute neuroinflammation, long-term memory function, and reactive astrogliosis were examined to assess therapeutic effects of DMF. RESULTS: DMF suppressed inflammatory activation of MG induced by LPS. DMF suppressed NF-κB activity through Nrf2-depedent and Nrf2-independent mechanisms in MG. DMF treatment reduced MG-mediated toxicity towards neurons. DMF suppressed brain-derived inflammatory cytokines in mice following peripheral LPS challenge. The suppressive effect of DMF on neuroinflammation was blunted in Nrf2 -/- mice. Importantly, DMF treatment alleviated long-term memory deficits and sustained reactive astrogliosis induced by peripheral LPS challenge. DMF might mitigate neurotoxic astrocytes associated with neuroinflammation. CONCLUSIONS: DMF treatment might protect neurons against toxic microenvironments produced by reactive MG and astrocytes associated with systemic inflammation