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Arthritis is a leading cause of disability in adults, which can be intensely incapacitating. The location and intensity of the pain is both subjective and challenging to manage. Consequently, patient-directed delivery of anti-inflammatories is an essential component of future therapeutic strategies for the management of this disorder. We describe the design and application of a light responsive red blood cell (RBC) conveyed dexamethasone (Dex) construct that enables targeted drug delivery upon illumination of the inflamed site. The red wavelength (650 nm) responsive nature of the phototherapeutic was validated using tissue phantoms mimicking the light absorbing properties of various skin types. Furthermore, photoreleased Dex has the same impact on cellular responses as conventional Dex. Murine RBCs containing the photoactivatable therapeutic display comparable circulation properties as fluorescently labelled RBCs. In addition, a single dose of light-targeted Dex delivery is 5-fold more effective in suppressing inflammation than the parent drug, delivered serially over multiple days. These results are consistent with the notion that the circulatory system be used as an on-command drug depot, providing the means to therapeutically target diseased sites both efficiently and effectively.

Exosomes (Exo) are a class of extracellular vesicles and involvement of stem cell-derived Exo in cardiac repair and cardioprotection is thought to be an important in the heart. Our HYPOTHESIS Is that specific microRNAs (miRs) from mesenchymal stem cell (MSC)-derived exosomes are actively and selectively sorted into Exo by RNA binding proteins and motifs on the miR, to serve specific functions of the Exo, including Cardioprotection. Methods: We characterized the miR populations of parental MSCs and their Exo via RNA Seq and confirmed by QRT-PCR the subpopulation of miRs that is increased in Exo vs . MSC cells. We then used Multiple Em for Motif Elicitation (MEME) Version 5.3.3 and determined the predicted conserved motifs. From these, we predicted RNA binding protein sites from the literature. In parallel, we performed mass spectrometry and western blot analyses to determine RNA binding proteins in MSC and Exo. Predicting that hnRNPA2B1 was a likely RNA binding protein for the new motif, we knocked out the cognate gene (CRISPR) in MSC and evaluated the KO Exo vs. the WT Exo by RNA Seq and QRT-PCR. We performed protein and RNA pulldowns, and EMSA to validate binding of hnRNPA2B1 to several of the miRs, and investigated the effects of these miRs on cell survival after simIR and in an in vivo mouse model of MI. Results: We found a set of eight miRs that are selectively concentrated in the MSC Exo. MEME software predicted a conserved binding motif of gAGu, which is close to canonical sites for binding of hnRNPA2B1 and hnRNPA1. We determined hnRNPA2B1 was in MSC and Exo and showed KO hnRNPA2B1 cells and Exo had no compensatory perturbation of other RNA binding proteins. The KO MSC Exo show reduction of the selective sorting of the miRs of interest. Pulldowns and binding assay results verify binding of hnRNPA2B1 to both miR-486a-5p and miR-122a. Finally, we showed that miR-486a-5p is protective in H9C2 cells submitted to simIR and results in significant 68% reduction of infarct size (n=7, P=0.0175) in vivo in association with repression of PDCD4 expression and apoptosis. Conclusions: We determined that a set of miRs is selectively concentrated in MSC Exo and demonstrated the necessity of hnRNPA2B1 in that process. This appears to involve a conserved RNA sequence motif (mutational analysis underway). A major miR affected is miR-486a-5p, which is strongly cardioprotective. Our results support that miR-486a-5p is selectively concentrated in MSC Exo and contributes to cardioprotection by reducing PDCD4 activity in apoptosis.

Timely reperfusion is still the most effective approach to limit infarct size in humans. Yet, despite advances in care and reduction in door-to-balloon times, nearly 25% of patients develop heart failure postmyocardial infarction, with its attendant morbidity and mortality. We previously showed that cardioprotection results from a skin incision through the umbilicus in a murine model of myocardial infarction. In the present study, we show that an electrical stimulus or topical capsaicin applied to the skin in the same region induces significantly reduced infarct size in a murine model. We define this class of phenomena as nociceptor-induced conditioning (NIC) based on the peripheral nerve mechanism of initiation. We show that NIC is effective both as a preconditioning and postconditioning remote stimulus, reducing infarct size by 86% and 80%, respectively. NIC is induced via activation of skin C-fiber nerves. Interestingly, the skin region that activates NIC is limited to the anterior of the T9−T10 vertebral region of the abdomen. Cardioprotection after NIC requires the integrity of the spinal cord from the region of stimulation to the thoracic vertebral region of the origin of the cardiac nerves but does not require that the cord be intact in the cervical region. Thus, we show that NIC is a reflex and not a central nervous system-mediated effect. The mechanism involves bradykinin 2 receptor activity and activation of PKC, specifically, PKC-α. The similarity of the neuroanatomy and conservation of the effectors of cardioprotection supports that NIC may be translatable to humans as a nontraumatic and practical adjunct therapy against ischemic disease. NEW & NOTEWORTHY This study shows that an electrical stimulus to skin sensory nerves elicits a very powerful cardioprotection against myocardial infarction. This stimulus works by a neurogenic mechanism similar to that previously elucidated for remote cardioprotection of trauma. Nociceptor-induced conditioning is equally potent when applied before ischemia or at reperfusion and has great potential clinically.

The impact of technology can be felt throughout the medical education continuum. From online learning environments in blended learning approaches to exclusively providing the preclinical curriculum online, there is a growing need to optimize the way that technology supports self-directed learning in the next generation of medical professionals. In this chapter, the authors address issues of best practice surrounding the development of virtual content for medical education. The information presented will be integral for medical education professionals, basic science/clinical faculty, and educational assessment specialists with an interest in the use of technology for contemporary medical education. The goal is to offer an overview of the theory and ethics behind adopting an online strategy for medical education. An emphasis is placed on developing best practices for presenting content, a comparison of blended and online-only approaches, and the ethical considerations necessary for the successful training of medical professionals online.

Optogenetic tools provide a level of spatial and temporal resolution needed to shed new light on dynamic intercellular processes. In this chapter we outline specific protocols for applying these tools to cell motility (optogenetic cofilin), apoptosis [optogenetic Bcl-like protein 4 (Bax)], and protein kinase-mediated signaling pathways [optogenetic cAMP-dependent protein kinase (PKA)]. The activity of these optogenetic species is regulated by the light-mediated dimerization of a cryptochrome/Cib protein pair, which controls the intracellular positioning of the protein of interest. The light induced recruitment of cofilin to the cytoskeleton is utilized for directed migration studies and filopodial dynamics. Light-triggered migration of Bax to the outer mitochondrial membrane induces cellular collapse and eventual apoptosis. Finally, the light-mediated movement of PKA to specific intracellular compartments offers the means to assess the consequences of PKA activity in a site-specific fashion via phosphoproteomic analysis.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease, and at least one-third of its patients relapse after treatment with the current chemotherapy regimen, R-CHOP. By gene-expression profiling, patients with DLBCL can be categorized into two clinically relevant subtypes: activated B-cell (ABC) and germinal center B-cell (GCB) DLBCL. Patients with the ABC subtype have a worse prognosis than those with GCB, and the subtype is defined by chronic, over-active signaling through the B-cell receptor and NF-κB pathways. We examined the effects of the Src family kinase (SFK) inhibitor dasatinib in a panel of ABC and GCB DLBCL cell lines and found that the former are much more sensitive to dasatinib than the latter. However, using multiplexed inhibitor bead coupled to mass spectrometry (MIB/MS) kinome profiling and Western blot analysis, we found that both subtypes display inhibition of the SFKs in response to dasatinib after both short- and long-term treatment. The MIB/MS analyses revealed that several cell-cycle kinases, including CDK4, CDK6, and the Aurora kinases, are down-regulated by dasatinib treatment in the ABC, but not in the GCB, subtype. The present findings have potential implications for the clinical use of dasatinib for the treatment of ABC DLBCL, either alone or in combination with other agents.

Ion channels play a major factor in maintaining cellular homeostasis but very little is known about the role of these proteins in cancer biology. In this work we have discovered that, the Kv11.3 (hERG3) a plasma-membrane potassium channel plays a critical role in the regulation of autophagy in a cancer cell model. We have found that pharmacologic stimulation of the Kv11.3 channel with a small molecule activator, NS1643 induced autophagy via activation of an AMPK-dependent signaling pathway in melanoma cell line. In addition, we have found that NS1643 produced a strong inhibition of cell proliferation by activating a cellular senescence program. Furthermore, inhibition of autophagy via siRNA targeting AMPK or treatment with hydroxychloroquine an autophagy inhibitor activates apoptosis in NS1643-treated cells. Thus, we propose that, Kv11.3 is a novel mediator of autophagy, autophagy can be a survival mechanism contributing to cellular senescence, and that use of a combinatorial pharmacologic approach of Kv11.3 activator with inhibitors of autophagy represents a novel therapeutic approach against melanoma.

Though experimental, stem cell transplantation has the potential to improve the condition of the heart after myocardial infarction. It does so by reducing infarct size and inducing repair of heart muscle and its blood supply. Mesenchymal stem cells (MSC) have been found to be effective in pre-clinical animal models and clinical trials, but the mechanisms by which they induce cardioprotection and repair are still not fully understood. Small extracellular vesicles known as exosomes are now recognized to be key mediators of beneficial MSC paracrine effects, and the concept that they transfer miRNA to change gene expression in recipient cells is of current therapeutic interest. We present complete deep miRNA sequencing of MSC exosome cargo, and found that of several cardioprotective miRNAs, miR-21a-5p was the most abundant. Because miR-21a-5p is a well-known cardioprotective miRNA, we investigated the hypothesis that MSC exosomes can cardioprotect the heart by increasing the level of miR-21a-5p in recipient cardiac cells, thereby downregulating expression of the pro-apoptotic gene products PDCD4, PTEN, Peli1 and FasL in the myocardium. Using miR-21 mimic transfection and treatment with wild type and miR-21a knockout MSC exosomes, we confirmed that exosomal miR-21a-5p is transferred into myocardium and is a major cardioprotective paracrine factor produced by MSCs acting via synergistic activity on multiple pathways. The data supports that residual cardioprotective effect may be due to other ncRNA or protein cargo. In silico analyses support that MSC exosomes may also contribute to angiogenesis, cell proliferation and other aspects of cardiac repair.

Because small-molecule activators of adenylyl cyclases (AC) affect ACs cell-wide, it is challenging to explore the signaling consequences of AC activity emanating from specific intracellular compartments. We explored this issue using a series of engineered, optogenetic, spatially restricted, photoactivable adenylyl cyclases (PACs) positioned at the plasma membrane (PM), the outer mitochondrial membrane (OMM), and the nucleus (Nu). The biochemical consequences of brief photostimulation of PAC is primarily limited to the intracellular site occupied by the PAC. By contrast, sustained photostimulation results in distal cAMP signaling. Prolonged cAMP generation at the OMM profoundly stimulates nuclear protein kinase (PKA) activity. We have found that phosphodiesterases 3 (OMM and PM) and 4 (PM) modulate proximal (local) cAMP-triggered activity, whereas phosphodiesterase 4 regulates distal cAMP activity as well as the migration of PKA's catalytic subunit into the nucleus.

In vitro but not in vivo evidence indicates that blockade of NF-κB is effective in reducing inflammation and production of IL-8. We hypothesized that the failure of in vitro experiments to predict in vivo outcome was due to the use of short time periods of observation and the use of single cytokines to stimulate NF-κB. HEK cells with a NF-κB reporter gene or CaCo-2 cells were stimulated with CM (IL-1-β; TNF-α, and IFN-γ) or individual cytokines in the presence and absence of NF-κB inhibitors, a STAT1 inhibitor, and/or a p38 MAPK inhibitor for periods up to 24 h. NF-κB activation, IL-8 production, and nitric oxide production were measured. CM-induced IL-8 production in HEK cells was additive to synergistic. CM enhanced production of IL-8 at 24 h but not 4 h was independent of NF-κB. The p38 inhibitor SB203580 and the STAT1 inhibitor EGCG blocked CM-induced IL-8 production at both early and late time periods. The NF-κB inhibitors PDTC and BAY11-7082 were found to increase CM-stimulated IL-8 production in Caco-2 cells at 24 h. Our data suggest an effective strategy to reduce IL-8 production is to block p38 or STAT1 rather than NF-κB.

Burn induces myeloid-derived suppressor cells (MDSCs), a heterogeneous population of immature polymorphonuclear neutrophils (PMNs) and monocytes, which protect against infection. Previous work from our laboratory demonstrated that inflammatory monocytes (iMos) were the major MDSC source of TNF-α in the postburn spleen, and we hypothesized that they were also the major source of postburn IL-10. To test this hypothesis, we examined cytokine production by postburn CCR2 knockout (KO) mice, which have fewer iMos than burn wild-type (WT) splenocytes, but equal numbers of PMNs and F4/80 macrophages. Using cell sorting and/or intracellular cytokine techniques, we examined IL-10 production by postburn PMNs and iMos. Finally, we compared IL-10 production by postburn PMNs and iMos with culture-derived MDSCs. Splenocytes from postburn CCR2 KO mice produced less IL-6 and TNF-α than WT burn splenocytes in response to LPS, but KO and WT burn splenocytes produced equal amounts of IL-10 in response to peptidoglycan. Depletion of PMNs from postburn splenocytes led to reductions in IL-10 and increases in IL-6 and TNF-α in response to peptidoglycan, but not in response to LPS. Sorting or intracellular cytokine techniques gave consistent results: Burn PMNs made more IL-10 than sham PMNs and also more IL-10 than burn or sham iMos. Polymorphonuclear neutrophil and iMos subpopulations from culture-derived MDSCs produced the same cytokine profiles in response to LPS and peptidoglycan as did the PMNs and iMos from postburn spleens: PMNs made IL-10, whereas iMos made IL-6. Finally, LPS-induced mortality of burn mice was made worse by anti-Gr-1 depletion of all PMNs and 66% of iMos from burn mice. This suggests that PMNs play a primarily anti-inflammatory role in vitro and in vivo.

Immature myeloid cells have been implicated as a source of postburn inflammation, and the appearance of these cells correlates with enhanced upregulation of hematopoiesis. The role of proliferative cells in postburn immune changes has not been directly tested. Gemcitabine, a ribonucleotide reductase inhibitor, has been shown to deplete proliferative immature myeloid cells in tumor models while sparing mature cells, leading to restored lymphocyte function and tumor regression. We treated burn mice at postburn day 6 (PBD6) with 120 mg/kg gemcitabine. On PBD8, splenocytes were taken and stimulated with LPS, peptidoglycan, or concanavalin A. The blood and spleen cell populations were enumerated by flow cytometry or automated cell counter. In addition, mice treated with gemcitabine were given LPS or infected with Pseudomonas aeruginosa at PBD8, and mortality was monitored. Gemcitabine depleted burn-induced polymorphonuclear leukocytes and inflammatory monocytes without affecting mature F4/80 macrophages. This was accompanied by reduced TNFα, IL-6, and IL-10 production by burn splenocytes. Burn splenocytes stimulated with mitogens exhibited increased nitric oxide production relative to sham mice. In vivo treatment of burn mice with gemcitabine blocked these burn-induced changes without damaging lymphocyte function. Treatment of burn mice with gemcitabine ameliorated burn-induced susceptibility to LPS and infiltration of polymorphonuclear leukocytes into the liver and lung. Finally, gemcitabine treatment blocked the protective effect of burn injury upon P. aeruginosa infection. Our report shows that proliferative cells are major drivers of postburn immune changes and provides evidence that implicates immature myeloid cells in these processes.

Previous studies indicate that the consumption of a high fat diet (HFD) 24h-2wks prior to the experience of ischemic injury can decrease the size of the infarcted area (normalized to the area at ri...

Ischemic preconditioning (IPC) is a protective stimulus whereby the heart undergoes a series of brief, non-lethal bouts of ischemia and reperfusion that protect it from subsequent prolonged ischemi...

Voltage-gated ion channels play a fundamental role in the generation and propagation of the cardiac action potential by acting synergistically to produce an ionic current across cellular membranes. Abnormalities of heart ion channel activities that lead to loss or gain of function (channelopathies) are often associated with disruption of the coordinated propagation of electrical activity of the cardiac myocytes and can generate fatal arrhythmogenesis. Drugs that act on cardiac ion channels have long been used to restore normal rhythm and conduction in patients affected by cardiac arrhythmias and offered to basic scientists the possibility to characterize distinct ion channel classes. This review will explore the mechanisms and role of the current anti-arrhythmic drugs used in the clinic, and discuss recent development on ion channel openers as potential anti-arrhythmic drugs.

Previous studies have demonstrated improvement of cardiac function occurs with acute consumption of a high-fat diet (HFD) after myocardial infarction (MI). However, no data exist addressing the effects of acute HFD upon the extent of injury after MI. This study investigates the hypothesis that short-term HFD, prior to infarction, protects the heart against ischemia-reperfusion (I/R) injury through NF-κB-dependent regulation of cell death pathways in the heart. Data show that an acute HFD initiates cardioprotection against MI (>50% reduction in infarct size normalized to risk region) after 24 h to 2 wk of HFD, but protection is completely absent after 6 wk of HFD, when mice are reported to develop pathophysiology related to the diet. Furthermore, cardioprotection after 24 h of HFD persists after an additional 24 h of normal chow feeding and was found to be dependent upon NF-κB activation in cardiomyocytes. This study also indicates that short-term HFD activates autophagic processes (beclin-1, LC-3) preischemia, as seen in other protective stimuli. Increases in beclin-1 and LC-3 were found to be NF-κB-dependent, and administration of chloroquine, an inhibitor of autophagy, abrogated cardioprotection. Our results support that acute high-fat feeding mediates cardioprotection against I/R injury associated with a NF-κB-dependent increase in autophagy and reduced apoptosis, as has been found for ischemic preconditioning.

Activation of NF-κB signaling and ensuing protective gene transcription in the heart is the basis for cardioprotection in the murine heart following an acute (24h) high fat diet (HFD). This protection is strongest after 2wks of high fat feeding and declines thereafter. The exact connection between acute high fat feeding and NF-κB activation is not yet fully understood. Long-term fat absorption in the intestine is associated with increased vascular permeability, decreased numbers of gram-positive bacteria, and cytokine release into lymphatic circulation. We considered the novel hypothesis that an acute HFD affects the intestinal microflora. Bacterial DNA from stool samples, luminal flushes, and mucosal scrapings were isolated from 24h high fat fed mice and changes in the phyla Firmicutes, Actinobacteria, Bacteroidetes, and Y-Proteobacteria measured by quantitative polymerase chain reaction. Our data indicates a significant increase in Actinobacteria in stool post high fat feeding. No statistically signific...

1 Department of Orthopedics, Hand and Microsurgical Center, The Second Affiliated Hospital of Harbin Medical University, Harbin, China 2 State-Province Key Laboratories of Biomedicine-Pharmaceutics, Harbin Medical University, Harbin, China 3 Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH, USA 4 Department of Orthopedics, University of Cincinnati College of Medicine, Cincinnati, OH, USA 5 Department of Anatomy, Harbin Medical University, Harbin, China

Probenecid is a highly lipid soluble benzoic acid derivative originally used to increase serum antibiotic concentrations. It was later discovered to have uricosuric effects and was FDA approved for gout therapy. It has recently been found to be a potent agonist of transient receptor potential vanilloid 2 (TRPV2). We have shown that this receptor is in the cardiomyocyte and report a positive inotropic effect of the drug. Using echocardiography, Langendorff and isolated myocytes, we measured the change in contractility and, using TRPV2(-/-) mice, proved that the effect was mediated by TRPV2 channels in the cardiomyocytes. Analysis of the expression of Ca(2+) handling and β-adrenergic signaling pathway proteins showed that the contractility was not increased through activation of the β-ADR. We propose that the response to probenecid is due to activation of TRPV2 channels secondary to SR release of Ca(2+).

Heat shock protein 70 (Hsp70) is well documented to possess general cytoprotective properties in protecting the cell against stressful and noxious stimuli. We have recently shown that expression of the stress-inducible Hsp70.3 gene in the myocardium in response to ischemic preconditioning is NF-κB-dependent and necessary for the resulting late phase cardioprotection against a subsequent ischemia/reperfusion injury. Here we show that the Hsp70.3 gene product is subject to post-transcriptional regulation through parallel regulatory processes involving microRNAs and alternative polyadenylation of the mRNA transcript. First, we show that cardiac ischemic preconditioning of the in vivo mouse heart results in decreased levels of two Hsp70.3-targeting microRNAs: miR-378* and miR-711. Furthermore, an ischemic or heat shock stimulus induces alternative polyadenylation of the expressed Hsp70.3 transcript that results in the accumulation of transcripts with a shortened 3′-UTR. This shortening of the 3′-UTR results in the loss of the binding site for the suppressive miR-378* and thus renders the alternatively polyadenylated transcript insusceptible to miR-378*-mediated suppression. Results also suggest that the alternative polyadenylation-mediated shortening of the Hsp70.3 3′-UTR relieves translational suppression observed in the long 3′-UTR variant, allowing for a more robust increase in protein expression. These results demonstrate alternative polyadenylation of Hsp70.3 in parallel with ischemic or heat shock-induced up-regulation of mRNA levels and implicate the importance of this process in post-transcriptional control of Hsp70.3 expression.

S U N D A Y 481 Anti-Histaminergic Responses On TRPV1 Channels Umesh Singh, Jonathan A. Bernstein, MD, Kristin Luther, Lauren Haar, W. Keith Jones; University of Cincinnati, Cincinnati, OH, Bernstein Allergy Group, Cincinnati, OH, University of Cincinnati Medical Center, Cincinnati, OH. RATIONALE: Activation of sensory neurons are known to be mediated by TRPV1 ion channels which may contribute to the pathophysiology of neurogenic inflammation. Recently we demonstrated that the topical antihistamine, azelastine (AZL), reported effective in non-allergic rhinitis (NAR), activates TRPV1 ion channels. The purpose of this study was to elucidate the effect of AZL on TRPV1 signaling. METHODS: Mice neuronal cells (CATH.a) were plated on glass coverslips and incubated (378C/5%CO2 324 hrs.). After loading cells with Ca2+ specific fluorescent dye Fluo-4 AM, changes in free cytosolic Ca2+([Ca2+]i), in response to AZL 30 mM and the TRPV1 agonist, Capsaicin 1 mM (CAP) , were assessed by confocal imaging. RT-PCR for mRNA expression of protein kinases (PKA and PKC) and phospholipase C (PLC) were performed to assess for TRPV1 responses recovering from desensitization. RESULTS: Sustained pre-treatment (;15 minutes) of CATH.a cells with CAP (1 mM) or AZL (30 mM) made these cells refractory to further increase in [Ca2+]i after subsequent applications of these agents indicating desensitization of TRPV1. After allowing for TRPV1 channel recovery (;25 minutes) from the desensitized state, treatment with either CAP (1 mM) or AZL (30 mM) resulted in a significant increase in [Ca2+]i which correlated with increased mRNA expression of the phosphorylating proteins PKA, PKC and PLC associated with re-sensitization of TRPV1. CONCLUSIONS: Sustained application of either CAP or AZL desensitize TRPV1 ion channels. This data could explain the therapeutic effect of AZL in NAR thought to be caused by neurogenic inflammation.