Fractalkine/CX3CL1 protects striatal neurons from synergistic morphine and HIV-1 Tat-induced dendritic losses and death

<p>Abstract</p> <p>Background</p> <p>Fractalkine/CX₃CL1 and its cognate receptor CX₃CR1 are abundantly expressed in the CNS. Fractalkine is an unusual C-X3-C motif chemokine that is important in neuron-microglial communication, a co-receptor for HIV infection, and can be neuroprotective. To assess the effects of fractalkine on opiate-HIV interactive neurotoxicity, wild-type murine striatal neurons were co-cultured with mixed glia from the striata of wild-type or <it>Cx3cr1 </it>knockout mice ± HIV-1 Tat and/or morphine. Time-lapse digital images were continuously recorded at 20 min intervals for up to 72 h using computer-aided microscopy to track the same cells repeatedly.</p> <p>Results</p> <p>Co-exposure to Tat and morphine caused synergistic increases in neuron death, dendritic pruning, and microglial motility as previously reported. Exogenous fractalkine prevented synergistic Tat and morphine-induced dendritic losses and neuron death even though the inflammatory mediator TNF-α remained significantly elevated. Antibody blockade of CX₃CR1 mimicked the toxic effects of morphine plus Tat, but did not add to their toxicity; while fractalkine failed to protect wild-type neurons co-cultured with <it>Cx₃cr1</it>^-/--null glia against morphine and Tat toxicity. Exogenous fractalkine also normalized microglial motility, which is elevated by Tat and morphine co-exposure, presumably limiting microglial surveillance that may lead to toxic effects on neurons. Fractalkine immunofluorescence was expressed in neurons and to a lesser extent by other cell types, whereas CX₃CR1 immunoreactivity or GFP fluorescence in cells cultured from the striatum of <it>Cx3cr1</it>^-/-(<it>Cx3cr1</it>^GFP/GFP) mice were associated with microglia. Immunoblotting shows that fractalkine levels were unchanged following Tat and/or morphine exposure and there was no increase in released fractalkine as determined by ELISA. By contrast, CX₃CR1 protein levels were markedly downregulated.</p> <p>Conclusions</p> <p>The results suggest that deficits in fractalkine-CX₃CR1 signaling contribute to the synergistic neurotoxic effects of opioids and Tat. Importantly, exogenous fractalkine can selectively protect neurons from the injurious effects of chronic opioid-HIV-1 Tat co-exposure, and this suggests a potential therapeutic course for neuroAIDS. Although the cellular mechanisms underlying neuroprotection are not certain, findings that exogenous fractalkine reduces microglial motility and fails to protect neurons co-cultured with <it>Cx3cr1</it>^-/-mixed glia suggest that fractalkine may act by interfering with toxic microglial-neuron interactions.</p

Participatory Process for Implementing a Colorectal Cancer Screening Intervention: an Action Plan for Local Sustainability

Background: Rigid protocols can hamper translation of evidence-based interventions from research to real-world settings. This investigation aimed to develop procedures for modifying the study protocol of a community-based participatory research (CBPR) project and to analyze the theoretical constructs that underlie this process. Methods: The research project is a dissemination and implementation study of the Educational Program to Increase Colorectal Cancer Screening (EPICS), an evidence-based intervention targeting African Americans in the United States. The study is being conducted in a partnership with community coalitions in 15 different cities. Each site initially presented unique issues that required modification of the study protocol. Results: In order to honor underlying CBPR theory, it was necessary to negotiate protocol changes with the community coalition at each site, while insuring preservation of the core elements of the intervention. Conclusions: We discuss the ways in which this represents a narrowing of the gap between CBPR and traditional research approaches

The Secreted Metalloprotease ADAMTS20 Is Required for Melanoblast Survival

ADAMTS20 (A disintegrin-like and metalloprotease domain with thrombospondin type-1 motifs) is a member of a family of secreted metalloproteases that can process a variety of extracellular matrix (ECM) components and secreted molecules. Adamts20 mutations in belted (bt) mice cause white spotting of the dorsal and ventral torso, indicative of defective neural crest (NC)-derived melanoblast development. The expression pattern of Adamts20 in dermal mesenchymal cells adjacent to migrating melanoblasts led us to initially propose that Adamts20 regulated melanoblast migration. However, using a Dct-LacZ transgene to track melanoblast development, we determined that melanoblasts were distributed normally in whole mount E12.5 bt/bt embryos, but were specifically reduced in the trunk of E13.5 bt/bt embryos due to a seven-fold higher rate of apoptosis. The melanoblast defect was exacerbated in newborn skin and embryos from bt/bt animals that were also haploinsufficient for Adamts9, a close homolog of Adamts20, indicating that these metalloproteases functionally overlap in melanoblast development. We identified two potential mechanisms by which Adamts20 may regulate melanoblast survival. First, skin explant cultures demonstrated that Adamts20 was required for melanoblasts to respond to soluble Kit ligand (sKitl). In support of this requirement, bt/bt;Kittm1Alf/+ and bt/bt;KitlSl/+ mice exhibited synergistically increased spotting. Second, ADAMTS20 cleaved the aggregating proteoglycan versican in vitro and was necessary for versican processing in vivo, raising the possibility that versican can participate in melanoblast development. These findings reveal previously unrecognized roles for Adamts proteases in cell survival and in mediating Kit signaling during melanoblast colonization of the skin. Our results have implications not only for understanding mechanisms of NC-derived melanoblast development but also provide insights on novel biological functions of secreted metalloproteases

International Society of Human and Animal Mycology (ISHAM)-ITS reference DNA barcoding database - the quality controlled standard tool for routine identification of human and animal pathogenic fungi

Human and animal fungal pathogens are a growing threat worldwide leading to emerging infections and creating new risks for established ones. There is a growing need for a rapid and accurate identification of pathogens to enable early diagnosis and targeted antifungal therapy. Morphological and biochemical identification methods are time-consuming and require trained experts. Alternatively, molecular methods, such as DNA barcoding, a powerful and easy tool for rapid monophasic identification, offer a practical approach for species identification and less demanding in terms of taxonomical expertise. However, its wide-spread use is still limited by a lack of quality-controlled reference databases and the evolving recognition and definition of new fungal species/complexes. An international consortium of medical mycology laboratories was formed aiming to establish a quality controlled ITS database under the umbrella of the ISHAM working group on "DNA barcoding of human and animal pathogenic fungi." A new database, containing 2800 ITS sequences representing 421 fungal species, providing the medical community with a freely accessible tool at http://www.isham.org and http://its.mycologylab.org/ to rapidly and reliably identify most agents of mycoses, was established. The generated sequences included in the new database were used to evaluate the variation and overall utility of the ITS region for the identification of pathogenic fungi at intra-and interspecies level. The average intraspecies variation ranged from 0 to 2.25%. This highlighted selected pathogenic fungal species, such as the dermatophytes and emerging yeast, for which additional molecular methods/genetic markers are required for their reliable identification from clinical and veterinary specimens.This study was supported by an National Health and Medical Research Council of Australia (NH&MRC) grant [#APP1031952] to W Meyer, S Chen, V Robert, and D Ellis; CNPq [350338/2000-0] and FAPERJ [E-26/103.157/2011] grants to RM Zancope-Oliveira; CNPq [308011/2010-4] and FAPESP [2007/08575-1] Fundacao de Amparo Pesquisa do Estado de So Paulo (FAPESP) grants to AL Colombo; PEst-OE/BIA/UI4050/2014 from Fundacao para a Ciencia e Tecnologia (FCT) to C Pais; the Belgian Science Policy Office (Belspo) to BCCM/IHEM; the MEXBOL program of CONACyT-Mexico, [ref. number: 1228961 to ML Taylor and [122481] to C Toriello; the Institut Pasteur and Institut de Veil le Sanitaire to F Dromer and D Garcia-Hermoso; and the grants from the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) and the Fundacao de Amparo a Pesquisa do Estado de Goias (FAPEG) to CM de Almeida Soares and JA Parente Rocha. I Arthur would like to thank G Cherian, A Higgins and the staff of the Molecular Diagnostics Laboratory, Division of Microbiology and Infectious Diseases, Path West, QEII Medial Centre. Dromer would like to thank for the technical help of the sequencing facility and specifically that of I, Diancourt, A-S Delannoy-Vieillard, J-M Thiberge (Genotyping of Pathogens and Public Health, Institut Pasteur). RM Zancope-Oliveira would like to thank the Genomic/DNA Sequencing Platform at Fundacao Oswaldo Cruz-PDTIS/FIOCRUZ [RPT01A], Brazil for the sequencing. B Robbertse and CL Schoch acknowledge support from the Intramural Research Program of the NIH, National Library of Medicine. T Sorrell's work is funded by the NH&MRC of Australia; she is a Sydney Medical School Foundation Fellow.info:eu-repo/semantics/publishedVersio

Adjunctive rifampicin for Staphylococcus aureus bacteraemia (ARREST): a multicentre, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Staphylococcus aureus bacteraemia is a common cause of severe community-acquired and hospital-acquired infection worldwide. We tested the hypothesis that adjunctive rifampicin would reduce bacteriologically confirmed treatment failure or disease recurrence, or death, by enhancing early S aureus killing, sterilising infected foci and blood faster, and reducing risks of dissemination and metastatic infection. METHODS: In this multicentre, randomised, double-blind, placebo-controlled trial, adults (≥18 years) with S aureus bacteraemia who had received ≤96 h of active antibiotic therapy were recruited from 29 UK hospitals. Patients were randomly assigned (1:1) via a computer-generated sequential randomisation list to receive 2 weeks of adjunctive rifampicin (600 mg or 900 mg per day according to weight, oral or intravenous) versus identical placebo, together with standard antibiotic therapy. Randomisation was stratified by centre. Patients, investigators, and those caring for the patients were masked to group allocation. The primary outcome was time to bacteriologically confirmed treatment failure or disease recurrence, or death (all-cause), from randomisation to 12 weeks, adjudicated by an independent review committee masked to the treatment. Analysis was intention to treat. This trial was registered, number ISRCTN37666216, and is closed to new participants. FINDINGS: Between Dec 10, 2012, and Oct 25, 2016, 758 eligible participants were randomly assigned: 370 to rifampicin and 388 to placebo. 485 (64%) participants had community-acquired S aureus infections, and 132 (17%) had nosocomial S aureus infections. 47 (6%) had meticillin-resistant infections. 301 (40%) participants had an initial deep infection focus. Standard antibiotics were given for 29 (IQR 18-45) days; 619 (82%) participants received flucloxacillin. By week 12, 62 (17%) of participants who received rifampicin versus 71 (18%) who received placebo experienced treatment failure or disease recurrence, or died (absolute risk difference -1·4%, 95% CI -7·0 to 4·3; hazard ratio 0·96, 0·68-1·35, p=0·81). From randomisation to 12 weeks, no evidence of differences in serious (p=0·17) or grade 3-4 (p=0·36) adverse events were observed; however, 63 (17%) participants in the rifampicin group versus 39 (10%) in the placebo group had antibiotic or trial drug-modifying adverse events (p=0·004), and 24 (6%) versus six (2%) had drug interactions (p=0·0005). INTERPRETATION: Adjunctive rifampicin provided no overall benefit over standard antibiotic therapy in adults with S aureus bacteraemia. FUNDING: UK National Institute for Health Research Health Technology Assessment

IHR (2005) Compliance: Laboratory Capacities and Biological Risks

This white paper results from research supported by the Naval Postgraduate School’s Project on Advanced Systems and Concepts for Countering Weapons of Mass Destruction (PASCC) via Grant No 244-13-1-0025 awarded by the NAVSUP Fleet Logistics Center San Diego. The views expressed in this report do not necessarily reflect the official policies of the Naval Postgraduate School nor does mention of trade names, commercial practices, or organizations imply endorsement by the U.S. Government.In 2005, the United States and the other Member States of the World Health Organization (WHO) agreed to a new approach to global health security. Recognizing that international agreements rooted in the nineteenth century no longer sufficiently addressed the health threats posed by novel, emerging, and reemerging pathogens such as severe acute respiratory syndrome (SARS) and highly pathogenic H5N1 avian influenza, the World Health Assembly (WHA) adopted the revised International Health Regulations [IHR (2005)].1 The revised IHR entered into force in June 2007. IHR (2005) obligate the now- 196 States Parties to develop the core capacities required to detect, assess, report, and respond to public health emergencies of international concern. The regulations cover biological, chemical, radiological/nuclear, and other threats to public health, regardless of origin (naturally, accidentally, or deliberately released)

Immediate Effects of Repetitive Magnetic Stimulation on Single Cortical Pyramidal Neurons.

Repetitive Transcranial Magnetic Stimulation (rTMS) has been successfully used as a non-invasive therapeutic intervention for several neurological disorders in the clinic as well as an investigative tool for basic neuroscience. rTMS has been shown to induce long-term changes in neuronal circuits in vivo. Such long-term effects of rTMS have been investigated using behavioral, imaging, electrophysiological, and molecular approaches, but there is limited understanding of the immediate effects of TMS on neurons. We investigated the immediate effects of high frequency (20 Hz) rTMS on the activity of cortical neurons in an effort to understand the underlying cellular mechanisms activated by rTMS. We used whole-cell patch-clamp recordings in acute rat brain slices and calcium imaging of cultured primary neurons to examine changes in neuronal activity and intracellular calcium respectively. Our results indicate that each TMS pulse caused an immediate and transient activation of voltage gated sodium channels (9.6 ± 1.8 nA at -45 mV, p value < 0.01) in neurons. Short 500 ms 20 Hz rTMS stimulation induced action potentials in a subpopulation of neurons, and significantly increased the steady state current of the neurons at near threshold voltages (at -45 mV: before TMS: I = 130 ± 17 pA, during TMS: I = 215 ± 23 pA, p value = 0.001). rTMS stimulation also led to a delayed increase in intracellular calcium (153.88 ± 61.94% increase from baseline). These results show that rTMS has an immediate and cumulative effect on neuronal activity and intracellular calcium levels, and suggest that rTMS may enhance neuronal responses when combined with an additional motor, sensory or cognitive stimulus. Thus, these results could be translated to optimize rTMS protocols for clinical as well as basic science applications

rTMS increases steady state current in cortical neurons.

<p>(A) Example voltage clamp traces of (top) stimulus and (bottom) responses (before and during TMS) in cortical neurons (traces have been filtered to remove the TMS artifact). The red dotted line in the response traces denotes the point at which steady state current was measured. (B) Population data of steady state current of cortical neurons at different voltages (Numbers in the bars indicate sample size). Error bars represent SEM.</p

TMS pulses induce transient inward currents at specific voltages.

<p>(A) Example voltage clamp traces of TMS induced responses in Model Cell (top panel, no neuron) and cortical neurons (bottom panel, TMS + neuron). (B) Average amplitudes of the TMS-induced current in cortical neurons (n = 10) at different voltages. Error bars represent SEM.</p

rTMS induces action potentials and reshapes spike timing.

<p>(A) Example trace of a neuron showing lower activation threshold during rTMS stimulation (i) 50 pA subthreshold stimulus step (ii) response before rTMS, (iii) response during rTMS (B) Example trace of a neuron showing the effect of rTMS on spike timing (i) 300 pA supratheshold stimulus step (ii) response before rTMS, (iii) response during rTMS. (C) Upper panel: Magnified view of an action potential before TMS (corresponding to grey box in B (ii)), Lower panel: Magnified view of a TMS artifact (arrow head, two pronged artifact) followed by an action potential during stimulation (corresponding to grey box in B (iii)), (D) Representative post stimulus time histogram (PSTH) of the response neuron in (B) during rTMS stimulation.</p