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Find video protocols related to scientific articles indexed in Pubmed.
Towards an understanding of why undergraduate teaching about delirium does not guarantee gold-standard practice-results from a UK national survey.
Age Ageing
PUBLISHED: 10-18-2014
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delirium is common and serious, yet frequently missed by medical staff. It is known that delirium is widely taught and examined in UK medical schools; however, what is taught, and how such teaching is delivered, remains unknown. The primary aim of this study was to determine the content of UK undergraduate medical education about delirium and establish how it is delivered. A secondary aim was to highlight and share examples of gold-standard teaching on delirium.
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Electromyographic Analysis of Responses to Third Person Intergroup Threat.
J Soc Psychol
PUBLISHED: 10-14-2014
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ABSTRACT This study attempts to replicate and extend the findings from Davis and Stephan's (2011) article investigating facial electromyographic (EMG) responses to individually directed or group directed realistic threat. Using news footage from the Columbine school shootings as a third person threatening stimulus, participants were instructed to view the clips while considering how they felt during the original events (individually-primed) or how students felt during the original events (group-primed). EMG analysis of activity levels of the medial frontalis and the corrugator supercilii indicated differential activation based on the instructions. Individually-primed participants experienced more fontalis activity and group-primed participants experienced more corrugator supercilii activity. These findings replicated the Davis and Stephan results and extended it to a third person-based intergroup threat.
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Preliminary Report of a Mathematical Model of Ventilation and Intrathoracic Pressure Applied to Prehospital Patients with Severe Traumatic Brain Injury.
Prehosp Emerg Care
PUBLISHED: 10-08-2014
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Abstract Background: Inadvertent hyperventilation is associated with poor outcomes from traumatic brain injury (TBI). Hypocapnic cerebral vasoconstriction is well described and causes an immediate and profound decrease in cerebral perfusion. The hemodynamic effects of positive-pressure ventilation (PPV) remain incompletely understood but may be equally important, particularly in the hypovolemic patient with TBI. Objective: Preliminary report on the application of a previously described mathematical model of perfusion and ventilation to prehospital data to predict intrathoracic pressure. Methods: Ventilation data from 108 TBI patients (76 ground transported, 32 helicopter transported) were used for this analysis. Ventilation rate (VR) and end-tidal carbon dioxide (PetCO2) values were used to estimate tidal volume (VT). The values for VR and estimated VT were then applied to a previously described mathematical model of perfusion and ventilation. This model allows input of various lung parameters to define a pressure-volume relationship, then derives mean intrathoracic pressure (MITP) for various VT and VR values. For this analysis, normal lung parameters were utilized. Separate analyses were performed assuming either fixed or variable PaCO2-PetCO2 differences. Ground and air medical patients were compared with regard to VR, PetCO2, estimated VT, and predicted MITP. Results: A total of 10,647 measurements were included from the 108 TBI patients, representing about 13 minutes of ventilation per patient. Mean VR values were higher for ground patients versus air patients (21.6 vs. 19.7 breaths/min; p < 0.01). Estimated VT values were similar for ground and air patients (399 mL vs. 392 mL; p = NS) in the fixed model but not the variable (636 vs. 688 mL, respectively; p < 0.01). Mean PetCO2 values were lower for ground versus air patients (30.6 vs. 33.8 mmHg; p < 0.01). Predicted MITP values were higher for ground versus air patients, assuming either fixed (9.0 vs. 8.1 mmHg; p < 0.01) or variable (10.9 vs. 9.7 mmHg; p < 0.01) PaCO2-PetCO2 differences. Conclusions: Predicted MITP values increased with ventilation rates. Future studies to externally validate this model are warranted.
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What is the Optimal Chest Compression Depth During Out-of-Hospital Cardiac Arrest Resuscitation of Adult Patients?
Circulation
PUBLISHED: 09-24-2014
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-The 2010 AHA guidelines suggested an increase in CPR compression depth with a target greater than 50 mm and no upper limit. This target is based upon limited evidence and we sought to determine the optimal compression depth range.
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The immune synapse clears and excludes molecules above a size threshold.
Nat Commun
PUBLISHED: 09-05-2014
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Natural killer cells assess target cell health via interactions at the immune synapse (IS) that facilitates signal integration and directed secretion. Here we test whether the IS also functions as a gasket. Quantitative fluorescence microscopy of nanometer-scale dextrans within synapses formed by various effector-target cell conjugates reveal that molecules are excluded in a size-dependent manner at activating synapses. Dextran sized ?4?nm move in and out of the IS, but access is significantly reduced (by >50%) for dextran sized 10-13?nm, and dextran ?32?nm is almost entirely excluded. Depolymerization of F-actin abrogated exclusion. Unexpectedly, larger-sized dextrans are cleared as the IS assembles in a zipper-like manner. Monoclonal antibodies are also excluded from the IS but smaller single-domain antibodies are able to penetrate. Therefore, the IS can clear and exclude molecules above a size threshold, and drugs designed to target synaptic cytokines or cytotoxic proteins must fit these dimensions.
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Factors affecting hospital charges after total shoulder arthroplasty: an evaluation of the National Inpatient Sample database.
J Shoulder Elbow Surg
PUBLISHED: 08-23-2014
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The number of total shoulder arthroplasties (TSA) performed in the United States increases yearly. At the same time, cost containment in health care continues to be a major concern. Therefore, it is imperative to identify specific variables that affect the cost of shoulder arthroplasty.
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The descriptive epidemiology of delirium symptoms in a large population-based cohort study: results from the Medical Research Council Cognitive Function and Ageing Study (MRC CFAS).
BMC Geriatr
PUBLISHED: 07-14-2014
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In the general population, the epidemiological relationships between delirium and adverse outcomes are not well defined. The aims of this study were to: (1) construct an algorithm for the diagnosis of delirium using the Geriatric Mental State (GMS) examination; (2) test the criterion validity of this algorithm against mortality and dementia risk; (3) report the age-specific prevalence of delirium as determined by this algorithm.
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Concordance between DSM-IV and DSM-5 criteria for delirium diagnosis in a pooled database of 768 prospectively evaluated patients using the delirium rating scale-revised-98.
BMC Med
PUBLISHED: 06-06-2014
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BackgroundThe Diagnostic and Statistical Manual fifth edition (DSM-5) provides new criteria for delirium diagnosis. We examined delirium diagnosis using these new criteria compared with the Diagnostic and Statistical Manual fourth edition (DSM-IV) in a large dataset of patients assessed for delirium and related presentations.MethodsPatient data (n¿=¿768) from six prospectively collected cohorts, clinically assessed using DSM-IV and the Delirium Rating Scale-Revised-98 (DRS-R98), were pooled. Post hoc application of DRS-R98 item scores were used to rate DSM-5 criteria. `Strict¿ and `relaxed¿ DSM-5 criteria to ascertain delirium were compared to rates determined by DSM-IV.ResultsUsing DSM-IV by clinical assessment, delirium was found in 510/768 patients (66%). Strict DSM-5 criteria categorized 158 as delirious including 155 (30%) with DSM-IV delirium, whereas relaxed DSM-5 criteria identified 466 as delirious, including 455 (89%) diagnosed by DSM-IV (P <0.001). The concordance between the different diagnostic methods was: 53% (¿¿=¿0.22) between DSM-IV and the strict DSM-5, 91% (¿¿=¿0.82) between the DSM-IV and relaxed DSM-5 criteria and 60% (¿¿=¿0.29) between the strict versus relaxed DSM-5 criteria. Only 155 cases were identified as delirium by all three approaches. The 55 (11%) patients with DSM-IV delirium who were not rated as delirious by relaxed criteria had lower mean DRS-R98 total scores than those rated as delirious (13.7¿±¿3.9 versus 23.7¿±¿6.0; P <0.001). Conversely, mean DRS-R98 score (21.1¿±¿6.4) for the 70% not rated as delirious by strict DSM-5 criteria was consistent with suggested cutoff scores for full syndromal delirium. Only 11 cases met DSM-5 criteria that were not deemed to have DSM-IV delirium.ConclusionsThe concordance between DSM-IV and the new DSM-5 delirium criteria varies considerably depending on the interpretation of criteria. Overly-strict adherence for some new text details in DSM-5 criteria would reduce the number of delirium cases diagnosed; however, a more `relaxed¿ approach renders DSM-5 criteria comparable to DSM-IV with minimal impact on their actual application and is thus recommended.
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Brief report: serpin Spi2A as a novel modulator of hematopoietic progenitor cell formation.
Stem Cells
PUBLISHED: 05-05-2014
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Prime regulation over hematopoietic progenitor cell (HPC) production is exerted by hematopoietins (HPs) and their Janus kinase-coupled receptors (HP-Rs). For HP/HP-R studies, one central challenge in determining specific effects involves the delineation of nonredundant signal transduction factors and their lineage restricted actions. Via loss-of-function studies, we define roles for an HP-regulated Serpina3g/Spi2A intracellular serpin during granulomyelocytic, B-cell, and hematopoietic stem cell (HSC) formation. In granulomyelocytic progenitors, granulocyte macrophage colony stimulating factor (GMCSF) strongly induced Serpina3g expression with Stat5 dependency. Spi2A-knockout (KO) led to 20-fold decreased CFU-GM formation, limited GMCSF-dependent granulocyte formation, and compromised neutrophil survival upon tumor necrosis factor alpha (TNF-?) exposure. In B-cell progenitors, Serpina3g was an interleukin-7 (IL7) target. Spi2A-KO elevated CFU-preB greater than sixfold and altered B-cell formation in competitive bone marrow transplant (BMT), and CpG challenge experiments. In HSCs, Serpina3g/Spi2A expression was also elevated. Spi2A-KO compromised LT-HSC proliferation (as well as lineage(neg) Sca1(pos) Kit(pos) (LSK) cell lysosomal integrity), and skewed LSK recovery post 5-FU. Spi2A therefore functions to modulate HP-regulated immune cell and HSC formation post-5-FU challenge.
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High-fidelity simulation training in advanced resuscitation for pharmacy residents.
Am J Pharm Educ
PUBLISHED: 04-25-2014
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To assess the impact of high-fidelity patient simulation on pharmacy resident knowledge, confidence, and competency with advanced resuscitation algorithms and interventions.
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Clinical practice guidelines for delirium management: potential application in palliative care.
J Pain Symptom Manage
PUBLISHED: 04-21-2014
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Delirium occurs in patients across a wide array of health care settings. The extent to which formal management guidelines exist or are adaptable to palliative care is unclear.
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Segmented filamentous bacteria: commensal microbes with potential effects on research.
Comp. Med.
PUBLISHED: 03-29-2014
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Segmented filamentous bacteria (SFB) are commensal bacteria that were first identified in the ilea of mice and rats. Morphologically similar bacteria occur in a broad range of host species, but all strains have been refractory to in vitro culture thus far. Although SFB were once considered innocuous members of the intestinal microbiota of laboratory rodents, they are now known to affect the development of the immune system in rodents and, subsequently, the phenotype of models of both enteric and extraintestinal disease. Therefore, SFB represent long-recognized commensal bacteria serving as an intercurrent variable in studies using rodent models of disease. Here we describe the basic biology of SFB and discuss the immunologic and physiologic effects of colonization with SFB, with particular attention to their effects on rodent models of disease. In addition, we propose that SFB represent only the 'tip of the iceberg' in our understanding of the influence of the microbiota on model phenotypes. As next-generation sequencing techniques are increasingly used to investigate organisms that are refractory to culture, we are likely to identify other commensal microbes that alter the models we use. This review underscores the need to characterize such host-microbe interactions, given that animal research represents a critical tool that is particularly vulnerable to scrutiny in an era of decreasing financial resources and increasing accountability for the use of animal models.
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Worsening Cognitive Impairment and Neurodegenerative Pathology Progressively Increase Risk for Delirium.
Am J Geriatr Psychiatry
PUBLISHED: 03-28-2014
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Delirium is a profound neuropsychiatric disturbance precipitated by acute illness. Although dementia is the major risk factor this has typically been considered a binary quantity (i.e., cognitively impaired versus cognitively normal) with respect to delirium risk. We used humans and mice to address the hypothesis that the severity of underlying neurodegenerative changes and/or cognitive impairment progressively alters delirium risk.
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Validation of the 4AT, a new instrument for rapid delirium screening: a study in 234 hospitalised older people.
Age Ageing
PUBLISHED: 03-02-2014
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to evaluate the performance of the 4 'A's Test (4AT) in screening for delirium in older patients. The 4AT is a new test for rapid screening of delirium in routine clinical practice.
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Delirium superimposed on dementia strongly predicts worse outcomes in older rehabilitation inpatients.
J Am Med Dir Assoc
PUBLISHED: 02-22-2014
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Delirium superimposed on dementia (DSD) is common in many settings. Nonetheless, little is known about the association between DSD and clinical outcomes. The study aim was to evaluate the association between DSD and related adverse outcomes at discharge from rehabilitation and at 1-year follow-up in older inpatients undergoing rehabilitation.
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Hospital cardiac arrest resuscitation practice in the United States: a nationally representative survey.
J Hosp Med
PUBLISHED: 01-22-2014
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In-hospital cardiac arrest (IHCA) outcomes vary widely between hospitals, even after adjusting for patient characteristics, suggesting variations in practice as a potential etiology. However, little is known about the standards of IHCA resuscitation practice among US hospitals.
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Statin use and risk of delirium in the critically ill.
Am. J. Respir. Crit. Care Med.
PUBLISHED: 01-15-2014
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Delirium is common in intensive care unit (ICU) patients and is a predictor of worse outcomes and neuroinflammation is a possible mechanism. The antiinflammatory actions of statins may reduce delirium.
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Ethical challenges and solutions regarding delirium studies in palliative care.
J Pain Symptom Manage
PUBLISHED: 01-07-2014
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Delirium occurs commonly in settings of palliative care (PC), in which patient vulnerability in the unique context of end-of-life care and delirium-associated impairment of decision-making capacity may together present many ethical challenges.
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Validation of a consensus method for identifying delirium from hospital records.
PLoS ONE
PUBLISHED: 01-01-2014
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Delirium is increasingly considered to be an important determinant of trajectories of cognitive decline. Therefore, analyses of existing cohort studies measuring cognitive outcomes could benefit from methods to ascertain a retrospective delirium diagnosis. This study aimed to develop and validate such a method for delirium detection using routine medical records in UK and Ireland.
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Human mesenchymal stromal cells deliver systemic oncolytic measles virus to treat acute lymphoblastic leukemia in the presence of humoral immunity.
Blood
PUBLISHED: 12-17-2013
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Clinical trials of oncolytic attenuated measles virus (MV) are ongoing, but successful systemic delivery in immune individuals remains a major challenge. We demonstrated high-titer anti-MV antibody in 16 adults with acute lymphoblastic leukemia (ALL) following treatments including numerous immunosuppressive drugs. To resolve this challenge, human bone marrow-derived mesenchymal stromal cells (BM-MSC) were used to efficiently deliver MV in a systemic xenograft model of precursor B-ALL. BM-MSCs were successfully loaded with MV ex-vivo, and MV was amplified intracellularly, without toxicity. Live-cell confocal imaging demonstrated viral hand-off between BM-MSC and ALL targets in the presence of antibody. In a murine model of disseminated ALL, successful MV treatment (judged by bioluminescence quantification and survival) was completely abrogated by passive immunization with high-titer human anti-MV antibody. Importantly, no such abrogation was seen in immunized mice receiving MV delivered by BM-MSCs. These data support the use of BM-MSCs as cellular carriers for MV in patients with ALL.
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MicroRNAs Transfer from Human Macrophages to Hepato-Carcinoma Cells and Inhibit Proliferation.
J. Immunol.
PUBLISHED: 11-13-2013
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Recent research has indicated a new mode of intercellular communication facilitated by the movement of RNA between cells. There is evidence that RNA can transfer between cells in a multitude of ways, including in complex with proteins or lipids or in vesicles, including apoptotic bodies and exosomes. However, there remains little understanding of the function of nucleic acid transfer between human cells. In this article, we report that human macrophages transfer microRNAs (miRNAs) to hepato-carcinoma cells (HCCs) in a manner that required intercellular contact and involved gap junctions. Two specific miRNAs transferred efficiently between these cells-miR-142 and miR-223-and both were endogenously expressed in macrophages and not in HCCs. Transfer of these miRNAs influenced posttranscriptional regulation of proteins in HCCs, including decreased expression of reporter proteins and endogenously expressed stathmin-1 and insulin-like growth factor-1 receptor. Importantly, transfer of miRNAs from macrophages functionally inhibited proliferation of these cancerous cells. Thus, these data led us to propose that intercellular transfer of miRNA from immune cells could serve as a new defense against unwanted cell proliferation or tumor growth.
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Nanoscale ligand spacing influences receptor triggering in T cells and NK cells.
Nano Lett.
PUBLISHED: 10-21-2013
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Bioactive nanoscale arrays were constructed to ligate activating cell surface receptors on T cells (the CD3 component of the TCR complex) and natural killer (NK) cells (CD16). These arrays are formed from biofunctionalized gold nanospheres with controlled interparticle spacing in the range 25-104 nm. Responses to these nanoarrays were assessed using the extent of membrane-localized phosphotyrosine in T cells stimulated with CD3-binding nanoarrays and the size of cell contact area for NK cells stimulated with CD16-binding nanoarrays. In both cases, the strength of response decreased with increasing spacing, falling to background levels by 69 nm in the T cell/anti-CD3 system and 104 nm for the NK cell/anti-CD16 system. These results demonstrate that immune receptor triggering can be influenced by the nanoscale spatial organization of receptor/ligand interactions.
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Specialty experience in geriatric medicine is associated with a small increase in knowledge of delirium.
Age Ageing
PUBLISHED: 10-16-2013
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delirium is underdiagnosed and undertreated. Understanding of delirium among doctors in medical and ICU settings has previously been shown to be low. We hypothesised that junior doctors who had gained experience in geriatrics, neurology or psychiatry may have an increased knowledge of delirium.
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The central role of the cytoskeleton in mechanisms and functions of the NK cell immune synapse.
Immunol. Rev.
PUBLISHED: 10-15-2013
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Natural killer (NK) cells discriminate between healthy and unhealthy target cells through a balance of activating and inhibitory signals at direct intercellular contacts called immune synapses. Rearrangements in the cellular cytoskeleton have long been known to be critical in assembly of immune synapses. Here, through bringing together the vast literature on this subject, the number of different ways in which the cytoskeleton is important becomes evident. The dynamics of filamentous actin are critical in (i) creating the nanometer-scale organization of NK cell receptors, (ii) establishing cellular polarity, (iii) coordinating immune receptor and integrin-mediated signaling, and (iv) directing secretion of lytic granules and cytokines. The microtubule network also is important in the delivery of lytic granules and vesicles containing cytokines to the immune synapse. Together, these data establish that the cytoskeleton acts as a central regulator of this complex and dynamic process - and an enormous amount of NK cell biology is controlled through the cytoskeleton.
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New horizons in the pathogenesis, assessment and management of delirium.
Age Ageing
PUBLISHED: 09-25-2013
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Delirium is one of the foremost unmet medical needs in healthcare. It affects one in eight hospitalised patients and is associated with multiple adverse outcomes including increased length of stay, new institutionalisation, and considerable patient distress. Recent studies also show that delirium strongly predicts future new-onset dementia, as well as accelerating existing dementia. The importance of delirium is now increasingly being recognised, with a growing research base, new professional international organisations, increased interest from policymakers, and greater prominence of delirium in educational and audit programmes. Nevertheless, the field faces several complex research and clinical challenges. In this article we focus on selected areas of recent progress and/or uncertainty in delirium research and practice. (i) Pathogenesis: recent studies in animal models using peripheral inflammatory stimuli have begun to suggest mechanisms underlying the delirium syndrome as well as its link with dementia. A growing body of blood and cerebrospinal fluid studies in humans have implicated inflammatory and stress mediators. (ii) Prevention: delirium prevention is effective in the context of research studies, but there are several unresolved issues, including what components should be included, the role of prophylactic drugs, and the overlap with general best care for hospitalised older people. (iii) Assessment: though there are several instruments for delirium screening and assessment, detection rates remain dismal. There are no clear solutions but routine screening embedded into clinical practice, and the development of new rapid screening instruments, offer potential. (iv) Management: studies are difficult given the heterogeneity of delirium and currently expert and comprehensive clinical care remains the main recommendation. Future studies may address the role of drugs for specific elements of delirium. In summary, though facing many challenges, the field continues to make progress, with several promising lines of enquiry and an expanding base of interest among researchers, clinicians and policymakers.
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Superresolution microscopy reveals nanometer-scale reorganization of inhibitory natural killer cell receptors upon activation of NKG2D.
Sci Signal
PUBLISHED: 07-25-2013
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Natural killer (NK) cell responses are regulated by a dynamic equilibrium between activating and inhibitory receptor signals at the immune synapse (or interface) with target cells. Although the organization of receptors at the immune synapse is important for appropriate integration of these signals, there is little understanding of this in detail, because research has been hampered by the limited resolution of light microscopy. Through the use of superresolution single-molecule fluorescence microscopy to reveal the organization of the NK cell surface at the single-protein level, we report that the inhibitory receptor KIR2DL1 is organized in nanometer-scale clusters at the surface of human resting NK cells. Nanoclusters of KIR2DL1 became smaller and denser upon engagement of the activating receptor NKG2D, establishing an unexpected crosstalk between activating receptor signals and the positioning of inhibitory receptors. These rearrangements in the nanoscale organization of surface NK cell receptors were dependent on the actin cytoskeleton. Together, these data establish that NK cell activation involves a nanometer-scale reorganization of surface receptors, which in turn affects models for signal integration and thresholds that control NK cell effector functions and NK cell development.
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Dynamics of natural killer cell receptor revealed by quantitative analysis of photoswitchable protein.
Biophys. J.
PUBLISHED: 07-02-2013
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Natural Killer (NK) cell activation is dynamically regulated by numerous activating and inhibitory surface receptors that accumulate at the immune synapse. Quantitative analysis of receptor dynamics has been limited by methodologies that rely on indirect measurements such as fluorescence recovery after photobleaching. Here, we report an apparently novel approach to study how proteins traffic to and from the immune synapse using NK cell receptors tagged with the photoswitchable fluorescent protein tdEosFP, which can be irreversibly photoswitched from a green to red fluorescent state by ultraviolet light. Thus, after a localized switching event, the movement of the photoswitched molecules can be temporally and spatially resolved by monitoring fluorescence in two regions of interest. By comparing images with mathematical models, we evaluated the diffusion coefficient of the receptor KIR2DL1 (0.23 ± 0.06 ?m(2) s(-1)) and assessed how synapse formation affects receptor dynamics. Our data conclude that the inhibitory NK cell receptor KIR2DL1 is continually trafficked into the synapse, and remains surprisingly stable there. Unexpectedly, however, in NK cells forming synapses with multiple target cells simultaneously, KIR2DL1 at one synapse can relocate to another synapse. Thus, our results reveal a previously undetected intersynaptic exchange of protein.
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Chronic mTOR activation promotes cell survival in Merkel cell carcinoma.
Cancer Lett.
PUBLISHED: 06-27-2013
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Merkel cell carcinoma (MCC) is an aggressive skin cancer with rising incidence. In this study, we demonstrate that mTOR activation and suppressed autophagy is common in MCCs. mTOR inhibition in two primary human MCC cell lines induces autophagy and cell death that is independent of caspase activation but can be attenuated by autophagy inhibition. This is the first study to evaluate mTOR and autophagy in MCC. Our data suggests a potential role of autophagic cell death upon mTOR inhibition and thus uncovers a previously underappreciated role of mTOR signaling and cell survival, and merits further studies for potential therapeutic targets.
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Early diagnosis and treatment of HIV infection: magnitude of benefit on short-term mortality is greatest in older adults.
Age Ageing
PUBLISHED: 05-14-2013
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the number and proportion of adults diagnosed with HIV infection aged 50 years and older has risen. This study compares the effect of CD4 counts and anti-retroviral therapy (ART) on mortality rates among adults diagnosed aged ?50 with those diagnosed at a younger age.
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Characteristics of patients with chronic exertional compartment syndrome.
Foot Ankle Int
PUBLISHED: 05-13-2013
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Chronic exertional compartment syndrome (CECS) is a condition that causes reversible ischemia and lower extremity pain during exercise. To date there are few large studies examining the characteristics of patients with CECS. This study aimed to present these characteristics by examining the largest published series of patients with a confirmed diagnosis of the disorder.
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Rituximab causes a polarization of B cells that augments its therapeutic function in NK-cell-mediated antibody-dependent cellular cytotoxicity.
Blood
PUBLISHED: 04-23-2013
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Rituximab, which binds CD20 on B cells, is one of the best-characterized antibodies used in the treatment of B-cell malignancies and autoimmune diseases. Rituximab triggers natural killer (NK)-cell-mediated antibody-dependent cellular cytotoxicity (ADCC), but little is known about the spatial and temporal dynamics of cell-cell interactions during ADCC or what makes rituximab potent at triggering ADCC. Here, using laser scanning confocal microscopy, we found that rituximab caused CD20 to cap at the B-cell surface independent of antibody crosslinking or intercellular contact. Unexpectedly, other proteins, including intercellular adhesion molecule 1 and moesin, were selectively recruited to the cap of CD20 and the microtubule organizing center became polarized toward the cap. Importantly, the frequency at which NK cells would kill target cells via ADCC increased by 60% when target cells were polarized compared with when they were unpolarized. Polarized B cells were lysed more frequently still when initial contact with NK cells occurred at the place where CD20 was capped. This demonstrates that the site of contact between immune cells and target cells influences immune responses. Together, these data establish that rituximab causes a polarization of B cells and this augments its therapeutic function in triggering NK-cell-mediated ADCC.
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The epidemiology of delirium: challenges and opportunities for population studies.
Am J Geriatr Psychiatry
PUBLISHED: 03-25-2013
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Delirium is a serious and common acute neuropsychiatric syndrome that is associated with short- and long-term adverse health outcomes. However, relatively little delirium research has been conducted in unselected populations. Epidemiologic research in such populations has the potential to resolve several questions of clinical significance in delirium. Part 1 of this article explores the importance of population selection, case-ascertainment, attrition, and confounding. Part 2 examines a specific question in delirium epidemiology: What is the relationship between delirium and trajectories of cognitive decline? This section assesses previous work through two systematic reviews and proposes a design for investigating delirium in the context of longitudinal cohort studies. Such a design requires robust links between community and hospital settings. Practical considerations for case-ascertainment in the hospital, as well as the necessary quality control of these programs, are outlined. We argue that attention to these factors is important if delirium research is to benefit fully from a population perspective.
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3-D stimulated emission depletion microscopy with programmable aberration correction.
J Biophotonics
PUBLISHED: 03-12-2013
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We present a stimulated emission depletion (STED) microscope that provides 3-D super resolution by simultaneous depletion using beams with both a helical phase profile for enhanced lateral resolution and an annular phase profile to enhance axial resolution. The 3-D depletion point spread function is realised using a single spatial light modulator that can also be programmed to compensate for aberrations in the microscope and the sample. We apply it to demonstrate the first 3-D super-resolved imaging of an immunological synapse between a Natural Killer cell and its target cell. (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).
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A peptide antagonist disrupts NK cell inhibitory synapse formation.
J. Immunol.
PUBLISHED: 02-04-2013
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Productive engagement of MHC class I by inhibitory NK cell receptors depends on the peptide bound by the MHC class I molecule. Peptide:MHC complexes that bind weakly to killer cell Ig-like receptors (KIRs) can antagonize the inhibition mediated by high-affinity peptide:MHC complexes and cause NK cell activation. We show that low-affinity peptide:MHC complexes stall inhibitory signaling at the step of Src homology protein tyrosine phosphatase 1 recruitment and do not go on to form the KIR microclusters induced by high-affinity peptide:MHC, which are associated with Vav dephosphorylation and downstream signaling. Furthermore, the low-affinity peptide:MHC complexes prevented the formation of KIR microclusters by high-affinity peptide:MHC. Thus, peptide antagonism of NK cells is an active phenomenon of inhibitory synapse disruption.
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Super-resolution microscopy of the immunological synapse.
Curr. Opin. Immunol.
PUBLISHED: 02-01-2013
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Deciphering the spatial organisation of signalling proteins is the key to understanding the mechanisms underlying immune cell activation. Every advance in imaging technology has led to major breakthroughs in unravelling how receptor and signalling proteins are distributed within the plasma membrane and how membrane signalling is integrated with endosomes and vesicular trafficking. Recently, super-resolution fluorescence microscopy has been applied to immunological synapses, gaining new insights into the nanoscale organisation of signalling processes. Here, we review the advantages and potential of super-resolution microscopy for elucidating the regulation of many aspects of immune signalling.
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Association of out-of-hospital advanced airway management with outcomes after traumatic brain injury and hemorrhagic shock in the ROC hypertonic saline trial.
Emerg Med J
PUBLISHED: 01-26-2013
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OBJECTIVE: Prior studies suggest adverse associations between out-of-hospital advanced airway management (AAM) and patient outcomes after major trauma. This secondary analysis of data from the Resuscitation Outcomes Consortium Hypertonic Saline Trial evaluated associations between out-of-hospital AAM and outcomes in patients suffering isolated severe traumatic brain injury (TBI) or haemorrhagic shock. METHODS: This multicentre study included adults with severe TBI (GCS ?8) or haemorrhagic shock (SBP ?70 mm Hg, or (SBP 71-90 mm Hg and heart rate ?108 bpm)). We compared patients receiving out-of-hospital AAM with those receiving emergency department AAM. We evaluated the associations between airway strategy and patient outcomes (28-day mortality, and 6-month poor neurologic or functional outcome) and airway strategy, adjusting for confounders. Analysis was stratified by (1) patients with isolated severe TBI and (2) patients with haemorrhagic shock with or without severe TBI. RESULTS: Of 2135 patients, we studied 1116 TBI and 528 shock; excluding 491 who died in the field, did not receive AAM or had missing data. In the shock cohort, out-of-hospital AAM was associated with increased 28-day mortality (adjusted OR 5.14; 95% CI 2.42 to 10.90). In TBI, out-of-hospital AAM showed a tendency towards increased 28-day mortality (adjusted OR 1.57; 95% CI 0.93 to 2.64) and 6-month poor functional outcome (1.63; 1.00 to 2.68), but these differences were not statistically significant. Out-of-hospital AAM was associated with poorer 6-month TBI neurologic outcome (1.80; 1.09 to 2.96). CONCLUSIONS: Out-of-hospital AAM was associated with increased mortality after haemorrhagic shock. The adverse association between out-of-hospital AAM and injury outcome is most pronounced in patients with haemorrhagic shock.
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All monofilament knots assume sliding conformation in vivo.
Dermatol Surg
PUBLISHED: 01-17-2013
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Surgeons are not always cognizant of the knots they tie. It has been thought that suture material does not determine what types of knots are tied.
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DHHC2 is a protein S-acyltransferase for Lck.
Mol. Membr. Biol.
PUBLISHED: 11-01-2011
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Lck is a non-receptor tyrosine kinase of the Src family that is essential for T cell activation. Dual N-terminal acylation of Lck with myristate (N-acylation) and palmitate (S-acylation) is essential for its membrane association and function. Reversible S-acylation of Lck is observed in vivo and may function as a control mechanism. Here we identify the DHHC family protein S-acyltransferase DHHC2 as an enzyme capable of palmitoylating of Lck in T cells. Reducing the DHHC2 level in Jurkat T cells using siRNA causes decreased Lck S-acylation and partial dislocation from membranes, and conversely overexpression of DHHC2 increases S-acylation of an Lck surrogate, LckN10-GFP. DHHC2 localizes primarily to the endoplasmic reticulum and Golgi apparatus suggesting that it is involved in S-acylation of newly-synthesized or recycling Lck involved in T cell signalling.
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An observational study to validate the Satisfaction Measure of the Injection of Growth Hormone Therapy (SMIGHTy) questionnaire.
Curr Med Res Opin
PUBLISHED: 09-16-2011
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The objective of this study was to psychometrically evaluate a tool to measure adult caregivers level of satisfaction with the delivery device used to administer injections of recombinant human growth hormone (rhGH) to a child - the Satisfaction Measure of the Injection of Growth Hormone Therapy (SMIGHTy*) questionnaire.
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A trial of an impedance threshold device in out-of-hospital cardiac arrest.
N. Engl. J. Med.
PUBLISHED: 09-02-2011
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The impedance threshold device (ITD) is designed to enhance venous return and cardiac output during cardiopulmonary resuscitation (CPR) by increasing the degree of negative intrathoracic pressure. Previous studies have suggested that the use of an ITD during CPR may improve survival rates after cardiac arrest.
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Early versus later rhythm analysis in patients with out-of-hospital cardiac arrest.
N. Engl. J. Med.
PUBLISHED: 09-02-2011
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In a departure from the previous strategy of immediate defibrillation, the 2005 resuscitation guidelines from the American Heart Association-International Liaison Committee on Resuscitation suggested that emergency medical service (EMS) personnel could provide 2 minutes of cardiopulmonary resuscitation (CPR) before the first analysis of cardiac rhythm. We compared the strategy of a brief period of CPR with early analysis of rhythm with the strategy of a longer period of CPR with delayed analysis of rhythm.
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Human NK cells differ more in their KIR2DL1-dependent thresholds for HLA-Cw6-mediated inhibition than in their maximal killing capacity.
PLoS ONE
PUBLISHED: 08-10-2011
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In this study we have addressed the question of how activation and inhibition of human NK cells is regulated by the expression level of MHC class I protein on target cells. Using target cell transfectants sorted to stably express different levels of the MHC class I protein HLA-Cw6, we show that induction of degranulation and that of IFN-? secretion are not correlated. In contrast, the inhibition of these two processes by MHC class-I occurs at the same level of class I MHC protein. Primary human NK cell clones were found to differ in the amount of target MHC class I protein required for their inhibition, rather than in their maximum killing capacity. Importantly, we show that KIR2DL1 expression determines the thresholds (in terms of MHC I protein levels) required for NK cell inhibition, while the expression of other receptors such as LIR1 is less important. Furthermore, using mathematical models to explore the dynamics of target cell killing, we found that the observed delay in target cell killing is exhibited by a model in which NK cells require some activation or priming, such that each cell can lyse a target cell only after being activated by a first encounter with the same or a different target cell, but not by models which lack this feature.
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Simulations of the NK cell immune synapse reveal that activation thresholds can be established by inhibitory receptors acting locally.
J. Immunol.
PUBLISHED: 06-20-2011
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NK cell activation is regulated by a balance between activating and inhibitory signals. To address the question of how these signals are spatially integrated, we created a computer simulation of activating and inhibitory NK cell immunological synapse (NKIS) assembly, implementing either a "quantity-based" inhibition model or a "distance-based" inhibition model. The simulations mimicked the observed molecule distributions in inhibitory and activating NKIS and yielded several new insights. First, the total signal is highly influenced by activating complex dissociation rates but not by adhesion and inhibitory complex dissociation rates. Second, concerted motion of receptors in clusters significantly accelerates NKIS maturation. Third, when the potential of a cis interaction between Ly49 receptors and MHC class I on murine NK cells was added to the model, the integrated signal as a function of receptor and ligand numbers was only slightly increased, at least up to the level of 50% cis-bound Ly49 receptors reached in the model. Fourth, and perhaps most importantly, the integrated signal behavior obtained when using the distance-based inhibition signal model was closer to the experimentally observed behavior, with an inhibition radius of the order 3-10 molecules. Microscopy to visualize Vav activation in NK cells on micropatterned surfaces of activating and inhibitory strips revealed that Vav is only locally activated where activating receptors are ligated within a single NK cell contact. Taken together, these data are consistent with a model in which inhibitory receptors act locally; that is, that every bound inhibitory receptor acts on activating receptors within a certain radius around it.
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Challenges to effective research in acute trauma resuscitation: consent and endpoints.
Shock
PUBLISHED: 04-26-2011
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Selection of study endpoints is one of the most important decisions in the design of effective clinical trials. Late mortality (e.g., 28 days) is an unambiguous endpoint, accepted by regulatory agencies, but it is viewed as problematic among researchers in the study of resuscitation for acute trauma injury with hemorrhagic shock. In February 2008, physicians, ethicists, statisticians, and research scientists from the military, academia, industry, the Federal Drug Administration, and the National Heart Lung and Blood Institute gathered to discuss the obstacles confronting the trauma community in their efforts to improve patient outcomes. The primary meeting objective was to generate preliminary suggestions for a series of follow-up meetings that will develop consensus guidelines for the design of large multicenter clinical trials. Twenty short presentations and discussions, summarized here, outlined the groups concerns and suggestions. Successful and failed, completed or ongoing, clinical studies provided insight as to endpoints that may be of value for future trauma and shock studies. In addition to the importance of appropriate endpoints in study design, other related topics were discussed, including trauma epidemiology, patient enrollment and inclusion criteria, community consultation and the difficulty of obtaining informed consent in acute trauma research, and the inclusion of quality of life in composite endpoints. The consensus was that more discussion was needed and that consideration of new endpoints for clinical trials in emergency trauma research was a worthwhile and necessary goal.
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Boltzmann energy-based image analysis demonstrates that extracellular domain size differences explain protein segregation at immune synapses.
PLoS Comput. Biol.
PUBLISHED: 04-19-2011
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Immune synapses formed by T and NK cells both show segregation of the integrin ICAM1 from other proteins such as CD2 (T cell) or KIR (NK cell). However, the mechanism by which these proteins segregate remains unclear; one key hypothesis is a redistribution based on protein size. Simulations of this mechanism qualitatively reproduce observed segregation patterns, but only in certain parameter regimes. Verifying that these parameter constraints in fact hold has not been possible to date, this requiring a quantitative coupling of theory to experimental data. Here, we address this challenge, developing a new methodology for analysing and quantifying image data and its integration with biophysical models. Specifically we fit a binding kinetics model to 2 colour fluorescence data for cytoskeleton independent synapses (2 and 3D) and test whether the observed inverse correlation between fluorophores conforms to size dependent exclusion, and further, whether patterned states are predicted when model parameters are estimated on individual synapses. All synapses analysed satisfy these conditions demonstrating that the mechanisms of protein redistribution have identifiable signatures in their spatial patterns. We conclude that energy processes implicit in protein size based segregation can drive the patternation observed in individual synapses, at least for the specific examples tested, such that no additional processes need to be invoked. This implies that biophysical processes within the membrane interface have a crucial impact on cell:cell communication and cell signalling, governing protein interactions and protein aggregation.
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Prospective evaluation of a clinical decision guideline to diagnose spinal epidural abscess in patients who present to the emergency department with spine pain.
J Neurosurg Spine
PUBLISHED: 03-18-2011
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A spinal epidural abscess (SEA) is rare but potentially devastating if not diagnosed early. Unfortunately, diagnostic delays and associated neurological deficits are common. The objectives of this analysis were to explore the use of a novel clinical decision guideline to screen patients who present to the emergency department (ED) with spine pain for SEA and to determine the diagnostic test characteristics of the erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) level in patients at risk for SEA.
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The need for standardized data reporting for prehospital airway management.
Crit Care
PUBLISHED: 03-14-2011
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Despite a widespread belief in the value of aggressive prehospital airway management, the therapeutic benefits of early tracheal intubation (TI) remain unclear. In fact, most attempts to elucidate the benefits of prehospital TI on outcome from traumatic brain injury and cardiopulmonary arrest have documented an increase in mortality associated with the procedure. While some degree of selection bias is likely present in these studies, the inherent adverse physiological effects of intubation and a high incidence of desaturation and subsequent hyperventilation may indicate a harmful effect of the procedure. This uncertainty regarding such a fundamental resuscitation procedure as TI underscores the need for standardized data reporting in prehospital airway management research. To this end, the Utstein prehospital airway conference proposed a set of variables that would move us in that direction. However, the present article by Lossius and colleagues documents how far we still have to travel before such standardization can be achieved. Only through these efforts can we elucidate the true benefits - or harm - of advanced airway management during critical resuscitation.
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Mechanisms for size-dependent protein segregation at immune synapses assessed with molecular rulers.
Biophys. J.
PUBLISHED: 03-08-2011
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Immunological synapses are specialized intercellular contacts formed by several types of immune cells in contact with target cells or antigen-presenting cells. A late-stage immune synapse is commonly a bulls-eye pattern of immune cell receptor-ligand pairs surrounded by integrin complexes. Based on crystal structures, the intermembrane distance would be ?15 nm for many immune cell receptor-ligand pairs, but ?40 nm for integrin-ligand pairs. Close proximity of these two classes of intermembrane bonds would require significant membrane bending and such proteins can segregate according to their size, which may be key for receptor triggering. However, tools available to evaluate the intermembrane organization of the synapse are limited. Here, we present what we believe to be a novel approach to test the importance of size in the intercellular organization of proteins, using live-cell microscopy of a size-series of fluorescently-labeled molecules and quantum dots to act as molecular rulers. Small particles readily colocalized at the synapse with MHC class I bound to its cognate natural killer cell receptor, whereas particles larger than 15 nm were increasingly segregated from this interaction. Combined with modeling of the partitioning of the particles by scaled-particle adsorption theory, these molecular rulers show how membrane-bending elasticity can drive size-dependent exclusion of proteins within immune synapses.
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A distinct subset of human NK cells expressing HLA-DR expand in response to IL-2 and can aid immune responses to BCG.
Eur. J. Immunol.
PUBLISHED: 02-27-2011
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Subsets of NK cells can have distinct functions. Here, we report that >25% of human peripheral blood NK cells express HLA-DR after culture with IL-2. This can be driven by an expansion of a small subset of NK cells expressing HLA-DR, in contrast to previous assumptions that HLA-DR is upregulated on previously negative cells. HLA-DR-expressing NK cells showed enhanced degranulation to susceptible target cells and expressed chemokine receptor CXCR3, which facilitated their enrichment following exposure to CXCL11/I-TAC. Suggesting HLA-DR-expressing NK cells have an important role in an immune response, stimulation of PBMCs with Mycobacterium bovis BCG (BCG) triggered expansion of this subset. Importantly, the magnitude of an individuals NK cell IFN-? response triggered by BCG was associated with the initial frequency of HLA-DR-expressing NK cells in PBMCs. More directly indicating the importance of HLA-DR-expressing NK cells, enriching the frequency of this subset in PBMCs substantially augmented the IFN-? response to BCG. Thus, HLA-DR expression marks a distinct subset of NK cells, present at low frequency in circulating blood but readily expanded by IL-2, that can play an important role during immune responses to BCG.
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The relationship between out-of-hospital airway management and outcome among trauma patients with Glasgow Coma Scale Scores of 8 or less.
Prehosp Emerg Care
PUBLISHED: 02-10-2011
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Airway management remains a fundamental component of optimal care of the severely injured patient, with endotracheal intubation representing the definitive strategy for airway control. However, multiple studies document an association between out-of-hospital intubation and increased mortality for severe traumatic brain injury.
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Remodelling of cortical actin where lytic granules dock at natural killer cell immune synapses revealed by super-resolution microscopy.
PLoS Biol.
PUBLISHED: 01-19-2011
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Natural Killer (NK) cells are innate immune cells that secrete lytic granules to directly kill virus-infected or transformed cells across an immune synapse. However, a major gap in understanding this process is in establishing how lytic granules pass through the mesh of cortical actin known to underlie the NK cell membrane. Research has been hampered by the resolution of conventional light microscopy, which is too low to resolve cortical actin during lytic granule secretion. Here we use two high-resolution imaging techniques to probe the synaptic organisation of NK cell receptors and filamentous (F)-actin. A combination of optical tweezers and live cell confocal microscopy reveals that microclusters of NKG2D assemble into a ring-shaped structure at the centre of intercellular synapses, where Vav1 and Grb2 also accumulate. Within this ring-shaped organisation of NK cell proteins, lytic granules accumulate for secretion. Using 3D-structured illumination microscopy (3D-SIM) to gain super-resolution of ~100 nm, cortical actin was detected in a central region of the NK cell synapse irrespective of whether activating or inhibitory signals dominate. Strikingly, the periodicity of the cortical actin mesh increased in specific domains at the synapse when the NK cell was activated. Two-colour super-resolution imaging revealed that lytic granules docked precisely in these domains which were also proximal to where the microtubule-organising centre (MTOC) polarised. Together, these data demonstrate that remodelling of the cortical actin mesh occurs at the central region of the cytolytic NK cell immune synapse. This is likely to occur for other types of cell secretion and also emphasises the importance of emerging super-resolution imaging technology for revealing new biology.
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A structural model of perfusion and oxygenation in low-flow states.
Resuscitation
PUBLISHED: 01-05-2011
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Recent investigations underscore the critical importance of ventilation strategies on resuscitation outcomes. In low perfusion states, such as cardiac arrest and traumatic shock, the rise in intrathoracic pressure that accompanies positive-pressure ventilation can significantly impede venous return and lead to a decrease in cardiac output. The optimal ventilation strategy in these "low-flow" states remains unclear.
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Cardiac arrest survival did not increase in the Resuscitation Outcomes Consortium after implementation of the 2005 AHA CPR and ECC guidelines.
Resuscitation
PUBLISHED: 01-04-2011
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We examined the effect of the 2005 American Heart Association guidelines on survival in the Resuscitation Outcomes Consortium (ROC) Cardiac Arrest Epistry.
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Role of decoy molecules in neuronal ischemic preconditioning.
Life Sci.
PUBLISHED: 01-04-2011
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Decoy receptors bind with TNF related apoptosis inducing ligands (TRAIL) but do not contain the cytoplasmic domains necessary to transduce apoptotic signals. We hypothesized that decoy receptors may confer neuronal protection against lethal ischemia after ischemic preconditioning (IPC).
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The effect of cross-training with adjustable airway model anatomies on laryngoscopy skill transfer.
Anesth. Analg.
PUBLISHED: 10-21-2010
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A problem with learning endotracheal intubation on airway mannequins is poor transfer of direct laryngoscopy skills from model to patient. We developed an airway model with adjustable anatomic features and investigated whether practicing on a model with frequent adjustments improved laryngoscopy skills transfer.
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Part 8: adult advanced cardiovascular life support: 2010 American Heart Association Guidelines for Cardiopulmonary Resuscitation and Emergency Cardiovascular Care.
Circulation
PUBLISHED: 10-20-2010
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The goal of therapy for bradycardia or tachycardia is to rapidly identify and treat patients who are hemodynamically unstable or symptomatic due to the arrhythmia. Drugs or, when appropriate, pacing may be used to control unstable or symptomatic bradycardia. Cardioversion or drugs or both may be used to control unstable or symptomatic tachycardia. ACLS providers should closely monitor stable patients pending expert consultation and should be prepared to aggressively treat those with evidence of decompensation.
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Out-of-hospital hypertonic resuscitation following severe traumatic brain injury: a randomized controlled trial.
JAMA
PUBLISHED: 10-07-2010
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Hypertonic fluids restore cerebral perfusion with reduced cerebral edema and modulate inflammatory response to reduce subsequent neuronal injury and thus have potential benefit in resuscitation of patients with traumatic brain injury (TBI).
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Latency and loss of pulse oximetry signal with the use of digital probes during prehospital rapid-sequence intubation.
Prehosp Emerg Care
PUBLISHED: 09-21-2010
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Prehospital personnel rely on timely and accurate pulse oximetry data when performing critical skills, such as rapid-sequence intubation (RSI). However, loss of signal may be a frequent occurrence in patients with poor peripheral perfusion. In addition, a delay or latency period in the timeliness of pulse oximetry data may exist with probes placed on the fingers.
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Delivering regional thrombolysis via a hub-and-spoke model.
J R Soc Med
PUBLISHED: 09-03-2010
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Audits in the United Kingdom and other countries show that only a small proportion of eligible stroke patients receive thrombolysis. Providing 24-hour thrombolysis cover presents major challenges in both infrastructure and staffing. One model for improving access is to provide out-of-hours cover in a regional centre but this may present problems including greater delays to hospital admissions.
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Prehospital airway and ventilation management: a trauma score and injury severity score-based analysis.
J Trauma
PUBLISHED: 08-12-2010
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Emergent endotracheal intubation (ETI) is considered the standard of care for patients with severe traumatic brain injury (TBI). However, recent evidence suggests that the procedure may be associated with increased mortality, possibly reflecting inadequate training, suboptimal patient selection, or inappropriate ventilation.
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Matched sizes of activating and inhibitory receptor/ligand pairs are required for optimal signal integration by human natural killer cells.
PLoS ONE
PUBLISHED: 08-09-2010
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It has been suggested that receptor-ligand complexes segregate or co-localise within immune synapses according to their size, and this is important for receptor signaling. Here, we set out to test the importance of receptor-ligand complex dimensions for immune surveillance of target cells by human Natural Killer (NK) cells. NK cell activation is regulated by integrating signals from activating receptors, such as NKG2D, and inhibitory receptors, such as KIR2DL1. Elongating the NKG2D ligand MICA reduced its ability to trigger NK cell activation. Conversely, elongation of KIR2DL1 ligand HLA-C reduced its ability to inhibit NK cells. Whereas normal-sized HLA-C was most effective at inhibiting activation by normal-length MICA, only elongated HLA-C could inhibit activation by elongated MICA. Moreover, HLA-C and MICA that were matched in size co-localised, whereas HLA-C and MICA that were different in size were segregated. These results demonstrate that receptor-ligand dimensions are important in NK cell recognition, and suggest that optimal integration of activating and inhibitory receptor signals requires the receptor-ligand complexes to have similar dimensions.
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Unusual case of small cell gastric carcinoma: case report and literature review.
Dig. Dis. Sci.
PUBLISHED: 06-20-2010
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Small cell carcinomas are among the most aggressive, poorly differentiated, and highly malignant of the neuroendocrine tumors (NETs). Of which, small cell gastric carcinoma is a rare small cell neuroendocrine tumor. The purpose of our study was to present this case and perform a comprehensive literature review.
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High plasma membrane lipid order imaged at the immunological synapse periphery in live T cells.
Mol. Membr. Biol.
PUBLISHED: 06-15-2010
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Cholesterol- and glycosphingolipid-enriched membrane lipid microdomains, frequently called lipid rafts, are thought to play an important role in the spatial and temporal organization of immunological synapses. Higher ordering of lipid acyl chains was suggested for these entities and imaging of membrane order in living cells during activation can therefore help to understand the mechanisms responsible for the supramolecular organization of molecules involved in the activation of T cells. Here, we employ the phase-sensitive membrane dye di-4-ANEPPDHQ together with a variety of spectrally-resolved microscopy techniques, including 2-channel ratiometric TIRF microscopy and fluorescence lifetime imaging, to characterize membrane order at the T cell immunological synapse at high spatial and temporal resolution in live cells at physiological temperature. We find that higher membrane order resides at the immunological synapse periphery where proximal signalling through the immunoreceptors and accessory proteins in microclusters has previously been shown to take place. The observed spatial patterning of membrane order in the immunological synapse depends on active receptor signalling.
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Multiple mechanisms downstream of TLR-4 stimulation allow expression of NKG2D ligands to facilitate macrophage/NK cell crosstalk.
J. Immunol.
PUBLISHED: 05-19-2010
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The activating receptor NKG2D recognizes proteins that are not normally expressed at the surface of most cells but are expressed during a cellular "stress" response (e.g., upon induction of the DNA damage pathway). This establishes recognition of "induced self" as an important strategy for surveillance of infections or tumor transformation. However, NKG2D ligands can also be induced on human macrophages by TLR stimulation, which has been far less studied. In this paper, we clarify that LPS, which ligates TLR-4, preferentially upregulated MICA and not MICB; CL097, which ligates TLR-7/8, upregulated both MICA and MICB; and polyinosinic-polycytidylic acid, which ligates TLR-3, upregulated neither. To probe how LPS stimulation triggers MICA expression, we determined that the stability of MICA mRNA was much longer than that of MICB mRNA, but neither was changed by LPS stimulation. This finding suggests that increased levels of MICA mRNA following LPS stimulation resulted from increased transcription. However, it was not sufficient for surface protein expression, which was controlled posttranscriptionally via a separate pathway involving the ataxia telangiectasia mutated/ataxia telangiectasia and Rad3 related kinases. Moreover, LPS stimulation decreased expression of microRNAs (miRNA)--miR-17-5, miR-20a, and miR-93--which target MICA, implicating a novel role for miRNAs in NKG2D ligand expression. Thus, TLR stimulation allows expression of NKG2D ligands through multiple pathways, including downmodulation of specific miRNAs.
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SH2 domain containing leukocyte phosphoprotein of 76-kDa (SLP-76) feedback regulation of ZAP-70 microclustering.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 05-13-2010
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T cell receptor (TCR) signaling involves CD4/CD8-p56lck recruitment of ZAP-70 to the TCR receptor, ZAP-70 phosphorylation of LAT that is followed by LAT recruitment of the GADS-SLP-76 complex. Back regulation of ZAP-70 by SLP-76 has not been documented. In this paper, we show that anti-CD3 induced ZAP-70 cluster formation is significantly reduced in the absence of SLP-76 (i.e., J14 cells) and in the presence of a mutant of SLP-76 (4KE) in Jurkat and primary T cells. Both the number of cells with clusters and the number of clusters per cell were reduced. This effect was not mediated by SLP-76 SH2 domain binding to ZAP-70 because SLP-76 failed to precipitate ZAP-70 and an inactivating SH2 domain mutation (i.e., R448L) on SLP-76 4KE did not reverse the inhibition of ZAP-70 clustering. Mutation of R448 on WT SLP-76 still supported ZAP-70 clustering. Intriguingly, by contrast, LAT clustering occurred normally in the absence of SLP-76, or the presence of 4KE SLP-76 indicating that this transmembrane adaptor can operate independently of ZAP-70-GADS-SLP-76. Our findings reconfigure the TCR signaling pathway by showing SLP-76 back-regulation of ZAP-70, an event that could ensure that signaling components are in balance for optimal T cell activation.
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Dynamics of subsynaptic vesicles and surface microclusters at the immunological synapse.
Sci Signal
PUBLISHED: 05-13-2010
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Imaging studies have identified clusters of kinases and adaptor proteins that serve as centers of signaling at the contact points between T cells and antigen-presenting cells (APCs). Here, we report that the kinase ZAP-70 and the adaptor proteins LAT and SLP-76 accumulated in separate clusters at the interface between T cells and coverslips coated with a stimulatory antibody against CD3, a component of the T cell antigen receptor complex. A fraction of LAT was detected in motile vesicles that repeatedly moved to surface microclusters of SLP-76 and the adaptor protein GADS (growth factor receptor-bound protein-related adaptor downstream of Shc), where they exhibited decreased motility. LAT molecules in which the residues tyrosine 171 and tyrosine 191 (which are required for the binding of LAT to GADS) were mutated to phenylalanine did not dwell at clusters of SLP-76. At immunological synapses, LAT-containing vesicles also colocalized with microclusters of SLP-76, as detected in experiments in which laser tweezers were used to position T cell-APC conjugates vertically for high-resolution imaging. Phosphorylation of LAT was most prominent when vesicular LAT colocalized with SLP-76. Indeed, the abundance of phosphorylated LAT within a microcluster of SLP-76 was greatest in those clusters that had more recent interactions with LAT-containing vesicles. Finally, negative signals by the inhibitory receptor ILT2 disrupted the assembly of SLP-76-containing microclusters. Together, these data show that the movement of LAT-containing vesicles is linked to the organization of protein microclusters and suggest an important role for vesicular LAT in the SLP-76 signalosome.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.