Cell-surface sensors for real-time probing of cellular environments

Author Manuscript 2012 August 1.The ability to explore cell signalling and cell-to-cell communication is essential for understanding cell biology and developing effective therapeutics. However, it is not yet possible to monitor the interaction of cells with their environments in real time. Here, we show that a fluorescent sensor attached to a cell membrane can detect signalling molecules in the cellular environment. The sensor is an aptamer (a short length of single-stranded DNA) that binds to platelet-derived growth factor (PDGF) and contains a pair of fluorescent dyes. When bound to PDGF, the aptamer changes conformation and the dyes come closer to each other, producing a signal. The sensor, which is covalently attached to the membranes of mesenchymal stem cells, can quantitatively detect with high spatial and temporal resolution PDGF that is added in cell culture medium or secreted by neighbouring cells. The engineered stem cells retain their ability to find their way to the bone marrow and can be monitored in vivo at the single-cell level using intravital microscopy.National Institutes of Health (U.S.) (Grant HL097172)National Institutes of Health (U.S.) (Grant HL095722)National Institutes of Health (U.S.) (Grant DE019191)National Institutes of Health (U.S.) (Grant NIAID 5RC1AI086152)Charles A. Dana FoundationAmerican Heart Association (Grant 0970178N)National Science Foundation (U.S.) (Graduate Fellowship

White matter signal abnormalities in former National Football League players

Introduction: Later-life brain alterations in former tackle football players are poorly understood, particularly regarding their relationship with repetitive head impacts (RHIs) and clinical function. We examined white matter signal abnormalities (WMSAs) and their association with RHIs and clinical function in former National Football League (NFL) players. Methods: Eighty-six clinically symptomatic former NFL players and 23 same-age reportedly asymptomatic controls without head trauma exposure underwent magnetic resonance imaging and neuropsychological testing. FreeSurfer calculated WMSAs. A cumulative head impact index quantified RHIs. Results: In former NFL players, increased volume of WMSAs was associated with higher cumulative head impact index scores (P = .043) and worse psychomotor speed and executive function (P = .015). Although former NFL players had greater WMSA volume than controls (P = .046), these findings are inconclusive due to recruitment of controls based on lack of clinical symptoms and head trauma exposure. Discussion In former NFL players, WMSAs may reflect long-term microvascular and nonmicrovascular pathologies from RHIs that negatively impact cognition

Proceedings of the 3rd Biennial Conference of the Society for Implementation Research Collaboration (SIRC) 2015: advancing efficient methodologies through community partnerships and team science

It is well documented that the majority of adults, children and families in need of evidence-based behavioral health interventionsi do not receive them [1, 2] and that few robust empirically supported methods for implementing evidence-based practices (EBPs) exist. The Society for Implementation Research Collaboration (SIRC) represents a burgeoning effort to advance the innovation and rigor of implementation research and is uniquely focused on bringing together researchers and stakeholders committed to evaluating the implementation of complex evidence-based behavioral health interventions. Through its diverse activities and membership, SIRC aims to foster the promise of implementation research to better serve the behavioral health needs of the population by identifying rigorous, relevant, and efficient strategies that successfully transfer scientific evidence to clinical knowledge for use in real world settings [3]. SIRC began as a National Institute of Mental Health (NIMH)-funded conference series in 2010 (previously titled the “Seattle Implementation Research Conference”; $150,000 USD for 3 conferences in 2011, 2013, and 2015) with the recognition that there were multiple researchers and stakeholdersi working in parallel on innovative implementation science projects in behavioral health, but that formal channels for communicating and collaborating with one another were relatively unavailable. There was a significant need for a forum within which implementation researchers and stakeholders could learn from one another, refine approaches to science and practice, and develop an implementation research agenda using common measures, methods, and research principles to improve both the frequency and quality with which behavioral health treatment implementation is evaluated. SIRC’s membership growth is a testament to this identified need with more than 1000 members from 2011 to the present.ii SIRC’s primary objectives are to: (1) foster communication and collaboration across diverse groups, including implementation researchers, intermediariesi, as well as community stakeholders (SIRC uses the term “EBP champions” for these groups) – and to do so across multiple career levels (e.g., students, early career faculty, established investigators); and (2) enhance and disseminate rigorous measures and methodologies for implementing EBPs and evaluating EBP implementation efforts. These objectives are well aligned with Glasgow and colleagues’ [4] five core tenets deemed critical for advancing implementation science: collaboration, efficiency and speed, rigor and relevance, improved capacity, and cumulative knowledge. SIRC advances these objectives and tenets through in-person conferences, which bring together multidisciplinary implementation researchers and those implementing evidence-based behavioral health interventions in the community to share their work and create professional connections and collaborations

Antibodies against endogenous retroviruses promote lung cancer immunotherapy

B cells are frequently found in the margins of solid tumours as organized follicles in ectopic lymphoid organs called tertiary lymphoid structures (TLS). Although TLS have been found to correlate with improved patient survival and response to immune checkpoint blockade (ICB), the underlying mechanisms of this association remain elusive. Here we investigate lung-resident B cell responses in patients from the TRACERx 421 (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy) and other lung cancer cohorts, and in a recently established immunogenic mouse model for lung adenocarcinoma. We find that both human and mouse lung adenocarcinomas elicit local germinal centre responses and tumour-binding antibodies, and further identify endogenous retrovirus (ERV) envelope glycoproteins as a dominant anti-tumour antibody target. ERV-targeting B cell responses are amplified by ICB in both humans and mice, and by targeted inhibition of KRAS(G12C) in the mouse model. ERV-reactive antibodies exert anti-tumour activity that extends survival in the mouse model, and ERV expression predicts the outcome of ICB in human lung adenocarcinoma. Finally, we find that effective immunotherapy in the mouse model requires CXCL13-dependent TLS formation. Conversely, therapeutic CXCL13 treatment potentiates anti-tumour immunity and synergizes with ICB. Our findings provide a possible mechanistic basis for the association of TLS with immunotherapy response

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Cerebrospinal Fluid Sulfonylurea Receptor-1 is Associated with Intracranial Pressure and Outcome after Pediatric TBI: An Exploratory Analysis of the Cool Kids Trial

Sulfonylurea receptor-1 (SUR1) is recognized increasingly as a key contributor to cerebral edema, hemorrhage progression, and possibly neuronal death in multiple forms of acute brain injury. SUR1 inhibition may be protective and is actively undergoing evaluation in Phase-2/3 trials of traumatic brain injury (TBI) and stroke. In adult TBI, SUR1 expression is associated with intracranial hypertension and contusion expansion; its role in pediatric TBI remains unexplored. We tested 61 cerebrospinal fluid (CSF) samples from 16 pediatric patients with severe TBI enrolled in the multicenter Phase-3 randomized controlled Cool Kids trial and seven non-brain injured pediatric controls for SUR1 expression by enzyme-linked immunosorbent assay. Linear mixed models evaluated associations between mean SUR1 and intracranial pressure (ICP) over the first seven days and pediatric Glasgow Outcome Scale-Extended (GOS-E Peds) over the initial year after injury. SUR1 was undetectable in control CSF and increased versus control in nine of 16 patients with TBI. Mean SUR1 was not associated with age, sex, or therapeutic hypothermia. Each 1-point increase in initial Glasgow Coma Score was associated with a 1.68 ng/mL decrease in CSF SUR1. The CSF SUR1 was associated with increased ICP over seven days (b = 0.73,  = 0.004) and worse (higher) GOS-E Peds score (b = 0.24,  = 0.004). In this exploratory pediatric study, CSF SUR1 was undetectable in controls and variably elevated in severe TBI. Mean CSF SUR1 concentration was associated with ICP and outcome. These findings are distinct from our previous report in adults with severe TBI, where SUR1 was detected universally. SUR1 may be a viable therapeutic target in a subset of pediatric TBI, and further study is warranted

Phase I randomized clinical trial of N-acetylcysteine in combination with an adjuvant probenecid for treatment of severe traumatic brain injury in children

<div><p>Background</p><p>There are no therapies shown to improve outcome after severe traumatic brain injury (TBI) in humans, a leading cause of morbidity and mortality. We sought to verify brain exposure of the systemically administered antioxidant N-acetylcysteine (NAC) and the synergistic adjuvant probenecid, and identify adverse effects of this drug combination after severe TBI in children.</p><p>Methods</p><p>IRB-approved, randomized, double-blind, placebo controlled Phase I study in children 2 to 18 years-of-age admitted to a Pediatric Intensive Care Unit after severe TBI (Glasgow Coma Scale [GCS] score ≤8) requiring an externalized ventricular drain for measurement of intracranial pressure (ICP). Patients were recruited from November 2011-August 2013. Fourteen patients (n = 7/group) were randomly assigned after obtaining informed consent to receive probenecid (25 mg/kg load, then 10 mg/kg/dose q6h×11 doses) and NAC (140 mg/kg load, then 70 mg/kg/dose q4h×17 doses), or placebos via naso/orogastric tube. Serum and CSF samples were drawn pre-bolus and 1–96 h after randomization and drug concentrations were measured via UPLC-MS/MS. Glasgow Outcome Scale (GOS) score was assessed at 3 months.</p><p>Results</p><p>There were no adverse events attributable to drug treatment. One patient in the placebo group was withdrawn due to adverse effects. In the treatment group, NAC concentrations ranged from 16,977.3±2,212.3 to 16,786.1±3,285.3 in serum and from 269.3±113.0 to 467.9±262.7 ng/mL in CSF, at 24 to 72 h post-bolus, respectively; and probenecid concentrations ranged from 75.4.3±10.0 to 52.9±25.8 in serum and 5.4±1.0 to 4.6±2.1 μg/mL in CSF, at 24 to 72 h post-bolus, respectively (mean±SEM). Temperature, mean arterial pressure, ICP, use of ICP-directed therapies, surveillance serum brain injury biomarkers, and GOS at 3 months were not different between groups.</p><p>Conclusions</p><p>Treatment resulted in detectable concentrations of NAC and probenecid in CSF and was not associated with undesirable effects after TBI in children.</p><p>Trial registration</p><p>ClinicalTrials.gov <a href="https://clinicaltrials.gov/ct2/show/NCT01322009" target="_blank">NCT01322009</a></p></div