Antibody-activation of connexin hemichannels in bone osteocytes with ATP release suppresses breast cancer and osteosarcoma malignancy.

Bone tissue represents the most frequent site of cancer metastasis. We developed a hemichannel-activating antibody, Cx43-M2. Cx43-M2, directly targeting osteocytes in situ, activates osteocytic hemichannels and elevates extracellular ATP, thereby inhibiting the growth and migration of cultured breast and osteosarcoma cancer cells. Cx43-M2 significantly decreases breast cancer metastasis, osteosarcoma growth, and osteolytic activity, while improving survival rates in mice. The antibody's inhibition of breast cancer and osteosarcoma is dose dependent in both mouse and human cancer metastatic models. Furthermore, Cx43-M2 enhances anti-tumor immunity by increasing the population and activation of tumor-infiltrating immune-promoting effector T lymphocytes, while reducing immune-suppressive regulatory T cells. Our results suggest that the Cx43-M2 antibody, by activating Cx43 hemichannels and facilitating ATP release and purinergic signaling, transforms the cancer microenvironment from a supportive to a suppressive state. Collectively, our study underscores the potential of Cx43-M2 as a therapeutic for treating breast cancer bone metastasis and osteosarcoma.

Editing the core region in HPFH deletions alters fetal and adult globin expression for treatment of ß-hemoglobinopathies.

Reactivation of fetal hemoglobin (HbF) is a commonly adapted strategy to ameliorate ß-hemoglobinopathies. However, the continued production of defective adult hemoglobin (HbA) limits HbF tetramer production affecting the therapeutic benefits. Here, we evaluated deletional hereditary persistence of fetal hemoglobin (HPFH) mutations and identified an 11-kb sequence, encompassing putative repressor region (PRR) to ß-globin exon-1 (ßE1), as the core deletion that ablates HbA and exhibits superior HbF production compared with HPFH or other well-established targets. PRR-ßE1-edited hematopoietic stem and progenitor cells (HSPCs) retained their genome integrity and their engraftment potential to repopulate for long-term hematopoiesis in immunocompromised mice producing HbF positive cells in vivo. Furthermore, PRR-ßE1 gene editing is feasible without ex vivo HSPC culture. Importantly, the editing induced therapeutically significant levels of HbF to reverse the phenotypes of both sickle cell disease and ß-thalassemia major. These findings imply that PRR-ßE1 gene editing of patient HSPCs could lead to improved therapeutic outcomes for ß-hemoglobinopathy gene therapy.

TGF-ß-dependent lymphoid tissue residency of stem-like T cells limits response to tumor vaccine.

TGF-ß signaling is necessary for CD8+ T cell differentiation into tissue resident memory T cells (TRM). Although higher frequency of CD8+ TRM cells in the tumor microenvironment is associated with better prognosis, TGF-ß-blockade typically improves rather than worsens outcomes. Here we show that in a mouse melanoma model, in the tumor-draining lymph nodes (TDLN) rather than in the tumors themselves, stem-like CD8+ T cells differentiate into TRMs in a TGF-ß and tumor antigen dependent manner. Following vaccination against a melanoma-specific epitope, most tumour-specific CD8+ T cells are maintained in a stem-like state, but a proportion of cells lost TRM status and differentiate into CX3CR1+ effector CD8+ T cells in the TDLN, which are subsequently migrating into the tumours. Disruption of TGF-ß signaling changes the dynamics of these developmental processes, with the net result of improving effector CD8+ T cell migration into the tumours. In summary, TDLN stem-like T cells transiently switch from a TGF-ß-dependent TRM differentiation program to an anti-tumor migratory effector development upon vaccination, which transition can be facilitated by targeted TGF-ß blockade.

Facilitators to healthy canteen policy implementation: A qualitative approach.

ISSUE ADDRESSED: Governments across the world use guidelines and policy to support improving the quality and nutrition in school canteens, yet little is known about what makes for success in supporting school canteens. This study aimed to investigate the factors influencing the implementation of a healthy school canteen policy. METHODS: A qualitative descriptive approach using interviews with a purposive sample of Victorian schools that had successfully implemented a healthy school canteen was conducted. Twelve interviews were conducted with principals (n = 4), assistant principal (n = 1), canteen managers (n = 5), food services manager (n = 1) and canteen staff members (n = 3) across six Victorian schools. Data were analysed using a content analysis approach. RESULTS: Three key themes explained the adoption of policy: Values - emphasising service over profit; Knowledge - understanding of nutrition and the policy; and Support - from within and external to the school. CONCLUSIONS: Implementation of school canteen policy is more likely to be achieved when a school can focus on the service and educative component of the policy and where there is a shared priority for healthy eating across the entire school community. SO WHAT?: Creating a culture of service and community engagement with a healthy school canteen may increase policy implementation and should be the focus of future health promotion efforts.

Preferential Expansion of Human CD34+CD133+CD90+ Hematopoietic Stem Cells Enhances Gene-Modified Cell Frequency for Gene Therapy.

CD34+CD133+CD90+ hematopoietic stem cells (HSCs) are responsible for long-term multilineage hematopoiesis, and the high frequency of gene-modified HSCs is crucial for the success of hematopoietic stem and progenitor cell (HSPC) gene therapy. However, the ex vivo culture and gene manipulation steps of HSPC graft preparation significantly reduce the frequency of HSCs, thus necessitating large doses of HSPCs and reagents for the manipulation. In this study, we identified a combination of small molecules, Resveratrol, UM729, and SR1 that preferentially expands CD34+CD133+CD90+ HSCs over other subpopulations of adult HSPCs in ex vivo culture. The preferential expansion enriches the HSCs in ex vivo culture, enhances the adhesion, and results in a sixfold increase in the long-term engraftment in NSG mice. Further, the culture-enriched HSCs are more responsive to gene modification by lentiviral transduction and gene editing, increasing the frequency of gene-modified HSCs up to 10-fold in vivo. The yield of gene-modified HSCs obtained by the culture enrichment is similar to the sort-purification of HSCs and superior to Cyclosporin-H treatment. Our study addresses a critical challenge of low frequency of gene modified HSCs in HSPC graft by developing and demonstrating a facile HSPC culture condition that increases the frequency of gene-modified cells in vivo. This strategy will improve the outcome of HSPC gene therapy and also simplify the gene manipulation process.

CD8+ Regulatory T Cell - A Mystery to Be Revealed.

Regulatory T cells (Treg) are essential to maintain immune homeostasis and prevent autoimmune disorders. While the function and molecular regulation of Foxp3+CD4+ Tregs are well established, much of CD8+ Treg biology remains to be revealed. Here, we will review the heterogenous subsets of CD8+ T cells have been named "CD8+ Treg" and mainly focus on CD122hiLy49+CD8+ Tregs present in naïve mice. CD122hiLy49+CD8+ Tregs, which depends on transcription factor Helios and homeostatic cytokine IL-15, have been established as a non-redundant regulator of germinal center (GC) reaction. Recently, we have demonstrated that TGF-ß (Transforming growth factor-ß) and transcription factor Eomes (Eomesodermin) are essential for the function and homeostasis of CD8+ Tregs. In addition, we will discuss several open questions regarding the differentiation, function and true identity of CD8+ Tregs as well as a brief comparison between two regulatory T cell subsets critical to control GC reaction, namely CD4+ TFR (follicular regulatory T cells) and CD8+ Tregs.

The downregulation of IL-18R defines bona fide kidney-resident CD8+ T cells.

Stepwise induction of CD69 and CD103 marks distinct differentiation stages of mucosal Trms. But the majority of non-mucosal Trm lacks CD103 expression. The expression of CD69 alone cannot faithfully define Trm cells in heavily vascularized non-mucosal tissues, such as the kidney. Here, we found that a subset of kidney Trms downregulated IL-18 receptor during differentiation. Via global transcriptional analysis and parabiosis experiments, we have discovered that the downregulation of interleukin-18 receptor (IL-18R) is associated with the establishment of tissue residency. Together with the expression of CD69, IL-18Rlo exclusively identify tissue-resident cells whereas IL-18Rhi population contains both tissue-resident and migratory ones. Local cytokines including transforming growth factor ß (TGF-ß) and interferon α (IFN-α)/ß as well as TGF-ß-dependent suppression of transcription factor Tcf-1 are essential for IL-18R downregulation during kidney Trm differentiation. Together, we identified a convenient surface marker to distinguish bona fide kidney-resident CD8+ T cells as well as underlying molecular mechanisms controlling this differentiation process.

Chromhidrosis: An Unusual Cause of Skin Discoloration.

ABSTRACT: Chromhidrosis is a rare condition characterized by the secretion of colored sweat. We report a case of a preadolescent healthy girl presenting with acute, recurrent blue discoloration of her armpits. The blue discoloration can be wiped off but recurs. As providers, it is useful to be familiar with this diagnosis and course of disease.

Manipulation of Developmental Gamma-Globin Gene Expression: an Approach for Healing Hemoglobinopathies.

ß-Hemoglobinopathies are the most common monogenic disorders, and a century of research has provided us with a better understanding of the attributes of these diseases. Allogenic stem cell transplantation was the only potentially curative option available for these diseases until the discovery of gene therapy. The findings on the protective nature of fetal hemoglobin in sickle cell disease (SCD) and thalassemia patients carrying hereditary persistence of fetal hemoglobin (HPFH) mutations has given us the best evidence that the cure for ß-hemoglobinopathies remains hidden in the hemoglobin locus. The detailed understanding of the developmental gene regulation of gamma-globin (γ-globin) and the emergence of gene manipulation strategies offer us the opportunity for developing a γ-globin gene-modified autologous stem cell transplantation therapy. In this review, we summarize different therapeutic strategies that reactivate fetal hemoglobin for the gene therapy of ß-hemoglobinopathies.

Innovative Approaches to Emergency Medical Services Fellowship Challenges.

INTRODUCTION: Since the development of an Accreditation Council of Graduate Medical Education (ACGME)-accredited emergency medical services (EMS) fellowship, there has been little published literature on effective methods of content delivery or training modalities. Here we explore a variety of innovative approaches to the development and revision of the EMS fellowship curriculum. METHODS: Three academic, university-based ACGME-accredited EMS fellowship programs each implemented an innovative change to their existing training curricula. These changes included the following: a novel didactic curriculum delivery modality and evaluation; implementation of a distance education program to improve EMS fellows' rural EMS experiences; and modification of an existing EMS fellowship curriculum to train a non-emergency medicine physician. RESULTS: Changes made to each of the above EMS fellowship programs addressed unique challenges, demonstrating areas of success and promise for more generalized implementation of these curricula. Obstacles remain in tailoring the described curricula to the needs of each unique institution and system. CONCLUSION: Three separate curricula and program changes were implemented to overcome specific challenges and achieve educational goals. It is our hope that our shared experiences will enable others in addressing common barriers to teaching the EMS fellowship core content and share similar innovative approaches to educational challenges.

Effectiveness of Prehospital Dual Sequential Defibrillation for Refractory Ventricular Fibrillation and Ventricular Tachycardia Cardiac Arrest.

Objective: Dual sequential defibrillation (DSD) - successive defibrillations with two defibrillators - offers a novel approach to refractory ventricular fibrillation (RVF) and tachycardia (VF/VT). While associated with rescue shock success, the effect of DSD upon out-of-hospital cardiac arrest (OHCA) is unknown. We evaluated the association of DSD with survival after refractory VF/VT OHCA. Methods: We used data from a large metropolitan fire-based EMS service. We included all adult OHCA during 2013-2016 with ≥3 standard defibrillations. Physicians authorized subsequent DSD use by two separate defibrillators (PhysioControl LIFEPAK® 12/15) with pads placed anterior-lateral and anterior-posterior. Evaluated outcomes included return of spontaneous circulation (ROSC), survival to hospital admission, survival to 72 hours, and survival to hospital discharge. Using multivariable logistic regression, we evaluated the association between defibrillation type and OHCA outcomes, adjusting for patient demographics and event characteristics. Results: We included 310 patients in the analysis, 71 patients receiving DSD and 239 receiving conventional defibrillation. Patient demographics and event characteristics were similar between both groups. ROSC was lower for DSD than standard defibrillation: 39.4% vs. 60.3%, adjusted OR 0.46 (95% CI: 0.25-0.87). There were no differences in survival to hospital admission (35.2% vs. 49.2%, adjusted OR 0.57 [95% CI: 0.30-1.08]), survival to 72 hours (21.4% vs. 32.3%, adjusted OR 0.52 [95% CI: 0.26-1.10]), or survival to hospital discharge (14.3% vs. 20.9%, adjusted OR 0.63 [95% CI: 0.27-1.45]). Conclusions: Compared with conventional defibrillation, DSD was associated with lower odds of prehospital ROSC. Defibrillation type was not associated with other OHCA endpoints. DSD may not be beneficial in refractory VF/VT OHCA.

Use of a Novel, Portable, LED-Based Capillary Refill Time Simulator within a Disaster Triage Context.

Introduction A simple, portable capillary refill time (CRT) simulator is not commercially available. This device would be useful in mass-casualty simulations with multiple volunteers or mannequins depicting a variety of clinical findings and CRTs. The objective of this study was to develop and evaluate a prototype CRT simulator in a disaster simulation context. METHODS: A CRT prototype simulator was developed by embedding a pressure-sensitive piezo crystal, and a single red light-emitting diode (LED) light was embedded, within a flesh-toned resin. The LED light was programmed to turn white proportionate to the pressure applied, and gradually to return to red on release. The time to color return was adjustable with an external dial. The prototype was tested for feasibility among two cohorts: emergency medicine physicians in a tabletop exercise and second year medical students within an actual disaster triage drill. The realism of the simulator was compared to video-based CRT, and participants used a Visual Analog Scale (VAS) ranging from "completely artificial" to "as if on a real patient." The VAS evaluated both the visual realism and the functional (eg, tactile) realism. Accuracy of CRT was evaluated only by the physician cohort. Data were analyzed using parametric and non-parametric statistics, and mean Cohen's Kappas were used to describe inter-rater reliability. RESULTS: The CRT simulator was generally well received by the participants. The simulator was perceived to have slightly higher functional realism (P=.06, P=.01) but lower visual realism (P=.002, P=.11) than the video-based CRT. Emergency medicine physicians had higher accuracy on portrayed CRT on the simulator than the videos (92.6% versus 71.1%; P<.001). Inter-rater reliability was higher for the simulator (0.78 versus 0.27; P<.001). CONCLUSIONS: A simple, LED-based CRT simulator was well received in both settings. Prior to widespread use for disaster triage training, validation on participants' ability to accurately triage disaster victims using CRT simulators and video-based CRT simulations should be performed. Chang TP , Santillanes G , Claudius I , Pham PK , Koved J , Cheyne J , Gausche-Hill M , Kaji AH , Srinivasan S , Donofrio JJ , Bir C . Use of a novel, portable, LED-based capillary refill time simulator within a disaster triage context. Prehosp Disaster Med. 2017;32(4):451-456.

Firearm Laws and Firearm Homicides: A Systematic Review.

Importance: Firearm homicide is a leading cause of injury death in the United States, and there is considerable debate over the effectiveness of firearm policies. An analysis of the effectiveness of firearm laws on firearm homicide is important to understand optimal policies to decrease firearm homicide in the United States. Objective: To evaluate the association between firearm laws and preventing firearm homicides in the United States. Evidence Review: We evaluated peer-reviewed articles from 1970 to 2016 focusing on the association between US firearm laws and firearm homicide. We searched PubMed, CINAHL, Lexis/Nexis, Sociological Abstracts, Academic Search Premier, the Index to Legal Periodicals and Books, and the references from the assembled articles. We divided laws into 5 categories: those that (1) curb gun trafficking, (2) strengthen background checks, (3) improve child safety, (4) ban military-style assault weapons, and (5) restrict firearms in public places and leniency in firearm carrying. The articles were assessed using the standardized Guide to Community Preventive Services data collection instrument and 5 additional quality metrics: (1) appropriate data source(s) and outcome measure(s) were used for the study, (2) the time frame studied was adequate, (3) appropriate statistical tests were used, (4) the analytic results were robust, and (5) the disaggregated results of control variables were consistent with the literature. Findings: In the aggregate, stronger gun policies were associated with decreased rates of firearm homicide, even after adjusting for demographic and sociologic factors. Laws that strengthen background checks and permit-to-purchase seemed to decrease firearm homicide rates. Specific laws directed at firearm trafficking, improving child safety, or the banning of military-style assault weapons were not associated with changes in firearm homicide rates. The evidence for laws restricting guns in public places and leniency in gun carrying was mixed. Conclusions and Relevance: The strength of firearm legislation in general, and laws related to strengthening background checks and permit-to-purchase in particular, is associated with decreased firearm homicide rates. High-quality research is important to further evaluate the effectiveness of these laws. Legislation is just 1 part of a multipronged approach that will be necessary to decrease firearm homicides in the United States.

Development of a Pediatric Mass Casualty Triage Algorithm Validation Tool.

BACKGROUND: Rapid, accurate evaluation and sorting of victims in a mass casualty incident (MCI) is crucial, as over-triage of victims may overwhelm a trauma system and under-triage may lead to an increase in morbidity and mortality. At this time, there is no validation tool specifically developed for the pediatric population to test an MCI algorithm's inherent capabilities to correctly triage children. OBJECTIVE: To develop a set of criteria for outcomes and interventions to be used as a validation tool for testing an MCI algorithm's ability to correctly triage patients from a cohort of pediatric trauma patients. METHODS: Expert opinion and literature review was used to formulate an initial Criteria Outcomes Tool (COT) that retrospectively categorizes pediatric (≤14 years of age) MCI victims based on resource utilization and clinical outcomes using the classic Red to Black MCI triage designations: Red - cardiopulmonary or mental status compromise needing intervention, Yellow - stable cardiopulmonary status but may require life or limb therapy, Green - minimally injured, and Black - deceased or likely to die given the circumstances. Using an anatomic approach, a list of criteria were defined and a modified-Delphi approach was used to create a summative COT that was reviewed by the American Academy of Pediatrics Disaster Preparedness Advisory Council. The resulting COT was independently applied to a weighted retrospective cohort of 25 pediatric victims from a single Level I trauma center by two reviewers to determine reproducibility. RESULTS: We created a Criteria Outcomes Tool (COT) with 47 outcomes and interventions to validate an MCI algorithm's triage designation. When the COT was applied to a cohort of 25 weighted pediatric charts, we identified the following resource utilization and outcome based triage designations: six Red, six Yellow, six Green, and seven Black triage outcomes. The 100% agreement was obtained between the two reviewers in each of the four categories. CONCLUSIONS: We designed an outcomes and resource utilization tool, the COT, to evaluate the ability of an MCI algorithm to correctly triage pediatric patients. Our tool has good reproducibility on initial study. KEY WORDS: pediatric; disaster; validation tools; triage algorithms; emergency.

Accuracy, Efficiency, and Inappropriate Actions Using JumpSTART Triage in MCI Simulations.

INTRODUCTION: Using the pediatric version of the Simple Triage and Rapid Treatment (JumpSTART) algorithm for the triage of pediatric patients in a mass-casualty incident (MCI) requires assessing the results of each step and determining whether to move to the next appropriate action. Inappropriate application can lead to performance of unnecessary actions or failure to perform necessary actions. Hypothesis/Problem To report overall accuracy and time required for triage, and to assess if the performance of unnecessary steps, or failure to perform required steps, in the triage algorithm was associated with inaccuracy of triage designation or increased time to reach a triage decision. METHODS: Medical students participated in an MCI drill in which they triaged both live actors portraying patients and computer-based simulated patients to the four triage levels: minor, delayed, immediate, and expectant. Their performance was timed and compared to intended triage designations and a priori determined critical actions. RESULTS: Thirty-three students completed 363 scenarios. The overall accuracy was 85.7% and overall mean time to assign a triage designation was 70.4 seconds, with decreasing times as triage acuity level decreased. In over one-half of cases, the student omitted at least one action and/or performed at least one action that was not required. Each unnecessary action increased time to triage by a mean of 8.4 seconds and each omitted action increased time to triage by a mean of 5.5 seconds. Discussion Increasing triage level, performance of inappropriate actions, and omission of recommended actions were all associated with increasing time to perform triage.

Comparison of Computerized Patients versus Live Moulaged Actors for a Mass-casualty Drill.

INTRODUCTION: Multiple modalities for simulating mass-casualty scenarios exist; however, the ideal modality for education and drilling of mass-casualty incident (MCI) triage is not established. Hypothesis/Problem Medical student triage accuracy and time to triage for computer-based simulated victims and live moulaged actors using the pediatric version of the Simple Triage and Rapid Treatment (JumpSTART) mass-casualty triage tool were compared, anticipating that student performance and experience would be equivalent. METHODS: The victim scenarios were created from actual trauma records from pediatric high-mechanism trauma presenting to a participating Level 1 trauma center. The student-reported fidelity of the two modalities was also measured. Comparisons were done using nonparametric statistics and regression analysis using generalized estimating equations. RESULTS: Thirty-three students triaged four live patients and seven computerized patients representing a spectrum of minor, immediate, delayed, and expectant victims. Of the live simulated patients, 92.4% were given accurate triage designations versus 81.8% for the computerized scenarios (P=.005). The median time to triage of live actors was 57 seconds (IQR=45-66) versus 80 seconds (IQR=58-106) for the computerized patients (P<.0001). The moulaged actors were felt to offer a more realistic encounter by 88% of the participants, with a higher associated stress level. CONCLUSION: While potentially easier and more convenient to accomplish, computerized scenarios offered less fidelity than live moulaged actors for the purposes of MCI drilling. Medical students triaged live actors more accurately and more quickly than victims shown in a computerized simulation.

Epidemiology of paediatric firearm injuries in the USA, 2001-2010.

OBJECTIVES: Firearm injuries to children and adolescents remain an important cause of morbidity and mortality in the USA. The objectives of this study were to describe the prevalence of and epidemiologic risk factors associated with firearm injuries to children and adolescents evaluated in a nationally representative sample of US emergency departments and ambulatory care centres. STUDY DESIGN: We performed a retrospective cross-sectional analysis of data from the National Hospital Ambulatory Medical Care Survey from 2001 to 2010. Firearm injury-related visits in patients 0-19 years old were identified using the International Classification of Diseases, Ninth Revision, Clinical Modification firearm injury codes. The primary outcome was the prevalence of firearm-related injuries. We used multivariate logistic regression to analyse demographic risk factors associated with these injuries. RESULTS: From 2001 to 2010, there were a total of 322 730 927 (95% CI 287 462 091 to 357 999 763) paediatric US outpatient visits; 198 969 visits (0.06%, 95% CI 120 727 to 277 211) were for firearm injuries. Fatal firearm injuries accounted for 2% of these visits; 36% were intentionally inflicted. There were increased odds of firearm injuries to men (OR 10.2, 95% CI 5.1 to 20.5), black children and adolescents (0-19 years) (OR 3.2, 95% CI 1.5 to 6.7) and adolescents 12-19 years old (all races) (OR 16.6, 95% CI 6.3 to 44.3) on multivariable analysis. CONCLUSIONS: Firearm injuries continue to be a substantial problem for US children and adolescents, with non-fatal rates 24% higher than previously reported. Increased odds for firearm-related visits were found in men, black children and those 12-19 years old.

Diagnosis and management of motor vehicle trauma in children: an evidence-based review.

Injuries from motor vehicle crashes are the leading cause of mortality in children aged 5 years and older in the United States. This review discusses common injuries in children after motor vehicle trauma and examines the evidence regarding the evaluation and treatment of pediatric patients involved in motor vehicle crashes. Both prehospital and emergency department care are discussed along with a differential diagnosis of the injuries most commonly seen in motor vehicle crashes. The various options for imaging modalities are also discussed in this review. A critical appraisal of the existing guidelines for the management of motor vehicle trauma and for the use of appropriate child-safety restraints is presented. Emergency clinicians will be able to use the patient's history and physical examination findings along with knowledge of common injuries to determine the most appropriate workup and treatment of pediatric patients who present with motor vehicle trauma.

M-protein-derived conformational peptide epitope vaccine candidate against Group A Streptococcus.

Identification of the most relevant epitopes is the initial challenge of peptide-based vaccine design. Chimeric conserved epitopes of the Group A Streptococcus (GAS) M-protein were used in the development of an anti-GAS vaccine candidate. Previously, these epitopes have incorporated a GCN4 peptide from yeast to maintain their native helical structure. Here, we designed a new peptide epitope based on the minimal B-cell epitope from GAS M-protein. This new epitope was able to adopt the desired helical conformation without the need for the foreign GCN4 flanking sequence. The selected epitope induced significant immune responses upon administration with external adjuvant, and when incorporated into the Lipid Core Peptide (LCP) system. Moreover, the antibodies produced against this epitope were able to recognize the native p145 sequence from M-protein.