Biological and genetic determinants of glycolysis: Phosphofructokinase isoforms boost energy status of stored red blood cells and transfusion outcomes.

Mature red blood cells (RBCs) lack mitochondria and thus exclusively rely on glycolysis to generate adenosine triphosphate (ATP) during aging in vivo or storage in blood banks. Here, we leveraged 13,029 volunteers from the Recipient Epidemiology and Donor Evaluation Study to identify associations between end-of-storage levels of glycolytic metabolites and donor age, sex, and ancestry-specific genetic polymorphisms in regions encoding phosphofructokinase 1, platelet (detected in mature RBCs); hexokinase 1 (HK1); and ADP-ribosyl cyclase 1 and 2 (CD38/BST1). Gene-metabolite associations were validated in fresh and stored RBCs from 525 Diversity Outbred mice and via multi-omics characterization of 1,929 samples from 643 human RBC units during storage. ATP and hypoxanthine (HYPX) levels-and the genetic traits linked to them-were associated with hemolysis in vitro and in vivo, both in healthy autologous transfusion recipients and in 5,816 critically ill patients receiving heterologous transfusions, suggesting their potential as markers to improve transfusion outcomes.

Platelet releasates mitigate the endotheliopathy of trauma.

BACKGROUND: Platelets are well known for their roles in hemostasis, but they also play a key role in thromboinflammatory pathways by regulating endothelial health, stimulating angiogenesis, and mediating host defense through both contact dependent and independent signaling. When activated, platelets degranulate releasing multiple active substances. We hypothesized that the soluble environment formed by trauma platelet releasates attenuates thromboinflammation via mitigation of trauma induced endothelial permeability and metabolomic reprogramming. METHODS: Blood was collected from injured and healthy patients to generate platelet releasates and plasma in parallel. Permeability of endothelial cells when exposed to trauma platelet releasates (TPR) and plasma (TP) was assessed via resistance measurement by Electric Cell-substrate Impedance Sensing (ECIS). Endothelial cells treated with TPR and TP were subjected to mass spectrometry-based metabolomics. RESULTS: TP increased endothelial permeability, whereas TPR decreased endothelial permeability when compared to untreated cells. When TP and TPR were mixed ex vivo, TPR mitigated TP-induced permeability, with significant increase in AUC compared to TP alone. Metabolomics of TPR and TP demonstrated disrupted redox reactions and anti-inflammatory mechanisms. CONCLUSION: TPRs provide endothelial barrier protection against TP-induced endothelial permeability. Our findings highlight a potential beneficial action of activated platelets on the endothelium in injured patients through disrupted redox reactions and increased antioxidants. Our findings support that soluble signaling from platelet degranulation may mitigate the endotheliopathy of trauma. The clinical implications of this are that activated platelets may prove a promising therapeutic target in the complex integration of thrombosis, endotheliopathy, and inflammation in trauma. LEVEL OF EVIDENCE: Prognostic/Epidemiological, Level III.

Trauma patients with type O blood exhibit unique multi-omics signature with decreased lectin pathway of complement levels.

BACKGROUND: Patients with type O blood may have an increased risk of hemorrhagic complications due to lower baseline levels of von Willebrand Factor (vWF) and factor VIII, but the transition to a mortality difference in trauma is less clear. We hypothesized that type O trauma patients will have differential proteomic and metabolomic signatures in response to trauma beyond vWF and FVIII alone. METHODS: Patients meeting the highest level of trauma activation criteria were prospectively enrolled. Blood samples were collected upon arrival to the emergency department. Proteomic and metabolomic (multi-omics) analyses of these samples were performed using liquid chromatography-mass spectrometry. Demographic, clinical, and multi-omics data were compared between patients with type O blood versus all other patients. RESULTS: There were 288 patients with multi-omics data; 146 (51%) had type O blood. Demographics, injury patterns, and initial vital signs and laboratory measurements were not different between groups. Type O patients had increased lengths of stay (7 vs. 6 days, p = 0.041) and a trend towards decreased mortality secondary to traumatic brain injury compared to other causes (TBI, 44.4 % vs. 87.5%, p = 0.055). Type O patients had decreased levels of mannose-binding lectin (MBL) and MBL associated serine proteases 1 and 2 which are required for the initiation of the lectin pathway of complement activation. Type O patients also had metabolite differences signifying energy metabolism and mitochondrial dysfunction. CONCLUSION: Blood type O patients have a unique multi-omics signature, including decreased levels of proteins required to activate the lectin complement pathway. This may lead to overall decreased levels of complement activation and decreased systemic inflammation in the acute phase possibly leading to a survival advantage, especially in TBI. However, this may later impair healing. Future work will need to confirm these associations, and animal studies are needed to test therapeutic targets. LEVEL OF EVIDENCE: Retrospective Comparative Study, Level IV.

Smoking primes the metabolomic response in trauma.

INTRODUCTION: Smoking is a public health threat because of its well-described link to increased oxidative stress-related diseases including peripheral vascular disease and coronary artery disease. Tobacco use has been linked to risk of inpatient trauma morbidity including acute respiratory distress syndrome; however, its mechanistic effect on comprehensive metabolic heterogeneity has yet to be examined. METHODS: Plasma was obtained on arrival from injured patients at a Level 1 trauma center and analyzed with modern mass spectrometry-based metabolomics. Patients were stratified by nonsmoker, passive smoker, and active smoker by lower, interquartile, and upper quartile ranges of cotinine intensity peaks. Patients were substratified by high injury/high shock (Injury Severity Score, ≥15; base excess, <-6) and compared with healthy controls. p Value of <0.05 following false discovery rate correction of t test was considered significant. RESULTS: Forty-eight patients with high injury/high shock (7 nonsmokers [15%], 25 passive smokers [52%], and 16 active smokers [33%]) and 95 healthy patients who served as controls (30 nonsmokers [32%], 43 passive smokers [45%], and 22 active smokers [23%]) were included. Elevated metabolites in our controls who were active smokers include enrichment in chronic inflammatory and oxidative processes. Elevated metabolites in active smokers in high injury/high shock include enrichment in the malate-aspartate shuttle, tyrosine metabolism, carnitine synthesis, and oxidation of very long-chain fatty acids. CONCLUSION: Smoking promotes a state of oxidative stress leading to mitochondrial dysfunction, which is additive to the inflammatory milieu of trauma. Smoking is associated with impaired mitochondrial substrate utilization of long-chain fatty acids, aspartate, and tyrosine, all of which accentuate oxidative stress following injury. This altered expression represents an ideal target for therapies to reduce oxidative damage toward the goal of personalized treatment of trauma patients. LEVEL OF EVIDENCE: Prognostic and Epidemiological; Level IV.

Biological and Genetic Determinants of Glycolysis: Phosphofructokinase Isoforms Boost Energy Status of Stored Red Blood Cells and Transfusion Outcomes.

Mature red blood cells (RBCs) lack mitochondria, and thus exclusively rely on glycolysis to generate adenosine triphosphate (ATP) during aging in vivo or storage in the blood bank. Here we leveraged 13,029 volunteers from the Recipient Epidemiology and Donor Evaluation Study to identify an association between end-of-storage levels of glycolytic metabolites and donor age, sex, and ancestry-specific genetic polymorphisms in regions encoding phosphofructokinase 1, platelet (detected in mature RBCs), hexokinase 1, ADP-ribosyl cyclase 1 and 2 (CD38/BST1). Gene-metabolite associations were validated in fresh and stored RBCs from 525 Diversity Outbred mice, and via multi-omics characterization of 1,929 samples from 643 human RBC units during storage. ATP and hypoxanthine levels - and the genetic traits linked to them - were associated with hemolysis in vitro and in vivo, both in healthy autologous transfusion recipients and in 5,816 critically ill patients receiving heterologous transfusions, suggesting their potential as markers to improve transfusion outcomes. Highlights: Blood donor age and sex affect glycolysis in stored RBCs from 13,029 volunteers;Ancestry, genetic polymorphisms in PFKP, HK1, CD38/BST1 influence RBC glycolysis;Modeled PFKP effects relate to preventing loss of the total AXP pool in stored RBCs;ATP and hypoxanthine are biomarkers of hemolysis in vitro and in vivo.

Mitigation of trauma-induced endotheliopathy by activated protein C: A potential therapeutic for postinjury thromboinflammation.

BACKGROUND: Activated Protein C (aPC) plays dual roles after injury, driving both trauma-induced coagulopathy (TIC) by cleaving, and thus inactivating, factors Va and VIIIa and depressing fibrinolysis while also mediating an inflammomodulatory milieu via protease activated receptor-1 (PAR-1) cytoprotective signaling. Because of this dual role, it represents and ideal target for study and therapeutics after trauma. A known aPC variant, 3K3A-aPC, has been engineered to preserve cytoprotective activity while retaining minimal anticoagulant activity rendering it potentially ideal as a cytoprotective therapeutic after trauma. We hypothesized that 3K3A-aPC would mitigate the endotheliopathy of trauma by protecting against endothelial permeability. METHODS: We used electric cell-substrate impedance sensing to measure permeability changes in real time in primary endothelial cells. These were cultured, grown to confluence, and treated with a 2 µg/mL solution of 3K3A-aPC at 180 minutes, 120 minutes, 60 minutes, 30 minutes prior to stimulation with ex vivo plasma taken from severely injured trauma patients (Injury Severity Score > 15 and BD < -6) (trauma plasma [TP]). Cells treated with thrombin and untreated cells were included in this study as control groups. Permeability changes were recorded in real time via electric cell-substrate impedance sensing for 30 minutes after treatment with TP. We quantified permeability changes in the control and treatment groups as area under the curve (AUC). Rac1/RhoA activity was also compared between these groups. Statistical significance was determined by one-way ANOVA followed by a post hoc analysis using Tukey's multiple comparison's test. RESULTS: Treatment with aPC mitigated endothelial permeability induced by ex vivo trauma plasma at all pre-treatment time points. The AUC of the 30-minute 3K3A-aPC pretreatment group was higher than TP alone (mean diff. 22.12 95% CI [13.75, 30.49], p < 0.0001) (Figure). Moreover, the AUC of the 60-minute, 120-minute, and 180-minute pretreatment groups was also higher than TP alone (mean diff., 16.30; 95% confidence interval [CI], 7.93-24.67; 19.43; 95% CI, 11.06-27.80, and 18.65; 95% CI, 10.28-27.02;, all p < 0.0001, respectively). Rac1/RhoA activity was higher in the aPC pretreatment group when compared with all other groups ( p < 0.01). CONCLUSION: Pretreatment with 3K3A-aPC, which retains its cytoprotective function but has only ~5% of its anticoagulant function, abrogates the effects of trauma-induced endotheliopathy. This represents a potential therapeutic treatment for dysregulated thromboinflammation for injured patients by minimizing aPC's role in trauma-induced coagulopathy while concurrently amplifying its essential cytoprotective function. LEVEL OF EVIDENCE: Prognostic and Epidemiological; Level III.

Mesenchymal Stem Cells Delivered Locally to Ischemia-Reperfused Kidneys via Injectable Hyaluronic Acid Hydrogels Decrease Extracellular Matrix Remodeling 1 Month after Injury in Male Mice.

The translation of stem cell therapies has been hindered by low cell survival and retention rates. Injectable hydrogels enable the site-specific delivery of therapeutic cargo, including cells, to overcome these challenges. We hypothesized that delivery of mesenchymal stem cells (MSC) via shear-thinning and injectable hyaluronic acid (HA) hydrogels would mitigate renal damage following ischemia-reperfusion acute kidney injury. Acute kidney injury (AKI) was induced in mice by bilateral or unilateral ischemia-reperfusion kidney injury. Three days later, mice were treated with MSCs either suspended in media injected intravenously via the tail vein, or injected under the capsule of the left kidney, or MSCs suspended in HA injected under the capsule of the left kidney. Serial measurements of serum and urine biomarkers of renal function and injury, as well as transcutaneous glomerular filtration rate (tGFR) were performed. In vivo optical imaging showed that MSCs localized to both kidneys in a sustained manner after bilateral ischemia and remained within the ipsilateral treated kidney after unilateral ischemic AKI. One month after injury, MSC/HA treatment significantly reduced urinary NGAL compared to controls; it did not significantly reduce markers of fibrosis compared to untreated controls. An analysis of kidney proteomes revealed decreased extracellular matrix remodeling and high overlap with sham proteomes in MSC/HA-treated animals. Hydrogel-assisted MSC delivery shows promise as a therapeutic treatment following acute kidney injury.

A proteomic analysis of NETosis in trauma: Emergence of serpinB1 as a key player.

BACKGROUND: Release of neutrophil extracellular traps (NETosis) may mediate postinjury organ dysfunction, but mechanisms remain unclear. The intracellular serine protease inhibitor (serpin) B1 is vital to neutrophil function and has been shown to restrict NETosis in inflammatory settings. In this study, we used discovery proteomics to identify the proteomic signature of trauma-induced NETosis. We hypothesized that serpinB1 would be a major component of this NET protein profile and associated with adverse outcomes. METHODS: This was a post hoc analysis of data collected as part of the COMBAT randomized clinical trial. Blood was collected from injured patients at a single Level I Trauma Center. Proteomic analyses were performed through targeted liquid chromatography coupled with mass spectrometry. Abundances of serpinB1 and known NETosis markers were analyzed with patient and injury characteristics, clinical data, and outcomes. RESULTS: SerpinB1 levels on emergency department (ED) arrival were significantly correlated with proteomic markers of NETosis, including core histones, transketolase, and S100A8/A9 proteins. More severely injured patients had elevated serpinB1 and NETosis markers on ED arrival. Levels of serpinB1 and top NETosis markers were significantly elevated on ED arrival in nonsurvivors and patients with fewer ventilator- and ICU-free days. In proteome-wide receiver operating characteristic analysis, serpinB1 was consistently among the top proteins associated with adverse outcomes. Among NETosis markers, levels of serpinB1 early in the patient's course exhibited the greatest separation between patients with fewer and greater ventilator- and ICU-free days. Gene Ontology analysis of top predictors of adverse outcomes further supports NETosis as a potential mediator of postinjury organ dysfunction. CONCLUSION: We have identified a proteomic signature of trauma-induced NETosis, and NETosis is an early process following severe injury that may mediate organ dysfunction. In addition, serpinB1 is a major component of this NET protein profile that may serve as an early marker of excessive NETosis after injury.

Platelet and cryoprecipitate transfusions from female donors improve coagulopathy in vitro.

BACKGROUND: Females are relatively hypercoagulable compared with males, with increased platelet aggregation and improved clot dynamics. However, sex differences in coagulation have not yet been considered in transfusion guidelines. Therefore, our objective was to evaluate hemostatic differences in sex concordant and sex discordant cryoprecipitate and platelet transfusions. We hypothesized that transfusion of blood products from female donors results in improved coagulopathy compared with male blood products. METHODS: This was a cohort study evaluating sex dimorphisms in coagulation assays and clotting factors in healthy volunteer plasma and cryoprecipitate. Sex dimorphisms in transfusions were evaluated using an in vitro coagulopathy model. Female or male platelets or single-donor cryoprecipitate was added to "recipient" whole blood after dilution of recipient blood with citrated saline to provoke a coagulopathic profile. Citrated native thromboelastography was then performed. Liquid chromatography/mass spectroscopy was performed on single-donor cryoprecipitate to evaluate sex dimorphisms in the proteome of cryoprecipitate. RESULTS: Females have an increased proportion of functional fibrinogen. Transfusion of female-donor platelets and cryoprecipitate induces a larger decrease in R time and greater increase in angle than male-donor platelets or cryoprecipitate. Female-donor cryoprecipitate has increased factor V and factor XIII compared with male cryoprecipitate, and comprehensive proteomics revealed sex differences in several proteins with potential immunological significance. CONCLUSION: Platelets and cryoprecipitate from female donors improve coagulopathy more than male blood products in vitro. Increased factor V and factor XIII activity as well as increased fibrinogen activity in female donors appears to drive this disparity. Sex differences in the proteome of cryoprecipitate may influence how transfusions modulate the thromboinflammation of trauma. The differing hemostatic profiles of female and male blood products suggest the potential role of sex-specific transfusions guidelines in hemostatic resuscitation.

Blockade of Osteoclast-Mediated Bone Resorption With a RANKL-Inhibitor Enhances Bone Formation in a Rat Spinal Fusion Model.

STUDY DESIGN: Rat spine fusion model. OBJECTIVE: The present study aimed to determine whether administration of osteoprotegerin (OPG) in a rat model of spinal fusion increases bone volume, bone density, and decreases osteoclasts in the fusion mass. SUMMARY OF BACKGROUND DATA: OPG is a soluble RANK-ligand inhibitor that blocks osteoclast differentiation and activation. This makes it a potential agent to control the remodeling process and enhance bone mass during spinal fusion. MATERIALS AND METHODS: Forty-eight male Sprague-Dawley rats received a one-level spinal fusion of L4-L5 with bone allograft. Rats were then divided into four groups according to initiation of treatment: (1) saline on day 0 (saline), (2) OPG on day 0 (OPG D0), (3) OPG on day 10 (OPG D10), and (4) OPG on day 21 (OPG D21) postsurgery. After their initial injection, rats received weekly subcutaneous injections of OPG (10 mg/kg) and were euthanized six weeks postsurgery. MicroCT analysis of the fusion site and histological analysis of bone surface for quantification of osteoclast lining was performed. RESULTS: Increased bone volume in the fusion site and around the spinous process was seen in OPG D0 and OPG D10 when compared with saline. Mean trabecular thickness was greater in all groups receiving OPG compared with saline, with OPG D0 and OPG D10 having significantly greater mean trabecular thickness than OPG D21. All OPG groups had less bone surface lined with osteoclasts when compared with Saline, with OPG D0 and OPG D10 having fewer than OPG D21. CONCLUSIONS: This study indicates that OPG inhibited osteoclast bone resorption, which led to greater bone at the fusion site. Future studies investigating OPG on its own or in combination with an osteogenic factor to improve spinal fusion outcomes are warranted to further elucidate its potential therapeutic effect.

Zone 1 REBOA in a combat DCBI swine model does not worsen brain injury.

BACKGROUND: Zone 1 resuscitative endovascular balloon occlusion of the aorta has been recommended for refractory shock after a dismounted complex blast injury for the austere combat scenario. While resuscitative endovascular balloon occlusion of the aorta should enhance coronary perfusion, there is a potential risk of secondary brain injury due to loss of cerebral autoregulation. We developed a combat casualty relevant dismounted complex blast injury swine model to evaluate the effects of resuscitative endovascular balloon occlusion of the aorta zone I on intracranial pressure and cerebral edema. We hypothesized that zone 1 aortic occlusion with resuscitative endovascular balloon occlusion of the aorta would increase mean arterial pressure transmitted in excessive intracranial pressure, thereby worsening brain injury. METHODS: 50 kg male Yorkshire swine were subjected to a combination dismounted complex blast injury model consisting of blast traumatic brain injury (50 psi, ARA Mobile Shock Laboratory), tissue injury (bilateral femur fractures), and hemorrhagic shock (controlled bleeding to a base deficit goal of 10 mEq/L). During the shock phase, pigs were randomized to no aortic occlusion (n = 8) or to 30 minutes of zone 1 resuscitative endovascular balloon occlusion of the aorta (zone 1 aortic occlusion group, n = 6). After shock, pigs in both groups received a modified Tactical Combat Casualty Care-based resuscitation and were monitored for an additional 240 minutes until euthanasia/death for a total of 6 hours. Intracranial pressure was monitored throughout, and brains were harvested for water content. Linear mixed models for repeated measures were used to compare mean arterial pressure and intracranial pressure between zone 1 aortic occlusion and no aortic occlusion groups. RESULTS: After dismounted complex blast injury, the zone 1 group had a significantly higher mean arterial pressure during hemorrhagic shock compared to the control group (41.2 mm Hg vs 16.7 mm Hg, P = .002). During balloon occlusion, intracranial pressure was not significantly elevated in the zone 1 aortic occlusion group vs control, but intracranial pressure was significantly lower in the zone 1 group at the end of the observation period. In addition, the zone 1 aortic occlusion group did not have increased brain water content (zone 1 aortic occlusion: 3.95 ± 0.1g vs no aortic occlusion: 3.95 ± 0.3 g, P = .87). Troponin levels significantly increased in the no aortic occlusion group but did not in the zone 1 aortic occlusion group. CONCLUSION: Zone 1 aortic occlusion using resuscitative endovascular balloon occlusion of the aorta in a large animal dismounted complex blast injury model improved proximal mean arterial pressure while not significantly increasing intracranial pressure during balloon inflation. Observation up to 240 minutes postresuscitation did not show clinical signs of worsening brain injury or cardiac injury. These data suggest that in a dismounted complex blast injury swine model, resuscitative endovascular balloon occlusion of the aorta in zone 1 may provide neuro- and cardioprotection in the setting of blast traumatic brain injury. However, longer monitoring periods may be needed to confirm that the neuroprotection is lasting.

Postinjury complement C4 activation is associated with adverse outcomes and is potentially influenced by plasma resuscitation.

BACKGROUND: Complement activation after trauma promotes hemostasis but is associated with increased morbidity and mortality. However, the specific pathways and downstream mediators remain unclear. Recently, the anaphylatoxin C4a has been shown to bind to thrombin receptors. While plasma-based resuscitation has been shown to modify the endotheliopathy of trauma, it may provide complement zymogens that fuel ongoing inflammatory cascades. We sought to characterize the activation of complement after injury and the effect of fresh frozen plasma (FFP) on this inflammatory response. We hypothesized that trauma induces C4 activation, which is associated with worse outcomes and influenced by FFP resuscitation. METHODS: Blood was collected from injured patients at a single level I trauma center enrolled in the Control of Major Bleeding after Trauma (COMBAT) randomized clinical trial. Proteomic analyses were performed through targeted liquid chromatography coupled with mass spectrometry. For the present observational study, concentrations of complement proteins were analyzed at multiple time points, compared between treatment groups, and correlated with outcomes. RESULTS: C4 activation occurred over the first 6 hours postinjury with peak activation 6 to 24 hours. Tissue hypoperfusion, defined as base deficit >10 mEq/L, and requirement for massive transfusion were associated with greater C4 activation. C4 activation was associated with mortality, multiple organ failure, and longer ventilator requirement. In addition, temporal trends of C1q, factor B, and C3 by outcome groups support the prevailing theory of primary classical pathway activation with alternative pathway amplification. Resuscitation with FFP over the first 6 hours was associated with increased C4 activation at 12 and 24 hours. CONCLUSION: C4 activation has an important inflammatory role postinjury, and FFP has the potential to augment this complement activation during resuscitation. LEVEL OF EVIDENCE: Prognostic/epidemiological, level III.

A 37-Year-Old Woman with Hypertrophic Cardiomyopathy with a Dual-Chamber Implantable Cardioverter-Defibrillator Requiring Percutaneous Transvenous Lead Extraction and Multidisciplinary Management.

BACKGROUND Percutaneous transvenous lead extraction (TLE) of cardiac implantable electronic devices can be performed with a high success rate. However, TLE has its limitations and challenges. Recognizing the challenges at an early stage during the procedure is vital for appropriate patient management. We present a challenging case of implantable cardioverter-defibrillator (ICD) lead extraction in which we aborted TLE in favor of elective surgical extraction (SE). This potentially prevented a major catastrophic complication of vascular tear, which would have required an emergent thoracotomy. CASE REPORT A 37-year-old woman with history of hypertrophic cardiomyopathy had a primary prevention dual-chamber ICD implant in 2001 and underwent right ventricular ICD lead revision in 2009 due to lead fracture. In 2019, she was again found to have right ventricular ICD lead malfunction. TLE was attempted, but no meaningful progression could be made despite using multiple extraction tools. Therefore, TLE was aborted in favor of SE. During elective SE, significant adhesions were noted, and the innominate vein was completely avulsed during removal of the leads, requiring venous reconstruction by the vascular surgery team. After SE and vascular reconstruction, an epicardial ICD system was placed, and the patient had an uneventful postoperative recovery. CONCLUSIONS This case report highlights the limitations of TLE and the importance of recognizing them in a timely manner. In all challenging cases, conversion to elective SE should be considered to avoid potential injuries warranting emergent surgical repair.

Sudden Death in the Young: Information for the Primary Care Provider.

There are multiple conditions that can make children prone to having a sudden cardiac arrest (SCA) or sudden cardiac death (SCD). Efforts have been made by multiple organizations to screen children for cardiac conditions, but the emphasis has been on screening before athletic competition. This article is an update of the previous American Academy of Pediatrics policy statement of 2012 that addresses prevention of SCA and SCD. This update includes a comprehensive review of conditions that should prompt more attention and cardiology evaluation. The role of the primary care provider is of paramount importance in the evaluation of children, particularly as they enter middle school or junior high. There is discussion about whether screening should find any cardiac condition or just those that are associated with SCA and SCD. This update reviews the 4 main screening questions that are recommended, not just for athletes, but for all children. There is also discussion about how to handle post-SCA and SCD situations as well as discussion about genetic testing. It is the goal of this policy statement update to provide the primary care provider more assistance in how to screen for life-threatening conditions, regardless of athletic status.

Anti-VEGF antibody delivered locally reduces bony bar formation following physeal injury in rats.

Physeal injuries can result in the formation of a "bony bar" which can lead to bone growth arrest and deformities in children. Vascular endothelial growth factor (VEGF) has been shown to play a role in bony bar formation, making it a potential target to inhibit bony repair tissue after physeal injury. The goal of this study was to investigate whether the local delivery of anti-VEGF antibody (α-VEGF; 7.5 µg) from alginate:chitosan hydrogels to the tibial physeal injury site in rats prevents bony bar formation. We tested the effects of quick or delayed delivery of α-VEGF using both 90:10 and 50:50 ratio alginate:chitosan hydrogels, respectively. Male and female 6-week-old Sprague-Dawley rats received a tibial physeal injury and the injured site injected with alginate-chitosan hydrogels: (1) 90:10 (Quick Release); (2) 90:10 + α-VEGF (Quick Release + α-VEGF); (3) 50:50 (Slow Release); (4) 50:50 + α-VEGF (Slow Release + α-VEGF); or (5) Untreated. At 2, 4, and 24 weeks postinjury, animals were euthanized and tibiae assessed for bony bar and vessel formation, repair tissue type, and limb lengthening. Our results indicate that Quick Release + α-VEGF reduced bony bar and vessel formation, while also increasing cartilage repair tissue. Further, the quick release of α-VEGF neither affected limb lengthening nor caused deleterious side-effects in the adjacent, uninjured physis. This α-VEGF treatment, which inhibits bony bar formation without interfering with normal bone elongation, could have positive implications for children suffering from physeal injuries.

Painful tumors of the skin: "CALM HOG FLED PEN AND GETS BACK".

Painful tumors of the skin present as dermal or subcutaneous nodules. They can originate from several sources: adipose tissue, cartilage degeneration, deposition of bone or calcium, eccrine glands, fibrous tissue, infiltration of benign (endometrium) or malignant (metastatic neoplasm) tissue, muscle, nerve, or vascular structures. Although pathologic evaluation of the lesion is necessary to determine the diagnosis, it is possible to make a reasonable differential diagnosis based on knowledge of prior tumors that have appeared as tender lesions. Two women with painful skin tumors - either osteoma cutis or an organizing thrombus - are described. Based on our clinical experience and review of the literature, 25 painful skin tumors were identified: chondrodermatitis nodularis helicis, angioendotheliomatosis, leiomyoma, metastases, hidradenoma, osteoma cutis, glomus tumor, fibromyxoma, leiomyosarcoma, eccrine angiomatous hamartoma, Dercum's disease, peizogenic pedal papule, neurilemmoma, angiolipoma, neuroma, dermatofibroma, granular cell tumor, endometriosis, thrombus, scar, blue rubber bleb nevus, angioma, calcinosis cutis, and keloid. An acronym - inspired by Charlotte's Web, a book that many children have read - that can be used as a memory aid for recalling the list of painful skin tumors is introduced: "CALM HOG FLED PEN AND GETS BACK".

Regenerative Medicine Approaches for the Treatment of Pediatric Physeal Injuries.

The physis, or growth plate, is a cartilaginous region at the end of children's long bones that serves as the primary center for longitudinal growth and characterizes the immature skeleton. Musculoskeletal injury, including fracture, infection, malignancy, or iatrogenic damage, has risk of physeal damage. Physeal injuries account for 30% of pediatric fractures and may result in impaired bone growth. Once damaged, cartilage tissue within the physis is often replaced by unwanted bony tissue, forming a "bony bar" that can lead to complications such as complete growth arrest, angular or rotational deformities, and altered joint mechanics. Children with a bony bar occupying <50% of the physis usually undergo bony bar resection and insertion of an interpositional material, such as a fat graft, to prevent recurrence and allow the surrounding uninjured physeal tissue to restore longitudinal bone growth. Clinical success for this procedure is <35% and often the bony bar and associated growth impairments return. Children who are not candidates for bony bar resection due to a physeal bar occupying >50% of their physis undergo corrective osteotomy or bone lengthening procedures. These approaches are complex and have variable success rates. As such, there is a critical need for regenerative approaches to not only prevent initial bony bar formation but also regenerate healthy physeal cartilage following injury. This review describes physeal anatomy, mechanisms of physeal injury, and current treatment options with associated limitations. Furthermore, we provide an overview of the current research using cell-based therapies, growth factors, and biomaterials in the different animal models of injury along with strategic directions for modulating intrinsic injury pathways to inhibit bony bar formation and/or promote physeal tissue formation. Pediatric physeal injuries constitute a unique niche within regenerative medicine for which there is a critical need for research to decrease child morbidity related to this injurious process.

Estradiol-mediated improvements in adipose tissue insulin sensitivity are related to the balance of adipose tissue estrogen receptor α and ß in postmenopausal women.

We recently demonstrated that short-term estradiol (E2) treatment improved insulin-mediated suppression of lipolysis in postmenopausal women, but to a greater extent in those who were late compared to early postmenopausal. In this follow-up study we tested whether subcutaneous adipose tissue (SAT) expression of estrogen receptors (ER) α and ß differs between early and late postmenopausal women. We further tested whether the balance of ERα to ERß in SAT determined the effect of E2 on SAT insulin sensitivity. The present study included 35 women who were ≤6 years past menopause (EPM; n = 16) or ≥10 years past menopause (LPM; n = 19). Fasted SAT samples were taken following 1-week transdermal E2 treatment or placebo (PL) in a random cross-over design. Samples were analyzed for nuclear/cytosolic protein content and mRNA expression using Western blot and qPCR, respectively. While ESR1 increased slightly (~1.4-fold) following E2 treatment in both groups, ERα and ERß protein expression did not differ between groups at baseline or in response to E2. However, the balance of ERα/ERß protein in the SAT nuclear fraction increased 10% in EPM compared to a 25% decrease in LPM women (group x treatment interaction, p<0.05). A greater proportion of ERα/ERß protein in the nuclear fraction of SAT at baseline (placebo day) was associated with greater reduction in SAT insulin resistance (i.e., better suppression of lipolysis, EC50) in response to E2 (r = -0.431, p<0.05). In conclusion, there do not appear to be differences in the proportion of adipose tissue ERα/ERß protein in late, compared to early, postmenopausal women. However, the balance of ERα/ERß may be important for E2-mediated improvement in adipose tissue insulin sensitivity. TRIAL REGISTRATION: Clinical Trials#: NCT01605071.

Age- and menopause-related differences in subcutaneous adipose tissue estrogen receptor mRNA expression.

OBJECTIVES: Changes in estrogen receptor (ER) expression likely underlie differential metabolic effects of estrogen in pre- and postmenopausal women. The aim of the current study was to determine whether ER gene expression in abdominal and femoral subcutaneous adipose tissue (SAT) was associated with age, menopause, or regional adiposity. METHODS: We studied pre- and post-menopausal (n=23 and 22, respectively; age 35-65y) normal weight (mean±SD; BMI 23.7±2.5kg/m2) women with similar total fat mass. Abdominal and femoral SAT ERα (ESR1) and ERß (ESR2) mRNA expression was determined by qPCR. RESULTS: Total fat mass did not differ between pre- and postmenopausal women (22.7±5.3vs. 21.7±5.3kg). Compared to premenopausal women, ESR1 and the ratio of ESR1 to ESR2 were lower (p≤0.05) in postmenopausal abdominal and femoral SAT. ESR1 and ESR1:ESR2 were inversely associated with age in abdominal SAT (r=-0.380 and r=-0.463, respectively; p<0.05) and femoral SAT (r=-0.353 and r=-0.472, respectively; p<0.05). ESR2 was not related to age or menopause. The inverse association between ESR1 and age persisted after adjusting for trunk fat mass, estradiol, or leptin. CONCLUSION: Among healthy pre- and postmenopausal women, increased age was associated with a decreased balance of ERα to ERß in abdominal and femoral subcutaneous adipose tissue.

Neural Crest Cells Contribute an Astrocyte-like Glial Population to the Spleen.

Neural crest cells (NCC) are multi-potent cells of ectodermal origin that colonize diverse organs, including the gastrointestinal tract to form the enteric nervous system (ENS) and hematopoietic organs (bone marrow, thymus) where they participate in lymphocyte trafficking. Recent studies have implicated the spleen as an anatomic site for integration of inflammatory signals from the intestine with efferent neural inputs. We have previously observed alterations in splenic lymphocyte subsets in animals with defective migration of NCC that model Hirschsprung's disease, leading us to hypothesize that there may be a direct cellular contribution of NCC to the spleen. Here, we demonstrate that NCC colonize the spleen during embryogenesis and persist into adulthood. Splenic NCC display markers indicating a glial lineage and are arranged anatomically adjacent to blood vessels, pericytes and nerves, suggesting an astrocyte-like phenotype. Finally, we identify similar neural-crest derived cells in both the avian and non-human primate spleen, showing evolutionary conservation of these cells.