Mitchell Dyer

Toll-like Receptor 4 Is Expressed on Intestinal Stem Cells and Regulates Their Proliferation and Apoptosis via the p53 Up-regulated Modulator of Apoptosis

Background: Factors that regulate intestinal stem cell (ISC) proliferation and apoptosis are unknown. Results: Toll-like receptor 4 (TLR4) is expressed on ISCs and regulates their proliferation and apoptosis, which is critical in the pathogenesis of necrotizing enterocolitis (NEC). Conclusion: TLR4 regulates ISC proliferation and apoptosis. Significance: This is the first study showing that ISC regulation by microbial receptors contributes to NEC pathogenesis. Factors regulating the proliferation and apoptosis of intestinal stem cells (ISCs) remain incompletely understood. Because ISCs exist among microbial ligands, immune receptors such as toll-like receptor 4 (TLR4) could play a role. We now hypothesize that ISCs express TLR4 and that the activation of TLR4 directly on the intestinal stem cells regulates their ability to proliferate or to undergo apoptosis. Using flow cytometry and fluorescent in situ hybridization for the intestinal stem cell marker Lgr5, we demonstrate that TLR4 is expressed on the Lgr5-positive intestinal stem cells. TLR4 activation reduced proliferation and increased apoptosis in ISCs both in vivo and in ISC organoids, a finding not observed in mice lacking TLR4 in the Lgr5-positive ISCs, confirming the in vivo significance of this effect. To define molecular mechanisms involved, TLR4 inhibited ISC proliferation and increased apoptosis via the p53-up-regulated modulator of apoptosis (PUMA), as TLR4 did not affect crypt proliferation or apoptosis in organoids or mice lacking PUMA. In vivo effects of TLR4 on ISCs required TIR-domain-containing adapter-inducing interferon-β (TRIF) but were independent of myeloid-differentiation primary response-gene 88 (MYD88) and TNFα. Physiological relevance was suggested, as TLR4 activation in necrotizing enterocolitis led to reduced proliferation and increased apoptosis of the intestinal crypts in a manner that could be reversed by inhibition of PUMA, both globally or restricted to the intestinal epithelium. These findings illustrate that TLR4 is expressed on ISCs where it regulates their proliferation and apoptosis through activation of PUMA and that TLR4 regulation of ISCs contributes to the pathogenesis of necrotizing enterocolitis.

A Critical Role for TLR4 Induction of Autophagy in the Regulation of Enterocyte Migration and the Pathogenesis of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) develops in response to elevated TLR4 signaling in the newborn intestinal epithelium and is characterized by TLR4-mediated inhibition of enterocyte migration and reduced mucosal healing. The downstream processes by which TLR4 impairs mucosal healing remain incompletely understood. In other systems, TLR4 induces autophagy, an adaptive response to cellular stress. We now hypothesize that TLR4 induces autophagy in enterocytes and that TLR4-induced autophagy plays a critical role in NEC development. Using mice selectively lacking TLR4 in enterocytes (TLR4ΔIEC) and in TLR4-deficient cultured enterocytes, we now show that TLR4 activation induces autophagy in enterocytes. Immature mouse and human intestine showed increased expression of autophagy genes compared with full-term controls, and NEC development in both mouse and human was associated with increased enterocyte autophagy. Importantly, using mice in which we selectively deleted the autophagy gene ATG7 from the intestinal epithelium (ATG7ΔIEC), the induction of autophagy was determined to be required for and not merely a consequence of NEC, because ATG7ΔIEC mice were protected from NEC development. In defining the mechanisms involved, TLR4-induced autophagy led to impaired enterocyte migration both in vitro and in vivo, which in cultured enterocytes required the induction of RhoA-mediated stress fibers. These findings depart from current dogma in the field by identifying a unique effect of TLR4-induced autophagy within the intestinal epithelium in the pathogenesis of NEC and identify that the negative consequences of autophagy on enterocyte migration play an essential role in its development.

Simultaneous extracorporeal membrane oxygenation and therapeutic plasma exchange procedures are tolerable in both pediatric and adult patients

Extracorporeal membrane oxygenation (ECMO) has been used in patients with pulmonary and/or cardiac disease. In rare circumstances, some patients may have to undergo simultaneous therapeutic plasma exchange (TPE). We sought to characterize simultaneous ECMO and TPE procedures at our institution.

Deep vein thrombosis in mice is regulated by platelet HMGB1 through release of neutrophil-extracellular traps and DNA

Venous thromboembolic (VTE) disease, consisting of deep venous thrombosis (DVT) and pulmonary embolism (PE) is a leading cause of morbidity and mortality. Current prophylactic measures are insufficient to prevent all occurrence in part due to an incomplete understanding of the underlying pathophysiology. Mounting evidence describes interplay between activation of the innate immune system and thrombus development. Recent work has demonstrated that platelet release of HMGB1 leads to increased microvascular complications following injury. Additionally, platelet HMGB1 was found to enhance DVT and increase the formation of neutrophil extracellular traps (NETs), although the role of HMGB1 induced NET release in thrombosis remains unexplored. Utilizing a transgenic mouse lacking HMGB1 specifically from platelets and megakaryocytes we now demonstrate the specific role of platelet-derived HMGB1 in acute and subacute/chronic venous thrombosis. Platelets account for the majority of circulating HMGB1 and HMGB1 deposition within the developing clot. The pro-thrombotic effect of platelet-derived HMGB1 is mediated through enhanced neutrophil recruitment, NET formation and specifically release of extracellular DNA during NET formation. Taken together, these data suggest that platelet HMGB1 mediated NET release is a primary regulator of DVT formation in mice.

In vitro characterization of SynthoPlate(™) (synthetic platelet) technology and its in vivo evaluation in severely thrombocytopenic mice.

Essentials Platelet transfusion suffers from availability, portability, contamination, and short shelf‐life. SynthoPlate™ (synthetic platelet technology) can resolve platelet transfusion limitations. SynthoPlate™ does not activate resting platelets or stimulate coagulation systemically. SynthoPlate™ significantly improves hemostasis in thrombocytopenic mice dose‐dependently.

Aged Human Stored Red Blood Cell Supernatant Inhibits Macrophage Phagocytosis in an HMGB1 Dependent Manner After Trauma in a Murine Model.

ABSTRACT Red blood cell transfusions in the setting of trauma are a double-edged sword, as it is a necessary component for life-sustaining treatment in massive hemorrhagic shock, but also associated with increased risk for nosocomial infections and immune suppression. The mechanisms surrounding this immune suppression are unclear. Using supernatant from human packed red blood cell (RBC), we demonstrate that clearance of Escherichia coli by macrophages is inhibited both in vitro and in vivo using a murine model of trauma and hemorrhagic shock. We further explore the mechanism of this inhibition by demonstrating that human-stored RBCs contain soluble high-mobility group box 1 protein (HMGB1) that increases throughout storage. HMGB1 derived from the supernatant of human-stored RBCs was shown to inhibit bacterial clearance, as neutralizing antibodies to HMGB1 restored the ability of macrophages to clear bacteria. These findings demonstrate that extracellular HMGB1 within stored RBCs could be one factor leading to immune suppression following transfusion in the trauma setting.

ADAMTS13: origins, applications, and prospects: ADAMTS13: APPLICATIONS AND PROSPECTS

ADAMTS13 is an enzyme that acts by cleaving prothrombotic von Willebrand factor (VWF) multimers from the vasculature in a highly regulated manner. In pathologic states such as thrombotic thrombocytopenic purpura (TTP) and other thrombotic microangiopathies (TMAs), VWF can bind to the endothelium and form large multimers. As the anchored VWF chains grow, they provide a greater surface area to bind circulating platelets (PLTs), generating unique thrombi that characterize TTP. This results in microvasculature thrombosis, obstruction of blood flow, and ultimately end‐organ damage. Initial presentations of TTP usually occur in an acute manner, typically developing due to an autoimmune response toward, or less commonly a congenital deficiency of, ADAMTS13. Triggers for TMAs that can be associated with ADAMTS13 deficiency, including TTP, have been linked to events that place a burden on hemostatic regulation, such as major trauma and pregnancy. The treatment plan for cases of suspected TTP consists of emergent therapeutic plasma exchange that is continued on a daily basis until normalization of PLT counts. However, a subset of these patients does not respond favorably to standard therapies. These patients necessitate a better understanding of their diseases for the advancement of future therapeutic options. Given ADAMTS13’s key role in the cleavage of VWF and the prevention of PLT‐rich thrombi within the microvasculature, future treatments may include anti‐VWF therapeutics, recombinant ADAMTS13 infusions, and ADAMTS13 expression via gene therapy.

Intravenous synthetic platelet (SynthoPlate) nanoconstructs reduce bleeding and improve ‘golden hour’ survival in a porcine model of traumatic arterial hemorrhage

Traumatic non-compressible hemorrhage is a leading cause of civilian and military mortality and its treatment requires massive transfusion of blood components, especially platelets. However, in austere civilian and battlefield locations, access to platelets is highly challenging due to limited supply and portability, high risk of bacterial contamination and short shelf-life. To resolve this, we have developed an I.V.-administrable ‘synthetic platelet’ nanoconstruct (SynthoPlate), that can mimic and amplify body’s natural hemostatic mechanisms specifically at the bleeding site while maintaining systemic safety. Previously we have reported the detailed biochemical and hemostatic characterization of SynthoPlate in a non-trauma tail-bleeding model in mice. Building on this, here we sought to evaluate the hemostatic ability of SynthoPlate in emergency administration within the ‘golden hour’ following traumatic hemorrhagic injury in the femoral artery, in a pig model. We first characterized the storage stability and post-sterilization biofunctionality of SynthoPlate in vitro. The nanoconstructs were then I.V.-administered to pigs and their systemic safety and biodistribution were characterized. Subsequently we demonstrated that, following femoral artery injury, bolus administration of SynthoPlate could reduce blood loss, stabilize blood pressure and significantly improve survival. Our results indicate substantial promise of SynthoPlate as a viable platelet surrogate for emergency management of traumatic bleeding.

Defining the Lethal Triad

Worldwide, trauma leads to millions of deaths and severe injuries each year. Analysis of the leading causes of death in trauma patients reveals that hemorrhage is one of the most common and preventable etiologies in the acute period [1]. For those that survive the initial insult, the cause of both morbidity and late mortality after trauma is, in part, excessive inflammation and a vicious cascade of coagulation abnormalities. Thus, traumatic hemorrhage can be broadly categorized into two groups: (1) early surgical bleeding and (2) coagulopathy. Surgical bleeding is secondary to the injury (e.g., splenic laceration, femur fracture), and treatment often involves mechanical control through surgical or interventional procedures. Coagulopathy following trauma presents in two distinct forms. It is now widely recognized that severe injury is characterized by a unique, endogenous coagulopathy, referred to as the acute traumatic coagulopathy (ATC) that may be present on admission in as many as 30% of injured patients [2]. This devastating condition presents a major obstacle in the care of trauma patients and is an evolving area of active research. The second form of coagulopathy following trauma is a consequence of iatrogenic factors and resuscitation. Previous resuscitation strategies involving large volumes of crystalloid and/or packed red blood cells in isolation (without blood component therapy) led to a dilutional coagulopathy. Although this remains a clinically important challenge, modern resuscitation strategies that limit crystalloid and focus on 1:1:1 ratio-based transfusion have limited this component substantially [3, 4]. In this chapter, we will focus on the previously named “lethal triad” which links coagulopathy with hypothermia and acidosis as major contributors to the ongoing hemorrhage despite control of surgical bleeding.

Uncontrolled Hemorrhagic Shock Modeled via Liver Laceration in Mice with Real Time Hemodynamic Monitoring

Uncontrolled hemorrhage is an important cause of preventable deaths among trauma patients. We have developed a murine model of uncontrolled hemorrhage via a liver laceration that results in consistent blood loss, hemodynamic alterations, and survival. Mice undergo a standardized resection of the left-middle lobe of the liver. They are allowed to bleed without mechanical intervention. Hemostatic agents can be administered as pre-treatment or rescue therapy depending on the interest of the investigator. During the time of hemorrhage, real-time hemodynamic monitoring via a left femoral arterial line is performed. Mice are then sacrificed, blood loss is quantified, blood is collected for further analysis, and organs are harvested for analysis of injury. Experimental design is described to allow for simultaneous testing of multiple animals. Liver hemorrhage as a model of uncontrolled hemorrhage exists in the literature, primarily in rat and porcine models. Some of these models utilize hemodynamic monitoring or quantify blood loss but lack consistency. The present model incorporates quantification of blood loss, real-time hemodynamic monitoring in a murine model that offers the advantage of using transgenic lines and a high-throughput mechanism to further investigate the pathophysiologic mechanisms in uncontrolled hemorrhage.