Necrosis Rather Than Apoptosis is The Dominant form of Alveolar Epithelial Cell Death In Lipopolysaccharide-Induced Experimental Acute Respiratory Distress Syndrome Model Alveolar epithelial cell (AEC) death, which is classified as apoptosis or necrosis, plays a critical role in the pathogenesis of acute respiratory distress syndrome (ARDS). In addition to apoptosis, some types of necrosis are known to be molecularly regulated, and both apoptosis and necrosis can be therapeutic targets for diseases. However, the relative contribution of apoptosis and necrosis to AEC death during ARDS has not been elucidated. Here, we evaluated which type of AEC death is dominant and whether regulated necrosis is involved in lipopolysaccharide (LPS)-induced lung injury, an experimental ARDS model. In the bronchoalveolar lavage fluid from the LPS-induced lung injury mice, both the levels of cytokeratin18-M65 antigen (a marker of total epithelial cell death) and -M30 antigen (an epithelial apoptosis marker) were increased. The M30/M65 ratio, which is an indicator of the proportion of apoptosis to total epithelial cell death, was significantly lower than that in healthy controls. Additionally, the number of propidium iodide-positive, membrane-disrupted cells was significantly higher than the number of TUNEL-positive apoptotic cells in the lung sections of lung injury mice. Activated-neutrophils seemed to mediate AEC death. Finally, we demonstrated that necroptosis, a regulated necrosis pathway, is involved in AEC death during LPS-induced lung injury. These results indicate that necrosis including necroptosis, rather than apoptosis, is the dominant type of AEC death in LPS-induced lung injury. Although further studies investigating human ARDS subjects are necessary, targeting necrosis including its regulated forms might represent a more efficient approach to protecting the alveolar epithelial barrier during ARDS. Address reprint requests to Kentaro Tojo, MD, PhD, Department of Anesthesiology and Critical Care Medicine, Yokohama City University, 3-9, Fukuura, Kanazawa-ku, Yokohama-city, Kanagawa 236-0004, Japan. E-mail: ktojo-cib@umin.net Received 13 March, 2019 Revised 14 April, 2019 Accepted 22 July, 2019 Competing interests: The authors declare that they have no competing interests. Disclosure of funding: This work was supported, in part, by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (17K17062, 17K11582). Supplemental digital content is available for this article. Direct URL citation appears in the printed text and is provided in the HTML and PDF versions of this article on the journal's Web site (www.shockjournal.com). © 2019 by the Shock Society

Kinetics of Ringer's Solution In Extracellular Dehydration and Hemorrhage Background.: Ringer's solution might be used treat volume depletion (extracellular dehydration) and hemorrhage, but there is no integrated view of how these fluid balance disorders influence the kinetics of the infused volume. Methods.: Acute dehydration (mean 1.7 L) was induced by repeated doses of furosemide (5 mg) in 10 healthy male volunteers, and 0.5 L and 0.9 L of blood was withdrawn in random order on different occasions in another 10 male volunteers, just before administration of Ringer's acetate solution. Infusions performed in the normovolemic state served as controls. Measurements of blood hemoglobin and urinary excretion were used to create volume kinetic profiles that were analyzed using mixed effects modeling software. Results.: Infusions over 15–30 min showed a marked distribution phase during which the plasma volume transiently increased by 50–75% of the administered volume. Dehydration and hemorrhage accelerated re-distribution but retarded the elimination; the half-life of the infused fluid increased from 36 to 51 min (mean) from 1 L of dehydration and to 95 min from 1 L of hemorrhage. Extravascular accumulation decreased with the dehydration volume and increased with the hemorrhage volume. Simulations show that 60% as much Ringer is needed to replace volume depletion amounting to 1 liter as compared to hemorrhage over a 2-hour period. A continued but slower drip after the initial fluid resuscitation prevents rebound hypovolemia. Conclusions.: Furosemide-induced dehydration and blood withdrawal in normotensive volunteers had modest effects on the Ringer's acetate kinetics. Urinary excretion was inhibited more by hemorrhage than by dehydration. Address reprint requests to Robert G. Hahn, MD, PhD, Professor of Anaesthesia & Intensive Care, Research Unit, Södertälje Hospital, 152 86 Södertälje, Sweden. E-mail: s: r.hahn@telia.com; robert.hahn@sll.se Received 8 May, 2019 Revised 18 July, 2019 Accepted 18 July, 2019 Conflicts of interest and sources of funding: The authors declare that they have no conflicts of interest to disclose. Only departmental funds were used. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Website (www.shockjournal.com). © 2019 by the Shock Society

Quality Control Measures and Validation in Gene Association Studies: Lessons for Acute Illness Acute illness is a complex constellation of responses involving dysregulated inflammatory and immune responses, which are ultimately associated with multiple organ dysfunction. Gene association studies have associated single-nucleotide polymorphisms (SNPs) with clinical and pharmacological outcomes in a variety of disease states, including acute illness. With approximately 4-5 million SNPs in the human genome and recent studies suggesting that a large portion of SNP studies are not reproducible, we suggest that the ultimate clinical utility of SNPs in acute illness depends on validation and quality control measures. To investigate this issue, in December 2018 and January 2019 we searched the literature for peer-reviewed studies reporting data on associations between SNPs and clinical outcomes and between SNPs and pharmaceuticals (i.e. pharmacogenomics) published between January 2011 to February 2019. We review key methodologies and results from a variety of clinical and pharmacological gene association studies, including trauma and sepsis studies, as illustrative examples on current SNP association studies. In this review article, we have found three key points which strengthen the potential accuracy of SNP association studies in acute illness and other diseases: 1) providing evidence of following a protocol quality control method such as the one in Nature Protocols (22) or the OncoArray QC Guidelines (21); 2) enrolling enough patients to have large cohort groups; and 3) validating the SNPs using an independent technique such as a second study using the same SNPs with new patient cohorts. Our survey suggests the need to standardize validation methods and SNP quality control measures in medicine in general, and specifically in the context of complex disease states such as acute illness. Address reprint requests to Yoram Vodovotz, Department of Surgery, University of Pittsburgh, W944 Starzl Biomedical Sciences Tower, 200 Lothrop St. Pittsburgh, PA 15213. E-mail: vodovotzy@upmc.edu Received 15 May, 2019 Revised 08 July, 2019 Accepted 08 July, 2019 Authors’ Contributions: MC: reviewed literature and wrote manuscript; RAN: wrote and reviewed manuscript, AJL: wrote and reviewed manuscript, FE: reviewed manuscript; YX: reviewed manuscript; AMK: reviewed manuscript; TRB: reviewed manuscript; YV: wrote and reviewed manuscript. Sources of Support: This work was supported by U.S. National Institutes of Health grant T32 GM075770. This work was also supported by the Office of the Assistant Secretary of Defense for Health Affairs through the Defense Medical Research and Development Program under awards W81XWH-18-2-0051 and W81XWH-15-PRORP-OCRCA. Opinions, interpretations, conclusions, and recommendations are those of the authors and not necessarily endorsed by the Department of Defense. © 2019 by the Shock Society

Rapid Communication: Cardiac and Skeletal Muscle Myosin Exert Procoagulant Effects Introduction: Trauma-induced coagulopathy (TIC) and the tissue injury-provoked procoagulant profile are prevalent in severely injured patients, but their mechanisms remain unclear. Myosin, exposed by or released from tissue injury, may play a role in promoting thrombin generation and attenuating fibrinolysis. The objective of the study is to examine the effects of cardiac and skeletal muscle myosins on coagulation in whole blood using thrombelastography (TEG). Materials and Methods: Whole blood was collected from healthy adult volunteers (n=8) and native TEGs were performed to evaluate the global coagulation response in the presence of cardiac or skeletal muscle myosin by measuring reaction (R) time (minutes), clot angle (o), and maximum amplitude (MA, mm). TEG measurements were compared using paired t-tests. Results: Cardiac and skeletal muscle myosins decreased R, from 10.8 min to 8.0 min (p<0.0001) and 6.9 min (p=0.0007), respectively. There were no effects observed on clot propagation (angle) or clot strength (MA) with myosin addition. In the presence of tPA, both cardiac and skeletal muscle myosins shortened R from 11.1 min to 8.62 min (p=0.0245) and 7.75 min (p=0.0027), respectively), with no changes on angle or MA. Conclusions: Cardiac and skeletal muscle myosins exhibit procoagulant effects in TEG assays. These whole blood TEG results support the hypothesis that cardiac and skeletal muscle myosins may be either pro-hemostatic or prothrombotic depending on context. Address reprint requests to Ernest E. Moore, Denver Health Medical Center, 777 Bannock St, MC0206, Denver CO 80204; E-mail: ernest.moore@dhha.org Received 6 June, 2019 Revised 25 June, 2019 Accepted 25 July, 2019 Conflict of Interest Statement: The authors Julia R Coleman, Jevgenia Zilberman-Rudenko, Jason M Samuels, Mitchell J Cohen, Christopher C Silliman MD PhD, Anirban Banerjee PhD, Angela Sauaia MD PhD, John H Griffin PhD, Hiroshi Deguchi MD PhD have no conflicts of interest related to this work to report. Ernest E Moore receives consumable support from Haemonetics and Instrumentation Laboratories, as well as holds a patent for the tPA-challenge TEG. Funding: Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health (T32 GM008315 and P50 GM049222). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or other sponsors of the project. © 2019 by the Shock Society

4-Phenylbutyrate Prevents Endoplasmic Reticulum Stress-Mediated Apoptosis Induced By Heatstroke In The Intestines of Mice Objective: Heatstroke can induce serious physiological dysfunction in the intestine. However, the underlying mechanisms of this condition are unknown, and therapeutic strategies are not available. In this study, we explored the role of endoplasmic reticulum (ER) stress signaling in this process and assessed whether pretreating mice with an inhibitor of ER stress could alleviate intestinal damage. Method: A heatstroke model was established in male mice. Mice were pretreated with 4-phenylbutyrate (4-PBA) before exposure to heat stress. Intestinal morphological changes were observed by hematoxylin and eosin (H&E) staining and transmission electron microscopy. The TUNEL assay was used to detect intestinal apoptosis. The expression of the ER stress-related proteins and apoptosis-related proteins was investigated by the Western blot assay. Result: Compared with control group, mice with heatstroke exhibited evidence of intestinal injury and epithelial apoptosis, accompanied by significantly increased expression of ER stress-related proteins in the intestines. The intestinal injury score and level of intestinal epithelial apoptosis were significantly reduced after administration of 4-PBA. Furthermore, the levels of the intestinal ER stress-related proteins GRP78, PERK, p-eIF2α, ATF4 and CHOP were decreased after 4-PBA treatment. Conclusion: Our results indicate that the ER stress-mediated apoptosis pathway is activated during heat stress-nduced intestinal injury. 4-PBA can inhibit heatstroke-induced intestinal ER stress and attenuate intestinal injury. We provide evidence that the beneficial effect of 4-PBA is closely related to the inhibition of ER stress-mediated apoptosis. These findings suggest that ER stress may be a novel therapeutic target in patients with heatstroke. Address reprint requests to Lei Su, Department of Intensive Care Medicine, General Hospital of Southern Theatre Command of PLA, Guangzhou, 510010, China. Key Laboratory of Hot Zone Trauma Care and Tissue Repair of PLA, General Hospital of Southern Theatre Command of PLA. 111 Liuhua Road, Guangzhou city, Guangdong, 510010, China; E-mail: slei_icu@163.com Received 24 April, 2019 Revised 9 May, 2019 Accepted 16 July, 2019 This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0 © 2019 by the Shock Society

Stem Cell Therapy and Hydrogen Sulfide: Conventional or Nonconventional Mechanisms of Action? Purpose: Hydrogen sulfide (H2S) has many beneficial biological properties, including the ability to promote vasodilation. It has been shown to be released from stem cells and increased by hypoxia. Therefore, H2S may be an important paracrine factor in stem cell mediated intestinal protection. We hypothesized that hydrogen sulfide created through conventional pathways would be a critical component of stem cell mediated intestinal protection following ischemic injury. Methods: Human bone marrow derived mesenchymal stem cells (BMSCs) were transfected with negative control siRNA (Scramble), or with siRNA to CBS, MPST, or CTH. Knockdown was confirmed with PCR and hydrogen sulfide gas assessed with AzMC fluorophore. Eight week old male mice then underwent intestinal ischemia for 60 mins, after which time, perfusion was restored. BMSCs from each of the above groups were then placed into the mouse abdominal cavity prior to final closure. After 24 hours, mice were reanesthetized and mesenteric perfusion was assessed by Laser Doppler Imaging (LDI). Animals were then sacrificed and intestines excised, placed in formalin, paraffin embedded, and stained with H & E. Intestines were then scored with a common mucosal injury grading scale. Results: PCR confirmed knockdown of conventional H2S producing enzymes (CBS, MPST, CTH). Hydrogen sulfide gas was decreased in MPST and CTH transfected cells in normoxic conditions but was not decreased compared to scramble in any of the transfected groups in hypoxic conditions. BMSCs promoted increased mesenteric perfusion at 24 hours post-ischemia compared to vehicle. Transfected stem cells provided equivalent protection. Histologic injury was improved with BMSCs compared to vehicle. CBS, MPST, and CTH knockdown cell lines did not have any worse histological injury compared to Scramble. Conclusion: Knocking down conventional H2S producing enzymes only impacted gas production in normoxic conditions. When cells were transfected in hypoxic conditions, as would be expected in the ischemic intestines, hydrogen sulfide gas was not depressed. These data, along with unchanged perfusion and histological injury parameters with conventional enzyme knockdown would indicate that alternative H2S production pathways may be initiated during hypoxic and/or ischemic events. Address reprint requests to Troy A. Markel, MD, Assistant Professor of Surgery, Indiana University School of Medicine, Riley Hospital for Children at IU Health, 705 Riley Hospital Dr., RI 2500, Indianapolis, IN 46202. E-mail: tmarkel@iupui.edu Received 17 June, 2019 Revised 8 July, 2019 Accepted 17 July, 2019 ARJ performed animal I/R experiments, protein isolation, histological grading and drafted the manuscript, NAD performed histological grading and statistical analysis, KRO performed hydrogen sulfide assays, TAM contributed critical ideas, assistance and manuscript advice. All authors provided critical revisions to the manuscript and assisted with its final preparation. Conflicts of Interest: TAM receives consultation fees from Scioto Biosciences and Onsite, LLC Funding: National Institutes of Health, NIDDK K08DK113226 (TAM) The Koret Foundation (TAM) Indiana University Biomedical Research Grant (KRO) The Department of Surgery at the Indiana University School of Medicine (TAM) © 2019 by the Shock Society

Direct Rivaroxaban-Induced Factor Xa Inhibition Proves to be Cardioprotective in Rats Background: Acute myocardial infarction is a leading cause of death worldwide. Though highly beneficial, reperfusion of myocardium is associated with reperfusion injury. While indirect inhibition of Factor Xa has been shown to attenuate myocardial ischemia-reperfusion (I/R) injury, the underlying mechanism remains unclear. Our study sought to evaluate the effect of rivaroxaban (RIV), a direct inhibitor of Factor Xa, on myocardial I/R injury and determine its cellular targets. Experimental Approach: We used a rat model of 40-minutes coronary ligation followed by reperfusion. RIV (3 mg/Kg) was given per os 1 hour before reperfusion. Infarct size and myocardial proteic expression of survival pathways were assessed at 120 and 30 minutes of reperfusion, respectively. Plasmatic levels of P-selectin and von Willebrand factor were measured at 60 minutes of reperfusion. Cellular RIV effects were assessed using hypoxia-reoxygenation (H/R) models on human umbilical vein endothelial cells and on rat cardiomyoblasts (H9c2 cell line). Key Results: RIV decreased infarct size by 21% (42.9% vs. 54.2% in RIV-treated rats and controls respectively, p < 0.05) at blood concentrations similar to human therapeutic (387.7 ± 152.3 ng/mL) levels. RIV had no effect on H/R-induced modulation of endothelial phenotype, nor did it alter myocardial activation of RISK and SAFE pathways at 30 min after reperfusion. However, RIV exerted a cytoprotective effect on H9c2 cells submitted to H/R. Conclusion: RIV decreased myocardial I/R injury in rats at concentrations similar to human therapeutic ones. This protection was not associated with endothelial phenotype modulation but rather with potential direct cytoprotection on cardiomyocytes. Address reprint requests to Laurent Macchi, MD, PhD, CHU Poitiers, Service d’hématologie biologique, 2, rue de la Milétrie, CS 90577, F-86021 POITIERS cedex. E-mail: laurent.macchi@chu-poitiers.fr Received 18 March, 2019 Revised 8 April, 2019 Accepted 10 July, 2019 This research was supported by a research grant from Bayer AG, Germany. Supplemental digital content is available for this article. Direct URL citation appears in the printed text and is provided in the HTML and PDF versions of this article on the journal's Web site (www.shockjournal.com). © 2019 by the Shock Society

Demographics, Treatments, and Outcomes of Acute Respiratory Distress Syndrome: The Focused Outcomes Research in Emergency Care in Acute Respiratory Distress Syndrome, Sepsis, and Trauma (Forecast) Study Purpose: Acute respiratory distress syndrome (ARDS) remains a major cause of death. Epidemiology should be continually examined to refine therapeutic strategies for ARDS. We aimed to elucidate demographics, treatments, and outcomes of ARDS in Japan. Methods: This is a prospective cohort study for ARDS. We included adult patients admitted to intensive care units through emergency and critical care departments who satisfied the American–European Consensus Conference (AECC) acute lung injury (ALI) criteria. In addition, the fulfillment of the Berlin definition was assessed. Logistic regression analyses were used to examine the association of independent variables with outcomes Results: Our study included 166 patients with AECC ALI from 34 hospitals in Japan; among them, 157 (94.6%) fulfilled the Berlin definition. The proportion of patients with PaO2/FIO2 ≤100, patients under invasive positive pressure ventilation (IPPV), and in-hospital mortality was 39.2%, 92.2%, and 38.0% for patients with AECC ALI and 38.9%, 96.8%, and 37.6% for patients with Berlin ARDS, respectively. The area of lung infiltration was independently associated with outcomes of ARDS. Low-mid tidal volume ventilation was performed in 75% of patients under IPPV. Glucocorticoid use was observed in 54% patients, and it was positively associated with mortality. Conclusions: Our study included a greater percentage of patients with ARDS with high severity and found that the overall mortality was 38%. The management of ARDS in Japan was characterized by high the utilization rate of glucocorticoids, which was positively associated with mortality. Address reprint requests to Seitaro Fujishima, MD, Center for General Medicine Education, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail: fujishim@keio.jp Received 13 April, 2019 Revised 11 July, 2019 Accepted 11 July, 2019 Funding source: This study was funded by the Japanese Association for Acute Medicine. Conflicts of interest: Dr. Fujishima reports grants and personal fees from Asahi Kasei Japan Co., personal fees from Thermofisher Diagnostics Co., grants from Chugai Pharmaceuticals Co. Ltd., grants from Daiichi-Sankyo Co., Ltd., grants from Pfizer, Inc., grants from Shionogi & Co., Ltd., outside the submitted work; Dr. Gando reports personal fees from Asahi Kasei America Cooperation, personal fees from Asahi Kasei Japan Cooperation, outside the submitted work; Dr. Abe reports grants from Grant-in-Aid for Challenging Exploratory Research JP 16K15388, personal fees from Asahi Kasei Pharma Co., personal fees from Japan Blood Products Organization, personal fees from Janssen Pharmaceutical K.K., outside the submitted work; Dr. Shiraishi reports personal fees from Nobelpharma Co., outside the submitted work; Dr. Okamoto reports personal fees from Asahi Kasei Japan Corporation, outside the submitted work; Dr. Sakamoto reports personal fees from Japan Blood Product Corporation, personal fees from Asahi Kasei Japan Corporation, outside the submitted work; Dr. Umemura reports personal fees from Asahi Kasei Pharma Corporation, personal fees from Japan blood products organization, outside the submitted work. The remaining authors have disclosed that they do not have any conflicts of interest. Supplemental digital content is available for this article. Direct URL citation appears in the printed text and is provided in the HTML and PDF versions of this article on the journal's Web site (www.shockjournal.com). © 2019 by the Shock Society

Military Supplement: Reassessment of The Need for An Oxygen Carrier For The Treatment of Traumatic Hemorrhage When Blood is Not An Option Approximately 10 years ago, the development of hemoglobin-based oxygen carriers (HBOC) was largely stalled after two large phase 3 clinical trials failed to achieve licensure primarily because the safety profile was viewed as unsatisfactory when HBOCs were compared to red cells. Concerns were also raised that HBOCs, as a class, had inherent toxicities. Since then, clinical experience with HBOC in expanded access programs and under licensure in South Africa have demonstrated that HBOCs can be used safely and effectively. In recent years, clinical studies have demonstrated that pre-hospital blood transfusion improves survival in severely injured patients with hemorrhage, especially when transport times are longer than 20 to 30 minutes. Yet, logistical constraints still limit use of blood products in the prehospital setting. As the urgent need for oxygen carrying capacity for trauma patients for whom red cells are not available is becoming much more apparent, it is imperative that we re-examine the possibility of using HBOCs when RBC transfusion is not an option. Address reprint requests to Anthony E. Pusateri, PhD, Director of Research and Chief Science Officer, U.S. Army Institute of Surgical Research, Ft. Sam Houston, Texas 78234. E-mail: anthony.e.pusateri.civ@mail.mil Received 24 May, 2019 Revised 12 June, 2019 Accepted 12 July, 2019 Conflicts of interest: A.E. Pusateri: none; E. Glassberg: none; R. B. Weiskopf: consults for HbO2 Therapeutics Disclaimers: A.E. Pusateri: The opinions or assertions of the author are his own and do not represent the official position of the US Army or the US Department of Defense. E. Glassberg: None; R.B. Weiskopf: None © 2019 by the Shock Society

Lactic Acidosis and The Role of Sodium Bicarbonate: A Narrative Opinion Lactic acidosis occurs commonly and can be a marker of significant physiologic derangements. However what an elevated lactate level and acidemia connotes and what should be done about it is subject to inconsistent interpretations. This review examines the varied etiologies of lactic acidosis, the physiologic consequences, and the known effects of its treatment with sodium bicarbonate. Lactic acidosis is often assumed to be a marker of hypoperfusion, but it can also result from medications, organ dysfunction, and sepsis even in the absence of malperfusion. Acidemia causes deleterious effects in almost every organ system but it can also have positive effects, increasing localized blood flow and oxygen delivery, as well as providing protection against hypoxic cellular injury. The use of sodium bicarbonate to correct severe acidemia may be tempting to clinicians, but previous studies have failed to show improved patient outcomes following bicarbonate administration. Bicarbonate use is known to decrease vasomotor tone, decrease myocardial contractility, and induce intracellular acidosis. This suggests that mild to moderate acidemia does not require correction. Most recently, a randomized control trial found a survival benefit in a subgroup of critically ill patients with serum pH levels <7.2 with concomitant acute kidney injury. There is no known benefit of correcting serum pH levels ≥ 7.2, and sparse evidence supports bicarbonate use <7.2. If administered, bicarbonate is best given as a slow IV infusion in the setting of adequate ventilation and calcium replacement to mitigate its untoward effects. Address reprint requests to Mona R. Rudnick, 234 Los Gatos Blvd., Los Gatos, CA 95030. E-mail: Mlr47@georgetown.edu Received 15 April, 2019 Revised 6 May, 2019 Accepted 10 July, 2019 Conflicts of Interest: The authors have no relevant conflicts of interest to report. © 2019 by the Shock Society