Daily Changes in The Expression of Clock Genes in Sepsis and Their Relation with Sepsis Outcome and Urinary Excretion of 6-Sulfatoximelatonin Background: Whereas the circadian system controls the daily production of melatonin and the daily activity of the immune system, increasing evidences support the association between circadian misalignment with the alterations in the immune response and melatonin rhythm during sepsis. The aim of this study was to analyze the daily changes in clock genes expression and the urinary excretion of 6-SM (6-sulfatoxymelatonin, the major melatonin metabolite), and their connection with the innate immune activity, oxidative status in blood, and clinical outcome during sepsis. Methods: Healthy volunteers, non-septic ICU patients, and septic ICU patients, were evaluated. The expression of bmal1, per2, clock, and cry1 genes was determined by PCR in blood; 6-SM was assessed in urine by ELISA; plasma cytokines IL-1β, IL-6, IL-8, TNFα, and IL-10 were determined by a multiplex array method, and lipid peroxidation (LPO) and protein oxidation (AOPP) by spectrophotometry. Hematological and biochemical data, and clinical scores of the patients, were also recorded. Results: Clock gene rhythm was maintained in non-septic patients but blunted in septic ones, whereas the innate immune and the oxidative stress responses were significantly higher in the latter. 6-SM excretion was also more elevated in septic than in non-septic patients, and it correlated with the degree of the immune response and oxidative status. 6-SM also correlated with SOFA and procalcitonin in the patients. Proinflammatory cytokines, LPO, and AOPP were normalized in the patients once recovered from sepsis. Conclusion: Our data suggest a relationship between clock genes rhythm disruption, the immune response and the oxidative status, with 6-SM acting as a compensatory response. ICU conditions are not a main clock disrupter because the significant differences found in the responses of septic versus non-septic patients under the same ICU environment. Address reprint requests to Carlos Acuña-Fernández, MD, Unidad of Anestesiology y Reanimación, Hospital Universitario de Canarias, Carreterra de Ofra, s/n, 38320 San Cristóbal de La Laguna, Santa Cruz de Tenerife, Spain. E-mail: cacufer89@gmail.com; Darío Acuña-Castroviejo, MD, PhD, Centro de Investigación Biomédica, Parque Tecnológico de Ciencias de la Salud, Universidad de Granada, Avenida del Conocimiento s/n; 18016 Granada, Spain. E-mail: dacuna@ugr.es Received 9 July, 2019 Revised 18 July, 2019 Accepted 26 July, 2019 Funding statement: This study has been funded by Instituto de Salud Carlos III through the projects PI13-00981, PI16–00519, and CB/10/00238 (Co-funded by European Regional Development Fund/European Social Fund) “Investing in your future”); the Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía, Spain (CTS-101), and from Pfizer S.L.U, Madrid, Spain. Conflicts of Interest: The authors declare no competing interests. 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

SIRT1 Mediates Septic Cardiomyopathy in a Murine Model of Polymicrobial Sepsis Background: Cardiac dysfunction, a common complication from severe sepsis, is associated with increased morbidity and mortality. However, the molecular mechanisms of septic cardiac dysfunction are poorly understood. SIRT1, a member of the sirtuin family of NAD+-dependent protein deacetylases, is an important immunometabolic regulator of sepsis, and sustained SIRT1 elevation is associated with worse outcomes and organ dysfunction in severe sepsis. Herein, we explore the role of SIRT1 in septic cardiac dysfunction using a murine model of sepsis. Methods: An in vitro model of inflammation in isolated H9c2 cardiomyocytes was used to confirm SIRT1 response to stimulation with lipopolysaccharide (LPS), followed by a murine model of cecal ligation and puncture (CLP) to investigate the molecular and echocardiographic response to sepsis. A selective SIRT1 inhibitor, EX-527, was employed to test for SIRT1 participation in septic cardiac dysfunction. Results: SIRT1 mRNA and protein levels in cultured H9c2 cardiomyocytes were significantly elevated at later time points after stimulation with LPS. Similarly, cardiac tissue harvested from C57BL/6 mice 36 hours after CLP demonstrated increased expression of SIRT1 mRNA and protein compared to sham controls. Administration of EX-527 18 hours after CLP reduced SIRT1 protein expression in cardiac tissue at 36 hours. Moreover, treatment with EX-527 improved cardiac performance with increased global longitudinal strain and longitudinal strain rate. Conculsions: Our findings reveal that SIRT1 expression increases in isolated cardiomyocytes and cardiac tissue after sepsis-inflammation. Moreover, rebalancing SIRT1 excess in late sepsis improves cardiac performance suggesting that SIRT1 may serve as a therapeutic target for septic cardiomyopathy. Address reprint requests to Lane M. Smith, MD, PhD, Department of Emergency Medicine, Meads Hall 2nd Floor, Medical Center Blvd., Winston Salem, NC 27157. E-mail: lmsmith@wakehealth.edu. Received 22 May, 2019 Revised 11 June, 2019 Accepted 26 July, 2019 © 2019 by the Shock Society

Letter to the Editor Regarding the Manuscript of Kasotakis et al “Histone Deacetylase 7 Inhibition in a Murine Model of Gram-negative Pneumonia-induced Acute Lung Injury” Shock. 2019 May 2. doi: 10.1097/SHK.0000000000001372. [Epub ahead of print] No abstract available

Histone Deacetylase 7 In Murine Gram-Negative Acute Lung Injury No abstract available

The Greater Omentum – A Vibrant and Enigmatic Immunologic Organ Involved in Injury and Infection Resolution Once thought of as an inert fatty tissue present only to provide insulation for the peritoneal cavity, the omentum is currently recognized as a vibrant immunologic organ with a complex structure uniquely suited for defense against pathogens and injury. The omentum is a source of resident inflammatory and stem cells available to participate in the local control of infection, wound healing, and tissue regeneration. It is intimately connected with the systemic vasculature and communicates with the central nervous system and the hypothalamic pituitary adrenal axis. Furthermore, the omentum has the ability to transit the peritoneal cavity and sequester areas of inflammation and injury. It contains functional, immunologic units commonly referred to as “milky spots” that contribute to the organ's immune response. These milky spots are complex nodules consisting of macrophages and interspersed lymphocytes, which are gateways for the infiltration of inflammatory cells into the peritoneal cavity in response to infection and injury. The omentum contains far greater complexity than is currently conceptualized in clinical practice and investigations directed at unlocking its beneficial potential may reveal new mechanisms underlying its vital functions and the secondary impact of omentectomy for the staging and treatment of a variety of diseases. Address reprint requests to Antonio De Maio, PhD, University of California San Diego, School of Medicine, 9500 Gilman Drive, #0739, La Jolla, CA 92093-0739. E-mail: ademaio@ucsd.edu Received 18 April, 2019 Revised 7 May, 2019 Accepted 26 July, 2019 Support: Support was provided by the National Institutes of Health (NIH) Grant R01 GM114473-01. The authors declare that they have no competing interests. © 2019 by the Shock Society

Pharmacokinetics of Tranexamic Acid Given as an Intramuscular Injection Compared to Intravenous Infusion in a Swine Model of Ongoing Hemorrhage Introduction: Tranexamic acid (TXA) improves survival in traumatic hemorrhage, but difficulty obtaining intravenous (IV) access may limit its use in austere environments, given its incompatibility with blood products. The bioavailability of intramuscular (IM) TXA in a shock state is unknown. We hypothesized that IM and IV administration have similar pharmacokinetics and ability to reverse in vitro hyperfibrinolysis in a swine controlled-hemorrhage model. Methods: Twelve Yorkshire cross swine were anesthetized, instrumented, and subjected to a 35% controlled hemorrhage, followed by resuscitation. During hemorrhage, they were randomized to receive a 1 g IV TXA infusion over 10 minutes, 1 g IM TXA in two 5 mL injections, or 10 mL normal saline IM injection as a placebo group to assess model adequacy. Serum TXA concentrations were determined using liquid chromatography-mass spectrometry, and plasma samples supplemented with tissue plasminogen activator (tPA) were analyzed by rotational thromboelastometry (ROTEM). Results: All animals achieved class III shock. There was no difference in the concentration-time areas under the curve (AUC) between TXA given by either route. The absolute bioavailability of IM TXA was 97%. IV TXA resulted in a higher peak serum concentration during the infusion, with no subsequent differences. Both IV and IM TXA administration caused complete reversal of in vitro tPA-induced hyperfibrinolysis. Conclusion: The pharmacokinetics of IM TXA were similar to IV TXA during hemorrhagic shock in our swine model. IV administration resulted in a higher serum concentration only during the infusion, but all levels were able to successfully correct in vitro hyperfibrinolysis. There was no difference in total body exposure to equal doses of TXA between the two routes of administration. IM TXA may prove beneficial in scenarios where difficulty establishing dedicated IV access could otherwise limit or delay its use. Address reprint requests to Marguerite W. Spruce, MD, General Surgery, 3225 58th St., Sacramento, CA 95820. E-mail: mfwinkler@ucdavis.edu Received 18 April, 2019 Revised 25 July, 2019 Accepted 25 July, 2019 Sources of Support: This work was partially funded by a grant from the Surgeon General of the Air Force Office of Air Force General Medical Education. Disclaimer: The animals involved in this study were procured, maintained, and used in accordance with the Laboratory Animal Welfare Act of 1966, as amended, and NIH 80–23, Guide for the Care and Use of Laboratory Animals, National Research Council. The views expressed in this material are those of the authors and do not reflect the official policy or position of the U.S. Government, the Department of Defense, the Department of the Air Force, or the University of California Davis. The work reported herein was performed under United States Air Force Surgeon General approved Clinical Investigation number FDG20180023A. This work was partially funded by a grant from the Surgeon General of the Air Force Office of Air Force General Medical Education. Intramuscular injection of Tranexamic acid is still investigational for the off-label use of severe bleeding. The authors report no 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

Antithrombin III Contributes to the Protective Effects of Fresh Frozen Plasma Following Hemorrhagic Shock by Preventing Syndecan-1 Shedding and Endothelial Barrier Disruption Background: Endothelial dysfunction during hemorrhagic shock (HS), is associated with loss of cell-associated syndecan-1 (Sdc1) and hyperpermeability. Fresh frozen plasma (FFP) preserves Sdc1 and reduces permeability following HS, although the key mediators remain unknown. Antithrombin III (ATIII) is a plasma protein with potent anti-inflammatory and endothelial protective activity. We hypothesized that the protective effects of FFP on endothelial Sdc1 and permeability are mediated, in part, through ATIII. Methods: ATIII and Sdc1 were measured in severely injured patients upon admission (N = 125) and hospital day 3 (N = 90) for correlation analysis. In vitro effects of ATIII on human lung microvascular endothelial cells (HLMVECs) were determined by pre-treating cells with vehicle, FFP, ATIII-deficient FFP, or purified ATIII followed by TNFα stimulation. Sdc1 expression was measured by immunostaining and permeability by electrical impedance. To determine the role of ATIII in vivo, male mice were subjected to a fixed pressure exsanguination model of HS, followed by resuscitation with FFP, ATIII-deficient FFP, or ATIII-deficient FFP with ATIII repletion. Lung Sdc1 expression was assessed by immunostaining. Results: Pearson correlation analysis showed a significant negative correlation between plasma levels of Sdc1 and ATIII (R = -0.62; p < 0.0001) in injured patients on hospital day 3. Also, in vitro, FFP and ATIII prevented TNFα-induced permeability (p < 0.05 vs TNFα) in HLMVECs. ATIII-deficient FFP had no effect; however, ATIII restoration reestablished its protective effects in a dose-dependent manner. Similarly, FFP and ATIII prevented TNFα-induced Sdc1 shedding in HLMVECs, however ATIII-deficient FFP did not. In mice, Sdc1 expression was increased following FFP resuscitation (1.7 ± 0.5, p < 0.01) vs. HS alone (1.0 ± 0.3), however, no improvement was seen following ATIII-deficient FFP treatment (1.3 ± 0.4, p = 0.3). ATIII restoration improved Sdc1 expression (1.5 ± 0.9, p < 0.05) similar to that of FFP resuscitation. Conclusions: ATIII plays a role in FFP-mediated protection of endothelial Sdc1 expression and barrier function, making it a potential therapeutic target to mitigate HS-induced endothelial dysfunction. Further studies are needed to elucidate the mechanisms by which ATIII protects the endothelium. Address reprint requests to Jessica C. Cardenas, PhD, 6431 Fannin St. MSB 5.214, Houston, TX 77030. E-mail: Jessica.C.Cardenas@uth.tmc.edu Received 24 May, 2019 Revised 11 June, 2019 Accepted 29 July, 2019 Sources of Funding: Departmental funds to JCC. Disclosures: The authors declare no conflicts of interest. © 2019 by the Shock Society

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