Alternative Complement Pathway Activation Provokes A Hypercoagulable State with Diminished Fibrinolysis Introduction: Several disease processes trigger prolonged activation of the alternative complement pathway. Crosslinks between complement activation and physiologic changes in platelets and neutrophils have been identified, but how this interplay alters the hemostatic potential in humans remains undefined. We hypothesize that activation of the alternative pathway triggers a hypercoagulable state. Methods: C3/C5 convertase Cobra Venom Factor (CVF, 10 Units/mL) was employed to activate the alternative complement pathway in whole blood. Complement inhibition was completed with inhibitors for C3/C3b (Compstatin, 25 and 50 μM), C3a receptor (SB290157, 300 nM, C3aR), and C5a receptor (W54011, 6 nM, C5aR). Coagulation was assessed using native thrombelastography which produces the following: reaction time (R time); angle; maximum amplitude (MA); percent fibrinolysis at 30-minutes post-MA (LY30). Results: Inhibition with C3aR and C5aR inhibitors did not alter clot formation (R time, 11.2 vs 11.6 min, p = 0.36), clot strength (MA, 52.0 vs 52.3 mm, p = 0.43) or fibrinolysis (LY30, 1.6 vs 4.0%, p = 0.19). Compstatin did not influence clot formation or clot strength but did induce a dose-dependent increase in fibrinolysis (control LY30 3.0 vs 7.8% and 12.4% for 25 and 50 μM respectively, p = 0.0002). CVF increased MA (58.0 vs 62.8 mm, p < 0.0001), decreased LY30 (2.3 vs 1.4%, p = 0.004), and increased R time (8.4 vs 9.9 min, p = 0.008). Compstatin reversed the effects of CVF, while C5a reversed only the change in LY30. Conclusions: C3 contributes to fibrinolysis, as inhibition with Compstatin enhanced fibrinolysis, and CVF cleavage of C3 decreased fibrinolysis. CVF also induced a hypercoagulable state with increased clot strength. Address reprint requests to Christopher C. Silliman, Research Laboratory, Bonfils Blood Center, 717 Yosemite Street, Denver, CO 80230, USA. E-mail: Christopher.Silliman@ucdenver.edu; Jason M. Samuels, MD, University of Colorado Denver - Anschutz Medical Campus, Aurora, CO United States. E-mail: Jason.Samuels@ucdenver.edu Received 28 June, 2019 Revised 18 July, 2019 Accepted 7 August, 2019 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). Financial Disclosures: The authors appreciate research support from Haemonetics with shared intellectual property. 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) and Department of Defense (USAMRAA, W81XWH-12-2-0028). 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. Presentation history: Portions of this data were presented at the 64th Annual International Society on Thrombosis and Haemostasis (ISTH) Scientific and Standardization Committee meeting Dublin, Ireland, July 18-21, 2018. © 2019 by the Shock Society

Inhibition of Lipolysis with Acipimox Attenuates Post-Burn White Adipose Tissue Browning and Hepatic Fat Infiltration Extensive burn injuries promote an increase in the lipolysis of white adipose tissue (WAT), a complication that enhances post-burn hypermetabolism, contributing to hyperlipidemia and hepatic steatosis. The systemic increase of free fatty acids (FFA) due to burn-induced lipolysis and subsequent organ fatty infiltration may culminate in multiple organ dysfunction and ultimately, death. Thus, reducing WAT lipolysis to diminish the mobilization of FFAs may render an effective means to improve outcomes post-burn. Here, we investigated the metabolic effects of Acipimox, a clinically approved drug that suppresses lipolysis via inhibition of hormone-sensitive lipase (HSL). Using a murine model of thermal injury, we show that specific inhibition of HSL with Acipimox effectively suppresses burn-induced lipolysis in the inguinal WAT leading to lower levels of circulating FFAs at 7 days post-burn (p < 0.05). The FFA substrate shortage indirectly repressed the thermogenic activation of adipose tissue following injury, reflected by the decrease in protein expression of key browning markers, UCP-1 (p < 0.001) and PGC-1α (p < 0.01). Importantly, reduction of FFA mobilization by Acipimox significantly decreased liver weight and intracellular fat accumulation (p < 0.05), suggesting that it may also improve organ function post-burn. Our data validate the pharmacological inhibition of lipolysis as a potentially powerful therapeutic strategy to counteract the detrimental metabolic effects induced by burn. Address reprint requests to Marc G. Jeschke, MD, PhD, Director Ross Tilley Burn Centre, Sunnybrook Health Sciences Centre, Division of Plastic Surgery, Department of Surgery, Department of Immunology, University of Toronto, Sunnybrook Research Institute, 2075 Bayview Ave., Rm. D704, Toronto, ON, CANADA, M4N 3M5. E-mail: marc.jeschke@sunnybrook.ca. Received 24 May, 2019 Revised 12 June, 2019 Accepted 9 August, 2019 Address for Reprints: Same as corresponding author, please see above. Conflicts of Interest and Source of Funding: This study was supported by – Canadian Institutes of Health Research # 123336. CFI Leaders Opportunity Fund: Project # 25407 NIH RO1 GM087285-01 Author Contributions: DB, RV, and CK performed experiments and wrote portions of the manuscript; CA and AA guided experiments and wrote portions of the manuscript; MJ guided the experiments and wrote portions of the manuscript. Competing Financial Interests statement: The authors have no competing financial interests to declare. © 2019 by the Shock Society

Pulmonary Vasodilation by Intravenous Infusion of Organic Mononitrites of 1,2-Propanediol in Acute Pulmonary Hypertension Induced by Aortic Cross Clamping and Reperfusion: A Comparison with Nitroglycerin in Anesthetised Pigs Introduction: Suprarenal aortic cross clamping (SRACC) and reperfusion may cause acute pulmonary hypertension and multiple organ failure. Hypothesis: The organic mononitrites of 1,2-propanediol (PDNO), an NO donor with a very short half-life, is a more efficient pulmonary vasodilator and attenuator of end-organ damage and inflammation without significant side effects compared to nitroglycerin and inorganic nitrite in a porcine SRACC model. Methods: Anesthetised and instrumented domestic pigs were randomised to either of four IV infusions until the end of the experiment (n = 10 per group): saline (control), PDNO (45 nmol kg−1 min−1), nitroglycerin (44 nmol kg−1 min−1), or inorganic nitrite (a dose corresponding to PDNO). Thereafter, all animals were subjected to 90 minutes of SRACC and 10 hours of reperfusion and protocolised resuscitation. Hemodynamic and respiratory variables as well as blood samples were collected and analysed. Results: During reperfusion, mean pulmonary arterial pressure and pulmonary vascular resistance were significantly lower, and stroke volume was significantly higher in the PDNO group compared to the control, nitroglycerin, and inorganic nitrite groups. In parallel, mean arterial pressure, arterial oxygenation, and fraction of methaemoglobin were similar in all groups. The serum concentration of creatinine and tumour necrosis factor alpha were lower in the PDNO group compared to the control group during reperfusion. Conclusions: PDNO was an effective pulmonary vasodilator and appeared superior to nitroglycerin and inorganic nitrite, without causing significant systemic hypotension, impaired arterial oxygenation, or methaemoglobin formation in an animal model of SRACC and reperfusion. Also, PDNO may have kidney-protective effects and anti-inflammatory properties. Address reprint requests to Kristofer F. Nilsson, Department of Cardiothoracic and Vascular Surgery, Örebro University Hospital, SE-70185 Örebro, Sweden. E-mail: kristofer-bo-ingemar.nilsson@regionorebrolan.se Received 13 June, 2019 Revised 6 August, 2019 Accepted 6 August, 2019 Conflicts of Interest and Source of Funding: Author KFN wishes to declare competing financial interest in the subject matter. Author CF wishes to declare financial interest in the clinical use of inhaled nitric oxide. The other authors declare no competing interests. This study was supported by grants from the Swedish Heart-Lung Foundation, the European Space Agency, the Fraenckel Foundation, the Lars Hierta Foundation, Karolinska Institutet, Region Örebro County ALF grants, The Swedish Society for Medical Research, and an unrestricted educational grant from CF Research and Consulting AB, Stockholm, Sweden. 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

Persistent Neuroinflammation and Brain Specific Immune Priming in A Novel Survival Model of Murine Pneumosepsis Pneumonia is the leading cause of sepsis and septic shock. Patients who survive pneumonia are vulnerable to long-term complications including increased risk of neurocognitive dysfunction. This study investigated the immune response and long-term complications of a non-surgical mouse model of Klebsiella pneumoniae pneumosepsis with antibiotic treatment. Pneumosepsis resulted in acutely enhanced expression of inflammatory cytokines, chemokines, and damage associated molecular patterns in the brain and spleen. Despite resolution of infection, murine pneumosepsis survivors demonstrated a deficit in exploratory locomotor behavior at two weeks. This was associated with brain specific persistent inflammatory gene expression and infiltrating myeloid cells in the brain. The brain inflammatory response was also primed in response to secondary challenge with lipopolysaccharide. The findings of this study demonstrate behavioral and inflammatory outcomes that parallel observations in other models of sepsis, but that have not previously been described in antibiotic treated pneumonia models, highlighting a common pathway to the development of chronic brain dysfunction in sepsis survival. Address reprint requests to Scott J. Denstaedt, MD, 4868 Biomedical Science Research Building 109 Zina Pitcher Pl. Ann Arbor, MI 48103. E-mail: sdenstae@med.umich.edu Received 19 June, 2019 Revised 10 July, 2019 Accepted 5 August, 2019 Financial support: National Institutes of Health grants T32HL00774921 (to SJD), MH116267 (to JLSS), K08NS101054 (to BHS), R01HL123515 (to TJS). Conflict of interest: The authors have no conflicts relevant to the present study. 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

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