US Army Medical Research Articles

Resuscitation Fluids Modulate Sterile Inflammation in a Rodent Model of Hemorrhagic Shock

Sterile inflammation may trigger an array of danger‐associated molecular patterns and activation of the vascular endothelium, leading to loss of vascular barrier, microvascular leakage, coagulation dysfunction, leucocyte extravasation and organ injury. In our rat hemorrhage model, we have demonstrated previously elevated cytokine levels in liver, kidney and lung along with circulating proteases and endothelial dysfunction following hemorrhagic shock (HS) and/or fluid resuscitation. We hypothesized that normal saline (NS) resuscitation after HS may sustain sterile inflammation by increasing leukocyte influx to tissues (transmigration) and net leukocyte recruitment (overall efficiency), which results in loss of barrier integrity and tissue injury, compared to HS alone or blood products. Anesthetized rats (n= 5–7/group) were bled 40% of blood volume and either left untreated (HS ONLY) for over 3h or resuscitated with NS, 45 ml/kg, or 5% Albumin (ALB), 15ml/kg. Shams rats (control) were subject to all procedures, except hemorrhage or resuscitation. Intravital microscopy was performed in over 100 cremaster venules to determine leukocyte transmigration and overall efficiency as well as blood flow, wall shear rate (WSR), endothelial glycocalyx thickness and vascular permeability. Post‐shock or post‐resuscitation blood and tissue samples were collected over 3h from onset of hemorrhage. Our HS model alone decreased blood flow and WSR more than 60% (vs. sham, P<0.05), and increased glycocalyx shedding, permeability as well as transmigrated leukocytes (vs. sham and ALB, P<0.05). High levels of transmigration post‐shock was sustained after NS (883 ± 433 transmigrated cells/mm2, vs. sham, P<0.05). Compared to HEM ONLY, IL‐1β, IL‐6 and TNF‐α levels in kidney were 2–3‐fold higher in NS group, as well as blood flow and WSR, glycocalyx shedding, permeability (P<0.05). The highest overall efficiency post‐shock was observed in NS group (224 ± 117 transmigrated cells/mm2 per 1,000 leukocytes/μL) vs. HS ONLY (164 ± 94, P=0.04), sham (113 ± 50, P<0.001) and albumin group. In contrast, ALB normalized glycocalyx, venular blood flow and WSR, as well as restored barrier function and decreased inflammation to sham levels, compared to HEM ONLY. Fluid administration presents a therapeutic conflict between hemodynamics stabilization and detrimental effects on the endothelial and microvascular function. Iatrogenic increase in leukocyte recruitment and migration into tissues after NS sustained sterile inflammation after hemorrhagic shock and thus presents some clinically relevant deleterious effects.Support or Funding InformationUSArmy Medical Research Materiel CommandThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Special Operations Force Risk Reduction: Integration of Expeditionary Surgical and Resuscitation Teams.

Hemorrhage in the presurgical setting has been the most significant cause of death on the battlefield. Damage control surgery (DCS) near the point of injury (POI) is not a new concept, but having conventional medical teams supporting Special Operations Forces (SOF) beyond robust military medical infrastructure is unique for the US military. The Expeditionary Resuscitative Surgical Team (ERST) was formed by the US Army Medical Command as a pilot team to fulfill a request for forces to provide DCS and personnel recovery near POI.

Abstract 4998: Breast tumor FOXA1 protein expression and reproductive characteristics among African-American and European-American women

Abstract Background: Forkhead box protein A1 (FOXA1) plays a key role in determining estrogen receptor (ER) function and mammary ductal development and may repress the basal cell phenotype during differentiation of breast epithelium. While reproductive factors are known to influence breast cancer risk depending on the subtypes, data on the association of tumor FOXA1 protein expression and reproductive characteristics are very limited. Methods: Tissue microarrays comprising surgical tumors from 638 women (466 African-American [AA] and 172 European-American [EA], aged 20-75 years) with primary breast cancer in the Women’s Circle of Health Study (WCHS) were analyzed for FOXA1 expression by immunohistochemistry and automated image analysis. In-person interviews were conducted to obtain data on demographics, medical and family histories, and reproductive and menstrual histories. Logistic regression was performed for FOXA1 positivity (>10% cells with strong staining) with menopausal status, age at menarche, age at first live birth, parity, and breastfeeding, adjusting for age at diagnosis, family history of breast cancer, and history of benign breast disease. Results: FOXA1 expression was higher in tumors from EA compared to those from AA women (80% vs. 70% positivity, P=0.011). FOXA1 expression was highly correlated with ER positivity (88% positive in ER+ and 26% in ER- breast cancer among AA women; 90% and 29% respectively among EA women; both P<0.001). Among AA women, a higher number of live births was associated with lower FOXA1 expression (odds ratio [OR]=0.45, 95% confidence interval [CI]=0.22-0.91 for 2 live births and OR=0.44, 95% CI=0.20-0.91 for ≥3 live births, versus nulliparous). However, the inverse association was not observed among EA women, potentially due to the small sample size. Conclusion: In AA women, a higher number of live births, which has been related to greater risk of ER- breast cancer, is associated with lower FOXA1 expression. Because FOXA1 promotes luminal and represses the basal differentiation, respectively, the effects of parity on expression of FOXA1 may be the link whereby it increases risk of ER- breast cancer. (Funding: NIH P01CA151135, R01CA100598, R01CA185623, P30CA072720, K07CA201334; US Army Medical Research and Material Command DAMD-17-01-1-0334; the Breast Cancer Research Foundation; and the Philip L. Hubbell family) Citation Format: Ting-Yuan David Cheng, Rochelle Payne Ondracek, Song Yao, Wiam Bshara, Thaer Khoury, Gary R. Zirpoli, Warren Davis, Elisa V. Bandera, Michael Higgins, Christine B. Ambrosone. Breast tumor FOXA1 protein expression and reproductive characteristics among African-American and European-American women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4998. doi:10.1158/1538-7445.AM2017-4998

Is Helicobacter pylori infection associated with chronic headache (specifically migraine)?

The opinions and assertions contained herein are those of the authors and are not to be construed as official or as reflecting the views of the US Army Medical Department, the Army at large, or the Department of Defense.

3D Integrated Tissue and Organ Printing System to Produce Human Body Parts with Structural Integrity

Tissue engineering and regenerative medicine strategies could offer new hope for patients with serious tissue injuries or end‐stage organ failure. Scientists are now applying the principles of cell transplantation, material science, and engineering to create biological substitutes that can restore and maintain normal function in diseased or injured tissues/organs. Among various three‐dimensional (3D) biofabrication technologies developed for the field of tissue engineering, 3D printing technology is one of the most attractive and powerful methods for use in constructing a structure that mimics native tissues, and thus many researchers are focusing on development of new printing technologies and applications. The hypothesis driving development of 3D bioprinting is that by precisely placing cells in relation to each other, an environment that encourages physiologically relevant cues both to and from these cells can be created, resulting in a tissue construct with functionality. Thus, we have developed 3D integrated tissue and organ printing (ITOP) system to allow construction of biological substitutes that mimic the structure and function of native tissues or organs. This system makes it possible to precisely place various cell types and biomaterials in a single 3D architecture. Our proof of concept experiments demonstrates the capabilities of our ITOP by fabricating structures of various tissue constructs of appropriate shape and size needed for clinical use. This novel organ printing system provides a leap forward in our ability to fabricate tissue constructs that could be used in translational applications.Support or Funding InformationThis work was supported, in part, by grants from the Armed Forces Institute of Regenerative Medicine (W81XWH‐08‐2‐0032), the Telemedicine and Advanced Technology Research Center at the US Army Medical Research and Material Command (W81XWH‐07‐1‐0718) and the Defense Threat Reduction Agency (N66001‐13‐C‐2027).

Epinephrine Does Not Influence Baroreflex Sensitivity During Lower Body Negative Pressure to Physiological Tolerance

BackgroundThe baroreflex plays an integral role in blood pressure maintenance by altering heart rate and sympathetic activity in response to changes in arterial pressure. Baroreflex sensitivity (BRS) has been shown to decrease linearly during progressive exposure to central hypovolemia as induced by lower body negative pressure (LBNP), and can be used as an index of decompensation status during hemorrhage. Circulating epinephrine is significantly elevated during high stress situations such as hemorrhage; however, it is unclear whether epinephrine influences BRS during progressive exposure to central hypovolemia. Based on previous work, we hypothesized epinephrine would attenuate any reduction in spontaneous cardiac baroreflex sensitivity during LBNP.MethodsTen young (35±3 yrs), healthy (BMI 24±1 kg/m2) men and women (6M/4F) underwent LBNP protocols on 2 separate days consisting of a 10‐min pre‐infusion baseline, 15‐min infusion period (randomized to saline or epinephrine, 0.1 μg/kg/min), 10‐min post‐infusion baseline, and 5 minutes of LBNP at −15, −30, −45, −60, −70, and −80 mmHg until the onset of presyncope (range= −30 to −80 mmHg). Presyncope was defined as a rapid decrease in systolic blood pressure greater than 15 mmHg, systolic blood pressure less than 80 mmHg, bradycardia, or voluntary termination due to presyncopal symptoms. Heart rate (HR; electrocardiogram) and blood pressure (BP; radial arterial catheter) were recorded continuously. Spontaneous cardiac BRS was determined using the sequence method (WinCPRS). Data are reported from both 10‐min baselines and from 1 minute at 20, 40, 60, 80, and 100 percent of each individual's total LBNP exposure time.ResultsWe observed no significant differences in total exposure time (1475±150 vs. 1415±79s; p=0.69) or cumulative stress index (820±135 vs. 699±69 min*mmHg; p=0.37) between saline and epinephrine visits, respectively. Baroreflex sensitivity did not change during post‐infusion baselines (ΔDown‐Down =−2±1 vs. −2±2 ms/mmHg; saline vs. epinephrine, respectively; p=0.09). Baroreflex sensitivity was significantly reduced with LBNP (p<0.01 main effect of LBNP). There was no difference between the change in baroreflex sensitivity during LBNP with saline or epinephrine infusion (ΔDown‐Down =−14±4 vs. −8±4 ms/mmHg, p=0.17).ConclusionsConsistent with previous work, spontaneous cardiac baroreflex sensitivity fell during LBNP. Contrary to our hypothesis, epinephrine infusion did not attenuate the fall in spontaneous cardiac baroreflex sensitivity observed during simulated blood loss. Because epinephrine did not alter the effect of LBNP on BRS, these data suggest that BRS may accurately reflect decompensation status during traumatic hemorrhage independent of elevated circulating epinephrine.Support or Funding InformationUS Army Medical Research and Materiel Command Contract W81XWH‐13‐2‐0038

The Mechanism of Inhibition of Botulinum Neurotoxin Type A by Two Quinolinol Compounds

The potential of Botulinum neurotoxins (BoNTs) to be used as biological weapons makes the need to identify small molecule inhibitors critical but it is a challenging target due to the large substrate‐enzyme interface. BoNTs are produced by the bacterium Clostridium botulinum in seven antigenically distinct serotypes (A‐G), with serotype A (BoNT/A) being the most potent toxin known to man. Recently, quinolinol‐based compounds have been identified as a promising starting point for effective BoNT/A inhibitors. Understanding the mechanism of inhibition by quinolinol compounds would facilitate inhibitor optimization studies. In this study, a Foster resonance energy transfer (FRET) based substrate of BoNT/A, SNAPtide, was used to study the mechanism of inhibition of BoNT/A endopeptidase activity by two new quinolinol compounds, MSU58 and MSU84, with IC50 values of 3.3 μM and 5.8 μM, respectively. Kinetic analysis and model discrimination analysis revealed both compounds to be competitive inhibitors of BoNT/A endopeptidase activity. Inhibition constants (KI) were found to be 3.2 μM for MSU58 and 6.2 μM for MSU84. The kinetic rate constant for the binding and release of substrate and inhibitor were also determined. These data indicate that these inhibitors bind in the active site of BoNT/A and that binding is mutually exclusive with the binding of the substrate. This study, is the first to report on the competitive inhibition of BoNT/A by a quinolinol group of compounds. These data help define the inhibitor binding pocket and, along with structure activity relationship studies, provide immediate direction for further compound synthesis.Support or Funding InformationNovartis Graduate Scholarship Award and US Army Medical Research (grant number W81XWH‐14‐C‐0090 S)

Measurement of the Compensatory Reserve Predicts Racial Differences in Orthostatic Tolerance

The Compensatory Reserve Index (CRI) has recently been established to accurately measure the body's integrated capacity to compensate for physiological conditions of reduced central blood volume, and predict hemodynamic decompensation. We previously demonstrated that African American (AA) women have a higher tolerance to orthostatic stress. Therefore, we tested the hypothesis that the CRI would identify racial differences in orthostatic tolerance prior to the onset of traditional signs and symptoms. We measured beat‐by‐beat blood pressure (BP) and heart rate (HR) in 23 AA (22 ± 1 years; 24 ± 1 kg/m2) and 31 Caucasian women (20 ± 1 years; 23 ± 1 kg/m2) during progressive lower body negative pressure (LBNP) to presyncope. Blood pressure waveforms were analyzed using a machine‐learning algorithm to derive the CRI at each LBNP stage; stroke volume (SV) was derived from the Finometer (Model flow). Resting mean arterial BP (AA, 78 ± 3 vs. Caucasian, 74 ± 2 mmHg) and HR (AA, 68 ± 2 vs. Caucasian, 65 ± 2 bpm) were similar between groups. The CRI progressively decreased during LBNP in both groups, however the CRI in Caucasian women was 4% lower than AA women at −15 mmHg LBNP and decreased to 21% lower at −50 mmHg LBNP (P<0.05). The rate of decline in SV during LBNP was greater in Caucasian women (ANOVA P<0.05), and differences were noted as early as −20 mmHg. However changes in BP and HR during LBNP were not different between groups. At presyncope, mean arterial BP (AA, 66 ± 3 vs. white, 65 ± 4 mmHg) and HR (AA, 102 ± 4 vs. white, 97 ± 3 bpm) were similar between groups, as was the change in SV (AA, Δ −33 ± 2 vs. white, Δ −35 ± 3 mL). Finally, the time to presyncope was 1.6 minutes longer in AA women. These data support the notion that greater tolerance to the orthostatic stress induced by LBNP in AA women can be explained by their greater reserve to compensate for progressive central hypovolemia compared to Caucasian women. An important clinical translation is that the CRI provides a more sensitive and specific measure for early assessment of differences in orthostatic tolerance in comparison to traditional vital signs and symptoms or changes in hemodynamics.Support or Funding InformationResearch supported by funding from the Combat Casualty Care Research Program of the US Army Medical Research and Materiel Command and NIH HL071159.

Comparison of Growth Supplements on the Differentiation of Adipose Derived Stem Cells from Burned and Non‐Burned Human Skin

Severe traumatic injuries, such as large total body surface area burns, limit the availability of autologous cells for wound repair. Previous experiments from our lab have demonstrated that adipose derived stem cells (ASCs) from burned debrided skin are viable, display an immunophenotype indicative of stemness, and are capable of tri‐lineage differentiation. Traditional tissue culture utilizes undefined, animal‐derived components that present numerous complications for the utilization of ASCs in human medicine. Accordingly, there is a need for defined, xeno‐free cell culture media. Human platelet lysate (hPL) is one potential media supplement for the replacement of fetal bovine serum in stem cell culture. In this study, burned human ASCs (BH‐ASCs) and ASCs derived from abdominoplasty (HAP‐ASCs) were compared in regard to the ability to proliferate and differentiate in media supplemented with traditional 10% FBS, 10% hPL, or grown in MesenPRO RS™. ASCs were obtained from the stromal vascular fractions of subcutaneous adipose tissue from burned and non‐burned skin. Cells were cultured in DMEM with 10% FBS or 10% hPL, or in MesenPRO RS™. Cells were induced to differentiate towards an adipogenic or osteogenic lineage at an early passage utilizing traditional differentiation media supplemented with FBS or 10% hPL. DMEM or αMEM was used for adipogenic and osteogenic lineages, respectively, with the addition of standard induction agents. Adipogenic differentiation was assessed after 14 days by Oil red O staining, triglyceride content, and expression of markers of adipogenesis, including PPARγ, leptin, and adiponectin. Osteogenic differentiation was assessed by alizarin red S staining and expression of markers of ostogenesis, including BMP‐2 and RUNX2. Results indicate that adipogenic and osteogenic differentiation appears to be influenced by both the ASC source (burned vs. non‐burned skin) and by the supplement in which they are grown and differentiated. Use of MesenPRO RS™ and supplementation with hPL appears to enhance differentiation towards both lineages over standard FBS. Prior experiments in our labs have indicated that both burn and non‐burn ACSs are capable of trilineage differentiation, however there is a need to characterize these cells, particularly the BH‐ASCs in xeno‐free media, with regard to their stemness and immunosuppresive capabilites if they are to be utilized in any capacity for regenerative medicine. Ongoing research efforts in our laboratory seek to address these issues to better define the utility of BH‐ASCs as a burn wound therapeutic.Support or Funding InformationThis work was possible due to the generous funding from the US Army Medical Research and Material Command and ORISE

The good, the bad and the ugly of nitric oxide, superoxide and peroxynitrite signaling during oxygen‐glucose deprivation in rat and arctic ground squirrel

Cerebral ischemia‐reperfusion (I/R) injury initiates a complex cascade of events, several of which involve the generation of nitic oxide (NO) and oxygen (O2•−) free radicals. NO and O2•− rapidly combine to form peroxynitrite (ONOO−) a potent oxidant and nitrating agent. Previous studies have revealed that the arctic ground squirrel (AGS; Urocitellus parryii) is a natural model of high tolerance to I/R injury, however, the mechanisms that contribute to the ability to tolerate this pathological scenario is still elusive. Here, we tested the hypothesis that tolerance to I/R injury modeled in an acute hippocampal slice preparation in AGS is modulated by reduced oxidative and nitrosative stress associated with oxygen glucose deprivation (OGD).Hippocampal slices (400 micron) from rat and AGS were subjected to oxygen glucose deprivation (OGD) to mimic I/R in vivo using a novel microperfusion technique. Injury consisted of exposing the brain slices to: 1) NO and O2•− donors with and without OGD; and 2) pretreatment of slices with inhibitors of NO, O2•− and ONOO− followed by OGD. In control slices, treatment was administered by switching to standard aCSF. Perfusates were collected every 15 min and analyzed for LDH release, an indicator of cell death; slices were analyzed for nitrosative and oxidative damage using the markers 3‐nitrotyrosine (3NT) and 4‐hydroxynonenal (4HNE).Results show the effect of NO, O2•− and ONOO− mediated injury in ischemic tolerant AGS and ischemic susceptible rat. 1) Effect of NO mediated Injury: An NO donor alone is not sufficient to cause cell death, but with OGD enhances cell death more in rat than in AGS; an inhibitor of nNOS attenuates OGD‐induced injury in rat but has no effect in AGS. 2) Effect of O2•− mediated injury: A O2•− donor alone causes slight injury in rat and even less injury in AGS. Combining the O2•− donor with OGD aggravates the injury in rat; a SOD mimetic attenuates OGD injury in rat but has no effect in AGS. 3) Effect of ONOO− mediated injury: A ONOO− inhibitor attenuates OGD injury in rat but has no effect in AGS. Rat also shows higher levels of 3NT and 4HNE after OGD insult than AGS. These data demonstrate that in rat both NO and O2•− are required for injury and point to ONOO− as the damaging agent. This same mechanism of OGD‐induced cell death is lacking in AGS supporting the hypothesis that resistance to oxidative and nitrosative stress contributes to tolerance to cerebral I/R injury in AGS.Support or Funding InformationThis work was supported by the US Army Medical research and Material Command, No 0517800; the National Institute of Neurological Disorder and Stroke, Nos. NS041069‐06 and R15NS070779; Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103395.

Quantitative Real Time PCR (qRT‐PCR) Evaluation of Pig Mitochondrial DNA Damage

Mitochondrial DNA (MtDNA) damage has generated much interest in recent years as a potential biomarker after ischemia/reperfusion and traumatic injury and in critical illness. MtDNA is particularly susceptible to damage due to is close proximity to the source of generation of reactive oxygen species during metabolism and its relatively low ability to repair DNA damage compared to nuclear repair mechanisms. Reduction in MtDNA damage appears to be a good indicator of a successful intervention. QRT‐PCR has been shown to be the most sensitive and accurate method to evaluate mtDNA damage requiring only nanogram amounts of DNA. While validated primer pairs for mtDNA damage for human and rodent mtDNA damage are available, comparable sets for pig have not previously been described, yet many studies in trauma and critical illness have utilized swine models. To select primer pairs for pigs, we aligned the pig mitochondrial DNA sequence to the pig genome in the UCSC Genome Browser and primers for regions not exhibiting overlap with the nuclear genome were selected with NCBI Primer3. Using the bleomycin injury model, porcine pulmonary artery endothelial cells obtained from Cell Biologics and cultivated in MCDB 131 medium were treated for 1 hr with bleomycin (10 μg/ml). Short and long sequences were tested with pig total DNA and demonstrated that one set detected specific induction of DNA lesions. The assay was able to detect 11 lesions per 10,000 base pairs. The short primer pairs also accurately predicted mitochondrial copy number DNA as corroborated for mtDNA by published results for the pig ND4 gene. We are currently employing this assay to detect mtDNA damage in a swine model of hemorrhagic shock.Support or Funding InformationFunded by US Army Medical Research Materiel Command

Tissue Inflammatory Responses in an Anesthetized Rat Hemorrhage Model Associated with Endothelial Dysfunction

We have demonstrated endothelial dysfunction such as reduction in glycocalyx thickness and increased permeability in venules in cremaster muscle following hemorrhage and fluid resuscitation with a crystalloid or a colloid in a rat model. The present study further characterizes the model by measuring indices of inflammation and oxidative and nitrosative stress in tissues from these animals. Anesthetized Sprague‐Dawley rats (n=4–5/group) were bled 40% of their blood volume, subjected to a 30 min shock period and resuscitated with limited volumes of normal saline (NS) or 5% albumin (A), or no treatment (Hem). At 1hr after resuscitation, rats were euthanized and liver, kidney, lung and skeletal muscle were collected and assayed for cytokines and select indices of nitrosative and oxidative stress. In liver and kidney, IL‐1β, IL‐6 and TNF‐α levels were 2–3‐fold higher in the NS than the A group and about 2‐fold higher than the Hem group. IL‐10 levels were also significantly higher in the NS group than the other groups. Similar responses were observed in lung from these groups, while in muscle, only IL‐1β and IL‐6 levels were significantly higher. In contrast, no significant differences were observed in all tissues in nitric oxide, total antioxidant or reduced glutathione concentrations nor xanthine oxidase activity among the groups. In this model of hemorrhagic shock, NS induced the highest inflammatory response, while 5% albumin was associated with the least response. Interestingly, these current observations were related to the degree of endothelial dysfunction observed previously. Taken together these data suggest that the endothelial dysfunction observed in hemorrhagic shock and fluid resuscitation is associated with an inflammatory response without a major nitrosative or oxidative stress component.Support or Funding InformationFunded by the US Army Medical Research Materiel Command

Energy restriction upregulates circulating myomiR expression in vivo and in vitro

Energy restriction (ER)‐induced weightloss attenuates the rate of protein synthesis, which over time can result inreductions in skeletal muscle mass. Recently, changes in the expression of circulating muscle‐specific microRNA (c‐myomiR)have been suggested to reflect rates of protein synthesis. However, whether an increased expression ofc‐myomiR are associated with lower rates of protein synthesis remains undefined. The present investigation sought to determine whether ER influences c‐myomiR expression, and if changes in c‐myomiR are associated with rates of protein synthesis both in vivo (humans) and in vitro (cultured myocytes). Sixteen older (64 ± 2 yrs) overweight (28.5 ±1.2 kg·m−2) males enrolled in this 35‐day controlled feedingtrial. A 7‐day weight maintenance (WM)period was followed by 28 days of 30% ER; whole‐body protein turnover (15N‐glycine)and c‐myomiR (miR‐1–3p, miR‐133a‐3p, miR‐133b, miR‐206) expression was assessed by RT‐qPCR at the conclusions of WM and ER. Participants lost 4.4 ± 0.3 kg body mass during ER (P < 0.05). Overall, ER upregulated c‐myomiR expression compared to WM (P < 0.05). Expression ofc‐myomiR was inversely associated (r= −0.700, P < 0.05) with whole‐body protein synthesis after ER. Similarly, assessing the expression of c‐myomiR obtained from media that C2C12myotubes were cultured; ER increased c‐myomiR expression by 2.9 ± 0.1 fold and lowered the protein synthesis rate by 2.1 ±0.2 fold compared to control. Corroboration of in vitro and in vivo findings indicates that increased expressions of c‐myomiR maybe noninvasive markers reflective of ER‐induced reductions in the rate protein synthesis.Support or Funding InformationThis material is based on the work supported by the U.S. Department of Agriculture(USDA), under agreement No. 58‐1950‐4‐003 and the US Army Medical Research and Material Command. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the USDA. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. The study was also supported by the Boston Claude D. Pepper Center Older American Independence Centers (OAIC;1P30AG031679). L.M.M is supported by T32NIDDK training grant # 5T32DK062032‐23. D.A.R. is supported by NIA K01 award # KAG047247A‐A1.

High Energy Expenditure and Negative Energy Balance Modulate Composition and Metabolism of the Gut Microbiota

Dietary intake and acute physiologic stress may modulate health in part by influencing interactions between the gut microbiome and host, but these relationships remain poorly characterized. This study explored relationships between gut microbiota composition and activity, and diet, gastrointestinal permeability and inflammation in Soldiers participating in a 4‐d, 51 km cross‐country ski march (SKI) characterized by high energy expenditure (6155 ± 515 kcal/d) and negative energy balance (2.7 ± 1.2 kg weight loss). During SKI, 73 Soldiers (20 ± 1 yr, BMI 23 ± 2 kg/m2) were provided 3 combat rations/d, or 3 rations/d supplemented with 1000 kcal/d from protein‐ or carbohydrate‐based snacks generating variability in energy, protein and carbohydrate intakes. Stool, blood and urine samples were collected from 26 Soldiers before and after SKI. Stool was analyzed for bacterial composition by 16S rDNA sequencing, and global metabolite profiling by UPLC‐MS/MS. Gastrointestinal permeability was assessed by dual sugar absorption test, and plasma lipopolysaccharide (LPS) and serum IL‐6 were measured as markers of microbial translocation and inflammation, respectively. Ordination, cluster and classification analyses of gut microbiota composition data indicated a pronounced alteration of gut microbiota composition during SKI independent of diet group. This effect was characterized by a more heterogeneous distribution of taxa abundances within samples following SKI (Shannon index, P = 0.04), and changes in the relative abundance of >50% of identified genera (Q < 0.10). Changes included increases in the relative abundance of several potentially deleterious and infectious genera, and decreases in the relative abundance of several genera thought to deter pathogen invasion, reduce inflammation and promote immunity. Prediction models linked changes in gut microbiota composition to 69 of 299 metabolites that were significantly altered in stool, with decreases in amino acid and nucleotide metabolites comprising the majority. Univariate analyses generally did not detect associations between changes in taxa relative abundance or concentrations of bacterially‐associated metabolites and energy or macronutrient intake, changes in gastrointestinal permeability, or changes in LPS and IL‐6 concentrations. These findings demonstrate pronounced changes in gut microbiota composition and metabolism in response to a multi‐stressor environment that appear to be independent of energy and macronutrient intakes within the ranges studied. The implications for health remain to be determined. The views expressed in this abstract are those of the authors and do not reflect official policy of the Dept. of Army, Dept. of Defense, or US Government.Support or Funding InformationFunded by US Army Medical Research and Material Command, the US Defense Health Program, and the Norwegian Defense Research Establishment agreement NO. W81XWH‐12‐0279.

Dietary Supplement Use in All United States Military Services

A2008 Institute of Medicine report (https://www.nap.edu/catalog/12095/use‐of‐dietary‐supplements‐by‐military‐personnel) noted that there was no clear picture of dietary supplement (DS) use in the military and recommended surveys to examine usage. Our study investigated the prevalence of DS use in the military services and demographic factors associated with use. Surveys were administered to active duty service members (SMs) in the Army (n=1,275), Air Force (AF, n=1,787), Navy (n=700), Marine Corps (MC, n=983) and Coast Guard (CS, n=1,059) located in the United States and overseas. The questionnaire listed both generic and specific DSs and included questions on demographics. Prevalence of use ≥1time/week was calculated and multivariate logistic regression was used to examine associations between any DS use (dependent variable) and various strata of demographics, with odds ratios (OR) and 95% confidence intervals (95%CIs)calculated. The overall prevalence of DS use was (%±standard error) 67±1% with use prevalences in the Army, AF, Navy, MC, and CG 59±1%, 66±1%, 71±2% 74±1% and 69±1%, respectively (p<0.01). Multivitamins/multi minerals were the most commonly used DS among all services (44±1%), followed by protein/aminoacids (31±1%), combination products (26±1%), and herbal substances (10±1%). Factors independently associated with DS use (≥1time/week) included female gender (OR [women/men]=1.35, 95%CIs=1.17–1.56), older age (OR [≥40/18–24 years]=1.46, 95%CIs=1.19–1.80), overweight body mass index (OR [25–29.9/<25 kg/m2]=1.19, 95%CIs=1.05–1.35), and military service (OR [Marines/Army]=1.72, 95%CIs=1.42–2.09). Thus, DS use among SMs differs based on specific demographics. Compared to the civilian data assessed in the National Health and Nutrition Examination Surveys (NHANES), SMs were much more likely to use DSs and used very different types of DSs, especially for proteins/amino acids and combination products which are often used to purportedly enhance physical performance.Support or Funding InformationWe are military service members (or employees of the U.S. Government). This work was prepared as part of our official duties. Title 17, U.S.C. §105 provides the “Copyright protection under this title is not available for any work of the United States Government.” Title 17, U.S.C. §101 defines a U.S. Government work as work prepared by a military service member or employee of the U.S. Government as part of that person's official duties. This research was supported by the Defense Health Agency under work unit no. N1335. The views expressed in this abstract are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of the Army, Department of the Air Force, Department of Veterans Affairs, Department of Defense, or the U.S. Government. Approved for public release; distribution unlimited. Human subjects participated in this study after giving their free and informed consent. This research has been conducted in compliance with all applicable federal regulations governing the protection of human subjects in research (NHRC.2013.0009).Supported by US Army Medical Research and Material Command. Funding from the Center Alliance for Nutrition Research.

Effect of Load Magnitude and Distribution on Lumbar Spine Posture in Active-duty Marines.

Repeated measures. The purpose of this study was to quantify the effect of operationally relevant loads and distributions on lumbar spine (LS) in a group of active-duty Marines. Low-back pain has been associated with heavy load carriage among military personnel. Although there are data describing the LS posture in response to load, the effect of varying load characteristics on LS posture remains unknown. Magnetic resonance images of Marines (n = 12) were acquired when standing unloaded and when carrying 22, 33, and 45 kg of load distributed both 50% to 50% and 20% to 80% anteriorly and posteriorly. Images were used to measure LS and pelvic postures. Two-way repeated-measures ANOVA and posthoc tests were used to compare LS posture across load magnitudes and distributions (α = 0.05). This project was founded by the US Army Medical Research Acquisition Activity, Award No. W81XWH-13-2-0043, under Work Unit No. 1310. No changes in LS posture were induced when load was evenly distributed. When load was carried in the 20% to 80% distribution lumbosacral flexion increased as a result of sacral anterior rotation and overall reduced lumbar lordosis. This pattern was greater as load was increased between 22 and 33 kg, but did not increase further between 33 and 45 kg. We observed that the inferior LS became uniformly less lordotic, independently of load magnitude. However, the superior LS became progressively more lordotic with increasing load magnitude CONCLUSION.: Postural adaptations were found only when load was carried with a posterior bias, suggesting that load-carriage limits based on postural changes are relevant when loads are nonuniformly distributed. Although the tendency would be to interpret that loads should be carried symmetrically to protect the spine, the relationship between postural changes and injury are not clear. Finally, the operational efficiency of carrying load in this distribution needs to be tested. 3.

Post-deployment screening for mental disorders and tailored advice about help-seeking in the UK military: a cluster randomised controlled trial

The effectiveness of post-deployment screening for mental disorders has not been assessed in a randomised controlled trial. We aimed to assess whether post-deployment screening for post-traumatic stress disorder (PTSD), depression, anxiety, or alcohol misuse was effective. We defined screening as the presumptive identification of a previously unrecognised disorder using tests to distinguish those who probably had the disorder from those who probably did not so that those people with a probable disorder could be referred appropriately, and assessed effectiveness and consequences for help-seeking by the odds ratio at follow-up between those receiving tailored help-seeking advice and those who received general mental health advice. We did a cluster randomised controlled trial among Royal Marines and Army personnel in the UK military after deployment to Afghanistan. Platoons were randomly assigned (1:1 initially, then 2:1) by stratified block randomisation with randomly varying block sizes of two and four to the screening group, which received tailored help-seeking advice, or the control group, which received general mental health advice. Initial assessment took place 6-12 weeks after deployment; follow-up assessments were done 10-24 months later. Follow-up measures were the PTSD Checklist-Civilian Version, Patient Health Questionnaire-9, Generalised Anxiety Disorder-7 scale, Alcohol Use Disorder Identification Test (AUDIT), and self-reported help-seeking from clinical and welfare providers comparing those receiving tailored advice and those receiving only general advice. All participants and all investigators other than the person who analysed the data were masked to allocation. The primary outcomes were PTSD, depression or generalised anxiety disorder, and alcohol misuse at follow-up. A key secondary outcome was assessment of whether post-deployment screening followed by tailored advice would modify help-seeking behaviour. Comparisons were made between screening and control groups, with primary analyses by intention to treat. This trial is registered with the ISRCTN Registry, number ISRCTN19965528. Between Oct 24, 2011, and Oct 31, 2014, 434 platoons comprising 10 190 personnel were included: 274 (6350 personnel) in the screening group and 160 (3840 personnel) in the control group. 5577 (88%) of 6350 personnel received screening and 3996 (63%) completed follow-up, whereas 3149 (82%) of 3840 received the control questionnaire and 2369 (62%) completed follow-up. 1958 (35%) of 5577 personnel in the screening group declined to see the tailored advice, but those with PTSD (83%) or anxiety or depression (84%) were more likely than non-cases (64%) to view the advice (both p<0·0001). At follow-up, there were no significant differences in prevalence between groups for PTSD (adjusted odds ratio 0·92, 95% CI 0·75-1·14), depression or anxiety (0·91, 0·71-1·16), alcohol misuse (0·88, 0·73-1·06), or seeking support for mental disorders (0·92, 0·78-1·08). Post-deployment screening for mental disorders based on tailored advice was not effective at reducing prevalence of mental health disorders nor did it increase help-seeking. Countries that have implemented post-deployment screening programmes for mental disorders should consider monitoring the outcomes of their programmes. The US Army Medical Research and Materiel Command-Military Operational Medicine Research Program (USAMRMC-MOMRP).

Corrigendum: Human antibody repertoire after VSV-Ebola vaccination identifies novel targets and virus-neutralizing IgM antibodies.

Nat. Med.; 10.1038/nm.4201; corrected online 15 November 2016 In the version of this article initially published online, the authors omitted acknowledging a funding agency and researchers for providing the pseudovirion neutralization assay data. The acknowledgments section has been revised to state,“We thank the US Army Medical Research Institute of Infectious Diseases team, including S.

The SOF Truths for Army Special Operations Forces Surgical Teams.

The US Army Special Operations Command and Army Medical Command are at a critical junction in Army medical training. Army Special Operations Forces (ARSOF) will receive Forward Resuscitative Surgical Teams (FRSTs) in the near future and must establish a training model to enable successful support for ARSOF operations. The military has been directed by Congress through the 2017 National Defense Authorization Act to embed trauma combat casualty care teams in civilian trauma centers. ARSOF FRSTs should be embedded in the nation's leading civilian trauma centers to build and sustain true expertise in delivering trauma care on the battlefield. The SOF Truths provide valuable insights into the required conditions for success of this new training paradigm.

Treatment of Bleeding in Severely Thrombocytopenic Patients with Transfusion of Dimethyl Sulfoxide (DMSO) Cryopreserved Platelets (CPP) Is Safe - Report of a Phase 1 Dose Escalation Safety Trial

Treatment of Bleeding in Severely Thrombocytopenic Patients with Transfusion of Dimethyl Sulfoxide (DMSO) Cryopreserved Platelets (CPP) Is Safe - Report of a Phase 1 Dose Escalation Safety Trial