US Army Medical Research Research Articles

Automated Evaluation of Microvascular Blood Flow and Oxygenation following Trauma and Hemorrhage in Rats

Studies evaluating blood flow and oxygen partial pressure (PO2) may not directly measure both parameters and may depend on investigator's selection of measuring sites. In addition, relevant systemic parameters may not be simultaneously recorded. Therefore, we implemented a new automated system for blood flow and PO2 acquisition in large tissue areas while collecting systemic information. In 10 animals anesthetized with isoflurane, cardio‐respiratory parameters were continuously recorded, in experiments lasting up to 4 h. Other data were collected at baseline, and after laparotomy and 30 min hemorrhage (40% of total blood volume). A cremaster muscle was spread over a thermostatically‐controlled pedestal fixed to a motorized stage. Sixteen noninvasive PO2 measurements using oxygen‐dependent phosphorescence quenching and fiber‐optics were performed during a computer‐controlled tissue scan. In the same areas used for PO2 measurements, microvascular blood flow was estimated employing laser speckle contrast imaging. Blood was sampled for extensive biochemistry and coagulation profiles. The system was used successfully by different operators. One set of flow and PO2 measurements was completed in less than 90 s. Changes in muscle flow correlated with local PO2 but several systemic parameters did not correlate with blood flow and PO2, emphasizing the importance of performing both local and systemic evaluations. System advantages include integration between local and multiple systemic parameters, unbiased data collection/analysis, easy implementation, improved performance, no need for customized programming, and simplified training compared to intravital microscopy.Support or Funding InformationSupported by US Army Medical Research and Materiel Command.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Efficacy of Naloxone as a Treatment for an Acute Exposure to Aerosolized Carfentanil

Carfentanil (CRF) is a powerful synthetic opioid that is approximately 100 times more potent than fentanyl and 10,000 times more potent than morphine. CRF originally was intended to be used as a sedative for big game animals in a veterinary setting, but is becoming increasingly common in the illicit drug trade. The increased use of CRF by people has put strain on first responders, who need to administer increased doses of naloxone (Naracan®, Evzio®), an FDA‐approved opioid antagonist, to counteract the effects. First responders may also worry about the potential of self‐exposure through inhalation or dermal contact with CRF. We set out to study the effectiveness of naloxone against a potentially lethal dose of inhaled CRF in male ferrets. Ferrets were implanted with telemetry devices to study cardiac parameters and exposed to aerosolized CRF in a whole‐body plethysmography chamber to record respiratory parameters. We observed profound depression of respiration which resulted in the animals becoming apneic 23% ± 9% of the time during exposure. We also observed cardiac abnormalities, in the form of premature junctional contractions (PJCs), in the ferrets during the apneic periods. At our acute exposure dose, which was lethal in 9% of our animals, ferrets were unresponsive and incapacitated for 105.6 ± 20.0 minutes following the end of exposure. Naloxone, when administered intramuscularly at human equivalent doses ranging from 4 to 40 mg, significantly reduced the time that ferrets were incapacitated following exposure. Although we saw no significant difference in the reduction of time that the animals were incapacitated between the treatment groups (average times between treatment and revival ranged from 6.5 to 9.7 minutes), higher doses of naloxone did revive a portion of the ferrets quicker than the lower doses (some as soon as 1.5–3.5 minutes post‐treatment). We also observed that naloxone was able to significantly reduce the amount of apneic episodes in CRF‐exposed ferrets and resolved the observed cardiac PJCs. Our results suggest that naloxone is a viable treatment against the effects of a potentially lethal dose of inhaled CRF in ferrets. These data have the potential to inform and aid treatment strategies for an acute inhalation exposure to synthetic opioids.Support or Funding InformationOpinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the US Army. This research complied with the Animal Welfare Act and implementing Animal Welfare Regulations and adhered to the principles noted in The Guide for the Care and Use of Laboratory Animals. This research was supported by DTRA RD‐CB. M.R. and N.A.A. were supported in part by an appointment to the Postgraduate Research Participation Program at the US Army Medical Research Institute of Chemical Defense administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the US Department of Energy and USAMRMC.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Blast-Related Traumatic Brain Injury: Current Concepts and Research Considerations.

Traumatic brain injury (TBI) is a well-known consequence of participation in activities such as military combat or collision sports. But the wide variability in eliciting circumstances and injury severities makes the study of TBI as a uniform disease state impossible. Military Service members are under additional, unique threats such as exposure to explosive blast and its unique effects on the body. This review is aimed toward TBI researchers, as it covers important concepts and considerations for studying blast-induced head trauma. These include the comparability of blast-induced head trauma to other mechanisms of TBI, whether blast overpressure induces measureable biomarkers, and whether a biodosimeter can link blast exposure to health outcomes, using acute radiation exposure as a corollary. This examination is contextualized by the understanding of concussive events and their psychological effects throughout the past century’s wars, as well as the variables that predict sustaining a TBI and those that precipitate or exacerbate psychological conditions.Disclaimer: The views expressed in this article are solely the views of the authors and not those of the Department of Defense Blast Injury Research Coordinating Office, US Army Medical Research and Development Command, US Army Futures Command, US Army, or the Department of Defense.

2493. Marburg Virus Disease: Virulence of Angola vs. Musoke Strain in Cynomolgus Macaques

BackgroundFrom 2004 to 2005, an outbreak of Marburg virus, a filovirus, in Angola led to a case-fatality rate of 90 percent. However, little information is available regarding the virulence of the Angola strain from this outbreak compared with the virulence of other strains. Therefore, we sought to assess time to selected outcomes in non-human primates (NHPs) experimentally infected with either Angola or Musoke Marburg strains.MethodsBetween 2012 and 2017, nine therapeutic trials at the US Army Medical Research Institute of Infectious Diseases were conducted in Macaca fascicularis monkeys challenged with 1 to 10,000 plaque forming units of Marburg virus administered intramuscularly. The current study population was comprised of 90 control NHPs, of which, 61 were administered Angola strain in four separate trials and 29 with Musoke strain in five trials. Clinical responses including development of rash and oral intake were collected following infection. The primary outcome of interest was time to death or euthanasia post-inoculation between strains evaluated using Cox proportional hazards regression. Secondary endpoints included time to development of a petechial rash and time to decreased appetite.ResultsFollowing Marburg virus challenge, all NHPs died and most NHPs experienced decreased food consumption (97%), and petechial rash (96%). The median time to death for Angola-infected NHPs was 8.9 days (25th, 75th percentiles: 7.9, 9.3), whereas Musoke-infected NHPs survived for a median of 10.0 days (25th, 75th percentiles: 9.0, 10.9) (Figure 1). Irrespective of strain, petechial rash was preceded by decreased food consumption by 0.7 days (SD 1.5) on average. Angola strain was associated with statistically significant earlier death (adjusted HR = 21.8; 95% CI: 8.9, 53.2), earlier development of petechiae (adjusted HR = 17.6; 95% CI: 7.0, 44.5) and earlier loss of appetite (adjusted HR = 5.8; 95% CI: 2.9,11.7).ConclusionThis was the first study to compare survival and clinical characteristics in NHPs between these strains. Despite sharing the similar genetic lineage, our data strongly supports increased virulence of Angola strain compared with Musoke strain. Pathophysiological mechanisms involved in increased virulence require further study. Disclosures All authors: No reported disclosures.

Non-trauma-focused meditation versus exposure therapy in veterans with post-traumatic stress disorder: a randomised controlled trial

Post-traumatic stress disorder (PTSD) is a complex and difficult-to-treat disorder, affecting 10-20% of military veterans. Previous research has raised the question of whether a non-trauma-focused treatment can be as effective as trauma exposure therapy in reducing PTSD symptoms. This study aimed to compare the non-trauma-focused practice of Transcendental Meditation (TM) with prolonged exposure therapy (PE) in a non-inferiority clinical trial, and to compare both therapies with a control of PTSD health education (HE). We did a randomised controlled trial at the Department of Veterans Affairs San Diego Healthcare System in CA, USA. We included 203 veterans with a current diagnosis of PTSD resulting from active military service randomly assigned to a TM or PE group, or an active control group of HE, using stratified block randomisation. Each treatment provided 12 sessions over 12 weeks, with daily home practice. TM and HE were mainly given in a group setting and PE was given individually. The primary outcome was change in PTSD symptom severity over 3 months, assessed by the Clinician-Administered PTSD Scale (CAPS). Analysis was by intention to treat. We hypothesised that TM would show non-inferiority to PE in improvement of CAPS score (Δ=10), with TM and PE superior to PTSD HE. This study is registered with ClinicalTrials.gov, number NCT01865123. Between June 10, 2013, and Oct 7, 2016, 203 veterans were randomly assigned to an intervention group (68 to the TM group, 68 to the PE group, and 67 to the PTSD HE group). TM was significantly non-inferior to PE on change in CAPS score from baseline to 3-month post-test (difference between groups in mean change -5·9, 95% CI -14·3 to 2·4, p=0·0002). In standard superiority comparisons, significant reductions in CAPS scores were found for TM versus PTSD HE (-14·6 95% CI, -23·3 to -5·9, p=0·0009), and PE versus PTSD HE (-8·7 95% CI, -17·0 to -0·32, p=0·041). 61% of those receiving TM, 42% of those receiving PE, and 32% of those receiving HE showed clinically significant improvements on the CAPS score. A non-trauma-focused-therapy, TM, might be a viable option for decreasing the severity of PTSD symptoms in veterans and represents an efficacious alternative for veterans who prefer not to receive or who do not respond to traditional exposure-based treatments of PTSD. Department of Defense, US Army Medical Research.

Serum GFAP and UCH-L1 for prediction of absence of intracranial injuries on head CT (ALERT-TBI): a multicentre observational study

More than 50 million people worldwide sustain a traumatic brain injury (TBI) annually. Detection of intracranial injuries relies on head CT, which is overused and resource intensive. Blood-based brain biomarkers hold the potential to predict absence of intracranial injury and thus reduce unnecessary head CT scanning. We sought to validate a test combining ubiquitin C-terminal hydrolase-L1 (UCH-L1) and glial fibrillary acidic protein (GFAP), at predetermined cutoff values, to predict traumatic intracranial injuries on head CT scan acutely after TBI. This prospective, multicentre observational trial included adults (≥18 years) presenting to participating emergency departments with suspected, non-penetrating TBI and a Glasgow Coma Scale score of 9-15. Patients were eligible if they had undergone head CT as part of standard emergency care and blood collection within 12 h of injury. UCH-L1 and GFAP were measured in serum and analysed using prespecified cutoff values of 327 pg/mL and 22 pg/mL, respectively. UCH-L1 and GFAP assay results were combined into a single test result that was compared with head CT results. The primary study outcomes were the sensitivity and the negative predictive value (NPV) of the test result for the detection of traumatic intracranial injury on head CT. Between Dec 6, 2012, and March 20, 2014, 1977 patients were recruited, of whom 1959 had analysable data. 125 (6%) patients had CT-detected intracranial injuries and eight (<1%) had neurosurgically manageable injuries. 1288 (66%) patients had a positive UCH-L1 and GFAP test result and 671 (34%) had a negative test result. For detection of intracranial injury, the test had a sensitivity of 0·976 (95% CI 0·931-0·995) and an NPV of 0·996 (0·987-0·999). In three (<1%) of 1959 patients, the CT scan was positive when the test was negative. These results show the high sensitivity and NPV of the UCH-L1 and GFAP test. This supports its potential clinical role for ruling out the need for a CT scan among patients with TBI presenting at emergency departments in whom a head CT is felt to be clinically indicated. Future studies to determine the value added by this biomarker test to head CT clinical decision rules could be warranted. Banyan Biomarkers and US Army Medical Research and Materiel Command.

Abstract 3242: Differences in stromal tumor-infiltrating lymphocytes in breast cancer from women of African and European ancestry after accounting for tumor characteristics

Abstract Background: Women of African ancestry (AA) have higher breast cancer mortality than other US racial/ethnic groups, attributed to a number of social and economic factors. However, breast tumors in AA women are more likely to present with higher grade and lacking estrogen receptor (ER) expression compared to those from women of European ancestry (EA), suggesting the importance of tumor factors in breast cancer disparities. Emerging evidence suggests that stromal tumor-infiltrating lymphocytes (TIL-str) have a role in immune response to therapy and prognosis of breast cancer. The objective of this study was to examine associations between risk factors for breast cancer and TIL-str, with consideration of tumor characteristics. Methods: We reviewed the hematoxylin and eosin slides from surgical or biopsy tumor blocks of invasive breast cancer from 1,385 cases (986 AA and 399 EA women, aged 20-75 years) in the Women's Circle of Health Study, a multicenter case-control study in New York and New Jersey. In-person interviews were conducted at diagnosis to obtain data on demographics, family histories, hormone use, and reproductive and menstrual histories. Anthropometric measurements were taken by trained staff. TIL-str were scored as 0%, 1%, and 10% to 100% in increments of 10%. Linear regressions were performed to estimate differences in TIL-str scores (β). Results: In univariate analysis, TIL-str was higher in tumors from AA than those from EA women overall (mean score = 21.5% vs. 12.5; P&amp;lt;0.001). TIL-str score was positively associated with higher grade, larger tumor size, and ER and progesterone receptor (PR) negativity in both AA and EA women (all P&amp;lt;0.05). In EA women, TIL-str score was also higher in more advanced than early stage tumors and human epidermal factor receptor 2 (HER2) positive than negative tumors (both P&amp;lt;0.05), but not in AA women. In multivariable analysis, AA compared to EA race was significantly associated with a higher TIL-str score (β = 5.7%, P&amp;lt;0.001), adjusting for age at diagnosis, body mass index, menopausal status, tumor grade, size, stage, and molecular subtypes defined by ER, PR, and HER2. There was no apparent association between TIL-str and history of breast cancer in first-degree relatives, history of benign breast disease, hormone use, reproductive factors, or body size. Conclusion: Self-reported African compared to European ancestry is independently associated with higher TIL-str scores in breast tumors. Our findings suggest that immune response may play a role in the differences in breast cancer biology and outcomes between AA and EA women. (Funding: The Breast Cancer Research Foundation; NIH P01CA151135, R01CA100598, R01CA185623, P30CA072720, K07CA201334; US Army Medical Research and Material Command DAMD-17-01-1-0334) Citation Format: Ting-Yuan D. Cheng, Rochelle Payne Ondracek, Song Yao, Warren Davis, Angela Omilian, Mateusz Opyrchal, Elisa V. Bandera, Michael J. Higgins, Christine B. Ambrosone, Thaer Khoury. Differences in stromal tumor-infiltrating lymphocytes in breast cancer from women of African and European ancestry after accounting for tumor characteristics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3242.

Anabolic signaling responses to exercise and recovery whey protein are suppressed at high altitude

Lowlanders sojourning to high‐altitude (HA) often experience muscle mass losses that are attributable to negative energy balance and potential dysregulation of intramuscular anabolic signaling. The combined effects of exercise and high‐quality protein ingestion can ameliorate the effects of short‐term, moderate negative energy balance (10‐d, ≤ 40 % total energy requirements) on intramuscular anabolic signaling at sea level (SL); however, the ability to sustain this effect at HA is unknown. This study examined the combined effects of HA (4300 m) and severe negative energy balance (21‐d, 70% of SL total energy requirements) on mTORC1 signaling responses after aerobic exercise and recovery whey protein (WP) ingestion in recreationally active, native lowlanders (n = 8 males). Total body mass and fat‐free mass were assessed using dual energy x‐ray absorptiometry before and after the 21‐d energy deficit at HA. Muscle biopsies of the vastus lateralis were obtained at SL, acute HA (3‐h HA exposure, day 0), and chronic HA (day 21), before (BASE) and after completing 80‐min of steady‐state aerobic exercise followed by a 2 mile time trial (POST), and 3 h into recovery (REC). In each trial, 25 g WP was consumed as a drink immediately after the POST biopsy. Intramuscular anabolic signaling was determined using Western blot and RT‐qPCR. Participants lost 7.5 ± 0.8 kg of total body mass (4.3 ± 1.0 kg of fat‐free mass, P &lt; 0.05). POST and REC p‐mTORSer2448, p‐70S6KSer424/421 and p‐rpS6Ser235/236 were 2‐, 23‐, and 7‐fold higher (P &lt; 0.05) than BASE at SL. At acute HA, POST p‐mTORSer2448, and POST and REC p‐70S6KSer424/421 were not different (P &gt; 0.05) from BASE and were 1‐ and 20‐fold lower than SL (P &lt; 0.05). However, POST and REC p‐rpS6Ser235/236 at acute HA was 4‐fold higher (P &lt; 0.05) than BASE and not different from SL. At chronic HA, POST and REC p‐mTORSer2448 and p‐70S6KSer424/421 were not different from BASE and were 1‐ and 19‐fold lower (P &lt; 0.05) than SL. POST and REC p‐rpS6Ser235/236 at chronic HA was 2‐fold higher (P &lt; 0.05) than BASE, but overall phosphorylation was 4‐fold lower (P &lt; 0.05) than SL. BASE HIF‐1α mRNA expression tended to be higher (P = 0.07) at acute HA than SL, whereas REC HIF‐1α mRNA expression at acute and chronic HA were lower (P &lt; 0.05) than SL. REDD1 mRNA expression and REDD1/14‐3‐3 coupling were not affected by study phase. These findings indicate that acute HA exposure induces intramuscular anabolic resistance that is further impaired with chronic HA exposure concomitant with severe negative energy balance, suggesting a mechanism for muscle loss at HA.Support or Funding InformationThis material is based on the work supported by US Army Medical Research and Material Command and the US Army Natick Soldier Research Development and Engineering Center. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the US Army.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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&lt;0.05), and increased glycocalyx shedding, permeability as well as transmigrated leukocytes (vs. sham and ALB, P&lt;0.05). High levels of transmigration post‐shock was sustained after NS (883 ± 433 transmigrated cells/mm2, vs. sham, P&lt;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&lt;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&lt;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.

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 (&amp;gt;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&amp;lt;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

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&lt;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&lt;0.05). The rate of decline in SV during LBNP was greater in Caucasian women (ANOVA P&lt;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 &lt; 0.05). Overall, ER upregulated c‐myomiR expression compared to WM (P &lt; 0.05). Expression ofc‐myomiR was inversely associated (r= −0.700, P &lt; 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 &gt;50% of identified genera (Q &lt; 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.