Army Medical Research Research Articles

Development of a Single Reaction PCR Genotyping Assay for Two Separate Gene Loci in Mice

There are many variables to consider when optimizing a polymerase chainreaction (PCR) assay for a specific gene—from methods of DNA extraction to choosing an effective combination of primers. A multiplex PCR assay analyzing two genes in the same reaction may require additional optimization. The objective of this experiment was to optimize a single PCR assay to effectively genotype C57BL/6 transgenic mouse strains in which two genes have been modified. By removing a portion of the Es1 gene, the expression of serum carboxylesterase was interrupted, resulting in serum carboxylesterase knock out (Es1 KO) mice. In another location, the gene expressing mouse acetylcholinesterase was replaced by a gene expressing human acetylcholinesterase, resulting in a human acetylcholinesterase knock in (AChEKI) mouse strain. There are several possible genotypes for each mouse strain: for Es1 KO mice the possibilities are wild type (WT +/+), knock out (KO −/−), or heterozygous (Het +/−), and for AChEKI mice the possibilities are human (H/H), mouse (M/M), or heterozygous (M/H). Optimization of the PCR genotyping assays for these strains was achieved by altering the method of DNA extraction, annealing temperatures, and testing multiple primer sets. Initially, DNA was extracted from mouse fecal matter, but this method was later determined to produce unreliable results in the subsequent PCR assays. DNA extracted from tail tissue was found to be much more reliable. Utilizing tail tissue DNA, a single pair of primers was found that could differentiate WT, KO, and Het alleles in the Es1 KO strain. The optimal annealing temperature for these primers was determined using a temperature gradient experiment. Primers for genotyping the AChE KI strain were selected and optimized in a similar manner. Finally, a multiplex PCR assay was developed allowing for the simultaneous determination of the genotype of both genes in mice.Support or Funding Information*This research was supported in part by an appointment to the Postgraduate Research Participation Program at the U.S. Army Medical Research Institute of Chemical Defense administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U. S. Army Medical Research and Materiel Command.The experimental protocol was approved by the Animal Care and Use Committee at the United States Army Medical Research Institute of Chemical Defense and all procedures were conducted in accordance with the principles stated in the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011), and the Animal Welfare Act of 1966 (P.L. 89‐544), as amended.Threat Reduction Agency – Joint Science and Technology Office, Medical S&T Division.The views expressed in this abstract are those of the author(s) and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evaluating the Pharmacodynamics and Protective Efficacy of Catalytic Bioscavenger following Subcutaneous Administration in Guinea Pigs

Previous studies determined the guinea pig was a viableanimal model for evaluating subcutaneous (s.c.)injection as an alternative route of administration of catalytic Bioscavengers:enzymes designed to render nerve agents non‐toxic in systemic circulation following exposure. Recent studies have focused on evaluating the pharmacodynamics (PD) and protective efficacy (PE) of the lead bioscavenger candidate, organophosphorus hydrolase (OPH) variant, YT, as both unmodified and modified (PEGylated) versions of the enzyme. A comparative study was conducted with YT and three progressively sized PEG‐YT variants (934, 935, and 936) following a single bolus s.c. dose (5 mg/kg). Bioavailability increased linearly with molecular size such that the largest sized PEG‐YT variant, 936, afforded the highest bioavailability, thus promoting 936 to subsequent PD and PEstudies. Dose dependence on bioavailability was also examined via three cohorts of guinea pigs receiving doses (5, 15, 20 mg/kg) of either YT or 936 in which a linear dose response was also found. Guinea pigs receiving either YT or 936 (5 mg/kg) via intramuscular (i.m.)or s.c. routes had superimposable PD profiles regardless of route, suggesting an identical absorption pathway for the drugs. Protective efficacy was also evaluated by exposing the animals to 2xLD50 of sarin (GB) 7 dayspost‐enzyme administration; only the animals receiving 936 (i.m. and s.c.) survived 24 hours post‐exposure. Periodic s.c. dosing strategies were also evaluated with 936 in which three injections were administered either 2 or 24 hours apart for a total dose of 15 mg/kg followed by a protective efficacy assessment 8 days post‐administration. Overall these data support the use of s.c. injection as a practical route of administration for catalytic bioscavenger with PE Gylation, synergistically enhancing both bioavailability and extending drug half‐life in circulation.Support or Funding InformationThe views expressed in this abstract are those of the author(s) and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government. The experimental protocol was approved by the Animal Care and Use Committee at the United States Army Medical Research Institute of ChemicalDefense and all procedures were conducted in accordance with the principlesstated in the Guide for the Care and Use of Laboratory Animals and the AnimalWelfare Act of 1966 (P.L. 89–544), as amended. This work was supported by the Defense Threat Reduction Agency‐Joint Science and Technology Office, MedicalS&T Division.*Thisresearch was supported in part by an appointment to the Postgraduate ResearchParticipation Program at the U.S. Army Medical Research Institute of ChemicalDefense administered by the Oak Ridge Institute for Science and Educationthrough an interagency agreement between the U.S. Department of Energy and USAMRMC.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Effects Of Ethynylestradiol‐3‐Sulfate (EE‐3‐S) On Blood Volume and Organ Function In Pigs With Severe Traumatic Hemorrhage

IntroductionThe beneficial effects of estrogens to improve survival from hemorrhage have been suggested in experimental studies of rodents and minipigs, as well as some clinical observations. However, the underlying possible protective mechanisms remain unclear. This study investigated the effects of ethynylestradiol‐3‐sulfate (EE‐3‐S) on blood volume and organ functions in pigs following severe traumatic hemorrhage and hypotensive resuscitation.MethodsTwenty anesthetized and instrumented pigs were randomized into saline (n=10) and EE‐3‐S(n=10) groups. Femur fracture was performed in each pig's left leg, followed byhemorrhage of 55% of estimated blood volume and 15 min shock period. Afterwards, pigs were hypotensively resuscitated with a small volume of saline(4 ml/kg) or EE‐3‐S (1 mg/kg at concentration of 1 mg/ml, plus 3 ml saline/kg). Indocyanine green dye (5 ml of 1 mg/ml) was injected before femur fracture and hemorrhage and 15 min after EE‐3‐S administration with serial blood samples taken accordingly to determine changes of blood volume. Pigs remained anesthetized and were monitored for 6 h or until death, and blood samples were taken every 2 h for measurements of blood gases and chemistry.ResultsAll baseline measurements are similar between the two groups. Femur fracture and hemorrhage reduced mean arterial pressure to 54±6% of baseline values and reduced blood volume from 104±4 ml/kg to 67±4 ml/kg (both p<0.05) and reduced Hctfrom 31±1% to 28±1% in the saline resuscitated group. Similar reductions were observed with EE‐3‐S resuscitation in blood volume (from 109±4 ml/kg to 68±3 ml/kg, p<0.05) and Hct (30±1% to 27±1%, p<0.05). Compared to baseline value, creatinine, asparate aminotransferase (AST), and lactate dehydrogenase(LDH) were elevated at 6 h or death from 1.3±0.1 mg/dL to 3.1± 0.2 mg/dL, 33±3 U/L to 98±16 U/L, and 450± 55 U/L to 645±122 U/L, respectively (all p<0.05). Similar increases were observed in creatinine (1.4±0.2 mg/dL to 2.9±0.1 mg/dL), AST (32±3 U/L to 135±46 U/L) and LDH (450±35 U/L to 501±95 U/L) in the EE‐3‐S group (all p<0.05). Survival time was similar between the saline (212±43 min) and EE‐3‐S (212±39 min) groups.ConclusionsFollowing severe traumatic hemorrhage and small volume resuscitation, administration of EE‐3‐S did not affect blood volume, organ function or short term survival in this model.Support or Funding InformationUS Army Medical Research and 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.

Gene Expression in the Rat Thalamus following Chronic Neuropathic Pain Development: mRNA sequencing analysis

Chronic pain afflicts roughly 44% of Service Members returning from Iraq and Afghanistan, and it is estimated that 100 million Americans suffer from this condition. The underlying molecular mechanisms of chronic pain development have remained elusive. The broad objective of this study is to identify molecular signals and indications of chronic pain in key brain regions during the onset and progression of neuropathic pain, in a rodent model. For this experiment, we collected tissue from five brain regions (thalamus, amygdala, cingulate cortex, insular cortex, and somatosensory cortex), as well as the L4–L6 spinal cord and L5 dorsal root ganglia, from rats subjected to the spared nerve injury (SNI) protocol, a model for chronic neuropathic pain. Tissue was collected at 1, 3, 7, 14 and 21 days post‐injury from each tissue and RNA extracted for mRNA sequencing (TruSeq total RNA protocol and Illumina HISCAN‐SQ platform). We present here the detailed mRNA sequencing workflow (pipeline) customized to provide the full complement of mRNA data (transcriptome) for multiple brain regions over the time course of the SNI protocol. In our initial analysis we focused on the assessment of the transcriptome of the thalamus, which is an important center of pain processing in the brain. We focused in particular on the 21 day time point, which is when the mechanical allodynia reaches its peak and therefore the most severe alterations in transcription are expected. In rats experiencing chronic pain relative to controls, we observed a reduction in the expression of several neurotransmitter receptors, such as CHRM4, CHRNB3, CHRNA5, CHRNB4, CNR1, ADORA2A, GPR85, BDKRB2, MC5R, DRD1, DRD2, and associated G proteins, such as GPR85, GNAT2, GNB3, GNG7, as well as the neurotransmitter TAC1. These findings suggest a global desensitization of neural signaling in the thalamus corresponding to chronic pain. Analysis of all time points and brain regions is underway and will provide a comprehensive picture of gene expression changes during the onset and progression of chronic pain.Research was conducted in compliance with the Animal Welfare Act, and all other Federal requirements. The views expressed are those of the authors and do not constitute endorsement by the U.S. Army.Support or Funding InformationThis work was supported by funding from the United States Army Medical Research and Material Command.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Reactive Skin Decontamination Lotion (RSDL) Maintains Its Effectiveness against Cutaneous Applications of VX Nerve Agent for at Least 5 Years Past Its Expiration Date

Reactive Skin Decontamination Lotion (RSDL) is the primary product the US military has fielded to decontaminate cutaneous exposures to chemical warfare agents. RSDL is extremely effective at protecting guinea pigs against cutaneous applications of the nerve agent VX. The 24‐hour cutaneous LD50 of VX in un‐anesthetized haired clipped guinea pigs is 140 µg/kg. When RSDL is used to remove VX two minutes after application the LD50 is 7,310 µg/kg. But RSDL is expensive to field and has a limited operational life. In the summer of 2017 we addressed the question of reducing costs by comparing the effectiveness of two expired (2012, 2014) lots of RSDL, to a lot that expires in April 2020. We applied the LD30 dose of 5,500 µg/kg to groups of n=12 and used the different batches of RSDL to remove the VX. There was no statistical difference in survival (n=10, 8 and 9) between the groups. The RSDL packets did change color as they aged, with the non‐expired RSDL having a bright yellow color, the RSDL that expired in January 2014 having a gray color and the RSDL that expired in April 2012 having a black color. Analysis of the various RSDL packets by proton, carbon and phosphorus NMR spectroscopy indicated that the difference in the component composition was not greater than 1%. To determine if protection was due to physical removal of VX we exposed two groups of n=4 to 5,500 µg/kg and 2,250 µg/kg of VX and used RSDL trainers to remove the VX. In the 5,500 µg/kg group all 4 animals died, and in the 2,250 µg/kg group 2 animals died. These results argue that RSDL can be effective past its expiration date and that the protection provided is not due entirely to physical removal.The views expressed in this abstract are those of the author(s) and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government. The experimental protocol was approved by the Animal Care and Use Committee at the United States Army Medical Research Institute of Chemical Defense and all procedures were conducted in accordance with the principles stated in the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act of 1966 (P.L. 89‐544), as amended.Support or Funding InformationThis research was supported by the Defense Threat Reduction Agency – Joint Science and Technology Office, Medical S&T Division.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Organophosphate Hydrolase (OPH) Designed as a Functional Monomer

Organophosphate hydrolase (OPH) mutants haveshown potential use as a medical countermeasure against a range of differentorganophosphorus compounds (OPs). OPH is typicallyexpressed in bacteria as a homodimer. Two separate monomers (~37kDa each) self‐assemble throughnon‐covalent bonding at the enzyme face close to the putative active site, andenzyme activity is dependent upon dimer formation. OPH homodimers do notsecrete expediently from mammalian cells and are often misfolded. This causespotential problems when trying to express the protein from a heterologous plasmidor viral delivery system. To enhance secretion of OPH from mammalian cells, wesought to increase protein solubility without catastrophic detriment toactivity and without addition of fusion proteins. To this end, we designed OPH tobe expressed as a functional monomer by joining two OPH subunits with a poly‐glycinelinker and expressing it as a single polypeptide in bacteria to evaluateactivity. We created the single polypeptide OPH with a tether 10 or 35 aminoacids in length between the two halves, and named them T10 and T35, respectively. Western blot analysis and paraoxon hydrolysis assays revealedthat T10 was being produced and there was a 71% loss in specific activity comparedto an untethered homodimer positive control. This was a surprise as we expectedT10 to have no enzymatic activity since the tether was too short to allowproper dimer formation. The T35 functional monomer (~76 kDa) was produced showing 29% loss of specific activity. Both T10 and T35 OPH showed high molecular weight aggregate formation greater than 250 kDa in dynamic light scattering and native polyacrylamide gels. Construction of a functional OPH monomer with activity against OPs is instrumental in designing secretable OPHs for adeno‐associatedviral expression. It may also prove useful in the development of chimeric OPHs for maintaining control over the composition of each OPH half, allowing blended activities against a combination of organophosphorus substrates. This is the first attempt known for producing OPH as a functional monomer.Support or Funding InformationThe views expressed in this abstract are those of the author(s) and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government. This research was supported by the Defense Threat Reduction Agency – Joint Science and Technology Office, Medical S&T Division. This research was supported in part by an appointment to the Postgraduate Research Participation Program at the U.S. Army Medical Research Institute of Chemical Defense administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and USAMRMC.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The use of classification tree analysis to assess the influence of surgical timing on neurological recovery following severe cervical traumatic spinal cord injury.

Post hoc analysis of prospectively collected data. Assess the influence of surgical timing on neurological recovery using classification tree analysis in patients sustaining cervical traumatic spinal cord injury. Hôpital du Sacré-Coeur de Montreal METHODS: 42 patients sustaining cervical SCI were followed for at least 6 months post injury. Neurological status was assessed from the American Spinal Injury Association impairment scale (AIS) and neurological level of injury (NLI) at admission and at follow-up. Age, surgical timing, AIS grade at admission and energy of injury were the four input parameters. Neurological recovery was quantified by the occurrence of improvement by at least one AIS grade, at least 2 AIS grades and at least 2 NLI. Proportion of patients that improved at least one ASIA grade was higher in the group that received early surgery (75 vs. 41 %). The proportion of patients that improved two AIS grades was also higher in the group that received early surgery (67 vs. 38 %). Finally, 30 % of the patients that received early decompression improved two NLI as compared with 0% in the other group. Early surgery was also associated with a non-statistically significant improvement in functional recovery. Neurological recovery of patients sustaining cervical traumatic spinal cord injury can be improved by early decompression surgery performed within 19 h post trauma. U.S. Army Medical Research and Material Command, Rick Hansen Institute.

Countering Zika Virus: The USAMRIID Response.

The United States Army Medical Research Institute of Infectious Diseases (USAMRIID) possesses an array of expertise in diverse capabilities for the characterization of emerging infectious diseases from the pathogen itself to human or animal infection models. The recent Zika virus (ZIKV) outbreak was a challenge and an opportunity to put these capabilities to work as a cohesive unit to quickly respond to a rapidly developing threat. Next-generation sequencing was used to characterize virus stocks and to understand the introduction and spread of ZIKV in the United States. High Content Imaging was used to establish a High Content Screening process to evaluate antiviral therapies. Functional genomics was used to identify critical host factors for ZIKV infection. An animal model using the temporal blockade of IFN-I in immunocompetent laboratory mice was investigated in conjunction with Positron Emission Tomography to study ZIKV. Correlative light and electron microscopy was used to examine ZIKV interaction with host cells in culture and infected animals. A quantitative mass spectrometry approach was used to examine the protein and metabolite type or concentration changes that occur during ZIKV infection in blood, cells, and tissues. Multiplex fluorescence in situ hybridization was used to confirm ZIKV replication in mouse and NHP tissues. The integrated rapid response approach developed at USAMRIID presented in this review was successfully applied and provides a new template pathway to follow if a new biological threat emerges. This streamlined approach will increase the likelihood that novel medical countermeasures could be rapidly developed, evaluated, and translated into the clinic.

Epidemiological Review of Francisella Tularensis: A Case Study in the Complications of Dual Diagnoses.

Introduction:Tularemia is a rare but potentially fatal disease that develops in numerous wild and domestic animals, including lagomorphs, rodents, cats, and humans. Francisella tularensis bacterium, the causative agent of tularemia, was identified by veterinary personnel at Fort Riley, Kansas during a routine post-mortum evaluation of a domestic feline. However, before formal diagnosis was confirmed, the sample was sent and prepared for rabies testing at the Department of Defense (DoD) U.S. Army Public Health Command Central (PHC-C), Food Analysis and Diagnostic Laboratory (FADL). This case report provides insight on how veterinarian staff and laboratory personnel can clinically manage esoteric, unexplained, or post-mortum examinations. The epidemiologic characteristics of tularemia, F. tularensis as an organism of military interest, potential laboratory management of F. tularensis, and clinical findings on a case of feline tularemia are discussed. It further raises questions as to whether or not dead animals should be treated as sentinels and be pre-screened for select agents, especially in instances of dual diagnoses.Methods:A necropsy was performed on the cat by the Fort Riley veterinarian, DNA extraction and PCR analyses were conducted by FADL microbiologists, histology and immunohistology analyses were conducted by the Kansas State Veterinary Diagnostic Laboratory, and feline tissue and blood were sent to the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) for confirmatory testing and strain identification of tularemia.Results:Tularemia was identified in the spleen of the cat by the Fort Riley veterinarian and during the histological sampling of the spleen by the Kansas State Veterinary Diagnostic Laboratory. A specific subsequent real-time polymerase chain reaction (RT-PCR) in vitro diagnostic detection of target DNA sequences of F. tularensis was conducted by the FADL microbiologists using a Joint Biological Agent Identification and Diagnostic System (JBAIDS) Tularemia Detection Kit to detect a presumptive qualitative result to detect tularemia in feline and blood samples. USAMRIID also performed RT-PCR and identified genomic DNA from F. tularensis Type A, (SPL15.013.02), thus confirming the FADL’s initial presumptive result of F. tularensis. USAMRIID attempted to culture F. tularensis from three samples (swab, feline tissue, and transfer pipette tip), but no growth consistent with F. tularensis was observed on the cysteine heart agar with sheep blood and antibiotics (CHAB) and chocolate (CHOC) plates. Discussions:Our case study of a dual diagnosis of presumptive F. tularensis and possible rabies exposure transmission from a pet cat to its owner provides insight on how veterinarian staff and laboratory personnel can clinically manage esoteric, unexplained, or post-mortum examinations. Our case study also demonstrates the obligation for cooperation between animal health, human health, and public health professionals in the management of zoonotic diseases.

Hyperkalemia in Combat Casualties: Implications for Delayed Evacuation.

Fixed facilities and rapid global evacuation ensured that delayed complications of trauma, such as hyperkalemia, occurred late in the evacuation chain where renal replacement therapies were available. However, future conflicts or humanitarian disasters may involve prolonged evacuation times. We sought to quantify one potential risk of delayed evacuation by assessing hyperkalemia in combat casualties. Retrospective study of military members admitted to intensive care units in Iraq and Afghanistan from February 1, 2002, to February 1, 2011. This study was approved by the U.S. Army Medical Research and Materiel Command Institutional Review Board. Demographics, injury severity score, burn injury, mechanism of injury, vital signs, creatinine, and potassium were collected. Logistic regression models were used to identify incidence and risk factors for hyperkalemia. Of 6,011 patient records, 1,472 had sufficient data to be included for analysis. Hyperkalemia occurred in 5.8% of patients. Those with hyperkalemia had higher injury severity scores, higher shock index, were more likely to have acute kidney injury, and were more likely to die. On multivariate analysis, acute kidney injury and shock index were significantly associated with the development of hyperkalemia. In a subgroup of patients with data on creatine kinase, rhabdomyolysis was associated with hyperkalemia in the univariate model, but was not significant after adjustment. Hyperkalemia occurred in 5.8% of patients in our cohort of critically injured combat casualties. The development of hyperkalemia was independently associated with acute kidney injury and shock index. In future conflicts, with prolonged evacuation times, mitigation strategies should be developed to treat hyperkalemia in casualties before arrival at definitive care.

Collaborative Systems Biology Projects for the Military Medical Community.

This pilot study was conducted to examine, for the first time, the ongoing systems biology research and development projects within the laboratories and centers of the U.S. Army Medical Research and Materiel Command (USAMRMC). The analysis has provided an understanding of the breadth of systems biology activities, resources, and collaborations across all USAMRMC subordinate laboratories. The Systems Biology Collaboration Center at USAMRMC issued a survey regarding systems biology research projects to the eight U.S.-based USAMRMC laboratories and centers in August 2016. This survey included a data call worksheet to gather self-identified project and programmatic information. The general topics focused on the investigators and their projects, on the project's research areas, on omics and other large data types being collected and stored, on the analytical or computational tools being used, and on identifying intramural (i.e., USAMRMC) and extramural collaborations. Among seven of the eight laboratories, 62 unique systems biology studies were funded and active during the final quarter of fiscal year 2016. Of 29 preselected medical Research Task Areas, 20 were associated with these studies, some of which were applicable to two or more Research Task Areas. Overall, studies were categorized among six general types of objectives: biological mechanisms of disease, risk of/susceptibility to injury or disease, innate mechanisms of healing, diagnostic and prognostic biomarkers, and host/patient responses to vaccines, and therapeutic strategies including host responses to therapies. We identified eight types of omics studies and four types of study subjects. Studies were categorized on a scale of increasing complexity from single study subject/single omics technology studies (23/62) to studies integrating results across two study subject types and two or more omics technologies (13/62). Investigators at seven USAMRMC laboratories had collaborations with systems biology experts from 18 extramural organizations and three other USAMRMC laboratories. Collaborators from six USAMRMC laboratories and 58 extramural organizations were identified who provided additional research expertise to these systems biology studies. At the end of fiscal year 2016, USAMRMC laboratories self-reported 66 systems biology/computational biology studies (62 of which were unique) with 25 intramural and 81 extramural collaborators. Nearly two-thirds were led by or in collaboration with the U.S. Army Telemedicine and Advanced Technology Research Center/Department of Defense Biotechnology High-Performance Computing Software Applications Institute and U.S. Army Center for Environmental Health Research. The most common study objective addressed biological mechanisms of disease. The most common types of Research Task Areas addressed infectious diseases (viral and bacterial) and chemical agents (environmental toxicant exposures, and traditional and emerging chemical threats). More than 40% of the studies (27/62) involved collaborations between the reporting USAMRMC laboratory and one other organization. Nearly half of the studies (30/62) involved collaborations between the reporting USAMRMC laboratory and at least two other organizations. These survey results indicate that USAMRMC laboratories are compliant with data-centric policy and guidance documents whose goals are to prevent redundancy and promote collaborations by sharing data and leveraging capabilities. These results also serve as a foundation to make recommendations for future systems biology research efforts.

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

Effects of Hypobaric and Normobaric Hypoxia on Myogenic and Proteolytic Gene Expression in Humans

Muscle mass is reduced during extended exposure to a hypoxic environment. Current research suggests that the physiological response to normobaric and hypobaric hypoxia may be different. It is currently unknown if these previously described differences extend to the skeletal muscle and transcriptional response regulating muscle mass. PURPOSE: To determine the effects of normobaric and hypobaric hypoxia on myogenenic and proteolytic gene expression. METHODS: Recreationally trained subjects (n = 15; age = 24 ± 4 y; VO2max = 3.60 ± 0.83 L · min-1) completed three trials of 60-min cycling at 70% of Wmax followed by 4-h of recovery at ambient control conditions (975 m), normobaric hypoxia (4,420 m), and hypobaric hypoxia (4,420 m). For each trial, a muscle biopsy was taken from the vastus lateralis before exercise and at the end of the 4-h recovery period for analysis of gene expression (RT-qPCR). RESULTS: There were no differences in the myogenic gene expression of MYOD (p = 0.713), MYF-5 (0.053), or MYOG (0.832) between trials. MYF-6 was higher after exercise (p = 0.002) regardless of trial. MSTN decreased from pre- to post-exercise (p < 0.001) in all conditions and was lower in hypobaric hypoxia compared to control condition (p = 0.02) and normobaric condition (p = 0.037). There were no differences in the proteolytic gene expression of atrogin-1 with exercise (p =0.811) or between trials (p = 0.419). However, FOXO3 (p = 0.009) and MuRF-1 (p < 0.001) gene expression increased with exercise but were not different between conditions (p = 0.543, p = 0.327, respectively). CONCLUSION: These data indicate that hypoxic recovery from exercise, regardless of whether normobaric or hypobaric, does not affect the expression of genes related to myogenesis and proteolysis with the exception of a modest attenuation of myostatin in hypobaric hypoxia. Funding provided by the Department of Defense United States Army Medical Research and Materiel Command (DOD USAMRMC: W81XWH-15-2-0075).

Effects of Hypobaric and Normobaric Hypoxia on Mitochondrial Related Gene Expression

Environmental stimuli such as temperature and hypoxia can influence cellular signaling in the skeletal muscle. Previously we have reported no changes in gene expression related to mitochondrial development with acute exposure to normobaric hypoxia. However, exposure to hypobaric hypoxia may elicit different physiological responses. PURPOSE: To determine the response of skeletal muscle mitochondrial related gene expression after 4-h exposure to normobaric hypoxia (NH), hypobaric hypoxia (HH) and normobaric normoxia (NN) after exercise. METHODS: Recreationally trained participants (n = 15, age: 24 ± 4 y, height: 178 ± 12 cm, weight: 72.47 ± 13.84 kg, body fat: 14 ± 7%, VO2max: 3.60 ± 0.83 L · min-1, Wmax: 274 ± 72 W) each completed three trials of 1-h cycling at 70% of Wmax. Following exercise, participants sat in an environmentally controlled chamber for a 4-h recovery period in NH (4,420 m), HH (4,420 m), or NN (975 m) environmental conditions. Blood oxygen saturation was measured using pulse oximetry at baseline, 30 min into exercise, immediately after exercise, and 30 min into each hour of recovery. Muscle biopsies were taken from the vastus lateralis pre-exercise and after a 4-h exposure period. Samples were analyzed using qRT-PCR to assess gene expression related to mitochondrial development. RESULTS: Arterial oxygen saturation was lower in HH and NH trials compared to the NN trial (p < 0.001) and lower in the HH compared to NH (p = 0.001). PGC-1α, GABPA, ERRα, and NRF1 mRNA were not different between the three conditions or from pre-exercise (p = 0.804, 0.650, 0.956, 0.563, respectively). TFAM mRNA increased in NH from pre-exercise to post-exercise (p = 0.036) and was higher than NN (p = 0.011). CONCLUSION: These data indicate that gene expression related to mitochondrial development is only marginally affected (TFAM) by the type of hypoxic environment after a 4-h treatment despite differences in arterial oxygen saturation. Funding provided by the Department of Defense United States Army Medical Research and Materiel Command (DOD USAMRMC: W81XWH-15-2-0075).

1074 POSTTRAUMATIC STRESS DISORDER, CANINE COMPANIONSHIP, AND SLEEP: PRELIMINARY FINDINGS

Large numbers of Veterans with post-deployment psychological disorders have reported improvements in sleep in association with canine companionship. Studies have also provided support for an association between canine companionship and lower baseline heart rate. We tested both possibilities in a sample of combat Veterans undergoing inpatient treatment for posttraumatic stress disorder (PTSD) and participating in a Service Animal Training Intervention in which they shared custody of a service canine with another Veteran. Alternating custody enabled within-subjects modeling of associations between canine companionship and sleep. 18 military Veterans with deployment-related PTSD underwent mattress actigraphy for up to four weeks yielding 493 nights of sleep data. Mattress actigraphy relies upon accelerometers embedded in a mattress topper. The intended sleep period is delimited manually by blind raters after which sleep parameters are calculated automatically. Participants also rated morning restedness on a five-point scale following a total of 329 nights. Whether or not participants had custody of their service canines was carefully tracked. Linear mixed modeling was applied to the longitudinal data, controlling for AHI, snoring, and body movements. A random intercept was specified for each participant. Modeling morning restedness detected only main effects of minutes of actigraphic quiescence (F(1,324) = 4.44, p = 0.036) and canine presence (F(1,313) = 3.89, p = 0.049), both predictors having positive coefficients. Modeling of sleep heart rate also detected main effects of actigraphic quiescence (F(1,476) = 5.38, p = 0.021) and of canine presence (F(1,472) = 5.55, p = 0.018), as well as an effect of snoring (F(1,478) = 13.23, p = 0.0003). Quiescence and canine presence were negatively associated with sleep heart rate, while snoring was positively associated. After accounting for other predictors, canine presence was associated with lower sleep heart rate and greater morning restedness in Veterans with PTSD. Preliminarily, these results are consistent with Veterans’ reports of positive effects of a familiar canine presence on their sleep. This research was supported by Contract W81XWH-15-2-0005 to Steven H. Woodward, PhD, from the Defense Health Program, Military Operational Medical Research Program, and U.S. Army Medical Research and Materiel Command.

0126 NEUROIMAGING OF CIRCUIT-SPECIFIC PROTEIN SYNTHESIS IN HUMAN SUBJECTS DURING SLEEP-DEPENDENT MEMORY CONSOLIDATION

The notion that memory consolidation depends on protein synthesis is based on demonstrations that protein synthesis inhibitors prevent consolidation. We sought to demonstrate directly that protein synthesis is affected during sleep-dependent memory consolidation. We used L-[1-(11)C]leucine positron emission tomography (PET) to measure circuit-specific brain protein synthesis during a daytime nap opportunity. Subjects completed the texture discrimination task (TDT) before and after the nap opportunity. We randomized 34 subjects to either wakefulness or sleep and counterbalanced training to either left or right visual field. In accord with the retinotopic specificity of the TDT, we considered ipsilateral V1 as the untrained within-subject control. Following training on the TDT, subjects underwent the PET scan with simultaneous polysomnography. V1 was drawn on magnetic resonance volumes and transferred to corresponding PET volumes to compute average rates of protein synthesis in left and right V1. If we disregard visual field trained (left, right), the Condition (wakefulness, sleep) x Local State (untrained, trained) interaction was not statistically significant (F[1,32]=0.07,p=0.79,f^2=0.002). Consideration of visual field trained indicates that the Condition x Visual Field Trained x Local State interaction was statistically significant (F[1,30]=9.08,p=0.005,f^2=0.30). Side-to-side differences were statistically significant only in the sleep condition: protein synthesis in the trained hemisphere was significantly higher if training was in left visual field and significantly lower if training was in right visual field. Another way to view these results is protein synthesis was higher in the right V1 during sleep-dependent memory consolidation regardless of the location of training. If we disregard visual field trained, our study could indicate sleep-dependent memory consolidation depends on processes other than protein synthesis such as synaptic renormalization. Consideration of visual field trained, however, may indicate that each hemisphere has inherent properties requiring either reduced or elevated protein synthesis for memory consolidation to occur during sleep. Intramural Research Program of the National Institute of Mental Health, Intramural Research Program of the National Institute of Neurological Disorders and Stroke, and Military Operational Medicine Research Program of the United States Army Medical Research and Materiel Command.

Anticonvulsant drug polytherapy stops status epilepticus and prevents neuronal loss in soman‐exposed rats

IntroductionWhen treatment of status epilepticus(SE) is delayed, seizures become self‐sustaining and refractory to benzodiazepine therapy. Chemical warfare nerve agents (CWNAs), such as soman(GD), increase acetylcholine through inhibition of acetylcholine sterase and can lead to SE if seizures are not treated quickly and controlled. Prolonged seizures may lead to extensive neuropathology, spontaneous recurrent seizures and long‐term performance deficits. To identify a better treatment against pharmacoresistant seizures caused by GD exposure, we are using combinations of drugs aimed at reversing the effects of maladaptive receptor trafficking that follows CWNA exposure.MethodsRats were implanted with telemetry transmitters for continuous monitoring of EEG, body temperature and activity. After surgical recovery, rats were exposed to 1.2 LD50 GD and treated 1 min later with atropine sulfate and the oxime HI‐6 and then 40 min after seizure onset with the anticonvulsant phenobarbital with or without the NMDA antagonist ketamine (PHE/KET), the benzodiazepine midazolam (PHE/MDZ) or a combination of all three drugs (PHE/KET/MDZ).Two weeks after exposure, rat brains were sectioned and stained for neuropathology assessments.ResultsTriple therapy with PHE/KET/MDZ reduced total time spent in seizures (SE +early recurrent seizures) in the first 72 h and reduced the number of spontaneous recurrent seizures compared to midazolam monotherapy. Tripletherapy also reduced the duration of toxic signs in the hours following GD‐exposure and prevented the development of hyperactivity that occurs in the weeks following exposure compared to GD/MDZ monotherapy. In addition, tripletherapy reduced loss of neurons in the piriform cortex and thalamus compared to GD/MDZ monotherapy.ConclusionTriple therapy with PHE/KET/MDZ hadanticonvulsant, antiepileptic, and neuroprotective effects compared with midazolammonotherapy in GD‐exposed rats. Triple therapy may be a highly effective approach against pharmacoresistant seizures, such as those caused by GD exposure, and may allow for administration of lower doses, resulting in fewer side effects than are often seen with large doses of individual drug therapies.Disclaimer: The views expressed in this abstract are those of the authors and do not reflect the official policy of the Department of the Army, Department of Defense, or the U.S. Government. The experimental protocol was approved by the Animal Care and Use Committee at the United States Army Medical Research Institute of Chemical Defense and all procedures were conducted in accordance with the principles stated in the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act of 1966 (P.L. 89‐544), as amended.Support or Funding InformationKatieWalker, Erica Kundrick and Sean O'Brien were supported in part by an appointment to the Research Participation Program for the U.S. Army Medical Research and Materiel Command administered by the Oak Ridge Institute for Science and Education through an agreement between the U.S. Department of Energy and U.S. Army Medical Research and Materiel Command. This research was supported by the NIH U01 to Dr. Claude Wasterlain.

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).

Intake of Nutrients Among Individuals at Risk for Suicide Enrolled in the Better Resiliency Among Veterans and Non‐Veterans with Omega‐3's (BRAVO) Clinical Trial

The relationship between diet quality and chronic conditions such as cardiovascular disease and cancer has been well‐studied, but less is known about the relationship between diet quality and mental health. Specifically, cross disciplinary scientific coalitions have identified suicide risk as a key area for nutrition research. The objective of this study was to assess intake of those nutrients of national concern for deficiency in the 2010 Dietary Guidelines (DG) for persons identified at high risk of suicide. The study sample included 111 adults in the BRAVO randomized trial who had recently attempted suicide or had high levels of diagnosed suicide ideation(Mean Beck Suicide Scale score 9.5 ± 6.6). Baseline diet was assessed by two 24‐hour recalls and one 30‐day food frequency questionnaire. We present the intake data from the initial in‐person 24‐hour recall using the Automated Multiple Pass Method compared to the Dietary Reference Intakes (DRI). Chi‐square or Fisher's exact tests were used to determine differences by demographic and clinical measures. Mean intakes for the DG key nutrients were: calcium: 1047.2 ± 596.4 mg;, potassium: 2605.8 ± 1083.2 mg, vitamin D: 5.5 ± 4.6 μg, and dietary fiber: 15.0 ± 8.7 g. Sample Population Dietary Intake Related to the Dietary Reference Intakes for Key Nutrients Calcium (mg) Potassium (mg) Vitamin D (μg) Dietary Fiber (g) % Did not meet DRI p‐value % Did not meet DRI p‐value % Did not meet DRI p‐value % Did not meet DRI p‐value Gender 0.0179 0.6469 0.0765 0.7327 Female 58.1 97.7 95.4 90.7 Male 33.8 94.1 83.8 92.6 Age 0.1912 0.3172 0.4211 0.3022 18–30 25.0 95.0 80.0 95.0 31–50 47.1 100 91.2 97.1 51+ 47.4 93.0 89.5 87.7 BSS* 0.5179 0.0112 0.7939 0.3832 Suicidal ideation 45.3 100 87.5 93.7 No suicidal ideation 39.1 89.1 89.1 89.1 BDI Depression** 0.1533 0.0874 0.8332 0.0270 Normal 35.0 85.0 85.0 80.0 Mild/borderline 39.3 100 85.7 96.4 Moderate 55.6 95.6 88.9 97.8 Severe/extreme 27.8 100 94.4 83.3 Beck Suicide Scale Beck Depression Inventory These data indicate that of the DG 2010 nutrients identified as at risk of deficiency for the nation, a significantly higher percentage of persons who have the highest scores on the standard measure of suicidality (BSS) do not meet the DRI for potassium while significantly more individuals with documented depression did not meet the DRI recommendations for fiber. These results indicate that individuals at risk for suicide have poor diet quality and may benefit from education on increasing diet quality as an important step in supporting mental health.Support or Funding InformationThe BRAVO study is sponsored by award #W81XH‐13‐2‐0015 from the Department of Defense (DoD), U.S. Army Medical Research and Materiel Command (USARMC), Congressionally Directed Medical Research Programs (CDMRP) through the U.S. Army Medical Research Acquisition authority (USAMRAA).

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