Army Medical Research Research Articles

Endothelial Cell DNA Integrity following Anoxia/Reoxygenation Injury

Ischemia/reperfusion injury (I/RI) is associated with trauma and various diseases including stroke, heart attack and hemorrhagic shock. This study investigated the extent of DNA damage in an in vitro system of hypoxia/reoxygenation that may be useful for screening for drugs to reduce this form of injury. The usual method of subjecting cells to reduced oxygen (O2) by purging with nitrogen is a slow process and does not reproduce the rapid local reduction in O2 that occurs in vivo during ischemia. Glucose oxidase (GOX), however, rapidly oxidizes extracellular glucose producing a very low O2 level (< 0.4 μM) within 5 min with only minor reductions in glucose levels. We previously showed 10 min of anoxia followed by reoxygenation of human umbilical vein endothelial cells (HUVEC) damaged various biomolecules, such as lipids, proteins, and DNA. In the present study, human umbilical vein endothelial cells (HUVEC) were subjected to GOX‐induced hypoxia for 10 min and returned to medium and normoxia for up to 6 hrs. The most commonly used biomarker of DNA oxidation, in epidemiological as well as experimental studies, is 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodGuo), however this methods requires large amounts of DNA. Here we used a high‐throughput assessment of DNA damage utilizing a high‐sensitivity long‐run real‐time PCR technique for DNA‐damage quantification in both the mitochondrial and nuclear genome. Following hypoxia cells were provided normoxia (3% oxygen) for up to 6 hours, fixed in 50% isopropanol and DNA extracted with DNAzol (MRC, Cincinnati, OH). The isolated DNA was subjected to real‐time polymerase chain reaction (RT‐PCR) with primers designed to amplify large and small regions of mitochondrial and nuclear DNA where the extent of template amplification is inversely proportional to the lesion frequency within a given DNA sequence. Detected lesions per 10kb DNA were determined. Compared to hydrogen peroxide induced DNA damage, the test conditions produced extensive damage to both mitochondrial and nuclear DNA that could readily be detected by 4 and 6 hours. These results were also correlated with HPLC analysis of 8‐oxoguanine that indicated that this biomarker to oxidative stress was enhanced about 50%. This in vitro system of hypoxia/reoxygenation can be used to further understand responses of cells to I/RI and identify drugs and potential therapeutic windows in vitro that may be translated to ameliorate ischemia/reperfusion injury in vivo.Support or Funding InformationFunded by US Army Medical Research Materiel Command

Informing the Historical Record of Experimental Nonhuman Primate Infections with Ebola Virus: Genomic Characterization of USAMRIID Ebola Virus/H.sapiens-tc/COD/1995/Kikwit-9510621 Challenge Stock "R4368" and Its Replacement "R4415".

The creation of licensed medical countermeasures against Select Agents such as Ebola virus (EBOV) is critically dependent on the use of standardized reagents, assays, and animal models. We performed full genome reconstruction, population genomics, contaminant analysis, and characterization of the glycoprotein gene editing site of historical United States Army Medical Research Institute of Infectious Diseases (USAMRIID) nonhuman-primate challenge stock Ebola virus Kikwit “R4368” and its 2014 replacement “R4415.” We also provide characterization of the master stock used to create “R4415.” The obtained data are essential to understanding the quality of the seed stock reagents used in pivotal animal studies that have been used to inform medical countermeasure development. Furthermore, these data might add to the understanding of the influence of EBOV variant populations on pathogenesis and disease outcome and inform attempts to avoid the evolution of EBOV escape mutants in response to current therapeutics. Finally, as the primary challenge stocks have changed over time, these data will provide a baseline for understanding and correlating past and future animal study results.

Small-molecule treatment for Ebola

When an Ebola outbreak began ravaging West Africa in 2013, doctors and scientists had few medicines to treat the thousands of people who would become infected with the deadly virus in the years to follow. In fact, no antiviral therapeutics have received regulatory approval or demonstrated clinical efficacy against Ebola to date. There might be new hope, though, thanks to researchers at Gilead Sciences, the U.S. Army Medical Research Institute of Infectious Diseases, the Centers for Disease Control & Prevention, and Boston University. Those scientists, who presented in San Diego, have developed a small molecule called GS-5734 that’s effective at treating monkeys infected with the Ebola virus (Nature 2016, DOI: 10.1038/nature17180). GS-5734 is a monophosphoramidate prodrug of an adenosine analog. Once the small molecule is administered, enzymes in the body cleave GS-5734’s monophosphoramidate and eventually replace it with a triphosphate. This metabolite, the researchers suspect, inhibits the virus’s RNA-dependent RNA

US Army blood program: 2025 and beyond.

In preparing to support the Army in 2025 and beyond, the Army Blood Program remains actively engaged with the research and advanced development of blood products and medical technology to improve blood safety and efficacy in conjunction with the US Army Medical Research and Materiel Command. National and International Blood Bank authorities have noted that the US Army research and development efforts in providing new blood products and improving blood safety operate on the cutting edge of technology and are transformational for the global blood industry. Over the past 14 years, the Army has transformed how blood support is provided and improved the survival rate of casualties. Almost every product or process developed by or for the military has found an application in treating civilian patients. Conflicts have many unwanted consequences; however, in times of conflict, one positive aspect is the identification of novel solutions to improve the safety and efficacy of the blood supply.

Bridging the Gap of Standardized Animals Models for Blast Neurotrauma: Methodology for Appropriate Experimental Testing.

Recent military combat has heightened awareness to the complexity of blast-related traumatic brain injuries (bTBI). Experiments using animal, cadaver, or biofidelic physical models remain the primary measures to investigate injury biomechanics as well as validate computational simulations, medical diagnostics and therapies, or protection technologies. However, blast injury research has seen a range of irregular and inconsistent experimental methods for simulating blast insults generating results which may be misleading, cannot be cross-correlated between laboratories, or referenced to any standard for exposure. Both the US Army Medical Research and Materiel Command and the National Institutes of Health have noted that there is a lack of standardized preclinical models of TBI. It is recommended that the blast injury research community converge on a consistent set of experimental procedures and reporting of blast test conditions. This chapter describes the blast conditions which can be recreated within a laboratory setting and methodology for testing in vivo models within the appropriate environment.

The Surprising Origins of a World War II-Era Mural That Was Nearly Lost.

Jack McMillen studied at the Minneapolis School of Art in the early 1930s and went on to work in the New Deal’s Federal Art Project as part of the Works Progress Administration. 1 One of McMillen’s murals still hangs on the wall of the U.S. Post Office in College Park, Georgia. Another McMillen mural is in storage at the Smithsonian. When the country entered World War II, McMillen was drafted at the age of 32 into the U.S. Army. Before he even finished boot camp he was put him in charge of a traveling art show being used to promote the sale of War Bonds (Fig. 1). McMillen traveled the country by train, setting up shop in major cities and small towns from the east to west coasts. In early 1944, immediately after basic training, McMillen quickly fell ill. He started to mysteriously lose weight, and after undergoing extensive tests at Walter Reed General Hospital, doctors determined he had a tumor obstructing his esophagus. The Army surgeons responsible for his care admitted they had never performed the kind of surgery needed to ensure his recovery, but the mass was successfully removed, and turned out to be benign. McMillen, like countless other soldiers of the day, recuperated at the Forest Glen Annex, a former girls’ school purchased by the Army for treating convalescing patients during World War II. It was here that Private McMillen painted the mural which now hangs prominently on display at the National Museum of Health and Medicine (NMHM). The semi-abstractionist painting features convalescing soldiers engaged in a wide range of activities on the Forest Glen campus, identifiable by its eclectic architecture. According to his son, Jack McMillen simply titled the original mural “Noon” (Fig. 2). In 1994, 50 years after McMillen finished the mural, NMHM learned that the painting had been bolted to a wall in a deteriorating building on the Forest Glen campus, with a leak in the ceiling. Despite extensive water damage, the generous support of the U.S. Army Office of the Surgeon General and the Borden Center for Excellence helped make its full restoration possible. Following the museum’s move to Forest Glen in 2011, the 7.1 ft × 10.6 ft mural earned a prominent spot in the National Museum of Health and Medicine, U.S. Army Medical Research and Materiel Command, 2500 Linden Lane, Silver Spring, MD 20910. doi: 10.7205/MILMED-D-15-00395 FIGURE 1. McMillen cherished this signed photograph he received from the movie actress Linda Darnell when she attended the U.S. Army’s traveling art show in Los Angeles in 1943. (Photo courtesy of Dr. Brian A. McMillen.)

Pathosphere.org: pathogen detection and characterization through a web-based, opensource informatics platform.

BackgroundThe detection of pathogens in complex sample backgrounds has been revolutionized by wide access to next-generation sequencing (NGS) platforms. However, analytical methods to support NGS platforms are not as uniformly available. Pathosphere (found at Pathosphere.org) is a cloud - based open - sourced community tool that allows for communication, collaboration and sharing of NGS analytical tools and data amongst scientists working in academia, industry and government. The architecture allows for users to upload data and run available bioinformatics pipelines without the need for onsite processing hardware or technical support.ResultsThe pathogen detection capabilities hosted on Pathosphere were tested by analyzing pathogen-containing samples sequenced by NGS with both spiked human samples as well as human and zoonotic host backgrounds. Pathosphere analytical pipelines developed by Edgewood Chemical Biological Center (ECBC) identified spiked pathogens within a common sample analyzed by 454, Ion Torrent, and Illumina sequencing platforms. ECBC pipelines also correctly identified pathogens in human samples containing arenavirus in addition to animal samples containing flavivirus and coronavirus. These analytical methods were limited in the detection of sequences with limited homology to previous annotations within NCBI databases, such as parvovirus. Utilizing the pipeline-hosting adaptability of Pathosphere, the analytical suite was supplemented by analytical pipelines designed by the United States Army Medical Research Insititute of Infectious Diseases and Walter Reed Army Institute of Research (USAMRIID-WRAIR). These pipelines were implemented and detected parvovirus sequence in the sample that the ECBC iterative analysis previously failed to identify.ConclusionsBy accurately detecting pathogens in a variety of samples, this work demonstrates the utility of Pathosphere and provides a platform for utilizing, modifying and creating pipelines for a variety of NGS technologies developed to detect pathogens in complex sample backgrounds. These results serve as an exhibition for the existing pipelines and web-based interface of Pathosphere as well as the plug-in adaptability that allows for integration of newer NGS analytical software as it becomes available.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-015-0840-5) contains supplementary material, which is available to authorized users.

Hard Lessons In Ebola Research

Last year, at the height of the Ebola outbreak, Sina Bavari and Travis Warren found their labs at the U.S. Army Medical Research Institute of Infectious Diseases ( USAMRIID) flooded with compounds for testing against the Ebola virus. Well-meaning drug companies hoping to help stem the crisis were offering anything that might have activity against Ebola to the government lab, which is one of only a handful of places in the country with a containment facility for screening molecules against the deadly virus. Bavari and Warren were happy for the help but also overwhelmed as well as uncertain whether anything they were getting would actually work. Most of the compounds were developed for other diseases. Bavari, who is chief scientific officer of USAMRIID, compared the screening effort to trying to catch apples by holding a basket under a peach tree. Fortunately, at least one apple seems to have fallen. In ...

Virtual Anatomy-1900.

Since the Renaissance, medical students have learned human anatomy by studying cadavers. An anatomist dissected a corpse while explaining the spatial relationships of different organs and their interaction within the human body. By the 19th century, an increasing supply of cadavers, from executed criminals or unclaimed bodies, allowed students to learn by performing their own dissections. Pocket anatomical books served as guides, but three-dimensional anatomical structures were difficult to convey via narrative and wood-cut drawings. Dr. Louis Auzoux (1797–1880) began manufacturing papiermâche anatomical models in 1827 (Fig. 1). Enlarged models also allowed a detailed depiction of anatomical structures referenced by an associated guidebook and could be “dissected” to allow the study of the relationship of internal structures. The models served as an adjunct for dissection, as noted during a demonstration in 1832: “. . . here it was to be investigated; by removal of a few parts, the situation of particular points might be quickly descried, and the memory might just receive, just before an operation, the most valuable assistance.” Over 75 years later, the Flexner Report emphasized the continued value of these models: “It is one thing to take the body to pieces; it is something else to conceive these severed and dissociated elements in stereoscopic relation; and it is a still further task to unravel the tissues themselves: hence, on the macroscopic side, the prominence now given to reconstruction through drawing and modeling, and the close study of charts and of cross sections, of models and of special preparations that form the indispensable teaching museum.” The Army Medical School, established in 1893 at the Army Medical Museum and Library, was a postgraduate medical school to train physicians their duties as medical officers. In preparation for the school’s establishment, the museum purchased 21 Auzoux models, which were treated as anatomical teaching specimens and assigned Anatomical Series numbers by museum curators. Between 1894 and 1916, an additional 42 models were purchased. These models document the school’s effort to provide a quality medical education using the latest instructive techniques. By developing man-made dissectible representations of human anatomy to prepare the medical student work on the human body, Dr. Auzoux established the intellectual basis for future medical simulators that prepare late 20th and early National Museum of Health and Medicine, U.S. Army Medical Research and Materiel Command, 2500 Linden Lane, Silver Spring, MD 20910. The National Museum of Health and Medicine, a Department of Defense museum founded as the Army Medical Museum in 1862, is committed to documenting the nation’s military medical heritage while sustaining a repository of contemporary medical technology to advance military medicine. This series of articles illuminates objects and collections of interest to AMSUS readers. For more about NMHM, visit www.medicalmuseum.mil. The contributor hereby certifies the contribution to be a work of the U.S. Government, which has been prepared by an officer or employee of the U.S. Government as part of his or her official duties, and therefore precluded from copyright protection under 17 USC §105. The contribution is in the public domain by operation of laws of the United States of America. The opinions and assertions contained herein are those of the author and do not necessarily represent the views of the National Museum of Health and Medicine or the Department of Defense (DoD), any of the military services, or other DoD components or any other government agencies, and does not constitute an endorsement by the DoD of any of the opinions expressed, or any information, products, or services contained therein. The contributor hereby certifies the contribution to be a work of the U.S. government, which has been prepared by an officer or employee of the U.S. government as part of his or her official duties, and therefore precluded from copyright protection under 17 USC §105. The contribution is in the public domain by operation of laws of the United States of America. doi: 10.7205/MILMED-D-15-00394 FIGURE 1. Kidney, Anatomie Clastique Du Docteur Auzoux (1903), M-550.10834. (Courtesy National Museum of Health and Medicine, Silver Spring, MD.)

PATTERNS OF BEHAVIORAL HEALTH SERVICES PROVIDED IN A 2012 NATIONAL SAMPLE OF U.S. OUTPATIENT SUBSTANCE USE DISORDER TREATMENT FACILITIES: IMPLICATIONS FOR THE TREATMENT OF CO-OCCURRING DISORDERS

s / Drug and Alcohol Dependence 156 (2015) e102–e182 e143 Hapten selection for heroin vaccines Gary R. Matyas2, Fuying Li1,3, Joshua Antoline1,3, Rashmi Jalah4, Oscar Torres4, Zoltan Beck4, Arthur Jacobson1,3, Carl Alving2, Kenner Rice1,3 1 Drug Design and Synthesis Section, National Institute on Drug Abuse, NIH, Bethesda, MD, United States 2 US Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD, United States 3 National Institute on Alcohol Abuse and Alcoholism, NIH, Bethesda, MD, United States 4 US Military HIV Research Program, Herny M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, MD, United States Aims: The aim of this study is to select a heroin hapten for a heroin vaccine. Heroin rapidly degrades to 6-acetylmophine and morphine after injection. We hypothesize that a heroin hapten can bedesigned that is chemically stable and can induce antibodies that bind to heroin and its metabolites. Methods: Seven different opioid haptens were synthesized. A mercaptopropanamide group was attached as the linker at the C3, C6or thebridgenitrogen. The acetyl groups of heroinwere replaced with acetamide or 2-oxypropyl groups. The haptens were attached to tetanus toxoid and mixed with liposomal lipid A. Mice were immunized with 3 doses every 3 weeks. The sera were assayed for hapten antibodies. The mice were challenged by the subcutaneous route with heroin (0.75–1mg/kg) and efficacy was assessed by nociception assays. Results: Anti-hapten titers ranged from 100,000 to 6,000,000. Mice immunizedwith haptens coupled at the bridge nitrogenwere only partially protected with a % maximal potential effect (%MPE) ≥50. Animals immunized with C3 position haptens were not challengeddue to lowantibody titers.Mice immunizedwith a C6 linked morphine hapten (MorHap) had a %MPE of 35. After optimization of the MorHap conjugation procedure, the %MPE was <10 and was maintained 9 weeks after the last vaccination. Conclusions: The best haptenwasMorHap,which induced high antibody titers that protected the mice from heroin challenge. MorHap was coupled at the C6 position, whereas bridge nitrogen linked haptens induced high titer antibodies, but only had moderate efficacy. Financial support: This work was supported by a Cooperative Agreement (W81XWH-07-2-067) between the Henry M. Jackson Foundation and the US Army Medical Research and Materiel Command; an Avant Garde award fromNIDA (1DP1DA034787-01); and the NIH Intramural Research Programs of NIDA and NIAAA. http://dx.doi.org/10.1016/j.drugalcdep.2015.07.390 PATTERNS OF BEHAVIORAL HEALTH SERVICES PROVIDED IN A 2012 NATIONAL SAMPLE OF U.S. OUTPATIENT SUBSTANCE USE DISORDER TREATMENT FACILITIES: IMPLICATIONS FOR THE TREATMENT OF CO-OCCURRING DISORDERS Pia M. Mauro, C. Debra M. Furr-Holden, Ramin Mojtabai Department of Mental Health, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD Aims: According to the 2012 National Survey on Drug Use and Health, of the 20.7 million adults meeting substance use disorder (SUD) criteria, 8.4 million (40.7%) had co-occurring mental illness; only 12.2% received specialty treatment for SUD. Facilities often identify theirmain treatment focus, but the categoriesmaynot fully describe clusters of services. To address this potential discrepancy, we empirically derived classes of specialty SUD outpatient treatment facilitiesusingpatternsofbehavioralhealth servicesprovided in the United States. Methods:Data were obtained from the 2012 National Survey of Substance Abuse Treatment Services (N-SSATS). The current study included community-based facilities offering outpatient treatment in the 50 states and the District of Columbia. Latent class analysis (LCA) was used to identify unobserved classes of facilities based on patterns of observed behavioral health services offered. Results: Of the 14,995 facilities participating in the 2012 NSSATS, 11,488 (76.6%) met inclusion criteria. Facility-identified primary treatment focus included SUD treatment (52.0%), mental health (7.5%), SUD and mental health (36.4%), and general health/other (4.1%). Over a third (38.9%) of facilities had special programs or groups for people with co-occurring disorders. Preliminary analyses indicated an 8-class structure distinguishing facilities beyond their primary treatment focus. These findings will be discussed, along with the relationship between empiricallyderived classes and treatment foci. Conclusions: Behavioral health comorbidities are highly prevalent in SUD treatment samples. Given the evolving healthcare environment, understanding the degree to which facilities nationwide provide treatment for co-occurring disorders has clinical and policy implications. Financial Support: Ms. Mauro and Dr. Furr-Holden receive funding from NIDA T32DA007292. http://dx.doi.org/10.1016/j.drugalcdep.2015.07.391 Changes in the pain analgesic and heroin epidemiology

Hapten selection for heroin vaccines

s / Drug and Alcohol Dependence 156 (2015) e102–e182 e143 Hapten selection for heroin vaccines Gary R. Matyas2, Fuying Li1,3, Joshua Antoline1,3, Rashmi Jalah4, Oscar Torres4, Zoltan Beck4, Arthur Jacobson1,3, Carl Alving2, Kenner Rice1,3 1 Drug Design and Synthesis Section, National Institute on Drug Abuse, NIH, Bethesda, MD, United States 2 US Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD, United States 3 National Institute on Alcohol Abuse and Alcoholism, NIH, Bethesda, MD, United States 4 US Military HIV Research Program, Herny M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, MD, United States Aims: The aim of this study is to select a heroin hapten for a heroin vaccine. Heroin rapidly degrades to 6-acetylmophine and morphine after injection. We hypothesize that a heroin hapten can bedesigned that is chemically stable and can induce antibodies that bind to heroin and its metabolites. Methods: Seven different opioid haptens were synthesized. A mercaptopropanamide group was attached as the linker at the C3, C6or thebridgenitrogen. The acetyl groups of heroinwere replaced with acetamide or 2-oxypropyl groups. The haptens were attached to tetanus toxoid and mixed with liposomal lipid A. Mice were immunized with 3 doses every 3 weeks. The sera were assayed for hapten antibodies. The mice were challenged by the subcutaneous route with heroin (0.75–1mg/kg) and efficacy was assessed by nociception assays. Results: Anti-hapten titers ranged from 100,000 to 6,000,000. Mice immunizedwith haptens coupled at the bridge nitrogenwere only partially protected with a % maximal potential effect (%MPE) ≥50. Animals immunized with C3 position haptens were not challengeddue to lowantibody titers.Mice immunizedwith a C6 linked morphine hapten (MorHap) had a %MPE of 35. After optimization of the MorHap conjugation procedure, the %MPE was <10 and was maintained 9 weeks after the last vaccination. Conclusions: The best haptenwasMorHap,which induced high antibody titers that protected the mice from heroin challenge. MorHap was coupled at the C6 position, whereas bridge nitrogen linked haptens induced high titer antibodies, but only had moderate efficacy. Financial support: This work was supported by a Cooperative Agreement (W81XWH-07-2-067) between the Henry M. Jackson Foundation and the US Army Medical Research and Materiel Command; an Avant Garde award fromNIDA (1DP1DA034787-01); and the NIH Intramural Research Programs of NIDA and NIAAA. http://dx.doi.org/10.1016/j.drugalcdep.2015.07.390 PATTERNS OF BEHAVIORAL HEALTH SERVICES PROVIDED IN A 2012 NATIONAL SAMPLE OF U.S. OUTPATIENT SUBSTANCE USE DISORDER TREATMENT FACILITIES: IMPLICATIONS FOR THE TREATMENT OF CO-OCCURRING DISORDERS Pia M. Mauro, C. Debra M. Furr-Holden, Ramin Mojtabai Department of Mental Health, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD Aims: According to the 2012 National Survey on Drug Use and Health, of the 20.7 million adults meeting substance use disorder (SUD) criteria, 8.4 million (40.7%) had co-occurring mental illness; only 12.2% received specialty treatment for SUD. Facilities often identify theirmain treatment focus, but the categoriesmaynot fully describe clusters of services. To address this potential discrepancy, we empirically derived classes of specialty SUD outpatient treatment facilitiesusingpatternsofbehavioralhealth servicesprovided in the United States. Methods:Data were obtained from the 2012 National Survey of Substance Abuse Treatment Services (N-SSATS). The current study included community-based facilities offering outpatient treatment in the 50 states and the District of Columbia. Latent class analysis (LCA) was used to identify unobserved classes of facilities based on patterns of observed behavioral health services offered. Results: Of the 14,995 facilities participating in the 2012 NSSATS, 11,488 (76.6%) met inclusion criteria. Facility-identified primary treatment focus included SUD treatment (52.0%), mental health (7.5%), SUD and mental health (36.4%), and general health/other (4.1%). Over a third (38.9%) of facilities had special programs or groups for people with co-occurring disorders. Preliminary analyses indicated an 8-class structure distinguishing facilities beyond their primary treatment focus. These findings will be discussed, along with the relationship between empiricallyderived classes and treatment foci. Conclusions: Behavioral health comorbidities are highly prevalent in SUD treatment samples. Given the evolving healthcare environment, understanding the degree to which facilities nationwide provide treatment for co-occurring disorders has clinical and policy implications. Financial Support: Ms. Mauro and Dr. Furr-Holden receive funding from NIDA T32DA007292. http://dx.doi.org/10.1016/j.drugalcdep.2015.07.391 Changes in the pain analgesic and heroin epidemiology

Alan Magill, ASTMH Immediate Past President, 1953-2015.

Alan Magill, ASTMH Immediate Past President, 1953-2015.

The Denver Serum Bank

At the University of Colorado, Dr. Gordon Meiklejohn pursed the study of influenza and other respiratory pathogens for an unbroken period of 40 years, under the auspices of the Commission on Influenza of the Armed Forces Epidemiological Board through a series of contracts with the U.S. Army Medical Research and Development Command. Sera, throat washings, and other specimens for diagnosis were sent to Dr. Meiklejohn's laboratory. After serologic and virologic studies were carried out, aliquots of sera and virus samples were logged in and frozen. Sera were stored at -20°C and virus specimens at -70°C. These specimens became known as the Denver Serum Bank. The Bank supported military research programs and other researchers nationally and internationally until the 1990s when lacking of funding and considerations of administration, space, and cost resulted in the destruction of all specimens.

Abstract 4017: Matrix manipulation affects attachment and growth of breast cancer cells in a bone-like microenvironment in vitro

Abstract Breast cancer notoriously metastasizes to the bone, but it is unknown why breast cancer cells colonize there. Breast cancer cells may become dormant in the skeleton until a stimulated to grow. The aim of this project is to better understand the role of the bone matrix in determining dormancy or growth. Breast cancer cells were co-cultured with MC3T3-E1 mouse osteoblasts that formed a collagenous matrix. The matrix was manipulated by fixation, decellularization, and by varying the medium estrogen levels as the osteoblasts grew. MDA-MB-231-BRMS1GFP (BRMS), a metastatic suppressed variant of the human MDA-MB-231 breast cancer cell line, was used because these cells do not grow in the bone, allowing for observation of growth or dormancy. BRMS were added to a matrix produced by differentiated MC3T3-E1 cells. BRMS proliferated more on a culture of osteoblasts and their matrix when fixed with 1% paraformaldehyde than on a live osteoblasts matrix. Why do the cells proliferate more on the fixed matrix? We ruled out the possibility that live osteoblasts and cancer cells competed for growth medium. Another possibility was that BRMS attach differently to the matrices with live or fixed osteoblasts. We compared attachment of BRMS to live, fixed, and decellularized matrices and to tissue culture plastic, and after 60 minutes it was seen that more BRMS cells attached to a fixed matrix compared to the other conditions. Fluorescent microscopy revealed that BRMS had many attachment points on a fixed matrix. On the live osteoblasts matrix, the cancer cells remained attached to one another instead of attaching to the matrix. Experiments will determine if the attachment explains the effect on proliferation by testing for attachment factors such as β-actin and focal adhesion kinase. Breast cancer is more prevalent in post-menopausal women. Estrogen production decreases with menopause, leading to the hypothesis that the effects of estrogen deprivation on bone cause metastasis. Osteoblasts were grown in the presence of ICI, an estrogen inhibitor preventing estrogen from binding. Osteoblasts grown with and without ICI were assayed for alkaline phosphatase (AP), stained with von Kossa (mineralization), and with ChondrexTM (collagenous and noncollagenous proteins). While AP and mineralization levels were unaffected by the estrogen inhibition, collagenous proteins production decreased. We will study breast cancer cell attachment to matrices grown without estrogen. It is hypothesized that proliferation will increase on a matrix with less collagen, similar to the increased proliferation seen on a fixed matrix. Work was supported by the U.S. Army Medical Research &amp; Materiel Command under W81XWH-12-1-0127, the Metavivor Research Foundation, Undergraduate Research Funds from the Eberly College of Science &amp; the Biochemistry &amp; Molecular Biology Summer Undergraduate Research Program. Citation Format: Shelby J. Foster, Donna M. Sosnoski, Andrea M. Mastro. Matrix manipulation affects attachment and growth of breast cancer cells in a bone-like microenvironment in vitro. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4017. doi:10.1158/1538-7445.AM2015-4017

Abstract 3265: Thrombopoietin and megakaryocytes in breast cancer metastasis to bone

Abstract Bone is one of the preferred sites for breast cancer cells to metastases. Once patients are diagnosed with skeletal metastases, the relative five-year survival rate falls from 90% to less than16%. Thrombocytosis is a sign of poor prognosis for survival, and thromboembolisms are one of the leading causes of death in all cancers patients. These conditions are due to high numbers and/or high platelet activity. Megakaryocytes (MKs) are responsible for platelet production, but also play a role in bone metabolism and skeletal homeostasis. We had determined in two mouse models of experimental metastasis (MDA-MB-231 in athymic mice, and 4T1.2 in Balb/c mice), that bone metastasis was associated with increased numbers of MKs. Under normal conditions MKs represent less than 1% of the total bone marrow cell population, but after analysis of the femurs of the mice with metastasis, we found at least a two-fold increase in the numbers of MKs present in the bone marrow in the MDA-MB-231mice. The Balb/c mice injected in the mammary glands with mouse 4T1.2 cells showed a four-fold increase in MKs in the spleen (extrameduallary hematopoiesis). Therefore we hypothesized that increased megakaryocytosis was linked to metastasis, and that decreasing the number of MKs would decrease metastasis. We transferred a thrombopoietin (TPO) expression deletion mutation to a Balb/c background and inoculated the mice with 4T1.2 cells. These TPO -/- mice showed approximately a 90% decrease in bone marrow MKs compared to the wild type mice. The numbers of blood platelets was also decreased by 90%. We tested our hypothesis by inoculation of 4T1.2LUC cells into the mammary gland, and then followed the course of the cancer and metastasis by luciferase imaging. Contrary to our hypothesis the TPO -/- mice developed metastasis to the bone more rapidly than the heterozygotes or wild type mice. Literature suggests that platelets interact with tumor cells to contribute to hematogenous metastasis and angiogenesis. In the TPO-/- mice with very few platelets, metastasis was more aggressive and developed more rapidly than in the wild type or the heterozygote mice. The next step will be to separate the role of the megakaryocytes from the platelets. This work was sponsored by the U.S. Army Medical Research and Materiel W81XWH-10-1-0253 Citation Format: Walter Jackson, Andrea M. Mastro, Donna M. Sosnoski. Thrombopoietin and megakaryocytes in breast cancer metastasis to bone. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3265. doi:10.1158/1538-7445.AM2015-3265

SU‐E‐T‐147: Beam Specific Planning Target Volumes Incorporating 4DCT for Pencil Beam Scanning Proton Therapy of Thoracic Tumors

Purpose:The purpose of this study is to determine whether organ sparing and target coverage can be simultaneously maintained for pencil beam scanning (PBS) proton therapy treatment of thoracic tumors in the presence of motion, stopping power uncertainties and patient setup variations.Methods:Ten consecutive patients that were previously treated with proton therapy to 66.6/1.8 Gy (RBE) using double scattering (DS) were replanned with PBS. Minimum and maximum intensity images from 4DCT were used to introduce flexible smearing in the determination of the beam specific PTV (BSPTV). Datasets from eight 4DCT phases, using ±3% uncertainty in stopping power, and ±3 mm uncertainty in patient setup in each direction were used to create 8*12*10=960 PBS plans for the evaluation of ten patients. Plans were normalized to provide identical coverage between DS and PBS.Results:The average lung V20, V5, and mean doses were reduced from 29.0%, 35.0%, and 16.4 Gy with DS to 24.6%, 30.6%, and 14.1 Gy with PBS, respectively. The average heart V30 and V45 were reduced from 10.4% and 7.5% in DS to 8.1% and 5.4% for PBS, respectively. Furthermore, the maximum spinal cord, esophagus and heart dose were decreased from 37.1 Gy, 71.7 Gy and 69.2 Gy with DS to 31.3 Gy, 67.9 Gy and 64.6 Gy with PBS. The conformity index (CI), homogeneity index (HI), and global maximal dose were improved from 3.2, 0.08, 77.4 Gy with DS to 2.8, 0.04 and 72.1 Gy with PBS. All differences are statistically significant, with p values &lt;0.05, with the exception of the heart V45 (p= 0.146).Conclusion:PBS with BSPTV achieves better organ sparing and improves target coverage using a repainting method for the treatment of thoracic tumors. Incorporating motion‐related uncertainties is essentialThis work was supported by the US Army Medical Research and Materiel Command under Contract Agreement No. DAMD17‐W81XWH‐07‐2‐0121 and W81XWH‐09‐2‐0174.

SU-D-304-05: Validation of Low-Dose-Tail Modeling for Proton Pencil Beam Spot Scanning Using a Quality Assurance Test Pattern

Purpose: The purpose of this manuscript is to demonstrate the utility of a comprehensive test pattern in validating calculation models of the low-dose tails of proton pencil beam scanning (PBS) spots. Such a pattern has been used previously for quality assurance purposes to assess spot shape and location, and for determining monitor units. Methods: In this study, a scintillation detector was used to measure the test pattern in air at isocenter for two proton beam energies (115 and 225 MeV) of two IBA universal nozzles (UN). Planar measurements were compared with calculated dose distribution based on the weighted superposition of spot profiles previously measured using a pair-magnification method. Results: Including the halo component below 1% of the central dose is shown to improve the gamma-map comparison between calculation and measurement from 94.9% to 98.4% using 2 mm/2% criteria for the 115 MeV proton beam of UN #1. In contrast, including the halo component below 1% of the central dose does not improve the gamma agreement for the 115 MeV proton beam of UN #2, due to the cutoff of the halo component at off-axis locations. When location-dependent spot profiles are used for calculation instead of spot profiles at central axis,more » the gamma agreement is improved from 98.0% to 99.5% using 2 mm/2% criteria. The cutoff of the halo component is smaller at higher energies, and is not observable for the 225 MeV proton beam for UN #2. Conclusion: In conclusion, the use of a comprehensive test pattern can facilitate the validation of the halo component of proton PBS spots at off axis locations. The cutoff of the halo component should be taken into consideration for large fields or PBS systems that intend to trim spot profiles using apertures. This work was supported by the US Army Medical Research and Materiel Command under Contract Agreement No. DAMD17-W81XWH-07-2-0121 and W81XWH-09-2-0174.« less

Muscle Damage, Inflammatory, And Performance Responses To Submaximal, Metabolically-Matched Load Carriage And Non-Weight Bearing Endurance Exercise

PURPOSE: Metabolic and mechanical stressors elicited during submaximal load carriage (LC) and non-weight bearing, conventional endurance (CE) exercise differ, potentially influencing patterns of post-exercise muscle damage, inflammation, soreness, and performance. This study characterized muscle damage, inflammation, soreness, and lower-body muscular performance during recovery from submaximal LC and CE. METHODS: Forty adults (mean ± SD, 22 ± 4 y, 80 ± 10 kg, 4.0 ± 0.5 L·min-1) were randomly assigned (n = 20 per group), to perform either a 90 min submaximal, (2.2 ± 0.1 L·m-1) LC (24 ± 3 kg load) or metabolically-matched non-weight bearing (cycle ergometry), CE bout. Muscle damage, inflammation, ratings of soreness (deltoids, gluteus, quadriceps, and gastrocnemius/soleus), and quadriceps isometric and isokinetic peak force (PF), rate of force development (RFD), and total work were measured at baseline, 3, 24, 48, and 72 h post-exercise. RESULTS: Myoglobin (ng/mL) peaked 3 h post-exercise (70 ± 45) and remained elevated 72 h post-exercise (31 ± 10) compared to baseline (27 ± 8; P < 0.05). Overall, mean myoglobin levels were higher (P < 0.05) with LC (43 ± 31) than CE (34 ± 17). Creatine kinase (IU/L) increased similarly 3, 24, and 48 h post-exercise for LC and CE, but remained elevated 72 h post-exercise (327 ± 128) compared to baseline (205 ± 173) for LC only (mode-by-time, P < 0.05). Lactate dehydrogenase increased to a peak 3 h post-exercise and returned to baseline levels by 24 h, regardless of mode (P < 0.05). Post-exercise (24 and 48 h) interleukin-6 and C-reactive protein levels, which increased post-exercise relative to baseline, were 54% and 61% higher, respectively, for CE than LC (mode-by-time, P < 0.05). Post-exercise quadriceps and gastrocnemius/soleus soreness were similar between LC and CE, whereas gluteal soreness was higher for CE than LC, and deltoid soreness was greater for LC than CE (mode-by-time, P < 0.05). Independent of exercise mode, PF at 60°/sec at 24 h and RFD at 48 h decreased 5% and 13% lower (P < 0.05), respectively. No other differences in performance were observed. CONCLUSION: Although muscle damage, inflammatory, and soreness responses to metabolically-matched LC and CE were different, neither submaximal exercise bout produced meaningful decrements in lower-body, maximal muscular performance. Supported by U.S. Army Medical Research and Material Command.

Net Protein Balance after Load Carriage Exercise is Enhanced by Amino Acid Supplementation

Essential amino acid (EAA) supplementation during conventional endurance (CE) exercise attenuates post-exercise protein breakdown (PB) and enhances net protein balance (NET). Whether consuming EAA during load carriage endurance (LC) exercise confers the same anabolic effect is not clear, as the metabolic and mechanical strain of LC could upregulate protein turnover and branched-chain amino acid oxidation (OX). This study examined whole-body protein turnover during recovery from LC and CE with and without EAA supplementation using the reciprocal-pool model. Forty adults (mean ± SD, 22 ± 4 y, 80 ± 10 kg, 4.0 ± 0.5 L•min-1) were randomly assigned to perform a 90 min LC (24 ± 3 kg load), or metabolically-matched (2.2 ± 0.1 L•m-1), CE (cycle ergometry) exercise bout, during which EAA (10 g EAA, 37% leucine) or nonnutritive control (CON) drinks were consumed. Post-exercise PB was lower and OX was higher for EAA (53 ± 13 and 23 ± 4 µmol·kg-1·h-1) than CON (102 ± 13 and 10 ± 1 µmol·kg-1·h-1), irrespective of exercise mode (P < 0.05). Similarly, NET was not influenced by exercise mode, but was higher (P < 0.05) for EAA (49 ± 8 µmol·kg-1·h-1) than CON (-3 ± 1 µmol·kg-1·h-1). No other differences in protein turnover were observed. These data confirm the protein-sparing effect of EAA supplementation during endurance exercise, and suggest that if the metabolic demands are equivalent, whole-body protein turnover responses to weighted and non-weight bearing endurance exercise are the same. Supported by US Army Medical Research and Material Command and US Army Combat Feeding Directorate.

Mechanism of Cell Death Induced by in Vitro Hypoxia/Reoxygenation Using Glucose Oxidase in Human Cells

Ischemia/reperfusion injury (I/RI) is associated with trauma and various diseases including stroke and heart attack. This study investigated the mode of cell death induced in an vitro system of hypoxia/reoxygenation that may be useful for screening for drugs to reduce I/RI. The usual method of subjecting cells to reduced oxygen (O2) by purging with nitrogen is a slow process and does not reproduce the rapid local reduction in O2 that occurs in vivo during ischemia. Glucose oxidase (GOX), however, rapidly oxidizes extracellular glucose producing a very low O2 level (&lt; 0.4 µM) within 5 min with only minor reductions in glucose levels. Human umbilical vein endothelial cells (HUVEC) and HeLa cells were subjected to GOX‐induced hypoxia for 10 min and returned to medium and normoxia for up to 6 hrs. Cells loaded with tetramethylrhodamine ethyl ester (TMRE) and subjected to hypoxia showed that the mitochondrial permeability transition pore was compromised by 3 hrs and mitochondria were losing the ability to retain TMRE. Significant propidium iodide (PI)‐positive HUVEC cells, an indicator of cell death,were evident by 6 hrs. Similar responses were observed in HeLa cells indicating a generalized necroptotic cell death. When catalase was included in the medium following GOX‐induced hypoxia, cell death was prevented implicating hydrogen peroxide as an important contributor to this injury. This in vitro system can be used to further understand responses of cells to I/RI and identify drugs and potential therapeutic windows in vitro that may be translated to ameliorate ischemia/reperfusion injury in vivo. Funded by US Army Medical Research Materiel Command.