Naval Medical Research Center

Silver Spring, MD, United States

Naval Medical Research Center

Silver Spring, MD, United States
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News Article | April 17, 2017
Site: www.prweb.com

Z-Medica, LLC, a leading developer and marketer of hemostatic devices, announces today that a portfolio of QuikClot® products has been approved by the National Health Surveillance Agency (ANVISA), the Brazilian regulatory agency responsible for medical devices. “Brazil is the largest market for Z-Medica products in South America,” says Z-Medica President and CEO Stephen J. Fanning. “This approval marks a major milestone achievement for Z-Medica, further expanding our global footprint. It is also very important to the people of Brazil, who will now have proven life-saving products available to help them stop severe bleeding.” The ANVISA approval covers all of Z-Medica’s QuikClot products, which will be marketed to Brazilian military, hospitals and emergency medical services. About Z-Medica, LLC Z-Medica, LLC is a medical device company founded in 2002 that develops fast acting, easy-to-use hemostatic products that stop bleeding wherever it occurs, making it possible to save lives and improve patient outcomes. Based on tests conducted by the Naval Medical Research Center and the U.S. Army Institute for Surgical Research, the Committee on Tactical Combat Casualty Care (CoTCCC) chose QuikClot Combat Gauze® as the hemostatic dressing of choice on the battlefield for compressible hemorrhage not amenable to tourniquet use or as an adjunct to tourniquet removal if evacuation time is anticipated to be longer than 2 hours. QuikClot® products are developed and manufactured in the United States. Z-Medica, LLC is a privately-held company based in Wallingford, CT. For more information, visit QuikClot.com and Z-Medica.com.


News Article | December 28, 2016
Site: www.prweb.com

Z-Medica, LLC, a leading developer and marketer of hemostatic devices, announces today that they have signed a three-year agreement to equip the New York City Police Department with life-saving QuikClot® Belt Trauma Kits™ (BTK). The BTK is designed to fit on an officer’s duty belt and contains Z-Medica’s QuikClot Combat Gauze®, a tourniquet, compression bandage and gloves which can be used by a police officer to administer emergency first aid to control bleeding until medical attention can be sought. According to the Hartford Consensus III, a set of guidelines established by the American College of Surgeons in collaboration with the medical community and federal government to create a protocol for national policy to enhance survivability from active shooter and intentional mass casualty events, uncontrolled hemorrhage is the single most preventable cause of death in these situations. Following a program initiated by the New York State Division of Homeland Security and Emergency Services (DHSES) several months ago, this initiative represents the largest standardization of advanced bleeding control kits ever deployed by a single city. The NYPD is the largest police force in the United States. “Our products have been battlefield tested and deployed with active military personnel in war zones for years,” stated Z-Medica’s President and CEO Stephen J. Fanning. “In the United States, QuikClot has become a standard product now used by law enforcement, emergency services and hospitals.” The three-year, $2.7 million agreement provides for BTKs and help with training personnel on how to use the kits. Training and deployment of the kits is expected to begin early 2017. About Z-Medica, LLC Z-Medica, LLC is a medical device company founded in 2002 that develops fast-acting, easy-to-use hemostatic products that stop bleeding wherever it occurs, making it possible to save lives and improve patient outcomes. Based on tests conducted by the Naval Medical Research Center and the U.S. Army Institute for Surgical Research, the Committee on Tactical Combat Casualty Care (CoTCCC) chose QuikClot Combat Gauze® as the hemostatic dressing of choice on the battlefield for compressible hemorrhage not amenable to tourniquet use or as an adjunct to tourniquet removal if evacuation time is anticipated to be longer than 2 hours. QuikClot® products are developed and manufactured in the United States. Z-Medica, LLC is a privately-held company based in Wallingford, CT. For more information, visit quikclot.com and z-medica.com.


WALLINGFORD, Conn., Dec. 5, 2016 (GLOBE NEWSWIRE) -- via PRWEB - Z-Medica, LLC, a leading developer and marketer of hemostatic devices, announces today that they have teamed up with The American College of Surgeons (ACS) to provide basic bleeding control instructor training to surgeons. Working with the ACS's Committee on Trauma, the "Bleeding Control Basic" course is a pilot program that fulfills the intent of the White House's "Stop the Bleed" national initiative which is designed to increase the public's awareness of how they can play a role in saving lives by applying basic bleeding control techniques until appropriate medical care is available. So far, more than 350 surgeons have participated in the training program, which instructs surgeons how to train laypersons on the basics of bleeding control. Those surgeons who participate in the program can then return to their communities and teach the basic bleeding control techniques. "Teaming up with the American College of Surgeons to provide this training was a natural fit for us," said Z-Medica President and CEO Stephen J. Fanning. "By providing the necessary tools, products and personnel to support this effort, Z-Medica continues its commitment to helping save lives." More information on the White House initiative and basic bleeding control can be found at http://www.bleedingcontrol.org. About Z-Medica, LLC Z-Medica, LLC is a medical device company founded in 2002 that develops fast-acting, easy-to-use hemostatic products that stop bleeding wherever it occurs, making it possible to save lives and improve patient outcomes. Based on tests conducted by the Naval Medical Research Center and the U.S. Army Institute for Surgical Research, the Committee on Tactical Combat Casualty Care (CoTCCC) chose QuikClot Combat Gauze® as the hemostatic dressing of choice on the battlefield for compressible hemorrhage not amenable to tourniquet use or as an adjunct to tourniquet removal if evacuation time is anticipated to be longer than 2 hours. QuikClot® products are developed and manufactured in the United States. Z-Medica, LLC is a privately-held company based in Wallingford, CT. For more information, visit quikclot.com and z-medica.com. This article was originally distributed on PRWeb. For the original version including any supplementary images or video, visit http://www.prweb.com/releases/2016/12/prweb13892700.htm


News Article | November 29, 2016
Site: www.eurekalert.org

LOS ALAMOS, N.M., Nov. 29, 2016 -- A new bioinformatics platform called Empowering the Development of Genomics Expertise (EDGE) will help democratize the genomics revolution by allowing users with limited bioinformatics expertise to quickly analyze and interpret genomic sequence data. Researchers at Los Alamos National Laboratory and their collaborators at the Naval Medical Research Center developed EDGE, which is described in a paper recently published in Nucleic Acids Research. "We realized that while next-generation sequencing instruments are becoming more widespread and more accessible to the average biologist or physician, the bioinformatics tools required to process and analyze the data were not as user-friendly or accessible," said Patrick Chain, of Los Alamos' Biosecurity and Public Health group and EDGE team lead. "Given the large number of applications where sequencing is now used, a robust bioinformatics platform that encapsulates a broad array of algorithms is required to help address questions a researcher may have. We sought to develop a web-based environment where non-bioinformatics experts could easily select what pipelines they need and rapidly obtain results and interact with their data." Stopping the spread of disease--from naturally occurring or manmade threats -- requires an in-depth understanding of pathogens and how they work. To this end, the ability to characterize organisms through accurately and rapidly comparing genomic data is an important part of Los Alamos' national security mission. Technology advancements have fueled the development of new sequencing applications and will flood current databases with raw data. A number of factors limit the use of these data, including the large number of associated software and hardware dependencies and the detailed expertise required to perform this analysis. To address these issues, Chain and his team have developed an intuitive web-based environment with a wide assortment of integrated and pioneering bioinformatics tools in pre-configured workflows, all of which can be readily applied to isolate genome sequencing projects or metagenomics projects. EDGE is a user-friendly and open-source platform that integrates hundreds of cutting-edge tools and helps reduce data analysis times from days or weeks to minutes or hours. The workflows in EDGE, along with its ease of use, provide novice next-generation sequencing users with the ability to perform many complex analyses with only a few mouse clicks. This bioinformatics platform is described as an initial attempt at empowering the development of genomics dxpertise, as its name suggests, for a wide range of applications in microbial research. EDGE has already helped streamline data analysis for groups in Thailand, Georgia, Peru, South Korea, Gabon, Uganda, Egypt and Cambodia, as well as within several government laboratories in the United States. The paper "Enabling the democratization of the genomics revolution with a fully integrated web-based bioinformatics platform" was published in Nucleic Acids Research in partnership with the Defense Threat Reduction Agency, the Naval Medical Research Center-Frederick and the Henry M. Jackson Foundation. Patrick Chain earned his master's of science in microbial genomics from McMaster University and his doctoral degree in molecular microbiology and molecular genetics at Michigan State University. He is currently leading the Bioinformatics and Analytics Team and the Metagenomics Program within the Biosecurity and Public Health group at Los Alamos National Laboratory. His background is in microbial ecology, evolution, genomics and bioinformatics, having spent the past 20 years using genomics to study various microbial systems, including the human microbiome, other environmental metagenomic communities, various isolate microbes or single cells, including bacterial and viral pathogens as well as fungal, algal, plant and animal systems. He currently leads a team of researchers whose charge is to devise novel methods, algorithms and strategies for the biological interpretation of massively parallel sequencing data. Los Alamos National Laboratory, a multidisciplinary research institution engaged in strategic science on behalf of national security, is operated by Los Alamos National Security, LLC, a team composed of Bechtel National, the University of California, BWXT Government Group, and URS, an AECOM company, for the Department of Energy's National Nuclear Security Administration. Los Alamos enhances national security by ensuring the safety and reliability of the U.S. nuclear stockpile, developing technologies to reduce threats from weapons of mass destruction, and solving problems related to energy, environment, infrastructure, health, and global security concerns.


News Article | February 22, 2017
Site: www.nature.com

The study was approved by the ethics committee of the medical faculty and the university clinics of the University of Tübingen and strictly adhered to Good Clinical Practice and the principles of the Declaration of Helsinki. The study is registered at ClinicalTrials.gov (https://clinicaltrials.gov/ct2/show/NCT02115516) and in the EudraCT database, number 2013-003900-38. The study was carried out under FDA IND 15862 and with approval of the Paul-Ehrlich-Institute. Volunteers were healthy, 18–45 years old, malaria-naive adults. The full list of inclusion and exclusion criteria is given in the clinical trial protocol (Supplementary Information). All volunteers, except those in the second part who received the every 5-day regimen, received 10 mg kg−1 or 620 mg chloroquine base (Resochin, Bayer) loading dose 2 days before the first dose of PfSPZ Challenge, whichever dose was less, followed by weekly doses of 5 mg kg−1 or 310 mg through 5 days after the last dose of PfSPZ Challenge. Volunteers who were immunized on days 0, 5, and 10 received chloroquine on days 0 (loading dose), 5, 10, and 15. Randomization was performed on the day of first immunization by an independent party through provision of sealed envelopes to the syringe preparation team members, who diluted PfSPZ Challenge12, 13, 34 or loaded placebo (normal saline) into syringes at an allocation ratio of 9:5, PfSPZ:placebo. Only the syringe preparation team was aware of allocation to the intervention and had no other role in the trial. The rest of the team remained blinded until completion of CHMI of group III. PfSPZ Challenge dose escalation for groups I, II, and III was staggered by at least four weeks and in each group three sentinel volunteers (PfSPZ:placebo, 2:1) received injections one to seven days before the main group. For group I, II, and III the three immunizations were given at 28-day intervals and CHMI by DVI of 3.2 × 103 PfSPZ was done 8–10 weeks following the last immunization. In the second part the three immunizations were administered at 14-day and 5-day intervals and CHMI was done at 10 weeks post immunization. Chloroquine concentrations were measured by mass spectrometry in the blood of selected volunteers on the day before CHMI to exclude carry-over from the immunization period. Following CHMI volunteers were regularly visited for 20 weeks. The primary efficacy endpoint was the proportion of volunteers with thick blood smears positive for Pf within 21 days of CHMI, primary safety endpoint was occurrence of related grade 3 or serious adverse events from first chloroquine dose until the end of the follow-up. We performed a randomized, placebo-controlled, double-blind trial in healthy, malaria-naive, 18–45 year olds (TÜCHMI-002; ClinicalTrials.gov ID: NCT02115516). Between 1 May and 4 July 2014, 42 volunteers were randomized to receive either three doses of normal saline (placebo) or 3.2 × 103 PfSPZ (PfSPZ Challenge12, 13, 34) (group I), 1.28 × 104 PfSPZ (group II), or 5.12 × 104 PfSPZ (group III) by DVI12, 13 at four-week intervals (Extended Data Fig. 2). All volunteers received oral chemoprophylaxis with chloroquine starting with a loading dose (10 mg kg−1 chloroquine base or 620 mg, whichever dose was less) two days before first PfSPZ inoculation followed by weekly maintenance doses (5 mg kg−1) through five days after last PfSPZ inoculation; total of 10 doses. Chloroquine is not active against SPZ or liver-stage parasites35 and Pf asexual blood-stage parasites leave the liver between days 5 and 6 following inoculation36; hence dosing five days following inoculation ensured high drug levels upon liver egress. Dose-escalation was staggered in four-week intervals and at each dose escalation the ratio of placebo-immunized to PfSPZ-CVac-immunized subjects was 5:9. Following PfSPZ dose escalation, accelerated regimens (14- and 5-day intervals) were assessed using essentially the same procedures. A full report will be published separately. Eight to ten weeks after last vaccine or placebo dose (51–67 days after last chloroquine dose), protective efficacy was assessed by CHMI using DVI with PfSPZ Challenge12, 13. Daily thick blood smears were performed as previously described37 from day 6 to 21, following each DVI for immunization and CHMI, during antimalarial treatment and at each late follow-up visit. Slides were considered positive when at least two readers detected two unambiguous parasites, each. A negative slide was defined as no observed parasites in the volume of blood required to detect with 95% probability less than 10 parasites per μl (~0.5 μl). In case of discordance, a third reading was performed. In addition, 1.2 ml blood was sampled in EDTA tubes (Sarstedt) for nucleic acid extraction at the same time-points. DNA extraction control 610 (Bioline) was added and total nucleic acid (DNA and RNA) was isolated from 0.5 ml blood using the QIAamp DNA blood mini kit (Qiagen) according to the manufacturer’s specifications but without addition of RNase. Parasitaemia was calculated from a standard curve generated from serially diluted (2–20,000 parasites per ml) Pf 3D7 ring-stage synchronized cultured parasites, counted by microscopy and cytometry. All purified nucleic acid samples were stored at –20 °C until time of use. Reverse transcription quantitative polymerase chain reaction (RT–qPCR) was performed using TaqMan RNA-to-CT 1-Step Kit using published primers and probes38, with a different fluorophore and addition of a minor groove binder (probe: VIC-ATGGCCGTTTTTAGTTCGTG-NFQMGB; primers: 5′-GCTCTTTCTTGATTTCTTGGATG-3′ and 5′-AGCAGGTTAAGATCTCGTTCG-3′). Reactions were done in 384 wells at 48 °C for 20 min (reverse transcription), 96 °C for 10 min (enzyme activation), and 45 cycles of 95 °C for 15 s, 62 °C 1 min. Samples were run as triplicates with no-template control, no-RT control and positive controls in the same plate. Amplification controls were assessed manually and cycle values (C ) were calculated with the second derivative maximum method (LightCycler 480 software; version 1.5.1.62). The assay was validated in accordance to MIQE guidelines38, 39 and had a lower limit of quantification of 3 parasites per ml. qPCR results were not reported to the clinical and microscopy teams during the study period to maintain blinding of the study. Sample size was calculated with the intention to show, with a power of 80% and a two-tailed alpha of 5%, a difference in proportion of infected volunteers of 25% or less of immunized volunteers and 99% in controls, allocated in a 2:1 ratio (8 PfSPZ:4 controls), allowing for one dropout in each group (9:5). Clinical data were captured on paper case report forms and transferred to an electronic database (OpenClinica; version 3.2) by double data entry. Efficacy data were reported as proportions (primary) and time to parasitaemia. Safety and tolerability data were listed and reported as summary statistics. Results of immunological assays were explored by post hoc analyses and used to generate hypotheses about correlates and immunological mechanisms of protection. Analyses were coded in R (version 3.2.3)40 when not otherwise stated. Where possible, estimate and 95% confidence interval are given. Box plots display median (middle line), 25th (lower hinge) and 75th (upper hinge) quartile. Whiskers extend to values within 1.5× the inter-quartile ranges of the lower and upper hinges, respectively. A two-sided P value less than 5% was considered statistically significant. Flow cytometry data were analysed with Pestle v1.7, SPICE v5.3 (ref. 41) and Prism 6 (GraphPad). Graphs were rendered in FlowJo, SPICE, and Prism. For vaccine immunogenicity, comparisons to pre-vaccine were performed using Wilcoxon signed rank test with Bonferroni correction for multiple comparisons or two-way ANOVA with Bonferroni correction, as specified in the text. Immune responses assessed at baseline, two weeks after final immunization, and at the time of CHMI were compared to outcome (parasitaemia or no parasitaemia) after CHMI. Assessment of immune responses that correlated with sterile protection was made using a stratified Wilcoxon test controlling for vaccine dose group as a covariate. Sera were assessed for vaccine-induced antibodies by ELISA (enzyme-linked immunosorbent assay), immunofluorescence assay, inhibition of sporozoite invasion assay and protein arrays representing 91% of the Pf proteome. ELISAs were performed for antigens first expressed in PfSPZ (PfCSP, PfSSP2/TRAP, PfCelTOS, PfMSP5, PfAMA1), early liver stages (PfEXP1 and PfLSA1) and late liver stages (PfMSP1 and PfEBA175). The ELISA methods for each antigen are described below. Recombinant (r) proteins used in ELISA assays are listed in Supplementary Table 7. 96-well plates (Nunc Maxisorp Immuno Plate) were coated overnight at 4 °C with 0.5 μg to 2.0 μg recombinant proteins (Supplementary Table 7) in 50 μl per well in coating buffer (KPL). Plates were washed three times with 2 mM imidazole, 160 mM NaCl, 0.02% Tween 20, 0.5 mM EDTA and blocked with 1% Bovine Serum Albumin (BSA) blocking buffer (KPL) containing 1% or 5% non-fat dry milk (Supplementary Table 7) for 1 h at 37 °C. Plates were washed three times and serially diluted serum samples (in triplicates) were added and incubated at 37 °C for 1 h. After three washes, peroxidase labelled goat anti-human IgG (KPL) was added at a dilution of 0.05 μg ml−1 to 0.2 μg ml−1 (Supplementary Table 7) and incubated at 37 °C for 1 h. Plates were washed three times, ABTS peroxidase substrate was added for plate development, and the plates were incubated for defined periods at 22 °C room temperature (Supplementary Table 7). The plates were read with a Spectramax Plus384 microplate reader (Molecular Devices) at 405 nm. The data were collected using Softmax Pro GXP v5 and fit to a 4-parameter logistic curve, to calculate the serum dilution at OD 1.0. A negative control (pooled serum from non-immune individuals from malaria free area) was included in all assays. The following positive control sera were used for the different antigens: serum from an individual with anti-PfCSP antibodies for PfCSP; pooled sera from individuals immunized with PfLSA-1 and PfEBA-175 subunit vaccines respectively for PfLSA1 and PfEBA175; pooled sera from volunteers from a malaria-endemic area (acquired from a blood bank in Kenya) for PfAMA1, PfEXP1, and PfMSP1. No positive control sera were available for PfMSP5, PfSSP2/TRAP or PfCelTOS. Samples were considered positive if the difference between the post-immunization OD 1.0 and the pre-immunization OD 1.0 (net OD 1.0) was ≥50 and the ratio of post-immunization OD 1.0 to pre-immunization OD 1.0 (ratio) was ≥3. Purified PfSPZ (NF54 strain) from aseptic Anopheles stephensi mosquitoes produced by Sanaria were resuspended in phosphate buffered saline (PBS (pH 7.4)) to obtain 5 × 103 PfSPZ per 40 μl. 40 μl (5 × 103 PfSPZ) were added to each well of Greiner cellstar clear-bottom black 96-well plates (Sigma-Aldrich). After addition of the suspension, plates were left at room temperature for 12–18 h for air-drying. 50 μl of sera diluted in PBS with 2% BSA were added to each well of the 96-well plate containing air-dried PfSPZ. Sera samples were added at twofold dilutions starting at 1:50. After adding samples, plates were incubated at 37 °C for 1 h. Plates were washed in PBS three times on an Aquamax Microplate washer. Alexa Fluor 488 conjugated goat anti-human IgG (Molecular Probes) was diluted to 1:250 in PBS with 2% BSA and 50 μl was added to each well. The plates were then incubated for 1 h at 37 °C. Plates were washed three times with PBS on an Aquamax Microplate washer. 100 μl PBS was added to each well. The plates were sealed using a plate sealer and stored in the dark at 4 °C until data acquisition. Samples were assessed by scanning the plates using an Acumen eX3 laser scanning imaging cytometer. The positive control was pooled human serum taken two weeks after the last immunization from 12 uninfected volunteers immunized 4 or 5 times with 1.35 × 105 PfSPZ Vaccine5. The Acumen image cytometer scans the entire surface area of each well in a 96-well plate and the fluorescence intensity values (arbitrary units) therefore represent the signal from all 5 × 103 PfSPZ that were seeded in each well. The data obtained from the Acumen image cytometer were plotted to fit a 4-parameter sigmoidal curve in GraphPad Prism software using serum dilution (log transformed) as the x axis variable and arbitrary fluorescence units (AFU) on the y axis. Over many iterations during development of this assay we have determined that sera from naive volunteers in the USA and Europe, including pre-immune sera, always register an arbitrary fluorescence value less than 2 × 105 even at the highest concentration (1:50 dilution, see above) used in this assay. Moreover, for sera that react to PfSPZ, 2 × 105 AFU falls in the exponential portion of their sigmoidal curves. Therefore, 2 × 105 was chosen as a threshold in the automated immunofluorescence assay and the results for each volunteer for antibodies to PfSPZ are reported as the reciprocal serum dilution at which fluorescence intensity was equal to 2 × 105 AFU. HC-04 (1F9) (ref. 42) cells (hepatocytes) were obtained from the Naval Medical Research Center. Master and working cell banks were produced, and after establishing they were free of mycoplasma, were quality control released. For the assay they were cultured in complete medium (10% FBS in DMEM/F12 with 100 units per ml penicillin and 100 μg per ml streptomycin; Gibco by Life Technologies) in Entactic-Collagen IV-Laminin (ECL)-coated 96-well clear bottom black well plates (Greiner Bio-One North America) at a density of 2.5 × 104 cells per well, and incubated for 24 h at 37 °C, 5% CO with 85% relative humidity. Twenty-four hours later cells were infected with 104 aseptic, purified, cryopreserved PfSPZ per well, without or with sera diluted in a 12-point series from subjects immunized with PfSPZ Vaccine. The assay control included PfSPZ added with media alone. All subjects were assessed at pre-immunization (baseline), post-immunization and pre-CHMI time points. Three hours later, PfSPZ that had not invaded the HC-04 cells were removed by washing three times with Dulbecco’s phosphate-buffered saline (DPBS), and the cultures were fixed using 4% paraformaldehyde for 15 min at room temperature. Differential immunostaining was performed to differentiate between PfSPZ inside the hepatocytes versus PfSPZ outside the hepatocytes. PfSPZ outside the hepatocytes were stained with an anti-PfCSP mAb (2A10, 6.86 μg ml−1) (Protein Potential LLC, with permission from New York University School of Medicine) conjugated with Alexa Fluor 633 (far-red) (1 μg ml−1; custom-conjugated at GenScript). The hepatocytes were then permeabilized with 0.1% Triton X-100 for 10 min at room temperature, and the PfSPZ inside the hepatocytes were stained with the anti-PfCSP mAb (2A10, 6.86 μg ml−1) conjugated with Alexa Fluor 488 (green; 1 μg ml−1, conjugated from Genscript). The numbers and intensity of infected hepatocytes (green only) were counted by scanning the plates using an Acumen eX3 laser scanning imaging cytometer. The data obtained from the Acumen image cytometer were plotted to fit a 4-parameter sigmoidal curve in GraphPad Prism software using serum dilution (log transformed) as the x axis variable and arbitrary fluorescence units on the y axis. 75% inhibition was interpolated from the sigmoidal curves as the reciprocal serum dilution at which the fluorescent intensity of infected wells with serum was 25% of the negative control without serum. The number of invaded PfSPZ scored in this assay in the absence of sera ranged from 400–600 (intensity of 1–3 × 106 fluorescence units) (4% to 6% of those added to the wells). A whole-proteome microarray with 91% coverage of the Pf proteome (PfWPM) was produced by Antigen Discovery, Inc. (ADI). Proteins were expressed as previously described43 from a library of Pf partial or complete open reading frames (ORFs) cloned into a T7 expression vector pXI using an in vitro transcription and translation (IVTT) system, the Escherichia coli cell-free Rapid Translation System (RTS) kit (5 Prime). The library was created through an in vivo recombination cloning process with PCR-amplified Pf ORFs, and a complementary linearized expressed vector transformed into chemically competent E. coli was amplified by PCR and cloned into pXI vector using a high-throughput PCR recombination cloning method described elsewhere44. Each expressed protein includes a 5′ polyhistidine (HIS) epitope and 3′ haemagglutinin (HA) epitope. After expressing the proteins according to manufacturer’s instructions, translated proteins were printed onto nitrocellulose-coated glass AVID slides (Grace Bio-Labs) using an Omni Grid Accent robotic microarray printer (Digilabs, Inc.). Microarray chip printing and protein expression were quality checked by probing random slides with anti-HIS and anti-HA monoclonal antibodies with fluorescent labelling. PfWPM chips contained 7,455 Pf peptide fragments, representing proteins from 4,805 unique genes, 302 IgG positive control spots and 192 spotted IVTT reactions without Pf ORFs (IVTT controls). For each PfWPM chip, 3 replicates were printed per microarray slide on 3 nitrocellulose pads. IgG-positive control spots were included as an assay control, whereas IVTT control spots were included as a sample-level normalization factor. Serum samples were diluted 1:100 in a 3 mg ml−1 E. coli lysate solution in protein arraying buffer (Maine Manufacturing) and incubated at room temperature for 30 min. Chips were rehydrated in blocking buffer for 30 min. Blocking buffer was removed, and chips were probed with pre-incubated serum samples using sealed, fitted slide chambers to ensure no cross-contamination of sample between pads. Chips were incubated overnight at 4 °C with agitation. Chips were washed five times with TBS-0.05% Tween 20, followed by incubation with biotin-conjugated goat anti-human IgG (Jackson ImmunoResearch) diluted 1:200 in blocking buffer at room temperature. Chips were washed three times with TBS-0.05% Tween 20, followed by incubation with streptavidin-conjugated SureLight P-3 (Columbia Biosciences) at room temperature protected from light. Chips were washed three times with TBS-0.05% Tween 20, three times with TBS, and once with water. Chips were air dried by centrifugation at 1,000g for 4 min and scanned on a ScanArray Express HT spectral scanner (Perkin-Elmer), and spot and background intensities were measured using an annotated grid file (.GAL). Data were exported and normalized using the IVTT control spots for statistical analysis in R40. Raw spot and local background fluorescence intensities, spot annotations and sample phenotypes were imported and merged in R, where all subsequent procedures were performed40. Foreground spot intensities were adjusted by local background by subtraction, and negative values were converted to 1. Next, all foreground values were transformed using the base 2 logarithm (log ). The dataset was normalized to remove systematic effects by subtracting the median signal intensity of the IVTT controls for each sample. As the IVTT control spots carry the chip, sample and batch-level systematic effects, but also antibody background activity to the IVTT system, this procedure normalizes the data and provides a relative measure of the specific antibody binding to the non-specific antibody binding to the IVTT controls (a.k.a. background). With the normalized data, a value of 0.0 means that the intensity is no different than the background and a value of 1.0 indicates a doubling with respect to background. A seropositivity threshold was established for each protein on the chip using the top 2.5th percentile of the pre-immunization samples for each protein. Reactive antigens were defined as those that had seropositive responses after immunization and before CHMI, but which did not show seropositive responses in the mock-immunization group. PBMCs were isolated by density-gradient centrifugation from heparinized whole blood. Assessment of cellular immune responses using multi-parameter flow cytometry was performed on PBMCs from cryopreserved samples at the completion of the study, as described6. In brief, PBMCs were thawed and rested in complete RPMI for 8 h followed by stimulation for 17 h with: (1) 1.5 × 105 viable, irradiated, aseptic, purified, cryopreserved PfSPZ from a single production lot; (2) PfSPZ Vaccine diluent (1% human serum albumin, HSA, CSL Behring); (3) 2 × 105 lysed RBC, >90% infected with late-stage schizonts (PfRBC) from a single production lot; or (4) 2 × 105 donor-matched uninfected erythrocytes from a single production lot. For the last 5 h of the stimulation, 10 μg ml−1 Brefeldin A (BD) was added to the culture. After stimulation, cells were stained as previously described45. The staining panels are shown in Supplementary Table 8 and the antibody clones and manufacturers are shown in Supplementary Table 9. Briefly, cells were surface stained with CCR7 at 37 °C for 20 min. Dead cells were identified by Aqua Live-Dead dye (Invitrogen), as per manufacturer’s instructions. This was followed by 15 min surface staining at room temperature for CD4, CD8, CD14, CD38, CD45RA, CD56, CD57, CD127, CD161, TCR-γδ, TCR-Vδ1, TCR-Vδ2, TCR-Vγ9, TCR-Vα7.2, CXCR6, or PD-1. Cells were washed, fixed, and permeabilized using Cytofix/Cytoperm kit (BD) and stained intracellularly for CD3, IFN-γ, IL-2, TNF-α, IL-4, IL-10, perforin, or Ki-67. Cells were washed, fixed in 0.5% paraformaldehyde, and measured on a modified LSR II (BD). Flow cytometry data were analysed using FlowJo v9.9 (Tree Star) blinded to vaccination group and CHMI outcome. All antigen-specific cytokine frequencies are reported after background subtraction of identical gates from the same sample incubated with the control antigen stimulation (HSA or uninfected erythrocytes). The data that support the findings of these studies are available in part on request from the corresponding author (S.L.H.) subject to restrictions. Some data are not publicly available, as they contain information that could compromise research participant privacy/consent.


News Article | December 19, 2016
Site: www.scientificcomputing.com

A new bioinformatics platform called Empowering the Development of Genomics Expertise (EDGE) will help democratize the genomics revolution by allowing users with limited bioinformatics expertise to quickly analyze and interpret genomic sequence data. Researchers at Los Alamos National Laboratory and their collaborators at the Naval Medical Research Center developed EDGE, which is described in a paper recently published in Nucleic Acids Research. “We realized that while next-generation sequencing instruments are becoming more widespread and more accessible to the average biologist or physician, the bioinformatics tools required to process and analyze the data were not as user-friendly or accessible,” said Patrick Chain, of Los Alamos’ Biosecurity and Public Health group and EDGE team lead. “Given the large number of applications where sequencing is now used, a robust bioinformatics platform that encapsulates a broad array of algorithms is required to help address questions a researcher may have. We sought to develop a web-based environment where non-bioinformatics experts could easily select what pipelines they need and rapidly obtain results and interact with their data.” Stopping the spread of disease—from naturally occurring or manmade threats—requires an in-depth understanding of pathogens and how they work. To this end, the ability to characterize organisms through accurately and rapidly comparing genomic data is an important part of Los Alamos’ national security mission. Technology advancements have fueled the development of new sequencing applications and will flood current databases with raw data. A number of factors limit the use of these data, including the large number of associated software and hardware dependencies and the detailed expertise required to perform this analysis. To address these issues, Chain and his team have developed an intuitive web-based environment with a wide assortment of integrated and pioneering bioinformatics tools in pre-configured workflows, all of which can be readily applied to isolate genome sequencing projects or metagenomics projects. EDGE is a user-friendly and open-source platform that integrates hundreds of cutting-edge tools and helps reduce data analysis times from days or weeks to minutes or hours. The workflows in EDGE, along with its ease of use, provide novice next-generation sequencing users with the ability to perform many complex analyses with only a few mouse clicks. This bioinformatics platform is described as an initial attempt at empowering the development of genomics expertise, as its name suggests, for a wide range of applications in microbial research. EDGE has already helped streamline data analysis for groups in Thailand, Georgia, Peru, South Korea, Gabon, Uganda, Egypt and Cambodia, as well as within several government laboratories in the United States. The paper “Enabling the democratization of the genomics revolution with a fully integrated web-based bioinformatics platform” was published in Nucleic Acids Research in partnership with the Defense Threat Reduction Agency, the Naval Medical Research Center-Frederick and the Henry M. Jackson Foundation.


Verdu E.F.,McMaster University | Riddle M.S.,Naval Medical Research Center
American Journal of Gastroenterology | Year: 2012

Acute infectious diarrhea is a frequent occurrence both in the developing world, where it results in considerable mortality, and in developed countries, where it accounts for a significant number of health visits, hospitalizations, and medical and non-medical losses. Recent evidence in basic, clinical, and epidemiological science domains has emerged that suggest that the burden caused by these infections is not limited to the acute illness, but may result in triggering or contributing to the pathogenesis of a number of chronic health problems. This review considers the breadth of this information for the purpose of consolidating what is currently known, identifying gaps in knowledge, and describing future directions and policy implications related to the chronic consequences of acute infectious diarrhea. A unifying hypothesis of this review is that infections may trigger a number of long-lasting changes in gut physiology and immunity that can increase the risk to a variety of chronic gastrointestinal diseases, particularly in genetically susceptible individuals.


Epstein J.E.,Naval Medical Research Center | Richie T.L.,Naval Medical Research Center
Current Opinion in Infectious Diseases | Year: 2013

Purpose of Review: The whole sporozoite (SPZ) vaccine platform provides the only established approach for inducing high-level sustained protective immunity in humans against malaria. We introduce this platform, highlight literature published since 2011, and discuss the challenges of further development. Recent Findings: There are three major approaches to development of a whole parasite vaccine to prevent malaria infection using the SPZ platform: radiation-attenuated sporozoites (irrSPZ), chemoprophylaxis with infectious sporozoites (CPS), and genetically attenuated parasites (GAPs). In all three, SPZ are administered to the vaccinee. All three protect animals against infection when administered by injection with a needle and syringe, and irrSPZ and CPS protect against Plasmodium falciparum malaria in humans when P. falciparum SPZ (PfSPZ) are administered by mosquito bite. Metabolically active, nonreplicating (radiation attenuated) aseptic, purified, cryopreserved PfSPZ (PfSPZ Vaccine), and infectious, aseptic, purified, cryopreserved PfSPZ administered with chemoprophylaxis (PfSPZ-CVac approach) administered by needle and syringe have entered clinical trials. Preliminary data indicate that the PfSPZ Vaccine is safe, well tolerated and highly protective when administered intravenously. Summary: With proof-of-concept now established for high-grade protection induced by parenteral administration of a whole sporozoite vaccine, pathways for further development are currently being defined. Demonstration of high-level, durable, cross-strain P. falciparum protection would set the stage for licensure of a vaccine that could lead to dramatic reductions in malaria morbidity and mortality, and eventually elimination of this ancient scourge. © 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins.


Richards A.L.,Naval Medical Research Center
FEMS Immunology and Medical Microbiology | Year: 2012

To determine the prevalence and distribution of rickettsial pathogens around the world, scientists have relied more and more upon molecular techniques in addition to serological and culture methods. The ease of use and sensitivity/specificity of molecular techniques such as quantitative real-time PCR assays and multilocus sequence typing have lead to an increase in reports of the detection and identification of new and old rickettsiae in previously known and in new endemic regions. These assays have been successfully used with clinical samples such as serum, blood, and tissue biopsies and with environmental samples such as arthropod vectors including ticks, fleas, lice, and mites, and blood and tissue specimens from small mammal collections and from wild and domestic large animals. These methods have lead to the detection of new and old rickettsial pathogens often in new locations leading investigators to suggest new regions of risk of these rickettsioses. © 2011.


Bevevino A.J.,Naval Medical Research Center
Clinical orthopaedics and related research | Year: 2014

Open calcaneus fractures can be limb threatening and almost universally result in some measure of long-term disability. A major goal of initial management in patients with these injuries is setting appropriate expectations and discussing the likelihood of limb salvage, yet there are few tools that assist in predicting the outcome of this difficult fracture pattern. We developed two decision support tools, an artificial neural network and a logistic regression model, based on presenting data from severe combat-related open calcaneus fractures. We then determined which model more accurately estimated the likelihood of amputation and which was better suited for clinical use. Injury-specific data were collected from wounded active-duty service members who sustained combat-related open calcaneus fractures between 2003 and 2012. One-hundred fifty-five open calcaneus fractures met inclusion criteria. Median followup was 3.5 years (interquartile range: 1.5, 5.1 years), and amputation rate was 44%. We developed an artificial neural network designed to estimate the likelihood of amputation, using information available on presentation. For comparison, a conventional logistic regression model was developed with variables identified on univariate analysis. We determined which model more accurately estimated the likelihood of amputation using receiver operating characteristic analysis. Decision curve analysis was then performed to determine each model's clinical utility. An artificial neural network that contained eight presenting features resulted in smaller error. The eight features that contributed to the most predictive model were American Society of Anesthesiologist grade, plantar sensation, fracture treatment before arrival, Gustilo-Anderson fracture type, Sanders fracture classification, vascular injury, male sex, and dismounted blast mechanism. The artificial neural network was 30% more accurate, with an area under the curve of 0.8 (compared to 0.65 for logistic regression). Decision curve analysis indicated the artificial neural network resulted in higher benefit across the broadest range of threshold probabilities compared to the logistic regression model and is perhaps better suited for clinical use. This report demonstrates an artificial neural network was capable of accurately estimating the likelihood of amputation. Furthermore, decision curve analysis suggested the artificial neural network is better suited for clinical use than logistic regression. Once properly validated, this may provide a tool for surgeons and patients faced with combat-related open calcaneus fractures in which decisions between limb salvage and amputation remain difficult.

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