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500 nm

ASSESSING THE EFFECT OF BIOLOGICAL AGENT INGESTION A SYSTEMS BIOLOGY BIOLOGICAL AGENT MODELING PLATFORM DR. AMANDA N. PAYNE, MICROBIOLOGIST, NAVAL SURFACE WARFARE CENTER-DAHLGREN. EPITHELIAL CELL MODEL. IN VITRO GUT MODEL. ZIHLER ET AL. 2011. GUT MICROBES. BACILLUS ANTHRACIS. 500 nm.

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500 nm

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  1. ASSESSING THE EFFECT OF BIOLOGICAL AGENT INGESTION A SYSTEMS BIOLOGY BIOLOGICAL AGENT MODELING PLATFORM DR. AMANDA N. PAYNE, MICROBIOLOGIST, NAVAL SURFACE WARFARE CENTER-DAHLGREN EPITHELIAL CELL MODEL IN VITRO GUT MODEL ZIHLER ET AL. 2011 GUT MICROBES BACILLUS ANTHRACIS 500 nm 2.00 µm DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.

  2. PROJECT SIGNIFICANCE AND RELEVANCE TO NSWCDD “For the life of me, I cannot understand why the terrorists have not attacked our food supply because it is so easy to do.” – 2004 SECRETARY THOMPSON • Aerosol agent detection, counter-proliferation and defeat is the major resource allocation area • Develop technical focus within critical science and engineering expertise to investigate risks associated with biological agent ingestion • Identify agent(s) of interest; Leverage Z21 expertise with Bacillus anthracis • Design and implement an in vitro systems biology modeling platform of ingested biological agent(s): • Inhalational exposure outcomes homogenous, highly lethal • Ingestional outcomes reportedly extremely variable; asymptomatic to fatal reported • Vaccination may not prove effective for gastrointestinally-acquired biological agents (e.g. Fransicellatularensis, KuoLee R. et al. 2007. Infect Immun, 75: 1651-1660) Why?? DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.

  3. GASTROINTESTINAL TRACT: ANATOMY AND FUNCTION • LARGEST ORGAN IN HUMAN BODY • DIVERSE AND DYNAMIC ENVIRONMENT: • LARGE pH RANGE • OXYGEN STRATIFICATION • REGIONS OF HIGH ENZYME ACTIVITY • CONSTANT MOVEMENT • HIGHLY ENERVATED • BIOLOGICAL AGENT DEFENSE PERSPECTIVE: • FACILITATES INGESTED AGENT ENTRY • SITE OF AGENT-HOST INTERACTION • LARGE INTESTINE MOST IMPORTANT SOURCE: CHILDREN’S HOSPITAL COLORADO DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.

  4. LARGE INTESTINAL IMPORTANCE IN MAMMALS REALITY: HIGHLY COMPLEX NETWORK OF CELLULAR AND IMMUNOGENIC ACTIVITY CONVENTIONAL THOUGHT LARGE TUBE WITH IN/OUTPUT DESTINED FOR NUTRIENT ABSORPTION • MOST HIGHLY COLONIZED HUMAN SITE: 1014 CFU G-1 • LARGEST MICROBIOME: “GUT MICROBIOTA” • LUMINAL VS. MUCOSAL NICHES DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.

  5. GUT MICROBIOTA: AN ORGAN WITHIN AN ORGAN PERTURBATION: ANTIBIOTICS, VACCINATION, PATHOGENS • GUT MICROBIOTA BASIC COMPOSITION: • >1000 SPECIES (BACTEROIDETES, FIRMICUTES, ACTINOBACTERIA, PROTEOBACTERIA) • DELICATE BALANCE, HIGHLY INDIVIDUAL • ORGANIZED INTO HIERARCHICAL NICHES • GUT MICROBIOTA BASIC FUNCTIONALITY: • DIGESTION • COMMUNICATION WITH GI EPITHELIUM • COMMUNICATION WITH CNS • EDUCATE IMMUNE SYSTEM • PATHOGEN EXCLUSION AND DEFEAT • DYSFUNCTION: • CHANGE IN COMPOSITION AND FUNCTIONALITY • LOSS OF NICHE • LOSS OF IMMUNOGENIC TOLERANCE • PATHOGEN INVASION INTO EPITHELIAL CELLS • SYSTEMIC DISEASE DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.

  6. ORGANISMAL SIGNIFICANCE OF BACILLUS ANTHRACIS AS BIOLOGICAL THREAT AGENT 2. VEGETATIVE CELL WITH POLY-D-GLUTAMYL CAPSULE (ESTABLISHMENT OF INFECTION) 2.00 µM SPORES HIGHLY CHEMICAL, ENZYME RESISTANT SURVIVE NUTRIENT STARVATION TOXIN: LETHAL FACTOR, EDEMA FACTOR, PROTECTIVE ANTIGEN • GERMINATION MOST SIGNIFICANT EVENT: TRANSITION TO VIABLE ORGANISM • FACTORS DICTATING BACILLUS ANTHRACIS SUCCESS AS FOOD-BORNE AGENT: • COMPETITION (GUT MICROBIOTA) • HOST CONTACT REQUIREMENT(S) • HOST FACTOR REQUIREMENT(S) DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.

  7. PROJECT OBJECTIVE AND KEY POINTS Project Objective Assess the impact of biological agent ingestion utilizing the commensal intestinal microbial community and related host-microbe interactions a benchmark of the potential risk for pathogenesis of ingested biological agent spores • KEY POINTS • GERMINATION: WHERE (GI LUMEN AND/OR MUCOSA), REQUIREMENTS • GUT MICROBIOTA COMMUNITY / FUNCTIONALITY IMPACT • HOST RESPONSE • AGENT RESPONSE (ANTIGENS, TRANSCRIPTOME) State-of-the-art technology = animal models (expensive, ethical impedance) and in in vitro gut models DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.

  8. EVALUATION OF BACILLUS ANTHRACIS AS BIOLOGICAL THREAT AGENT: IN VITRO MODEL DESIGN AND PARAMETERS • I. Gastric Conditions: • pH 2 peptone water • 600 U pepsin • Flow rate: 0.5 mL min-1 • II. Small Intestinal Conditions: • pH adjusted to 6.5 • 4% bile salt; 2% bile salt • 0.2% (w/v) pancreatin for an additional 120 min. • Pump flow rate: 1 mL min-1 Pump Temp Stir Enzyme Solutions Three-stage in vitro gut model Single-Stage in vitro gut model KEY IN VITRO MODEL PARAMETERS • Continuous Fermentation* • pH control • Anaerobic control • Temperature control • Complex Medium *System retention rate: 1/D µ = D, where µ is the growth rate and D the dilution factor. D = f/v, where D is the dilution rate, f is the flow rate and v equals the total volume Payne et al.2012. Trends Biotechnol DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.

  9. GUT MICROBIOTA GROWTH MEDIUM REQUIREMENTS DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.

  10. NSWCDD IN VITRO GUT MODEL OPERATIONAL SETUP EPPENDORF BIOFLO 115 BIOREACTOR CO2 regulator (ANAEROBIC CONTROL) SPENT MEDIUM (REACTOR OUTFLOW) SINGLE STAGE MODEL PROXIMAL COLON (pH 5.7) GUT MICROBIOTA + GASTRIC AND SMALL INTESTINAL-TREATED B. ANTHRACIS SPORES SODIUM HYDROXIDE (pH CONTROL) FRESH MEDIUM CAVEAT: MODEL DESIGN PRECLUDES INCLUSION OF HOST FACTORS; MODELS ARE OF LUMINAL GI PROCESSES DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.

  11. COMBINED IN VITRO GUT AND IN VITRO HUMAN CELL MODELS: SYSTEMS BIOLOGY APPLICATION IN VITRO GUT MODEL IN VITRO HUMAN CELL MODELS INFLAMMATION + PATHOGEN ADHESION INVASION GENE EXPRESSION CHANGES CELL MODEL DIVERSITY 1. MONOLAYER 2. M-CELL MODEL 2. 3-D CELL MODEL CACO-2 EPITHELIAL CELL B CELL MACROPHAGE DENDRITIC CELLS COLON CELL LINES: CACO-2, HT-29, T84 DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.

  12. IN VITRO GASTRIC AND SMALL INTESTINAL-TREATMENT INDUCES PHYSICAL CHANGES IN B. ANTHRACIS SPORES SEM Images of Treated B. anthracisSterne Spores TEM Images of Treated B. anthracisSterne Spores Control (Untreated) Gastric Treated Control (Untreated) Gastric Treated 2.00 µm 2.00 µm 100 nm Gastric and Small Intestinal Treated Significant exosporium wrinkling Gastric and Small Intestinal 500 nm Effect disappears after small intestinal treatment (resembles control again) 1.00 µm 100 nm TREATMENT DOES NOT IMPACT SPORE VIABILITY DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.

  13. IN VITRO GUT MODEL RESULTS: B. ANTHRACIS IN CULTURE FAIL TO GERMINATE EXPERIMENT 1 EXPERIMENT 2 LACK OF GERMINATION: COMPETITION (GUT MICROBIOTA) pH GERMINATION REQUIREMENTS GUT MICROBIOTA: CHANGE IN COMMUNITY CHANGE IN FUNCTIONALITY DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.

  14. B. ANTHRACIS GASTRIC AND SMALL INTESTINAL-TREATED SPORES ALSO FAIL TO GERMINATE WITHOUT GUT MICROBIOTA DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.

  15. B. ANTHRACIS GASTRIC AND SMALL INTESTINAL-TREATED SPORES ALSO FAIL TO GERMINATE UNDER AEROBIC CONDITIONS IN FERMENTATION MEDIUM DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.

  16. B. ANTHRACIS GERMINATION IS WILD-TYPE WITHOUT ENZYMATIC TREATMENT CONDITIONS: 37°C 4 HOUR DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.

  17. PROJECT OUTLOOK • CONTINUE EFFORT TO UNDERSTAND B. ANTHRACIS GERMINATION IN GI TRACT: • IN VITRO M-CELL MODEL EXPERIMENTS • IN VITRO GUT MODEL AT DIFFERENT pH • INVESTIGATE IMMUNE RESPONSE OF B. ANTHRACIS SPORES • CYTOKINE/CHEMOKINE ASSAYS • ELECTRON AND CONFOCAL MICROSCOPY OF CELLS IN CONTACT WITH SPORES • CORRELATE RESULTS WITH INHALATIONAL DATA • EXAMINE DIFFERENT B. ANTHRACIS STRAINS (PASTEUR) • COMBINE EFFORT WITH NRL TO DEVELOP FOOD-BORNE AGENT DETECTION PLATFORM DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.

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