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Biotechnology Applications

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The 1950s GM Firebird III

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Public Health - Sanitation

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Public Health - Vaccination Polio

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Allergy and Asthma Epidemic in Developing Countries Devereux G. 2006. Nature Rev Immunol 6;869-874.

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Hygiene Hypothesis early childhood infections inhibit the tendency to develop allgergic disease

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Helminth Infections & Allergies: Inverse Correlation % Population*Helminth Infection** Allergic Disease*** Globally ~ 25%~ 2% United States < 1%~ 13% * United States Census (www.census.gov) ** Clin Exp Immunol, 2002. 128(3): p. 398-404 *** Lancet, 1998. 351(9111): p. 1225-32 Helminth infections are associated with a decreased incidence of allergic disease in industrialized areas

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The Hygiene Hypothesis

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Hygiene Hypothesis - Infection history makes a difference

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Many Helminth Species Have an Obligate Lung Stage Geohelminths Schisto Filariasis HOOKWORM ASCARIS SCHISTOSOMA Malaria

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INFECTION Skin penetration L3 larvae enter circulation Reach lungs within a day PULMONARY PHASE L3 molt into L4 Larvae Spend less than 48hrs in lung Crawl/coughed up trachea Swallowed down esophagus ADULT PHASE L4 reach small intestine Molt into adult stage worms Patency by day 7 RESOLUTION Normal BALB/c will expel the worms within 11 days SCID maintain infection

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Outline Part I: Innate responses to Nb in the lungs - Gene array, PCR Part II: Persistent alterations in the immune environment of the lung - Flow cytometry Part III: Molecular basis for helminth-induced dampening of inflammation

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Innate Immune Responses to Nb in the Lung Experimental Design

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Major Gene Array Platforms Bead Array Illuminia Spotted Array Agilent GeneChipAffymetrix

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General Scheme Affymetrix GeneChip Array Image Courtesy of Bioteach DNA RNA Protein >5,500,000 features per chip

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Innate Source of Th2 Cytokines in the Lung lungintestinelungintestine

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Nb Infection Innately Induced AAM Genes lungintestinelungintestine

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Polymerase Chain Reaction - PCR Thermal Vents Thermus aquaticus T. aquaticus DNA Polymerase 30 rounds of amplification = >1,000,000,000 molecules

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Real Time RT PCR results

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Alternatively Activated Macrophage Alternatively Activated Macrophages

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T Cell Help is Required for AAAM Maintenance

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Observations: Nb induces cellular/molecular changes through day 12 PI Helminth-induced suppression of reactivity to secondary challenge (Wohlleben et al. 2004; Wilson et al. 2005; Yang et al. 2006) : Hypotheses: Helminth infection results in permanent alterations to the lungs immunological environment

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Nb Induces an Altered Lung Environment Experimental Design 2-413-1536

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Granular Macrophages Contain Surfactant day 36 PI

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Flow cytometry antibody-based detection of surface molecules

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Day 36 PI AMs are CD11c +, F4/80 +, Mrc1 mid & MHC II hi day 36 PI

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Surface Phenotype of CD11c + AAAM CD11c+ AAAM up-regulate: MHC I & II PDL-1CD80/CD86CD40CD1d Day 0 2 4 8 13 CD11c MHC I MHC II PDL-I PDL-2 CD80 CD86 CD40 CD1d ICAM-1

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AAAM are Highly Phagocytic Ova- Nb+Nb- Ova+* CytoB Ova-Alexa 448 at 2 g/ml for 30 min CytoB - cytochalasin B at 800 g/ml Ova-Alexa 448 CD11c APC CD11c APC Ova-Alexa 448 22.59.2 5.2 3.5 0.070.02

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CD11c + AM Expression Profile AAM (arg1, fizz1, Ym1/2, trem2b) Scavenger Receptors ( clecsf5/6/8/10, marco, mgl1/2, msr1/2 ) Chemokines ( ccl4/8/9/11/12/17/24 ) Cell Cycle Lipid Metabolism ( ppar gamma, cd68, apobec1, ch25h ) 194797 392 76 277 59524 day 2 PI (606) day 2 PI (606) day 4 PI (1,674) day 4 PI (1,674) day 8 PI (1,252) day 8 PI (1,252) Up-regulated >2-fold

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Altered Lung Environment: Hypotheses: The Nb-altered lung environment results in a modulation in responses to secondary challenges - HDM allergen challenge Gene array, PCR, histology, lung function

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Similar Numbers, Different Genes are Up-regulated

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Nb-Altered Lung Down-Regulates Many More Genes

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Nb-Altered Lung Decreases Airway Hyperresponsiveness

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Altered Lung Environment: Dampened Responses to Allergen Key Findings: Persistent changes resulted in a reduction in the overall inflammatory response to allergen challenge in the lungs of Nb-infected animals Distinct gene expression profiles in the lungs of NbHDM vs HDM mice Lower fold-increase of transcription of Th1 & Th2 cytokines in NbHDM mice Resistance in the airways is significantly depressed in the lungs of NbHDM mice

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Hygiene Hypothesis early childhood infections inhibit the tendency to develop allgergic disease

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Alternatively Activated Alveolar Macrophages IL-21 NKT ? ? CD1d (PDL 1/2 ?) IL-6

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JHU Gene Array Core Facility Anne Jedlicka Meg Mintz Jason Bailey Amanda Dziedzic Acknowledgments Collaborators Joe Urban (USDA) Andrea Keane-Myers (NIH) Jamie Lee (Mayo Clinic) Funding / Support NIH NHLBI (U01 HL66623) NIH Training Grant (T32AI007417) Hegner, Cort Root Memorial Scholarship Martin Frobisher Fellowship Scott Lab Josh Reece Mark Siracusa Kiwon Park Sarah Coffman Ellen Tarr JHU Sabra Klein Mike Overstreet Fidel Zavala Mark Soloski Bill Spannhake Wayne Mitzner Jon Fallica Doug Norris Vern Carruthers Brian Schofield Paul Fallon Theresa Southard Cory Brayton