This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ***Please note the Tables and Figures mentioned below would not reproduce in this format. Please see attachments sent with the paper copy.*** Work in year 4 of the COBRE was focused on exploring new avenues for human cryptosporidiosis research as well as development of new immunology assays to compliment this upcoming work. Establishment of functional immunology assays: Using specimens from clinical research studies on Cryptosporidium and live attenuated S. Typhi vaccines, we have established functional antibody and cellular assays for evaluation of human pathogens and toward standardization for use in a future human immunology core facility. Four new functional antibody assays have been developed using S. typhi as a model pathogen: opsonization/ phagocytosis (Figure 1);bacterial killing post-phagocytosis;measures of phagocytic index (PI, Figure 2);and a lysosome-colocalization assay. For cellular immunology, we have developed a seven-color flow cytometry assay to evaluate intracellular cytokines pre-and post-infection or vaccination (Figure 3). A B-cell memory ELISPOT assay has also been formally developed. All these assays are now in use for studying live attenuated dengue vaccines and will be used for future study of Cryptosporidium infection. Other assays, specifically for the study of viral pathogens, have been developed. These include standardized plaque reduction and neutralization assays (PRNT) using Vero cells (mosquito cell line) for Dengue serotype 1-4, West Nile, St. Louis Encephalitis and Yellow Fever viruses. Viral amplication assays (Dengue serotypes 1-4) have also been developed. New Cryptosporidium studies: opportunities for further Cryptosporidium work are anticipated to come to fruition in year 5 of the COBRE. First, opportunities for Cryptosporidium immunology are available in Dhaka, Bangladesh as part of a large birth cohort of children. Presently, 400 children have been enrolled and 12% have evidence of Cryptosporidium infection by 6 months of life. Clinical data, human DNA, peripheral lymphocytes and sera are available, making this an ideal setting for continuing our work into human immune responses to this infection. The work from this cohort will focus on three major avenues: genome-wide associations to expand our observations of genetic susceptibility to cryptosporidiosis;expanded and functional work on mannose-binding lectin (MBL) and other collectins/surfactants which function as an innate components in immunity to Cryptosporidium infection and evaluation of cellular immune responses to infection using 7 or 8-color flow cytometry and ELISPOT assays (see above).Secondly, we are initiating enrollment of a large cohort of persons with end-stage AIDS and Cryptosporidium infection (Uganda). We will be confirming the MBL association with Cryptosporidium in this population.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Exploratory Grants (P20)
Project #
5P20RR021905-05
Application #
8167731
Study Section
Special Emphasis Panel (ZRR1-RI-8 (01))
Project Start
2010-07-01
Project End
2011-06-30
Budget Start
2010-07-01
Budget End
2011-06-30
Support Year
5
Fiscal Year
2010
Total Cost
$168,613
Indirect Cost
Name
University of Vermont & St Agric College
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
King, Benjamin R; Samacoits, Aubin; Eisenhauer, Philip L et al. (2018) Visualization of Arenavirus RNA Species in Individual Cells by Single-Molecule Fluorescence In Situ Hybridization Suggests a Model of Cyclical Infection and Clearance during Persistence. J Virol 92:
Ziegler, Christopher M; Bruce, Emily A; Kelly, Jamie A et al. (2018) The use of novel epitope-tagged arenaviruses reveals that Rab5c-positive endosomal membranes are targeted by the LCMV matrix protein. J Gen Virol 99:187-193
Hasan, Muhammad M; Teixeira, Jose E; Huston, Christopher D (2018) Invadosome-Mediated Human Extracellular Matrix Degradation by Entamoeba histolytica. Infect Immun 86:
King, Benjamin R; Hershkowitz, Dylan; Eisenhauer, Philip L et al. (2017) A Map of the Arenavirus Nucleoprotein-Host Protein Interactome Reveals that Junín Virus Selectively Impairs the Antiviral Activity of Double-Stranded RNA-Activated Protein Kinase (PKR). J Virol 91:
Bonney, Elizabeth A; Howard, Ann; Krebs, Kendall et al. (2017) Impact of Immune Deficiency on Remodeling of Maternal Resistance Vasculature 4 Weeks Postpartum in Mice. Reprod Sci 24:514-525
King, Benjamin R; Kellner, Samuel; Eisenhauer, Philip L et al. (2017) Visualization of the lymphocytic choriomeningitis mammarenavirus (LCMV) genome reveals the early endosome as a possible site for genome replication and viral particle pre-assembly. J Gen Virol :
Bonney, Elizabeth A (2017) Alternative theories: Pregnancy and immune tolerance. J Reprod Immunol 123:65-71
Ziegler, Christopher M; Eisenhauer, Philip; Kelly, Jamie A et al. (2017) A proteomic survey of Junín virus interactions with human proteins reveals host factors required for arenavirus replication. J Virol :
Sateriale, Adam; Miller, Peter; Huston, Christopher D (2016) Knockdown of Five Genes Encoding Uncharacterized Proteins Inhibits Entamoeba histolytica Phagocytosis of Dead Host Cells. Infect Immun 84:1045-1053
Nock, Adam M; Wargo, Matthew J (2016) Choline Catabolism in Burkholderia thailandensis Is Regulated by Multiple Glutamine Amidotransferase 1-Containing AraC Family Transcriptional Regulators. J Bacteriol 198:2503-14

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