Until recently, C. difficile associated diarrhea (CDAD) occurred as an opportunistic infection in older hospitalized patients on antibiotics. Currently, CDAD has become much more worrisome because of widespread increases in its frequency and severity throughout the US and Canada due to the emergence of hypertoxinogenic, binary toxin-producing, quinolone-resistant (Bl) strains. Furthermore, life threatening CDAD is now affecting healthy younger people in communities, even without recent antibiotic use. Even worse, current treatment consisting of more antibiotics is failing and often prolongs shedding or relapses. Hence we urgently need novel approaches that block toxin-induced colitis to prevent the progression of colitis produced by C. difficile toxin(s)) without further disruption of protective flora. This application from the University of Virginia brings together our experienced enterics laboratory team with basic science colleagues in toxin pathogenesis and in drug development, and a pharmaceutical partner (Adenosine Therapeutics, LLC). We shall address the emerging quinolone-resistant C. difficile colitis with an innovative antitoxic approach that is supported by pilot data demonstrating considerable promise. We propose to investigate three types of anti-inflammatory agents alone and in combination: adenosine A2A receptor agonists that block leukocyte activation;A2B antagonists that block cytokine production by epithelial and mast cells, and the pro-absorptive injury repairing agent, alanyl-glutamine. The first three closely interrelated specific aims are designed to determine how these novel therapeutics regulate intestinal cell targets to block toxin-induced apoptosis, inflammation, and secretion. We plan to: 1) use new human intestinal epithelial cell organoid and C57BL/6 murine models to define the targets of purified toxins A and B as well as B-variant and new Bl strain supernatants;2) use C57BL/6 mice with adenosine receptor knockouts and Cre/loxp targeted cellular AaA receptor deletions to define the relevant adenosine targets to inhibition of toxin effects;and 3) use the information gained in aims 1 and 2 to test combinations of A2A agonists, A2B antagonists and alanyl-glutamine, alone and in combination, in animal models in order to prepare for clinical trials of these novel drug therapies for increasingly serious CDAD in humans.
Our fourth aim i nvolves discovery efforts towards developing more highly absorbed AaR agonists and the synthesis of the proposed A2A, agonists and A2B antagonists necessary to support this grant.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project--Cooperative Agreements (U01)
Project #
5U01AI075526-04
Application #
7922544
Study Section
Special Emphasis Panel (ZAI1-AR-M (M1))
Program Officer
Wachtel, Marian R
Project Start
2007-08-01
Project End
2012-07-31
Budget Start
2010-08-01
Budget End
2011-07-31
Support Year
4
Fiscal Year
2010
Total Cost
$726,199
Indirect Cost
Name
University of Virginia
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
065391526
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
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Moore, John H; Pinheiro, Caio C D; Zaenker, Edna I et al. (2015) Defined Nutrient Diets Alter Susceptibility to Clostridium difficile Associated Disease in a Murine Model. PLoS One 10:e0131829
Rodrigues, Raphael S; Oliveira, Renato A C; Li, Yuesheng et al. (2013) Intestinal epithelial restitution after TcdB challenge and recovery from Clostridium difficile infection in mice with alanyl-glutamine treatment. J Infect Dis 207:1505-15
Warren, Cirle A; van Opstal, Edward J; Riggins, Mary S et al. (2013) Vancomycin treatment's association with delayed intestinal tissue injury, clostridial overgrowth, and recurrence of Clostridium difficile infection in mice. Antimicrob Agents Chemother 57:689-96
Li, Yuesheng; Figler, Robert A; Kolling, Glynis et al. (2012) Adenosine A2A receptor activation reduces recurrence and mortality from Clostridium difficile infection in mice following vancomycin treatment. BMC Infect Dis 12:342
Warren, Cirle A; Li, Yuesheng; Calabrese, Gina M et al. (2012) Contribution of adenosine A(2B) receptors in Clostridium difficile intoxication and infection. Infect Immun 80:4463-73
Warren, Cirle Alcantara; Calabrese, Gina M; Li, Yuesheng et al. (2012) Effects of adenosine AýýýA receptor activation and alanyl-glutamine in Clostridium difficile toxin-induced ileitis in rabbits and cecitis in mice. BMC Infect Dis 12:13
Kolling, Glynis L; Wu, Martin; Warren, Cirle A et al. (2012) Lactic acid production by Streptococcus thermophilus alters Clostridium difficile infection and in vitro Toxin A production. Gut Microbes 3:523-9
Warren, Cirle A; Guerrant, Richard L (2011) Pathogenic C difficile is here (and everywhere) to stay. Lancet 377:8-9
Pawlowski, S W; Calabrese, G; Kolling, G L et al. (2010) Murine model of Clostridium difficile infection with aged gnotobiotic C57BL/6 mice and a BI/NAP1 strain. J Infect Dis 202:1708-12

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