Inhibition of Chlamydial Infection by Ivermectin Figure 3. Ivermectin is not cytotoxic to epithelial cells. (A) HeLa cells were cultured in the presence of the indicated concentrations of ivermectin or vehicle (0.2% ethanol), supernatants were then centrifuged at 500 g for 5 minutes, and adherent cells were removed with trypsin/EDTA and gently recombined with supernatant pellets. Cell viability was determined by trypan blue exclusion, evaluated using a hemocytometer. (B) Supernatants from the cultures described in panel A, and from wells treated with 2% Triton X-100 30 minutes prior to collection time points, were combined and evaluated for LDH activity using a Roche Cytotoxicity Detection Kit per the manufacturer’s instructions. Shown are means plus standard deviations for 3 independent experiments. doi:10.1371/journal.pone.0048456.g003 Author Contributions Conceived and designed the experiments: MAP DMO. Performed the experiments: MAP VWL IO CM. Analyzed the data: MAP CM. References 1. Burg RW, Miller BM, Baker EE, Birnbaum J, Currie SA, et al. (1979) Avermectins, new family of potent anthelmintic agents: producing organism and fermentation. Antimicrob Agents Chemother 15: 361–367. 2. Egerton JR, Ostlind DA, Blair LS, Eary CH, Suhayda D, et al. (1979) Avermectins, new family of potent anthelmintic agents: efficacy of the B1a component. Antimicrob Agents Chemother 15: 372–378. 3. Miller TW, Chaiet L, Cole DJ, Cole LJ, Flor JE, et al. (1979) Avermectins, new family of potent anthelmintic agents: isolation and chromatographic properties. Antimicrob Agents Chemother 15: 368–371. 4. Chabala JC, Mrozik H, Tolman RL, Eskola P, Lusi A, et al. (1980) Ivermectin, a new broad-spectrum antiparasitic agent. J Med Chem 23: 1134–1136. 5. Omura S, Crump A (2004) The life and times of ivermectin - a success story. Nat Rev Microbiol 2: 984–989. 6. Taylor MJ, Hoerauf A, Bockarie M (2010) Lymphatic filariasis and onchocerciasis. Lancet 376: 1175–1185. 7. Chosidow O, Giraudeau B, Cottrell J, Izri A, Hofmann R, et al. (2010) Oral ivermectin versus malathion lotion for difficult-to-treat head lice. N Engl J Med 362: 896–905. 8. Currie BJ, McCarthy JS (2010) Permethrin and ivermectin for scabies. N Engl J Med 362: 717–725. 9. Bavoil PM, Hsia R-C, Ojcius DM (2000) Closing in on Chlamydia and its intracellular bag of tricks. Microbiol 146: 2723–2731. Contributed reagents/materials/analysis tools: DMO. Wrote the paper: MAP DMO. 10. Fields KA, Hackstadt T (2006) The Chlamydia Type III Secretion System: Structure and Implications for Pathogenesis. In: Bavoli PM, Wyrick PB, editors. Chlamydia: Genomics and Pathogenesis. 219–233. 11. Wyrick PB (2000) Intracellular survival by Chlamydia. Cell Microbiol 2: 275–282. 12. Belland R, Ojcius DM, Byrne GI (2004) Chlamydia. Nat Rev Microbiol 2: 530– 531. 13. Baraka OZ, Mahmoud BM, Marschke CK, Geary TG, Homeida MM, et al. (1996) Ivermectin distribution in the plasma and tissues of patients infected with Onchocerca volvulus. Eur J Clin Pharmacol 50: 407–410. 14. Gokbulut C, Cirak VY, Senlik B, Aksit D, Durmaz M, et al. (2010) Comparative plasma disposition, bioavailability and efficacy of ivermectin following oral and pour-on administrations in horses. Vet Parasitol 170: 120–126. 15. Adelsberger H, Lepier A, Dudel J (2000) Activation of rat recombinant alpha(1)beta(2)gamma(2S) GABA(A) receptor by the insecticide ivermectin. Eur J Pharmacol 394: 163–170. 16. North RA (2002) Molecular physiology of P2X receptors. Physiol Rev 82: 1013– 1067. 17. Pettengill MA, Marques-da-Silva C, Avila ML, d’Arc Dos Santos Oliveira S, Lam VW, et al. (2012) Inhibition of Chlamydia trachomatis Infection in Epithelial Cells Due to Stimulation of P2X4 Receptors. Infec Immun in press. 18. Pettengill MA, Lam VW, Ojcius DM (2009) The danger signal adenosine induces persistence of chlamydial infection through stimulation of A2b receptors. PLoS One 4: e8299. PLOS ONE | www.plosone.org 4 October 2012 | Volume 7 | Issue 10 | e48456