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Amebic dysentery and amebic liver abscess caused by the protozoan parasite Entamoeba histolytica are important causes of morbidity and mortality worldwide.1 Interestingly, the prevalence of amebic liver abscess and amebic dysentery is between 5- and 7-fold higher in men than women.2 The reason for the greater susceptibility to disease in men is unknown, particularly because there does not seem to be a difference between men and women in the rates of intestinal colonization by E. histolytica.2 To invade through the intestinal mucosa and survive within the portal circulation to establish infection within the liver, E. histolytica trophozoites must resist killing by complement.1 We tested the hypothesis that differences between men and women in the efficiency of complement-mediated killing of amebic trophozoites might explain the varying susceptibility of the sexes to amebic liver abscess and amebic colitis.
We obtained serum samples from 100 consecutively enrolled healthy volunteers from an area that is not endemic for amebiasis, and examined the efficiency of their sera in killing amebic trophozoites. Among the volunteers were 66 women and 34 men, who provided 84 samples (16 were technically unsuitable for study) for analysis. Informed consent was obtained from all individuals, and this study was approved by the Washington University Institutional Review Board and Human Research Protection Office. Serum samples were screened for anti-amebic antibodies using immunoblotting;3 no samples in this population were positive.
Complement-mediated killing of axenically cultured amebic trophozoites was measured by FACS assay using a modified version of the method of Hamelmann and others.4 In brief, the serum samples were centrifuged at 5000 x g, and the supernatant aliquoted and stored at –70°C. Heated-inactivated serum (56°C for 30 min) was prepared as a control. E. histolytica HM1:IMSS trophozoites were harvested at the logarithmic phase of growth were chilled on ice for 10 minutes, pelleted by centrifugation at 500 x g for 5 minutes, washed twice with cold phosphate-buffered saline (PBS), and resuspended in PBS at cell concentration of 2 x 106/mL; 35 µL of the amoeba were mixed with 50 µL NHS and PBS to a final volume of 100 µL. The tropohozoites at a final cell concentration of approximately 5 x 105/mL were incubated for 30 minutes at 37°C, then 20 µL 0.2M EDTA was added to stop the reaction. After centrifugation at 950 x g for 2 minutes, the trophozoites were resuspended in 200 µL PBS including 0.01M EDTA and 0.5 µg/mL propidium iodide (PI, Sigma, Saint Louis, MO). The samples were incubated in the dark at room temperature for 5 minutes, and lysis was determined using flow cytometry (FACSCalibur, BD Biosences, Franklin Lake, NJ). Five thousand events were counted, and the data was analyzed with FACSscan Research Software. As previously reported, the viable trophozoites show a high green self-fluorescence monitored in channel (FL1, 530 nm) (see gated populations in Figure 1A
) whereas killed or damaged trophozoites are seen as a population with high fluorescence in channel 3 (FL3, 650 nm) indicating PI staining, and as populations that have lost both autofluorescence and PI uptake.4 Specific lysis was calculated as previously described.4
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and Table 1). Heat-inactivated serum (lacking complement activity) was significantly less efficient in killing trophozoites than untreated serum, but did have greater killing than buffer alone (Table 1), suggesting there is a small component of complement-independent killing of trophozoites by serum. Mean trophozoite killing was not different for heat-inactivated serum from men and women, indicating that the complement-independent component of serum killing does not differ between the sexes (Table 1). Killing occurred via complement, as the addition of EDTA that blocks total complement activity (Table 1), significantly inhibited killing by both female and male serum samples. The mean activity of serum from men and women did not differ in either alternative pathway or classic pathway mediated hemolysis of sensitized sheep red blood cells (data not shown). Although our sample size was relatively small (51 Caucasians, 25 African Americans, and 8 Asians) we did not see an independent effect of race/ethnicity on the efficiency of serum in killing trophozoites, nor was age (50 and above compared with under 50) a factor among men, women, or the entire population (data not shown).
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Received November 21, 2007. Accepted for publication February 17, 2008.
Acknowledgments: The authors thank Xioachun Zhang for outstanding technical assistance and Erin Melhop for excellent discussions. This work is dedicated to the memory of Jian Guo, an outstanding colleague and friend.
Financial support: This work was supported by NIH Grants AI30084 and U54AI057160, the Midwest Regional Center of Excellence in Biodefense and Emerging Infectious Diseases Research.
* Address correspondence to Samuel L. Stanley, Jr., Department of Medicine, Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Avenue, Campus Box 8051, Saint Louis, MO 63110. E-mail: stanleys{at}wusm.wustl.edu 
Authors’ addresses: Margaret Snow, Minghe Chen, and Samuel L. Stanley, Jr., Department of Medicine, Washington University School of Medicine, 660 S. Euclid Avenue, Campus Box 8051, Saint Louis, MO 63110, Tel: 314-362-1070, Fax: 314-362-3525, E-mails: Maggiesnow{at}sbcglobal.net, mchen{at}id.wustl.edu, and stanleys{at}wusm.wustl.edu. John Atkinson, Department of Medicine, Washington University School of Medicine, 660 S. Euclid Avenue, Campus Box 8045, Saint Louis, MO 63110, Tel: 314-362-8391, Fax: 314-362-1366, E-mail: jatkinso{at}im.wustl.edu.
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Deceased.