The E3 ubiquitin ligase EDD is an adverse prognostic factor for serous epithelial ovarian cancer and modulates cisplatin resistance in vitro.

Despite a high initial response rate to first-line platinum/paclitaxel chemotherapy, most women with epithelial ovarian cancer relapse with recurrent disease that becomes refractory to further cytotoxic treatment. We have previously shown that the E3 ubiquitin ligase, EDD, a regulator of DNA damage responses, is amplified and overexpressed in serous ovarian carcinoma. Given that DNA damage pathways are linked to platinum resistance, the aim of this study was to determine if EDD expression was associated with disease recurrence and platinum sensitivity in serous ovarian cancer. High nuclear EDD expression, as determined by immunohistochemistry in a cohort of 151 women with serous ovarian carcinoma, was associated with an approximately two-fold increased risk of disease recurrence and death in patients who initially responded to first-line chemotherapy, independently of disease stage and suboptimal debulking. Although EDD expression was not directly correlated with relative cisplatin sensitivity of ovarian cancer cell lines, sensitivity to cisplatin was partially restored in platinum-resistant A2780-cp70 ovarian cancer cells following siRNA-mediated knockdown of EDD expression. These results identify EDD as a new independent prognostic marker for outcome in serous ovarian cancer, and suggest that pathways involving EDD, including DNA damage responses, may represent new therapeutic targets for chemoresistant ovarian cancer.

Recovery of Cryptosporidium oocysts and Giardia cysts from source water concentrates using immunomagnetic separation.

Immunomagnetic separation (IMS) procedures for the simultaneous isolation of Cryptosporidium oocysts and Giardia cysts have recently become available. We validated Dynal's GC-Combo IMS kit using source water at three turbidity levels (5000, 500 and 50 nephelometric turbidity units [ntu]) obtained from different geographical locations and spiked with approximately 9--11 (oo)cysts per ml. Mean recoveries of Cryptosporidium oocysts and Giardia cysts in deionized water were 62% and 69%, respectively. In turbid water matrices, mean recoveries of Cryptosporidium oocysts were between 55.9% and 83.1% while mean recoveries of cysts were between 61.1% and 89.6%. Marginally higher recoveries of the heat inactivated (oo)cysts were observed (119.4% Cryptosporidium oocysts and 90.9% Giardia cysts) in deionized water when compared with recoveries of viable (oo)cysts (69.7% Cryptosporidium oocysts and 79% Giardia cysts). Age of (oo)cysts on recoveries using the GC-Combo IMS kit demonstrated no effects up to 20 months old. Recovery of Giardia cysts was consistent for isolates aged up to 8 months (81.4%), however, a significant reduction in recoveries was noted at 20 months age. Recoveries of low levels (5 and 10 (oo)cysts) of Cryptosporidium oocysts and Giardia cysts in deionized water using IMS ranged from 51.3% to 78% and from 47.6% to 90.0%, respectively. Results of this study indicate that Dynal's GC-Combo IMS kit is an efficient technique to separate Cryptosporidium/Giardia from turbid matrices and yields consistent, reproducible recoveries. The use of fresh (recently voided and purified) (oo)cysts, aged (oo)cysts, viable and heat-inactivated (oo)cysts indicated that these parameters do not influence IMS performance.

Recovery and viability of Cryptosporidium parvum oocysts and Giardia intestinalis cysts using the membrane dissolution procedure.

Previously, the cellulose acetate membrane filter dissolution method was reported to yield Cryptosporidium parvum oocyst recoveries of 70.5%, with recovered oocysts retaining their infectivity. In contrast, high spike doses (approximately 1 x 10(5) Cryptosporidium parvum oocysts and Giardia intestinalis cysts) yielded recoveries ranging from 0.4% to 83.9%, and 3.2% to 90.3%, respectively, in this study. Recoveries with low spike doses (approximately 100 (oo)cysts) continued to demonstrate high variability also. Efforts to optimize the method included increased centrifugation speeds, suspension of the final concentrate in deionized water for organism detection on well slides, and analysis of the entire concentrate. A comparison of two monoclonal antibodies was also conducted to identify potential differences between antibodies in detection of organisms. Archived source and finished water samples were spiked, yielding variable recoveries of C. parvum oocysts (11.8% to 71.4%) and G. intestinalis cysts (7.4% to 42.3%). Effects of organic solvents used in the membrane dissolution procedure on the viability of recovered (oo)cysts was determined using a fluorogenic vital dyes assay in conjunction with (oo)cyst morphology, which indicated > 99% inactivation. These data indicate that the membrane dissolution procedure yields poor and highly variable (oo)cyst recoveries, and also renders the majority of recovered organisms non-viable.

An evaluation of the Gelman Envirochek capsule for the simultaneous concentration of Cryptosporidium and Giardia from water.

The Gelman Envirochek capsule is a membrane device for the simultaneous concentration of Cryptosporidium oocysts and Giardia cysts from water. Samples are filtered through a Supor polyethersulphone membrane with a 1 micron absolute pore size. (Oo)cysts are mechanically eluted from the membrane fibre using a wrist action shaker and a nonionic detergent and concentrated by centrifugation. The concentrate can be further processed using any separation technique to separate the target organisms from other debris. This method enables multiple samples to be processed within 1 h. Recoveries from seeded tap and source water samples were in excess of 70% for Cryptosporidium and 80% for Giardia.