Table 1.
In vitro cell culture conditions for EC proliferation experiments.
Figure 1.
Proliferation of LECs in response to filarial ES products.
5×104 LECs were plated in a 96 well plate in EBM-2MV and allowed to adhere for 24 hours. Cells were starved for 24 hours in MCBD media and then stimulated with or without VEGF (10 ng/mL) or a dilution series of filarial ES products from 1∶10 to 1∶100 in MCDB media containing 5% NHS for 72 hours. Thymidine incorporation was measured. Data seen here is one representative LEC proliferation experiment. Various cell lines, cell numbers, starvation conditions, media types and serum types were utilized for optimization of this assay but we never detected reproducible proliferation rates in response to ES products.
Figure 2.
Cytokine production by LECs in response to filarial ES products.
3×105 LECs were plated in EGM-2MV media and allowed to come to confluence. Cells were then stimulated in EGM-2MV media with or without ES (1∶10), LPS (100 ng/mL) or TNFα (10 ng/mL) for 72 hrs and supernatants were harvested and assessed for cytokine production including IL-1β (A), TNFα (B), IL-8 (C), IL-6 (D) and IFNγ (E) by luminex bead technology. Experiments were completed in triplicate and this figure includes representative data from one experiment.
Figure 3.
Growth factor production by LECs in response to filarial ES products.
2×104 LECs were plated in EGM-2MV media and allowed to adhere for 24 hours. Cells were then starved for 24 hours in MCDB-131 media prior to stimulation for 24 hours in EGM-2MV media with or without ES (1∶10) or TNFα (10 ng/mL). Supernatants were harvested and assessed for growth factor production including VEGF-C (A) and VEGF-D (B) by ELISA. Experiments were completed in triplicate and this figure includes representative data from one experiment.
Figure 4.
Surface expression of adhesion molecules in response to worm ES products.
LECs were grown to confluence in EGM-2MV media then cells were stimulated for 24 hours in EGM-2MV media with or without ES (1∶10) or TNFα (10 ng/mL). LECs were harvested and subjected to flow cytometry analysis for the expression of ICAM-1 or VCAM-1. This figure includes representative data from one experiment.
Table 2.
Incubation of LECs with Brugia ES fails to induce cellular activation as measured by a variety of parameters.