The cuboidal ECs seen in B. malayi-infected nude mice suggest that a multiplication of LECs may also contribute to lymphangiectasia. Phthalylsulfacetamide However, this phenomenon could not be reproduced in vitro with human umbilical vein endothelial cells. We repeated these in vitro experiments with LECs, but we were still not able to detect an increase in EC proliferation in response to worm ES products under the culture conditions that we employed. In these experiments, we used ES products from adult female worms because of their greater abundance compared to ES products of male worms. We should also note that the ES products used to stimulate LECs do contain ES products of microfilariae, as this parasite stage is also L-Ascorbyl 6-palmitate released by the female worm in vitro at the same time that the female worm secretes ES products. We did not attempt to separate the adult female and microfilarial ES products as both may be found in the infected lymphatic vessels potentially inducing lymphatic dilation. We reasoned that ES products from both stages would be best to reproduce the biological environment in vivo. In preliminary experiments, we also attempted to co-culture living adult worms with LEC monolayers, but the active movement of the living adult worms disrupted the LEC monolayer, preventing our efforts to address the direct effect of the living parasites on LEC function. In our hands, we were not able to demonstrate reproducible LEC proliferation in response to the positive control VEGF. The lack of a robust in vitro proliferation response to VEGF is not uncommon and many responses only exhibit,50% increase over ECs stimulated in media alone. VEGF did not induce a significant increase in the proliferation of LECs even at lower seeding numbers, other serum concentrations or other stimulation times suggesting VEGF may not be the best positive control for in vitro proliferation studies using LECs, but may be better suited for vascular ECs. The lack of proliferation seen in the HMVEC-dLy cells upon stimulation with VEGF may result from the commercial optimization of culture conditions for these cells with VEGF. According to the manufacturer��s instructions, these primary LECs require VEGF for routine culture so the ability of VEGF to induce LEC proliferation may be diminished. In general and in our cultures, the lack of LEC proliferation is most likely related to the stringency of the culture conditions required for cell growth. However, Bennuru and Nutman demonstrated microfilariae-induced LEC differentiation as measured by tubule formation, suggesting that further investigation is needed to address the potential role of microfilariae in altering lymphatic pathology. In parallel experiments, microfilariae crude worm extract stimulated the production of various lymphangiogenic and immunologic mediators