A preliminary assessment of the feasibility of evaluating promising antifilarials in vitro against adult Onchocerca volvulus.

The suitability of motility indices and tetrazolium-based colorimetric assays for the determination of the viability of adult Onchocerca volvulus after in vitro exposure to potential macrofilaricides has been examined. Experimentation showed that both techniques could be applied to adult O. volvulus, although the variability between individual worms necessitated the use of large experimental groups. The potential of using cut anterior tips of female O. volvulus for screening was also investigated. These were shown to give reasonably consistent motility indices, and drug effects were discernible even after 72 h in vitro culture. Application of these viability criteria to studies on the short-term in vitro survival of intact male and female O. volvulus incubated in Eagles MEM plus serum, under 5% CO2 in air, showed this medium to be suboptimal with a greater than 50% loss of worm viability within 144 h of nodulectomy. Males isolated by the collagenase technique were shown to be significantly less viable than dissected males, by both motility indices and tetrazolium reduction. The results highlight the need to use either dissected males, or in the case of females, the need to minimize exposure to collagenase solution. A possible mechanism for selecting a more uniformly viable female worm population is discussed. Examination of the in vitro effects of CGP 20376 using these viability criteria/assay systems showed some delayed suppression of worm motility, but after 120 h in vitro CGP 20376 was not macrofilaricidal against male or female O. volvulus. Male worms were also implanted subcutaneously into gerbils.(ABSTRACT TRUNCATED AT 250 WORDS)

Nephrotoxicity assessment by measuring cellular ATP content. I. Substrate specificities in the maintenance of ATP content in isolated rat nephron segments.

To clarify the characteristics of cellular ATP synthesis in individual nephron segments for assessing nephrotoxicity of chemicals, cellular ATP content was measured by the luciferin/luciferase system under various conditions using intact nephron segments isolated from male Sprague-Dawley rats. Increasing the duration of collagenase treatment of kidney slices significantly lowered the cellular levels of ATP newly synthesized from 2 mM glutamine in PST at 37 degrees C over 30 min (p less than 0.01). The tubular incubation time significantly affected the cellular ATP content in the early and middle portions (S2) of the proximal tubule (p less than 0.05 and p less than 0.01, respectively) over 20 min and in the late proximal tubule over 10 min. Among numerous substrates tested, such as D-glucose, glutamine, pyruvate, DL-lactate, and beta-hydroxybutyrate, the substrate utilization for maintaining cellular ATP content was entirely variable according to each nephron segment. Pyruvate and glutamine were the best substrates in the proximal tubule. On the other hand, ATP production from glutamine was less than that from the other substrates in the distally located nephron segments: medullary and cortical thick ascending limbs of Henle's loop (MAL and CAL, respectively), distal tubule, cortical and medullary collecting tubules (CCT and MCT, respectively). In general, glucose, pyruvate, and lactate appear to be equivalent in maintaining ATP content in the distal segments of renal tubules. A monovalent cation ionophore, monensin, at 10 micrograms/ml decreased the cellular ATP content in MAL, CAL, and MCT significantly. Mercuric chloride (HgCl2) was used as a model compound to study nephrotoxicity by investigating its effects on cellular ATP metabolism in microdissected nephron segments. HgCl2 at 1 x 10(-6) M significantly decreased ATP content only in S2 (p less than 0.05), clearly demonstrating S2 to be the most sensitive segment within the nephron. These results indicate that measurement of cellular ATP content would be a useful method forecasting the intrarenal toxic site and potency of possible nephrotoxic chemical compounds.

Adult human saphenous vein endothelial cells: assessment of their reproductive capacity for use in endothelial seeding of vascular prostheses.

Autogenous endothelial seeding (AES) of vascular prostheses (VP) using venous endothelial cells (EC) reduces platelet-VP interactions and improves patency rates in small caliber VP in dogs. To conserve patients' veins for use in coronary or limb bypass surgery, human trials of AES should require proof that adequate numbers of EC with the growth capacity to cover VP can be harvested from acceptably small pieces of peripheral vein. EC were isolated from excess saphenous vein segments remaining after coronary bypass surgery by filling veins with 0.1% CLS II collagenase at 37 degrees C for 15 min and removing EC by flushing the veins with culture medium. EC were cultured on fibronectin-coated dishes in medium 199 with 30% human serum and 300 micrograms/ml of endothelial cell growth factor. These cells grew to form confluent monolayers, and were identified as EC by tests for factor VIII antigen. Veins from 53 patients with a mean age of 55.8 +/- 9.8 (SD) years yielded vein segments with an average area of 1.9 +/- 0.6 cm2, from which an average of 5.3 +/- 2.8 X 10(4) cells were removed per cm2 of vein area. EC in culture underwent 14.3 +/- 1.4 population doublings with an average population doubling time of 1.8 +/- 0.3 days (N = 14 cultures), which allowed an 100-fold increase in cell number to occur in 11 to 12 days. These data suggest that the EC available from small vein segments in adult humans have the growth capacity to cover areas comparable in size to the luminal areas of VP commonly used in arterial surgery.