Effects of the excretory/secretory products of Trichostrongylus colubriformis on the growth of different cell lines.

The effects of the excretory/secretory (ES) products of the parasitic nematode Trichostrongylus colubriformis were examined on the proliferation of seven cell lines derived from a digestive or non-digestive origin. The excretory/secretory products of T. colubriformis were incorporated in the culture medium of the different cell lines and cell proliferation was measured by means of the 5-bromo-2'-deoxy-uridine (Brdu) assay. An increase in cell numbers was found with the three epithelial intestinal cells (RIC, IEC-6, IRD-98) and with epithelial kidney cells (MDCK). In contrast, an inhibition in the proliferation of epithelial ovarian cells (CHO) and fibroblasts (3T3) was observed with the addition of the excretory/secretory products and no effect was detected on the cell growth of hepatocytes (HepG2). These data are discussed with respect to the tissue specificity of the existing mitogenic effect of the worms on the intestinal crypt cells during parasitism.

Role of acetylcholinesterase (AChE) secreted by parasitic nematodes on the growth of the cell line from epithelial origin HT29-D4.

The excretory-secretory (E-S) products of the parasitic nematodes Trichostrongylus colubriformis and Nematodirus battus were found to modify the in vitro proliferation of the tumorous colic HT29-D4 cell line of epithelial origin. A characteristic feature of these E-S products is the presence of a high level of acetylcholinesterase (AChE) activity, the biological significance of which remains unclear. To determine a possible role of AChE on cell growth, the enzyme was purified from E-S products using edrophonium chloride. Purity was confirmed by polyacrylamide gel electrophoresis, using silver and Karnovsky stains, before assessing its effects on cell proliferation. The purified AChE was incorporated at different concentrations in a culture medium of HT29-D4 cells. A mitogenic effect was shown for low concentrations (0.1-14 units). By contrast, an inhibitory effect was noted at high concentrations (35-1400 units). Furthermore, polyclonal antibodies were prepared and depletion of AChE in E-S products by immunoprecipitation or affinity chromatography resulted in a partial or total disappearance of the stimulatory effect of cell growth. Thus, the results form this in vitro study suggest a modulatory role for AChE secreted by nematode parasites on the proliferation of epithelial cells of the host.

Characterization of acetylcholinesterase secreted by the trichostrongyle nematode parasites of ruminants.

Acetylcholinesterase (AChE) secreted by seven different ruminant trichostrongyles was studied in vitro. AChE activity was particularly high (84 and 160 x 10(-3) M.g-1.min-1) in the excretion products of Nematodirus spathiger and N battus, moderate (3 and 5 x 10(-3) M.g-1.min-1) for Trichostrongylus colubriformis and T vitrinus and low (0.9 x 10(-3) M.g-1.min-1) for Teladorsagia circumcincta. No activity was observed with Haemonchus contortus and Cooperia curticei. At 4 degrees C, 80% of AChE activity was maintained over 72 h except for T circumcincta where a loss of 50% was observed after 24 h. At 37 degrees C, N spathiger and T colubriformis maintained an activity over 72 h, but for the other species, a loss of 50% was observed after 24 h. The molecular weights of the AChE from the different species, estimated by gel filtration (Sephadex S300HR), ranged between 64 and 150 kDa. The coefficients of sedimentation estimated by sucrose density gradient ranged between 4.8 S and 7.8 S and corresponded to a monomeric hydrophilic form (G1). For T vitrinus, an amphiphilic form was suspected.

Effects of the excretory/secretory products of six nematode species, parasites of the digestive tract, on the proliferation of HT29-D4 and HGT-1 cell lines.

The excretory/secretory (ES) products of the nematode parasite Trichostrongylus colubriformis have been found to increase the in vitro proliferation of the epithelial cell line HT29-D4. To assess the specificity of this effect, ES products from other trichostrongyle species were tested on colonic (HT29-D4) and gastric (HGT-1) tumour cell lines. Adult worms of six different nematode species, parasites of the stomach or the small intestine of ruminants, were incubated in vitro in Dulbecco's Modified Eagle's Medium for 24 h. The conditioned media were then added at different concentrations to the culture medium of the two cell lines. A stimulation of the HT29-D4 cell growth occurred with the ES products of two parasite species of the small intestine, at the concentrations of 0.1 microgram protein/ml (Trichostrongylus vitrinus) and 1.0-5.0 micrograms/ml (Cooperia curticei). Inversely, a decrease in cell number was observed with the ES products of another intestinal species, Nematodirus battus at concentrations of 1.0-5.0 micrograms/ml. With the ES products of the abomasal nematodes, a proliferation of HT29-D4 cells was obtained at 0.25-5.0 micrograms/ml with ES products of Teladorsagia circumcincta but no significant effect was observed for Haemonchus contortus. On the tumoral gastric cell line HGT-1, the ES products from the 6 nematode species gave a similar stimulative effect. These in vitro results suggest that nematode parasite species secrete or excrete component(s) which could affect the epithelial regeneration of the host digestive tract.

Stimulation of HT29-D4 cell growth by excretory/secretory products of the parasite nematode Trichostrongylus colubriformis.

The presence of the nematode parasite Trichostrongylus colubriformis in the small intestine is associated with an increase in epithelial renewal. To assess the possible role of excretory/secretory products from the worm on cell proliferation, adult Trichostrongylus colubriformis were incubated in vitro in Dulbecco's Modified Eagle's Medium for 24 h and the conditioned medium was added to the culture medium of the transformed epithelial cell line HT29-D4. A stimulation of the HT29-D4 cell growth was ascertained at concentrations of 0.25-1.0 micrograms protein/ml using counts of cell numbers, the MTT method and incorporation of tritiated thymidine. An increased incorporation of tritiated thymidine was also observed with the excretory/secretory products from T. colubriformis fourth stage larvae at 1.0 microgram/ml. Dialysis of the medium conditioned by the worms indicated that the molecular weight of the factor is greater than 8000 Daltons in size. Heat treatment, acid hydrolysis and precipitation by trichloracetic acid of the conditioned medium resulted in the disappearance of the proliferative effect while treatment with trypsin partially depleted the stimulative activity. These results suggest that T. colubriformis produce some protein factor which could increase the epithelial regeneration in the host small intestine.

The computer-assisted interpretation of copolymer mass spectra.

A computer program called MSCOPOL has been developed to aid in the interpretation of copolymer mass spectra. The program reads the mass spectrum, calculates the most likely monomer masses via correlation or Fourier transform methods, determines possible end group masses based on the monomer masses, and can then search monomer and end group data bases for likely chemical moieties. Refinement of the end group result is possible by calculation of the monomer ratio and degree of polymerization as a function of end group mass. The program is written in Microsoft Visual Basic and runs on an IBM compatible PC. Applications are shown for polystyrene, poly(N-vinyl pyrrolidone/vinyl acetate), and poly(ethylene oxide/propylene oxide).