Parasite-derived Chemotaxins

E/S products of adult parasites contained chemotactic activity for both neutrophils and eosinophils (Tables 24 and 25). The two batches of H. contortus adult E/S products both showed ECF and NCF activity in all tests and, of the three adult O. circumcincta preparations, one showed both activities and the other two either NCF or ECF activity. The reason for the less consistent activity in some O. circumcincta E/S products may be a better survival of adult H.

contortus than O. circumcincta in the incubation media used, hence a greater secretion of chemicals. In support of this, in an in vitro study of a HeLa cell vacuolating agent, H. contortus more consistently produced the agent in E/S products than did O. circumcincta (PRZEMECK 2003). It was noted that there were no viable worms in batch 20 at the end of the 18.5 h of incubation, suggesting that, although visibly moving during the early incubation periods,

5. Discussion

these worms may not have been metabolically very active from the start of the incubation.

Another factor may be the number of worms in the incubation medium. Batch 22, which contained only weak ECF activity, had a much greater worm density (500/ml) than all other incubates. Density-dependent effects have been observed in other studies: at high worm density, the production of the HeLa cell vacuolating agent declined (PRZEMECK 2003) and the rate of acid excretion by incubated parasites is similarly affected (H. SIMPSON, pers. comm.).

Little chemotactic activity for both neutrophils and eosinophils was present in larval E/S products, particularly of O. circumcincta (Table 23). Of the tests of three L3 H. contortus preparations, two of three were positive for ECF and one of two for NCF. The only batch of O. circumcincta larval E/S products with chemotactic activity (both ECF and NCF) was batch 17, which had been generated in the anaerobic cabinet after an incubation period of 7-14 days, at which stage most larvae should already have developed from L3 to L4. A possible explanation for the weak chemotactic potential of most larval E/S preparations may lie in the very low secretory activity of third-stage larvae in vitro, which has been confirmed by GAMBLE and MANSFIELD (1996), who found that secretion/excretion of significant amounts of protein in H. contortus cultures did not occur until larvae had moulted to the fourth stage. More experiments employing culture fluids obtained under anaerobic conditions and after a prolonged incubation period would be needed to evaluate if this incubation method stimulates larvae to be metabolically more active than larvae cultured in an atmosphere of 5% CO2 in air, which is the most common incubation technique for these parasites (SCHALLIG et al. 1994; GAMBLE and MANSFIELD 1996).

The less pronounced chemotactic activity for both neutrophils and eosinophils of larval than adult E/S products may be due to the higher metabolic activity of adult worms, which have been found to release a range of chemical substances

5. Discussion

(Section 2.5.). This increased activity also shows in the higher amounts of protein measured in adult E/S products (Section 4.2.). The example of a concentration-dependent migration pictured in Figures 4 and 5 is characteristic of a chemotactic response (WILKINSON 1974). At high concentrations of a chemotactic factor, the number of migrating cells usually levels off or even declines, which may be due to inactivation of cells confronted with high amounts of the stimulating agent.

The effect of a uniform concentration of chemotactic substance was studied in the chemokinesis tests included in many experiments. There is evidence for chemokinetic activity in several experiments. It was less frequently observed than chemotaxis, but it may be an indication that cellular migration is indeed a mixture of chemotactic and chemokinetic movement, as suggested by some authors (KELLER and SORKIN 1966; WILKINSON 1974). If the cell movement were due only to chemotaxis, the chemotactic factor would not stimulate a migratory response above background migration when present in the same amounts on both sides of the filter, whereas a chemokinetic substance increases the unspecific movement of cells, resulting in cell counts above random movement under these circumstances. The discussion about the relation between chemotaxis and chemokinesis is ongoing (Section 2.6.1.) and the strict separation of these terms may not be relevant for the effects of a substance on cellular locomotion in vivo.

The extracts of both larval and adult H. contortus and O. circumcincta showed chemotactic activity for neutrophil and, to a lesser extent, eosinophil granulocytes (Tables 26 to 29). The response occurred in a concentration-dependent fashion, as demonstrated with extract dilution series. In several experiments with fractionated O. circumcincta larval extracts, it was not possible to identify a particular fraction with chemotactic activity, which appeared in all molecular weight fractions.

5. Discussion

Many investigations into the presence of parasite-derived chemotactic factors have employed parasite extracts, since they are easier to obtain than E/S products. Extracts also contain higher amounts of total protein, but the majority of these substances may have no chemotactic activity and could possibly ‘mask’

the actual chemotactic factor, making its identification more difficult. On the other hand, there could also be a range of substances with chemotactic activity in vitro in extracts, which would normally not make contact with host tissues. To establish the importance of such factors for cellular accumulation in vivo, the presence of the chemotaxin found in an extract should also be investigated in E/S preparations of the same parasite.