In order to identify genes that are differentially expressed as a consequence of oxidative stress due to paraquat we used the differential display technique to compare mRNA expression patterns in Caenorhabditis elegans . A C.elegans mixed stage worm population and a homogeneous larval population were treated with 100 mM paraquat, in parallel with controls. Induction of four cDNA fragments, designated L-1, M-47, M-96 and M-132, was confirmed by Northern blot analysis with RNA from stressed and unstressed worm populations. A 40-fold increase in the steady-state mRNA level in the larval population was observed for the L-1/M-47 gene, which encodes the detoxification enzyme glutathione S-transferase. A potential stress-responsive transcription factor (M-132) with C2H2-type zinc finger motifs and an N-terminal leucine zipper domain was identified. The M-96 gene encodes a novel stress-responsive protein. Since paraquat is known to generate superoxide radicals in vivo , the response of the C.elegans superoxide dismutase (SOD) genes to paraquat was also investigated in this study. The steady-state mRNA levels of the manganese-type and the copper/zinc-type SODs increased 2-fold in the larval population in response to paraquat, whereas mixed stage populations did not show any apparent increase in the levels of these SOD mRNAs.

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The search for appropriate vaccine candidates and drug targets against onchocerciasis has so far been confronted with several limitations due to the unavailability of biological material, appropriate molecular resources, and knowledge of the parasite biology. To identify targets for vaccine or chemotherapy development we have undertaken two approaches. First, cDNA expression libraries were constructed from life cycle stages that are critical for establishment of Onchocerca volvulus infection, the third-stage larvae (L3) and the molting L3. A gene discovery effort was then initiated by random expressed sequence tag analysis of 5,506 cDNA clones. Cluster analyses showed that many of the transcripts were up-regulated and/or stage specific in either one or both of the cDNA libraries when compared to the microfilariae, L2, and both adult stages of the parasite. Homology searches against the GenBank database facilitated the identification of several genes of interest, such as proteinases, proteinase inhibitors, antioxidant or detoxification enzymes, and neurotransmitter receptors, as well as structural and housekeeping genes. Other O. volvulus genes showed homology only to predicted genes from the free-living nematode Caenorhabditis elegans or were entirely novel. Some of the novel proteins contain potential secretory leaders. Secondly, by immunoscreening the molting L3 cDNA library with a pool of human sera from putatively immune individuals, we identified six novel immunogenic proteins that otherwise would not have been identified as potential vaccinogens using the gene discovery effort. This study lays a solid foundation for a better understanding of the biology of O. volvulus as well as for the identification of novel targets for filaricidal agents and/or vaccines against onchocerciasis based on immunological and rational hypothesis-driven research.

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