The effect of γ-radiation and heat shock on protein synthesis and antioxidant enzymes in the gastrointestinal parasite, Heligmosomoides polygyrus

Inlernational Journal Jbr Parasirology. Vol. 26, No. 4, pp. 353-361, 1996 Q 1996 Australian Society for Parasitology. Published by Elsevier Science Ltd. Printed in Great Britain OOM7519/96 $15.00+000 SOO20-7519(96)00018-5 Copyright

The Effect of y-Radiation and Heat Shock on Protein Synthesis and Antioxidant Enzymes in the Gastrointestinal Parasite, Heligmosomoides polygyvus RICHARD

J. PLEASS* and ALBERT

E. BIANCO

Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA U.K. (Received

27 September

1995;

accepted

22 January

1996)

Abstract-Pleass and antioxidant

R. J. & Bianco A. E. 1996. The effect of y-radiation and heat shock on protein synthesis enzymes in the gastrointestinal parasite, Heligmosomoides polygyrus. International Journal for Parasitology 26: 353-361. Protein synthesis and antioxidant enzyme activities were investigated in y-irradiated (300 Gy) and heat shocked (42’C) larval stages of the gastrointestinal parasite, Heligmosomoides pofygyrus bukeri (H. polygyrus). No qualitative or quantitative differences were observed in the incorporation of (“S)-methionine into somatic proteins of unirradiated or irradiated exsheathed thud-stage (L3) larvae at either 37’C or 42°C. The rate of protein synthesis doubled in L3 stages maintained at 42°C compared with 37’C, irrespective of whether the larvae had been irradiated or not. The composition of excretory/secretory (ES) proteins varied between u&radiated and irradiated exsheathed L3 larvae maintained under identical conditions. Prominent heat-inducible proteins of 26 and 17 kDa were synthesised and excreted at 42°C by both m&radiated and irradiated L3 stages. No major differences in protein synthesis could be detected between u&radiated and irradiated fourth-stage (L4) larvae. Temperature elevation significantly reduced protein synthesis in L4 stages, most notably in unirradiated parasites. Heat-inducible proteins were not detected in response to either irradiation or temperature elevation in L4 larvae. Immune sera recognised a similar spectrum of antigens in both unirradiated and irradiated L4 somatic and ES preparations and reacted with antigens from irradiated L4 parasites with less intensity than with antigens from unirradiated L4 larvae. Catalase was the only antioxidant enzyme examined with activity that changed significantly in irradiated parasites, being reduced to approximately 36% of normal levels in irradiated L4 stages. No significant difference existed between irradiated and unirradiated parasites in the levels of activity of superoxide dismutase and glutathione reductase. Copyright 0 1996 Australian Society for Parasitology. Published by Elsevier Science Ltd. Key words: Heligmosomoides reductase; gamma radiation;

polygyrus bakeri; protein synthesis;

antioxidants; heat shock.

INTRODUCTION

Most studies on irradiation of pathogenic organisms have been conducted with a view to the development or analysis of live, attenuated vaccines. Successfulvaccination of animals has been *To whom correspondence should be addressed at: Department of Pathology, University of Dundee Medical School, Ninewells Hospital, Dundee, Scotland, DDl 9SY. E-mail: [email protected]. 353

superoxide

dismutase;

catalase:

glutathione

achieved with irradiated protozoans (Nussenzweig, Vanderberg & Most, 1969), digeneans (Bickle et al., 1985), cestodes (IAEA, 1967) and nematodes (Behnke & Robinson, 1985). Several theories have been advanced to explain why irradiated parasites are more efficacious than normal parasites at eliciting protective immunity. These include radiation induced overproduction of larval antigens (Agyei-Frempong 8~Catty, 1983), delayed parasite development resulting in prolonged antigen presentation at a constant

354

R. J. Pleass

& A. E. Bianco

rate of antigen production (Urban & Romanowski, 1985), underproduction of larval antigen leading to secondary consequences (Wales, 1989), or radiation induced damage of antigen leading to alterations in surface antigenicity (Politz et aZ., 1990). y-Radiation can influence a variety of metabolic processes in living cells and tissues (Van Sonntag, 1987). Amongst these, the transcription and synthesis of heat shock proteins (HSPs) and antioxidant enzymes as a result of oxidative injury from y-rays are especially well documented examples (Von Sonntag, 1987; Halliwell & Gutteridge, 1989). Since HSPs and antioxidant enzymes are known immunogens (Selkirk ef al, 1989), it is possible that protective immunity arises from an over expression of these molecules. On the other hand, underproduction of these proteins could lead to an accumulation of aberrant molecules in irradiated parasites, which in the absence of functional repair systemsmight lead to heightened immunogenicity. Heligmosomoides polygyrus is one of the best characterised models of parasitic nematode infections. It has been used extensively to study protective immunity engendered by radiation-attenuated infective larvae (Hagan, Behnke & Parish, 1981; Pleass& Bianco, 1995). Investigations of the host response to irradiated larvae have defined many aspects of immunity in this model (Behnke, 1987) and have revealed that it is primarily the exsheathed L3 and the L4 larvae that induce a protective response (Wahid & Behnke, 1992). Nevertheless, little is known of the effects of y-radiation on biosynthetic activity or the stress response in post-infective larvae of H. polygyrus.

Here we have examined changes in irradiated organisms in respect of protein synthesis and the production of heat inducible proteins or antioxidant enzymes, with the aim of narrowing down the above theories. Experiments were carried out at 42°C so as to stimulate a “classical stress response” thereby providing a positive control to enable us to make statements about the effects of y-radiation on this stress response. We were interested to know if radiation compounded the response or abrogated it.

MATERIALS

AND

METHODS

Parasite and hosts. Laboratory strain H.polygyrus was used throughout. The parasite was maintained in inbred CBAKa mice. The methods employed for parasite maintenance and recovery of larval stages have all been described elsewhere (Behnke & Wakelin, 1977). Irradiation H.polygyrus

of larvae. were exposed

Infective, third stage larvae of to 300 Gy of y-radiation from a

Cobalt 60 source as previously described (Pleass & Bianco, 1995). In this study, parasites were exposed to 300 Gy based on the observation that this is the optimal level of y-radiation to use in the preparation of H.polygyrus larvae for vaccination experiments (Hagan et al., 1981; and our unpublished observations). Exsheathment of L3 stages and extraction of L4 stages. L3 stages were exsheathed in vivo following the protocol of Ey (1988). Worms were washed in multiple changes of PBS containing 400 IU/ml penicillin/streptomycin. They were finally resuspended in Ml99 medium (Sigma) and adjusted to the desired working concentration. Normal or irradiated developing L4 larvae were collected according to Ey, Prowse & Jenkins (1981). Intestines were placed in 15 ml of Ml99 culture medium and incubated for 1 h at 37°C. Emerging larvae were sexed, counted and transferred to 1.5 ml microfuge tubes for washing in culture medium supplemented with 400 IU/ml penicillin/streptomycin. Preparation and culturing of L3 and L4 stages for in vitro metabolic labelling. Exsheathed L3 stages and 5-day-old L4 stages were cultured to a density of 50,000 and 2OO/ml of culture medium respectively, containing 50 &i of (35S)methionine. Parasites were cultured for 24 h at 37°C or 42°C with an atmosphere of 5% CO, in air. After incubation, samples were chilled on ice and centrifuged to pellet the worms. The culture supematants were passed through a 0.22 pm filter, mixed with 0.01% v/v of protease inhibitor cocktail (2 mM phenyhnethylsulphonylfluoride, ImM ethylene diamine tetra-acetic acid, ImM ethylene glycol bis (2-aminoethylether)-N,N,h”,N’-tetra-acetic acid, 0. ImM N-tosylamide-L-phenylalanine chloromethylketone, 0.2mM N-a-p-tosyl+lysyl choloromethylketone HCI) and stored at -70°C. The worms were washed in fresh culture medium and stored at - 70°C. Protein preparation. Parasite proteins were extracted in 30 pl electrophoresis sample buffer (3% SDS, 62 mM Tris-HCI pH 6.8, 15% glycerol) containing 5% /3-mercaptoethanol. Samples were incubated for 5 min at IOOY, centrifuged at 13,000 r.p.m. for 5 min and the supematant collected for electrophoresis. Protein extracts corresponding to 1000 L3 or 15 L4 stage worms were loaded per track of an 8-20% SDS-PAGE gradient gel. These loadings were determined in preliminary experiments designed to achieve equivalence amongst tracks based on the intensity of Coomassie Blue staining. Radiolabelled ES proteins were loaded at 100,000 TCA-precipitable c.p.m. per gel track. For immunoprecipitations, L4 stages were broken up on ice in a Dounce homogeniser containing 700 ul of PBS with 1.5% n-octyl glucoside and protease inhibitor cocktail. The sample was vortexed and kept on ice for 1 h with intermittent vortexing. The parasite suspension was then centrifuged at 10,000 g for 30 min at 4°C and the supematant, equivalent to 100,000 c.p.m., used for immunoprecipitations. For ES preparations, the material was concentrated by ultrafiltration using an Amicon apparatus fitted with PM-IO membranes.

y-Radiation

and protein synthesis in Heligmosomoides

Immunoprecipitation experiments. Parasite lysates or ES were precleared by the addition of 100 ~1 Protein-A Sepharose (packed gel) and centrifuged at 30,000 g for 30 min at 4°C. Supernatant fractions equivalent to 100,000 c.p.m. were reacted with 6 ~1 of antisera raised against irradiated larval infections and left overnight at 4°C. Three microlitres of goat anti-mouse immunoglobulin was added to samples and left on ice for 1 h. Immune complexes were precipitated with Protein A-Sepharose for 1 h on ice, washed extensively in buffer (0.5% Triton X-100,0.1% SDS, 50 mM Tris-HCl pH 7.2, 0.5 M NaCl, 20 mM LiCl) and dissociated by heating to 1OOC for 5 min in electrophoresis sample buffer. All preparations were centrifuged for 5 min at 16,000 g to remove insoluble material, and were subjected to electrophoresis at 20 mA on S-20% polyacrylamide gradient gels with standard molecular weight markers. Gels were fixed and stained with Coomassie Brilliant Blue, immersed in AmplifyTM for 30 min and dried for fluorography. Determination of antioxidant enzyme activity. Worms were homogenised in PBS over ice using a sterile ground glass homogeniser and were disrupted further by 10 cycles of sonication at 4°C over an interval of 1 h. Crude enzyme extracts were centrifuged for 15 min at 16,000 g at 4°C. The resulting supernatant was used for enzyme assays and for protein determination by the Coomassie Brilliant Blue method of Bradford (1976). SOD activity was assayed by the method of McCord & Fridovich (1969), and that of CAT and GR by methods provided by Sigma. All assays were standardised using commercially available enzymes. For SOD, one unit of enzyme activity was defined as that amount which causes 50% inhibition of ferricytochrome C reduction by superoxide at pH 7.8, observed at 550 nm. For CAT, one unit of enzyme activity was the amount of protein required to break down 3.45 ~Mol of H,O, in 1 min at pH 7, observed at 240 nm. For GR, one unit of activity was that required to reduce 1.O~Mol of oxidised glutathione per min at pH 7.6, observed at 340 nm. Results are presented as units per mg protein or as units per individual worm. Stafistical analysis. The results are presented as group mean values i standard deviations (S.D.). The nonparametric Kruskall-Wallis statistic H was used to establish whether significant differences existed between treatment groups. If significant, specific groups were compared by the Mann-Whitney U test. Where appropriate, the MannWhitney U statistic was used to assessdifferences in antioxidant levels between different worm extracts. A value of PcO.05 was considered to be significant. All analyses were performed using Minitab@.

RESULTS Protein

synthesis

by irradiated

and unirradiated

355

polygyrus

asjudged by scintillation counting or somatic protein profiles (Fig. la) at either 37°C or 42°C. The rate of protein synthesis doubled with an increase in temperature from 37°C to 42°C for both unirradiated (37°C 4860 f 462 c.p.m.; 42°C 10,734 f 801 c.p.m.) and irradiated (37°C 4635 % 384 c.p.m.; 42-C, 11,016 f 570 c.p.m.) larvae. The increased protein synthesis was reflected by an increase in production of numerous proteins at the higher temperature, most notably of 200, 94, 89, 84, 77, 70, 64, 62, 60, 58, 38, and 30 kDa (Fig. la). The increased synthesis of these temperature inducible somatic proteins was unaffected by radiation. In contrast, a number of differences existed in the profiles of labelled ES proteins from unirradiated and irradiated parasites (Fig. lb). At 37°C the ES profiles were essentially similar, except parasites overproduced molecules of 200 and 80 kDa in unirradiated and irradiated worms respectively. At 42°C synthesis or secretion of ES proteins with molecular weights of 200, 105, 85, 80, 64, 58, 43, 40, 38, 35 and 33 kDa were markedly depressed in irradiated parasites. Both unirradiated and irradiated worms synthesised 2 low molecular weight ES proteins of 26 and 17 kDa at 42°C. These were absent from cultures maintained at 37°C (Fig. lb) and their synthesis was unaffected by radiation. Protein synthesis by irradiated L4 stages

and unirradiated

At either 37°C or 42°C irradiation had little effect on radioactive counts incorporated into somatic proteins with the exception of irradiated female worms cultured at 37°C which had 50% fewer counts per worm than unirradiated females (2275 & 259 c.p.m. vs 1095 f 365) despite their counts per pm3 of tissue being similar. However, no qualitative differences occurred in the pattern of labelled polypeptides (Fig. 2, cf. lanes 1 and 3 and lanes 5 and 7). Although male worms did not differ significantly, protein synthesis, as measured by methionine incorporation, was reduced in all female worms cultured at 42°C compared with worms cultured at 37°C. This was most significant in unirradiated females (37°C 2275 * 259 c.p.m.; 42°C 321 f 145; P
and irradiated

Immunoprecipitations of somatic L4 extracts of unirradiated or irradiated parasites with immune No qualitative or quantitative differences were sera (IMS), chronic infection sera (CIS) and naive observed in the incorporation of [?S]-methionine mouse sera (NMS) were analysed. IMS and CIS both into unirradiated or irradiated exsheathed L3 stages, reacted with antigens of molecular weights 69, 64,43,

exsheathed L3 stages

356

R. J. Pleass & A. E. Bianco

60 kDa

(b)

12

116 94-

26

30 20

Fig. 1. Fluorograms of [35S]-methionine labelled proteins synthesised by unirradiated and irradiated exsheathed L3 larvae of H.polygyrus during 36h of in vitro culture at 37°C or 42°C. (a) Somatic protein preparation. (b) ES proteins in culture supernatant. Lane 1, unirradiated L3 / 37°C; lane 2, irradiated L3 / 37’C; lane 3, unirradiated L3 / 42°C; lane 4, irradiated L3 / 42’C; lane 5, unirradiated L3 ES I 37°C; lane 6, irradiated L3 ES I 37’C; lane 7, unirradiated L3 ES / 42°C; and lane 8, irradiated L3 ES I42’C. 1000 L3 stages or 100,000 c.p.m. of L3 ES proteins were electrophoresed per lane.

40, 32, 30, 18.5, 17.5 and 10 kDa both in female (Fig. 3) and male somatic extracts. As similar results were obtained with worms of either sex, only data from immunoprecipitations with female worm antigen are shown. In addition, IMS recognised 2 additional bands of 201 and 43 kDa in unirradiated females (Fig. 3, lane 2), which were absent in unirradiated males and irradiated parasites of either sex. Both groups of sera immunoprecipitated

smaller quantities

of the immunodominant antigens from irradiated larvae compared with unirradiated parasites (Fig. 3, cf. lanes 2 and 3 against lanes 6 and 7). The effect of irradiation activities of H.polygyrus

on antioxidant

enzyme

Measurements were made of SOD, CAT, and GR in PBS soluble-extracts of both unirradiated

activity

and irradiated larvae following a 5-day period of development from the L3 to L4 stage in vivo. Representative data from 1 of 3 experiments are given in Table 1 There were no detectable differences in the levels of SOD (Mann-Whitney U statistic =5, P 0.05) or GR (Mann-Whitney U statistic =8.5, P 0.05) activity in unirradiated or irradiated L4 larvae. However, CAT activity was significantly reduced in irradiated worms (Mann-Whitney U statistic=l.50, PcO.05) compared with unirradiated parasites. On an individual worm basis, irradiated L4 stages had approx. 2.5x lessCAT than unirradiated L4 parasites. On the same basis, adult worms had markedly higher levels of CAT and SOD than L4 worms (Mann-Whitney U statistics, P dO.05). Adult female extracts possessed higher enzyme activities than adult male worms. SOD enzyme activity was confirmed by analyses of

y-Radiation and protein synthesis in Heligmosomoides

12

kDa

3

4

polygyrus

351

5678

200

116 94 66

30 20 14

Fig. 2. Fluorograms of [%I-methionine labelled somatic proteins synthesised by mm-radiated or irradiated L4 larvae of H. polygyrw during 24h of in vitro culture. (a) Female worms, (b) male worms. Lanes 1 and 5, unirradiated J-4 I 37°C; lanes 2 and 6, unirradiated L4 I42”C; lanes 3 and 7, irradiated L4 I 37°C; lanes 4 and 8. irradiated L4 I42”C. Extracts of 15 L4 stages were electrophoresed per lane.

SOD from unirradiated and irradiated L4 stages on composition of ES proteins varied between unirradisubstrate gels (results not shown). SOD activity was ated and irradiated L3 stages, especially in parasites detected under non-reducing conditions at 96,43 and subjected to the additional stress of temperature 36 kDa, and no observable differences were seen elevation. Although no major differences in somatic or ES protein synthesis could be detected between between unirradiated and irradiated parasites. unirradiated and irradiated L4 stages, immune sera recognised antigens from irradiated L4 parasites with DISCUSSION less intensity than unirradiated L4 larvae. y-Radiation had no effect on somatic protein These results are similar to studies of protein marsoni synthesis by exsheathed L3 larvae. However, the synthesis in irradiated Schistosoma

358

R. J. Pleass & A. E. Bianco

kDa

5

6

7

8

Fig. 3. Immunoprecipitations of [35S]-methionine labelled somatic antigens from female L4 stages. (a) Unirradiated LA larvae, (b) irradiated L4 larvae. Lanes 1 and 5, total somatic preparation; lanes 2 and 6, immune mouse sera (IMS); lanes 3 and 7, chronic infection sera (CIS); lanes 4 and 8, naive mouse sera (NMS). Proteins were loaded at 100,000 c.p.m. per gel track. Table l-Mean in unirradiated

( f S.D.) specific activities of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) and irradiated L4 stage and adult worm PBS soluble-extracts (n =5). Activities are expressed as units per mg protein (U/mg) and units per worm (U/worm)

Life-cycle stage

SOD (U/mg)

SOD (U/worm)

CAT (U/mg)

CAT (U/worm)

GR (IJ/mg)

Unirradiated IA Irradiated L4 Unirradiated adult female Unirradiated adult male

1.6 (0.2) 1.4 (0.1) 7.4 (0.7) 4.5 (0.6)

0.004 0.002 0.04 0.02

7.7 5.0 9.4 7.8

0.02 0.007 0.09 0.07

0.15 0.13 Not Not

schistosomula (Wales, Kusel & Jones, 1992). These authors showed that “mechanically transformed’ schistosomuhawhose cercariae were irradiated at 200 Gy had a transient inhibition (up to 80%) of protein

(0.8) (1.4) (0.7) (1.0)

(0.02) (0.03) done done

synthesis which recovered to normal levels after 72 h in culture. They concluded that a partial inhibition of protein synthesis may contribute to the heightened immunogenicity of these larvae. Other authors have

y-Radiation and protein synthesis in

been unable to demonstrate by biochemical (Simpson 1985), morphological (Mastin, Biclcle & Wilson, 1985) or immunological means (Vieira et aZ., 1987) differences between normal and irradiated schistosomes,or filarial parasites (Devaney, Bancroft & Egan, 1993). The effect of radiation on nonproteinaceous components has received little attention in the majority of studies. However, a recent paper by Wales et al. (1993) gives strong evidence that changes in carbohydrate antigens consequent upon U.V.irradiation may be important in generating the enhanced immunogenicity of irradiated cercariae in Smansoni infections. The reduced size of irradiated parasites (Pleass8t Bianco, 1995) clearly does not stem entirely from defects in protein synthesis, but from targets overridingly more sensitive to radiation than proteins. Individual nematode life-cycle stages are remarkably radioresistant and doses of 900-1600 Gy do not markedly alter parasite metabolism (Ciordia & Bizzell, 1960; Gomberg & Gould, 1953). However, the differentiation of larvae is much more sensitive, and doses of 100-300 Gy may perturb parasite development (Behnke, Parish 8r Hagan, 1980). It may therefore, be that developmental and/or regulatory genes and their products are more radiosensitive than structural “house-keeping” genes and proteins. As regulatory elements are relatively minor constituents of total cellular proteins, this may explain why major changes in protein synthesis were not observed in irradiated larvae following analysis by one dimensional SDS-PAGE. Nevertheless, several changes were observed in the profile of ES products synthesised andlor released by irradiated parasites. Possibly, these include molecules involved in defence against host attack, or in other biologically important functions, the lack of which may underlie the failure of irradiated larvae to complete development and establish chronic infections. Amongst the molecules that were elaborated and appeared in the ES following temperature elevation to 42°C were 2 proteins of 26 and 17 kDa. As these appeared to be strictly heat-inducible, it is tempting to conclude that they are small HSPs, as described by others from a wide variety of parasitic and nonparasitic organisms (Devaney et al., 1992; Jecock & Devaney, 1992; Tweedie et al., 1993). Nevertheless, their appearance in the secretions is not what would be expected for classicalHSPs, which generally occur in the nucleus or cytoplasm of the cell (Polla, 1991). Artefactual release of these products from damaged cells seems unlikely because the worms remained active and apparently healthy in cultures maintained at 42°C during the course of these experiments. Moreover, no proteins of equivalent molecular et al.,

Heligmosomoides

polygyrus

359

weights were detected in extracts of the somatic tissues, suggesting that these molecules are secreted rapidly and in quantitative amounts. Further work will be necessary to establish whether these are related to small HSPs, or to some other class of heat-inducible protein. Amongst the large and diverse family of HSPs and related stressproteins, different molecules are induced and expressedby different stressorswhich perform a variety of cellular tasks. The importance of many HSPsis based on their capacity to associatewith other proteins, regulating their tertiary structures and translocation, and thereby altering their fate and function in the cell. Neither radiation nor temperature shock gave rise to the expected increasein somatic tissuesof conventional stress protein synthesis or to elevated antioxidant enzyme activity. However, the prediction that aberrant and more antigenic proteins would therefore accumulate in irradiated larvae was not supported by immunoprecipitation analyses, based on comparative analyses of unirradiated and irradiated parasites maintained at either 37°C or 42°C. The apparent failure of stress responses to be activated in irradiated parasites may make them less able to cope with attack from reactive oxygen intermediates produced in their tissuesby ionising radiation, or by host immune effector cells.Irradiated parasites, lessable to prevent or repair damage, would be likely to succumb to the host’s immune response and die. Since few differences were observed between irradiated and unirradiated L4 larvae in terms of protein synthesis and antigenicity, we believe that the reason irradiated larvae were more immunogenic than normal larvae in earlier studies (Hagan et aL, 1981) was not due to an effect of the radiation per se but simply because unirradiated larvae unlike irradiated larvae were able to develop to adulthood and therefore the mice experienced the immunodepressive activities of the adults (Pleass& Bianco, 1994). The level of radiation used in this study (300 Gy) was insufficient to cause appreciable loss of antioxidant enzyme activity through direct damage to the enzymes (Coggle, 1983). Reductions observed in CAT activity in irradiated parasites therefore, most probably reflect a decrease in tissue levels of this enzyme. This observation is interesting in light of the apparent importance of CAT in maintaining H.poZygyrus infections, and the reported discrepancy with Nippostrongylus brasiliensis infections (Smith & Bryant, 1986). Comparative studies of the 2 species have revealed that H.poZygyrus possessesthree times the level of CAT relative to N. brasiliensis, and it may be that this enables the worm to resist H,O, induced damage and avoid expulsion from the gut, as occurs with N. brasiliensis in rats.

R. J. Pleass & A. E. Bianco

360

The tinding that adults of H.polygyrus possess higher SOD activities than larvae contrasts with the majority of gastrointestinal hehninths studied (Knox & Jones, 1992). Higher enzyme activities in larvae have been associated with the requirement to counteract oxidative stress associated with the mucosal inflammatory response to developing worms. The elevated SOD activities in adult H.polygyrus might reflect the importance of this enzyme in maintaining chronicity of infection. It has been speculated that antioxidant enzymes may even be involved in the suppression of host protective immunity by adult worms (Behnke, Barnard & Wake& 1992). The results of this study suggest that y-radiation at levels used in the generation of attenuated larvae for vaccination experiments do not elicit major changes in protein synthesis by H.polygyrus. Moreover, the data do not support the notion that irradiated larvae are more immunogenic than unirradiated parasites (Hagan et al., 1981), or that they overexpress antigens that are the target of responses in vaccinated hosts (Agyei-Frempong dr Catty, 1983). It should be recognised that minor antigenic products, or nonproteinaceous components might be involved, and these would not have been detected in the present investigation. Nevertheless, it has been demonstrated recently that unirradiated larvae of H.polygyrus can stimulate high levels of host resistance, but only if the sensitising infection is eliminated with anthehninthic prior to challenge (Pleass & Bianco, 1994). This contrasts with earlier findings (Hagan et al., 1981) and implies that the induction of immunity is not dependent on properties peculiar to the irradiated larvae. Instead, it may be that the prevention of adult worm maturation is a more important factor than the immunogenicity of larvae in the vaccine efficacy of radiation attenuated parasites. If this is so, the focus of future investigations should turn to adult worm biosynthesis in relation to the well established phenomenon of immunomodulation of the hostprotective response by H.polygyrus infections (Pleass & Bianco, 1994). Acknowledgements-R.J.P. was supported by a S.E.R.C. studentship. A.E.B was supported by a Senior Basic Biomedical Fellowship from the Wellcome Trust. We thank Dr Peter Clay at the Dept of Chemical Engineering, Imperial College for use of the Cobalt-60 source. REFERENCES

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