Humoral and cellular responses to homologous extracts of Nematospiroides dubius and Nippostrongylus brasiliensis

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AND CELLULAR REiSPONSES TO HOMOLO~US EXTRACTS OF N~~ATOS~IROI~~S DUBI~S AND NIPPOSTRONGYLUS BRASILIENSIS PATRICIA PRICE* and KEVEN J. TURNER?

* Department of Microbiolo~, t Clinical Imm~olo~

University of Western Australia, Nedlands, 6009, Western Australia Unit, Princess Margaret Hospital, Subiaco, 6008, Western Australia

(Received 21 June 1985: in revisedfonn 22 January 1986)

Abstract-PRICE P. and TURNERK. J. 1986. Humoral and cellular responses to homologous extracts of

Nematospiroidesdubius and Nippostrongyks brasilierks. InternationalJournal for Parasitology16: 601606. Humoral and cellular responses to homologous parasite extracts were studied in C57BL mice infected with Ne~~s~iro~es debts or Nippostron~i~ brasihensis,to determine whether these parasites induced specific i~~osuppression which might facilitate their survival. IgG and IgM titres to adult, excretorysecretory, larval and egg antigens from N. dubius and adult antigen from N. brasiliensisincreased progressively for several weeks, irrespective of parasite rejection. Delayed-type hypersensitivity responses to the same antigens peaked after about 2 weeks and then remained constant in N. dubius-infected mice, but declined after the rejection of N. brasiliensh. The specific lymphoproliferative responses of spleen cells to these extracts reached peak values 1-3 weeks after infection and were anti-Thy 1.2-sensitive. They then declined sharply, but remained above the levels seen in uninfected mice. These results should be considered in the inte~retation of any investigations into the stable host-parasite relationship which exists during a primary N. dubius infection. INDEX KEY WORDS: Nematospiroides dubius; Nippostrongyh brasiliensis; homologous immune responses; delayed-type hypersensitivity; ELISA assay; lymphoproliferation; host-parasite relationship; parasite antigens.

INTRODUCI’ION PREVIOUS studies from our laboratory have been con-

cerned with the role of non-specific immunodepression in the survival of intestinal hehninths (e.g. Price & Turner, 1983b, c, 1984). However a number of parasites are known to induce specific i~unosuppression. For example, Kwa & Mak( 1980) demonstrated depressed delayed-type hypersensitivity (DTH) responses to homologous antigens in patients with filariasis, whilst heterologous responses were normal. The parallel depression of lymphoproliferative responses to filarial antigens was associated with adherent suppressor cells and serum factors (Piessens, Ratiwayanto, Tuti, Palmieri, Peissens, Koiman & Dennis, 1980), rather than, cyclophospamide-sensitive T suppressor cells (Katz & Larnmie, 1984). However, there is indirect evidence suggesting that regulatory T cells contribute to the depression of homologous lymphopro~ferative responses in sheep infected with the intestinal nematode, ~ae~o~c~~ conto~ (e.g. Adams, 198 3). The aim of this paper was to determine whether the non-specific immunosuppressive effects of Nematospiroides dubius had parallels in homologous responses. This has a clear basis in experiments by Cayzer & Dobson (1983) and Behnke, Hannah &

Pritchard (1983) which demonstrated resistance to reinfection in mice maintaining adult N. dubius. Similar results have been reported with N. dubiztsinfected mice challenged with other parasites (e.g. Hagan & Wakelin, 1982). However, the present study adopted a different approach. Humoral, DTH and l~phoproliferative responses to parasite extracts were correlated with parasite development and rejection in mice infected with N. dubius or Nippostrongyh brasiliensis . This approach is especially valid in view of the likelihood that the development of host-protective vaccines may depend on thecharacterization of specific cellular responses to parasite antigens in terms of the subsets of T cells which are activated and the antigens involved. MATERIALS AND METHODS Animals and parasites. Male SPF C57BL/6J mice were obtained from the Animal Resources Center (Murdoch University, W.A.) and rn~nt~n~ under barrier conditions throughout the experiments. The methods used to transmit and maintain the-parasites (N. dubius and N. brasiliensis) have been described oreviouslv price &Turner, 1983aj.The infective larvae will be denote2 kern L3 and Nip L3, reipectively. Parasiteextracts.Adult N. dubius and N. brasiliensis(ratderived) were collected in warm phosphate-buffered saline 601

602

P. PKI~Eand K. J.

(PBS)inamodifiedBaerman’sApparatus,washedandstored frozen in distilled water. These will be denoted Nem Ext and Nip Ext, respectively. Excretory-secretory (ES) products and egg extract were prepared by incubating washed adult N. &&us worms overnight at 37°C in PBS containing 10,000 i.u./ml penicillin. The eggs were separated from the supernatant (ES products) by cent~~gati~n. Adult worms, eggs and infective larvae (“Nem L3 Ext”) were disrupted by sonication on ice in 2-s bursts. Complete disruption of adult worms occurred within 30 s.Eggs and larvaeremainingintact after 2 min sonication were removed by centrifugation. The resultant preparations were sterilized by millipore filtration and stored at -70°C. The protein content of all extracts was estimated by a Coomassie Blue Dye-Binding assay relative to a bovine serume albumin standard (Read & Northcote, 198 1). Analysisof humoral responses. An ELISA assay for nonparasite antigens was described in an earlier publication (Price & Turner, 198 3~). The only modification required for the reproducible measurement of anti-parasite responses was that the plates were coated with antigen by overnight incubation only. The optimal levels of parasite protein for sensitization (determined by the titration of immune and non-immune sera) were 22pg Nem Ext protein ml-‘, 4 pg ES protein ml--‘, 1.2 pug Nem L3 Ext protein ml-‘, 3.3 hg egg protein ml-’ and 14 pg Nip Ext protein ml-‘. DTH responses. Responses were induced by challenging one ear with IO@ parasite extract and the other with 10 ,ul PBS,viaaSO gaugeneedle.~ediffetencein thickness of the two ears was measured as units X IO-?mm with a Moore and Wright micrometer 24 h later. Lymphoproliferativeresponses. Methods employed for the preparation and culture of spleen and mesenteric lymph node (MLN) suspensions are -described elsewhere @ice & Turner. in press\. Triplicate 200 u 1cultures were established with 6 X l&OS ceils in’mouse-buffered RPMI, supplemented with 4 X lo-‘hn Z-metcaptoethanol and 0.5% autologous serum. Parasite extracts were added in 10 p 1PBS and the cultures were incubated for 72 h prior to pulsing with 18.5 KBq ‘H-thymidine (Amersham, U.K.). They were harvested for liquid scintillation counting after a further 18 h. The anti-Thy 1.2 sensitivity of selected lymphoproliferation responses we&determined-by incubation ofspleen or lymph node cells (37°C. 45 mint with monoclonal anti-Thv 1.2 (Olac, U.K.j and fresh guihea-pig serum. The efficac; and specificity of these reagents was verified by Trypan Blue exclusion and mitogen-induced lymphoproliferation using spleen, thymus and bone marrow preparations.

RESULTS Stage-speci.c humoral responses in infected mice To determine whether ail stages of the parasites’ life cycle contained antigens capable of inducing detectable humoral responses, pooled sera from mice infected orally with 300 Nem L3 were assayed for IgG and IgJkl antibodies to Nem Ext, ES products and Nem L3 Ext and IgG antibodies to egg extract. Egg counts confirmed that the parasite persisted throughout the experiment at close to peak levels. A standard pooled serum from long-term infected mice was assigned a value of 1000 units of IgG and IgM directed against each extract, so the values recorded in Fig. 1 reflect the levels of antibody to that extract in the

‘I‘URNEK

12,

IgG

I..

._,

. .

‘.

12( Ig M 2 ..E

210

‘b-----______________

5 20

II/

@%.

=n

3

4.

t

. 20

. 40 60 80 XXI lie Time after infection (days1

140

160

FIG. 1. Phase-specific humoral responses to N. dubius extracts. Log, ELISA units derived from pooled sera from groups of approx. five mice infected orally with 300 Nem L3 on day 0. Samples were titrated on ELISA plates coated with Nem Ext (+), ES products (+), Nem L3 Ext (-A-> or egg extract (-0 -). IgM titres to egg extract were not recorded.

standard serum and cannot be compared with those recorded with other extracts. Significant IgG and IgM responses of broadly similar kinetics were recorded with all four extracts. Comparisons between early and late bleeds for each extract showed that IgG titres to egg extract and ES products developed only after the adult worms were established and egg production was initiated Nem L3 Ext yielded proportionately higher titres early in the infection. IgM titres to ES products were maintained in long-term infected mice, whilst titres to Nem Ext and Nem L3 Ext declined by half (one log, unit). To investigate the effects of the route of infection on the development of humoral responses to N. dubius, pooled sera from groups of six oally or intravenously (iv) infected mice were titrated against Nem Ext, ES products and Nem L3 Ext. In a second experim~nt,responsestoor~i~ectionwerecompared with those induced by intraperitoneal (ip) immunization with 1 mg Nem Ext in 4 mg Al(OH), (see Table 1). Following iv infection, only the IgG response to Nem L3 Ext was higher than that developed by an oral infection. This is a logical outcome of systemic larval migration. I~u~zation with Nem Ext induced

Homologous responses during helminth infections

603

TABLE ~--STAGE-SPECIFIC HUMORALRESPONSESTO N. dUbiUSExTRACTS I&G

N. dubius

IgM

Nem Ext

ES

NemL3 Ext

Expt. 1: Controls Nem L3 (oral) Nem L3 (iv)

2.4 6.3 5.3

1.7 4.6 3.5

4.7 6.9 8.4

4.9 11.1 9.6

6.4 8.4 9.1

6.0 9.0 8.3

Expt. 2: Controls Nem L3 (oral) Nem Ext (ip)

2.0 4.8 4.1

1.0 2.8 2.0

5.0 8.5 7.5

4.3 10.6 9.6

4.7 8.5 9.1

4.8 8.4 8.0

NemExt

ES

NemL3Ext

Log, ELISA units derived from pooled sera from groups of six mice.

Expt. 1: 250 Nem L3 administered on day (- 18). Expt. 2: 250 Nem L3 or 1 mg Nem Ext-Al(OH), administered on day (- 10). measurable titres, ‘which could be detected with all extra& tested. DTH responses to N. dubius extracts Mice infected with N. dub& for 3 or 6 weeks were

challenged with extracts of the parasite for the measurement of specific DTH responses (seeTable 2). Egg extract developed similar responses in control and infected mice, so this data is omitted from the presentation. NemExt, Nem L3 Ext and ES products induced significant responses in both groups of infected mice. The apparent increases between the third and sixth week were not statistically significant (ttest, P> 0.05). Specific lymphoproliferative responses to N. dubius extracts The aim of these experiments was to determine the relationship between increases in the sizes of the spleen

and MLN in infected mice and the development of parasite-specific cellular responses. Spleen and MLN cell suspensions were prepared initially from mice sacrificed 1 or 3 weeks after oral infection and from uninfected controls. Lymphoproliferative responses recorded after culture with Nem Ext or ES products are shown in Fig. 2. Background lymphoproliferative responses (no extracts) were elevated 1 week after infection. However, this effect did not equal the dramatic specific responses seen when parallel cultures were stimulated with Nem Ext or ES products. Lymphoproliferation recorded in the presence of these agents decreased between the first and third weeks, but TABLE 2-DTH

Extract Nem Ext ES products Nem L3 Ext

background responses also declined, so the corrected values for Nem Ext-induced proliferation remained constant and ES-induced responses increased slightly. Spleen cell yields increased three-fold in the first week of infection and thereafter remained constant. Hence it appears that both the specific and non-specific responses to N. dubius developedduringthefirstweek of infection in the spleen. The average size of the MLN was 1.7,2.8 or 4.4 X 10’ cells in control mice and mice infected for 1 or 3 weeks, respectively. Hence, unlike the spleen, this organ increased in size at a fairly constant rate for the first 3 weeks. Specific lymphoproliferative responses to N. dubius extracts reached peak levels 1 week after infection and thereafter remained constant in cells from this organ. To extend the time course of this experiment and determine whether T cells were involved in the specific lymphoproliferative responses to Nem Ext, ES products and Nem L3 Ext, aliquots of spleen preparations derived from mice infected with N. dubius 3 or 6 weeks earlier, and from uninfected controls were pretreated with anti-Thy 1.2 plus complement or complement alone and cultured with the parasite extracts (see Table 3). Nem Ext induced a large specific response which was highly sensitive to anti-Thy 1.2. This declined by approx. 25% between the third and sixth week of infection. In this experiment ES products elicited a higher response from uninfected mice than was usual. However, the difference in the specific response between controls and 3-week-infected mice was of the same order of magnitude as that described in Fig. 2. The decrease between the third and sixth weeks

RESPONSESTO EXTRACTSOF N.

dubius

Dose/ear (pg protein)

Uninfected mice

Nem L3 (-3 weeks)

Nem L3 (-6 weeks)

5.3 0.2 0.3

2.8 f 1.6 2.9 + 1.4 3.0 + 1.2

8.1 & 1.1 9.4 + 1.6 12.5 f 4.5

11.9 + 2.7 14.0 + 2.3 17.8 f 3.2

Mean DTH responses (-I-s.E.) derived from groups of six mice 24 h after ear challenge with 10~1 N. dubius extracts. Mice were infected orally with 250 Nem L3 at the times indicated.

P. PRICE and K. J. TURNER

604

lymphoproliferative responses are declining slowly in the spleen (Table 3) but are stable in the MLN (Fig. 2 and unpublished observation).

80

Correlation between humoral and cellular responses to N. brasiliensis

Groups of mice were infected subcutaneously with 600 Nip L3 for 7,lO or 20 days before being sacrificed for the measurement of humoral, DTH and splenic lymphoproliferative responses to an adult worm extract (Nip Ext). Uninfected controls were included as usual and the results are summarized in Fig. 3. Egg counts confirmed that the parasite was rejected between 8 and 10 days after infection. Both IgG and IgM titres were detectable by day 7, increased significantly during the rejection phase and showed little change thereafter. Individual variation was minor with this assay (Fig. 3A). In contrast, DTH responses to ear challenge with 10 pg Nip Ext (3 pg protein) were highly variable (as were individual egg counts) and declined rapidly after parasite rejection. Total cell counts from the spleen and MLN were not affected by N. brasiliensis. The lymphoproliferative responses of spleen cells to Nip Ext were low when parasite rejection began on day 7, increased to peak levels on day 10 and thereafter decreased only slightly. There was a detectable specific proliferative response in the MLN on day 7, but other times were not tested.

56

F 040 x -32 z (7 c 24 $ ;r 16

P g

8

0

1

2 3 Time after

0 Infection

1

2

3

(weeks)

to N. dubius responses FIG. 2. Lymphoproliferative and MLN preparations. Mean extracts in spleen DPM X 10-r values (~s.E.) derived from triplicate 200 ~1 cultures of spleen or MLN cells pooled from groups of mice infected orally with 250 Nem L3 on day 0. Key: Unstimulated cultures (0); Nem Ext, 1.75pg protein/well (0); ES

products, 0.6 pg protein/well (A).

DISCUSSION

was similar to that seen with Nem Ext and the responses were again highly sensitive to anti-Thy 1.2. Nem L3 Ext induced relatively low specific lymphoproliferative responses which declined by the sixth week of infection. Preliminary experiments showed these to be approx. 75% anti-Thy l.%-sensitive. Hence it is established that by the sixth week of a stable N. dubius infection, specific humoral responses (IgG and IgM) are still rising (Fig. l), DTH responses are approximately constant (Table 2) and T-cell TABLE

3-LYMPHOPROLIFERATIVE

Protein/ Extract Blank Nem Ext ES products Nem L3 Ext

well (pg)

The only previous studies of humoral responses N. dubius by an ELISA assay were described

to

by Cayzer & Dobson (1983). This group did not measure IgG and IgM separately or consider stage-specific antigens. These facilities in the ELISA assay have been exploited in the prsent system to show that mice infected with N. dubius develop IgG and IgM titres corresponding to antigens found in Nem Ext, Nem L3 Ext and homologous ES products and egg extract. A degree of cross-reaction between these extracts was evident from the titres to ES products and RESPONSES

Uninfected mice

1.4 0.20 0.07

4 * 0.2 11 f 0.8 48 f 3 2152

0.20 0.07

6 + 0.5 4 + 0.3

TO

N. dubius

EXTRACTS

Nem L3 (-3 weeks)

Nem L3 (-6 weeks)

lo+ 1 125,3 (85) 99 ?C3 (84) 81+2

5.0 * 0.6 104+7 (92) 97f5 (88) 62 +_ 2

16.2 + 2 13 + 0.3

8 + 0.2 I * 0.7

Mean DPM X 10mi values (*se.) derived from triplicate 200~1 cultures of spleen cells, from groups of five normal or N. dub&-infected mice, stimulated with homologous extracts. Figures in parentheses represent the percentage of the response sensitive to anti-Thy 1.2 (i.e. calculated from stimulated minus unstimulated values for anti-Thy 1.2 plus complement treated cells/stimulated minus unstimulated values for cells treated with complement alone).

Homologous responses during helminth infections

I

~ 0

4 8 12 16 20 --~(MLNI Time after infection (days1

FIG. 3. Accumulated humoral and cellular response to N. brusiliensis infection. Groups of six mice were infected (SC)with 600 rat-derived Nip L3 on day 0. A: Log, GM antiNip Ext ELISA units (+s.E.) corrected by subtraction of titres recorded in uninfected mice; IgG (O), IgM (0). B: Mean DTH responses (+s.E.) to Nip Ext ear challenge (3 pg protein). C: Mean DPM X lop3 values (~s.E.) derived from triplicate 200 ,uI cultures of spleen cells (solid symbols) or MLN cells (open symbols). Key: 2.2 ,~g Nip Ext protein/ well (0,0); 0.2 pugNip Ext protein/well (A, A); unstimulated

cultures (0,

n).

Nem L3 Ext induced by immunization with Nem Ext (Table 1). However, the importance of stage-specific antigens was illustrated by the more rapid increase in IgG titres to Nem L3 Ext after infection (compared with responses to egg antigen or ES products) and the higher titres to Nem L3 Ext seen in iv-infected mice. This is consistent with reports that antibodies to stagespecific antigens of infective and migratory larvae and adult N. dub&, N. brasiliensis and T. spiralis develop at different rates during primary infections (Pritchard, Maizels, Behnke & Appleby, 1984; Maizels, Meghji & Ogilvie, 1983; Phillip, Taylor, Parkhouse & Ogilvie, 198 1, respectively). The ELISA assay could be of considerable value in the extension of these studies to identify host-protective antigens. Our investigation also represent the first systematic study of DTH and lymphoproliferation responses to

605

extracts of N. dub&s. Splenic lymphoproliferative responses to Nem Ext and ES products peaked 1 week after infection and thereafter declined slowly. In contrast the MLN continued to accumulate cells able to respond to parasite antigens in vitro and parasitespecific humoral and DTH responses increased steadily throughout the period of study. Day, Howard, Prowse, Chapman & Mitchell (1979) reported shortlived DTH responses to N. dubius and N. brasiliensis ES products in infected mice. The difference between our finding with N. dubius (Table 2) and their results cannot be explained by differences in the duration of infection in the two systems. They also demonstrated a progressive increase in ES product-specific antibody to both parasites by a gel diffusion assay, which is consistent with the present results. In our system N. brasiliensis induced clear humoral and lymphoproliferative responses to adult antigen which persisted after the rejection of the parasite. However, DTH responses were as variable as the egg counts and declined as soon as the worms were eliminated (Fig. 3), as observed in the study by Day ef al. (1979). Proliferative responses to an extract equivalent to ES products from N. brasiliensis worms have been demonstrated, coincident with primary and secondary parasite rejection, in spleen and MLN preparation from mice infected with 1000 Nip L3 (Vickery, Klein & Friedman, 1981). These kinetics are comparable to those obtained in the present study with a primary infection. Since the lymphoproliferative responses to N. dubius antigens were anti-Thy 1.2-sensitive, they may be related to the T-cells able to transfer immunity to this parasite, such as those described by Williams & Behnke (1983). Prowse (1981) demonstrated that MLN cells from mice infected with N. dubius three times produced interleukin 2 in the presence of adherent peritoneal cells and parasite antigen. The response was considered to be a measure of parasiteantigen-specific T recognition, probably involving helper T cells. The requirement for peritoneal cells may indicate that T cell-mediated rejection mechanisms are limited in vivo by the availability of macrophages within the MLN. Subsequent investigations (Prowse, 1982) showed short-lived interleukin 2 production in the peritoneal cavity and draining lymph nodes during a primary infection. This response developed and subsided before the development of resistance to re-infection, but it could have a role in the persistence of the primary infection. In conclusion, it is now clear that there are several helminths which exert non-specific immunosuppression whilst inducing cellular and humoral responses to homologous antigens. From the literature, S. rat& and T. spiralis can be placed on this list (Genta, Ottesen, Gem & Neva, 1983; Barriga, 1975) and from the present study, N. dubius can be added. Here parasite-mediated immunomodulation clearly does not involve a total ablation of immune responsiveness, or even a suppression of the expression of responses to parasite antigens, as has been demonstrated with

P. P~tct and K. J. TURN&R

606

filaria and H. con~ortus (Piessens etal., 1980; Adams, 1983, respectively). Rather there must be complex regulatory processes involving many aspects of the immune system which selectively limit those responses which could mediate rejection. Elucidation of these mecha~sms is essential for the intological control of parasitic disease. Acknowledgements-‘This

study was supported by the Princess Margaret Hospital Children’s Medical Research Foundation &d represents publication No. 221 of the Clinical Immunology Research Unit of the Princess Margaret Children’s Medical Research Foundation. REFERENCES ADAMS D. B. 1983. Investigations with Dexamethasone

of the processes which moderate immunity against the nematode, Haemonchus conforms, in sheep. Australian

Jot~rnuI of Expe~menta~ Biology and Medical Science 6 1: 345-353. BARRIGA0.0. 197.5. Selective immunodepression in mice by Trichinella spiralis extracts and infections. Cellular Immunology 17: 306-309. BEHNKE J. M., HANNAHJ. & PRITCHAID D. I. 1983. Nematospiroides dubius in the mouse: evidence that adult

worms depress the expression of homologous immunity. Parasite ~mmunoio~

5: 398-408.

CAYZERC. J. R. & DOBSONC. 1983. Suppression of antibody production in mice given multiple concurrent infections with Nematospiroides dubius. International Journal for Parasitology 13: 61-65. DAY K. P., HOWARDR. J., PROWSES. J., CHAPMAN C. B. & MITCHELLG. F. 1979. Studies on chronic versus transient intestinal nematode infections in mice. I. A comparison of responses to excretory/secretory (ES) products of Nippostron~~us bras&e&s & Nematosp~roides dubius worms. Parasite Immunology 1: 217-239. GENTA R. M., OYTESENE. A., GEM A.A. & NEVA F. A.

1983. Immunologic responses to experimental Strongyloidiasis in rats. Zeirschrifi fur Parasitenkunde 69: 667”

,

675.

HAGABP. & WAKELI.N D. 1982. ‘~emutosp~roides dubius: Effect of infection on lymphocyte responses to Trichinei~a spiralis in mice. Experimental Parasitology 54: 157-165. KATZ S. P. & LAMMI~P. J. 1984. Effect of cyclophosphamide on the immune responsiveness of jirds infected with Brugia pahangi. Infection and Immunity 43: 753-755. KWA B. H. & MAK J. W. 1980. Specific depression of cellmediated immunity in Malayan filariasis. Trunsactions of the Royal Society for Tropical Medicine and Hygiene 74: 522-527. MAIZEL~ R. M., MEGHJIM. & OG~LVIE B. M. 1983.

Restricted sets of parasite antigens from the surface of different stages and sexes of the nematode parasite Nippostrongvlus brasiliensis. immunology 84: 107- 12 1.

PHILLIPM., TAYLORP. M., PARKHOUSE R. M. E. & OGILVIE B. M. 1981. Immune response to stage-specific surface antigens of the parasitic nematode Trichinella spiralis. JournalofExperimentalMedicine 154:210-215. PIESSENSW. F., RATIWAYANTO S., TUTI S., PALMIE~ J. H.,

PEISSENSP., KOIMA.N I. & DENNISD. T. 1980. Antigenspecific suppressor cells and suppressor factors in human filariasis with Brugia malayi. New England Jooumal of Medicine 302: 833-837. PRICE P. & TURNER K. J. 1983a. Immunolorical

consequences of intestinal helminth infections in C57BL mice. The effects on lymphoid tissue and reticuloendothelial function. Australian Journal ofExperimental Biology and Medical Science 6 1: 37 l-382. PRICE I? & TURNER K. J. 1983b. Immunological consectuences of intestinat helminth infections in C57BL m&e. Humoral responses to polyvinyl pyrrolidone. Australian Journal of Experimental

Biology and Medical

Science 61: 383-396. PRICE P. & TURNER K. J. 1983~. Immunological consequences of intestinal helminth infections in C57BL mice. Natural and induced responses to sheep erythrocytes. Australian Journal of Experimental Biology and Medical Science 6 1: 397-406.

PRICE P. & TURNER K. J. 1984. Immunological consequences of intestinal helminth infections in C57BL mice. Humoral responses to ovalbumin. Parasite immunology 6: 499-508. PRICEP. & TURNER K. J. In press. Immunological consequences of intestinal hel~nth infections. Cellular and anamnestic responses to ovalbumin. Australian Journalof Experimental Biology and Medical Science.

PRITCHARDD. I., MAIZELS R. M., BEHNKE J. M. & APPLEBYP. 1984. Stage-specific antigens of Nematospiroides dub&.

Immunology

53: 325-335.

PROWSES. J. 198 1. Lymphokine (interleukin 2) secretion as a measure of T cell recognition of parasite antigens. Australian Journal ofExperimental Science 59: 695-705.

Biology and Medical

PROWSES. J. 1982. Interleukin 2 secretion by sensitized cells from mice infected with Nemafospiroides dubius: Kinetics and specificity of the response. Parasite Immunology

4: 363-372.

READ S. M. & NORTHCCTED. H. 1981. Minimization of variation in the response to different proteins of the Coomassie Blue G dye-binding assay for protein. Analytical Biochemistry 116: 53-64. VICKERYA. C., KI.EIFI T. W. & FRIEOMANH. 1981. Modulation of lymphoid cell blastogenic responsiveness to mitogens by Nippostrongylus brasiliensis infection. Proceedings of the Society of Experimental Biological ~~~e~~~cine 168: 3337. WILLIAMS D. .I. & BE~NKE J. M. 1983. Host protective

antibodies and serum immunoglohulin isotypes in mice chronically infected or repeatedly immunized with the nematode parasite Nematospiroides dubius. Immunology 48: 37-47.

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