The course of Plasmodium chabaudi chabaudi infections in interferon-gamma receptor deficient mice

The course of Plasmodium chabaudi chabaudi infections in

interferon-gamma receptor deficient mice

NICOLAS FAVRE1, BERNHARD RYFFEL2, GERARD BORDMANN1 & WERNER RUDIN1

1Swiss Tropical Institute, PO Box, Socinstrasse 57, CH-4002, Basel, Switzerland2 Department of Immunology, Medical School, University of Cape Town, South Africa

SUMMARY

Interferon-gamma receptor (IFN-gR) deficient miceparasitized with blood-stage Plasmodium chabaudichabaudiwere used to assess the anti-malarial activity ofinterferon-gamma (IFN-g). There was no significantdifference in the parasitaemia between the two types ofmice during the first peak of parasitaemia. However,IFN-gR deficient mice displayed an increased leucocytosisand a high mortality rate, whereas all of the wild type micesurvived. IFN-gR deficient mice, unlike wild type mice,developed a pronounced second parasitaemia peak, 9 to11 days after the first one, with a parasitaemia of up to 65%associated with mortality. Furthermore, increased serumlevels of nitric oxide (NO) were only found in wild type miceat the peak of parasitaemia, whereas it remained at back-ground levels in IFN-gR deficient mice. Parasite-specificantibody production was not significantly different inIFN-gR deficient mice, as compared to wild type mice. Inaddition, both wild type and IFN-gR deficient mice wereequally protected upon reinfection. These results indicate adelayed development of protective immunity and imply acrucial function for the IFN-gR in the control of blood stagemalaria during the initial three weeks of infection.

Keywords malaria, interferon-gamma receptor deficiency,Plasmodium chabaudi chabaudi,interferon-gamma

INTRODUCTION

The mechanisms of an effective protective immuneresponse toPlasmodium chabaudi chabaudiare not com-pletely understood. Interferon-gamma (IFN-g), a pluripotentinflammatory cytokine, has been shown to be produced inmice during infections with plasmodia (Slade & Langhorne1989, Taylor-Robinson & Phillips 1994). It has beendemonstrated that IFN-g together with TNF-a are criticalin the development of resistance to blood stage infectionswith Plasmodium chabaudi(Jacobs, Radzioch & Stevenson1996). Furthermore, monoclonal anti-IFN-g antibody treat-ment impaired the ability of the host to limit parasitemultiplication but did not abrogate the development ofacquired immunity resulting in control and elimination ofan acute infection (Medinget al. 1990, Stevensonet al.1990). A recent study by Tsujiet al. (1995) investigated thecourse of infections withPlasmodium chabaudi adami(556KA) and ofPlasmodium yoelii yoelii(17X NL strain)in IFN-g receptor (IFN-gR) deficient mice. A moderatelyprolonged parasitaemia was found in IFN-gR deficient miceas compared to wild type mice.

In the present report, we analysed the course of blood-stage infections in IFN-gR deficient mice parasitized withPlasmodium chabaudi chabaudi(Pcc). In addition, theeffects of the disease on haematopoiesis and developmentof immunity in IFN-gR deficient and wild type mice and theappearance ofPcc-specific antibodies were compared.

MATERIALS AND METHODS

Animals

Wild type and IFN-gR deficient mice (129/Sv/Ev XC57BL/6), 8–10 weeks of age, previously described in(Huanget al. 1993), were used for the experiments. Theywere housed under specific pathogen-free conditions.

Parasite Immunology, 1997:19: 375–383

q 1997 Blackwell Science Ltd 375

Correspondence: Werner RudinReceived: 2 January 1997Accepted for publication: 21 May 1997

Parasites and infection

Plasmodium chabaudi chabaudiAS was a kind gift fromDr D.Walliker (Edinburgh, Scotland, UK). Parasites weremaintained throughout the experiments by blood passage inIFN-gR deficient mice. Infections were performed by i.p.injection with 105 parasitized red blood cells. Parasitaemiawas scored on Giemsa-stained tail-blood smears. Standardhaemograms were performed daily with 10ml fresh hepa-rinized tail-blood on a Sysmex microcellcounter F-500(Digitana AG, Zurich, Switzerland).

Reagents

Bovine serum albumin (BSA) was purchased fromBoehringer Mannheim (Rotkreuz, Switzerland). Affinitypurified alkaline phosphatase (ALPH)-labelled goat anti-mouse IgM, IgG1, IgG2a, IgG2b and IgG3 were used(Southern Biotech. Ass. Inc., Bioreba, Basel, Switzerland).

Plasmodium chabaudi chabaudiserum free supernatantwas prepared using a protocol modified from that previouslydescribed (Taylor-Robinson & Phillips 1994). Briefly, wildtype mice were infected with 106 Plasmodium chabaudichabaudi parasitized erythrocytes. At parasitaemia peak(40–50%), blood was collected and washed in Hanks’balanced salt solution (HBSS; Life Technologies, Basel,Switzerland). Leucocytes were removed by centrifugationover a LRM (lymphocyte removal medium) gradient,according to the manufacturer’s protocol (Organon Teknika,Durham, NC, USA). 2× 109 Plasmodium chabaudichabaudiparasitized erythrocytes were cultivated in 20 mlserum free Iscove’s Modified Dulbecco’s Medium (IMDM;Life Technologies, Basel, Switzerland) with 200 U/mlnystatin (Boehringer Mannheim, Rotkreuz, Switzerland)for 30 h at 378C in a 3% O2, 4% CO2, 93% N2 atmosphere.Culture were centrifuged 10 min at 800g and supernatantswere used diluted 1:10 in PBS for ELISA plate coating.

Pcc-specific antibodies ELISA

Plates (Immulon 4B, Dynatech, Embrach, Switzerland)were coated with 100ml/well Plasmodium chabaudi cha-baudiserum free supernatant overnight at 48C. After wash-ing with PBSþ 0.05% Tween 20 (PBS-T), plates wereblocked for 60 min with PBSþ 1% BSA (PBS-BSA) andrinsed with ddH2O. Sera were diluted in PBS-BSA andincubated in the plates for 4 h at 378C. After washing withPBS-T, 100ml ALPH-anti mouse Ig isotypes antibodies,diluted 1:1000 in PBS-BSA, were added and the plates wereincubated for 3 h at 378C. The plates were washed withPBS-T and 1 mg/ml p-nitrophenylphosphate (p-NPP; Bio-Rad, Glattbrugg, Switzerland) in carbonate buffer (pH 9.6)

was added. The A405 was measured using an ELISA reader.For each serum sample, linear regression of the opticaldensity was performed over four different dilutions, aftersubtraction of the corresponding values obtained with serafrom naive mice. Titres were calculated as the dilution forwhich the mean optical density was equivalent to the meanoptical density of the background. NO data and antibodytitres were from three different experiments. Mice weresacrificed and sera collected at day 0 (naive), day 10 (firstparasitaemia peak), day 17 (between parasitaemia peaks),day 21 (second parasitaemia peak in IFN-gR deficient mice)and day 26 (after second parasitaemia peak).

Measurements of nitric oxide (NO)

As no NO¹2 was detectable in serum, NO¹

3 was reducedusing nitrate reductase (Boehringer Mannheim, Rotkreuz,Switzerland) as previously described (Rudinet al. 1997).Briefly, 25ml serum were incubated with 10ml enzyme perwell and 50ml Tris-nicotinamide-adenine dinucleotidephosphate for 1 h at 378C. 10ml LDH/pyruvate solutionwas added followed after 15 min by the addition of 100mlGriess reagent. The A540 was measured using an ELISAreader.

Statistics

Results are given as means of at least four animals6 stand-ard deviation. Differences between wild type and IFN-gRdeficient mice were analysed by Student’s t-test.P # 0.005was considered as being significant.

RESULTS

Survival in IFN- gR deficient mice

We asked whether IFN-gR deficiency has an influence onthe resistance toPlasmodium chabaudi chabaudiinfection,usually non-lethal for mice of this genetic background (ownobservations). As shown in Figure 1c, 100% of the wildtype mice (16/16) survived a primaryPcc infection while77% of the infected IFN-gR deficient mice (17/22) suc-cumbed. These data have been pooled from three differentexperiments where the course of infection was studied, andno mice were sacrificed. Interestingly, the mortality duringthe second parasitaemia peak (3/8: 37.5%) was only slightlylower than the mortality observed during the first peak (14/22: 63.5%). Therefore, protective immunity was not yetachieved by the time of the second peak, 18 to 22 days afterinfection.

N.Favreet al. Parasite Immunology

q 1997 Blackwell Science Ltd,Parasite Immunology, 19, 375–383376

Volume 19, Number 8, August 1997 Course ofP. chabaudi chabaudiinfections in IFN-gR deficient mice

q 1997 Blackwell Science Ltd,Parasite Immunology, 19, 375–383 377

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Figure 1 Time course ofPlasmodium chabaudi chabaudiinfection. Parasitaemia course in (a) wild type mice (n ¼ 4) and (b) IFN-gR deficientmice (n ¼ 8) infected with 105 P.c.cparasitized RBC i.p. at day 0. Data from one representative experiment, each mouse is representedindividually. (c) Survival in wild type mice (open squares;n ¼ 16) and IFN-gR deficient mice (closed squares;n ¼ 22) afterP.c.c.infection.Pooled data from three independent experiments.B IFNR deficient;A Wild type.

Parasitaemia, anaemia, leukocytosis and splenomegaly

We investigated whether IFN-gR deficiency has anyinfluence on parasitaemia and on haematological alterationsin Pcc infected mice. Figures 1a and 1b show the course ofparasitaemia in wild type and IFN-gR deficient mice fromone representative experiment. There was no significantdifference in parasitaemia between the two groups ofmice, at day 10, time of the first peak (IFN-gR deficientmice: 40.0% 6 5.9%; wild type mice: 38.8% 6 9.1%).However, IFN-gR deficient mice developed a markedsecond parasitaemia peak, 9 to 11 days later. The percent-ages of infected red blood cells during this second peak werecomparable to the levels reached during the first peak(37.5% 6 18.9%). Wild type mice only developed aminor second parasitaemia peak, that rarely reached 0.7%(0.23% 6 0.21%; P ¼ 0.005). For both types of mice, nofurther parasitaemia peak was observed, up to day 60 afterinfection.

Severe anaemia is a common complication of malariainfection. However, there is often no close association ofanaemia and parasitaemia (Clarket al. 1987, Miller et al.1989, Phillips & Pasvol 1992, Taylor-Robinson & Phillips1992, Stevenson & Tam 1993, Miller, Good & Milon 1994).

The haematological values are summarized in Fig. 2. Theextent of the anaemia was similar for both types of miceduring the first peak. In parallel with parasitaemia, thenumber of erythrocytes decreased again at day 19 inIFN-gR deficient mice only.

Leucocytosis peaked at day 12, two days after the firstparasitaemia peak. In IFN-gR deficient mice, the leuco-cytosis was significantly higher than in wild type mice(102.6 6 13.3 × 103 WBC/ml, 61.6 6 7.7 × 103 WBC/ml,respectively). Only in IFN-gR deficient mice a secondincrease of leucocyte counts (81.2 6 19.1 × 103 WBC/ml,wild type mice: 12.2 6 1.9 × 103 WBC/ml) was foundfollowing the second increase of parasitaemia.

Interestingly, splenomegaly was less marked in IFN-gRdeficient mice, as compared to wild type mice (0.38 6

0.04 g, 0.86 6 0.11 g respectively;n ¼ 4) at day 10,whereas mice had similar body weights (24.41 6 0.72 g,22.88 6 1.07 g respectively). At the time of the secondpeak, there was no difference between IFN-gR deficientmice and wild type mice in spleen weights (0.21 6 0.01 g,0.26 6 0.05 g respectively; n ¼ 4) and body weights(25.38 6 2.65 g, 22.69 6 1.8 g respectively). Spleensfrom non-infected mice weighted between 0.08 and 0.09 gfor both wild type and IFN-gR deficient mice.



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Figure 2 Time course of haematological values duringPlasmodium chabaudi chabaudiinfection. (a) Erythrocytes (×106/ml) and (b) leucocytes(×103/ml) numbers in tail-blood of wild type mice (n ¼ 4) and IFN-gR deficient mice (n ¼ 8) infected with 105 P.c.c.parasitized RBV i.p. at day0. Values were determined daily in 10ml blood. Data from one representative experiment, each mouse is represented individually and symbolscorrespond to Fig. 1.

NO levels in serum

Increased production of nitric oxide (NO) during blood-stage malaria has been shown to correlate with protectionagainst infection in mice (Jacobs, Radzioch & Stevenson1995, Taylor-Robinson, Severn & Phillips 1996). In addi-tion, significant production of nitric oxide is confined to abrief period of time, corresponding to the peak of theparasitaemia (Taylor-Robinson & Phillips 1994, Jacobset al. 1995, Stevensonet al. 1995, Taylor-Robinsonet al.1996). We assessed NO production by measuring the levelsof nitrate (NO¹

3 ) in sera at the time of the different peaks. Asshown in Figure 3, increased levels of nitrate were onlydetectable in the sera from wild type mice at the time of theparasitaemia peak at day 10 (1906 118mM NO¹

3 , day 0:42 6 10mM NO¹

3 ), but not in IFN-gR deficient mice (day10: 586 16mM NO¹

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ever, the increase in NO production was not significant inwild type mice.

Antibody titres

Clearance of the blood-stages of the parasite has beenshown to be antibody dependent (Langhorne, Simon &Meding 1990). We investigated whether IFN-gR deficiencyhas any influence on the appearance ofPcc specific anti-bodies. As summarized in Figure 4, parasite-specific anti-body production was similar in IFN-gR deficient mice andin wild type mice at the time points analyzed. A rise in IgMproduction was observed in IFN-gR deficient mice after thesecond parasitaemia peak. At day 10, the time of the first

parasitaemia peak, IgG2a, IgG2b and IgG3 productions werereduced in IFN-gR deficient mice, as compared to wild typemice, whereas IgG1 productions were comparable.

Resistance to secondary infection

We further investigated whether both mouse types wereprotected against secondary infection (Taylor-Robinson &Phillips 1992, D’Imperio Limaet al. 1996). Five IFN-gRdeficient mice, which had survived primary infections, werematched with wild type mice and challenged by i.p. injec-tion with 105 Pccparasitized red blood cells, at least 80 daysafter the primary infection.

There were neither significant differences in blood values(data not shown) nor in parasitaemia for both types ofmice. Both types developed a weak parasitaemia, peakingat day 11 (IFN-gR deficient: 0.94% 6 1.72%; wild type:0.28% 6 0.41%). No second parasitaemia peak wasobserved neither in wild type nor in IFN-gR deficientmice and all mice survived the challenge. Thus bothmouse types were similarly protected against secondaryinfections by acquired immunity.

DISCUSSION

Interferon-gamma (IFN-g), a pluripotent inflammatorycytokine, has been shown to be produced in mice duringinfections with malaria parasites and to play, together withTNF-a, a critical role in the development of resistance toblood stage infection withPlasmodium chabaudi(Slade &Langhorne 1989, Medinget al. 1990, Stevensonet al. 1990,Taylor-Robinson & Phillips 1994, Tsujiet al. 1995, Jacobset al. 1996). To confirm these observations and to investi-gate the effects of IFN-gR deficiency on haematopoiesisduring malaria, we analysed the course of blood-stageinfection of Plasmodium chabaudi chabaudiAS and ofblood values in IFN-gR deficient mice.

Our results show a similar parasitaemia course in bothtypes of mice for the first part of the infection. However,IFN-gR deficiency allowed the appearance of an importantsecond parasitaemia peak. Parasite-specific antibody pro-duction was slightly reduced in IFN-gR deficient mice, forthe IgG2a, 2b and 3 isotypes, at the time of the firstparasitaemia peak. Nitric oxide production was impairedin IFN-gR deficient mice, as compared to wild type mice.Furthermore, we observed a high mortality rate in IFN-gRdeficient animals, whereas wild type animals survived. Inaddition, our study showed that IFN-gR deficiency has noeffect on the development of anaemia. Leucocytosis wasmore pronounced in IFN-gR deficient mice.

There are apparent discrepancies between our resultsand those published by other groups (Medinget al. 1990,



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Figure 3 Serum nitrate (NO¹3 ) levels duringPlasmodium chabaudichabaudiinfection. Sera were obtained from wild type mice andIFN-gR deficient mice by heart puncture at day 0 (naive mice), day10 (first parasitaemia peak), day 17 (between parasitaemia peaks),day 21 (second parasitaemia peak in IFN-gR deficient mice), and day26 (after second parasitaemia peak). Serum NO¹

3 levels weredetermined, after enzymatic reduction, by Griess reaction. Eachcolumn represent one individual mouse.A Wild type;B IFNgRdeficient.



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Figure 4 Production of parasite-specific immunoglobulin isotypes (a) IgM, (b) IgG1, IgG2a, IgG2b and IgG3, duringPlasmodium chabaudichabaudiinfection. Sera were obtained from wild type mice and IFN-gR deficient mice by heart puncture at the same time points as indicated inFigure 3. Each column represent one individual mouse.A Wild type;B IFNgR deficient.

Stevensonet al. 1990, Tsujiet al. 1995, Jacobset al. 1996).Meding et al. (1990) reported a significantly higher firstparasitaemia peak in mice treated with monoclonal anti-IFN-g antibodies (R4-6A2 and AN-18.17.24). This effecthas also been reported by Stevensonet al. (1990) usingDB-1 and R4-6A2 monoclonal anti-IFN-g antibodies.However, Jacobset al. (1996) using DB-1 monoclonalanti-IFN-g antibody did not observe significant differencesin the parasitaemia course of blood-stageP.c.c. infections.Working with Plasmodium chabaudi adamiinfections,Tsuji et al. (1995) observed a slight delay in the develop-ment of the parasitaemia in IFN-gR deficient mice, ascompared to wild type animals.

The most important discrepancy is the high mortalityrate observed in our experiments whereas all mice survivedin the experiments of other groups (Stevensonet al. 1990,Tsuji et al. 1995, Jacobset al. 1996).

These discrepancies could have several explanations.First, mice with other genetic backgrounds or, in the caseof Tsuji et al. (1995) another parasite strain, have been usedfor these investigations.

Secondly, the use of antibodies to investigate the role ofIFN-g does most probably not impair all paracrine andautocrine effects of IFN-g as IFN-gR deficiency does.Furthermore, IFN-gR deficient mice might have adaptedalternative pathways to attenuate the effects of their heredi-tary deficiency. This might not be the case in mice treatedwith antibodies upon infection and may explain somediscrepancies such as their parasitaemia being comparableto wild type mice.

The appearance of a high second parasitaemia peak hasalready been reported inPcc-infected C57BL/6 mice treatedwith neutralizing anti-IL-12 monoclonal antibody (Yap,Jacobs & Stevenson 1994). We have also observed thisphenomenon with p40, IL-12 sub-unit, deficient mice(manuscript in preparation). These observations and theresults presented above support the hypothesis that part ofthe IL-12 protection in murine malaria acts through IFN-g

(Stevensonet al. 1995).Differences in splenomegaly during malaria have been

previously reported. Jacobset al. (1996) observed adecrease in spleen weight in mice treated with anti-TNF-a

but not in mice treated with anti-IFN-g antibodies alone.Our results show a role for IFN-gR in the development ofsplenomegaly, as spleen weights of IFN-gR deficient miceare reduced, as compared to wild type mice. In IFN-gRdeficient mice, the redistribution of cells in the spleen andother organs, e.g. liver, could be disturbed (Kumararatneetal. 1987). Cells remaining in the blood circulation instead ofpopulating the spleen or the liver, due to the lack of IFN-gRdepending signals, could partly explain the increased leuko-cytosis in these mice, as compared to wild type mice. In

addition, the reduced splenomegaly could be due to the lackof a specific proliferation triggered through the IFN-g

receptor. The observed differences in splenomegaly mightresult from a combination of these two factors. Less markedsplenomegaly, as compared to wild type, has also beenobserved in IFN-gR deficient mice injected with IL-12(Caret al. 1995).

The differences observed in nitric oxide (NO) productionby both mouse strains are not surprising, as NO is known tobe produced by macrophages upon IFN-g stimulation. Thereare reports indicating that NO might play a role in protectionagainst blood stage malaria infections (Rockettet al. 1994,Stevensonet al. 1995, Jacobset al. 1996, Taylor-Robinsonet al. 1996). Since the differences in NO production werenot very marked, the differences in the infection courseobserved between IFN-gR deficient and wild type mice inthe above experiments might not be due to the lack of NOproduction. It is more likely that the lack of NO productionis a consequence of an ill-functioning Th1-type immuneresponses due to IFN-gR deficiency.

In the present study, we did not observe importantdifferences in the production of parasite-specific antibodyduring the primary blood-stage infections described.D’Imperio Lima et al. (1996) described a correlationbetween a large IFN-g production and a predominance ofIgG2a and IgG3 antibody classes in primary malaria infec-tions in mice. Our results indicate a higher production of theIgG isotypes IgG2a, IgG2b, and IgG3, at the time of the firstpeak by wild type mice, as compared to IFN-gR deficientmice. Except for IgG3, these differences were not signifi-cant. Moreover, from day 17 onwards, IgG levels weresimilar in both IFN-gR deficient and wild type mice. Thissuggests that switching to different antibody isotype pro-duction, such as IgG2a, might not be solely dependent on theproduction of IFN-g by T-cells. Upon secondary infection,D’Imperio Lima and his colleagues observed a productionof IL-4 and IL-5 and an Ig-isotype pattern restricted to IgG1.We observed an IgG1 production at the time of the firstparasitaemia peak that was similar for both mice types.However, we found IFN-gR deficient mice to have higheramounts of serum IgG1 after their second parasitaemiapeak, than observed in the sera of wild type mice. Thiscould be due to a more important production of IL4 by theIFN-gR deficient mice, as the absence of IFN-g signalling,known to down-regulate Th2-type cytokines, could enhancethe production of IL-4. Others have shown that IFN-gRdeficiency can modulate the humoral isotype antibodyproduction pattern (Coyleet al. 1996) and that highamounts of IgG2a were only produced by wild type micein their experimental system. IgG2a has been proposed tobe a protective anti-plasmodial immunoglobulin isotype(Waki et al. 1995). These facts might explain the observed



increased susceptibility of the IFN-gR deficient mice toPlasmodium chabaudi chabaudiinfection and the protectionobserved against secondary infections.

The appearance of a second parasitaemia peak in theblood, whereas the first peak has been controlled, suppose,at least, two different mechanisms to fight the parasite.Control of the parasite by a Th1-type immune response,followed by clearance of the parasite from the blood, by aTh2-type immune response, has been reported by others(Langhorneet al. 1990, von der Weid & Langhorne 1993,Yapet al. 1994, Taylor-Robinson 1995). Our results suggestthat in the absence of IFN-g signalling, a partial control ofthe parasitaemia during the first peak of parasitaemia is stillpossible, despite high mortality. However, IFN-gR defi-ciency seems to hinder the clearance and control of theparasite, thus allowing the appearance of a second para-sitaemia peak, apparently similar to the first one, as indi-cated by the different parameters measured. It is possiblethat the absence of IFN-g signalling allows the parasite toescape the immune system during the first parasitaemia peakby hiding in an organ, e.g. spleen or liver, as the bloodparasitaemia values diminish to 0 to 1%, similarly asobserved in wild type animals. Release of parasites fromthis reservoir could then permit the appearance of a secondpeak. An ill-functioning of the natural killer cell (NK)population, due to the lack of an amplification loop throughthe IFN-gR, is also a possible explanation to our results,as NK have been postulated to be protective inPlasmo-dium chabaudiinfections (Kitaguchiet al. 1996). Anotherexplanation could be antigenic variation (Robertset al.1992, Smithet al. 1995, Gardneret al. 1996). The parasitecould be able to develop new antigenic variants, takingadvantage of a delayed elimination by the immune systemimpaired by the absence of IFN-g signalling. However, notyet at the first parasitaemia peak but at least by the time ofthe resolution of the second peak, the mice have acquired animmune protection similar to the one observed in wild typemice, as no other parasitaemia peak is observed and as theyare equally protected against secondary infections.

In conclusion, IFN-gR deficient mice have a delayeddevelopment of protective immunity. They are, however,able to resolve blood-stage malaria after three weeks ofinfection.

In addition,Plasmodium chabaudi chabaudiinfection inIFN-gR deficient mice seems to be a promising model tohighlight some of the key steps in the development ofimmunity during murine malaria.

ACKNOWLEDGEMENTS

We thank Professor M.Aguet (ISREC, Epalinges, Lausanne)for the kind gift of the IFN-gR deficient mice. Thanks to Ms

I. Herde for her technical assistance, to Mrs K.Gysin for herexcellent work at the animal station and to Drs H.-P.Beckand G.Pluschke for helpful discussions and critical review ofthe manuscript.

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The course of Plasmodium chabaudi chabaudi infections in interferon-gamma receptor deficient mice