INFECTION AND IMMUNITY, Jan. 1986, p. 141-146 Vol. 51, No. 10019-9567/86/010141-06$02.00/0Copyright © 1986, American Society for Microbiology

Plasmodium berghei Malaria: Effects of Acute-Phase Serum andErythrocyte-Bound Immunoglobulins on Erythrophagocytosis by Rat

Peritoneal MacrophagesBERYL J. PACKERt AND JULIUS P. KREIER*

Department of Microbiology, The Ohio State University, Columbus, Ohio 43210

Received 24 June 1985/Accepted 9 October 1985

Acute-phase serum (APS) collected from Plasmodium berghei-infected rats inhibited phagocytosis oftrypsinized rat erythrocytes and of erythrocytes from P. berghei-infected rats. Macrophages (M4) incubatedwith APS or heat-aggregated acute-phase serum (HAAPS) for 6 h, followed by 18 h incubation in serum-freemedium, exhibited significantly higher levels of phagocytosis than M4 similarly cultured but with normal ratserum. When APS was present at the time of assay, it inhibited erythrophagocytosis by M+ which had beenin culture for 0 or 24 h. M4 activation by HAAPS was inhibited by 2-deoxy-D-glucose, which suggests thatactivation by HAAPS is Fc-receptor mediated. Adsorption of APS with staphylococcal protein A abrogated theability of APS to inhibit phagocytosis and that of HAAPS to effect M4 activation, suggesting that immunecomplexes are involved in both processes. Surface-bound immunoglobulins eluted from erythrocytes of P.berghei-infected rats promoted phagocytosis of trypsinized erythrocytes by HAAPS-activated M4 but not byresting M+. These results indicate that the immunoglobulins which bind to infected or damaged erythrocytesduring malarial infections promote erythrophagocytosis by activated M4 and that the immune complexes inserum from rats with acute malaria may inhibit erythrophagocytosis early in the infection but may, over time,induce changes in the M4 which later facilitate erythrophagocytosis.

Malaria, caused by parasites of the genus Plasmodium, isone of the most important diseases in the world today.Wernsdorfer (36) estimated that malaria causes the deaths ofone million infants and children annually. Despite the impor-tance of malaria, the immunological mechanisms that enablethe host to control plasmodial infection have not been clearlyidentified. Immune mechanisms directed against infectederythrocytes have been proposed (38), as have mechanismsdirected against the merozoite when it passes from oneerythrocyte to another (2, 5, 6, 8, 20). It is likely that bothhumoral and cellular responses are important in controllinginfection (16, 22, 23, 26).Both immunoglobulin G (IgG) and IgM antibodies are

bound to erythrocytes (RBCs) of individuals with malaria(12, 18, 19, 34, 38, 39). Some of the IgM antibodies on theRBCs are cold reactive types, specific for lipids exposed byproteolytic degradation of the RBC membranes. They can beeluted by incubation at 37°C (7, 15, 18, 27). Attempts todemonstrate that antibody eluted from RBCs of infected ratsenhances erythrophagocytosis by resting macrophages ei-ther in vitro (5, 9) or in vivo (17, 28) have failed.

Ingestion of immune complexes or heat-aggregated immu-noglobulins triggers activation of macrophages (MO) (24).Immune complexes are present in the plasma of malariousmice (13, 33), humans (10), and rats (3). This paper describesexperiments which provide indirect evidence that immunecomplexes may initially protect plasmodia from phago-cytosis by blocking Fc-receptors, but that later in the courseof the disease immune complex-mediated activation of M4Xmay facilitate phagocytosis of opsonized RBCs. Phago-cytosis of RBCs opsonized with antibodies by MX activatedby ingestion of immune complexes may thus in part account

* Corresponding author.t Present address: Department of Microbiology and Immunology,

Temple University School of Medicine, Philadelphia, PA 19140.

for the anemia and drop in parasitemia that occur at the'crisis" period of plasmodial infection.

MATERIALS AND METHODS

Animals. Inbred Fisher 344 rats (CDF; Charles RiverBreeding Laboratories, Inc., Wilmington, Mass.) weighingfrom 150 to 200 g were used as the source of rat serum, RBCs,and peritoneal M+~. Swiss albino mice were used to maintainP. berghei.

Parasites. P. berghei berghei (strain from Walter ReedArmy Institute of Research) was stored in liquid nitrogen.Frozen stock parasites were thawed and used to infect Swissalbino mice as needed. The strain from the frozen stock waspassed in mice no more than twice during these studies. Ratswere infected with 107 fresh, infected mouse RBCs by tailvein injection.Serum collection, storage, and preparation. Rats were

anesthetized with ether, and blood was collected by cardiacpuncture. All sera were pooled, filter sterilized, and stored at-20°C. Acute-phase serum (APS) is defined here as serumwhich was collected and pooled 14 days postinoculationwhen infected rats had parasitemias ranging from 28 to 33%.Heat-aggregated normal rat serum (HANRS) and heat-aggregated acute-phase serum (HAAPS) were prepared byheating the sera at 63°C in a water bath for 30 min just beforeuse.M4 harvest, staining, and counting. Normal rats were

exsanguinated while anesthetized. The M4X were obtained byfilling the peritoneal cavity with 30 ml of sterile RPMI-1640(GIBCO Laboratories, Grand Island, N.Y.) which had beensupplemented with 25 mM HEPES (N-2-hydroxethyl-piperazine-N'-2-ethanesulfonic acid; Sigma Chemical Co.,St. Louis, Mo.), 25 U of penicillin per ml, 25 jig of strepto-mycin per ml, and sodium bicarbonate to give a pH of 7.0.Cells were collected through the peritoneal wall into a

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sterile syringe and washed, and 2 x 106 viable cells wereplated onto 35-mm plastic tissue culture dishes. The cellswere incubated for 1 h at 37°C in a humidified, 7% CO2chamber; then nonadherent cells were removed by vigorouswashing with sterile phosphate-buffered saline (PBS; pH7.2). The adherent cells, which were >98% esterase posi-tive, were used immediately as normal or resting MX unlessstated otherwise. All phagocytosis assays were conducted at37°C for 30 min in a humidified chamber with 7% CO2 in air.All treatments were run in triplicate for each study, andexperiments were repeated three times.

After each assay, plates were washed vigorously with PBSto remove RBCs which were not internalized, methanolfixed, and stained with Giemsa. Between 300 and 400 M4Xwere counted for each plate, the number of M4 containingone or more RBCs was determined microscopically, and theresults are reported as the percent of phagocytic MX +standard deviation (SD). Statistical analyses were madeusing the Student t-test. The use of a hypotonic solution tolyse bound RBCs was precluded, because this treatmentsometimes caused mast cell degranulation and lysis of theMX monolayers.RBC harvest and preparation. Normal or infected rats

were anesthetized, then exsanguinated by cardiac punctureinto an equal volume of cold Alsever's solution. The RBCswere washed in cold PBS and then resuspended in RPMI-1640 to a concentration of 108 per ml. A two-ml amount ofthe RBC suspensions was added to the M4 monolayers toprovide an RBC M4~ratio of 100:1 in all experiments. FreshRBC suspensions were prepared for each assay. InfectedRBCs (IRBCs) were collected from rats having a 25 to 35%parasitemia, which was determined by counting 10,000Giemsa-stained RBCs on a thin smear.

Uninfected RBCs (NRBCs) were trypsinized (TRBCs) bysuspending 0.2 ml of washed, packed cells in 4 ml of a 0.25%trypsin solution in PBS. The suspension was placed in awater bath at 37°C for 15 min with occasional mixing. TheTRBCs were then washed thoroughly before use.

Effects of APS on phagocytosis by M4 after various culturetimes. M4 were harvested and plated as described, exceptthat 10% heat-inactivated fetal calf serum was added to theculture medium. M4 monolayers that were assayed imme-diately are described as normal, resting, or zero-hour cul-tures. These cultures were used for baseline measures of thepercent of normal MX that were phagocytic after varioustreatments.M4 cultured 24 h were incubated with 3 ml of RPMI-1640

containing 10% fetal calf serum. Monolayers were washedjust before each assay, and M4 were incubated withNRBCs, IRBCs or TRBCs prepared as previously de-scribed. Rat RBC suspensions either contained no serum orwere supplemented with 10% heat-inactivated normal ratserum (NRS) or 10% heat-inactivated APS.M4 activation using serum from acutely infected rats.

Based on the observations that heat-aggregated IgG orimmune complexes can induce MO activation within 6 h (24),studies were conducted to determine whether MX exposedto HAAPS, APS, NRS or HANRS for 6 h would becomeactivated. Freshly prepared M4 monolayers were incubatedat 37°C with 7% CO2 for 6 h in 2 ml of RPMI-1640 supple-mented with 10% NRS, APS, HANRS, or with HAAPS.After 6 h, the monolayers were washed with PBS andincubated in serum-free RPMI-1640 for an additional 18 h, topermit sufficient time for the M4X to ingest bound aggregatesand to regenerate Fc receptors (25). Monolayers were thenwashed, and suspensions of NRBCs, IRBCs, or TRBCs

prepared as described earlier were placed on the monolay-ers, incubated, methanol-fixed, stained, and counted.

Inhibition of Mo activation with 2-dG. RPMI-1640 contain-ing 50 mM 2-deoxy-D-glucose (2-dG) was used to determinewhether HAAPS activation of M4 was mediated through Fcreceptors, since it has been reported that 2-dG inhibits thephagocytosis of IgG aggregates or of IgG-antigen complexeswhich bind to Fc receptors (21). Fresh M4 monolayers wereincubated for 45 min in RPMI-1640 containing 2-dG beforethe addition of serum-supplemented medium. Medium con-taining 10% HAAPS and 50 mM 2-dG was then placed on theM4i monolayers. Cells were incubated for 6 h, washed, andthen incubated for 18 h in serum-free RPMI-1640. Monolay-ers treated with 2-dG but without HAAPS for the same timewere checked for viability at 24 h by trypan blue dyeexclusion. Positive controls without 2-dG were also run.

Titration of RBC surface IgG and IgM by passive hemag-glutination. IRBCs or NRBCs, pooled from two to fiveanimals, were collected in cold Alsever's solution (50%[vol/vol]) and held at 4°C for 1 h to promote binding of coldhemagglutinins to the RBCs. Cells were then washed threetimes in cold PBS and resuspended to a 50% concentration.One half of each suspension was held at 4°C (nonelutedRBCs) and the other half was held at 37°C (eluted RBCs) for40 min. Cells were then centrifuged and supernatants wereheld for further analysis. Eluted and noneluted RBCs weresuspended in cold PBS at a 0.5% concentration. Hema-cytometer counts were used to adjust the cells to an equalcell concentration. All suspensions were held at 4°C beforeuse.Doubling dilutions of goat anti-rat IgG (Kirkegard and

Perry Laboratories, Gaithersburg, Md.) were made withcold PBS, as were dilutions of rabbit anti-rat IgM (MilesLaboratories, Inc., Elkhart, Ind.). The one-half dilutionscontained 25 ,ug of anti-rat IgG antibody or 1.5 mg of proteinin the anti-IgM preparation (the antibody concentration wasnot specified). Tests were run in duplicate with 0.1 ml of theantisera dilutions and 0.1 ml of the eluted or non-elutedIRBCs or NRBCs. Sets of tubes were incubated at 4°C andat 37°C, and titers were recorded at 4 h. The titer representsthe highest dilution of antiserum causing erythrocyte agglu-tination.

Eluate preparation and storage. Studies were conducted todetermine whether immunoglobulins eluted from IRBCscould facilitate phagocytosis ofTRBCs by HAAPS-activatedM¢. The term eluate refers to the supernatant that wasremoved from IRBCs which had been held at 37°C for 40min. Procedures used were those described by Kreier et al.(18). To compensate for some loss of eluted immunoglob-ulins during handling, the eluate was concentrated to onehalf the original volume of blood collected, using XM100ADiaflo ultrafiltration membranes (Amicon Corp., Lexington,Mass.) to retain and concentrate molecules with molecularweights greater than 100,000. Samples were prepared on iceand then the concentrated eluate was filter sterilized andfrozen at -20°C until used. Dilutions of eluate were preparedin RPMI-1640 and comprised 10% of the final 2-ml volume ofTRBC suspensions used in the assays. Presence of both ratIgG and IgM in the eluate was verified by immunoelectro-phoresis.

Adsorption of immune complexes from APS and HAAPSwith protein A. Protein A-binding staphylococci (The En-zyme Center, Walden, Mass.) were washed repeatedly insterile PBS and then were pelleted by centrifugation. Pelletswere resuspended in APS or HAAPS twice, 10 min eachtime, at room temperature. Adsorption times were limited to

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10 min, since immune complexes or antibody aggregatesshould bind protein A in this time, while most monomericIgG binds more slowly (14). Adsorbed serum samples werecentrifuged to pellet the staphylococci to which the com-plexes were bound; then all samples were filter-sterilized andassayed immediately. M4 cultured 24 h in medium contain-ing 10% fetal calf serum were used to determine whetheradsorption of APS with protein A would abrogate inhibitionof phagocytosis.

RESULTSEffects of NRS and APS on phagocytosis of RBCs by rat

peritoneal Mp cultured for various times. There was a slightincrease in phagocytosis of NRBCs with increasing time ofculture of M+ in the presence of NRS. The level ofphagocytosis of IRBCs and of TRBCs was consistentlygreater than that of NRBCs. The level of phagocytosis ineach case was increased by cultivation of the M4 for 24 h.The addition of APS to the cultures at the time of assay forphagocytic capacity at 24 h significantly inhibited phag-ocytosis of IRBCs and of TRBCs, as compared to thepercent of phagocytic m4 observed when NRS was used (P< 0.05). The inhibitory effect of APS was concentrationdependent. At the low concentrations of APS, a greaterproportion of M4~incubated for 24 h phagocytized IRBCsand TRBCs than did MX at 0 h (Fig. 1), which indicates thatlow levels of culture-induced activation occur. Adsorption ofAPS with protein A abrogated the inhibition (data notshown).

Effect of culture for 6 h in the presence of APS and HAAPSon erythrophagocytic capacity of rat peritoneal M4 after anadditional 18 h cultivation. Cultivation for 6 h in APS orHAAPS, followed by 18 h cultivation in serum-free RPMI-1640, increased phagocytic capacity of M4X over that of M4treated similarly but with NRS or HANRS in the initial 6 hincubation. The increases were apparent with all types ofRBCs tested, but phagocytosis of IRBCs and TRBCs wasalways greater than phagocytosis of normal rat RBCs (Fig.2). The percent of M4 incubated with HAAPS whichphagocytosed IRBCs or TRBCs was significantly higher (P< 0.05) than was observed with all other treatments.

Relationship of RBC-bound immunoglobulins to parasit-

10_

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10 NRS 5%NRS 10%APS 5%APS 2.5%APS

24 hr

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1 2 3 1 2 3 1 2 3 1 2 3NRS APS HANRS HAAPS

FIG. 2. Effect of culture for 6 h in the presence of NRS, APS,HANRS, or HAAPS on erythrophagocytosis by rat peritoneal M4after an additional 18-h cultivation in serum-free medium. PercentM4 ingesting NRBCs, 1; IRBCs, 2; or TRBCs, 3. Bars represent themean number of phagocytic M4 ± SD.

emia in rats infected with P. berghei. Both IgG and IgM weredetected on surfaces of pooled, washed RBCs from infectedrats by a passive hemagglutination test (Table 1). No RBC-bound IgG was detected on days 3 or 6 postinfection, even inanimals having 10 to 13% parasitemias. Erythrocytes fromrats which had a 55 ± 5% parasitemia 9 days postinfectionhad surface IgG and IgM titers of 32 and 512, respectively.Those rats which did not reach a 51 + 5% parasitemia until21 days postinfection had a higher IgG titer and a lower IgMtiter than rats tested 9 days postinfection. The IgG titer inanimals having a 31 ± 5% parasitemia at 15 days postinfec-tion was 16, and rats of this same group had an IgM titer of8. Pooled RBCs from rats which had 0% parasitemia by 23days postinfection had no detectable surface IgG or IgM. Inno case was hemagglutination observed at 37°C.

Effect of immunoglobulins eluted from the surfaces of RBCsof P. berghei-infected rats on phagocytosis of TRBCs byHAAPS-activated M+. RBCs from infected rats having hightiters of RBC-bound IgG and IgM were held at 37°C to elutebound globulins. The eluates were concentrated and thenadded to suspensions of TRBCs. The mixture was thenadded to MX cultures. Activated M4X in the cultures ingestedfew RBCs when held at 4°C under any conditions (data notshown). When assayed at 37°C, however, the number of M(ingesting TRBCs in the absence of eluate was significantlylower than the proportion of M4X ingesting TRBCs that weresupplemented with 10% eluate (Fig. 3). Addition of 5%eluate also enhanced phagocytosis. Enhancement of pha-

TABLE 1. The relationship of RBC-bound IgG and lgM toparasitemia in rats infected with P. bergheia

No. of Days tIgG IgManimals post- Parasitemia titer titerpooled infection Paaiei Lgj (g/l

2 3 1.5 0 03 6 12.0 0 05 9 55.0 32 5126 15 31.0 16 82 21 51.0 64 1610 23 0.0 0 0

a Each time point represents the mean value of blood samples that were

pooled from two to five animals.

lvubm o Orb n;>;IW70Ptr;>070 r'O &--70 %r-O

FIG. 1. Effects of NRS and APS on erythrophagocytosis by M4cultured 0 or 24 h before assay. Percent M4 ingesting NRBCs, 1;IRBCs, 2; or TRBCs, 3. Bars represent the mean number ofphagocytic M4 + SD.

* %-.LI a

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0

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% ELUATE IN CULTURESFIG. 3. Effect of immunoglobulins eluted from the surfaces of

RBCs of P. berghei-infected rats on phagocytosis of TRBCs byHAAPS-activated M+. Bars represent the mean number of phago-cytic M4 ± SD.

gocytosis above control levels by both 10 and 5% eluate wassignificant (P < 0.05). Enhancement was abrogated bydiluting eluate to 2.5% or less. Eluate did not enhancephagocytosis by resting M4 at any of the concentrationstested (data not shown).

Effect of 2-dG on activation of M4 by cultivation withHAAPs. MO that were cultured for 6 h in medium containing50 mM 2-dG and 10% HAAPS, followed by 18 h culture inRPMI-1640 were not activated. This was indicated by thesignificantly lower phagocytic capacity of these M4o (P <0.05) for NRBCs, TRBCs, and IRBCs than of M4X similarlyincubated but without 2-dG (Fig. 4). Cultures incubated withand without 2-dG contained similar numbers of viable M4X as

determined by microscopic examination and trypan blueexclusion.

20

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010

NO NO NO2 dG 2 dG +2dG 2 dG

+2dG +2dG

NRBC IRBC TRBCFIG. 4. Effect of 2-dG on M4o activation by HAAPS. The bars

indicate the mean percent ± SD of M4 ingesting NRBCs, IRBCs, or

TRBCs. Cultures were incubated with 2-dG for 2 h before theaddition of HAAPS, and 2-dG was present during the 6-h incubationwith HAAPS. Cultures were then incubated 18 h in serum-freemedium with no 2-dG present.

1 2 3 1 2 3 1 2 3NRBC I RBC TRBC

FIG. 5. Effect of adsorption of HAAPS with protein A onactivation of M4 by cultivation with HAAPS. The bars indicate themean percent ± SD of M4 ingesting NRBCs, IRBCs, or TRBCs.The M4 cultured with HANRS, 1; HAAPS, 2; or HAAPS adsorbedwith protein A, 3.

Effect of absorption of HAAPS with protein A on activationofM+ by cultivation with HAAPs. M4 that were cultured for6 h in medium containing HAAPs which had been adsorbedwith protein A, followed by 18 h of culture in RPMI-1640,were less activated than M4o similarly treated but withunadsorbed HAAPS. This is indicated by the observationthat a significantly higher percentage (P < 0.01) of M4incubated with nonadsorbed HAAPS than with adsorbedHAAPS engulfed NRBCs, TRBCs, or IRBCs (Fig. 5). Ad-sorption of complexes was perhaps not complete becausephagocytes cultured with adsorbed HAAPS were moreactive than control MX incubated with HANRS.

DISCUSSIONReports of parasitized and nonparasitized RBCs in M4 of

birds, monkeys, mice, and rats suffering from acute malariahave long susggested the importance of erythrophagocytosisin clearance of parasitemia (4, 35, 40). Massive erythro-phagocytosis generally has a sudden onset and is associatedwith a precipitous decline in parasitemia and rapidly devel-oping anemia. The stage of the disease in which these latterevents occur is called the crisis (reviewed in reference 16;29). It has generally been assumed that an immune responseis responsible for the crisis, but there is no consensus amongmalariologists on the mechanism producing it (1).

This study and many others (12, 18, 19) have shown that asparasitemia develops, much of the erythron acquires a coatof immunoglobulin. As parasitemia develops, first antigens(31, 32) and then later immune complexes (3, 10, 13) appearin the plasma.Immune complexes from malaria-infected mice block

erythrophagocytosis in vitro (33, 34). In this study, we notonly have confirmed that immune complexes inhibitphagocytosis of opsonized RBCs, but also we have shownthat the complexes induced activation of resting M+,thereby enhancing their capacity to phagocytize enzyme-damaged RBCs coated with antibodies eluted from RBCs ofrats with malaria. The development of agglutinins during theacute anemia phase of malarial infection which can bindTRBCs may be related to exposure of RBC antigens whichare also exposed as a result of proteolytic modification ofRBCs during a natural infection (18). These may be autoan-tibodies, some of which have previously been shown to beIgM types (7, 18). Jayawardena et al. (11) reported that mice

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incapable of producing IgM autoantibodies to enzyme-damaged RBCs developed more severe parasitemias afterPlasmodium yoelii infection than did mice that were capableof producing such autoantibodies. Both types of mice finallycontrolled their infections when they developed IgG antibod-ies specific for P. yoelii. Green and Packer (8) recentlyreported that rheumatoid factors enhance agglutination ofPlasmodiumfalciparum schizonts in vitro. It is possible thatautoantibodies against infected or damaged RBCs act in asimilar manner to facilitate erythrophagocytosis. Both IgGand IgM were present in the eluates that were found toenhance erythrophagocytosis by HAAPS-activated M( inthis study. Significantly higher levels of phagocytosis ofIRBCs and TRBCs than NRBCs may also indicate that thespecificity of these immunoglobulins is for altered antigenson the RBC membrane. Such alterations may occur due todeposition of parasite antigens on membrane surfaces or tomechanical or proteolytic damage to RBCs during malarialinfection.The observation that HAAPs caused higher levels of

activation than did APS is consistent with the observation ofPestel et al. (24) that IgG aggregates or immune complexescan induce M4 activation and that the degree of activationincreases when larger heat-aggregated globulins are present.It has also been shown that chemically cross-linked IgGoligomers have higher affinity for Fc receptors than doesmonomeric IgG, and that affinity increases with increasingIgG polmerization (30). Aggregation of immune complexeson the M4 surface appears to be necessary for the efficientendocytosis and clearance of immune complexes in vivo (forreview, see the study by R. G. Q. Leslie [18a]). Heat-induced aggregates may thus have higher affinity for Fcreceptors and may also cross-link MX surface receptors topromote efficient clearance of the complexes.These various observations are compatible with the hypo-

thesis that crisis occurs when MX become activated byingestion of immune complexes or aggregated immunoglob-ulins and acquire the ability to phagocytose RBCs coatedwith autoantibodies. In this scenario the defense of theparasite, based at least in part on a "smoke screen" (37) ofsoluble antigens and blocking immune complexes, is dis-solved when M4X become activated after ingesting immunecomplexes. The host response is thus modified by contactwith components of the defense of the parasite, such asimmune complexes, in such a way as to negate their smokescreen effect. It is important to note that this anti-erythrocyte-based system is only one part of the host de-fense. The study of Jayawardena et al. (11), for example,shows clearly the importance of the more direct antiparasitesystem in host defense. Antiparasite-IgG was required tocontrol infections in mice that lacked the IgM autoantibody-based antierythrocyte system; and mice which producedboth IgM and IgG experienced milder infections than didmice which lacked the ability to produce 1gM.

ACKNOWLEDGMENTS

This work was supported in part by a World Health Organizationgrant M2/181/151.We thank Greg Harvey for assistance in the preparation of this

manuscript.

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