Vol. 47, No. 1INFECTION AND IMMUNITY, Jan. 1985, p. 247-2520019-9567/85/010247-06$02.00/0Copyright © 1985, American Society for Microbiology

Cytotoxicity of Human Serum for Leishmania donovaniAmastigotes: Antibody Facilitation of Alternate Complement

Pathway-Mediated KillingDAVID L. HOOVER,"* MELVIN BERGER,2 MONTY H. OPPENHEIM,1 WAYNE T. HOCKMEYER,1 AND

MONTE S. MELTZER' 3

Department ofImmunology, Walter Reed Army Institute of Research' and Department of Allergy and Immunology,2 andDepartment ofDermatology, Walter Reed Army Medical Center, Washington, D.C. 20307

Received 3 July 1984/Accepted 24 September 1984

Mechanisms that mediate recovery from leishmanial infection have not been fully characterized but aregenerally believed to involve interactions between T lymphocytes and macrophages. A major role forserum-mediated effector mechanisms in the protection of humans from reinfection with Leishmania, however,has not been ruled out. In this report, amastigotes of L. donovani were incubated with dilutions of serum fromnormal subjects and from patients with kala-azar. Normal serum was cytotoxic for parasites at a dilution of-1:20. Cytotoxicity did not occur in the presence of EDTA, was abolished by heating serum to 56°C for 30 min,and was not diminished by prior adsorption of normal serum with parasites at 0°C. Killing proceeded normallyin the presence of magnesium-ethylene glycol-bis(13-aminoethyl ether)-N, N-tetraacetic acid, however, and wasfully effected by C2-deficient serum. These studies indicated that killing of amastigotes, unlike that ofpromastigotes, was mediated via the alternate pathway of serum complement. In further studies, cytotoxicityof normal serum was enhanced three- to fivefold by factors in patient serum. This enhanced cytotoxicity alsoproceeded via the alternate complement pathway. Factors that enhanced cytotoxicity were characterized asparasite-specific immunoglobulin G: they eluted with immunoglobulin G on column chromatography, wereadsorbed by immobilized staphylococcal protein A, and were not removed from the parasite surface byextensive washing. Thus, infection of individuals with L. donovani resulted in the production of a new,qualitatively and quantitatively distinct immune mechanism directed against the amastigote form of theparasite, namely, antibody-directed, alternate complement pathway-mediated cytotoxicity. These resultsprovide a mechanistic framework for a role of humoral factors in human resistance to reinfection with L.donovani.

The protozoan parasite Leishmania donovani causes asevere human disease known as visceral leishmaniasis, orkala-azar. Infection is acquired from sandfly bites afterinjection of the extracellular, promastigote form of theparasite into the skin. Promastigotes enter macrophages andconvert to the intracellular, amastigote form. Amastigotesreplicate and burst their host cell. Parasites are then releasedinto the extracellular space and disseminate to liver, spleen,lymph nodes, and bone marrow. This dissemination isassociated with the clinical syndrome of fever, hepatospleno-megaly, weight loss, and pancytopenia. Without specificantimicrobial therapy, patients die of infection; treated sur-vivors, however, are immune to reinfection (12).Mechanisms that mediate immunity to Leishmania spp.

have not been fully characterized but are generally believedto involve interactions between T lymphocytes and macro-phages. This belief is supported by certain phenomena thatoccur in infected patients and animal models: (i) humanvisceral leishmaniasis progresses despite extremely hightiters of specific anti-parasite antibody; (ii) delayed cutane-ous hypersensitivity to leishmanin, a preparation of For-malin-killed organisms, is depressed when visceral disease isactive, but returns to responsive after successful treatmentand recovery; and (iii) lymphoid cells, but not serum,adoptively transferred from immune mice can provide pro-tection from subsequent intravenous or subcutaneous chal-

* Corresponding author.

lenge with parasites (11). Despite these observations, amajor role for serum-mediated effector mechanisms in pro-tection of humans from reinfection with L. donovani has notbeen ruled out. Indeed, in a mouse system, immune serummarkedly augments the protection against Leishmaniatropica induced by adoptively transferred immune lymphoidcells (10). Moreover, processes that effect recovery frommany established infections in humans may not necessarilybe those that mediate resistance to reinfection. For example,chickenpox, caused by varicella-zoster virus, is usually aminor illness of healthy children. Infection can be preventedby transfer of immune serum, even in patients whose cell-mediated defenses are severely compromised (3). Once theviral infection is established, however, administration ofimmune serum does not affect the clinical course; further-more, in individuals who lack adequate cellular immuneresponses, severe disease frequently ensues despite thepresence of specific antibody (1). Thus, antibodies to vari-cella-zoster virus are sufficient to prevent acquisition ofinfection, but play little, if any, role in extinguishing estab-lished disease.

In this report, we show that factors present in high titer insera of individuals with kala-azar significantly augment kill-ing of L. donovani amastigotes by normal human serum.This killing is directed by specific antibody and mediated bythe alternate complement pathway. These results provide amechanistic framework for the role of humoral factors inhuman resistance to reinfection with L. donovani.

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248 HOOVER ET AL.

MATERIALS AND METHODS

Leishmania. L. donovani, Sudan strain 2-S, was propa-gated in Syrian hamsters (Charles River Laboratories, Wil-mington, Del.). Amastigotes were harvested in RPMI 1640with 25 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer (GIBCO, Grand Island, N.Y.)from minced infected spleens after tissue homogenization ina Ten Broeck grinder and washed twice after centrifugationat 200 x g and twice after centrifugation at 2,000 x g (4).Leishmania tropica major, NIH 173, was propagated ininfected foot pads of BALB/cJ mice, (Jackson Laboratories,Bar Harbor, Maine). Amastigotes were harvested as previ-ously described (8), except that RPMI 1640 without serumwas used throughout the harvest procedure. Parasites werestained with toluidine blue 0 and counted in a hemacytome-ter. Amastigotes (107 in 1.0 ml) were added to tubes andpelleted by centrifugation at 1,000 x g.Serum. Serum previously collected from three patients

who were chronically infected with L. donovani in Kenyawas heated at 56°C for 30 min before testing. These serahad high titers (1:320 to 1:1280) of anti-leishmanial antibodydetected by complement fixation with promastigotesas targets (data not shown; W. T. Hockmeyer, B. T.Wellde, C. L. Sabwa, D. H. Smith, P. H. Rees, and P. A.Kager, Ann. Trop. Med. Parasitol., in press). Serum col-lected from normal volunteers was either used on the day ofcollection or stored at -70°C and thawed immediatelybefore testing.Serum treatment of amastigotes. Amastigotes were sus-

pended in 0.6 ml of various dilutions of serum in RPMI 1640and incubated at 37°C for 40 min. After incubation, amasti-gotes were washed twice in RPMI and suspended in 0.8 ml ofassay medium (RPMI 1640, 10% heat-inactivated fetal bo-vine serum [GIBCO], 50 ,ug of gentamicin per ml). In certainexperiments, amastigote suspensions were incubated withserum diluted with an equal volume of phosphate-bufferedsaline containing EDTA (10 mM final concentration) orMgCl2 (2.5 mM) and ethylene glycol-bis(,-aminoethyl ether)-N,N,N',N',-tetraacetic acid (10 mM), all at pH 7.4. Toanalyze the time course of cytotoxicity, we suspendedamastigotes in 0.3 ml ofRPMI and placed them at 37°C for 15min. Then, 0.3 ml of diluted serum was added, and cultureswere returned to the incubator. At appropriate intervals,ice-cold EDTA (10 mM final concentration) was added, andcultures were placed on ice before being washed twice withRPMI 1640.

Estimation of parasite viability. Analysis of amastigotekilling required a reliable and accurate method to determinethe viability of amastigote preparations. As detailed previ-ously (5), we estimated viability by allowing amastigotes toconvert to promastigotes at 26°C in the presence ofhydroxurea. Hydroxyurea permits transformation of amast-igotes to enlarged, flagellated or nonflagellated forms butinhibits replication of promastigotes after conversion. Amas-tigotes were diluted 1:10 in conversion medium (1:1 Cunn-ingham SM [2] medium/RPMI 1640-30% FBS-50 ,ug ofgentamicin per ml) with 0.2 mg/ml hydroxyurea. Afterincubation at 26°C for 72 h, parasites were stained withtoluidine blue 0 and counted in a hemacytometer. Amast-igotes that did not convert remained small and rounded, incontrast to the larger, elongated conversion intermediates,which were easily distinguished from amastigotes after 72 hof incubation (6).

Statistical analysis. All data were analyzed for statisticalsignificance by Student's t test.

CD I

CD

CD

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2.5- FRESH+ 1150 CONTROL SERUM

0*

c405 /135 /45 5/I

DILUTION OF FRESH OR A SERUM

FIG. 1. Enhancement of immune serum of normal serum-medi-ated cytotoxicity. Amastigotes (107) were incubated at 37°C for 40min with dilutions of fresh (unheated) normal serum with a 1:50dilution of heat-inactivated (A) immune serum or heat-inactivatednormal serum. Viability of treated amastigotes was estimated byconversion to promastigotes. Points along the fresh serum dose-re-sponse curve differed significantly (P < 0.05) from controls and fromeach other through a dilution of 1:135.

RESULTS

We have previously shown that certain factors in freshnormal human serum were strongly cytotoxic to amastigotesof L. tropica (5). These cytotoxic serum factors were gener-ated during specific antibody-independent interaction of theparasite with components of the alternate complement path-way. We further showed that under identical conditions,amastigotes of L. donovani were substantially less suscepti-ble to this complement-mediated cytotoxicity: cytotoxicityagainst L. donovani amastigotes required a higher concen-tration of normal human serum than that needed for L.tropica. Moreover, even at a 1:2 dilution, only half of the L.donovani amastigotes were killed, compared with 95% of L.tropica amastigotes (5). Experiments presented in Fig. 1showed that the relatively weak cytotoxic activity of normalhuman serum against L. donovani amastigotes was signifi-cantly increased by addition of a 1:50 dilution of immuneserum from patients with kala-azar. The cytotoxic activity ofnormal serum plus immune serum against L. donovaniamastigotes was more than threefold greater than that ofnormal serum plus nonimmune control serum throughout thenormal serum dose-response curve. The cytotoxic activityof normal serum with and without additional immune serumwas abrogated by prior heating of normal serum at 56°C for30 min.

This substantial enhancement by immune serum of normalhuman serum-mediated killing of L. donovani amastigotes

INFECT. IMMUN.

SERUM CYTOXICITY FOR L. DONOVANI 249

coc-

ui05

C 2

25-

L.donovani, 1:20 FRESH + 1:50 CONTROL SERUM

Ltopicea, 1:20 FRESH + 1:50 CONTROL SERUM_______ _______---a

L.donovani, 1:20 FRESH + 1:50 IMMUNE SERUM

15 30 60

TIMEMIN

FIG. 2. Kinetics of serum cytotoxicity. Amastigotes (10') of L.tropica were incubated at 37°C with a 1:20 dilution of fresh normalserum and a 1:50 dilution of heat-inactivated normal serum. Thesame number of L. donovani amastigotes was incubated with fresh(unheated) serum and a 1:50 dilution of either normal or immuneserum. After 1 to 60 min, an equal volume of ice-cold 20 mM EDTAwas added. The viability of the treated amastigotes was estimatedby conversion to promastigotes and expressed as a percentage of thenumber of viable organisms at time zero. Points along the two lowercurves are significantly different (P < 0.05) from points on the uppercurve at all comparable points.

was further documented by analysis of the time course forcytotoxicity (Fig. 2). About 50% of the amastigotes werekilled by factors in normal serum after 20 min of incubationat 37°C. Addition of immune serum significantly increasedboth the level and rate of cytotoxicity. At the plateau,one-fifth as many amastigotes survived in cultures withimmune serum as in cultures with control serum; the rate ofcytotoxicity was also increased fourfold in cultures withimmune serum. Indeed, the rate and degree of cytotoxicityseen with L. donovani amastigotes incubated in normalserum plus immune serum were then comparable to thoseobserved with L. tropica amastigotes cultured in normalserum alone (Fig. 2).

Activity in immune serum that increased normal serum-mediated cytotoxicity against L. donovani amastigotes waspresent in high titers (Fig. 3). In two separate experiments,dilutions of immune serum were added to concentrations ofnormal human serum that by itself effected minimal cyto-toxicity (25% in Fig. 3A and 10% in Fig. 3B). Addition ofimmune serum increased this level of cytotoxicity three- toeightfold; the activity in immune serum was present througha 1:3,000 dilution. Titers of activity at least this high werefound in sera of three different patients with kala-azar (datanot shown). It should be noted that at the dilutions tested,i.e., .1:50, heat-inactivated immune serum by itself had noeffect on amastigote viability.The preceding experiments clearly show that factors in

immune sera increased both the degree and the rate ofcytotoxicity against L. donovani amastigotes by normal

serum. The nature of these factors was clarified in furtherexperiments. (i) Enhancing activity was not removed fromthe surface of amastigotes by washing; one-fourth of theconcentration of normal serum was required to kill parasitesincubated with immune serum compared with those incu-bated with control serum before washing and treatment withnormal serum (data not shown). (ii) Factors coeluted withimmunoglobulin G during gel chromatography (Fig. 4); infact, activity was limited to the immunoglobulin G region ofthe column. (iii) Activity was removed by prior adsorptionwith staphylococcal protein A immobilized on Sepharose4B. Immune serum adsorbed with protein A-Sepharoseenhanced cytotoxicity of normal serum by only 4% at a 1:640dilution. In contrast, the same concentration of unadsorbedimmune serum or immune serum adsorbed with Sepharosealone enhanced killing by 47 and 46%, respectively. Immuneserum (1 ml of a 1:10 dilution) was adsorbed three times with108 amastigotes at 4°C in 5 experiments, with no change indose-response of the serum activity (data not shown). Nev-ertheless, the experiments outlined above strongly suggestedthat the cytotoxicity-enhancing activity in immune serumwas parasite-specific immunoglobulin G. Our failure to ad-sorb activity suggests that antibody may be of low affinity ordoes not require stable binding to the amastigote to enhancekilling.A previous report showed that the insect-associated,

promastigote form of L. donovani was killed by normalserum (9). Killing was directed by natural, possibly cross-reacting antibody and was mediated by the classical comple-ment pathway. This was not the case with L. donovaniamastigotes. For example, cytotoxic activity against amas-tigotes was not removed from normal serum by prior adsorp-tion with the parasite (Fig. 5). Cytotoxicity in normal serumwas abrogated, however, by heating at 56°C for 30 min (Fig.1), and was therefore likely to involve one or more comple-ment components. This likelihood was examined further byanalysis of the effects of chelating agents on serum-mediatedcytotoxicity (Fig. 6). Cytotoxicity by normal serum with and

100-

CONTROL SERUM

75

Co2CONTROL SERUM

CD

SE 50

IMMUNE SERUM

25-

o A Bp3600 1200 '400 i3200 '800 >200

FIG. 3. Titration of enhancing activity in immune serum. L.donovani amastigotes were incubated at 37°C for 40 min with a 1:20(A) or a 1:50 (B) dilution of fresh (unheated) normal serum anddilutions of immune serum. Viability of treated amastigotes wasdetermined by conversion to promastigotes. Points along the curvesof immune serum differ significantly (P < 0.05) from those of normalserum through a 1:1,200 dilution in (A) and a 1:3,200 dilution in (B).

VOL. 47, 1985

250 HOOVER ET AL.

1gM

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IgG

-2.0

F..

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FRACTION NUMBERFIG. 4. Fraction of enhancing activity by gel chromatography. Serum was passed over a calibrated BioGel ASM column and eluted with

phosphate-buffered saline. Fractions were pooled as indicated. Fractions (1:6 dilution) were incubated with L. donovani amastigotes and a1:20 dilution of fresh (unheated) serum. Viability, expressed as a percentage of the number of parasites surviving incubation with fresh serumplus phosphate-buffered saline diluted 1:6, was estimated by conversion to promastigotes. Survival of amastigotes incubated with fresh serumand material from fractions 116 to 134 was significantly less (P < 0.05) than that of parasites incubated with fresh serum plusphosphate-buffered saline or material from other fractions.

without immune serum was completely inhibited by EDTAbut not by magnesium-ethylene glycol-bis(3-aminoethylether)-N,N,N',N',-tetraacetic acid. These results suggestedthat the major pathway for cytotoxicity against L. donovaniamastigotes by normal serum with or without immune serumwas mediated by the alternate complement cascade. Toconfirm this hypothesis, we examined the cytotoxic effectsof serum from C2-deficient patients on L. donovani amast-igotes (Fig. 7). Serum from C2-deficient patients was cyto-toxic to L. donovani amastigotes at a level comparable tothat seen with normal serum; in both instances, cytotoxicitywas increased fourfold by addition of a small amount ofimmune serum. Thus, normal human serum-mediated cyto-toxicity against L. donovani amastigotes in the presence orabsence of immune serum was effected via the alternatecomplement cascade.

DISCUSSIONOur data show that heat-stable factors in sera of patients

with kala-azar markedly enhance the cytotoxicity of normalserum against amastigote forms of L. donovani. Theseobservations require us to reconsider our concepts of therole of humoral factors in host immunity to L. donovani.Previous studies by Pearson and associates have shown thatpromastigotes, the insect-associated form of L. donovani,are killed by factors in normal human serum (9). Cytotoxic-ity, in that instance, was mediated by the classical comple-ment cascade and was initiated by adsorbable factors, pre-sumably cross-reacting, natural antibodies present in sera of

normal subjects. The possible protective role of these cyto-toxic but ubiquitous antipromastigote antibodies is unclear.It is possible that antibodies directed against the (usually)intracellular, amastigote form will be more protective thanthose directed against the promastigote form.The studies described in this report enhance that possibil-

ity by documenting a novel interaction between L. donovaniand factors in human serum. Killing of amastigotes, unlikethat of promastigotes, is not mediated by the classicalcomplement pathway; L. donovani amastigotes induce a lowlevel of cytotoxicity mediated by direct activation of thealternate complement pathway. Moreover, in contrast to theantibody-directed killing of promastigotes, killing of amas-tigotes by normal serum is not mediated by adsorbablefactors. Alternate pathway-mediated cytotoxicity, however,is significantly increased by antibodies that develop duringnatural infection. These findings document limited andnonprotective serum-mediated immune mechanisms di-rected against the amastigote form that are present in normalindividuals. Infection of such individuals by L. donovani,however, generates in serum a new, qualitatively and quan-titatively distinct immune response, i.e., antibody-directed,alternate complement pathway-mediated cytotoxic activity.Previously infected persons are also immune to reinfection(12). It is possible, then, that production of antibodiescapable of enhancing serum cytotoxicity toward the amast-igote stage is a crucial requirement for effective antiparasiticimmunity. If so, strategies for vaccine development shouldbe focused accordingly.

INFECT. IMMUN.

SERUM CYTOXICITY FOR L. DONOVANI 251

A NORMAL

CONTROL SERUM

A.

CIOI.-

CD

Cc,X 50-

25-

2 5 -

SERUM DILUTIONFIG. 5. Cytotoxicity of adsorbed serum for L. donovani. Amas-

tigotes were incubated for 40 min at 37°C with unadsorbed serum orserum that had been adsorbed five times with 5 x 107 amastigotes at0°C. Parasite viability was estimated by conversion to promasti-gotes. Curves for fresh serum differ significantly (P < 0.05) fromcontrol curve but do not differ from each other.

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+FRESH NORMAL CONTROL

+ SERUMCONTROL SERUM

T 3

FRESH C2DEFICIENT

EMUINEFRESH NORMAL SERUM

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FIG. 7. Killing of L. donovani amastigotes by C2-deficient se-rum. Amastigotes (107 were incubated with dilutions of fresh(unheated) normal or C2-deficient serum and a 1:50 dilution ofheat-inactivated normal (control) or immune serum for 40 min at37°C. The viability of treated amastigotes was estimated by conver-sion to promastigotes. The viability of the amastigotes was signifi-cantly less (P < 0.05) in the presence of normal and C2-deficientsera than in the presence of heated serum. Normal and C2-deficientserum did not differ from each other in cytotoxic effect.

Quite apart from considerations of vaccine development,one can raise innumerable questions regarding basic mech-anisms of serum toxicity toward amastigotes. At the dilu-tions studied in this report, serum cytotoxicity towardpromastigotes, in contrast to that toward amastigotes, islargely mediated by the classical complement pathway (9).This implies that transformation of the parasite from promas-tigote to amastigote stage dramatically alters its interactionswith serum complement. No longer do natural antibodiesmediate cytotoxicity; instead, amastigotes directly activatethe alternate complement pathway. Moreover, antibodiespresent in the serum of infected patients markedly enhancecytotoxicity toward amastigotes; this enhancement is medi-ated exclusively via the alternate pathway. Studies with

FIG. 6. Effect of complement inhibitors on killing of L. donovaniamastigotes by human serum. Amastigotes (107 were incubated at37°C for 40 min with a 1:50 dilution of heat-inactivated normal(control) or immune serum plus a 1:20 dilution of fresh (unheated) orheat-inactivated normal serum in phosphate-buffered saline, 10 mMEDTA in phosphate-buffered saline, or 10 mM ethylene glycol-bis(3-aminoethyl ether)-N,N,N',N'-tetraacetic acid plus 2.5 mM MgCl2in phosphate-buffered saline, all at pH 7.4. The viability of treatedamastigotes was estimated by conversion to promastigotes. Theviability of the amastigotes incubated with fresh serum (with controlor immune serum) in the presence of PBS or MgEGTA wassignificantly (P < 0.05) different from that of amastigotes in heatedserum or EDTA groups.

A + CONTROL A + IMMUNE FRESH +

SERUM SERUM CONTROL SERUMFRESH +

IMMUNE SERUM

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252 HOOVER ET AL.

chelating agents and C2-deficient serum clearly indicatedthat the classical pathway made no significant contributiontoward antibody-directed serum killing of amastigotes. Thecharacteristics of amastigotes or antibodies or both that leadto alternate rather than classical pathway activation andcytotoxicity remain unknown and are the subject of contin-ued interest by our group.More crucial questions address the mechanism by which

antibody-facilitated alternate pathway-mediated killing oc-curs. Recent work indicates that antibodies, even minus theFc portions, may focus and concentrate the interactions ofalternate complement components with the cell wall andcapsule of gram-negative bacteria. This influence of antibod-ies on the geometry of complement attachment to target maybe more important than its influence on the rate of comple-mnent deposition in determining subsequent cytotoxicity bythe membrane attack complex (7). The relationship of theseobservations to ours is not yet known. To facilitate anti-leishmanial cytotoxicity, must the entire antibody be present,or can fragments serve as well? Is rate, or geometry, moreimportant in our system? Where in the process of comple-ment, cell, and antibody interaction does enhancement ofcytotoxicity toward amastigotes occur? How efficient isalternate pathway-mediated killing compared with the morefamiliar classical pathway-mediated process? Does preferen-tial interaction of serum with amastigotes via the alternatepathway favor the parasite or the host? The answers to thesequestions will require substantial investigation. Such stud-ies, however, should shed considerable light on the nature ofthe host-parasite relationship in this immunologically exceed-ingly complex disease.

ACKNOWLEDGMENTS

We thank Carl H. Hammer and Michael M. Frank for the gift ofthe C2-deficient serum, Rufus Gore and Joseph Williams for provid-ing amastigotes, and David Wynn for excellent technical assistance.

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and J. Smith. 1972. Prevention of varicella in high risk children:a collaborative study. Pediatrics 50:718-722.

2. Cunningham, I. 1977. New culture medium for maintenance oftsetse tissues and growth of trypanosomatids. J. Protozool.24:325-329.

3. Dolin, R., R. C. Reichman, M. H. Mazur, and R. H. Whitley.1978. Herpes zoster-varicella infections in immunosuppressedpatients. Ann. Intern. Med. 89:375-388.

4. Dwyer, D. M. 1976. Antibody-induced modulation ofLeishmaniadonovani surface membrane antigens. J. Immunol. 117:2081-2091.

5. Hoover, D. L., M. Berger, C. A. Nacy, W. T. Hockmeyer, andM. S. Meltzer. 1984. Killing of Leishmania tropica amastigotesby factors in normal human serum. J. Immunol. 132:893-897.

6. Hoover, D. L,, and C. A. Nacy. 1983. Analysis of macrophageinteractions with cryopreserved amastigotes of Leishmaniatropica. Infect. Immun. 41:1363-1367.

7. Joiner, K. A., R. C. Goldman, C. H. Hammer, L. Leive, andM. M. Frank. 1983. Studies on the mechanism of bacterialresistance to complement-mediated killing. VI. IgG increasesthe bactericidal efficiency of C5b-9 for E. coli 0111B4 by actingat a step before C5 cleavage. J. Immunol. 131:2570-2575.

8. Nacy, C. A., M. S. Meltzer, E. J. Leonard, and D. J. Wyler.1981. Intracellular replication and lymphokine-induced destruc-tion of Leishmania tropica in C3H/HeN mouse macrophages. J.Immunol. 127:2381-2386.

9. Pearson, R. D., and R. T. Steigbigel. 1980. Mechanism of lethaleffect of human serum upon Leishmania donovani. J. Immunol.125:2195-2201.

10. Preston, P. M., and D. C. Dumonde. 1976. Experimental cuta-neous leishmaniasis. V. Protective immunity in subclinical andself-healing infection in the mouse. Clin. Exp. Immunol.23:126-138.

11. Rezai, H. R., J. Farrell, and E. L. Soulsby. 1980. Immunologicalresponses of L. donovani infection in mice and significance of Tcell in resistance to experimental leishmaniasis. Clin. Exp.Immunol. 40:508-514.

12. Zuckerman, A. 1975. Current status of the immunology of bloodand tissue protozoa. I. Leishmania. Exp. Parasitol. 38:370-400.

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