AA(4 1 H P . ERIZATION OF ANTIBDY DEPENDENT KILLING OFIRP P SURAES BY MACRUPHAGESfUl ALBERT EINSTEIN COL OF

MDIE BRON N Y D0 R OSENS RE IC E AL .APR 83 im

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Characterizatian of Antiboy-Dependent Killing

of Trypinosoms by Nacrophages

0 Annual Ssw-y Report

David L. Rosenstreich, M.D. and

Hellen C. Greenblatt, Ph.D.

April, 1983

Supported by

U.S. ARMY MEDICAL RESEARCH AND DEVELOPMENT COMMND

FORT DETRICK, FREDERICK, WYLAND 21701

ak-

Contract No. DAND17-81-C-1196

Albert Einstein College of MedicineBronx, New York 10461

Approved for public release; distribution unlimited.

The findings in this report are not to be construed as an official Department

of the Army position unless so designated by other authorized documents.

84 12 17 010

SECURITY CLASSIFICAION OF THIS PAGE (111% 800 Sa.. __________________

REPORT DOCUMENTATION PACE a"__________________

111a. OOVT ACCESSSN O . NEI5CArALGOC MONOER

4. Vt t~rL9(w tSMW0 S. Type or REPORT & 11011110 COv61119

CHARACTERIZATION OF ANTIBODY-DEPENDENT KILLING 15 January 1982-14 January 19MOF TRYPANOSOMES BY MACROPHAGES ______________

0. PCXsNW**m auG1. REPORT IOUND94

David L. Rosenstreich, M.D.Hellen C. Greenblatt, Ph.D. DAMD17-81-C-1196

0. PQm.RuOQR OuG1ANIZATION %&Me AND LON112 10. *gA.2 9 EjE~ITPR4OCol. TASAlbert Einstein College of Medicine1300 Morris Park Avenue 62770A.3Ml62770A87I.AE.074Bronx, New York 10461 _____________

If. CONTROLgm LIOOFIE eS NA0 AND AGASEIS IL. REPORT OATS

U.S. Army Medical Research &Development Command April, 1983Fort Detrick IS. "UnOXIS OF PAGEsFrederick, Maryland 21721 AM -SGOAMS_________

14 IMM T 1111 " WIMIC "AN 0. A0 1141111141011101.W.11111011111 j100 IS. S6CURITV CLASS. (of Oft e e

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S4. bOSIMIOUTION STAT9mENT We SWO xwm"-

Approved for public release - distribution unlimited

IT. DIueTINOUTO4 STATEMEaNT (or WHO 4681NO meow"d 0 M00611 ". lIt 611141111 ROWN)

te. S11PS IV NTN so" E= . werse e f08~m~ id' Vm@

Trpnsm rhodesiense; trypanosomes; African trypanosomiasis; protozoa,jpiiii tes macrophages; phagocytosis; antibody

SEE N4EXT PAGE

4 T-- e I, 1 .07-

gcMTV CLGPICAUU OF T1408 PAOC(Ehm Oe GPOn.

ABSTRACT

In the presence of antibody, macrophages bind trypanosomes in vitro.The cellular and humoral components of this assay have been studT'-edidefined. In general, such antibody must be specific for the trypanosomevariant. However, WRATat-14 and WRATat-15 variants, obtained after vectortransmission of WRATat-I.1, mediated of WRATat-1 trypanosomes to macrophages.Using a large panel of anti-WRATat-l.1 specific monoclonal antibodies, aclose correlation was found between the ability to mediate macrophage bindingin vitro, and in vivo trypanosome neutralization. The relative efficacy ofvarious immunogTo-Tn subclasses was analyzed. Monoclonal antibodies of allsubclasses were able to mediate trypanosome binding by macrophages. However,when polyvalent anti-WRATat-300 (Wellcome) serum from a rabbit infected 4weeks was fractionated into isotype specific-immunoglobulins, only the cold-precipitable portion of IgM was functional. The role of complement was alsoanalyzed. It was confirmed that complement enhances macrophages binding oftrypanosomes in the presence of limiting dilutions of antiserum. There was nospecies specificity in the action of complement since dog, human, guinea pigand mouse sera could be used as complement sources.4 In the presence of C4deficient complement, or sera depleted of C2 and Factor B, binding wassubstantially decreased as compared to that found in normal serum. Binding inC5 deficient sera was not effected. These findings suggest that complementmediated binding of trypanosomes by antibody required components of bothpathways, but did not require terminal components.

I

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charecterizatimof et' toyDpeeiet Killing

ef Trwuemsmes by berphages ;f-eso

uAmm I Smmm# Repar IDlC TAOUhaflfounflOd 0

David L. Rosenstreich, M.D. and 1wDistribution/_

liellen C. &aoenlatt, Ph.D. Avalability COdeS-Avail and/or

Nlat special

April. 1983

Supported by

U.S. Army MEDICAL RESEARCH1 AND DEVELOPMENT COMMAND

FORT DETRICK. FREDERICK. MARYLAND 21701

Contract No. DAN1781-C-1196

Albert Einstein College of Mdicine

Bronx, New York 10461

Approved for public release; distribution unlimited.

The findings In this report are not to be construed as an official Department

of the ArvW position unless so designated

by other authorized documents.

K 3

f

20. SUI Y

In the presence of antibody, macrophages bind trypanosomes in vitro. Thecellular and humoral components of this assay have been studied and defined.In general, such antibody must be specific for the trypanosome variant.However, WRATat-14 and WRATat-15 variants, obtained after vector transmissionof WRATat-.1, mediated binding of WRATat-l trypanosomes to macrophages.Using a large panel of anti-RTat-1.1 specific monoclonal antibodies, a closecorrelation was found between the ability to mediate macrophage binding invitro, and in vivo trypanosome neutralization. The relative efficacy ofvarious i wo--Toulin subclasses was analyzed. Monoclonal antibodies of allsubclasses wer able to mediate trypanosome binding by macrophages. However,when polyvalent anti-WRATat-300 (Wellcom) serum from a rabbit infected 4weeks was fractionated into isotype specific-immunoblobulins, only the cold-precipitable portion of [gN was functional. The role of complement was alsoanalyzed. It was confirmed that complement enhances macrophage binding oftrypanosomes in the presence of limiting dilutions of antiserum. There was nospecies specificity in the action of complement since dog, human, guinea pigand mouse sera could be used as complement sources. In the presence of C4deficient coo lement, or sera depleted of C2 and Factor 8, binding wassubstantially decreased as compared to that found in normal serum. Binding inCS deficient sera was not effected. These findings suggest that complementmediated binding of trypanosomes by antibody required components of bothpathways, but did not require terminal components.

94

e . •,W0_...

FII I-

In conducting the research described in this report, the investigator(s)adhered to the "Guide for the Care and Use of Laboratory Animals," prepared bythe Comittee on Care and Use of Laboratory Animals of the Institute ofLaboratory Animal Resources, National Research Council (OHEW Publication 1o.(NIH) 78-23, Revised, 1978).

Citations of comercial organizations and trade names in this report donot constitute an official Department of the Arwt endorsement or approval ofthe products or services of these organizations.

.1

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k

, S

Table of Costents

Page

DO 1473 ..........................................................4........

Abstract .................................................................. 2

Title Page .............................................................. .. 3

Summary ................................................................... 4

Foerd ................................................... 5

Table of Contents ............................................ 6-7

Text of Annual Report - Contract Goals and Progress

(a) Determination of the requirement for variant specific antibody .... 8

(b) Analyses of the ability of nonoclonal antibodies specific for

discrete trypanosome antigenic determinants to mediate

macrophage-trypanosome binding .................................. 9

(c) Deterination of the immunoglobulin class dependence of binding ... 9

(d) The effect of specific coMalenot pathways and components ........ 10

(e) Characterization of the macrophage functions and receptors

active in this system .......................................... 12

TABLES

Table 1: Effect of monoclonal antibody, and sera obtained from infected

mice and monkeys on the binding of variant trypanosomes...13

Table It: Ability of monoclonals to neutralize jn vivo

and mediate in vitro binding to macrophages ............... 14

Table III: Effect of fractions isolated from rabbit anti-trypanosome

serum to mdiate binding of trypansoms ................... 15

Table IV: Ability of complement deficient and intact sera to enhance

binding of trypanosomes to macrophages under conditions

of limited mounts of heat-activated1 anti-trypenosml .

antibody .................................................. 16

Table V: House macrophage lines to be tested for trypanosome bnding.176

References ........................... ..... 18-19

Distribution List .................... .........20

7

Characterization of Antibody-Oependent Killing of Trypanosoms by Placrophages.

Contract No. OIW17-81-C-1196

NOW REPORT

This contract was initiated September 1981 to characterize the phenomenonof macrophage binding of opsonized T. rhodesiense (Greenblatt et al 1983).The contract had the following goal-.

(a) Determine the requirement for variant specific antibody.(b) Analyze the ability of monoclonal antibodies specific for discrete

trypanosome antigenic determinants to mediate macrophage-trypanosomebinding.

(c) Determine the immunoglobulin class dependence of this binding.(d) Analyze the effect of specific complement pathways and components.(e) Characterize the macrophage functions and receptors active in this

system.

Progress was made in the following areas:

(a) Determination of the requirement for variant specific antibody

Injection of a clone of trypanosomes into mice will result in anantiserum that is specific for that clone (Campbell. et al 1979). Antibody-mediated, complement dependent killing of trypanosome4s {vitro requiresvariant-specific antibody (Diggs and Toussaint, personaTCommication) as doother antibody-dependent immune responses directed against trypanosomes. Someinvestigators have demonstrated that only variant-specific antibody wiWlmediate the binding of T. S!aiense to macrophages (Takayanagi and Nakatake,1977). However, Cook (1TO98) found that antigen-specificity of rabbit immunesera was not a factor in the binding of T. brucei Etat 4 or T. brucel 427 torabbit peritoneal cells. It was therefore necessary to test h eneed forspecificity of antibody in mediating the attachment and internalization oftrypanosomes to macrophages.

The results of a series of experiments to determine whether trypanosomebinding to murine macrophages required variant-specific antibody aresummarized in Table I. Normal mouse sera were unable to mediate binding ofany trypanosomes, regardless of which trypanosome variant was used. Rabbitanti-WRATat-300 (Welicomo) and the monoclonal antibody directed againstARTat-l could only mediate binding of their respective trypanosomes. VConclusions: These data suggested that antibody directed against a specificvariant type was required to mediate binding of these parasites tomacrophages.

There Is also evidence for cross-reactivity between antibodies. W1ATat-14 and WRATat-l5 are two variant antigen types obtained after vectortransmission of WRATat-I.1 (Esser jjal 1982). Sera from three monkeysinjected with these new variants wer able to mediate binding of WRATat-ltrypanosomes to mouse resident peritoneal macrophages. Such cross reactivitybetween various trypanosome strains my be useful for the development of newdiagnostic assays since only a primary antigenic type and/or antibody directedagainst the type, would be required.

8

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MAL= ,

(b) Analysis of the abilit of monoclonal antibodies specific fordiscrete trypanosome antigenic determinants to mediate macrophage-trypanosomebinding

Klaus Esser (WRAIR, Washington, D.C.), has developed about 40 hybridomasby fusing WRATat-l.1 spleen cells from infected mice to immunoglobulln-secreting myeloma lines (Sacks, et al 1982). These hybridomas generatemonoclonal antibodies which reac-w'Ih variant antigen types oftrypanosomes. The majority of these products are capable of neutralizingparasite infectivity (Table 1I).

Each of the monoclonal antibodies were screened over a large range ofconcentrations, and in the presence or absence of enhancing levels ofcomplement in the macrophage assay. A careful microscopic analysts was thenperformed for each individual monoclonal antibody and the percentage ofmacrophages binding trypanosomes was calculated.

Table 11 lists the monoclonal antibodies assayed, their ability tomediate binding of trypanosomes to macrophages in vitro, and their ability toneutralize Infection in vivo. There was a close-positve correlation betweenthe ability to mediate binding of WRATat-l trypanosomes to resident peritonealcells and the ability of the monoclonal antibody to opsonize trypanosomes forin vivo neutralization; 91% of the monoclonal antibodies could both mediate-Tnar- and neutralize in vivo.

Two IgG1 monoclonal antibodies, 6.7H1l and 16.3F1.4, were able to mediatebinding especially when enhanced by the presence of complement; they were nothowever protective iniv. (Klaus Esser, personal comunication).Monoclonal antibodyT15.25.1 presented difficulties suggesting that twoseparate lots, with different potencies may be involved. The first lot of15.2145.1 tested was unable to mediate binding. However, a second lot producedgood binding. When neutralization studies were carried out with 15.2145.1results were negative or ambivalent. Currently it is typed as a monoclonalantibody incapable of neutralization in vivo; however, if different lots wereassayed this negative designation migET N--riversed.

Monoclonal antibodies of subclasses tested including: 1gM, 1gG), IgG2a,and IgG2b, were able to mediate binding of trypanosomes to macrophages. Theredid not appear to be any correlation between the imunoglobulin class and itsfunctitonal actitvity.

(c) Determination of the imunoglobulin class deoendence of binding

Dr. Jeenan Tseung of WRAIR, Washington, D.C. provided us with antibodysubclasses of rabbit anti-WaATat-300 (Wellcome) strain of T. rhodestn.Serum was collected from an infected rabbit over a 4 week perTod, and antibodysubclasses prepared by sequential monium sulfate precipitation (35-40%),cold precipitation, ion exchange chromatography on OEAE-cellulose, andaffinity chromatography on imobilized staphylococcal protein A.

These fractions were demonstrated to be Isotype specific Ig, 1ga, and$IgG2b, and were demostrated to have no contamination with 1gA. The cold-

precipitable portion of 1gM was able to mediate binding of trypanosomes to W%of the macrophages both in the presence or absence of complement

, • 9

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I

(Table I1). None of the other fractions were able to promote anyattachment. These data suggest that only the 1gM fraction of rabbit anti-WRATat-300 was functional in this assay. Other reports have indicated thatantibodies of both IgM and IgG specific to trypanosomes appear within 3 weeksof infection (Seed et al 1969, Musoke et al 1981, Cook 1981). However, Musokeet al (1981) determineTthat along with--i, the IgGI isotype, not the IgG2,was responsible for clearance of parasites in cattle which is consistent withour in vitro findings. We did not have any IgGl fractions to assay so thequest-nFfIgGl specificity for the parasite cannot be resolved. This datadiffers from our finding using the monoclonal antibodies (section b, above).In those experiments, it was found that monoclonal antibodies of all isotypeswere capable of mediating binding to macrophages. Such diversity may reflectthe nature of the monoclonal antibody generating system rather than asituation found "in nature". Whether only specific Ig subclasses arefunctional against trypanosomes is an important question, and will requireadditional experimentation for its resolution.

(d) The effect of specific complement pathways and components

Introduction

The comlement cascade of serum consists of functionally inactiveprecursor molecules. Activation is the term designated for the process whichpermits these proteins to cooperate in a specific sequence. Activation of C3is one of the central events of the complement cascade and occurs via theclassical and/or alternative pathways. Enzymes generated during the cascadecleave the C3 molecule, producing several biologically active molecules (C3a,C3b and C3d), and initiating the terminal comlement cascade (activation ofC5-C9).

The components of the classical complement system and the late actingterminal proteins are designated numerically as Cl, C4, C2, C3, C5, C6, C7,C8, and Cg. The classical pathway is initiated by an antibody-antigenreaction which activates the sequence, Cl, C4, C2, and finally C3. Cleavageterminal sequence is triggered(either via the classic or alternativepathways) lysis of the particle binding complement may occur.

The second pathway by which C3 can be cleaved and the terminal pathway(C5-Cg) initiated, is the alternative pathway. When this system isfunctional, the classic pathway may be by-passed resulting in little or noconsumtion of Cl, C4, or C2. The alternative pathway functions by activatingFactor B in the presence of magnesium ions. Cleavage of B results in anenzyme that is capable of activating C3 which in turn will cleave C5-C9 (theterminal sequence).

The Fc portion of antigen-antibody complexes bind to specific receptorson macrophages. The Fc receptors of macrophages are triggered to initiatephagocytosis of IgG-coated and perhaps IgM-coated (Silverstein et al 1977,Roubin and Zolla-Pazner 1979) particles. These receptors also pper to havean imeunoregulatory role (M4organ et &1 1983). There are other sets ofreceptors on macrophages that bind fragments of 3. Such binding, via C3breceptors, is thought to enhance particle ingestion via Fc receptors(Ehlenberger and Mussenzweig, 1977). Also under specific conditions,particles coated only with C3b will be readily phagocytized (Griffin and

10

ul11inax 1981).

1) Role of complement In enhanced binding of trypanosomes to macrophages inthe presence of limiting dilutions of antibody

In previous work (Greenblatt et a] 1983) it was demonstrated that immuneserum whic had been heat-treated to i-nactivate complement, mediatedprogressively lower percentages of tfypanosome binding to macrophages as theantiserum was diluted to l0' to 40- . Upon the addition of rat complement tothese samples, macrophages again bound high numbers of trypanosomes. Thesedata suggested that in the presence of limiting dilutions of antiserum, ratcomplement enhances the binding of trypanosomes to macrophages.

Therefore. in order to further investigate the role of complement in theincreased binding of trypanosomes to peritoneal macrophages, the complementthat is normally present in serum had to be inactivated. Heat-inactivation ofserum at 530C for 90 min destroys many complement proteins and inactivates thecomplement pathway. Sera were treated in this manner and tested for theirability to mediate binding of trypanosomes. In the presence of limitingdilutions of heat inactivated sera only 22% of the macrophages boundtrypanosomes (Table IV). When these sera were supplemented with ratcomplement, 58% of the macrophages bound parasites. Guinea pig, dog, human,and mouse sera could also be used as complement sources (Table IV). Thesefindings indicate that complement enhances macrophage binding of trypanosomesin the presence of limiting dilutions of antiserum. In addition, there is nospecies specificity in this action of complement.

2) Role of terminal, classical and alternative pathway components

The requirement for specific complement components in the binding assaywas determined by testing sera that were lacking individual components.

There has been demonstrated a requirement for antibody, alternativepathway and terminal components for the killing of T. rhodesiense in vitro(Flemings and Diggs 1978). Therefore it was possilTeEhaiTthe killing ofparasites was mediated by macrophage complement components released andactivated by parasites at the surface of the macrophages.

Lysis at the macrophage surface has been shown to be triggered in othersystems by specific IgG fractions (Ralph 1980; Walker 1977). Lysis cascadestypically require terminal components including CS. Mouse sera deficient inCS was utilized to determine the need for terminal complement components inmediating killing of trypanosomes by macrophages. In the presence of normalmouse serum, 60% of the macrophages bound trypanosomes, as compared to 79%binding in the presence of CS deficient serum (Table IV).

Also, guinea pig serum deficient in C4 components of the classicalpathway (C40) was compared to normal guinea pig serum for its ability toenhance uptake under conditions of limited levels of antibody. This serumonly partially restored the binding activity of antibody (Table IV). In thepresence of C40 serum only 44% of the macrophages bound trypanosomes comparedwith 68% binding in the presence of intact guinea pig serum (Table IV).

Treatment of serum at SOOC for 20 min partially decreases levels of C2

- ': I1

(of the classical pathway) and destroys Factor B (of the alternativepathway). When such heat-treated complement was added to heat-inactivatedserum, binding activity was only partfilly restored (from 22% to 41%)(Table IV).

Conclusions: Complement mediated binding of trypanosomes by antibodyrequires components of both pathways, but does not require terminalcomponents. The most plausible interpretation is that such enhancement ismediated via macrophage COb receptors.

(e) Characterization of the macrophage functions and receptors active inthis system

We have begun to screen a nuumber of normal and mutant mouse macrophagecell lines for their ability to bind trypanosomes. Currently, we aredetermining the growth characteristics and adherence characteristics of theselines. Several of these lines exhibit stable, well defined abnormalities(Table V). Using mutant cell lines which lack specific surface receptors orare metabolically abnormal may provide insight into the mechanisms by whichmacrophages clear trypanosomes.

12

TABLE I

Effect of mmoclonal antibody, and sera obtained from Infected niceawd mnkeys an te binding of variant trypanosomes

WRATat-300 WRATat-186

Serum (Wellcome) WRATat-l (EATRO 1886)

Normal mouse sera

Rabbit anti-WRATat-300

Monoclonal 2.157.1 +i Iqonke antR t-RA-14

Monkey pre-b leed

Monkey antt-WATat-14 +

Monkey ani-kIATat-15+

1i

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TABLE II

Ability of mnoclonals to neutralize In vivo midmdiate in vitro binding to mcibaW

In vivo MacrophageeutraTization Binding Class

31.4D1.6 0 0 IgM32.1A5.5 + + IgM32.2A1.1 + + IgM32.2A4.1 + + IgM32.3F1.6 + + 1gM

2.187.1 + +IgG1*6.7Hl1 0 +IgGl6.8C1.2 + IgG16.1105 + +IgGl16.2A12.2 + +IgGl

'16.3F1.4 0 +IgGl

2.108.1 0 OIgG2a12.3D3.4 + +IgG2a12.4F3.1 + +IgG2a16.1A5.3 +I- +IgG2a16.1A8.1 + +IgG2a32.1A3.1 0 OIgG2a32.165.1 + +IgG2a

4.G9.1 0 OIgG2b

12.2E7.2 + +1q63*15.2H5.1 +Ig63

31.465.1 0 01g6332.381.5 0 01gG332.4C1.5 0 OIg 332.3812.1 +

*Indicates discrepancy between in vivo and in vitro findings.

14

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TAKE III

Effect of fractions Isolated fru rabit mti-trypamosome seisto mediate binding of trypamsainms

% Binding of Trjypanosoets to Macrophage

Cold Non-coldPrec ipitable Precipitable

Hyperlnmne rabbit strum (83%)

19Mwithout cowpleuent 85 0with complemenit 76 0

1q62 awith or without complemenlt 0 0

qG62bwith or without complement 0 0

TAILE TV

M111ity of C¢Wmpwt deficient Md intact sara to enhancebinding of trAmosomes to mrq*agms under conditions of

limited a tI of IhIt-tst;4OA mtf-trypanosoml antibody

Treated Rat Complement % Attachment530C/90 mini 22 t 6.5

500C/20 urin2 41 + 2.8

Media 21 + 1.6

Intact Sera

rat 58 + 5.6guinea pig 68 ± 2.0dog 68 + 13human 54 1.5mouse 69 ± 15

Complement Deficient Sera

C4 deficient guinea pig 44 + 4.5C5 deficient mouse 79 ; 7

1Anti-tryanosome antibody was heat-inactivated at 530C/90 min to disrupt

the cop1ement cascade.

2Coplemnt was heated at SO°C for 20 min to destroy Factor 8 and C2.

16

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TAKE V

bue nrphage lines to be teste for trypansw binding

Line Functional Characteristics

J774.2 Fc mnediated phagocytosis

J774.1.11 Defective in 1962a phagocytosisnormal binding of monomeric IgG2a

J774.3.4 Depressed levels of IgG2a andIgG2b phagocytosis

CTRN 1 Lacks CAMP dependent proteink ise

17

KEFERENCES

Cambel1, G.H., K.M. Esser, B.T. Wellde and C.L. Diggs. 1979. Isolation andcharacterization of a new serodeme of Trvlanosoma rhodesiense. Am. J. Trop. Med. Hyg.28:974-983.

Cook, R.M. 1981. Attachment of Trypanosoma bruce for rabbit peritoneal exudate cells.Int. J. Parasitol. 11:149-156.

Ehlenberger, A.G. and V. Nussenzweiq. 1977. The role of membrane receptors for C3b andC3d in phagocytosis. J. Exp. Red. 145: 357-371.

Esser, K.M., N.J. Schoenbechler and J.8. Gingrich. 1982. Trypanosoma rhodesiense bloodforms express all antigen specificities relevant to protection against metacyclic (insectform) challenge. J. imunol. 129:1715-1718.

Flemings, B. and C. Diggs. 1978. Antibody-dependent cytotoxicity against Trypanosomarhodesiense mediated through an alternative comlement pathway. Infect. Imun. 19:928-933.•

Greenblatt, H.C., C.L. Diggs and M. Aikawa. 1983. Antibody dependent phagocytosis ofTrypanosoma rhodesiense by murine macrophages. Am. J. Trop. Med. Hyg. 32:1-12.

Griffin, E.M. and P.J. Mu1linax. 1981. Augmentation of macrophage complement receptorfunction in vitro III C3b receptors that promote phagocytosis migrate within the plane ofthe macrop"age--p asma membrane. J. Exp. Md. 154:291-305.

MIrgan, E.L., J.E. Shields, C.S. Campbell, R.L. Barton, G.A. Koppel and W.O. Weigle.1983. Synthetic Fc peptide-mediated regulation of the Immune response. .Characterization of the immunomodulating properties of a synthetic 23-amino acid peptidederived from the sequence of the CH3 domain of human IgGl. J. Exp. Med. 157:947-956.

Musoke, A.J., V.M. Nantulya, A.F. Barbet, F. Kironde and T.C. McGuire. 1981. Bovineimmune response to African trypanosomes: specific antibodies to variable surfaceglycoproteins of Tr anosoma bjc. Parasite Immunol. 3:97-106.

Ralph, P., 1. Nakoinz, 8. Diamond and 0. Yenton. 1980. All classes of mrine [gGantibody mediate macrophage phagocytosis and lysis of erythrocytes. J. Imunol. 125:1885-1888.

Roubin, R. and S. Zolla-Pazner. 1979. Markers of macrophage heterogeneity. I. Studiesof macrophages from various organs of normal mice. 9:972-978.

Sacks, D.L., K.M. Esser and A. Sher. 1982. Immunization of nice against Africantrypanasomiasis using anti-idiotypic antibodies. J. Exp. Mad. 155:1108-1119.

Seed, J.R., R.L. Cornille, E.L. Risby &nd A.A. Gem. 1969. The presence of agglutinatingantibody in the 19H im oglobulin fraction of rabbit antistrum during eaxwtntalAfrican trypanosomiasis. .:283-292.

Silverstein, S.C., R.N. Steinmmn and Z.A. Cohn. 1977. Endocytosis. Ann. Rev. Stochem.46:669-722.

is

Takaymnagi, T. and Y. Nakatake. 1977. Trywoao sgjljq e: The binding activity ofantiserum to macrophages. Exp. ParasitoT _Z:Z-Z.Walker, W.S. 1977. Mediation of macrophage cytolytic and phagocytic activities byantibodies of different classes and class specific Fc-receptors. J. Inmunol. 119;367-373.

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DISTRIBUTION LIST

12 copies DirectorWalter Reed Army Institute of ResearchWalter Reed Army Medical CenterAttn: SGRD-UWZ-CWashington, D.C. 20012

4 copies CommanderU.S. Army Medical Research and Development CommandAttn: SGRD-RPMFort DetrickFrederick, MO 21701

12 copies Defense Technical Information Center (OTIC)Attn: DTIC-DDCameron StationAlexandria, VA 22314

I copy DeanSchool of MedicineUniformed Services University of the Health Sciences4301 Jones Bridge RoadBethesda, MD 20014

1 copy ComandantAcademy of Health Sciences, U.S. ArmyAttn: AHS-CDMFort Sam Houston, TX 78234

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