Inrernsrional JournalforParasifology Primed in Great Britain

Vol. 20, No. 6, pp. 725-730,

1990 0

OOZO-7519/W 13.M) + 0.00 Pergamon Press plc Sociery for Parasitology

1990 Au.waliim

ANTIBODY-MEDIATED CYTOTOXIC EFFECTS IN VITRO AND IN VW0 OF RAT CELLS ON INFECTIVE LARVAE OF BRUGIA MALA YI R. CHANDRASHEKAR,*

U. R. RAot

and D. SUBRAHMANYAM~

Research Center, Pharma Division, Hindustan Ciba-Geigy Ltd, Goregaon East, Bombay 400 063, India (Received 21 November 1989; accepted 5 March 1990) A~S~~C~-CHANDRASHEKAR R., RAO U. R. and SUBRAHMANYAM D. 1990. Antibody-mediated cytotoxic effects in vitro and in vivo of rat cells on infective larvae of Brugia malayi. International Journal for Parasitology 20: 725-730. Albino rat macrophages and neutrophils in the presence of immune serum adhered to and promoted killing of Brugiu malayi infective larvae in vitro. At a similar cell-target ratio,

macrophages were more potent than neutrophils in inducing cytotoxic response to the larvae. Eosinophils were also effective in killing but only at a high cell-target ratio. The activity in the immune serum could be absorbed to and eluted from a Protein A-Sepharose column suggesting involvement of IgG antibody in the reaction. An indirect fluorescent antibody test confirmed the presence of IgG on the surface of larvae incubated in immune serum. Infective larvae were attacked by host cells within micropore chambers 1624 h after implantation into immunized rats. Further, a strong cytotoxic response to the larvae was seen when they were introduced intraperitoneally into immune rats indicating the role of antibody and cells in vivo. We suggest that antibody-dependent cellular cytotoxicity may represent an important mechanism of parasite killing in an immune host. INDEX KEY WORDS: cytotoxicity, IgG.

Brugia

malayi: infective

INTRODUCTION have previously shown that rat neutrophils, macrophages and to a lesser extent eosinophils kill microfilariae of Brugia species in vitro and in vivo in the presence of antibody and/or complement (Chandrashekar, Rao, Subrahmanyam, Hopper, Nelson & King, 1984; Chandrashekar, Rao, Parab & Subrahmanyam, 1985). Thus, in vitro studies have been used to determine the possible mechanisms involved in innate resistance as well as acquired immunity to larval filarial worms. Studies on host immune mechanisms against infective third stage larvae of filarial parasites are of special importance WE

since they represent the initial infective stage for the human host and anti-parasite mechanisms to them have important consequences for preventing infection. However, there is very little information available on immune reactions operating against infective larvae (Higashi & Chowdhury, 1970; Sim, Kwa & Mak, 1982; Haque, Ouaissi, Santoro, Des Moutis & Capron,

* Present address and address for correspondence: Infectious Diseases Division, Jewish Hospital, Washington University Medical Center, 216 S. Kingshighway, St. Louis, MO 63110, U.S.A. t Present address: College of Public Health, University of South Florida, Tampa, FL 33612, U.S.A. 4 Present address: Swiss Tropical Institute, Socinstrasse 57, Basel, Switzerland.

larvae; macrophages;

neutrophils;

eosinophils;

1982; Chandrashekar, Rao & Subrahmanyam, 1985). All these prior studies have demonstrated the role of antibody and/or complement in in vitro cytotoxic response to the larvae. To date there is no clear cut evidence whether similar mechanism operate in vivo. The susceptibility or resistance of a host depends on such mechanisms that operate in vivo once the parasite

enters the host. We report here experiments which demonstrate the role of antibody-dependent cellular cytotoxicity (ADCC) both in vitro and in vivo in killing the infective

larvae

of the human

filarial

parasite,

Brugia malayi.

MATERIALS AND METHODS Animals andparasites. Three- to 4-week old male Mastomys natalensis (‘GRA’ Gissen strain) and 4-week old male albino

rats (‘Wistar’ strain) bred by conventional methods at the vivarium of this Research Center were used in the study. Mastomys were infected with B. malayi as described by Singer, LImmler & Kimmig (1981). B. malayi infective third stage larvae (L3) were recovered from Aedes aegypti (‘SS strain) that were fed 2 weeks earlier on Mastomys infected with B. maluyi. Infective larvae were isolated by the Baennann technique (Ash and Riley, 1970). Seru. Normal serum from rat @IRS) was freshly prepared before each experiment. Immune serum was obtained from albino rats that received three intramuscular injections (15 days apart) of sonicates of 5-7 x 10’ L3, each time, in Freunds complete adjuvant. The animals were bled a week after the last immunization, by cardiac puncture, and immune serum (actively immunized rat serum, AIRS) 725

R. CHAN~~AS~~KAR, U. R. Rno and D.

726

isolated. Where indicated, the immune serum was heated at 56°C for 3 h to inactivate implement and heat-labile factors. EDTA and EGTA were used at a final ~n~ntration of 2 mr.r. AIRS was treated with Stophyt~oc~~ aureus cells FANSGRBIN (Calbiochem, San Diego, CA)] and anti-rat IgG antiserum (Cappel Research Products, Malvern, PA) as described elsewhere (Chandrashekar, Ran, Parab & Subrabmanyam, 1985). Fractionation of immune serum. IgG was purified from immune serum by affinity chromatography on a Protein ASepharose CLdB column (Pharmacia, Uppsala, Sweden) as described previously (Chandrashekar, Rao & Subrahmanyam, 1985). For isolation of IgM, the immune serum was passed through a prota~n~~pharo~ column and was efuted using 0.08 M-phosphate buffer containing 1.1 M-sodium chloride (Hudson % Hay, 1980). The IgG and IgM fractions thus obtained were tested for purity in immun~ff~on using goat anti-rat IgG and IgM (Cappel), respectively. C&s. Rat neutrop~ls, m~rop~ges and eosinaphils were purified from peritoneal exudates induced by i~~a~~toneal injection of casein or oil (Mehta, Sindhu, Subrahmanyam, Wopper & Nelson, 1982). The viability of the cells was more than 90% as judged by trypan blue exclusion. Smears of various cell types were prepared using a cytospin centrifuge (Shrurdon) stained with May-Griinwald-Giemsa, and the morphology and purity were assessed by microscopy. Cytotoxicity assay. The cytotoxicity assay was carried out as described previously (Chandrashekar, Rao & Subr~bm~vam, 19851.Briefi~. 10 L3 in 50 ul of RPMI-1640 (Gibco) were in&bat& with 5.x 10SceIts in 50 ~1 of RPMI1640 and 50 ul of fNRS and/or AIRS (50 uit (final volume 200 ~1) in a-flat-bottom 96lwell m&titer ‘plate (Costar, Cambridge, MA). Controf cultures contained L3 and sera, but no effector cells. ~tero~ore chambers. Micropore chambers were assembled using 14 x 2 mm plexiglass rings (Millipore, Bedford, MA) and 0.3 or 3.0 pm Nucleopore chemotactic membranes

(Nu&opore, Pleasanton, CA). The chambers containing 10

~~~~~~~NYAM

L3 wereimplanted subentaneously in normal and immunized rats, through an incision of 2-3 cm and placed dorsolaterally and the skin was sutured (Weiss & Tanner, 1979). After 16,24 or 48 h, the chambers were taken out and the contents were examined microscopically. The nature of migrated cells in the chambers was analysed by cytospin analysis as described above. RESULTS interactions with L3 in vitro B. malayi L3 were incubated at 37°C for 16 h with a

Rat cell-antibody

cell suspension containing either pure neutrophils, or eosinophils or macropbages at different e&&or-target ratios and AIRS at a final dilution of 1:4. The resuhs OR adherence and cytotoxicity are shown in Table 1. ~a~rophages were the most effective cell ~pulation in adhering to and killing the larvae foltowed by neutrophifs. At the end of I6 h incubation, neutrophils induced 41% cytotoxicity compared to 76% by macrophages. Rosinophils in the presence of AIRS readily adhered to the larvae but could induce only 28% killing during the same duration of incubation. Factors involved in ADCC to L3

The factors involved in neutrophil-mediated cytotoxicity to L3 were investigated using AIRS after various treatments. Heat treatment of AIRS at 56’C for 3 h did not significantly affect the ADCC caused by neutrophils indicating the role for heat stable factor(s) {Table 2). However, when AIRS was treated with EDTA at a finai ~n~entration of 2 mM, it completely blocked ADCC indicating a possible role for ions in the reaction. Pretreatment of AIRS with 2 mM-EGTA enhanced the cytotoxicity for reasons not apparent at

TABLE I-AIRS-MEDMTBD IU\TCELLULAR INTERACTIONS WITHB. malayi L3 in vitro

Cells (% purity)

Cells per L3 (x 103)

Neutrophils

(95)

5 ::

Control @IRS)

50 50

Macrophages (97)

5 15 25 50

Control (fhrRS) Eosinophils

(95) Control (MRS)

% Adherence*

% Cytotoxicity*

19 f 36 * 52 f 80 f 42 i

5 6 8 7 5

10 St 3 12 f 7 17 f 6 41 f 13 12 rt 5

46 f 72 f 81 f 92 zt 54f

6 8 6 6 II

II 41 50 76 38

*4 It9 f 3 f: 6 f 8

5

24 f 6

0

25 15 50 50

35 f 810 55* 74 f 10 4019

20: 6 28 rf: 6 8rt2

* Values are mean f S&M. of three experiments; % adherence, % of L3 with cells adhering; % cytotoxieity, % of L3 dead due to cell adherence (total cessation of motility}. Incubations at 37-C for 16 h. MRS. Fresh normal rat serum.

ADCC to 8. muluyi

~rurn~~~a~ent

infective larvae

% Adherence” Ii

727

% Cytotoxieity’

MRS

36f

AIRS hiAIRS (56’C, 3 h) hiAIRS (WC, 3 h) + MRS AIRS + EDTA (2 mM) AIRS + EGTA (2 mM) hiAIRS (WC, 3 h) + EGTA (2 mM)

?I jl 7 59 f 4 63 f 4 0 95 k 5

47 f 6 43 i 1 50 f 9 0 93 f 6

9Zt6

76 f 6

65f

12

* Values are mean f S.E.M.of four experiments; % adherence, % of L3 with cells adhering; % cytotoxicity, % of L3 dead due to cell adherence (total cessation of motility). Incubations were at 37°C For 16 h. Neutrophi-target ratio, SO,O@h 1. hi, Heat-inactivated; fNRS, fresh normal rat serum; AIRS, a&i&y immunized rat serum.

TABLE 3-N~TUREO~~ANT~~ODYINIIOLVEDIN Treatment of AIRS None Protein A Anti-rat IgG Anti-rat IgM Depletion of IgGt Depletion of IgM$ Purified IgG PurifiedIgG f MRS Purified IgM FuririedIgM + MRS

ADCCroB.

% Adherence* 88 f 2 0 0 80 f 6 24 f 4 a9 4 6 4Of7 84k 11 0 38f If

mukzyiL3 % Cytotoxicity’ 695 0 0 57% 0 57 f 18 jr 63 i 0 14 i

11 14 14 6 9 8

+ Values are mean f S.E.M.ofthree experiments; % adherence, % ofL3 with celis adhering; % cytotoxieity, % of L3 dead due to cell adherence (total cessation of m&My). t Depletion by Protein A-Sepharose. 1 Depletion by protamine-Sepharose. Incubations were at 37°C for 16 h. Macrophage-target ratio, 50,000: I. fNRS, Fresh normal rat serum; AIRS, actively immunized rat serum.

present. A similar observation was made with heatinactivated AIRS. AIRS, when treated with S. aweus cells, completely blocked ADCC. Similarly, AIRS treated with anti-rat IgG antiserum also failed to promote ADCC indicating the role of IgG in the reaction (Table 3). Nowever, AIRS treated with anti-rat IgM did not block ADCC. Purified IgG was found to be effective in kilhng L3 but greater activity co&d be obtained in the presence of fNRS. IgM fraction was not active in ADCC, though a weak cytotoxic response was seen on addition of fNRS, probably an effect of complement in the reaction. Indirect fluorescent antibody test also revealed the presence of IgG on the surface of L3 incubated in AIRS and fluorescein-conjugated anti-rat IgG. L3 incubated in AIRS failed to show fluorescence with fluoresceinconjugated anti-rat IgM. Thus these data confirm that IgG antibody is invotved in ADCC reaction. Fate of L3 ~~t~~~rn&Zrl$XB=c &W&e?5 In order to study whether similar ADCC can occur irzviw, a micropore chamber technique was employed. Ten L3 in Iscoves Modified Dutbeccos Medium

(IMDM; Gibco) were introduced into chambers covered with Nucleopore membranes. These were implanted into two groups of rats, one being the control and the other immunized with B. m&yd L3, which was the source of immune serum. The chambers were taken out at f 6,24 and 48 h and ADCC mediated by the migrated cells was recorded (Table 4). L3 were attacked by host ceils within the chambers covered with membranes of 3.0 pm pore size only 16-24 h after implantation. By 16 h, nearly 58% of L3 were killed and after 24 h, 92% killing was observed compared to 20 and 46% in normal rats at these time intervals. This clearly shows the role of antibody and cells in inducing cytotoxic response to L3 in viva. Cytospin analysis of the contents of the chambers showed that the major cell types involved in in viva ADCC were macrophages followed by polymorpbs. Macrophages appeared activated and were flattened against the farvai cuticle. There was also a significant in~~tration of lymphocytes into the chambers. L3 were unaffected when enclosed in 0.3 pm chambers due to the absence of cell migration both in normal and immunized rats.

128

R. CHANLIRASHEKAR, Ll. R. RAO and D. SUBRAHMANYAM

Normal(n = 6) Immunized~ (n =i 6)

16h

% Cytotoxicity* at 24b

48h

20 i 5 58 f 11

46if4 92 i 6

49 i 6 100

* Values are mean f s.E.M.; % cytotoxicity, % L3 dead due to cell adherence (total cessation of motility). t Rats were immunized with E. malnyi L3 as described in the Materials

and Methods. TABLE S-ADHERENCE

ANDCYTOTOXIC~TY OF PERITONEALCELLS TO 3. w&yi I~T~PER~~~L~l~TORA~

Rats 16 Normal (n = 6) Immunized (n = 6)

% Adherence* % Cytotoxicity* ‘??Adherence* % Cytotoxicity*

36 i 26f 74 * 60 f

14 18 6 7

L3

WHEN INIECTED

Time conrse (h) 24

48

56f 18 34sr 11 100 88 f 6

78 f 12 40 f 8 100 100

* Mean f s.E.M.; % adherence, % of L3 with cells adhering; % cytotoxicity, % of L3 dead due to cell adherence (total cessation of motility). The results are based on the number of L3 recovered. Rats were immunized with B. malayiL3 as described in the Materials and Methods.

Fifty L3 in IMDM were injected intra~~toneally into normal and immunized rats. After 16,24 and48 h, the peritoneal cavity was washed with heparinized IMDM. The effect of peritoneal cells on the parasites was recorded by cytospin analysis. A marked adherence of cells at 16 h was observed in immunized rats with a mortality of 60% (Table 5). This cellular adherence and killing progressively increased for up to 48 h when no live L3 could be recovered. In contrast, only 40% killing was seen in normal rats by 48 h due to complement activation. The major types involved in in vivo peritoneal ADCC were macrophages and neutrophils. There was very little accumulation of eosinophils on the infective larvae, both in normal and immunized rats. DiSCUSSION There is increasing evidence in the study of resistance to nematodes in general and to filariae in particular, that stage-specific antigens may be associated with the surface of these worms. The evidence for this is especially striking in the case of microfilatiae where studies with Diro$lori~ immitis,

Brugia puhangi, Litomosoides carinii and Acanthocheilonema viteue show that antibodies to the microfilarial surface appear in the circulation of the host when microfilariae are cleared from the blood (Wang, 1964; Sub~hm~yam, Rao, Mehta 62 Nelson, 1976; Weiss & Tanner, f979). In contrastto the apparent association of antibodies to the surface antigens of microfilariae and their disappearance from the bkmd,

the immune responses against L3 have not been clearly defined. Antibodies to the surface of L3 have been described but unlike the antibodies to the mierofilarial surface, they have not been reIated to the resistance to this stage of the worm. It appears that cell-mediated immune responses may be of more importance in immunity to these stages (Piessens, McGreevy, Ratiwayanto, McGreevy, Piessens, Koiman, Saraso & Dennis, 1980). In the present study, the immune mechanisms operating on the cuticle of B. malayi L3 have been studied in vitro and in viva. Serum from albino rats immunized with sonicated homogenates of the parasites was used as the source of antibodies. The IgG fraction of the immune serum was highly active in inducing a cytotoxic effect on L3. This &G-mediated killing was enhanced by fNRS, though a sign&ant Ievel of killing occurred with heat-inactivated AIRS as well. The weak cytotoxic effect observed with the IgM fraction in the presence of f&IRS may be a complement effect. B. ma&G L3 are susceptibie to cellular killing in vitro in the presence of complement alone (Chandrashekar, Rao, Parab & Subrahmanyam, 1986). The in vitxa ADCC reported by Sim et al. (1982) was complement independent. This is in contrast to the observation with B. pahungi L3 where complement of MRS was required for absolute killing (Chandrashekar, Rao t Subrahmanyam, 1985). Thus, the variations in requirement of complement suggest that factors required for cytotoxieity seem to be dependent on the parasite species involved. Several nir vitro studies conducted to determine the possible mechanisms involved in innate resistance as

ADCC to B. malayi infective larvae

well as acquired immunity to larval filarial worms indicate the importance of ADCC phenomenon. The relevance of these in v&o observations to in viva killing mechanisms is difficult to assess. While the potential importance of the various reactants studied in vitro can be assessed, many of the in vitro systems cannot possibly mimic the in vivo situation. However, the present study indicates the role of antibody and cells in vivo in inducing cytotoxic response to the infective larvae. L3 were attacked by host macrophages and polymorphs within the micropore chambers 16-24 h after implantation into immunized rats. Similar observations were made when L3 were introduced into their peritoneal cavity. Abraham, Weiner & Farrell (1986) observed that A. viteae L3 were killed in diffusion chambers 10 days after implan~tion into infected jirds. They concluded that there was a necessity for a developmental change for generating susceptibility to immune attack and that fresh L3 from ticks were not susceptible to cellular attack even in the presence of antibody. However, in our study, L3 recovered from mosquitoes were susceptible to ADCC both in vitro and in vivo. The present study clearly shows that a compromise between in vitro and in vivo observations on larval destruction involves the use of micropore chambers implanted subcutaneously. These chambers provide a more physiological milieu for larval growth and survival than in vitro cultures and still permit analysis of host effector mecha~sms (Weiss & Tanner, 1979; Rajasekariah, Monteiro, Netto, Deshpande & Subrahmanyam, 1989). The infiltrating cell populations provoked by the parasites in these chambers differ from those in the peripheral blood and might reflect a situation close to that found normally in vivo. At a similar cell-target ratio, macrophages were more potent in killing the larvae in the presence of immune serum. However, macrophages and neutrophils were equally active against B. pahangi L3 in the presence of antibody (Chandrashekar, Rao & Subrahmanyam, 1985). Eosinophils could not induce cytotoxicity to L3 comparable to the other two cell types tested in the present study. A similar observation has been made in the A. viteue-rat system where macrophages were required to kill the parasites (Haque et al., 1982). The authors reported that in vitro adherence of macrophages to A. viteae L3 required the simultaneous presence of eosinophils. However, in our study, both cell types were capable of independently adhering to the larvae. Neutrophils, macrophages and eosinophils are all known to possess surface receptors for the Fc piece of IgG (Wong & Wilson, 1975; Rabellino & Metcalf, 1975). Interestingly enough, we found macrophages to be the predominant cell type involved in in viva ADCC. The variety of cell types reported in studies ofcell-adherence to nematodes is at present puzzling; but probably indicates the existence of a number of m~ha~sms for parasite removal dependent upon the status of host-parasite interaction. Macrophages have been reported to be

729

involved in in vivo ADCC to L. curinii microfilariae (Nelson, Subr~~yam, Rao & Mehta, 1976) and to ~jpetalone~a setariosum adults (Woes & McLaren, 1982). Thus the available evidence strongly suggests that ADCC effector mechanisms may be of great importance in mediating immunity to L3. Mehta, Subrahmanyam & Sindhu (1981) reported that sonicated antigens of L3 of L. carinii could induce resistance to infection in albino rats. Thus these studies warrant intensive investigation on the isolation of functional antigens from the larval stages which confer such an effective resistance to filarial infection, for possible use as vaccines. REFERENT ABRAHAMD., WEINERD. J. & FARRELLJ. P. 1986. Protective responses of the jird to larval Dipetalonema viteae. Zmmunology57: 165-l 69. ASH L. R. & RILEYJ. M. 1970. Development of subperiodic Brugia maiayi in the jird, Meriones unguiculatus,with notes on infections in other rodents. Journal of Parasitology 56:

969-973. CHANDRASHEKAR R., RAo U. R., SUBRAHMANYAM D., HOPPER K., NELSOND. S. & KING M. 1984. Bruaia oahanai: serumdependent cell-mediated reactions to _sheathed and exsheathed microfilariae. Zmmunology53: 41 I-417. CHANDRASHEKAR R., RAOU. R., PARABP. 3. & SUBRAHMANVAM D. 1985. Brugia malayi: serum-dependent cell-mediated reactions to microfilariae. Southeast Asian Journal of Trolled Medicine and Public Health 16: 15-21. CHAND~SHE~R R., RAo U. R. & SUB~HMANYAMD. 1985. Serum-dependent cell-mediated reactions to Brugia pahangi infective larvae. Parasite Zmmunology7: 633-642. CHANDRASHEKAR R., RAo U. R., PARABP. 3.8 SUBRAHMANYAM D. 1986. Brugia malayi: rat cell-interactions with infective larvae mediated by complement. Experimental Parasitology

62: 362-369. HAQUE A., OUAISSI M. A., SANTOROF., DES MOUTIS I. & CAPRONA. 1982. Complement-mediated leukocyte adherence to infective larvae of Dipetabnema viteae: requirement of eosinophil or eosinophil products in effecting macrophage adherence. Journal of Immunology 129: 2219-2225. HIGASHIG. I. & CHOWDHURYA. B. 1970. Zn vitro adhesion of eosinophils to infective larvae of Wuchereria bancrofti. Zmm~ology 19: 65-83. HUDSON L. & HAY F. C. 1980. AfFinity chromato~aphy. In: Practical Zmmu~o~ogy,pp. 221-242. Blackwell Scientific Publications, Oxford. MEHTA K., SUBRAHMANYAM D. & SINDHU R. K. 1981. Immunogenicity of homogenates of the developmental stages of Litomosoides carinii in albino rats. Acta Tropica

38: 319-324. MEHTAK., SINDHUR. K., SUBRAHMANYAM D., HOPPERK. &

NELSOND. S. 1982. &E-dependent cellular adhesion and cytotoxicity to Litomosoides carinii microfilariae-nature of effector cells. Chnical and Experimental Immunology 48: 478484. NELSON D. S., SUBRAHMANYAM D., RAo Y. V. B. G. & MEHTA K. 1976. Cellular morphology in pleural exudate of albino rats infected with Litomosoides carinii. Transactions of the

Boyat Society of TropicalMedicine and Hygiene 70: 254255. W. F., MCGREEW P. B., RA~IWAYANTO S.,

PIESSENS

MCGREEVYM., P~E~SENSP. W., KOIMANI., SARASOJ. S. &

R. CHANDRASHEKAR, U. R. RAO and D.

730

DENNISD. T. 1980. Immune responses in human infections with Erugia malayi: co-relation of cellular and humoral reactions to microfilarial antigens with clinical status. American 563-570.

Journal of Tropical Medicine

and Hygiene 29:

RABELLINOE.

SUBRAHMANYAM

and Hygiene 76: 362-369. SUBRAHMANYAMD.,

RAOY. V. B. G., MEHTAK. & NEWN D. S. 1976. Serum-dependent adhesion and cytotoxicity of cells to Litomosoides carinii microfilariae. Nature (London) 260: 529-530. Wuss N. &TANNERM. 1979. Studies on Dipetalonema viteae (Filarioidea). 3. Antibody-dependent cell-mediated destruction of microfilariae in vivo. Trowwnedizin und

M. 8c METCALF D. 1975. Receptors for C3 and IgG on macrophage, neutrophil and eosinophil colony cells grown in vitro. Journal of Immunology 115:688-692. Parasitologic 30: 73-80. &A~IXA~H G. R., MONTEIRO Y. M., Nm~o A., DESHPANDE WONGL. & WILXINJ. D. 1975. The identification of Fc and L. & SUBRAHMANYAM D. 1989. Protective immune responses C3 receptors on human neutrophils. Journal of Immunowith trickle infections of the third stage tilarial larvae, logical Methodr 7: 69-76. Wuchereria bancrofti in mice. Clinical and Experimental WONGM. M. 1964. Studies on microtilaremia in dogs. II. Immunology 78: 292-298. Levels of microfilaremia in relation to immunologic SANGBRI., LXMMLER G. & KIMM~G P. 1981. Filarial infections responses of the host. American Journal of Tropical of Mastomys natalensis and their relevance for Medicine and Hygiene 13: 66-77. experimental chemotherapy. Acta Tropica 38: 277-288. WORMSM J. & MCLAREND. J. 1982. Macrophage-mediated SIM B. K. L., KWA B. H. & MAK J. W. 1982. Immune damage to fllarial worms (Dipetalonema setariosum) in vivo. responses in human Brugia malayi infections: serumJournal of Helminthology 56: 235-241. dependent cell-mediated destruction of infective larvae in vitro. Transactions of the Royal Society of Tropical Medicine