INFECTION AND IMMUNITY, Sept. 1989, p. 2771-2775 Vol. 57, No. 90019-9567/89/092771-05$02.00/0Copyright X3 1989, American Society for Microbiology

Effects of Gamma Interferon on Syntheses of DNA and Proteins byEntamoeba histolytica

CARLOS CASTELLANOS, CELSO RAMOS, AND LIBRADO ORTIZ-ORTIZ*Departamento de Inmunologia, Instituto de Investigaciones Biomedicas, Universidad Nacional Aut6noma de Mexico,

04510 Mexico City, D.F., Mexico

Received 21 February 1989/Accepted 6 June 1989

To define the participation of cell-mediated immunity in resistance to amebic infection through the action ofsoluble mediators or lymphokines (LKs), including gamma interferon (IFN--y), we studied their effect onEntamoeba histolytica. Supernatants from cultures of lymphoid cells, which had been stimulated in vitro withconcanavalin A and were rich in lymphokines (LRSNs), and recombinant IFN-y were used. LRSN andrecombinant IFN-y inhibited the growth of E. histolytica trophozoites in vitro. These LKs did not show acytotoxic effect on the ameba, but they did inhibit rather significantly protein and DNA syntheses of theprotozoa. Interestingly, LRSN incubated at 4°C in the presence of trophozoites lost the ability to inhibit thereplication of vesicular stomatitis virus. IFN-y inactivated at pH 2 had no effect on DNA synthesis by theameba, thus suggesting that IFN-y is responsible for the observed inhibition of parasite growth. Furthermore,the IFN-,y inhibitory effect was abolished by a monoclonal antibody specific for this LK. The results suggest thatIFN-y may participate in protection against amebiasis infection through the activity of mediators released bylymphocytes during infection.

The participation of cell-mediated immunity (CMI) duringinfection by Entamoeba histolytica has been shown both invitro and in vivo (13). However, its role as a mechanism ofresistance to amebic infection has not been demonstrated.Numerous studies have been carried out in which it wasobserved that treatment of experimental animals with immu-nosuppressive drugs or extirpation of their primary or sec-ondary lymphoid organs reduced the resistance of the host toamebic infection in all cases (1, 5, 6, 22). In an attempt todefine the role of CMI in amebiasis, direct cytotoxicitystudies have been done by various workers. Such studieshave shown that when the trophozoites of E. histolyticawere confronted with peripheral lymphocytes from patientsin the early stages of amebic liver abscess, the amebasphagocytized the lymphocytes, whereas lymphocytes frompatients recovering from the abscess killed the amebas (9).Similar results were obtained in experiments carried out inhamsters. In these studies, the immune systems of animalsinoculated with ameba antigen were able to destroy the E.histolytica trophozoites, whereas infected animals were un-able to mount a significant cytotoxic effect on the parasite(7).

It has recently been reported that, upon direct contact,activated human macrophages and cytotoxic T lymphocytesexercise a significant lethal effect on E. histolytica (17, 19). Ithas also been observed both in vitro and in vivo thatperitoneal exudate cells and lymphocytes from the mesen-teric lymphatic ganglia, either from gerbils with cecal ame-biasis or from those immunized with amebic extract, exer-cise a greater cytopathic effect on nonpathogenic amebastrains than on pathogenic strains (2). It is possible that CMIparticipates in resistance to amebic infection through theaction of soluble mediators or lymphokines (LKs) becausethe cytotoxicity mediated by T lymphocytes is geneticallyrestricted (24).With this aim, we examined the effect on E. histolytica of

said LKs, particularly gamma interferon (IFN--y), which has

* Corresponding author.

been implicated in the mechanisms of resistance to otherparasites (10, 11). In this study, supernatants (SNs) fromcultures of lymphoid cells which had been stimulated in vitrowith concanavalin A (ConA), a lectin known to favor theformation of these mediators (16, 23), and recombinantIFN--y were used. The results indicate that IFN--y has aninhibitory effect on the syntheses ofDNA and proteins by E.histolytica.

MATERIALS AND METHODS

Animals. Male BALB/c mice 6 to 8 weeks of age from acolony maintained at the Instituto de Investigaciones Bio-mddicas, Universidad Nacional Aut6noma de Mdxico wereused.

E. histolytica. Seventy-two-hour cultures of E. histolyticaHM1:IMSS were used throughout. This strain was main-tained in axenic culture medium (3).

Cell preparation. Cell suspensions were prepared by minc-ing the spleen in Hanks balanced salt solution and filteringthe resulting mixtures through nylon mesh at 4°C. The cellfiltrate was centrifuged at 200 x g, and the packed cells weresuspended in culture medium consisting of RPMI 1640(GIBCO Laboratories, Grand Island, N.Y.) supplementedwith 10% heat-inactivated fetal calf serum, 5 x 10-5 M2-mercaptoethanol, 1 mM glutamine, 100 U of penicillin perml, and 100 ,ug of streptomycin per ml. Cell viability wasdetermined by the trypan blue exclusion test and cell numberwas determined by hemacytometer count (15).

Preparation of LRSN. We used the SNs from cultures ofspleen cells which had been stimulated with ConA, asdescribed previously (15), as the source of LK-rich SN(LRSN). In brief, spleen cells from normal mice werecultured at a density of 5 x 106 cells per ml in the presenceof ConA (10 jig/ml) in supplemented RPMI 1640. After thecells had been incubated for 48 h at 37°C in humidified 5%CO, the cultures were centrifuged at 10,000 x g for 20 min.To the resulting SNs was added 0.1 M methyl mannoside.

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Cell-free SNs were frozen at -20°C until used. Assays todetermine lymphocyte proliferation (15) and inhibition ofvesicular stomatitis virus plaque formation in Vero cells (4)were run in parallel to quantitate the ConA stimulatingactivity. Only those LRSNs which had been obtained fromcells responding to ConA under the conditions previouslydescribed (15) and which had an inhibitory effect on viralplaque formation were used. Inhibition of plaque formationby undiluted LRSN was equivalent to 1,000 U of IFN-y(DNAX Research Institute, Palo Alto, Calif.) per ml. Con-trol SN consisted of the SNs obtained from spleen cellcultures which had not been stimulated with ConA butotherwise similarly processed; it will hereafter be referred toas SCCSN.Recombinant IFN-y. IFN-y was obtained through the

generosity of Albert Zlotnik of DNAX Research Institute. Ithad been produced by transfection of murine IFN--y cDNAinto Cos 7 monkey kidney cells. Its activity was confirmed asdescribed previously (4). Control SN consisted of the SNsobtained from mock-transfected Cos 7 monkey kidney cellsand will hereafter be referred to as MKCSN.

Incubation at pH 2. LRSN or IFN-y was dialyzed for 24 hagainst 0.1 M glycine-HCl-0.15 M NaCl buffer at pH 2.0,followed by dialysis for 48 h against phosphate-bufferedsaline (PBS) at pH 7.2 and a final dialysis against completemedium. Control SN was dialyzed against PBS and completemedium (25).

Effect of LRSN or IFN-y on E. histolytica trophozoites. Toexamine the effect of LRSN or IFN-y on cultures of E.histolytica trophozoites, 4 ml of undiluted LRSN, 0.2 ml ofdifferent numbers of IFN--y units, or the appropriate SCCSNor MKCSN control was added to tubes containing variousconcentrations of trophozoites in axenic culture medium, asindicated in each experiment. In all cases, the final volumewas 13 ml. The tubes were incubated at 37°C, and theviability and number of trophozoites were determined atdifferent intervals, as described elsewhere (14).The effect of the LKs on the synthesis of ameba proteins

was determined by adding 0.5 mCi of [3H]leucine (142Ci/mmol; Amersham, Amersham, United Kingdom) to thecultures 12 h before harvesting the trophozoites. Trophozo-ites were harvested at certain intervals as indicated in eachexperiment and were washed three times with PBS at pH 7.2by centrifugation at 200 x g for 10 min. The pellet wassuspended in PBS containing 5 mM phenylmethylsulfonylfluoride (Sigma Chemical Co., St. Louis, Mo.), 5 mM methylmaleimide (Sigma), and 1 mM EDTA (disodium salt). Thesuspension was frozen and thawed five times, and a 100-,lIportion was treated with 0.5 ml of 25% trichloroacetic acid,followed by 0.5 ml of Casamino Acids (Difco Laboratories,Detroit, Mich.). The mixture was incubated at 4°C after 30min, filtered through a glass-fiber filter, dried, and counted ina liquid scintillation counter (12).The effect of the LKs on the synthesis of ameba DNA was

determined by adding 14 jiCi of [3Hlthymidine ([3H]TdR;20.0 Ci/mmol; Dupont, NEN Research Products, Boston,Mass.) to the cultures 18 h before harvesting the trophozo-ites. Trophozoites were harvested at certain intervals, asindicated in each case, with a cell harvester (Brandell). Theglass-fiber beads were counted in a liquid scintillationcounter (21).

Absorption of IFN-y by E. histolytica trophozoites. Absorp-tion studies of IFN-,y by E. histolytica trophozoites wereperformed by incubating 4,000 U of IFN--y and 2 x 105trophozoites at 4°C for 60 min. Afterward, the mixture wascentrifuged at 200 x g for 5 min and the SN was tested for

IFN--y activity. This activity was measured in both absorbedand nonabsorbed materials by inhibition of vesicular stoma-titis virus plaque formation in Vero cells as describedelsewhere (4).

Inhibition of IFN-,y activity on E. histolytica trophozoites bya monoclonal antibody specific to IFN-,y. A monoclonalantibody specific for IFN-y (20), kindly provided by RobertD. Schreiber, Department of Pathology, Washington Uni-versity School of Medicine, St. Louis, Mo., was used tofurther delineate the role of IFN-y on the inhibition of DNAsynthesis by E. histolytica. For this purpose, undilutedLRSN (4 ml) or IFN--y (4,000 U, 0.2 ml) was mixed with themonoclonal anti-IFN-y (150 p.g/60 [lA) and incubated for 60min at 37°C. Afterward, the effect of such antibody-treatedLKs on DNA synthesis by the trophozoite (100,000 tropho-zoites in axenic culture medium in a final volume of 13 ml)was measured by [3H]TdR incorporation as describedabove.

IL-2 activity assay. The assay method used was thatdescribed by Gillis et al. (8). In brief, a cytotoxic T-cell lineobtained from one C57BL/6 mouse spleen was maintained inculture for long periods through the use of T-cell growthfactor (interleukin-2 [IL-2J) (8). These cells were washed inbalanced salt solution, suspended in 0.1 ml of supplementedRPMI 1640 to a concentration of 104 cells, and added to eachwell of a 96-well microplate (model 3596; Costar, Cam-bridge, Mass.). Serial dilutions of the SNs to be tested (0.1ml) from the ConA-stimulated spleen cell cultures wereadded to these cells with alpha methyl mannoside at a finalconcentration of 0.1 M and incubated for 24 h under cultureconditions. Thereafter, each well was pulsed with [3H]TdRat 1 p.Ci/p.mol (specific activity, 6.7 Ci/[Lmol; Dupont). After18 h of incubation, the cells were harvested and the incor-poration of [3H]TdR was measured in a beta counter(Packard Instrument Co., Inc., Rockville, Md.).

RESULTSEffect of LRSN or IFN--y on the viability and growth of E.

histolytica. Culturing trophozoites with either LRSN or IFN-y for different periods of time did not have any effect on theviability of the trophozoites. Viability was always greaterthan 90%. However, after 72 h of incubation, the number ofamebas containing undiluted LRSN or 4,000 U of IFN-yshowed a significant decrease (P < 0.05) compared with thatobserved for ameba growth in axenic culture medium (Fig.1). Growth of the ameba in SN control groups was notdifferent from that observed in the axenic medium. Theamount of IFN-y tested was 4,000 U, since preliminaryexperiments showed this dose was the minimal concentra-tion that inhibits growth. On the other hand, volumes ofLRSN greater than 4 ml added to axenic culture mediumdramatically affected growth of the parasite (data notshown). Therefore, in subsequent experiments, only undi-luted LRSN (4 ml) and 4,000 U of IFN-y were used.

Effect of LRSN or IFN-,y on protein synthesis by E. histolyt-ica. Experiments performed to determine the effect of LRSNon protein synthesis by E. histolytica trophozoites indicatedthat after 48 h of culture, LRSN significantly inhibited thelevels of protein synthesis (P < 0.005), whereas controlSCCSN did not. This effect was observed throughout theexperiments (Fig. 2A). In the same manner, IFN-y signifi-cantly inhibited protein synthesis by the ameba (P < 0.01)(Fig. 2B). On the other hand, the MKCSN control group hadno effect on protein synthesis by the ameba.

Effect of LRSN or IFN--y on DNA synthesis byE. histolytica.When E. histolytica trophozoites were cultured in the pres-

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100

0

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24 48 72

HOURS OF INCUBATIONFIG. 1. Effect of LRSN or IFN-y on the growth of E. histolytica

trophozoites. Four milliliters of undiluted LRSN (0) or 4.000 U ofIFN-y (A) was added to tubes containing 105 amebas in a finalvolume of 13 ml. The cultures were incubated at 37°C. and thenumber of amebas was determined. The results show that the LKssignificantly decreased the number of amebas compared with thoseobserved in either axenic culture medium (0). SCCSN (O). or

MKCSN (E). Each value represents the mean standard error of11 experiments. The P value was determined by the Student t test.

ence of LRSN, it was observed that DNA synthesis was

altered. Thus, after 48 h of culture in the presence of LRSN.DNA synthesis began to diminish to statistically significantlevels (P < 0.02) which were maintained after 72 h of culture.Control SCCSN did not show any effect on DNA synthesisin ameba cultures (Fig. 2A). When IFN-y was used in similarexperiments, the same significant decrease in DNA synthe-sis was observed after 48 and 72 h of culture (P < 0.01) whencompared with its MKCSN control (Fig. 2B).

Effect of pH on the inhibition of DNA synthesis by LRSN or

IFN--y. To study the effect that pH had on the inhibition ofDNA synthesis by LRSN, the LRSN was adjusted to pH 2for 24 h and was thereafter readjusted to pH 7.0, as de-scribed above. Four milliliters of the LRSN thus treated wasincubated in the presence of E. histolyticca trophozoites (seeabove). The LRSNs which had been maintained at pH 2.0for 24 h lost their capacity to inhibit DNA synthesis in E.histolytica trophozoites (Table 1). This effect was not ob-served in the similarly treated control SCCSN groups; thesegroups showed normal growth of amebas compared withtrophozoite growth in ameba medium alone.

Since LRSN lost its activity when adjusted to pH 2.0, itwas of interest to determine whether the same treatment hadany effect on the inhibition of DNA synthesis induced byIFN-y. IFN-y (4,000 U, 0.2 ml) incubated with E. histolvticatrophozoites (see above) for 48 and 72 h inhibited DNAsynthesis. This effect was lost when the IFN-y had beenincubated for 24 h at pH 2.0 (Table 2). DNA synthesis ofamebas in control groups in which the MKCSN medium was

used was not inhibited. In a previous experiment, we foundthat IFN--y similarly incubated for 24 h but maintained at pH7.2 did not lose its capacity to inhibit the growth of E.

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HOURS OF INCUBATIONFIG. 2. Effect of LRSN or IFN-y on protein and DNA syntheses

by E. Iiistolvytica trophozoites. (A) Effect of LRSN (0) or its SNcontrol (0) on protein (----) or DNA (-) synthesis was determinedby measuring the incorporation of [3H]leucine or [3H]TdR. respec-tively. by E. hiistolytica trophozoites as described in Materials andMethods. (B) Effect of IFN-y (0) or its MKCSN control (0) on

protein (----) or DNA (-) synthesis was studied similarly. The figureshows that either LRSN or IFN-y inhibited the levels of protein andDNA syntheses after 48 h of culture. Each value represents themean standard error of five experiments for 13H]leucine incorpo-ration; each value represents the mean + standard error of sixexperiments for [3H]TdR incorporation.

Ihistolwia after dialysis; its activity on the amebas was

similar to that of the untreated IFN--y (data not shown).Absorption of IFN--y by E. histolytica trophozoites. Absorp-

tion studies were done to determine whether E. histoltwi(trophozoites contain a receptorlike activity that enablesthem to interact with IFN-y. The results showed that thetrophozoites were able to absorb IFN-y activity from bothLRSN and recombinant IFN-y, as judged by the inability ofsuch absorbed material to inhibit the growth of vesicularstomatitis virus which is sensitive to IFN-y (Table 3).To test the specificity of the absorption procedure, an-

other LK known to be present in LRSN, namely, IL-2, was

assessed in the absorbed LRSN. The results indicated that

TABLE 1. Effect of pH on DNA synthesis by LRSN"

Cul- l3HITdR incorporation (cpm/ml) by E. histolvtica trophozoitesture pH 2 incubation

time Control LRSN

(h) SN Control SN LRSN

48 7.175 1,195 3.805 + 240 5,700 + 455 6,315 + 1.17072 12.000 + 255 7,500 460 11,505 1,265 12,320 + 1,265

"The effect of LRSN (4 ml) on the synthesis of ameba DNA was

determined by adding 14 LCi of (3H]TdR (20.0)0 Ci/mmol) to the culture(54.000 trophozoites per 9 ml of medium) 18 h before harvesting the tropho-zoites. Trophozoites were harvested at 48 and 72 h with a cell harvester(Brandel). The glass-fiber beads were counted in a liquid scintillation counter.Each value represents the mean + standard error of five experiments. The Pvalues for the differences in the means of LRSN and control SN are P < 0.02and P < 0.01 for trophozoites cultured for 48 and 72 h, respectively; whenLRSN and control SN were incubated at pH 2. the means were notstatistically significantly different at both culture times.

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TABLE 2. Effect of pH on the inhibition of DNAsynthesis by IFN--y"

Cul- [3H]TdR incorporation (cpm/ml) by E. histolytica trophozoitesture pH 2 incubationtime Control IFN-Hy(h) SN Control SN IFN--y

48 17,690 ± 542 10,513 + 362 21,100 ± 198 19,500 ± 25672 36,100 ± 710 29,446 + 1,998 29,950 ± 550 34,360 ± 626

a The effect of IFN--y (0.2 ml) on the synthesis of ameba DNA wasdetermined by adding 14 ,uCi of [3H]TdR (20.0 Ci/mmol) to the cultures(76,800 trophozoites per 12.8 ml of medium), 18 h before harvesting thetrophozoites. They were processed as described in footnote a of Table 1. Eachvalue represents the mean + standard error of five experiments. The P valuesfor the differences in the means of IFNy and control SN are P < 0.01 fortrophozoites cultured for 48 and 72 h; when IFN-y and control SN wereincubated at pH 2, the means were not statistically significantly different atboth culture times.

IL-2 activity was similar in both the absorbed (41,959 ±11,255 cpm/20 ,lI) and nonabsorbed (42,216 + 2,940 cpm/20jil) LRSN, as evidenced by the similar results in the incor-poration of [3H]TdR by an IL-2-dependent T-cell line. Theseresults further support the ability of E. histolytica trophozo-ites to interact and inactivate IFN-y specifically.

Abrogation of IFN-,y activity on E. histolytica trophozoitesby a monoclonal antibody specific to IFN--y. The effect of amonoclonal antibody specific to IFN--y on the inhibition ofDNA synthesis achieved by LRSN or IFN-,y on E. histolyt-ica trophozoites was evaluated. Thus when either LRSN orIFN--y was treated with the monoclonal anti-IFN--y, itsability to inhibit DNA synthesis was abolished. EitherLRSN or IFN--y inhibited the [3H]TdR incorporation by theamebas (Fig. 3). However, when the LKs were previouslytreated with monoclonal anti-IFN--y, the capacity to inhibitDNA synthesis was abrogated.

DISCUSSIONThe results reported herein indicate that lymphoid cells

can be stimulated nonspecifically by ConA, resulting in theliberation of soluble factors or LKs that inhibit the growth oftrophozoites of E. histolytica. The controls showed thatinhibition of growth was not due to the consumption ofnutrients in the culture medium. This inhibition of amebagrowth could not be attributed to lysis of the protozoathrough the action of soluble toxic factors because reducedviability of the parasite throughout the course of the exper-iment was not observed, in agreement with previous reports(18). The data suggest that LRSN, particularly IFN--y, inter-fered with the metabolism of the ameba, as was demon-

TABLE 3. Inhibition of viral plaque formation by LRSN orIFN-y previously absorbed with E. histolytica trophozoites"

Material Plaque formation (%)assayed Nonabsorbed Absorbed

SN control 100.0 100.0LRSN 16.1 111.0SN control 100.0 100.0IFN-y 10.2 107.1

a LRSN (1 ml) or IFN--y (1,000 U) was absorbed with 2 x 105 E. histolyticatrophozoites at 4'C for 60 min. IFN-y activity was measured in both absorbedand nonabsorbed materials by inhibition of vesicular stomatitis virus-inducedcytopathic effect in Vero cells. Monolayers were challenged with 104 viralparticles. The inhibition in viral plaque formation induced by the nonabsorbedLKs was significantly different from that obtained with the control SNs (P <0.05). On the other hand, the absorbed LKs did not inhibit viral plaqueformation. Each value represents the mean of three experiments.

Group kica Ction SN LK2 Antitiwnelir) Control IFN-f

2

3

48 + -

_ IFN-7- IFN-7'

4 72

5

6

7

89

72

+ 3-

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- IFN-7 +

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- LRSN +

0 4 8 12 I

3H-TdR INCORPORATION (cpm, 10 3/mI)

FIG. 3. Effect of a monoclonal antibody specific to IFN--y on theinhibition of DNA synthesis achieved by LRSN or IFN-y on E.histolytica trophozoites. E. histolytica trophozoites incorporated[3H]TdR when grown in the presence of either SCCSN or MKCSNcontrols (groups 1, 4, and 7). However, IFN--y (groups 2 and 5) orLRSN (group 8) inhibited [3H]TdR incorporation by the amebas.Nevertheless, when IFN-y (groups 3 and 6) or LRSN (group 9) werepreviously treated with anti-IFN--y, their ability to inhibit DNAsynthesis was abolished. Each value represents the mean ± stan-dard error of four experiments.

strated by the significant reduction in the incorporation of[3H]leucine and [3H]TdR. IFN-y was shown to be involvedin this interference since, when LRSN or IFN-y itself wasincubated at pH 2.0 for 24 h, the inhibitory effect wasabrogated. Furthermore, that IFN--y was responsible for theinhibition of DNA synthesis is strongly supported by theabrogation of such activity by means of the monoclonalantibody specific for this LK.The effect of the LKs, particularly IFN--y, was manifested

at 48 h (Fig. 2). It should be noted that the effects of IFN--yon the syntheses of proteins and DNA did significantlyreduce the number of amebas in this period. At 72 h ofculture, the alteration observed in the syntheses of proteinsand DNA induced by both LKs brought about a reduction inthe number of amebas. It is possible that this effect on thesynthesis of DNA may be a consequence of the inhibition ofproteins necessary for cell division.The absorption of IFN--y by E. histolytica trophozoites

suggests that the parasite possessed a specific recognitionmechanism for IFN--y, possibly mediated by a receptorlikemolecule. This statement is further supported by the factthat the same procedure did not modify the presence in theLRSN of another LK, namely, IL-2. Work is in progress todetermine whether absorption of IFN--y by the ameba is dueto the presence of a specific receptor.The participation of CMI as a mechanism of resistance in

infections caused by E. histolytica has been documented.Thus, there have been reports that macrophages activatedwith LKs exercise a cytotoxic effect on trophozoites of E.histolytica that is a contact-dependent, antibody-indepen-dent mechanism, involving oxidative and nonoxidative pro-cesses (19). It has further been reported that the lympho-cytes of patients who recovered from amebic liver abscesswere cytotoxic to amebic trophozoites, possibly through asecreted cell product (9). In a later report, it was shown thatcytotoxic T lymphocytes, generated by incubation of Tlymphocytes from normal healthy individuals in the pres-ence of phytohemagglutinin, could kill virulent amebas. Theeffector cells responsible for the observed cytotoxicity car-ried the CD8 phenotype (J. G. Cox, R. A. Salata, and J. I.

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Ravdin, Clin. Res. 32:365A, 1984). Furthermore, T lympho-cytes from patients cured of amebic liver abscess also killedamebas but only when activated with amebic protein, indi-cating that an antigen-specific activation of cytotoxic activityof T lymphocytes from patients occurs in invasive amebiasis(17). These studies indicate that CMI response with demon-strable afferent and efferent expressions may represent, inthe long run, the most important defense mechanism of thehost against infection by E. histolytica.

In 1976, Guerrero (9) observed that SNs from lympho-cytes obtained from individuals who had been cured ofamebic liver abscess had a cytopathogenic effect on amebas.He suggested that substances liberated by lymphocytescould be cytotoxic for amebas and therefore involved inresistance through a CMI mechanism. Our results indicatethe involvement of substances distinct from those reportedby Guerrero, since under our experimental conditions, wedid not observe toxic effects which altered the viability of theameba. It is interesting that this finding indicates that a directcontact between lymphocytes and the ameba is not neces-sary to inhibit growth of the protozoa. This characteristicmay allow the lymphocyte to bias the genetic restrictionimposed in T-cell-mediated cytotoxicity phenomena (24).

IFN--y has been shown to be useful in other systems, forexample, in the inhibition of simian malaria. IFN-y com-pletely suppressed experimental infection with Plasmodiuimcynomologi B sporozoites in rhesus monkeys. Interestingly,this treatment had no protective effect against trophozoite-induced infections, suggesting that the IFN--y effect waslimited to the exoerythrocytic stage of parasitic develop-ment. At the present time, we are doing in vivo experimentsto determine if IFN-y can protect against intrahepatic chal-lenge with E. histolytica in the hamster model.The function of IFN--y described herein appears indepen-

dent of their well-known activating effect on macrophageswhich, once activated, are capable of killing amebas inaxenic culture. This report, to our knowledge, is the first toindicate that the products liberated by spleen cells, espe-cially IFN--y, exercise an inhibitory effect on the synthesesof proteins and DNA by a parasite, in this case amebas,thereby suggesting that IFN--y is involved in the mechanismof resistance to infection by E. histolytica.

ACKNOWLEDGMENTS

This work was supported in part by grant PCSABNA-020830 fromCONACYT and by a grant from the Agency for InternationalDevelopment.We thank Janice Frankowski Pawlak and Esperanza Ruiz-Garcia

for the preparation of this manuscript.

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2. Chadee, K., E. Meerovith, and F. Moreau. 1985. In vitro and invivo interaction between trophozoites of Entamoeba histolyticaand gerbil lymphoid cells. Infect. Immun. 49:828-832.

3. Diamond, L. S., D. R. Harlow, and C. C. Cunnuck. 1978. A newmedium for the axenic cultivation of Entamoeba histolytica andother Entamoeba. Trans. R. Soc. Trop. Med. Hyg. 72:431-432.

4. Friedman, R. M., R. K. Maheshwari, F. T. Jay, and C. Czarn-iecki. 1980. Mechanism of interferon inhibition of viruses thatbud from the plasma membrane. Ann. N.Y. Acad. Sci. 350:533-544.

5. Ghadirian, E., and E. Meerovitch. 1981. Effect of immunosup-pression on the size and metastasis of amebic liver abscess inhamsters. Parasite Immunol. 3:329-338.

6. Ghadirian, E., and E. Meerovitch. 1981. Effect of splenectomyon the size of amoebic liver abscess and metastatic foci inhamsters. Infect. Immun. 31:571-573.

7. Ghadirian, E., and E. Meerovitch. 1982. In vitro amoebicidalactivity of immune cells. Infect. Immun. 36:243-246.

8. Gillis, S., M. M. Ferm, W. Ou, and K. A. Smith. 1978. Cellgrowth factor: parameters of production and quantitative mi-croassay for activity. J. Immunol. 120:2027-2032.

9. Guerrero, M., D. Rios, and L. Landa. 1976. Interaction betweentrophozoites of Entamoeba histolytica and lymphocytes ofpatients with invasive amebiasis, p. 529-539. In B. Sepulvedaand L. S. Diamond (ed.), Proceedings of the InternationalConference on Amebiasis. Instituto Mexicano del Seguro So-cial, Mexico City.

10. Maheshwari, R. K., C. W. Czarniecki, G. P. Dutta, S. K. Puri,B. N. Dhawan, and R. M. Friedman. 1986. Recombinant humangamma interferon inhibits simian malaria. Infect. Immun. 53:628-630.

11. McCabe, R. E., B. J. Luft, and J. S. Remington. 1984. Effect ofmurine interferon gamma on murine toxoplasmosis. J. Infect.Dis. 150:961-962.

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