R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d i c i n a T r o p i c a l 2 0 ( 4 ) : 1 9 3 - 1 9 8 , O u t - D e z , 1 9 8 7
A R T I G O S
E F F E C T S O F L E V A M I S O L E O N E X P E R I M E N T A L I N F E C T I O N S B Y
P L A S M O D I U M B E R G H E I I N M I C E
En r i q u e M e l e n d e z C. 1 e A n t o n i a n a U. Kr e t t li2
L e v a m i s o l e ( p h e n y l i m i d o t h i a z o l ) , c o n s i d e r e d a s tr o n g im m u n o s ti m u la n t, w h e n a d m i n i s t e r e d to h e a lt h y S w i s s m i c e d i d n o t c a u s e a s i g n i f i c a n t in c r e a s e in -the w e ig h t o f th e i r th y m u s , l i v e r a n d s p le e n , e v e n th o u g h th e d r u g w a s u s e d a t d i f f e r e n t ti m e s b e f o r e r e m o v i n g s u c h o r g a n s . H i g h d o s e s o f d r u g u s e d in th e 4 - d a y p r o p h y l a c t i c s c h e m e h a d n o a n t i m a l a r i a l e ffe c t. H o w e v e r , w h e n g i v e n to m a l a r i a in f e c te d m ic e 2 4 h o u r s b e fo r e , a t th e s a m e tim e , a n d 2 4 h o u r s a f t e r th e in o c u la t i o n o f a c h lo r o q u i n e - s e n s it i v e o r a c h lo r o q u i- n e - r e s is ta n t s t r a i n o/PIasm odium berghei s m a l l d o s e s o f th e d r u g in d u c e d a s o m e w h a t d e c r e a s e d p a r a s i t e m i a , th e d o s e o f 1 m g / k g b o d y w e i g h t b e f o r e th e in o c u lu m b e in g th e b e s t s c h e m e . T h e m o r t a l i t y r a t e s b y m a l a r i a in th e l e v a m i s o l e t r e a t e d g r o u p s w e r e a l s o d e l a y e d a l t h o u g h a l l m ic e f i n a l l y d ie d . T h e d a t a s u g g e s t t h a t l e v a m i s o l e m a y d i s p l a y a s t i m u l a n t e f f e c t o n th e d e p r e s s e d im m u n e r e s p o n s e c a u s e d b y m a la r ia .
K ey words: Malaria. P l a s m o d i u m b e r g h e i . Chloroquine-resistant strain. Leva- misole. Immunostimulation.
M alaria induced by P . b e r g h e i in rodents or by other plasmodia, including those in human species, decreases the immune response o f infected vertebrates against unrelated antigens and causes an increased susceptibility to concomitant infections (Review in 6). Experimental studies have demonstrated a reduction in the B lymphocytes and the total number of cells in peripheral lymph nodes of mice infected with P . b e r g h e i as well as a substancial decrease of thymus weight and rates of thymus T cells7. Furthermore, a decreased number of circulating lymphocytes in chil dren with malaria has been observed313. A progressive infiltration of plasma cells in periarteriolar areas of the spleen of infected mice was shown during the immu- nosupression period9. Recent studies also show that the lymphocyte response to purified P l a s m o d i u m f a l c i p a r u m antigen during acute malaria caused by
this parasite is significantly reduced. However, the res ponse to other unrelated mitogens (lectins) or to tuber culin antigen was not suppressed by malaria1. Some drugs and biological agents have been used as
immu-1. Sección de Parasitología, Escuela de Medicina, Univer- dad Centro Occidental “ Lisandro A lvarado” , A partado 400, Barquisimeto, Venezuela.
2. Departam ento de Parasitología, ICB, Universidade F e deral de M inas Gerais and Centro de Pesquisas René Rachou, F IO C R U Z , Belo Horizonte, M G , Brasil. Reprint requests to A .U . Krettli. Av. Augusto de Lima 1715. 30 190 Belo Horizonte, M G , Brazil.
P art of this work was presented at the Congress of Para sitology, Equador, 1985. W ork supported by CNPq-Brazil.
Recebido para publicação em 3/11/86.
nostimulants, such as cyclophosphamide, which may give the animal some protection against malaria2.
Levamisole is also considered an immunopoten- tiating agent although the drug was originally used for its therapeutic value against helminths (filarias) and some protozoal infections (leishmaniasis) (Review in 11). The improvement o f the immune functions by the drug has been attributed to the enhancement o f cell activity and to an increased number of total T cells when they were depleted. Since the effectiveness of many prophylactic and therapeutic drags can be in creased by the host immune response11 and since m alaria is immunosuppressive, we have investigated the immunopotentiating activity of levamisole in mala ria. In parallel, we have also determined a possible change in the total weight of the thymus, spleen and liver, organs involved in the immune response, after treatment of normal mice with levamisole.
M A T E R IA L A N D M ETH O D S
P a r a s i t e s - Parasites o f the erythrocytic cycle of P . b e r g h e i N K 6 5 14 sensitive to chloroquine, and, a chloroquine-resistant strain (CR) were maintained by weekly blood passages in female Swiss mice with 18 to 20 grams weight and a standard inoculum. The C R strain was drug-induced in the laboratory5 and frozen at - 70°C for 6 years before use. I t was then kept in mice under chloroquine action to maintain its resistan ce to the drug.
M e l e n d e z H , K r e t t l i A . E f f e c t s o f l e v a m i s o l e o n e x p e r i m e n t a l i n f e c t i o n s b y Plasmodium berghei i n m i c e . R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d i c i n a T r o p i c a l 2 0 : 1 9 3 - 1 9 8 , O u t - D e z , 1 9 8 7
stained blood smears and the total number of red blood cells (RBC) in a N eubauer chamber. After the desired dilution in sodium citrate 107 parasitized RBC were given to each animal by intraperitoaeal route (i.p.). Parasitemia was determined by blood smears every 48 hours until the animal death. M ortality was daily
monitored. J
A d m i n i s t r a t i o n o f l e v a m i s o l e - Levamisole § hydrochloride, a imidothiazolic derivate kindly pro- 1 vided by Johnson & Johnson Laboratories (Campinas,
São Paulo), was dissolved in distilled water to give 0.25 ml per mice p.o. Groups of 6 or 7 animals inocu lated with/*, b e r g h e i received levamisole in the doses of 0.1 , 1 or 1 0 mg/kg of body weight, either as a single dose or on 2 consecutive days according to the fol lowing scheme: a) 24 hours before the inoculum; b) concomitant to the inoculum; c) 24 hours after ino culation. The two criteria used to evaluate the immu- nostimulant effect of the drug were parasitemia curves and mortality in both control untreated and experimen tal groups.
W ith the P . b e r g h e i CR strain a dosage of 1 mg/kg per 1 or 2 consecutive days before the inoculum was used.
L iv e r , s p le e n , a n d th y m u s w e ig h t a f t e r le v a m i s o l e - Groups of 3 o r 4 treated or untreated normal mice were used to evaluate the effect oflevamisole on the weight of the lymphoid organs; 0.1 , 1 or 1 0 mg/kg were used in either a single dose or on 2 consecutive days. Animals were sacrificed at 2 4 ,4 8 and 72 hours after the drug administration according to each sche me. Each animal was anesthetized with sulfuric ether and total bleeding was done sectioning the vascular axilar plexus. Then their spleens, livers and thymuses were carefully removed and immediately weighed on a Mettler precision balance (Mo-H31AR).
C h e m o th e r a p e u ti c a c t i v i t y o f l e v a m is o l e
a g a i n s t P . b e r g h e i - To determine a possible chemo therapeutic activity of levamisole, groups of 7 mice inoculated i.p. with 107P . b e r g h e i parasitized RBC were treated with 4 consecutive daily doses of 25, 50, 100 and 150 mg/kg. Blood smears were examined on the 5 th and 7 th days after inoculation, in both control and treated groups, to determine the rate of parasite mia. M ortality was daily monitored.
S t a t i s t i c a n a l y s i s - Student’s “ t” test was used to determine the significance of the differences among the means in the liver, spleen and thymus weights; a X2 test was used to determine the significance of the differences among parasitemia means.
RESU LTS
T h e r a p e u t ic a c t i v i t y o f l e v a m i s o l e - There was no differences in parasitemia by-P. b e r g h e i in levami sole treated or untreated groups (Fig. 1). M ortality
50 .
40
0 25 50 100 150
DOSES OF LEVAMISOLE ( m g/ k g) DAILY
F i g u r e 1 - Parasitem ia rates in mice on days 5 ( D ) and 7 ( D ) after i.p. inoculation with P l a s m o d i u m b e r g h e i
(101 infected RBC per mouse) and treatment, by oral route, with different doses of levamisole, during 4 consecutive days, beginning 24 hours after parasite inoculation. Vertical lines indicate the standard error of the mean (n = 6). There was no statistical difference between parasitemia in control and treated groups.
rates of 1 0 0% were observed on all groups by day 1 0 after inoculation. Therefore, levamisole was ineffecti ve against P . b e r g h e i.
L e v a m i s o l e e f f e c t o n l y m p h o i d o r g a n s o f n o r m a l m ic e - W hen either a single dose oflevamisole or two of 0.1, 1 or 1 0 mg/kg were given to normal mice their thymus and spleen weights were in general above the normal values although only with the higher dose of drug, given 48 or 72 hours before, this increased size was more clearly detected. However, the differences were not statistically significant on either the liver, spleen or thymus weights during the treatm ent(Fig. 2). Since there were rather large individual variations with some of the animals this might have affected the mean of the groups.
M e l e n d e z H , K r e t t l i A . E f f e c t s o f l e v a m i s o l e o n e x p e r i m e n t a l i n f e c t i o n s b y Plasmodium berghei i n m i c e . R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d i c i n a T r o p i c a l 2 0 : 1 9 3 - 1 9 8 , O u t - D e z , 1 9 8 7
F i g u r e 2 - M eans and standard deviations o f thymus, liver and spleen weights o f normal untreated mice ( □ ) or mice previously treated with le vamisole by oral route in different doses, given 24 ( O ), 48 ( • ) or 72 hours (A ) before, in two consecutive days. There was no statistical difference between con trol and treated mice.
DAYS AFT ER INOCULAT ION O F R b e r g h e i
F i g u r e 3 - M ean parasitemia by P l a s m o d i u m b e r g h e i (strain N K 65) in groups of mice inoculated by intraperi- toneal route with 10? parasitized RBC. In solid lines are the groups treated with different doses (mg/kg) oflevamisole ( • = 0.1; A = 1.0; ▲ = 10) given once 24 hours before inoculation and in dotted line the untreated mice.
M e l e n d e z H , K r e t t l i A . E f f e c t s o f l e v a m i s o l e o n e x p e r i m e n t a l i n f e c t i o n s b y Plasmodium berghei i n m i c e . R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d i c i n a T r o p i c a l 2 0 : 1 9 3 - 1 9 8 , O u t - D e z , 1 9 8 7
E f f e c t o f l e v a m i s o l e o n a c h l o r o q u in e - r e s is t a n t ( C R ) P . b e r g h e i s t r a i n - A reduction in the parasitemia was found after administering levamisole at 1 mg/kg dose, the only dose used, before the infection by C R P . b e r g h e i strain. Parasitemia was also reduced in other groups treated with either chloroquine alone or levamisole plus chloroquine. There was no significant reduction in the mortality rates in the experimental groups although they were delayed by levamisole treatment.
D ISC U SSIO N
In the present work we show that levamisole is not active against P . b e r g h e i even though doses close to the toxic levels have been used. The thymus and spleen weights were increased by the drug used in normal mice (1 - 1 0 mg/kg) but the statistical analysis show no significant difference in relation to untreated mice. The later result has also been observed by Hoebeke and Franchi4 though in their experiments they worked with animals ranging from 20 to 25 g of w eight The weight variation in our mice ranged from 18 to 2 0g allowing therefore a more precise evaluation of the organ weights.
W hen levamisole was administered before the inoculation of P . b e r g h e i strain N K 65, the drug pro duced a reduction o f parasitemia which was more pro nounced between the 4th and 8th day of infection, and using 1 mg/kg dose for two consecutive days. Thus it was apparently this dose which better stimulated the immunity in the infected animals. The decreased para sitemia observed after levamisole treatment may have been a result of an increased phagocytosis with des truction of the plasmodia. Some authors have proved that this drug causes a significant stimulation of RBC phagocytosis4 8.
After the 8* day o f P . b e r g h e i inoculation leva misole did not alter parasitemia possibly because its highest immunostimulant effect was present during the first 48 hours after drug administration10. Indeed, le vamisole administered at the same time or after inocu lation of parasites, did not alter the parasitemias, as reported by other authors12.
Based on our results and because levamisole is a already used in humans we suggest that levamisole could be used in human malaria in careful studies, initially with/*, v iv a x , since this parasite is not usually lethal. It may improve treatment of the disease by chloroquine and eventually help the difficult task of con trolling malaria parasitemias in the P . f a l c i p a r u m CR strains, now largely spread in all endemic areas in Brazil.
R ESU M O
L e v a m i s o l ( f e n i l i m i d o t i a z o l ) , c o n s id e r a d o urn p o t e n t e im u n o e s t im u la n te , q u a n d o a d m i n i s t r a d o a
c a m u n d o n g o s s u íç o s n ã o c a u s o u a u m e n to s ig n if ic a n
-te n o s p e s o s d o tim o , f i g a d o o u b a ç o , a p e s a r d e a d r o g a t e r s i d o u s a d a e m d if e r e n t e s te m p o s a n t e s d a r e m o ç ã o d e s s e s ó r g ã o s . D o s e s e l e v a d a s d a d r o g a u s a d a s n o e s q u e m a p r o f i l á t i c o d e 4 d i a s n ã o ti v e r a m e f e ito a n ti - m a lá r ic o . E n t r e t a n t o q u a n d o d a d a a c a m u n d o n g o s
c o m m a lá r ia , 2 4 h o r a s a n te s , a o m e s m o te m p o o u 2 4
h o r a s a p ó s in o c u la ç ã o d e u m a c e p a d e Plasmodium berghei c lo r o q u in a s e n s ív e l o u u m a c e p a c lo r o q u in a -r e s is te n te o l e v a m i s o l -r e d u z iu , a i n d a q u e d is c -r e t a
m e n te , a p a r a s i t e m i a n o s g r u p o s tr a ta d o s , s e n d o a d o s e d e 1 m g / k g o m e lh o r e s q u e m a . F o i o b s e r v a d o t a m b é m a t r a s o n a m o r t a l i d a d e p o r m a l á r i a n o s g r u p o s t r a t a d o s c o m o le v a m is o l . N o e n ta n to , t o d o s
o s a n i m a i s m o r r e r a m . O s d a d o s s u g e r e m q u e o le v a m i s o l te m e f e ito im u n o e s t im u la n te , a i n d a q u e d i s c r e to , n a r e s p o s ta im u n e d e a n im a is , d e p r i m i d a p e l a
m a lá r ia .
Palavras chaves: Malária. P l a s m o d i u m b e r g h e i. Cepa cloroquina-resistente. Levamisol. Imunoesti mulante.
A C K N O W L E D G E M E N T S
We acknowledge the assistance of Argenis Perozo and Robert Smith with the manuscript and CNPq-Brazil for financial support
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