R e v is ta d a S o c ie d a d e B ra sile ira d e M ed icin a T ro p ica l 2 7 (l): 1 9 -2 4 , ja n -m a r , 1994.
E F F E C T S O F S E V E R E P R O T E IN R E S T R IC T IO N I N L E V E L S O F P A R A S IT E M IA A N D I N M O R T A L IT Y O F M IC E A C C U T E L Y I N F E C T E D W IT H T R Y P A N O S O M A C R U Z I
Nildete G .L. Gomes, Fausto E .L. Pereira, Gisele G.S. Domingues and Jesse R. Alves
A d u lt m ice w ere su b m itted to d ifferen t d eg ree s o f p ro te in restriction f o r fiv e w e eks (4.75, 9 .5 ,1 4 .2 5 a n d 19% o f p ro te in in isocaloric d ie ts with no rm a l co n ten t o f m in era l a n d vitam ins), b ein g su b seq u en tly in fected w ith tw o stra in s o /T rypanosom a cruzi.’ 10? tryp o m a stig o tes o f Y s tra in o r 10* tryp o m a stig o tes o f CL strain. The sa m e d ie t w a s m a in ta in ed f o r a ll a n im a ls a n d th e infectio n w a s fo llo w e d up by evalu ation o f b lo o d p a ra sites, m ortality a n d intensity o f lesions in th e h ea r t a n d skeleto n m uscle. Only se ve re p ro te in restriction (4.75% ) in d u c ed d e c re a se in resista n ce to th e infection with both the Y a n d CL stra in s o f T . cruzi, w hich re su lte d in h ig h er p a ra sitem ia a n d m orta lity. The inflam m atory lesions in h ea rt a n d skeleto n m u scle w ere less exten sive in g ro u p s with se vere p ro te in restriction despite the in crea sed n um ber o f p a ra site in m u scle cells. D ep ressio n o f im m une m echanism s co u ld b e re sp o n sib le fo r th e red u ced re sista n ce a n d re d u ce d in flam m ato ry reaction after T. cruzi infection in severely p ro te in re stricted anim als.
K ey-w o rd s: M aln utritio n. C hagas ’ disease. T rypanosom a cruzi.
C linical and epidem iological observations s u g g e s t th a t m a ln u tritio n , m a in ly p ro te in d e p riv a tio n , p lays an im p o rtan t ro le in the developm ent o f infections which include protozoal d is e a s e s 2 8. A lth o u g h m o s t re p o r ts sh o w enhancement o f infections in malnourished patients o r experim ental anim als, some observations have shown increased resistance to malaria infection in rats that w ere subm itted to protein restriction3 10. M oreover, while protein deprived mice infected w ith L e i s h m a n i a m e x i c a n a showed arrest o f lesion grow th during the five initial weeks o f infection, with subsequent development o f non-healing lesions, norm ally nourished mice developed progressively healing lesions9.
The effects o f m alnutrition on the evolution o f Chagas’ disease has not yet been evaluated. There are few reports showing enhancement of parasitemia
; and m ore severe lesions in the heart o f infected rats
submitted to diets with deficient amounts of thiamine, riboflavine, pantothenate or pyridoxine16 17 1819.
D epartam ento de Patologia, C entro Biomédico, Universidade Federai do Espírito Santo. V itória, ES.
A d d re ss to: D r. Fausto E .L . Pereira. Centro B iom édíco/U FES. C aixa Postal 780, 29060-970 V itoria, ES, Brasil.
R ecebido para publicação em 05/11/92.
O th e r a sp e cts h a v e b e e n e v a lu a te u as th e dem onstration that protein m alnutrition delays the recovery o f cardiac adrenalin in chronic experimental Chagas’ disease in rats5.
B ecause the few in fo rm atio n s ab o u t the relationship between nutrition and Chagas’ disease, w e decide to investigate the course o f T r y p a n o s o m a c r u z i infection (parasitemia, m ortality and intensity o f lesions in heart and skeleton muscle) in norm ally nourished (19 % o f protein) and in m ice m aintained on isocaloric diets w ith 14.25, 9 .5 o r 4.75% o f protein.
M ATERIAL A ND M ETHOD S
Animials and T r y p a n o s o m a crazistrains. Adult m ale outbred albino mice w eighing 23-25g, w ere used in thgexperim ents. The infection was done by i.p . route with two strains o f T r y p a n o s o m a c r u z i:
G o m es N G L , P ere ira F E L, D o m in g u es G C S, A lv es JR . E ffects o f se v e re p r o te in re stric tio n in le ve ls o f p a r a s ite m ia a n d in m o rta lity o f m ice a ccu te fy in fe c ted withT r y p a n o s o m a c r u z i. R ev ista d a S o c ied a d e B ra sile ira d e M e d ic in a T ro p ic a l 2 7 :1 9 -2 4 , ja n -m a r , 1994.
D iets used a n d ex p erim en tal design. The diets (19% , 14.25% , 9.5% an d 4.7 5% o f protein) differred only in the am ount o f protein (soy bean protein supplem ented w ith D-m ethionine), being o th e rw ise e q u iv a le n t and w ere ad m n istere d a d l i b i t u m (Table 1).
Table 1 - B a sic co m p o n en ts u se d in diets.
Com ponents (%)
Total protein (% )
19.00 14.25 9.50 4.75
Soy bean protein' 23.75 17.81 11.90 6.75
Saccharose2 60.00 65.00 71.00 76.00
Corn oil3 2.00 2.00 2.00 2.00
Fibers4 10.00 10.00 10.00 10.00
M ethionin 0.24 0.20 0.10 0.08
M ineral m ix5 5.00 5.00 5.00 5.00
Vitamin mix5 0.10 0.10 0.10 0.10
1: isolated soy bean protein (80% protein). SANBRA, SP, B razil; 2: com m ercial sugar; 3: co m oil M azzola, Refinações d e Milho Brasil, SP, Brazil; 4 : peel rice pow der; 5: mineral and vitamin mix Roche, s upplemented according the Nutrients Requirem ents for Laboratory Anim als, National Academ y o f Sciences, W ashington, D C , 1978.
E ight groups o f mice (ten per group), were m aintained on these diets for five weeks, and infected w ith the Y strain (four groups) or CL strain (four groups) o f 71 c r u z i . As the Y strain induces a lethal infection after the second week, the groups infected w ith this strain w ere maintained on the same diet until the animals spontaneously died. For the CL strain the inoculum used induces an acute infection w ith low m ortality. F or this reason the CL infected groups w ere m aintained in the diets for 30 days after infection, when all the survivors were sacrificed. Control non-infected mice o f each dietary group w ere kept sim ultaneously w ith the infected groups. Food consum ption was randomly verified by w eighing the food given, the food remaining and the am ount o f food spilt.
Follow u p o f th e infection. All mice were w eighed weekly. Parasitem ia was evaluated by the m ethod described by B rener1 w ith three or two days intervals after infection, respectively for CL or Y strain.
H istopathology. The infected animals which died spontaneously, as well those sacrificed at the end o f the experiment, w erenecropsied. Fragm ents o f heart and skeleton muscle (thigh muscles) were fixed in isotonic Bouin (Bouin liquid diluted at 1:4 in saline) for at least 48h, embedded in paraffin and stained routinely.
S ta tistic a l m e th o d s. W hen necessary the
S tudent’s t test was used for com parison o f
parasitemia and the W ilcoxon test was used for comparison o f mortality. U nless otherw ise stated the significance level was p < 0.05.
RESULTS
The animals maintained in the 19 % protein diet had w eight gain before the infection, but there was discrete weigth loss during the period o f infection. The protein deprived, non infected groups, had no weight gain (moderate protein restriction o r 14.25 %) or a weight loss no higher than 30% o f the initial body weight. A fter infection there was w eight loss, more accentuated in the groups w ith severe protein restriction (9.5 and 4.75 % o f protein). N one o f the control non infected mice died spontaneously during the follow up o f infected groups.
Parasitemia was higher in groups mantained on severe protein restriction (4.75 %) for both CL and Y strains. T he groups m aintained on moderate protein deprivation presented parasitem ia sim ilar to that o f norm ally nourished groups (Figures 1 and
2
).The m ortality o f mice infected w ith the Y strain was higher and earlier in the severe protein restricted group (4.75% protein). F o r the other protein restricted groups the m ortality was sim ilar to that i observed in norm ally nourished anim als (Figure 3). In mice infected w ith the CL strain m ortality was observed until the 30th day o f infection and was significantly higher in the group w ith severe protein restriction (4.75% o f protein). The groups w ith moderate protein restriction presented m ortality similar to the norm ally nourished group (Figure 4).
The microscopic study o f the groups infected with the Y strain, all o f which died spontaneously, showed no differences in the lesions o f heart and skeleton muscle. Scarce inflam m atory foci w ere observed and parasites in the muscle cells were infrequent, both in norm ally nourished and in
G o m es N G L , P e r e ir a F E L , D o m in g u e s G C S, A lv e s JR . E ffe cts o f se v e r e p r o te in r e stric tio n in le v e ls o f p a r a s ite m ia a n d in m o rta lity o f m ice a ccu te ty in fe c te d w ith T rypanosom a cruzi. R e v ista d a S o c ie d a d e B ra sile ira d e M e d ic in a T ro p ic a l 2 7 :1 9 -2 4 , ja n - m a r , 1994.
G om es N G L , P e r eira F E L, D o m in g u es G CS, A lv e s JR . E ffe c ts o f se v e r e p r o te in re stric tio n in le v e ls o f p a r a s ite m ia a n d in m o rta lity o f m ice a cc u tely in fe cted w ithT r y p a n o s o m a c r u z i. R ev ista d a S o cie d a d e B ra sile ira d e M ed ic in a T ro p ica l 2 7 :1 9 -2 4 , ja n -m a r , 1994.
protein deficient groups. In the groups infected w ith the CL strain there was a higher frequency o f parasites in the muscle cells o f the severe protein restricted group although the inflammatory lesions seemed to be less extensive than those observed in norm ally nourished groups (19% protein) or in groups w ith m oderate protein deprivation.
DISCUSSION
The results showed that only severe protein restriction (4.75 % of protein) increased parasitemia and m ortality after T r y p a n o s o m a c r u z i infection in mice. Less severe or moderate protein restriction did not modify the evolution o f infection with T.
c r u z i at least in respect to parasitemia, m ortality and
inflam m atory lesions in the heart and skeleton muscle. It is possible that the duration o f protein restriction before infection - 5 weeks - had not been sufficient to interfere with the mechanisms of resistance to infection in the groups submitted to less severe protein restriction (14.75 and 9.5% of protein).
Protein deprivation can induce reduction in cell mediated and hum oral immune response that is related to the severity o f protein restriction and the period o f time on restricted diet4. M oderate protein restriction, m ainly for short periods, not only m aintains but can enhance the immune response, specially cellular mechanisms6. Our results agree w ith these observations showing that the more severe the protein restriction the more severe the reduction in defense m echanism s against the infection w ith T. c r u z i .
Despite the increased num ber o f parasites in the muscle cells o f the 4.75% protein group infected w ith the CL strain, the inflammatory reaction was less severe than in the other groups infected w ith the same strain. This m ild inflammatory reaction could be due to the reduction o f cell mediated immunity induced by severe protein restriction. It is well know n that cell mediated immune mechanisms play an im p o rta n t ro le on th e p a th o g e n e sis o f inflam m atory lesions in Chagas’ disease1112131415.
F urther studies are necessary to elucidate the immune mechanisms against T. c r u z i in protein restricted animals.
RESUM O
C am undongos alb inos adultos fo r a m su b m etid o s a d ietas com d iferen tes con cen tra çõ es d e p ro te ín a (4,7 5% , 9 ,5 % , 14,25% e 19% d e p ro te ín a em d ietas isocalóricas com q u a ntidades norm ais d e m in era is e vitam inas) durante cinco sem a n a s e em se g u id a in fectados co m d u a s cepas d o T r y p a n o s o m a cru zi.' l ( f i trip o m a stig o ta s d a cepa Y ou 10* tripom astigota s d a cepa CL. Os anim ais fo r a m m antidos com as m esm a s d ietas e a infecção f o i seguida com avalia ção d a p a ra sitem ia , d a m ortalidade e d a s lesões no co ra çã o e no m ú scu lo esquelético. Som ente a restrição p ro té ic a se v e ra (4,7 5 % d e p ro te ín a ) induziu redução significante d a resistência à infecção com as d u a s cep a s utilizadas, o q u e f o i d em o n stra d o p e la m a io r p a ra sitem ia e m a io r m o rta lid a d e. A s lesões inflam atória s no co ra çã o e no m ú scu lo esq u elé tic o fo ra m m enos extensas nos anim ais su b m etid o s à d esn u triçã o p ro téic a severa a p esa r d o m a io rp a ra sitism o d a s célu la s m usculares nesses anim ais. A d ep ressã o d a resposta im unitária celu la r induzida p e la d esn u triçã o p ro té ic a seria resp o n sá ve l p e la dim inuição d a resistên cia ao p a ra sita e p e la red u çã o d a in ten sid a d e d a reação
inflam atória observada.
P ala vras-chaves: D esnutrição. D o en ça d e Chagas. T r y p a n o s o m a c r u z i.
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