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 ic i n a T r o p i c a l 2 4 ( 4 ) : 2 3 5 - 2 4 3 , o u t - d e z , 1 9 9 1
L IV E R A N D S E R U M S O L U B L E P R O T E IN C H A N G E S A N D
P A T H O M O R P H O L O G Y I N U N D E R N O U R I S H E D M IC E W IT H
A C U T E S C H IS T O S O M IA S IS M A N S O N I
E r id a n M . C o u tin h o , F red erico G . C . A b a th , L u cila P . C . G . d e F r e ita s, A u re n i C . S a lza n o , M a ria A . L a p a , F lo risb ela S. C a m p o s a n d E lia n e B . M e lo .
B o d y , l i v e r a n d s p l e e n w e i g h t s ; h is t o p a t h o l o g y o f th e l i v e r , s p l e e n a n d i n t e s t i n e s ;
h e p a t i c a n d s e r u m s o l u b l e p r o t e i n s c h a n g e s w e r e t h e p a r a m e t e r s s t u d i e d in u n d e r n o u r i s h e d
S w i s s a l b i n o m i c e e x p e r im e n ta ll y in f e c t e d w i th S. m an so n i. N o n - i n f e c t e d d e f i c i e n t a n i m a b
h a d l o w e r l i v e r / b o d y w e i g h t a n d s p l e e n / b o d y w e i g h t r a t i o s a s c o m p a r e d t o th e c o n t r o l s
( 2 2 .6 0 % c a s e i n g r o u p ) . I n f e c t e d m i c e s h o w e d h i g h e r v a l u e s r e g a r d l e s s t h e t y p e o f d ie t.
U n d e r n o u r i s h e d i n f e c t e d s u b g r o u p s h o w e d a p e r s i s t e n t e x u d a t i v e p e r i o v u l a r r e a c t i o n i n th e
li v e r . S o l u b l e h e p a t i c p r o t e i n s c o n t e n t a n d s e r u m p r o t e i n f r a c t i o n s a p p e a r e d t o b e l o w e r in
th e d e f i c i e n t i n f e c t e d m ic e . A s i g n i f i c a n t d i f f e r e n c e w a s d e t e c t e d in t h e g a m m a g l o b u l i n
f r a c t i o n b e t w e e n i n f e c t e d a n d n o n - i n f e c t e d a n im a ls f e d th e c o n t r o l d i e t w i t h h i g h e r v a l u e s
f o r t h e f o r m e r . O u r d a t a s u g g e s t t h a t t h e e f f e c t s o f m a l n u t r i t i o n , p e r s e , a r e s o m e t i m e s m o r e
d e t r i m e n t a l t o t h e h o s t th a n t h o s e d u e to M a n s o n ' s s c h i s t o s o m i a s i s .
K e y - w o r d s : S c h isto so m a m a n so n i. M a l n u t r i t i o n . M i c e . P r o te in s . '
The interrelationships between malnutrition and M anson1 s schistosomiasis have been studied in this
laboratory for several years'01' 12,314151617:s,l)20.
The use o f purified and/or semisynthetic diets for laboratory anim als is an artificial way of reproducing clinical, biochemical and pathological findings seen in infected hum an populations under field conditions. A diet based on human staple foods usually ingested in N orheast Brazil (in areas w here schistosom iasis is endemic) was devised. This diet, referred as “Regional Basic D iet” (RBD), w hen given to wealing mice and rats, induces alterations resem bling those found in the m arasmatic clinical form of protein -
energy m alnutrition10 12 25 32 3 3 w hich is highly
prevalent in N ortheast Brazil.
In a previous paper (Coutinho e ta l, 1991: in press) several indicators o f protein nutritional
Centro de Pesquisas Aggeu M agalhães/Fundação Oswaldo Cruz e Departamento de Nutrição/Centro de Ciências da Saúde, Universidade Federal de Pernambuco, Recife, PE, Brazil.
Address f o r c o r re s p o n d e n ce : D ra. Eridan M . Coutinho - Centro de Pesquisas Aggeu Magalhães/FIOCRUZ. Caixa Postal 7472, 50730. Recife, PE, Brazil. Recebido para publicação em 19/08/91.
status w ere studied in undernourished Swiss albino mice fed RBD and infected experim entally w ith 5.
m a n s o n i . The purpose o f the present investigation
was to study the effects o f RBD on liver and serum soluble proteins and tissue m orphology in the same groups o f animals.
M ATERIAL A ND M ETHODS
A n i m a l s . W eanling, Swiss albino m ice o f both
sexes w ere maintained on the three diets (RBD, 7.82% casein and 22.60% casein) for 63 days. Twenty mice w ere studied in each diet and divided into infected and non-infected subgroups.
Animals were housed individually in w ire mesh
c a g e s , and maintained at 2 3 ±2 °C w ith a 12 hour lig h t/12 hour dark cycle.
D i e t s . The com position o f RBD is show n in
Table 1. The two control diets (Tables 2 and 3) contained casein at tw o levels: 7.82% (control I) and 22.60% (control II). The control diets were supplem ented w ith m ineral salts and v itam in m ixture, according to Tagle & D onoso31. The diets w ere given in pellet form.
I n f e c ti o n . A S c h i s t o s o m a m a n s o n i strain isolated
C o u li n h o E M , A b a t h F G C , F r e it a s L P C G , S a l z a n o A C , L a p a M A , C a m p o s F S , M e l o E B . L i v e r a n d s e r u m s o l u b l e p r o t e i n c h a n g e s a n d p a t h o m o r p h o l o g y in u n d e r n o u r is h e d m i c e w i th a c u t e s c h i s t o s o m i a s i s m a n s o n i . 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 4 : 2 3 5 - 2 4 3 , o u t - d e z , 1 9 9 1
T a b l e 1 - C o m p o s i t i o n o f t h e R e g i o n a l B a s i c D i e t ( R B D ) - g / l O O g
Components Amounts in Proteins Carbohy Fats Minerals Fibers
g/lOOg drates
Kidney beans (Phaseolus vulgaris) 18.34 3.96 10.66 0.24 0.57 1.09
Manioc flour (Manihot esculenta) 64.81 0.82 48.59 0.12 0.43 5.64
Salted and dried meat (Charque) 3.74 2.74 0.43 0.21 0.06
-Fat from salted and dried meat 0.35 - - 0.35 -
-Sweet potato (Iponaea batatas) 12.76 0.30 9.99 0.03 0.20 0.48
Total 100.00 7.82 69.67 0.95 1.26 7.21
NDpCal % = 5.97
T a b l e 2 - C o m p o s i t i o n o f t h e c o n t r o l d i e t I - g / 1 0 0 g
Components Amounts in
g/100g
Proteins Carbohy
drates
Fats Minerals Fibers
Casein 8.87 7.06
_
_
.
Soybean oil 8.00 - - 8.00 .
-Water-soluble vitamins 1.00 - - - . .
Fat-soluble vitamins 1.00 - - 1.00 .
-Salt mixtures 2.50 - - - 2.50
-Cellulose 3.50 - - - . 3.50
Corn starch 75.13 7.76 74.37 - -
-Total 100.00 7.82 74.37 9.00 2.50 3.50
Supplemented with mineral salts and vitamin mixtures according to Tsgle31.
T a b l e 3 - C o m p o s i t i o n o f t h e c o n t r o l d i e t I I -
g/ i oog
Components Amounts in
g/100g
Proteins Carbohy
drates
Fats Minerals Fibers
Casein 27.80 22.09
_
.
Soybean oil 13.00 - - 13.00 -
-Water-soluble vitamins 1.00 - - - -
-Fat-soluble vitamins 1.00 - - 1.00 -
-Salt mixtures 2.50 - - - 2.50
-Cellulose 4.00 - - - - 4.00
C om starch 50.70 0.51 50.19 - -
-Total 100.00 22.60 50.19 14.00 2.50 4.00
C o u tin h o E M , A b a t h F G C , F r e i t a s L P C G , S a l z a n o A C , L a p a M A , C a m p o s F S , M e l o E B . L i v e r a n d s e r u m s o l u b l e p r o t e i n c h a n g e s a n d p a t h o m o r p h o l o g y in u n d e r n o u r is h e d m ic e w it h a c u t e s c h i s t o s o m i a s i s m a n s o n i. 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 4 : 2 3 5 - 2 4 3 , o u t- d e z , 1 9 9 1
Brazil) and m aintained regularly in our laboratory was used. Each animal was infected percutaneously
w ith 80 cercariae shed from B i o m p h a l a r i a g l a b r a t a
reared and infected in the laboratory. (Infections up to 100 o r 150 cercariae per m ouse are well tolerated27 M).
E x p e r i m e n t a l p r o t o c o l . Twenty - one days old
weanling mice weighing 9 to 12 g received tap
w ater and diets a d l i b i t u m . The body weight was
determined every week and the food intake was recorded every day.
Subgroups o f 5 mice per control diet were submitted to a pair-feeding trial. So, the daily food intake o f control mice (diets II and III) was restricted to th e a d lib itu m intake of RBD - fed animals (diet I).
S o l u b l e S e r u m a n d L i v e r p r o t e i n s
d e t e r m i n a t i o n s . Blood was collected by aspiration
from the axillary vein, allow ed to clot and immediately centrifuged at 1.000 x g for 5 minutes. Total protein concentration in serum was determined by the biuret reaction23. Electrophoretic studies were carried out in cellulose acetate according to the microzonal system. The liver soluble proteins were determined in homogenates from pieces o f the right lobe o f the liver, according to Lowry et ali26 and expressed in m g/total liver weight.
M o r p h o l o g i c a l s t u d i e s M ice were sacrificed by
cervical dislocation. Liver, spleen and both small and large intestines samples were routinely fixed in 10% buffered formalin, parafin embedded, cut at a six m icra thickeness and stained by haematoxylin- eosin and M a llo ry 's trichrom e stains1.
Liver samples w ere otherwise silver stained by Gomori 's technique5 to evidentiate the reticulin fibers. Frozen sections w ere stained w ith Sudan III to evalute the distribution and morphological quantification o f hepatic triglycerides.
S t a t i s t i c a l a n a l y s i s Student's “t ” - test and the
analysis o f variance w ere used to assess the significance o f the data. A statistically significant difference w as indicated by a p < 0 .0 5 .
RESULTS
Q u a n t i t a t i v e s o l u b l e p r o t e i n s c h a n g e s
- C h a n g e s in s e r u m s o l u b l e p r o t e i n s . Values
for total serum proteins w ere significantly low er in
the infected animals fed the two low - protein diets. Significant differences w ere always found between mice fed the two low -protein diets as compared to those fed the 22.60% casein diet
regardless S . m a n s o n i infection. T he album in
fraction had a sim ilar behaviour. The album ins/ globulins (A/G) ratio was decreased in the infected casein-fed groups. An apparent, but not significant, slight distortion was detected in the infected RBD - fed mice as compared to the casein-fed animals. Values for alpha-1 and alpha-2 globulins fractions were significantly low er in RBD - fed infected mice as compared to non-infected ones fed the same diet. Also, the alpha-2 globulin fraction was low er for the infected group fed 7.82% casein. Only in these anim als the beta-globulin fraction w as s ig n if ic a n tly h ig h e r . T h e v a lu e s fo r gam maglobulins w ere significantly higher in the serum o f the infected controls (Table 5).
- C h a n g e s in l i v e r s o l u b l e p r o t e i n s . L iv e r
proteins measured w ith the F olin - phenol reagent showed a trend to low er values in infected animals under both low -protein diets (RBD and 7.82% casein), although these differences w ere no t statistically significant.
S ig n ifican t re su lts w ere d etected am ong experimental low -protein and control groups w ith and w ithout infection, as well as betw een casein groups (Table 4).
M o r p h o l o g i c a l f i n d i n g s
- T o t a l b o d y w e i g h t , l i v e r a n d s p l e e n w e i g h t s/
b o d y w e i g h t r a t i o s . At the end o f the experim ent, a significant differences was detected in the total body w eight for low -protein fed m ice, w ith and w ithout infection, the low est values for RBD - fed infected animals. Infection did not seem to affect the body w eight o f the control group II (Table 4).
The mean value for liver w eights and their standard error for the experim ental groups are shown in Table 4. Values for lives w eight/total body weight ratio were higher in infected mice. These results w ere statistically significant, except in the 7.82% casein - fed group.
C o u ti n h o E M , A b a t h F G C , F r e i t a s L P C G , S a l z a n o A C , L a p a M A , C a m p o s F S M e l o E B . L i v e r a n d s e r u m s o l u b l e p r o t e i n c h a n g e s a n d p a t h o m o r p h o l o g y in u n d e r n o u r is h e d m ic e w it h a c u t e s c h i s t o s o m i a s i s m a n s o n i . R e v i s t a d a
C o u tin h o E M , A b a t h F G C , F r e i t a s L P C G , S a l z a n o A C , L a p a M A , C a m p o s F S , M e l o E B . L i v e r a n d s e r u m s o l u b l e p r o t e i n c h a n g e s a n d p a t h o m o r p h o l o g y in u n d e r n o u r is h e d m ic e w it h a c u t e s c h i s t o s o m i a s i s m a n s o n i . 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 4 : 2 3 5 - 2 4 3 , o u t- d e z , 1 9 9 1
experim ental groups.
A gain, significant results were obtained for both low -protein fed groups w hen compared to control group II, the highest values for infected anim als. But results w ere not significant when non-infected mice in the two low -protein groups w ere com pared to each other.
- H i s t o p a t h o l o g y . In infected RBD-fed mice, the liver parenchym a showed several periovular granulom as, some o f them w ith a predominantly histiocytic cellular reaction (eggs w ith a still preserved m iracidium ), other w ith an exudative reaction in w hich neutrophils and many eosinophils could be detected. Periovular microabscesses, as well as a lasting exudative celullar reaction around both viable and non-viable eggs, could be seen F igure 1). V acuolar degeneration and diffuse steatosis, particularly o f the portal type, w ere the lesions m ost frequently observed.
In the intestines, particularly in the jejuno-ileal seg m en t, s e v e ral fib ro tic a n d /o r h is tic y tic granulom as w ere seen in the lamina propria o f the mucosal layer. H ow ever, they were more abundant in the subm ucosa, w here ova agglom erated, inducing an extensive reaction progressing to fibrosis in the wall o f the small intestine (Figure
2). Also, a marked eosinophylic infiltration around retained eggs in the submucosa layer could be detected.
T he spleen showed a poorly deliniated w hite pulp, low cellular density o f the periarteriolar lymphatic sheaths and M alpighian follicles w ithout reactive centers.
Hepatic lesions in infected mice fed the 7 .8 2 % casein diet (control I) w ere sim ilar to those reported for RBD-fed infected mice. H ow ever steatosis seemed to be m ilder, exudation o f periovular granulomas less apparent and collagenisation o f the egg granulomas less delayed in this subgroup.
In infected mice fed a 22.60% casein diet (control II), the liver parenchym a, as a w hole, had anormal aspect, except by the presence o f periovular schistosomotic granulomas w ith a higher cellularity w hile other granulom as w ere predom inantly o f the productive type, w ith many histiocytes, fibroblasts and collagen fibers (Figure 3). Sometimes a focus
o f acute coagulative necrosis beside small p ie c e s
o f the eggshell and a peripheral histiocytic reaction could be seen. Eventually, afew cells w ith fat droplets could be detected in the liver parenchyma.
C o u tin h o E M , A b a th F G C , F r e ita s L P C G , S a lz a n o A C , L a p a M A , C a m p o s F S , M e l o E B . L iv e r a n d s e r u m s o l u b le p r o t e i n c h a n g e s a n d p a t h o m o r p h o lo g y in u n d e r n o u r is h e d m ic e w ith a c u te s c h is to s o m ia s is m a n s o n i. R e v is t a d a
S o c ie d a d e B r a s i l e i r a d e M e d ic in a T r o p i c a l 2 4 : 2 3 5 - 2 4 3 , o u t- d e z , 1 9 9 1
central coagulative necrosis, could be seen in a hyperem ic red pulp. Periovular granulomas were seen in every layer o f the small and large intestinal w alls. H ow ever, they seemed to be m ore abundant in the subm ucosa, w here they joined each other
and induced a diffuse process o f fibrosis.
C o u tin h o E M , A b a t h F G C , F r e i t a s L P C G , S a l z a n o A C , L a p a M A , C a m p o s F S , M e l o E B . L i v e r a n d s e r u m s o l u b l e p r o t e i n c h a n g e s a n d p a t h o m o r p h o l o g y in u n d e r n o u r is h e d m ic e w i th a c u t e s c h i s t o s o m i a s i s m a n s o n i . 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 ic i n a T r o p i c a l 2 4 : 2 3 5 - 2 4 3 , o u t - d e z , 1 9 9 1
(RBD and 7.82% casein diet).
N o clear-cut differences could be detected regarding the spleen and intestine lesions in all the experim ental groups.
DISCUSSION
The public health importance o f the relationship between schistosom iasis and nutritional status in hum an populations is still unclear30. M ost o f the stu d ies on these in tera ctio n s have involved experim ental animals and the use o f purified and/ or sem isynthetic diets2. On the other hand, it is w id e ly a c c e p te d th a t s c h is to s o m ia s is and m alnutrition are m utually interactive problems, that is, both act synergistically21, although other reports say ju s t the opposite2.
A ccording to our findings, the liver soluble proteins changes showed a trend to low er values in infected mice fed both low -protein diets (RBD and 7.82% casein control diet) as compared to infected anim als fed the 22.60% casein control diet.
A lthough the hepatic protein content seems to be better expressed in relation to the hepatic D N A 9, it can also be expressed in relation to the total liver w eight28 as in the present paper. So, one can speculate that due to a probable increase in the am ount o f oviposition and to the formation of periovular granulom as w ith a m ore intensive inflam m atory reaction, the high protein content in the liver o f the control group represents the amount of a non-specific protein pool in the organ, which is resposible for the higher values obtained in this group w hen com pared to the group o f non-infected mice fed the same diet. Probably, the hight values for liver weights in non-infected mice fed the 22.60 % casein diet reflect the better quality o f the
dietary protein and, in the infected subgroup, S .
m a n s o n i infection also accounts for this increased
weight.
Sim ilarly, the changes in the serum protein levels w ere m ore commonly seen in the low protein-fed groups, particularly in RBD-fed mice. On the other hand, a progressive increase in the beta and gam m aglobulin fractions, w ith negligible or in e x is te n t ch anges in th e alb um in s and alphaglobulin concentrations, w ould be expected in the serum o f control mice22.
The liver w eight/total body w eight and the spleen w eight/total body w eight ratios w ere higher in the infected mice, regardless the type o f dietary intake, indicating the im portant role played by schistosomiasis in the occurrence o f liver and spleen enlargements in the course o f the disease.
Histologically, reduction in cellularity and a lasting exudative periovular reaction seem to occur more commonly in R BD-fed m ice as com pared to casein-fed ones w here m ost o f the periovular granulomas were o f the productive (non-exudative) type. These findings were previously reported1618 and seem to be related to some factors, such as a higher deposition o f im mature eggs and/or a delay o f their m aturation in the tissues.
It has been dem onstrated3 4 that a chronic restriction o f protein and calories by the use o f semisynthetic diets induces, in experim entally infected mice, a decrease in egg-laying and a reduction of the granulom atous reaction in the tissues o f the definitive host. It is also know n that severe acute protein m alnutrition suppresses the h o s t 's cellular im m unity in schistosom iasis, causing a dramatic decrease in the granulom atous reaction1.
The formation of granulom as around eggs in the h o s t's tissues is inhibited by several types o f
nutritional déficiences4 u . The form er authors
showed that the egg load in the liver decreases as dietary protein is decreased, inhibition being greatest w ith a 50 % calorie-deficient diet. Akpom and W arren4 found that a significant proportion of the eggs recovered from m ice fed 4% protein deficient and 50 % calorie deficient diets was not viable.
There were no significant differences in splenic and intestinal lesions detected in our three groups o f animals.
C o u tin h o E M , A b a t h F G C , F r e i t a s L P C G , S a l z a n o A C , L a p a M A , C a m p o s F S , M e l o E B . L i v e r a n d s e r u m s o l u b l e p r o t e i n c h a n g e s a n d p a t h o m o r p h o l o g y in u n d e r n o u r is h e d m i c e w i th a c u t e s c h i s t o s o m i a s i s m a n s o n i . 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 4 : 2 3 5 - 2 4 3 , o u t - d e z , 1 9 9 1
is the m ain aggravating cause o f the disease, studies on their characteristics and evolution are im portant topics fo r investigations8.
Further researches are recommended to estimate the extent to w hich im provem ent o f the nutritional status o f the host may contribute for decreasing the effects o f schistosom iasis in mice and possibily in h u m an beings resid in g in endem ic areas o f underdeveloped countries.
RESU M O
N o p r e s e n t e e s t u d o o s p e s o s c o r p o r a l , h e p á tic o e
e s p lé n i c o ; a m o r f o lo g i a p a t o l ó g i c a d o f í g a d o , b a ç o e
in t e s tin o s ; e a s m o d i f i c a ç õ e s d a s p r o t e í n a s s o lú v e is
h e p á ti c a s e s é r i c a s , f o r a m o s p a r â m e t r o s in v e s tig a d o s
e m c a m u n d o n g o s a lb in o s S u íç o s d e s n u tr id o s e in f e c ta d o s
c o m S. mansoni. O s a n im a is d e s n u tr id o s n ã o in f e c ta d o s
a p r e s e n ta r a m r e l a ç õ e s f í g a d o / p e s o c o r p o r a l e b a ç o !
p e s o c o r p o r a l c o m v a l o r e s m e n o r e s d o q u e o s a n im a is
c o n tr o le s ( g r u p o c a s e in a a 2 2 , 6 0 % ) . C a m u n d o n g o s
in f e c t a d o s m o s tr a r a m e s s e s ín d ic e s m a is e l e v a d o s ,
in d e p e n d e n te m e n te d o tip o d e d ie ta . O s u b g r u p o d e
c a m u n d o n g o s d e s n u tr id o s in f e c ta d o s a p r e s e n to u , n o
f í g a d o , r e a ç ã o p e r i o v u l a r e x s u d a tiv a p e r s is te n te . O
c o n t e ú d o d e p r o t e í n a s s o lú v e i s n o f í g a d o e n o s o r o t a m b é m m o s t r o u - s e r e d u z i d o n o s c a m u n d o n g o s
in f e c ta d o s d e s n u tr id o s . D i f e r e n ç a s i g n i f i c a tiv a f o i
d e t e c t a d a q u a n to à s g a m a g l o b u li n a s , c o m p a r a n d o - s e
a n im a is in f e c ta d o s c o m n ã o in f e c ta d o s a lim e n ta d o s
c o m a d i e t a c o n tr o le II, v a l o r e s m a is a lto s o c o r r e n d o
n o g r u p o d o s in f e c ta d o s . E s p e c u la - s e q u e o s e f e ito s d a
m á n u t r iç ã o p o d e m s e r m a is p r e ju d ic ia is a o h o s p e d e ir o
d o q u e a q u e le s p r o v o c a d o s p e l o S. m a n s o n i.
P a l a v r a s - c h a v e s : S chistosom a m ansoni.
D e s n u t r iç ã o . P r o t e í n a s . C a m u n d o n g o .
ACKNOW LEDGEM ENTS
W e are indebted to M iss L ígia Pedrosa for her critical review and M iss M aria do Carmo Lacerda for typping the m anuscript.
REFEREN CES
1. Akpom C A. Suppression of granuloma formation
around S c h is to s o m a m a n s o n i eggs in severe protein
malnutrition: the role of the egg. Transactions of
the Royal Society of Tropical Medicine and Hygiene 75:444-446, 1981.
2. Akpom CA. S ch isto so m iasis: n u tritio n a l implications. Reviews of Infectious Diseases 4:776- 782, 1982.
3. Akpom CA, Warren KS. The inhibition of
granuloma formation around S c h is to s o m a m a n s o n i
eggs. VI. Protein, calorie and vitamin deficiency. American Journal of Pathology 79:435-452,1975. 4. Akpom CA, Warren KS. Calorie and protein
malnutrition in chronic murine Schistosomiasis mansoni: effect on the parasite and the host. The Journal of Infectious Diseases 132:6-14, 1975. 5. Behmer OA, Tolosa EMC, Freitas Neto AG.
Manual de técnicas para histologia normal e patológica. Editora da Universidade de São Paulo, São Paulo, 1976.
6. Beisel WR. Role of nutrition in immune system diseases. Comprehensive Therapy 13:13-19,1987. 7. Brito T , Ho shino-Shimizu S, Silva LC, Kanamura H, Costa CM, Pinto PS. Immunopatology of
experimental schistosome (S . m a n s o n i ) egg
granulomas in mice - possible defence mechanisms mediated by local immune complexes. Journal of Pathology 140:17-28, 1983.
8. Chen MG, Mott KE. Progress in assessment of
morbidity due to S c h is to s o m a m a n s o n i infection
(A review of recent literature). Tropical Diseases Bulletin 85:1-56, 1988.
9. Compbell RM, Kosterlitz HW. The effectof dietary protein on the turnover o f phospholipids, ribonucleic acid and desoxyribonucleic acid in the liver. Journal of Biological Chemistry 175:989- 990, 1948.
10. Coutinho EM. Relações hospedeiro - parasito na esquistossomose mansônica, em função da Dieta Básica Regional (estudo epidemiológico e anátomo- patológico). Tese de doutorado, Centro de Ciências da Saiíde da Universidade Federal de Pernambuco, Brasil, 1976.
11. C outinho EM . Estado n u tricio n a l e
esquistossomose. Revista da Sociedade Brasileira de Medicina Tropical XIII:91-96, 1979-1980. 12. Coutinho EM. Patobiologia da desnutrição nas
doenças parasitárias. Memórias do Instituto Oswaldo Cruz 75:63-76, 1980.
13. Coutinho EM, Barbosa FS, Barbosa JM, Pessoa D, Pinto RF, Oliveira PA, Rodrigues BA. Inquérito clínico-nutricional e antropométrico preliminar em áreas endêm icas de esquistossom ose mansônica, no nordeste do Brasil. Revista da Sociedade Brasileira de Medicina Tropical VI.-211- 236, 1972.
C o u tin h o E M , A b a t h F G C , F r e i t a s L P C G , S a l z a n o A C , L a p a M A , C a m p o s F S , M e l o E B . L i v e r a n d s e r u m s o l u b l e p r o t e i n c h a n g e s a n d p a t h o m o r p h o l o g y in u n d e r n o u r is h e d m ic e w i th a c u t e s c h i s t o s o m i a s i s m a n s o n i . 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 4 : 2 3 5 - 2 4 3 , o u t- d e z , 1 9 9 1
alimentares e suas repercussões sobre a biologia do hospedeiro, na esquistossomose experimental
(S . m a n s o n i) do camundongo albino. In: Anais da 3 * Jornada Científica da Fundação Oswaldo Cruz, Rio de Janeiro p.454-455, 1985.
15. Coutinho-Abath E. Influence of protein intake on
the penetration o f cercariae o f S c h is to s o m a m a n s o n i
in the skin of normal and experimentally infected mice. Revista do Instituto de Medicina Tropical de São Paulo 4:230-241, 1962.
16. Coutinho-Abath E. Observações preliminares sobre o padrão dietético em áreas endêmicas de esquistossomose mansônica. Jornal Brasileiro de Medicina 8:339-344, 1964.
17. Coutinho-Abath E. Esquistossomose mansônica e nutrição. Revista Brasileira de Malariologia e Doenças Tropicais XVI:233-253., 1964.
18. Coutinho-Abath E, Amaral JA, Barbosa JM. Alterações bioquímicas na esquistossomose mansônica humana, com especial referência ao metabolismo lipídico. Jornal Brasileiro de Medicina 11:157-168, 1966.
19. Coutinho-Abath E, Espírito Santo M, Barbosa JM, Mello SA. Padrão alimentar em áreas endêmicas de Esquistossomose mansônica, no Nordeste do Brasil. Revista Brasileira de Malariologia e Doenças Tropicais 16:553-589, 1964.
20. Coutinho-Abath E, Magalhães Filho A, Barbosa JM. Lesões hepáticas no camundongo albino
experimentalmente infestado por S c h is to s o m a
m a n s o n i e submetido a dietas de diferente teor
protéico. Revista do Instituto de Medicina Tropical de São Paulo 4:311-322, 1962.
21. De Witt WB. Experimental Schistosomiasis mansoni in mice maintained on nutritionally deficient diets. I. Effects of a Torula yeast ration deficient in factor 3, vitamin E, and cystine. Journal of Parasitology 43:119-128, 1957. 22. De Witt WB, W arren KS. Hepato-splenic
schistosomiasis in mice. The American Journal of Tropical Medicine and Hygiene 8:440-446,1959. 23. Gornall AG, Bardawell CG, David MJBC.
Determination of serum protein by means of the biuret reaction. Journal of Biological Chemistry 177:751, 1949.
24. K: auft RF, Warren KS. The effect of calori and protein malnutrition of both the parasite and the host in acute murine schistosomiasis mansoni. The Journal of Infectious Diseases 120:560-575,1969.
25. Lago ES, Pessoa DCNP, Teodósio NR, Cabral FilhoJE. Longevity and growth curveof chronically malnourished rats fed the Regional Basice Diet
(RBD). I n : Proceedings of the 2nd International
Symposium of Nutrition, Recife p .40-41, 1988. 26. Lowry OH, Rosenbrough NG, Farr AL, Randall
RJ. Protein measurement with the Folin - plenol
reagent. Journal of Biological Chemistry 193:265- 275, 1951.
27. Moore DV, Yolles TK, Meleney HE. A comparison of common laboratory animals as experimental
hosts for S c h i s t o s o m a m a n s o n i . Journal of
Parasitology 35:156-170, 1949.
28. Ponce PN, Osorio MB, Aragdn FZ. DNA proteinas en el higado de ratas alimentadas com maiz: importância de expressar la concentración de proteinas hepáticas en función del DN A. Archivos Latino-Americanos de N utrición 31:471-484,1981. 29. Reis MG, Andrade ZA. Functional significance of
periovular granuloma in schistosomiasis. Brazilian Journal of Medical Research 20:55-62, 1987. 30. Stephenson LS, Latham M C, M lingi B.
Schistosomiasis and human nutrition. Cornell International Nutrition Monograph Series n° 16, New York p. 1-21, 1986.
31. Tagle MA, Donoso G. Net protein utilization determined in short and long-term experiments with rats. Journal of Nutrition 87:173-178, 1965. 32. Teodósio NR, Lago ES, Cabral Filho JE, Castro
RM, Varela RM, Campos FACS, C osta FBR,
Guedes RCA, Silva AT. Dietary deficiency and malnutrition - 2. Effects of malnutrition induced by the Regional Basic Diet (RBD) on development
and growth. In: Proceedings ofthe 1st International
Symposium of Nutrition, Recife p.39-40, 1986. 33. Teodósio NR, Romani SAM, Lágo ES. Dietary
deficiencies and malnutrition. 1- An experimental dietetic model. In: Proceedings of the 1st International SymposiumofNutrition, Recifep.38, 1986.
34. Warren KS. The influence of treatment on the development and course of murine hepatosplenic Schistosomiasis mansoni. Transactions ofthe Royal Society of Tropical Medicine and Hygiene 56:510- 519, 1986.
35. W arren KS, Domingo EO, Cowan R B T .
