EXPERIMENTAL
PARASITOLOGY
73,
117-126(1991)
Schistosoma mansoni: Angiotensin Converting Enzyme Activity in Mice under the Influence of Praziquantel and/or Captopril M.T. KHAYYAL,S.SALEH, A. A. METWALLY,* S. S.BOTROS,* AND M.R. MAHMOUD* Departments
of Pharmacology, Faculty
of Pharmacy, Cairo University, and *Theodor Biiharz Research Institute, Embaba, Giza, Egypt
KHAYYAL, M. T., SALEH, S., METWALLY, A. A., 1991. Schistosoma mansoni: Angiotensin converting
BOTROS,
S.S.,
MAHMOUD,
M.R.
enzyme activity in mice under the influence of praziquantel and/or captopril. Experimental Parasitology, 73, 117-126. Murine schistosomiasis is usually associated with hepatic granulomatous lesions together with high serum and granuloma angiotensin converting enzyme (ACE) activity. Praziquantel (PRZ) which is known to reduce granuloma size was studied to show whether this effect is related to changes in ACE activity. Furthermore, captopril was studied to show whether by inhibiting ACE activity, the drug could also affect granuloma size. PRZ, captopril, and their combination led to significant reduction in liver granuloma. However, in normal mice, captopril was shown to increase rather than decrease serum ACE. The decrease in ACE activity by PRZ was correlated with its curative effect in infected mice. However, in experimentally induced pulmonary granulomata, the drug reduced granuloma size without affecting ACE activity of either serum or granuloma. It may be concluded that reduction in ACE activity may be beneficial as far as diminution of granuloma size is concerned and irrespective of whether there is an active infection or not. The possible use of Captopril as an antihypertensive in bilharzial infections associated with hypertension would probably not adversely affect the granulomatous lesions. 0 1991 Academic Press, inc. INDEX DESCRIPTORS: Schistosoma mansoni; Trematoda; Angiotensin converting enzyme (ACE); Liver granuloma; Praziquantel; Captopril; Serum Ace inhibitors; Histopathology; Lung granuloma; Oogram; Lesions; Enzyme
Murine Schistosoma mansoni has been shown to be associated with the development of granulomas in the liver and the intestine of infected animals. Such granulomas have high ACE activity which seems to be dependent on the size of the granulomata and chronicity of infection (Weinstock and Boros 1981, 1982). The macrophages surrounding the granulomas also have high ACE activity which could possibly contribute to the elevated serum ACE activity of the infected animals (Weinstock and Boros 1982). Inhibition of ACE activity by effective drugs, e.g., captopril, has been shown by Weinstock and Boros (1981, 1982) to reduce granufoma size too, but the effect appeared to be reversible upon drug withdrawal (Boomsma et al. 1981; Wein-
stock and Boros 1981; Forslund et al. 1982). A reduction in granuloma size and cellularity was also observed in infected hamsters treated with praziquantel (James et al. 1977; El-Hawey et al. 1986; El-Ibiary et al. 1986). Mehlhorn et al. (1982) also reported the involution of liver granulomas after treating infected mice effectively with the drug. Furthermore, in the absence of an active infection, it was observed that the lung granulomas from normal and sensitized mice injected intravenously with schistosome eggs also show high ACE activity (Weinstock et al. 1979). In humans, S. mansoni infection has been shown to increase serum ACE activity, while treatment was shown to reduce it (Grivaux et al. 1986). ACE inhibitors are frequently used to treat hypertension clinically (Ondetti et al. 117 0014-4894/91$3.00 Copyright 6 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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KHAYYALETAL.
1977, Rubin et al. 1978). The present experimental study was conducted to show whether the use of captopril would influence the curative process initiated by specific anti-schistosomal therapy as far as the granulomatous lesions are concerned. Captopril has therefore been tested together with praziquantel on the ACE activity and size of liver granulomata caused by active infection as well as in lung-induced granulomata in noninfected animals. Furthermore, the effect on serum ACE has been studied in both normal and infected animals under the influence of praziquantel and/or captopril. MATERIALS
Animals
AND METHODS
Used and Methods of Infection
Laboratory-bred albino mice, 18-20 g each, were infected subcutaneously with 60 cercariae of an Egyptian strain of S. mansoni as described by Peters and Warren (1969). They were used 7 weeks after infection.
Drugs and Dosage Praziquantel (Bayer AG, Leverkusen, FRG) was given orally in a dose of 500 mg kg- ‘ body weight for 2 consecutive days. This was shown to be the effective curative dose in experimentally S. mansoni-infected mice (Gonnert and Andrews 1977).The drug was given as a suspension in 2% Cremophor EL (Sigma Chemical Company, St. Louis, MO). Captopril (E. R. Squibb & Sons, Inc. Princeton, NJ) was given orally in a dose of 100 mg kg- ’ day for 4 weeks. This dose for this length of time has been used by other authors for studying the effect of ACE inhibition (Weinstock and Boros 1981; Weinstock and Ehrinpreis 1981).The drug was given in the form of a 0.1% aqueous solution.
Experimental
Design
This study involved experiments carried out on granuloma from S. mansoni-infected mice and others conducted on lung-induced granulomata from noninfected animals.
A. Experiments Carried Out on Granuloma from S. mansoniInfected Animals Infected animals were divided into four groups, each consisting of 10-20 mice and subjected to the following treatment regimens:
Group I: The animals were given captopril daily for 4 weeks. The mice were sacrificed 24 hr after 1, 14, and 28 doses. Group 2: The mice were given praziquantel for 2 consecutive days. The animals were sacrificed 24 hr. 2 weeks, and 4 weeks after the last dose. Group 3: Animals in this group were treated with captopril and praziquantel. Praziquantel was given for 2 successive days. Captopril treatment was started at the same time and continued for 4 weeks. Animals were sacrificed 24 hr, 2 weeks, and 4 weeks after the last dose of praziquantel (i.e., 24 hr after the last dose of captopril). Group 4: In this group. the mice were given 2% Cremophor EL in the same volume used for praziquantel gavage daily for 2 days and served as concurrent controls to the praziquantel-treated groups. Animals were sacrificed at 24 hr, 2 weeks, and 4 weeks after the last dose. In another set of experiments, normal (noninfected) animals were also subdivided into four groups and subjected to the same treatment regimens described above. Procedure at autopsy. Mice were killed by decapitation, and the blood was collected. The serum was separated by centrifugation at 3000 rpm for 10 min and stored frozen at - 20°C for the assay of ACE activity. Infected animals, after decapitation, were perfused with saline (Pellegrino and Sigueira 1956), and the worms were collected separately from the liver and portomesenteric veins and counted. Livers were then removed and divided into three portions, one portion was used for ova counting, one for isolation of granulomata and, one fixed in 10% formalin for histopathological examination of granulomata and determination of granuloma size. The intestine was also removed for ova counting and oogram studies. Ova counting and oogram studies. A portion of liver or intestine was digested overnight in 4% KOH for counting S. mansoni eggs, as described by Cheever (1968). A fragment of intestine (1 cm long) was cut off from each mouse, dried on a filter paper, and then spread on a slide for microscopical examination. One hundred ova in each fragment were examined, counted, and classified according to their degree of development (Pellegrino ef ~1. 1962). Isolation ofgranulomas. Granulomas from the liver of lung were isolated by the method of Pellegrino and Brenner (1956). Subsequently, they were washed three times with Hanks’ balanced salt solution (HBSS), packed at 3000 g for 5 min at 20°C to remove all supernatant, and frozen at -70°C. Under these conditions, there is no change in ACE activity for up to two months (Weinstock and Boros 1981). Assay
of ACE
acrivity
and protein
concentration,
To determine the ACE activity in isolated granulomas, granulomas were ground in a mortar with a pestle for 2 min (200 mg tissue/3 ml HBSS) on ice and subse-
ACE ACTIVITY quently sonicated on ice for 30 sec. One aliquot was used for determination of protein concentration using Folin Ciocalteu phenol reagent (Fisher Scientific Co., New York) (Lowry et al. 1951), while the other ahquot was used for determination of ACE activity using hippuryl-L-histidyl I-leucine substrate (Sigma Chemical Co., St. Louis, MO 63178) according to the method described by Hurst and Lovell-Smith, (1981). Granuloma ACE activity was calculated as nmoles/min/mg protein. Determination of grunuloma size. Isolated circumscribed granulomas containing parasite ova were located microscopically after staining with haematoxyhn and eosin in the histologic sections of the liver or lung. Their diameters were measured with an image-splitting eye piece (Vicker’s Instrument, Wobum, MA) by determining the mean of two measurements made across perpendicular axes. The mean diameter of at least 50 lesions from the animals of each group was measured. The “size” of the granuloma was calculated in terms of the volume of each lesion according to the formula (Boros and Warren 1970):
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tween groups. P values less than 0.05 were considered significant. RESULTS
Effect of S. mansoni Infection on ACE Activity
Treatment of normal animals with captopril resulted in a gradual rise in serum ACE activity, amounting to an increase of 28 and 38% of control values after daily administration for 2 and 4 weeks, respectively. On the other hand, praziquantel administered for 2 days did not affect ACE activity and, when given with captopril, did not influence the latter’s effect on that parameter (Fig. 1). Seven weeks after infection with S. mansoni, the serum ACE was found to have been raised by nearly 48% and continued to rise steadily to reach 65% during the Volume of granuloma = (radius)3 X 4 X i following 4 weeks. Treatment of the infected animals with captopril either alone B. Experiments Carried out on for 4 weeks or in combination with praziLung-Induced Granulomata quantel tended to normalize the raised levInduction of lung granulomas was carried out acels. Praziquantel on its own for 2 days was cording to the method of Moore et al. (1977). Ova also found to reduce the high serum ACE obtained from livers of g-week-old 5. mansoniactivity, the effect being first observed 2 infected mice were suspended in phosphate-buffered saline in a concentration of 8000 eggs/ml. The suspen- weeks after stoppage of treatment and sion (0.5 ml) was injected into the tail vein of 8week-old normal mice using a tuberculin syringe with a 21-gauge needle. The ova embolized in the lungs induced focal granulomas. Sixteen days after ova injection, the animals were divided into two groups of 15-20 mice each, one receiving praziquantel in a dose of 500 mg kg-’ dayy’ for two consecutive days and the other was given 2% Cremophor EL and served as a control. Both groups were sacrificed by decapitation 16 days after the end of treatment, blood was collected and the serum was separated and stored frozen at -2O“C until required for estimation of ACE activity. The lung was dissected out and divided into two portions. One portion was fixed in 10% formalin and processed for light microscopy with haematoxylin-eosin stain to determine the size of the granuloma, while the other was used for isolation of lung granuloma according to the method of Pellegrino and Brenner (1956). The granulomas were then stored frozen at - 70°C until required for determination of ACE activity and protein concentration.
Statistical Analysis Data were subjected to the Student’s t test for unpaired data to determine significant differences be-
2 2 ,E 2
320
300
t
5 a 280 z a: 280 s c6 240. .s :: 220 z E 200I., 5 v)
7
14
Observatiin
period
,
,
21
28
in days
FIG. 1. Effect of treatment with captopril (A) (100 mg kg-’ day-‘), praziquantel (0) (500 mg kg-’ day-‘), and their combination (A) on ACE activity (n mol/min/ml) in the serum of normal mice; ACE activity of normal control (0). Each point represents the mean of 10-20 observations; vertical lines indicate SE of the mean.
120
KHAYYAL
ET AL.
becoming more manifest after 4 weeks (Fig. 2). In infected animals, the granulomas deposited in the liver showed a gradually increasing level of ACE activity, which became especially marked 11 weeks after infection. Both praziquantel and captopril either individually or in combination markedly reduced the ACE activity in the granulomas nearly to the same extent (Fig. 3). This effect was associated with a marked gradual reduction in granuloma size which reached nearly one-third the original size after 4 weeks of treatment with each of the regimens tested (Fig. 4). Parasitological
Findings
Praziquantel caused a marked reduction in worm burden associated with a significant decrease in egg load in both liver and intestine. The oogram showed no viable eggs. The number of ova per gram of intestine was also greatly reduced (Table I). Captopril did not affect the number of
FIG. 3. Effect of treatment with captopril (A) (100 mg kgg’ day-r), praziquantel (0) (500 mg kg-’ day- ‘), and their combination (A) on ACE activity (n mol/min/mg protein) in liver granuloma of infected mice; ACE activity of infected control (0). Each point represents the mean of 10-20 observations; vertical lines indicate SE of the mean.
schistosomes or their distribution and egg load. Histopathological
e
340
I-
1
$t 280. a E ‘E 260. e 2 240. vj .5 .c 220. B 4” g 200. 4 IL B
r 2
a. Infected animals. Histpathological examination of the liver revealed the presence I
aJ300 .E’ I
Findings
-
6oob-‘--------L ------t-d f-0 I\ \ 500
7 Observation
14 period
21
28
in days
FIG. 2. Effect of treatment with captopril (A) (100 mg kg ’ day-‘), praziquantel (0) (500 mg kg- ’ day-‘), and their combination (I?!.)on ACE activity (n moliminiml) in the serum of infected mice; ACE activity of infected control (0). Each point represents the mean of l&20 observations; vertical lines indicate SE of the mean.
Observation
period
in days
FIG. 4. Effect of treatment with captopril (A) (100 mg kg- ’ day- ‘). praziquantel (0) (500 mg kg-’ day- ‘), and their combination (A) on hepatic granuloma size (mm3 x IO-“); granuloma size of infected control (0). Each point represents IO-20 observations; vertical lines indicate SE of the mean.
ACE ACTIVITY
121
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TABLE
I
Effect of Captopril (100 mg kg-’ Day-’ for 4 Weeks), Praziquantel(500 mg kg-’ Dayy’ for 2 Days), and Their Combination on Some Parasitological Parameters Groups Items Worm burden Liver egg load/g (X 103) Intestine egg load/g (X 103) % Mature ova % Dead ova
Control
Captopril
Praziquantel
Combination
14.8 -c 2.2 14.8 t 2.6 16.4 2 3.0 43.0 -t- 5.3 19.1 f 4.1
12.8 _’ 2.3 12.1 2 2.4 15.3 * 2.8 29.3 k 3.2 31.5 k 6.1
1.3 k 0.3* 4.7 2 1.5* 1.2 ” 0.3* 0.4 2 0.4 99.6 _+0.4
1.3 f 0.4* 6.1 2 1.5* 1.0 ?Z0.3* 9.9 * 5.1 90.6 k 5.1
Note. Animals were sacrificed 24 hr after captopril treatment or 4 weeks after praziquantel treatment. * Significant difference from the corresponding control at P g 0.05.
of large granulomata with pronounced cel- lowing infection with S. mansoni (Weinstock and Boros 1981; Weinstock et al. lularity consisting mainly of eosinophils and a smaller number of neutrophils, lym- 1981; Weinstock and Blum 1983) and shows phocytes, plasma cells, macrophages, and that granulomas isolated from liver of intibroblasts at 7 weeks after infection. How- fected mice contain high ACE activity ever, with progression of the infection at 9 which may be released into the circulation and 11 weeks there was a slight decrease in resulting in raised serum ACE activity. the average size of granulomas with less There was a close correlation between cellularity indicating a spontaneous small ACE activity of liver granulomas and cirbut progressive decrease in the granuloma culating serum ACE activity and liver gransize with time. Figure 5 shows a granuloma uloma size. This was also evident after after 11 weeks of infection for comparative treatment with captopril, praziquantel, or their combination. However, it was shown purposes with granulomas after treatment. Treatment with captopril (Fig. 6) or with that with the progress of infection, there is praziquantel (Fig. 7) alone or in combina- a gradual diminution of granuloma size tion (Fig. 8) resulted in a progressive reduc- while the serum ACE activity continues to tion of the granuloma size to about 60% of rise. This suggests that ACE may particiits original size at the end of the experimen- pate in modulation of the granuloma size tal period and caused disintegration of ova. (Weinstock and Boros 1981; Weinstock et b. Experimentally induced lung granu- al. 1981). The source of raised ACE activity loma. Praziquantel did not cause any signif- in schistosomiasis appears to be the macrophages that surround the schistosomal icant change in the level of ACE activity either in serum or in the isolated pulmonary granuloma (Hinman et al. 1979; Hara et al. granuloma (Fig. 9). However, the size of 1981; Weinstock et al. 1981, Weinstock and the granuloma was significantly reduced by Boros 1982). Although treatment with eiabout 40% (Fig. 9), when observed 16 days ther praziquantel or captopril individually after treatment with the drug. The histo- resulted in a reduction in ACE activity as pathological picture of the lung granulo- well as a reduction in granuloma size in the mata before and 16 days after treatment liver, there was no evidence that the comwith praziquantel is shown in Figs. 10 and bination of both drugs was additive, reflecting possibly on the independent nature of 11, respectively. the mode of action of each. DISCUSSION To investigate whether the effect of The present study conBrms earlier obser- praziquantel was a consequence of the anvations that serum ACE activity rises fol- tischistosomal effect of the drug on the
KHAYYAL
ET AL.
FIG. 5. Liver of a mouse after 11 weeks of infection. The figure shows S. mansoni ova with fibrocellular granulomas. (Haematoxylin-Eosin, x 197.5). FIG. 6. Liver of mouse treated with captopril(lO0 mg kgg’ day-’ for 4 weeks) showing a marked reduction in the size and cellularity of the granulomas as compared to controls. (Haematoxylin-Eosin, x 197.5). FIG. 7. Liver of a mouse 4 weeks after treatment with praziquantel(500 mg kg- ’ day-’ for 2 days) showing a marked reduction in the size and cellularity of the granulomas (Haematoxylin-Eosin, x 197.5).
ACE ACTIVITY
IN MICE
123
FIG. 8. Liver of a mouse treated with a combination of captopril(lO0 mg kg- r day- ’ for 4 weeks), and praziquantel (500 mg kg- ’ day-’ for 2 days), showing a marked reduction in the size and cellularity of the granulomas. (Haematoxylin-Eosin, x202.5).
adult worms and a cure from active infection or whether the effect was a direct one affecting the granuloma per se, it was tested in an experimentally induced lung granuloma. Praziquantel was found to reduce granuloma size in the same way as was previously reported by Botros et al., (1984). The reduction in lung granuloma size was not accompanied by a change in ACE activity in the serum or in the granulomata. Histopathological examination of the suppressed granulomata did not show any diminution in the number of macrophages, which as previously mentioned are a major source of ACE activity. This would seem to suggest that the effect of the drug in reducing ACE activity in infected animals is probably a consequence of reducing the worm burden. Furthermore, this would lead one to conclude that the active live worms contribute at least in part to the raised level of serum ACE activity. Captopril when given alone did not alter the number of schistosomes, their distribution, or the egg load. Furthermore, the drug did not affect the reduction in worm burden or in liver egg load induced by praziquantel.
Captopril caused a decrease in liver granuloma ACE activity and reduced its size. Weinstock and Boros (1981) suggested that the drug does not appear to have an antiinflammatory effect on the grounds that it did not suppress the size of lung granuloma induced by methylated bovine serum albumin-coupled sepharose beads, which is an artificial hypersensitivity granuloma that has no ACE activity. The suppression of granuloma size in infected animals was at-
+20 l
10
-50 -60
ACE in Serum
Granuloma
size
FIG. 9. Effect of praziquantel treatment (500 mg kg- ’ day- ‘) on ACE activity in serum and lung granuloma and on the size of pulmonary granuloma 2 weeks after treatment. *Significant difference from the corresponding control at P < 0.05.
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ET AL.
FIG. 10. Pulmonary granuloma of a control mouse representing the mean diameter at 32 days after iv injection of S. munsoni ova, and showing the presence of periovular cellular inflammatory reaction (Haematoxylin-Eosin, x202.5). FIG. 11. Pulmonary granuloma of a mouse treated with praziquantel(500 mg kg- ’ day r for 2 days). Treatment was started 16 days after induction of the lung granuloma, and autopsy was 16 days after end of treatment (Haematoxylin-Eosin, x202.5).
tributed to specific ACE inhibition (Weinstock and Blum, 1985). It would therefore seem from the present study that captopril does not adversely affeet the anti-schistosomal activity of praziquantel and indeed tends to diminish the size of the granulomata. Treatment with
captopril did not influence ACE levels or granuloma sizes in praziquantel-treated mice. The implications of the present findings, particularly with respect to the potential use of captopril to control hypertension associated with schistosomiasis infections, have yet to be resolved.
ACE ACTIVITY
REFERENCES BOOMSMA, F., DEBRUYN, J. E. B., DERKX, F. H. M., AND SCHALEKAMP, M. A. D. H. 1981. Opposite effects of captopril on angiotensin - 1 converting enzyme “activity” and “concentration” relation between enzyme inhibition and long-term pressure response. Clinical Science 60, 491498. BOROS, D. L., AND WARREN, K. S. 1970. Delayed hypersensitivity granuloma formation and dermal reaction induced and elicited by a soluble factor isolated from S. mansoni eggs. Journal of Experimental Medicine 132, 488-507. BOTROS, S. S., METWALLY, A. A., AND KHAYYAL, M. T. 1984. The immunological aspects of praziquantel in unsensitized mice with experimentally induced schistosome pulmonary granuloma. Trunsactions of the Royal Society of Tropical Medicine and Hygiene 78, 569-572. CHEEVER, A. M. 1968. Conditions affecting the accuracy of potassium hydroxide digestion technique for counting Schistosoma mansoni eggs in tissue. Bulletin of the World Health Organization 39, 328-331. EL-HAWEY, A. M., HASSAN, I., EL-IBIARY, S., AND MASSOLJD, A. M. 1986. Histopathological changes and experimental Schistosoma mansoni before and after praziquantel therapy. Journal of the Egyptian Society of Parasitology 16(l), 117-125. EL-IBIARY, S. A., EL-HAWEY, A. M., MAHMOUD, I. H., AMER, A. M., AND KAMAL EL-DIN, M. 1986. The study of histopathological changes in experimental schistosomiasis before and after praziquantel therapy. Journal of the Egyptian Society of Pathology S(l), 141-152. FORSLUND, T., FYHRQUIST, F., GRONHAGEN-RISKA, C., AND TIKKANEN, I. 1982. Induction of angiotensin converting enzyme with the ACE inhibitory compound MK- 42 1. in the rat lung. European Journal of Pharmacology 80, 121-125. GONNERT, R., AND ANDREWS, P. 1977. Praziquantel, a new broad spectrum antischistosomal agent. Zeitschrift fuer Parasitenkunde 52, 12%150. GRIVAUX, M., PIERON, R., LANCASTRE, R., BENETEAU, B., AND BAUMANN, F. S. 1986. Angiotensin converting enzyme and schistosomiasis. Medicine et les Maladies Infectieuses 16[2(1)1, 72-77. HARA, A., FUKUYAMA, K., AND EPSTEIN, W. L. 1981. Angiotensin converting enzyme and other enzymes in livers of mice with experimental schistosomiasis. Experimental Molecular Pathology 35, 199-210. HINMAN, L. M., STEVENS, C., METTHAY, R. A., GEE, J. B. 1979. Angiotensin convertase activities in human alveolar macrophages. Effect of cigarette smoking and sarcoidosis. Science 205, 202-203.
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HURST, P. L., AND LOVELL-SMITH, C. J. 1981. Optimized assay for serum angiotensin-converting enzyme activity. Clinical Chemistry 47(12), 2048-2052. JAMES, C., WEBBE, G., AND NELSON, G. S. 1977. The susceptibility to praziquantel of Schistosoma haematobium in the baboon (Papio. anuhis) and of Schistosomn japonicum in the vevert monkey. Zeitschrift fuer Parasitenkunde 52, 179-194. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. 1951. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265-275. MEHLHORN, H., FRENKEL, J. K., ANDREWS, P., AND THOMAS, H. 1982. Light and electron microscopic studies on Schistosoma mansoni granulomas of mouse livers following treatment with praziquantel. Tropenmedizin und Parasitologie 33, 229-239. MOORE, D. L., GROVE, D. I., AND WARREN, K. S. 1977. The Schistosoma mansoni egg granuloma quantitation of cell populations. Journal of Pathology 121, 41-50. ONDETTI, M. A., RUBIN, B., AND CUSHMAN, D. W. 1977. Design of specific inhibitors of A.C.E. New class of orally active antihypertensive agents. Science 196, 441-444. PELLEGRINO, J., AND BRENNER, 2. 1956. Method of isolating Schistosome granulomas from mouse liver. Journal of Parasitology 42(6), 564. PELLEGRINO, J., OLIVERA, C. A., FARIA. J., AND CUNHA, A. S. 1962. New approach to the screening of drug in experimental Schistosomiasis mansoni in mice. American Journal of Tropical Medicine and Hygiene 11, 201-215. PELLEGRINO, J., AND SIQUEIRA, A. F. 1956. Tecnica para perfusae para colhejta de schistosoma mansoni: Infestadas. Revista Brasileira de Malariol. 8, 589597. PETERS, P. A., AND WARREN, K. S. 1969. A rapid method of infecting mice and other laboratory animals with Schistosomiusis mansoni subcutaneous injection. Journal of Parasitology 55, 558. RUBIN, B., LAFFAN, R. J., KOTLER, D. G., O’KEEFE, E. H., DEMAIO, D., AND GOLDBERG, M. E. 1978. SQ 14,225 (D-3-mercapto-2-methyl propanoyl-Lproline) a novel orally active inhibitor of angiotensin - 1 converting enzyme. Journal of Pharmacology and Experimental Therapeutics 204, 271-280. WEINSTOCK, J. V., AND BLUM, A. M. 1983. Isolated liver granulomas of murine S. mansoni contain components of the angiotensin system. Journal of Immunology 131(5), 2529-2532. WEINSTOCK, J. V., AND BLUM, A. M., 1985. Effect of granuloma modulation induced by regulatory T-lymphocyte activity on angiotensin II/III production by granuloma macrophages in murine S. mansoni. Cellular Immunology 94(2), 558-567.
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J. V., AND BOROS, D. L. 1981. Alteration of the granulomatous response in murine schistosomiasis by the chronic administration of captopril, an inhibitor of angiotensin-converting en81, 953-958. zyme. Gastroenterology WEINSTOCK, J. V., AND BOROS, D. L. 1982. Production of angiotensin - 1 converting enzyme by liver granuloma formation of Schistosomiasis mansoni infected mice. Gasfroenterology 82, 106110. WEINSTOCK, J. V., BOROS, D. L., AND GEE, J. B. 1979.Angiotensin converting enzyme activity in isolated granulomas of mice with Schistosomiasis mansoni. Gastroenterology 77, A46. WEINSTOCK,
ET AL. J. V., BOROS, D. L., AND GEE, J. B. 1981.Enhanced granuloma angiotensin - 1 converting enzyme activity associated with modulation in murine schistosomiasis. Gastroenterology 81, 48-
WEINSTOCK,
53. J. V., AND EHRINPREIS, M. N. 1981. Effect of SQ 14,225, an inhibitor of angiotensin - 1 converting enzyme on the granuloma response to Schistosomiasis mansoni eggs in mice. Journal of Clinical Investigation 67(4), 931-936.
WEINSTOCK,
Received 11 June 1990; accepted with revision 8 December 1990
PARASITOLOGY
73,
117-126(1991)
Schistosoma mansoni: Angiotensin Converting Enzyme Activity in Mice under the Influence of Praziquantel and/or Captopril M.T. KHAYYAL,S.SALEH, A. A. METWALLY,* S. S.BOTROS,* AND M.R. MAHMOUD* Departments
of Pharmacology, Faculty
of Pharmacy, Cairo University, and *Theodor Biiharz Research Institute, Embaba, Giza, Egypt
KHAYYAL, M. T., SALEH, S., METWALLY, A. A., 1991. Schistosoma mansoni: Angiotensin converting
BOTROS,
S.S.,
MAHMOUD,
M.R.
enzyme activity in mice under the influence of praziquantel and/or captopril. Experimental Parasitology, 73, 117-126. Murine schistosomiasis is usually associated with hepatic granulomatous lesions together with high serum and granuloma angiotensin converting enzyme (ACE) activity. Praziquantel (PRZ) which is known to reduce granuloma size was studied to show whether this effect is related to changes in ACE activity. Furthermore, captopril was studied to show whether by inhibiting ACE activity, the drug could also affect granuloma size. PRZ, captopril, and their combination led to significant reduction in liver granuloma. However, in normal mice, captopril was shown to increase rather than decrease serum ACE. The decrease in ACE activity by PRZ was correlated with its curative effect in infected mice. However, in experimentally induced pulmonary granulomata, the drug reduced granuloma size without affecting ACE activity of either serum or granuloma. It may be concluded that reduction in ACE activity may be beneficial as far as diminution of granuloma size is concerned and irrespective of whether there is an active infection or not. The possible use of Captopril as an antihypertensive in bilharzial infections associated with hypertension would probably not adversely affect the granulomatous lesions. 0 1991 Academic Press, inc. INDEX DESCRIPTORS: Schistosoma mansoni; Trematoda; Angiotensin converting enzyme (ACE); Liver granuloma; Praziquantel; Captopril; Serum Ace inhibitors; Histopathology; Lung granuloma; Oogram; Lesions; Enzyme
Murine Schistosoma mansoni has been shown to be associated with the development of granulomas in the liver and the intestine of infected animals. Such granulomas have high ACE activity which seems to be dependent on the size of the granulomata and chronicity of infection (Weinstock and Boros 1981, 1982). The macrophages surrounding the granulomas also have high ACE activity which could possibly contribute to the elevated serum ACE activity of the infected animals (Weinstock and Boros 1982). Inhibition of ACE activity by effective drugs, e.g., captopril, has been shown by Weinstock and Boros (1981, 1982) to reduce granufoma size too, but the effect appeared to be reversible upon drug withdrawal (Boomsma et al. 1981; Wein-
stock and Boros 1981; Forslund et al. 1982). A reduction in granuloma size and cellularity was also observed in infected hamsters treated with praziquantel (James et al. 1977; El-Hawey et al. 1986; El-Ibiary et al. 1986). Mehlhorn et al. (1982) also reported the involution of liver granulomas after treating infected mice effectively with the drug. Furthermore, in the absence of an active infection, it was observed that the lung granulomas from normal and sensitized mice injected intravenously with schistosome eggs also show high ACE activity (Weinstock et al. 1979). In humans, S. mansoni infection has been shown to increase serum ACE activity, while treatment was shown to reduce it (Grivaux et al. 1986). ACE inhibitors are frequently used to treat hypertension clinically (Ondetti et al. 117 0014-4894/91$3.00 Copyright 6 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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1977, Rubin et al. 1978). The present experimental study was conducted to show whether the use of captopril would influence the curative process initiated by specific anti-schistosomal therapy as far as the granulomatous lesions are concerned. Captopril has therefore been tested together with praziquantel on the ACE activity and size of liver granulomata caused by active infection as well as in lung-induced granulomata in noninfected animals. Furthermore, the effect on serum ACE has been studied in both normal and infected animals under the influence of praziquantel and/or captopril. MATERIALS
Animals
AND METHODS
Used and Methods of Infection
Laboratory-bred albino mice, 18-20 g each, were infected subcutaneously with 60 cercariae of an Egyptian strain of S. mansoni as described by Peters and Warren (1969). They were used 7 weeks after infection.
Drugs and Dosage Praziquantel (Bayer AG, Leverkusen, FRG) was given orally in a dose of 500 mg kg- ‘ body weight for 2 consecutive days. This was shown to be the effective curative dose in experimentally S. mansoni-infected mice (Gonnert and Andrews 1977).The drug was given as a suspension in 2% Cremophor EL (Sigma Chemical Company, St. Louis, MO). Captopril (E. R. Squibb & Sons, Inc. Princeton, NJ) was given orally in a dose of 100 mg kg- ’ day for 4 weeks. This dose for this length of time has been used by other authors for studying the effect of ACE inhibition (Weinstock and Boros 1981; Weinstock and Ehrinpreis 1981).The drug was given in the form of a 0.1% aqueous solution.
Experimental
Design
This study involved experiments carried out on granuloma from S. mansoni-infected mice and others conducted on lung-induced granulomata from noninfected animals.
A. Experiments Carried Out on Granuloma from S. mansoniInfected Animals Infected animals were divided into four groups, each consisting of 10-20 mice and subjected to the following treatment regimens:
Group I: The animals were given captopril daily for 4 weeks. The mice were sacrificed 24 hr after 1, 14, and 28 doses. Group 2: The mice were given praziquantel for 2 consecutive days. The animals were sacrificed 24 hr. 2 weeks, and 4 weeks after the last dose. Group 3: Animals in this group were treated with captopril and praziquantel. Praziquantel was given for 2 successive days. Captopril treatment was started at the same time and continued for 4 weeks. Animals were sacrificed 24 hr, 2 weeks, and 4 weeks after the last dose of praziquantel (i.e., 24 hr after the last dose of captopril). Group 4: In this group. the mice were given 2% Cremophor EL in the same volume used for praziquantel gavage daily for 2 days and served as concurrent controls to the praziquantel-treated groups. Animals were sacrificed at 24 hr, 2 weeks, and 4 weeks after the last dose. In another set of experiments, normal (noninfected) animals were also subdivided into four groups and subjected to the same treatment regimens described above. Procedure at autopsy. Mice were killed by decapitation, and the blood was collected. The serum was separated by centrifugation at 3000 rpm for 10 min and stored frozen at - 20°C for the assay of ACE activity. Infected animals, after decapitation, were perfused with saline (Pellegrino and Sigueira 1956), and the worms were collected separately from the liver and portomesenteric veins and counted. Livers were then removed and divided into three portions, one portion was used for ova counting, one for isolation of granulomata and, one fixed in 10% formalin for histopathological examination of granulomata and determination of granuloma size. The intestine was also removed for ova counting and oogram studies. Ova counting and oogram studies. A portion of liver or intestine was digested overnight in 4% KOH for counting S. mansoni eggs, as described by Cheever (1968). A fragment of intestine (1 cm long) was cut off from each mouse, dried on a filter paper, and then spread on a slide for microscopical examination. One hundred ova in each fragment were examined, counted, and classified according to their degree of development (Pellegrino ef ~1. 1962). Isolation ofgranulomas. Granulomas from the liver of lung were isolated by the method of Pellegrino and Brenner (1956). Subsequently, they were washed three times with Hanks’ balanced salt solution (HBSS), packed at 3000 g for 5 min at 20°C to remove all supernatant, and frozen at -70°C. Under these conditions, there is no change in ACE activity for up to two months (Weinstock and Boros 1981). Assay
of ACE
acrivity
and protein
concentration,
To determine the ACE activity in isolated granulomas, granulomas were ground in a mortar with a pestle for 2 min (200 mg tissue/3 ml HBSS) on ice and subse-
ACE ACTIVITY quently sonicated on ice for 30 sec. One aliquot was used for determination of protein concentration using Folin Ciocalteu phenol reagent (Fisher Scientific Co., New York) (Lowry et al. 1951), while the other ahquot was used for determination of ACE activity using hippuryl-L-histidyl I-leucine substrate (Sigma Chemical Co., St. Louis, MO 63178) according to the method described by Hurst and Lovell-Smith, (1981). Granuloma ACE activity was calculated as nmoles/min/mg protein. Determination of grunuloma size. Isolated circumscribed granulomas containing parasite ova were located microscopically after staining with haematoxyhn and eosin in the histologic sections of the liver or lung. Their diameters were measured with an image-splitting eye piece (Vicker’s Instrument, Wobum, MA) by determining the mean of two measurements made across perpendicular axes. The mean diameter of at least 50 lesions from the animals of each group was measured. The “size” of the granuloma was calculated in terms of the volume of each lesion according to the formula (Boros and Warren 1970):
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tween groups. P values less than 0.05 were considered significant. RESULTS
Effect of S. mansoni Infection on ACE Activity
Treatment of normal animals with captopril resulted in a gradual rise in serum ACE activity, amounting to an increase of 28 and 38% of control values after daily administration for 2 and 4 weeks, respectively. On the other hand, praziquantel administered for 2 days did not affect ACE activity and, when given with captopril, did not influence the latter’s effect on that parameter (Fig. 1). Seven weeks after infection with S. mansoni, the serum ACE was found to have been raised by nearly 48% and continued to rise steadily to reach 65% during the Volume of granuloma = (radius)3 X 4 X i following 4 weeks. Treatment of the infected animals with captopril either alone B. Experiments Carried out on for 4 weeks or in combination with praziLung-Induced Granulomata quantel tended to normalize the raised levInduction of lung granulomas was carried out acels. Praziquantel on its own for 2 days was cording to the method of Moore et al. (1977). Ova also found to reduce the high serum ACE obtained from livers of g-week-old 5. mansoniactivity, the effect being first observed 2 infected mice were suspended in phosphate-buffered saline in a concentration of 8000 eggs/ml. The suspen- weeks after stoppage of treatment and sion (0.5 ml) was injected into the tail vein of 8week-old normal mice using a tuberculin syringe with a 21-gauge needle. The ova embolized in the lungs induced focal granulomas. Sixteen days after ova injection, the animals were divided into two groups of 15-20 mice each, one receiving praziquantel in a dose of 500 mg kg-’ dayy’ for two consecutive days and the other was given 2% Cremophor EL and served as a control. Both groups were sacrificed by decapitation 16 days after the end of treatment, blood was collected and the serum was separated and stored frozen at -2O“C until required for estimation of ACE activity. The lung was dissected out and divided into two portions. One portion was fixed in 10% formalin and processed for light microscopy with haematoxylin-eosin stain to determine the size of the granuloma, while the other was used for isolation of lung granuloma according to the method of Pellegrino and Brenner (1956). The granulomas were then stored frozen at - 70°C until required for determination of ACE activity and protein concentration.
Statistical Analysis Data were subjected to the Student’s t test for unpaired data to determine significant differences be-
2 2 ,E 2
320
300
t
5 a 280 z a: 280 s c6 240. .s :: 220 z E 200I., 5 v)
7
14
Observatiin
period
,
,
21
28
in days
FIG. 1. Effect of treatment with captopril (A) (100 mg kg-’ day-‘), praziquantel (0) (500 mg kg-’ day-‘), and their combination (A) on ACE activity (n mol/min/ml) in the serum of normal mice; ACE activity of normal control (0). Each point represents the mean of 10-20 observations; vertical lines indicate SE of the mean.
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ET AL.
becoming more manifest after 4 weeks (Fig. 2). In infected animals, the granulomas deposited in the liver showed a gradually increasing level of ACE activity, which became especially marked 11 weeks after infection. Both praziquantel and captopril either individually or in combination markedly reduced the ACE activity in the granulomas nearly to the same extent (Fig. 3). This effect was associated with a marked gradual reduction in granuloma size which reached nearly one-third the original size after 4 weeks of treatment with each of the regimens tested (Fig. 4). Parasitological
Findings
Praziquantel caused a marked reduction in worm burden associated with a significant decrease in egg load in both liver and intestine. The oogram showed no viable eggs. The number of ova per gram of intestine was also greatly reduced (Table I). Captopril did not affect the number of
FIG. 3. Effect of treatment with captopril (A) (100 mg kgg’ day-r), praziquantel (0) (500 mg kg-’ day- ‘), and their combination (A) on ACE activity (n mol/min/mg protein) in liver granuloma of infected mice; ACE activity of infected control (0). Each point represents the mean of 10-20 observations; vertical lines indicate SE of the mean.
schistosomes or their distribution and egg load. Histopathological
e
340
I-
1
$t 280. a E ‘E 260. e 2 240. vj .5 .c 220. B 4” g 200. 4 IL B
r 2
a. Infected animals. Histpathological examination of the liver revealed the presence I
aJ300 .E’ I
Findings
-
6oob-‘--------L ------t-d f-0 I\ \ 500
7 Observation
14 period
21
28
in days
FIG. 2. Effect of treatment with captopril (A) (100 mg kg ’ day-‘), praziquantel (0) (500 mg kg- ’ day-‘), and their combination (I?!.)on ACE activity (n moliminiml) in the serum of infected mice; ACE activity of infected control (0). Each point represents the mean of l&20 observations; vertical lines indicate SE of the mean.
Observation
period
in days
FIG. 4. Effect of treatment with captopril (A) (100 mg kg- ’ day- ‘). praziquantel (0) (500 mg kg-’ day- ‘), and their combination (A) on hepatic granuloma size (mm3 x IO-“); granuloma size of infected control (0). Each point represents IO-20 observations; vertical lines indicate SE of the mean.
ACE ACTIVITY
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TABLE
I
Effect of Captopril (100 mg kg-’ Day-’ for 4 Weeks), Praziquantel(500 mg kg-’ Dayy’ for 2 Days), and Their Combination on Some Parasitological Parameters Groups Items Worm burden Liver egg load/g (X 103) Intestine egg load/g (X 103) % Mature ova % Dead ova
Control
Captopril
Praziquantel
Combination
14.8 -c 2.2 14.8 t 2.6 16.4 2 3.0 43.0 -t- 5.3 19.1 f 4.1
12.8 _’ 2.3 12.1 2 2.4 15.3 * 2.8 29.3 k 3.2 31.5 k 6.1
1.3 k 0.3* 4.7 2 1.5* 1.2 ” 0.3* 0.4 2 0.4 99.6 _+0.4
1.3 f 0.4* 6.1 2 1.5* 1.0 ?Z0.3* 9.9 * 5.1 90.6 k 5.1
Note. Animals were sacrificed 24 hr after captopril treatment or 4 weeks after praziquantel treatment. * Significant difference from the corresponding control at P g 0.05.
of large granulomata with pronounced cel- lowing infection with S. mansoni (Weinstock and Boros 1981; Weinstock et al. lularity consisting mainly of eosinophils and a smaller number of neutrophils, lym- 1981; Weinstock and Blum 1983) and shows phocytes, plasma cells, macrophages, and that granulomas isolated from liver of intibroblasts at 7 weeks after infection. How- fected mice contain high ACE activity ever, with progression of the infection at 9 which may be released into the circulation and 11 weeks there was a slight decrease in resulting in raised serum ACE activity. the average size of granulomas with less There was a close correlation between cellularity indicating a spontaneous small ACE activity of liver granulomas and cirbut progressive decrease in the granuloma culating serum ACE activity and liver gransize with time. Figure 5 shows a granuloma uloma size. This was also evident after after 11 weeks of infection for comparative treatment with captopril, praziquantel, or their combination. However, it was shown purposes with granulomas after treatment. Treatment with captopril (Fig. 6) or with that with the progress of infection, there is praziquantel (Fig. 7) alone or in combina- a gradual diminution of granuloma size tion (Fig. 8) resulted in a progressive reduc- while the serum ACE activity continues to tion of the granuloma size to about 60% of rise. This suggests that ACE may particiits original size at the end of the experimen- pate in modulation of the granuloma size tal period and caused disintegration of ova. (Weinstock and Boros 1981; Weinstock et b. Experimentally induced lung granu- al. 1981). The source of raised ACE activity loma. Praziquantel did not cause any signif- in schistosomiasis appears to be the macrophages that surround the schistosomal icant change in the level of ACE activity either in serum or in the isolated pulmonary granuloma (Hinman et al. 1979; Hara et al. granuloma (Fig. 9). However, the size of 1981; Weinstock et al. 1981, Weinstock and the granuloma was significantly reduced by Boros 1982). Although treatment with eiabout 40% (Fig. 9), when observed 16 days ther praziquantel or captopril individually after treatment with the drug. The histo- resulted in a reduction in ACE activity as pathological picture of the lung granulo- well as a reduction in granuloma size in the mata before and 16 days after treatment liver, there was no evidence that the comwith praziquantel is shown in Figs. 10 and bination of both drugs was additive, reflecting possibly on the independent nature of 11, respectively. the mode of action of each. DISCUSSION To investigate whether the effect of The present study conBrms earlier obser- praziquantel was a consequence of the anvations that serum ACE activity rises fol- tischistosomal effect of the drug on the
KHAYYAL
ET AL.
FIG. 5. Liver of a mouse after 11 weeks of infection. The figure shows S. mansoni ova with fibrocellular granulomas. (Haematoxylin-Eosin, x 197.5). FIG. 6. Liver of mouse treated with captopril(lO0 mg kgg’ day-’ for 4 weeks) showing a marked reduction in the size and cellularity of the granulomas as compared to controls. (Haematoxylin-Eosin, x 197.5). FIG. 7. Liver of a mouse 4 weeks after treatment with praziquantel(500 mg kg- ’ day-’ for 2 days) showing a marked reduction in the size and cellularity of the granulomas (Haematoxylin-Eosin, x 197.5).
ACE ACTIVITY
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123
FIG. 8. Liver of a mouse treated with a combination of captopril(lO0 mg kg- r day- ’ for 4 weeks), and praziquantel (500 mg kg- ’ day-’ for 2 days), showing a marked reduction in the size and cellularity of the granulomas. (Haematoxylin-Eosin, x202.5).
adult worms and a cure from active infection or whether the effect was a direct one affecting the granuloma per se, it was tested in an experimentally induced lung granuloma. Praziquantel was found to reduce granuloma size in the same way as was previously reported by Botros et al., (1984). The reduction in lung granuloma size was not accompanied by a change in ACE activity in the serum or in the granulomata. Histopathological examination of the suppressed granulomata did not show any diminution in the number of macrophages, which as previously mentioned are a major source of ACE activity. This would seem to suggest that the effect of the drug in reducing ACE activity in infected animals is probably a consequence of reducing the worm burden. Furthermore, this would lead one to conclude that the active live worms contribute at least in part to the raised level of serum ACE activity. Captopril when given alone did not alter the number of schistosomes, their distribution, or the egg load. Furthermore, the drug did not affect the reduction in worm burden or in liver egg load induced by praziquantel.
Captopril caused a decrease in liver granuloma ACE activity and reduced its size. Weinstock and Boros (1981) suggested that the drug does not appear to have an antiinflammatory effect on the grounds that it did not suppress the size of lung granuloma induced by methylated bovine serum albumin-coupled sepharose beads, which is an artificial hypersensitivity granuloma that has no ACE activity. The suppression of granuloma size in infected animals was at-
+20 l
10
-50 -60
ACE in Serum
Granuloma
size
FIG. 9. Effect of praziquantel treatment (500 mg kg- ’ day- ‘) on ACE activity in serum and lung granuloma and on the size of pulmonary granuloma 2 weeks after treatment. *Significant difference from the corresponding control at P < 0.05.
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FIG. 10. Pulmonary granuloma of a control mouse representing the mean diameter at 32 days after iv injection of S. munsoni ova, and showing the presence of periovular cellular inflammatory reaction (Haematoxylin-Eosin, x202.5). FIG. 11. Pulmonary granuloma of a mouse treated with praziquantel(500 mg kg- ’ day r for 2 days). Treatment was started 16 days after induction of the lung granuloma, and autopsy was 16 days after end of treatment (Haematoxylin-Eosin, x202.5).
tributed to specific ACE inhibition (Weinstock and Blum, 1985). It would therefore seem from the present study that captopril does not adversely affeet the anti-schistosomal activity of praziquantel and indeed tends to diminish the size of the granulomata. Treatment with
captopril did not influence ACE levels or granuloma sizes in praziquantel-treated mice. The implications of the present findings, particularly with respect to the potential use of captopril to control hypertension associated with schistosomiasis infections, have yet to be resolved.
ACE ACTIVITY
REFERENCES BOOMSMA, F., DEBRUYN, J. E. B., DERKX, F. H. M., AND SCHALEKAMP, M. A. D. H. 1981. Opposite effects of captopril on angiotensin - 1 converting enzyme “activity” and “concentration” relation between enzyme inhibition and long-term pressure response. Clinical Science 60, 491498. BOROS, D. L., AND WARREN, K. S. 1970. Delayed hypersensitivity granuloma formation and dermal reaction induced and elicited by a soluble factor isolated from S. mansoni eggs. Journal of Experimental Medicine 132, 488-507. BOTROS, S. S., METWALLY, A. A., AND KHAYYAL, M. T. 1984. The immunological aspects of praziquantel in unsensitized mice with experimentally induced schistosome pulmonary granuloma. Trunsactions of the Royal Society of Tropical Medicine and Hygiene 78, 569-572. CHEEVER, A. M. 1968. Conditions affecting the accuracy of potassium hydroxide digestion technique for counting Schistosoma mansoni eggs in tissue. Bulletin of the World Health Organization 39, 328-331. EL-HAWEY, A. M., HASSAN, I., EL-IBIARY, S., AND MASSOLJD, A. M. 1986. Histopathological changes and experimental Schistosoma mansoni before and after praziquantel therapy. Journal of the Egyptian Society of Parasitology 16(l), 117-125. EL-IBIARY, S. A., EL-HAWEY, A. M., MAHMOUD, I. H., AMER, A. M., AND KAMAL EL-DIN, M. 1986. The study of histopathological changes in experimental schistosomiasis before and after praziquantel therapy. Journal of the Egyptian Society of Pathology S(l), 141-152. FORSLUND, T., FYHRQUIST, F., GRONHAGEN-RISKA, C., AND TIKKANEN, I. 1982. Induction of angiotensin converting enzyme with the ACE inhibitory compound MK- 42 1. in the rat lung. European Journal of Pharmacology 80, 121-125. GONNERT, R., AND ANDREWS, P. 1977. Praziquantel, a new broad spectrum antischistosomal agent. Zeitschrift fuer Parasitenkunde 52, 12%150. GRIVAUX, M., PIERON, R., LANCASTRE, R., BENETEAU, B., AND BAUMANN, F. S. 1986. Angiotensin converting enzyme and schistosomiasis. Medicine et les Maladies Infectieuses 16[2(1)1, 72-77. HARA, A., FUKUYAMA, K., AND EPSTEIN, W. L. 1981. Angiotensin converting enzyme and other enzymes in livers of mice with experimental schistosomiasis. Experimental Molecular Pathology 35, 199-210. HINMAN, L. M., STEVENS, C., METTHAY, R. A., GEE, J. B. 1979. Angiotensin convertase activities in human alveolar macrophages. Effect of cigarette smoking and sarcoidosis. Science 205, 202-203.
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HURST, P. L., AND LOVELL-SMITH, C. J. 1981. Optimized assay for serum angiotensin-converting enzyme activity. Clinical Chemistry 47(12), 2048-2052. JAMES, C., WEBBE, G., AND NELSON, G. S. 1977. The susceptibility to praziquantel of Schistosoma haematobium in the baboon (Papio. anuhis) and of Schistosomn japonicum in the vevert monkey. Zeitschrift fuer Parasitenkunde 52, 179-194. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. 1951. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265-275. MEHLHORN, H., FRENKEL, J. K., ANDREWS, P., AND THOMAS, H. 1982. Light and electron microscopic studies on Schistosoma mansoni granulomas of mouse livers following treatment with praziquantel. Tropenmedizin und Parasitologie 33, 229-239. MOORE, D. L., GROVE, D. I., AND WARREN, K. S. 1977. The Schistosoma mansoni egg granuloma quantitation of cell populations. Journal of Pathology 121, 41-50. ONDETTI, M. A., RUBIN, B., AND CUSHMAN, D. W. 1977. Design of specific inhibitors of A.C.E. New class of orally active antihypertensive agents. Science 196, 441-444. PELLEGRINO, J., AND BRENNER, 2. 1956. Method of isolating Schistosome granulomas from mouse liver. Journal of Parasitology 42(6), 564. PELLEGRINO, J., OLIVERA, C. A., FARIA. J., AND CUNHA, A. S. 1962. New approach to the screening of drug in experimental Schistosomiasis mansoni in mice. American Journal of Tropical Medicine and Hygiene 11, 201-215. PELLEGRINO, J., AND SIQUEIRA, A. F. 1956. Tecnica para perfusae para colhejta de schistosoma mansoni: Infestadas. Revista Brasileira de Malariol. 8, 589597. PETERS, P. A., AND WARREN, K. S. 1969. A rapid method of infecting mice and other laboratory animals with Schistosomiusis mansoni subcutaneous injection. Journal of Parasitology 55, 558. RUBIN, B., LAFFAN, R. J., KOTLER, D. G., O’KEEFE, E. H., DEMAIO, D., AND GOLDBERG, M. E. 1978. SQ 14,225 (D-3-mercapto-2-methyl propanoyl-Lproline) a novel orally active inhibitor of angiotensin - 1 converting enzyme. Journal of Pharmacology and Experimental Therapeutics 204, 271-280. WEINSTOCK, J. V., AND BLUM, A. M. 1983. Isolated liver granulomas of murine S. mansoni contain components of the angiotensin system. Journal of Immunology 131(5), 2529-2532. WEINSTOCK, J. V., AND BLUM, A. M., 1985. Effect of granuloma modulation induced by regulatory T-lymphocyte activity on angiotensin II/III production by granuloma macrophages in murine S. mansoni. Cellular Immunology 94(2), 558-567.
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J. V., AND BOROS, D. L. 1981. Alteration of the granulomatous response in murine schistosomiasis by the chronic administration of captopril, an inhibitor of angiotensin-converting en81, 953-958. zyme. Gastroenterology WEINSTOCK, J. V., AND BOROS, D. L. 1982. Production of angiotensin - 1 converting enzyme by liver granuloma formation of Schistosomiasis mansoni infected mice. Gasfroenterology 82, 106110. WEINSTOCK, J. V., BOROS, D. L., AND GEE, J. B. 1979.Angiotensin converting enzyme activity in isolated granulomas of mice with Schistosomiasis mansoni. Gastroenterology 77, A46. WEINSTOCK,
ET AL. J. V., BOROS, D. L., AND GEE, J. B. 1981.Enhanced granuloma angiotensin - 1 converting enzyme activity associated with modulation in murine schistosomiasis. Gastroenterology 81, 48-
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53. J. V., AND EHRINPREIS, M. N. 1981. Effect of SQ 14,225, an inhibitor of angiotensin - 1 converting enzyme on the granuloma response to Schistosomiasis mansoni eggs in mice. Journal of Clinical Investigation 67(4), 931-936.
WEINSTOCK,
Received 11 June 1990; accepted with revision 8 December 1990
