Annals of Tropical Medicine & Parasitology
ISSN: 0003-4983 (Print) 1364-8594 (Online) Journal homepage: http://www.tandfonline.com/loi/ypgh19
Experimental schistosomiasis mansoni: modulation of granulomas by inhibition of collagen cross-link formation. Preliminary report M. Giboda, J. Ženka, I. Juliš & J. Vítovec To cite this article: M. Giboda, J. Ženka, I. Juliš & J. Vítovec (1992) Experimental schistosomiasis mansoni: modulation of granulomas by inhibition of collagen cross-link formation. Preliminary report, Annals of Tropical Medicine & Parasitology, 86:6, 631-636, DOI: 10.1080/00034983.1992.11812719 To link to this article: https://doi.org/10.1080/00034983.1992.11812719
Published online: 15 Nov 2016.
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Citing articles: 5 View citing articles
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Annals of Tropical Medicine and Parasitology, Vol. 86, No.6, 631-636 (1992)
Experimental schistosomiasis mansoni: modulation of granulomas by inhibition of collagen cross-link formation. Preliminary report BY M. GIBODA,]. ZENKA Institute ofParasitology, Czechoslovak Academy of Sciences, BramJovska 31, CS -3 70 OS Ceske Budejovice, Czechoslovakia
I. JULIS Department of Pathology, Third Medical Faculty, Charles University, Prague, Czechoslovakia AND].
VfTOVEC
Imtitute of Parasitology, Czechoslovak Academy of Sciences, Branisovska 31, CS-370 OS Ceske Budijovice, Czechoslovakia Received 2 June 1992, Revised 28 September 1992, Accepted 28 September 1992
/1-Aminopropionitrile (BAPN) is an inhibitor of the lysyl oxidase required for cross-link formation in collagen maturation. The efficacy of BAPN, alone or in association with the anti-schistosoma! drug, praziquantel (PZQ), was primarily assessed by measuring the reduction in liver and intestinal egg loads in murine schistosomiasis mansoni. Depending on the treatment group (PZQ, BAPN, BAPN + PZQ), organspecific effects were observed using microscope image analysis. Most notable was the relatively small size of granulomas in the livers of BAPN-treated mice, which contrasted with the relatively large size and irregular shape of the granulomas in the intestinal tissues of these mice. Mice treated with the combination ofBAPN and PZQhad decreased liver and spleen weights, and a significant reduction in the number of eggs trapped in both the liver (86%) and the intestine (99·1 %), compared with untreated mice and those given PZQalone. The lowest number ofliving eggs/g of tissue in both the liver and intestine was recorded in the combined BAPN + PZQ-treated group. These results suggest that the concurrent treatment of infected mice with PZQand BAPN enhances the release of eggs trapped in the intestine and also results in a significant reduction ofliver egg load. The mechanism by which BAPN reduces the number of liver granulomas in PZQ-treated mice is currently being investigated.
The pathology of schistosomiasis is based on the host tissues' immunological reaction to the parasite eggs. Therefore, reduction of the number of eggs trapped in the tissues is a reasonable therapeutic goal for treatment of schistosomiasis in man. Granuloma formation around the eggs, and ensuing fibrosis, involve an increased synthesis 0003-4983/92/060631 + 06 $08.00/0
of extracellular components of connective tissue, particularly collagen and glycosaminoglycans (Grimaud 1987a). It is possible to affect, pharmacologically, the post-translation steps of collagen biosynthesis. Inhibition of maturation (polymerization) of collagen has so far been the most successful and powerful method of interfering with the physical © 1992 Liverpool School of Tropical Medicine
632
GIBODA ET AL.
properties of fibrotic structures. In the course of maturation the collagen fibrils are stabilized by lysine-derived intermolecular cross-links. The intermolecular cross-links play a crucial role in determining the characteristic physical and chemical properties of the collagen (Ricard-Bium and Ville, 1988). Lysyl oxidase is the enzyme required for cross-link formation in collagen and its activity is irreversibly inhibited by fJaminopropionitrile (BAPN). The successful experimental trials of Peacock and Madden (1966), Craver et a/. (1968) and Davis et a/. ( 1972), who employed BAPN to enhance wound healing in animals, lead to the application of BAPN in controlled clinical studies (Peacock and Madden, 1966; Keiser and Sjoerdsma, 1967). The rational for using BAPN in the treatment of Schistosoma mansoni infection was to decrease the rigidity and increase the solubility of newly produced collagen in the periovular granulomas and thereby promote the release of eggs trapped in the tissues. PZQ primarily kills adult worms, but also mature eggs (Richards et a/., 1989) and will prevent the saturation of host tissues with eggs laid by S. mansoni females. Our hypothesis, therefore, was that the combination of BAPN and PZQ would be more effective than PZQ alone in reducing the tissue egg load and resulting pathology. Particular attention was paid to the quantitative microscopic evaluation of the granulomatous response induced by eggs which accumulated in the liver and intestine of treated mice.
MATERIALS AND METHODS Experimental Animals and Treatments Each of 40, young adult, outbred, ICR mice, each weighing 20-30 g, were transcutaneously infected with 200 cercariae of the Puerto Rican strain of S. mansoni. They were then divided into four equal groups. The control, group I, was not treated. Each mouse in group II was given BAPN (5 mg in 0·5 ml saline by subcutaneous injection) daily for 20 days from day 40 post-infection (when parasite eggs were present in the intestine and liver). Each mouse in group
III was given two 800 mg kg- 1 doses ofPZQby intraoesophageal probe (Ruppel et a!., 1991), one on day 45 post-infection (p.i.) and one on day 49. Group IV mice were given both BAPN and PZQ, using the same protocols as for groups II and III. Parasitology The rate of schistosome egg excretion via the faeces was estimated for days 57-59 p.i. by the Kato-Katz technique, using commercial, OVO/FEC kits (Boehringer Mannheim). On day 60, mice were anaesthetized with Pentobarbital (Spofa, Prague), perfused through the portal vein to estimate worm infestation (Smithers and Terry, 1965) and then autopsied. Oograms were obtained by the method of Pellegrino et a/. (1962). From each mouse, two crush tissue slides were prepared from the liver and one each from the small and large intestines. A total of 200 eggs from each mouse was counted and each mature egg classified as living or dead (Pellegrino et a!., 1962). Tissue egg counts were estimated after digestion of I g of liver and 0· 5 g each of small and large intestines in 5% (w/v) KOHfor 16hoursat 37°C(Cheever, 1968). Histology and Microscope Image Analysis Portions of the livers and small intestines of each mouse were fixed in 10% buffered formaldehyde, embedded, sectioned (6 J.!m) and stained with haematoxylin, Giemsa or the van Gieson method to permit diagnostic and quantitative microscopy (Bancroft and Cook, 1984) of seven to 49 granulomas from each sample. The area, perimeter, shape [as the factor: (area/perimeter 2) x 4 n] and radius (mean distance from the centre of gravity to the border, measured on eight projections) of each granuloma were measured using an Olympus CUE2 microscope image analysis system. The differences in the results from each group were assessed by the KolmogorovSmirnov test and Student's t-test. RESULTS The effects of the different drug treatments on host-response, worm recovery, and tissue egg
SCHISTOSOMIASIS GRANULOMA MODULATION
633
TABLE!
Mean ( ± s.o.) worm recoveries, host response and tissue egg counts from four groups of 10 mice infected with Schistosoma mansoni and untreated (control) or treated with fJ-aminopropionitrile ( BAPN), praziquantel ( P ZQ), or both drugs Treatment Parameter
Control
BAPN
PZQ
BAPN+PZQ
11-4±5·1 34±24 22·7
5-4±4·2 6·5±4-4 6·0
0·12 0·0 0·06
0·0 0·0 0·0
35·0±4·9 12·8±2·8 2·4±0·4
37·8±3·8 10·8± 1·0 1·5 ± 0·3**
47·9 ± 7·7* 10·3±1·2 1·8±0·7
43·2±7·1*t 6·8 ± 0·8***ttt 0· 7 ± 0· 2***tt
1600±570 1780± 1320 119±30
1890±840 1440± 1150 82±56
1500±880 840±800 0·0
220 ± 1OO***tt 19± 16**t 0·0
WORM RECOVERIES
No. of female No. of male Proportion of total cercariae applied (%) MOUSE RESPONSE
Weight of mouse (g) Liver weight(% of body weight) Spleen weight(% of body weight) EGG COUNTS
Liver (eggs g l) Intestine (eggs g ·· 1) Faeces (eggs g- 1)
Significance of differences between treatment groups and controls (shown for mouse response results and tissue egg counts only): *P
ISSN: 0003-4983 (Print) 1364-8594 (Online) Journal homepage: http://www.tandfonline.com/loi/ypgh19
Experimental schistosomiasis mansoni: modulation of granulomas by inhibition of collagen cross-link formation. Preliminary report M. Giboda, J. Ženka, I. Juliš & J. Vítovec To cite this article: M. Giboda, J. Ženka, I. Juliš & J. Vítovec (1992) Experimental schistosomiasis mansoni: modulation of granulomas by inhibition of collagen cross-link formation. Preliminary report, Annals of Tropical Medicine & Parasitology, 86:6, 631-636, DOI: 10.1080/00034983.1992.11812719 To link to this article: https://doi.org/10.1080/00034983.1992.11812719
Published online: 15 Nov 2016.
Submit your article to this journal
View related articles
Citing articles: 5 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ypgh19
Annals of Tropical Medicine and Parasitology, Vol. 86, No.6, 631-636 (1992)
Experimental schistosomiasis mansoni: modulation of granulomas by inhibition of collagen cross-link formation. Preliminary report BY M. GIBODA,]. ZENKA Institute ofParasitology, Czechoslovak Academy of Sciences, BramJovska 31, CS -3 70 OS Ceske Budejovice, Czechoslovakia
I. JULIS Department of Pathology, Third Medical Faculty, Charles University, Prague, Czechoslovakia AND].
VfTOVEC
Imtitute of Parasitology, Czechoslovak Academy of Sciences, Branisovska 31, CS-370 OS Ceske Budijovice, Czechoslovakia Received 2 June 1992, Revised 28 September 1992, Accepted 28 September 1992
/1-Aminopropionitrile (BAPN) is an inhibitor of the lysyl oxidase required for cross-link formation in collagen maturation. The efficacy of BAPN, alone or in association with the anti-schistosoma! drug, praziquantel (PZQ), was primarily assessed by measuring the reduction in liver and intestinal egg loads in murine schistosomiasis mansoni. Depending on the treatment group (PZQ, BAPN, BAPN + PZQ), organspecific effects were observed using microscope image analysis. Most notable was the relatively small size of granulomas in the livers of BAPN-treated mice, which contrasted with the relatively large size and irregular shape of the granulomas in the intestinal tissues of these mice. Mice treated with the combination ofBAPN and PZQhad decreased liver and spleen weights, and a significant reduction in the number of eggs trapped in both the liver (86%) and the intestine (99·1 %), compared with untreated mice and those given PZQalone. The lowest number ofliving eggs/g of tissue in both the liver and intestine was recorded in the combined BAPN + PZQ-treated group. These results suggest that the concurrent treatment of infected mice with PZQand BAPN enhances the release of eggs trapped in the intestine and also results in a significant reduction ofliver egg load. The mechanism by which BAPN reduces the number of liver granulomas in PZQ-treated mice is currently being investigated.
The pathology of schistosomiasis is based on the host tissues' immunological reaction to the parasite eggs. Therefore, reduction of the number of eggs trapped in the tissues is a reasonable therapeutic goal for treatment of schistosomiasis in man. Granuloma formation around the eggs, and ensuing fibrosis, involve an increased synthesis 0003-4983/92/060631 + 06 $08.00/0
of extracellular components of connective tissue, particularly collagen and glycosaminoglycans (Grimaud 1987a). It is possible to affect, pharmacologically, the post-translation steps of collagen biosynthesis. Inhibition of maturation (polymerization) of collagen has so far been the most successful and powerful method of interfering with the physical © 1992 Liverpool School of Tropical Medicine
632
GIBODA ET AL.
properties of fibrotic structures. In the course of maturation the collagen fibrils are stabilized by lysine-derived intermolecular cross-links. The intermolecular cross-links play a crucial role in determining the characteristic physical and chemical properties of the collagen (Ricard-Bium and Ville, 1988). Lysyl oxidase is the enzyme required for cross-link formation in collagen and its activity is irreversibly inhibited by fJaminopropionitrile (BAPN). The successful experimental trials of Peacock and Madden (1966), Craver et a/. (1968) and Davis et a/. ( 1972), who employed BAPN to enhance wound healing in animals, lead to the application of BAPN in controlled clinical studies (Peacock and Madden, 1966; Keiser and Sjoerdsma, 1967). The rational for using BAPN in the treatment of Schistosoma mansoni infection was to decrease the rigidity and increase the solubility of newly produced collagen in the periovular granulomas and thereby promote the release of eggs trapped in the tissues. PZQ primarily kills adult worms, but also mature eggs (Richards et a/., 1989) and will prevent the saturation of host tissues with eggs laid by S. mansoni females. Our hypothesis, therefore, was that the combination of BAPN and PZQ would be more effective than PZQ alone in reducing the tissue egg load and resulting pathology. Particular attention was paid to the quantitative microscopic evaluation of the granulomatous response induced by eggs which accumulated in the liver and intestine of treated mice.
MATERIALS AND METHODS Experimental Animals and Treatments Each of 40, young adult, outbred, ICR mice, each weighing 20-30 g, were transcutaneously infected with 200 cercariae of the Puerto Rican strain of S. mansoni. They were then divided into four equal groups. The control, group I, was not treated. Each mouse in group II was given BAPN (5 mg in 0·5 ml saline by subcutaneous injection) daily for 20 days from day 40 post-infection (when parasite eggs were present in the intestine and liver). Each mouse in group
III was given two 800 mg kg- 1 doses ofPZQby intraoesophageal probe (Ruppel et a!., 1991), one on day 45 post-infection (p.i.) and one on day 49. Group IV mice were given both BAPN and PZQ, using the same protocols as for groups II and III. Parasitology The rate of schistosome egg excretion via the faeces was estimated for days 57-59 p.i. by the Kato-Katz technique, using commercial, OVO/FEC kits (Boehringer Mannheim). On day 60, mice were anaesthetized with Pentobarbital (Spofa, Prague), perfused through the portal vein to estimate worm infestation (Smithers and Terry, 1965) and then autopsied. Oograms were obtained by the method of Pellegrino et a/. (1962). From each mouse, two crush tissue slides were prepared from the liver and one each from the small and large intestines. A total of 200 eggs from each mouse was counted and each mature egg classified as living or dead (Pellegrino et a!., 1962). Tissue egg counts were estimated after digestion of I g of liver and 0· 5 g each of small and large intestines in 5% (w/v) KOHfor 16hoursat 37°C(Cheever, 1968). Histology and Microscope Image Analysis Portions of the livers and small intestines of each mouse were fixed in 10% buffered formaldehyde, embedded, sectioned (6 J.!m) and stained with haematoxylin, Giemsa or the van Gieson method to permit diagnostic and quantitative microscopy (Bancroft and Cook, 1984) of seven to 49 granulomas from each sample. The area, perimeter, shape [as the factor: (area/perimeter 2) x 4 n] and radius (mean distance from the centre of gravity to the border, measured on eight projections) of each granuloma were measured using an Olympus CUE2 microscope image analysis system. The differences in the results from each group were assessed by the KolmogorovSmirnov test and Student's t-test. RESULTS The effects of the different drug treatments on host-response, worm recovery, and tissue egg
SCHISTOSOMIASIS GRANULOMA MODULATION
633
TABLE!
Mean ( ± s.o.) worm recoveries, host response and tissue egg counts from four groups of 10 mice infected with Schistosoma mansoni and untreated (control) or treated with fJ-aminopropionitrile ( BAPN), praziquantel ( P ZQ), or both drugs Treatment Parameter
Control
BAPN
PZQ
BAPN+PZQ
11-4±5·1 34±24 22·7
5-4±4·2 6·5±4-4 6·0
0·12 0·0 0·06
0·0 0·0 0·0
35·0±4·9 12·8±2·8 2·4±0·4
37·8±3·8 10·8± 1·0 1·5 ± 0·3**
47·9 ± 7·7* 10·3±1·2 1·8±0·7
43·2±7·1*t 6·8 ± 0·8***ttt 0· 7 ± 0· 2***tt
1600±570 1780± 1320 119±30
1890±840 1440± 1150 82±56
1500±880 840±800 0·0
220 ± 1OO***tt 19± 16**t 0·0
WORM RECOVERIES
No. of female No. of male Proportion of total cercariae applied (%) MOUSE RESPONSE
Weight of mouse (g) Liver weight(% of body weight) Spleen weight(% of body weight) EGG COUNTS
Liver (eggs g l) Intestine (eggs g ·· 1) Faeces (eggs g- 1)
Significance of differences between treatment groups and controls (shown for mouse response results and tissue egg counts only): *P
