MINORU MIYASATO, HIROKI IIDA, NAOKI YOSHIOKA AND HIROSHI TSUTSUMI. Department of Parasitology, Kurume University School of Medicine, Kurume, 830 Japan

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1 THE KURUME MEDICAL JOURNAL Vol. 29, p , 1982 Experimental Pulmonary Embolm with Sc htosoma japonicum Eggs I. Htological Observations on Tsue Reactions to S japonicum Eggs Lycopodium Powder MINORU MIYASATO, HIROKI IIDA, NAOKI YOSHIOKA AND HIROSHI TSUTSUMI Department Parasitology, Kurume University School Medicine, Kurume, 830 Japan Received for publication November 9, 1982 Summary: Granuloma lycopodium was macroscopically microscopically compared with Schtosoma japonicum s to determine wher lycopodium power could be used as a substitute for s. s were olated from liver intestine s 8 weeks infection with S. japonicum. Approximately 100, 000 s or 200,000 lycopodia were injected into s each through auricular vein. s were removed 14, 28, Paraffinembedded s s were stained with hematoxylin-eosin, azanmallory luna stains. Tubercula were grossly observed in s 4 s at 14 ; one which was a lycopodia charactertics lycopodium tuberculum were similar to charactertics tuberculum. Microscopically, two types reactions developed s. One was very severe or was mild or absent. severe s were large in size at 14, but y gradually became smaller, although y were still present 86. s with mild reactions were very small in size at 14, diminhed in size with time, dappeared by 86. With all types injections, majority inflammatory cells in were mononuclear cells with some eosinophils pseudoeosinophils. Giant cells were ten observed at 14 y slowly increased in number. giant cells were observed in most at 86. Scar was seen at charac - tertics lycopodium were very similar to charactertics except that lycopodium was smaller in size had more giant cells, rate destruction absorption giant cells in tsue was also slower. Although lycopodia did not induce as severe a reaction as s, it can be assumed that lycopodium could be used as substitute for S. japonicum s under certain conditions. Key words : Schtosoma japonicum-pulmonary embolm-lycopodium -schtosomal tubercle Introduction Granuloma S japonicum s a very severe lesion. Many studies se s in human autopsies experimental animals have been publhed. was considered to be induced by leucocytic throm - bos due to obstruction microvasculature by s (Nakayama, 1910), or 107

2 1 08 MIYASATO, ET AL. by mechanical obstruction toger with production a toxin by s (Fujinami, 1916; Kiyono Murakami, 1917). tous reaction may involve an allergic mechanm since htological findings were similar to those tuberculos (Sakurabayashi, 1955). In recent studies, Von Lichtenberg described Ascar su s or Schtosoma mansoni s. Th appeared to be due to an elemental immune process rar than a modified "foreign body reaction" because was enhanced in sensitized hosts (Lichtenberg Meckbel, 1962; Lichtenberg, 1963; Meckbel Lichtenberg, 1962). Warren (1978) suggested that tous reaction to S. mansoni s was a cellmediated immunological reaction. Extensive studies on SEA (Schtosoma antigen) or relationship between SEA have been completed (Andrade Barka, 1962; Kloetzel, 1967; Boros Warren, 1970). purpose present study to compare S. japonicum s with that lycopodia, to investigate wher lycopodium could be used as a substitute for S. japonicum s. experiments pulmonary fibros due to S japonicum s are now difficult to do because insufficient s are being olated. Materials Methods S. japonicum s were olated by a modification method Smirs (1960) from liver intestine white s which had been exposed 8 weeks previously to 500-1,000 cercariae a Japanese strain S. japonicum obtained from infected Oncomelania nosoplwra. s were triple-washed with penicillin G (50, 000 IU), n with sterile saline. Approximately 100,000 s were suspended in 10 ml 10% glucose, injected into s white s through auricular vein. effects lycopodium powder ( Japanese Pharmacopoeia II, 1962) were compared to effects s. As a lycopodium was approximately half size an, a total 200,000 lycopodia (0.05 g in weight), which were triple-washed suspended in 10 ml 5 % glucose, were injected. 15 female white s weighing about 2.5 kg were grou - ped, as shown in Table 1. Eight animals were injected with s seven were injected with lycopodia. At 14, 28, injection, s, in group at least two, were anestized with pentobarbital, n sacrificed by severing carotic artery. Paraffin-embedded tsue from each was cut into serial s 7 1um in thickness, stained with hematoxylin-eosin. In addition, four s were stained with azan -mallory for collagen fibers with (1946) for eosonophils. Results I. Macroscopic findings s were normal in all s except No. 1, 2, 3 9 which showed evidence tubercula. Investigations se s are shown in Table 2. In three animals at 14 injection (s No. 1, 2 3), a large number tubercula were diffusely dtributed in all lobes. tubercula were approximately 1-2 mm in diameter, firm in constency, greyh white in color bordered from surroundings. t u bercula No. 2 were scattered extensively along edge s. In one 14 lycopodia injection ( No. 9), a tuberculum developed in right lower lobe at which had charactertics that were similar to tubercula. II. Microscopic findings At each experimental period, condition emboli in tsue tsue reactions to emboli, especially

3 PULMONARY EMBOLISM WITH S. JAPONICUVI EGGS 109 TABLE 1 Experimental plan S. J. E. : Schtosoma japonicum. Lyco. : Lycopodium. TABLE 2 Macroscopic findings; s were normal in all s except No.1, 2, 3 9. s were investigated primarily for extence tuberculum 1. s were pinkh in color, st in constency with a smooth surface. A large number tubercula were present. cut surface each tuberculum was about 1-2 mm in diameter, firm in constency greyh white in color. y were bordered from surroundings. 2. A large number tubercula were diffusively scattered at edges s. tubercula were similar to those in No findings resembled those in No A tuberculum was present in right lower lobe. tuberculum was similar to those in No. 1.

4 110 MIYASATO, ET AL, TABLE 3 Microscopic findings: tsue reaction to embolus, cellular compostition tuberculum, condition embolus 14 emboli : very severe (more than 8 times embolus diameter in size). : severe (4-8 times embolus diameter in size).+ : moderate (1-4 times embolus diameter in size). } : slight -: absent TABLE 4 Microscopic findings: tsue reaction embolus, cellular composition tuberculum, condition embolus 28 emboli injection

5 PULMONARY EMBOLISM WITH S. JAPONICUM EGGS 111 TABLE 5 Microscopic findings: tsue reaction embolus, cellular composition tuberculum, condition embolus 84 or 86 emboli injection, were investigated. htological findings are described in Table 3, 4 5. olated s were composed viable s, degenerated s shells. When y were all injected into s, different types tous reactions occurred. In present study, two types reaction were observed. One was very severe, or was very mild or absent. following de - scribes htological findings two types reaction to s a reaction to lycopodium. 1. Tsue reaction to embolus 1) Egg tuberculum inducing severe reactions 14 injection, schtosomal s were found in following sites : arterial intima lumina al - veolar septa. se s were 6-12 times as large as diameters. re were many inflammatory cells in s. Most cells were mononuclear including lymphocytes, macrophages, htiocytes epiloid cells. remaining cells were polymorphonuclear, including eosinophils pseudoeosinophils. Giant cells were ten seen near s. connective tsue had proliferated slightly in. However, almost never developed into a scar. Necrotic lesions in were not detected (Fig. 1-A). At 28, s appeared to have a two layered structure. An internal layer was composed mainly htiocytes giant cells, an external layer was composed connective tsues inflammatory cells. size cellular composition were similar to at 14, however, giant cells were more widely dtributed in at 28 (Fig. 2-D). s declined in size, but still were present at 86. was composed primarily mononuclear cells eosinophils. Giant cells were observed in most. Scars in were found with

6 112 MIYASATO, Fig. 1. A cells that re Fig. no a very 2. D loma slight. reaction. S. japonicum s cells from 28 lycopodium. at 14. localized lycopodia in S. with at no E, ~100). inflammatory pulmonary artery. severe reaction. 14. to granufrom are E, ~100). Th re differences reaction a closely japonicum Th B small remarkable F inflammatory are C. number cells. re large composition are 28 japonicum a Th in S. with mononuclear cellular inflammatory differences lycopodium remarkable AL. lycopodia. japonicum 14 eosinophils lycopodium. mild few 14 S. with are resembles primarily 14 composed E ET s 28 very

7 PULMONARY Fig. loma 84 present. 3. G canalization diminhed, still embolic present. localized vessel Th but lycopodia action. EMBOLISM 1 At served 86 can be s small seen. tubercles 14, s were obat same site where schtosomal s were detected. size was about 4-8 times as large as lycopodium diameter. cellular composition lycopodium closely resembled that ; however eosinophils pseudoeosinophils were rare, giant cells were numerous when compared with. After 28, had same appearance as 14. After 84, s were reduced in size, but y were still present. most common cells were mononuclears, followed by eosinophils. Scar was observed at all experimental periods, especially at 84 (Fig. 1-B, Fig. 2-E Fig. 3-H). capillary. 86 No reaction to granu- Scar Th size. in 113 reaction. diminhed a severe H EGGS S. japonicum with in S.JAPONICUM or without remnant s. scars were irregular in shape, were formed by loose or dense connective tsues with round cell infiltration (Fig. 3-G). 2) Lycopodium WITH S. japonicum with it mild found re. Re- E, ~400). 3) Egg tubercles s induced were mild localized reactions in small pulmonary arteries at 14, seen with or without an inflammatory reaction m. re were very few inflammatory cells. re were a few mono nuclears, giant cells polymorphonuclears including mainly eosinophils in. remarkable After 28, re were no differences from granu- loma at 14. rare at 86 Fig. 3-I). 2. Condition 1) s Granuloma (Fig. 1-C, Fig. 2-F was embolus inducing a severe reaction At 14, most s were intact or almost intact. A few s were destroyed by inflammatory cells. By 28, s were destroyed, usualy only shells or s could be stained with eosin. Calcified s were occasionally found in capillary. alveolar septa even 86

8 114 MIYASATO, ET AL. 2) Lycopodium All lycopodium were intact At 86, some lyco - podium were calcified or destroyed in s. 3) s inducing a mild reaction s were eir calcified or destroyed leaving shells at 14. re were only shells by Dcussion Many studies on schtosomal lesions induced by worms or ova have been experi - mentally clinically pursued. Nakayama (1910) htopathologically classified schtosomal tubercula into two types : type 1 chiefly consting infiltration cells, type 2 consting granulation tsue. It was concluded that tubercula were produced by mechanical stimulation to tsue by. I3shiyama (1953) concluded that tubercula were induced by a toxin from because re was difference in reaction to viable s reaction to non-viable s. A similar experiment was carried out by Lichtenberg (Lichtenberg Raslavicious, 1967) who observed that heat killed s produced pseudotubercles a similar cell composition reaction as whole live s, but pseudotubercles were lesser in size duration. In present study, two kinds reaction to s were detected. One was very severe s remained intact or nearly intact. or was very mild or absent s were calcified or destroyed, including shells. former may be due to viable s, latter to non - viable s. tsue re - action to s differs depending on condition, thus it can't necessarily be concluded that schtosomal tubercla are induced by mechanical stimulation at tsue by s. It probable that pathogenes schtosomal tubercula involves not only mechanical stimulation, but also a complex interaction between host immune system producing materials. Warren et al. (1967) suggested that basic pathogenes S. mansoni attributable to an immunologic reaction with delayed hypersensi - tivity (Warren et al. 1967, 1968, 1969, 1974). y demonstrated a tous response against a soluble SEA preparation (Boros Warren, 1970). In contrast, S japonicum appears to resemble mechanm foreign body, that, a non - immunologic reaction due to activation chemical mediators inflammation (Warren Domingo, 1970; Warren Boros, 1975). Recent studies have emphasized uniqueness S. mansoni since dominant cell type in lesion was eosinophil. Studies Colley demonstrated that depletion thymus-dependent lymphocytes abolhed anti-sea lymphocyte blastogenic response in vitro major peak peripheral blood eosinophilia in vivo (Colley 1971, 1975). Phillips h associates indicated that eosinophilia, tous hypersensitivity ultimate host morbidity in S. mansoni were dependent upon thymic dependent lymphocyte function (Phillips et al. 1977). Egg producing materials, which were indicated as enzymic activities phospholipid (And - rade Barka, 1962; Kloetzel, 1977), have been described for S. mansoni s. Thus although pathogenes S. mansoni has been studied elucidated, pathogenes S. japonicum has remained unknown in many respects. In present study, lycopodium resembled except that lycopodium was smaller in size, number giant cells was higher, rate destruction absorption in tsue was slower

9 PULMONARY EMBOLISM WITH S. JAPONICUM EGGS 115 development scar was more rapid when compared with. lycopodia did not induce as severe a reaction as. Kiyono Murakami (1917) htopathologically classified tsue reaction to lycopodium into three stages. first was a cellular infiltration stage which occurred within 24 hours intravenous cellular infiltration was localized microvasculature obstructed by lycopodium. Most inflammatory cells were polymorphonuclear cells lymphocytes. second was a cellular proliferation stage which occurred between 24 hours approximately 10. Compared with first stage, htiocytes, lymphocytes epilial cells increased, but polymorphonuclears decreased, giant cells began to be observed in. third was a scar stage which occurred at more than 10 Scar consting lymphocytes, htiocytes, fibroblasts collagen fibers developed. Hosokawa osokawa et al. 1954) described morphological changes blood vessels in s with lycopodium embolm. was composed fibroblasts, large mononuclear cells foreign body giant cells. Kiyono Hosokawa recognized lycopodium as one foreign bodies. Relation to th point, tsue reactions to lyco - podium in our study are worthy dcussion. presence two kinds lycopodium, with without inflammatory reactions it, occurrence a more severe reaction to lycopodium than to shells non-viable s were observed. shell non-viable can be regarded as foreign bodies. However, it not just that lycopodium a foreign body, because reaction to lycopodium different from reaction to shell non-viable. Since lycopodium consts dryed Lyoopodium clavatum spores, it can be assumed that charactertics which lycopodium possesses originally are maintained in glucose suspension. se charactertics have not yet been determined, biochemically or immunologically. An experimental study portal embolm with agents which were similar in size to S. japonicum, has been performed by Tsutsumi et al. (Tsutsumi et al. 1972) to elucidate mechanm liver fibros. y demonstrated that lycopodium in particular produced a htological pattern resembling that S japonicum. Our study revealed that lycopodia was similar to that S japonicum s, although lycopodia induced less intense htological reaction than S japonicum s. In conclusion, lycopodium could be used as a substitute for S. japonicum to some extent in experi - ments embolm. Acknowledgements : Th investigation was partially supported by a Grant-in-Aid for Special Research Promotion, Mintry Education, Science Culture ; Project No entitled "Fundamental Studies on Control Tropical Parasitic Deases". References ANDRADE, Z. A. BARKA, T. (1962). Htochemical observation on experimental schtosomias mouse. Am. J. Trop. Med. Hyg. 11, BoRos, D. L. WARREN, K. S. (1970). Delayed hypersensitivity type dermal reaction induced elicited by a soluble factor olated from Schtosoma mansoni s. J. Exp. Med. 132, COLLEY, D. G. (1971). Schtosomal antigen induced lymphocyte blastogenes in experimental murine Schtosoma mansoni infection. J. Immunol. 115, DABIS, B. H., MAHMOUD, A. A. F. WARREN, K. S. (1974). Granulomatous hypersensitivity to Schtosoma mansoni s in thymectomized bursectomized chickens. J. Immunol. 113, DOMINGO, E. O. WARREN, K. S. (1968). inhibition Schi-

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