EXPERIMENTALPARASITOLOGY 75, t-9(1992)

Trichinella

spiralis: Modifications of the Cuticle of the Newborn Larva during Passage through the Lung

FABRIZIO BRUSCHI, *,'STEFANO *Institute of General Pathology, flnstifute of Clinical Physiology,

S~LFANELLI,~ANDRUBEN

A. BINAGHIS

Universi@ of Perugia, Policlinico Monteluce, 06100 Peru&a, Italy; C.N.R., Via Savi 8, 56100 Pisa, Italy; and #C.N.R.S., Paris, France

BRUSCHI, F., SOLFANELLI, S., AND BINAGHI, R. A. 1992. Trichinella spiralis: Mod&ations of the cuticle of the newborn larva during passage through the lung. Experimental Parasitology 75, l-9. A scintigraphic method was developed to study the distribution of radioactivity after iv injection of “‘I-labeled Trichinella spiralis newborn larvae into normal rats. It was found that the radioactivity was immediately retained in the lungs and thereafter slowly released, with a mean transit time in excess of 9 hr, as calculated by image analysis. At various times after iv injection of newborn larvae into normal mice, the lungs were removed and parasites were recovered and counted. Fifty to seventy percent of the larvae injected were recovered after 30 set, between 10 and 30% after 1 min, and less than 4% at 15 min. These results indicate that during the very rapid passage of newborn larvae through the lungs, labeled components of the cuticle are detached and retained. It is suggested that the modifications produced in the cuticle of the newborn larva during its passage through the lung may increase its resistance to the nonspecific defense mechanisms of the host. Q 1992Academic press,arc. INDEX DESCRIPTORS AND ABBREVIATIONS: Trichinella spiralis; Newborn larvae (NBL); Nematode; Parasitic; Scintigraphy; Radiolabeled parasites: y Camera; Lungs; Rat; Mice.

lymphatic vessels and migrates through the The fate of Trichinelfa spiralis newborn thoracic duct and the central venous circularvae (NBL), after their passage through lation. Wang and Bell’s data (1986a) show the intestinal wall, presents a number of ill- unequivocally that most newborn larvae defined aspects among which is the exact pass through two capillary filters; the heroute of transit to striated muscle and the patic and the pulmonary. These authors estime spent in completing this passage. timate that migration time from the small Blood circulation, connective tissue and intestine to the thoracic duct and the portal body fluids, and lymph-blood pathways vein is less than 1 hr and observed that the have been alternatively considered as pos- “fastest newborn larvae” can migrate sible routes (Herrick and Janeway 1909; through the pulmonary or the hepatic capillary beds in just 1 min. These observations Berntzen 1965; Harley and Gallicchio indicate the shortest migration times but 1971). Recently it has been shown (Wang and they do not allow a precise estimation of the Bell 1986a) that most of the larvae passing average migration time of the whole newthrough the intestinal mucosa enter the born population. Binaghi and collaborators (1981) have albloodstream and not the lymphatic vessels, ready demonstrated retention of radioactivcontrary to a generally accepted belief (Despommier 1976). And then, via the por- ity in the lungs after iv injection of radiolatal vein, newborn larvae enter the liver, beled newborn larvae in normal mice. The while a very small portion (-2%) enters the radioactivity in the lungs of these animals, killed at different intervals, decreased slowly, suggesting a prolonged residence of * To whom correspondence should be addressed. the larvae. It has also been observed that iv INTRODUCTION

1 OOM-4894/92 $5.00 Copyright Q 1992 by AcademicF'ress,Inc. All rights of reproduction

in any form reserved.

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BRUSCHI, SOLFANELLI,

injected NBL can remain trapped in the lungs (Wang and Bell 1986b). Evaluation of the exact transit time through the lungs is relevant not only for a clear characterization of the parasite life cycle in the migratory stage, but assumes greater importance in view of recent results which have shown modifications of NBL surface structure during the first hours of life (Jungery et al. 1983). These changes seem relevant regarding the host immune response to the parasite (Ortega-Pierres et al. 1984; Gansmtiller et al. 1987). In this study we employed scintigraphic techniques to define how long radioactivity remained in the lungs and to follow it dynamically in the different areas of the body. Concurrently, we estimated the number of newborn larvae present in the lungs at various times by actually counting them after recovery from the dissected tissue.

AND BINAGHI

chloramine-T (Markwell 1982) (Iodo-beads, Pierce, Rockford, IL) (Expts 3 and 4). Two beads were kept in contact for 2 min (Expt 3) and one bead for 5 min (Expt 4) with about 50,000 NBL and 13’1(Sorin, Saluggia, Italy, 0.8-l mCi activity). Then, parasites were washed four times in saline solution at pH 7.4. When the labeled larvae were incubated at 37°C in MEM medium, 70 to 100% of them showed normal motility. The infectivity of the radiolabeled larvae was tested by injecting them iv into normal mice and counting the number of muscle larvae found 30 days later. The percentage of recovery was similar (not lower than 80%) to that found in mice injected in the same way with unlabeled newborn larvae. Scintigraphic study. Animals were anesthetized with a mixture of droperidol(1.25 mg/kg, bw) phentanyl (0.025 mg/kg), and ketamine (25 mg/kg) administered im 5-10 mitt before injection. Small doses of the same drugs were subsequently administered as required. This permitted recording of the activities of the injected isotopes for a period ranging from 1 to 4 hr without affecting the geometry of the recorded areas. Infectivity of NBL in anesthetized animaIs, treated with the same mixture, was 80 + 5% (n = 3) that of NBL inoculated in saline-treated mice. To check the effect of anesthetics injected in the animals on larvae MATERIALS AND METHODS motility, NBL were incubated with anesthetized rat Animals. For the scintigraphic studies we used male serum or saline-treated rat serum, at 50% dilution, at 37°C. The motility of NBL, evaluated microscopically, outbred SpragueDawley rats, of 250400 g body wt. The animals were maintained, in groups of three to was present in 100% larvae in both cases, at 3, 10,30, four, in polystyrene cages. Food and water were pro- 60, and 240 min and at 18 and 25 hr incubation. Ten thousand 13’I-NBL were injected iv in normal vided ad fibitum and a diurnal rhythm of light was maintained. BALB/cfHad/Se mice of both sexes, aged rats. The activities were between 9 and 17 uCi for the between 2 and 4 months, were used for maintenance of NBL labeled with chloramine-T and about 2&50 uCi the parasite. To obtain T. spirulis adult worms, out- for parasites labeled by Iodo-beads. The animals were bred Sprague-Dawley rats of both sexes, 3O@lOOg followed for 1 hr in Expts 1 and 2 and their activities were recorded every 10 min for a period of 120 sec. In body wt, were used. Parasites. A strain of T. spirulis has been main- Expt 3 the animal was followed dynamically (i.e., the tained in our laboratory since 1970by passage in mice activities were registered continuously for periods of infected with -00 muscle larvae per OS(reference 45 min) for 4.5 hr. In this experiment we also recorded 15 hr after parasite injection. code, MSUS/WG/6YISSSl). In Expt 4 the animal was continuousIy followed for NBL were obtained in vitro from cultured adult worms isolated from intestines of rats infected 6 days 1.5 hr. To evaluate lung perfusion, normal rats were previously with 7000-8000 muscle larvae per OS, ac- injected with human albumin microspheres, l-10 pm in diameter, labeled with 99mTc (TCKJ, Sorin) (Sancording to the method of Dennis et al. (1970). Adult tolicandro et al. 1971). A I-mCi activity was injected worms were cultured in Eagle’s MEM with Earle’s salts (GIBCO, Paisley, Scotland) with 10% heat- per animal. Controls were performed by iv injecting inactivated fetal calf serum (Flow, Milan, Italy) and carrier-free 13iI (500&i activity) or i3’I human albupenicillin (200 U/ml), streptomycin (100 pg/ml), and min (S.A.R.I., Sorin, 22-p,Ci activity) in normal rats. The distribution of isotopes and their relative activfimgizone (0.4 U/ml). NBL were separated from adult worms after 3 hr incubation at 37”C, washed twice in ity in the different body parts of the animal were recorded with a y camera, Toshiba GCA 402, using a Eagle’s MEM, and immediately used for inoculation high resolution collimator for tests performed with or radiolabeling. Radiolubeling. NBL were labeled with “‘I (13’1- *mTc isotope (energy peak at 140 kV) and a high enNBL) by the chloramine-T method, previously used in ergy collimator for tests performed with 13’1isotope similar experiments (Binaghi et al. 1981) (Expts 1 and (energy peak at 360 kV). In both cases we used a 20% window on the emission peak. Data were registered 2) and by polystyrene beads covalently bound with

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on-line with a Medusa computer (SEPA, Milan, Italy) connected to the y camera and elaborated to calculate the activities present in the different areas of the animal’s body (head, chest, abdomen, periphery) so as to follow distribution of the isotopes. In different areas (of equal size) the mean transit times of i (Desgrez ef al. 1977) for radioactivity in i3’I-NBL and 13’1experiments were calculated by the equation

NEWBORN

LARVAE

IN LUNGS

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noted at this time and this continued during the period of observation. The total activity was rather diffuse in all parts of the animal body 48 set after injection and analysis revealed equilibrium between chest and abdomen (Fig. 2B). The behavior of 1311labeled human albumin was very similar to that of the carrier-free isotope and no relet/2 ;=vant retention was observed anywhere 0.693 (data not shown). In the case of 1311-labeled where t/2 = l/K and K = the linear regression curve parasites a completely different distribution slope. In the thoracic areas 0 time was taken at the pattern was observed (Figs. 3 and 4). In all moment of highest activity. four experiments the activity was concenRecovery of newborn larvae from the lungs. This was done at different times after iv inoculation. The trated in the lungs immediately after injection. In Expts 1 and 2 we found a much lungs were homogenized in MEM medium containing heparin (IO U/ml), in a Potter-Elvehjem. One volume higher thoracic activity (1310-1026 and of the suspension was then mixed with 4 vol of distilled 5752-4988 counts, respectively) than in the water and after 1 min 4 vol of MEM medium at 2~ abdominal area (230-235 and 479-611 concentration was added. This treatment resulted in counts), during the period of observation complete lysis of the red cells, without impairing the (about 1 hr). viability of the recovered larvae, as shown by their motility. After concentrating by centrifugation at 1000 In Expt 3 after 1 min the activity was g for 5 min, the number of larvae was calculated by concentrated in the lungs and was slowly counting the aliquots under a microscope. Controls decreased during the following 16 min were made by adding a known number of NBL to a (Figs. 4A and 4B). The lung activity still lung from normal animals, before homogenization. The larvae counted were 60-70% of the actual number remained higher than that of the other aradded. The difference was due to larvae destroyed eas, without significant increases in abdoduring the procedure or the difficulty in viewing them men or head, after 76 min (Fig. 4B). No among the lung debris. important variations were observed in peInjections ofNBL in the left ventricle of the heart. A ripheral activity during the observation pel-ml syringe with a 25-g needle was used. The animal was firmly held in the hand of one operator while an- riod. In Expt 3 we recorded the activity 15 other one performed the injection in the left side of the hr after injection: substantial activity was chest at the level of the cardiac projection area. When found in the thoracic area (25% of the initial the needle reached the left ventricle blood appeared in value) and fair activity was found in the the syringe and the injection was then immediately liver and head (see Fig. 1D). performed. At the end of the injection gentle aspiration The results of Expt 4 are given in Figs. confirmed that the needle was always in the left ventricle. 4C and 4D. They confirm all the aspects of Expt 3. In fact, retention of activity in the RESULTS lung was immediately observed, followed Human albumin microspheres (HAMby a slow decrease, during the period of 99mTc) were immediately trapped by the observation. However, during the recordlungs after iv injection; the lungs were ing period, activity in the lungs was always clearly visible (Fig. 1A). Carrier-free 1311iv higher than that observed in the abdomen. injected diffused very quickly throughout No relevant increase was observed in the the body (Fig. 1B) and no lung retention periphery. was observed (Fig. 2A). On the contrary, Figure 5 gives the analysis of lung activafter 7 set radioactivity in the abdomen was ities for Expts 1 and 2. The mean transit higher than in the thoracic area. A gradual time of radioactivity through the lungs was increase in the head area (thyroid) was 9.7, 10.3, 11.2, and 9.2 hr in Expts 1, 2, 3,

BRUSCHI,

SOLFANELLI,

AND

BINAGHI

FIG. 1. Scintigraphic distribution in normal rats of HAM--rnTc (A), r3iI (B), and “‘I-NBL (C and D). L, lungs; H, head; A, abdomen; P, periphery; i.p., injection point. The black line is a sketch of the animal’s outline. In A (5 min after injection) only the lungs are visible; in B (5 min after injection) the whole animal’s body presents activity and the head area (thyroid) is clearly visible. In C (60 min after injection) the activity is concentrated in the thoracic regions with less activity in the abdomen and no activity in the head. The high activity in C in the lower part of the animal is only due to the injection point. In D (15 hr after injection) the thyroid is clearly visible.

FIG. 2. Dynamic registration (A) and area analysis (B) of a normal rat injected with carrier-free ‘3’I. (A) Dynamic registration of the first % set after injection of i3iI in a normal rat. The whole body is clearly visible at 48 set (1 frame/6 set). (B) Analysis of the activity of thoracic (L), abdomen (A), head (H), and periphery (P) areas during a 192-set period of observation. At 48 set the equilibrium between chest and abdomen and between head and periphery is already established. Time in frames.

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FIG. 3. Distribution and trend of activity over the diierent areas after 13’1-NBL injection. The rat silhouette (left) shows the distribution of activity recorded from 0 to 60 min and is the sum of activities over 1 hr. The trend of activity (right) was calculated over four areas of the same size (corresponding to 100pixels); L, A, and H, the same as in legend to Fig. 2, and i.p., injection point.

and 4, respectively. The mean transit time of carrier-free 1311was 0.17 hr (Table I). Different experiments were performed in normal mice at various times after iv NBL injection. When the animals were immediately killed (less than 30 set) after iv injection a considerable proportion of larvae was recovered, 31.5 + 9% (mean ? SD; IZ = 6). After 1 min recovery was 19.5 + 13.4 (n = 6) and at 15 min 2.2 2 1% (n = 6) of the injected NBL were found. These values must be corrected for loss inherent in the technique employed, but it is clear that it only takes a few minutes for NBL to pass through the lungs. We performed recovery experiments with labeled NBL, and at 10 min from iv injection 6 ? 5% (n = 3) of injected larvae were recovered. We estimated the degree of recirculation by injecting NBL into the left ventricle of normal rats and we observed that a very small percentage of these (less than 4%) is subsequently found in the lungs after 30 set or 10 min (data not shown). DISCUSSION

In many helminthic infections there is transit through the lungs by migrant stages (Spencer 1977; Befus et al. 1984; Wilson 1990).

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The principal observations from the present experiments are that living i3iI newborn larvae, intravenously injected, pass through the lungs very quickly, in a matter of a few minutes, but leave their labeled cuticular proteins in the lungs, which remain there for a period of many hours. The scintigraphic study showed clearly that the radioactivity is immediately trapped in the lungs, confirming the first observations of Binaghi et al. (1981) who counted the total radioactivity in the lungs of mice killed at various times after injection with labeled newborn larvae. The scintigraphic method has the advantage of permitting continuous measurements of the radioactivity in the same animal and in different areas of the body. In this way, individual variation is eliminated and the distribution of the radioactivity can be followed for long periods. Labeling with r3’I was performed in conditions not affecting the viability of the newborn larvae. The use of polystyrene beads with covalently bound chloramine-T gave a particular advantage since iodination can be stopped by simply separating the parasites from the beads without adding other reagents that might possibly affect the larvae. The different controls performed indicate that the radioactivity found in the lungs is due to labeled proteins and not to free iodine. In fact, both carrier-free i3’I and labeled human albumin diffuse very quickly throughout the animal’s body, disappear rapidly from the lungs, and are subsequently concentrated in the liver and thyroid. A large amount of albumin is in fact retained by the endothelial intercellular junctions, but small amounts cross the endothelium to reach the interstitium by pinocytotic transport (Jeffery and Con-in 1984). The larval suspension injected contained practically no free iodine and no sign of deiodination was observed during the first minutes. The thyroid area became active only many hours after parasite injection (Fig. 1D) and the liver area only after more

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SOLFANELLI,

AND

BINAGHI

FIG. 4. Examples of dynamic registration (A and C) and area analysis (B and D) of two normal rats injected with r3rI-NBL. (A and C) Dynamic registration of the first 16 min from injection of labeled parasites. During this period the activity remains concentrated in the lungs with little diffusion outside (1 frame/60 set). (B and D) Analysis of area activities shows that the lung activity is always higher than that of abdominal activity. No important variation between head (H) and periphery (P) is observed.

than 1 hr from injection. This cannot be attributed to the presence of soluble-labeled proteins in the larval suspension, but is best interpreted as being due to the uptake of labeled cuticular components released by the newborn larvae. The mean transit time (7) through a limited selected area of the lungs, calculated by dynamic registration, varied from 9.2 to 11.2 hr, irrespective of the labeling method and the time of observation. Even if the observation periods were restricted to only a few hours, finding significant activity in the lungs (compared to abdominal activity) 15 hr after injection of labeled larvae con-

firms the long permanence of the radioactivity in this organ. All these observations suggest that proteins of the cuticle of the larvae are detached and retained during its short passage through the lungs. This happens very quickly, since after only 15 min most of the NBL have already left the lung to reach the muscle where they develop inside the nurse cell (Despommier 1990). Modifications of the cuticular structure, probably involving the release of protein components, have already been described by Gansmtiller et al. (1987). It has been demonstrated that these modifications take place in vitro in the few hours following

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1 (13’INBL) 2 (13’INBL) 3 (13’INBL) 4 (13’INBL)

f” (hours)

Observation time (minutes)

0.17 9.72 10.30 11.28 9.21

306 60 60 270’ 96

0 Mean time of transit through the lungs; f = t/2 where t/2 = l/K and K is the linear regression 0.693 curve slope. b The observation period of “‘I was 30 min because at this time equilibrium of activities in the different areas had already been reached. ’ The t was computed over a period of 75 min.

no2

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IN LUNGS

TABLE I Mean Transit Times Through the Lungs of Radioactivity after 13’1Carrier Free or Labeled Newborn Larvae Injection

l

RAT

LARVAE

30

41 TIME

52

60

(min)

5. Analysis of lung retention in Expts 1 and 2. The registrations were performed with six to seven recordings, respectively, over 120-set periods from Time 0 to 63 min (Expt 1) or 60 min (Expt 2). Rat 1: a = 87.7, b = -0.0017, r = -0.945. Rat 2: a = 98.7, b = -0.0016, r = -0.975. FIG.

birth. But in the present case, during migration through the lungs, release of radiolabeled components is very fast and occurs within a few minutes. We have not yet been able to reproduce this phenomenon in vitro by culturing labeled newborn larvae in the presence of a suspension of homogenized normal lung (data not shown). Wang and Bell (1986b) have studied recirculation of newborn larvae in the rat and conclude that a “proportion” migrates within the vascular system for several hours and a smaller population extravasates in nonmuscular tissue but can reenter the circulatory system via the lymphatics/ blood. According to our results, recirculation of the labeled larvae only involves a small percentage of larvae, thus confirming the results of Wang and Bell (1986b) and this cannot account for the radioactivity remaining in the lungs.

The finding that the newborn larvae cuticle is modified during passage through the lung could have important implications. Gansmtiller and collaborators (1987) showed that newborn larvae cultured in vitro for 20 hr became resistant to an immune attack from normal peritoneal cells activated by specific antibodies and it has been demonstrated that a cuticular antigen of the larvae only a few hours old was the target of an immune cytotoxic reaction (Ortega-Pierres et al. 1984). It is tempting to hypothesize that the modifications produced in vitro in the first hours of life would occur much more rapidly in vivo as suggested by the release of labeled components. Preliminary results show that 2-hrold newborn larvae injected into the tail vein, which immediately go to the heart and lungs, are more infective (66.9 -+ 18.4%; mean + SD) than larvae injected into the left ventricle (51.1 + 19.1%). These latter larvae are disseminated throughout the body and reach their muscular niche without having passed through the lungs. Since the degree of recirculation observed is very low, it is possible that most of the larvae injected into the left ventricle never leave the muscle tissue, and therefore are not ex-

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posed to whatever process might occur in the lung. It can be hypothesized that the cuticular changes observed in vitro during the first hours of life (Jungery et al. 1983; Gansmiiller ef al. 1987)are similar to those produced in the lung, and reported here, and that they increase the infectivity of the newborn larvae. It has been shown that only 25-35% of newborn larvae injected directly into the muscle are able to develop and of these 60% remain in the site (Despommier er al. 1975). It is possible that cuticular modifications of the newly born larvae which increase their resistance to the nonspecific host defence may take place quickly during passage through the lungs. Our results demonstrate that, after entering the circulation, T. spiralis newborn larvae pass very rapidly through the lung barrier and their cuticle is modified during this passage. The modification observed involves the release of 1311-labeledcomponents, probably induced by the interaction with pulmonary cells. At the moment we are not able to say by which mechanism labeled products are retained in the lungs; adhesion of proteins to the capillary endothelium, which on the contrary does not seem to involve human albumin, and rapid passage to the interstitium by pinocytotic transport are two possibilities. The first mechanism could trigger platelet activation which would be responsible for the lung perfusion defects observed during experimental infection in monkeys (Bruschi et al. 1989). The exact biological significance of the parasitelung interaction and its relevance to the parasite life cycle and the hostparasite relationship is as yet still not understood, but we can hypothesize that passage through the lungs is a proparasite mechanism as regards a primary infection. ACKNOWLEDGMENTS We are grateful to Professor C. Giuntini, Dr. P. Sal-

AND BINAGHI vadori, and Mr. S. Antongiovanni for their helpful collaboration. We are indebted to S. M. Venturiello, S. Costantini, and G. Giambartolomei for performing some of the experiments of newborn larvae recovery from the lungs. This work was partially financially supported by the Italian MURST. REFERENCES BEFUS, D., EGWANG,T., AND GAULDIE, .I. 1984. Inflammatory and immune responses to parasites. In “Immunology of the Lung and Upper Respiratory Tract” (J. Bienenstock, Ed.), pp. 264-281. McGraw-Hill, New York. BERNTZEN, A. K. 1%5. Comparative growth and development of Trichinella spiralis in vitro and in vivo, with a redescription of the life cycle. Experimental Parasitology 16, 74406. BINAGHI, R. A., PERRUDET-BADOUX,A., AND BousSAC-ARON, Y. 1981. Mechanisms of immune defence against Trichinella spiralis newborn larvae. In “Trichinellosis V” (C. W. Kim, E. J. Ruitenberg, and J. S. Teppema, Eds.), pp. 85-89. Reedbooks, Chertsey. BRUSCHI, F., SOLFANELLI, S., ALESSANDRONI, P., Pozro, E., AND GIUNTINI, C. 1989. A lung perfusion study in Trichinella spiralis infected monkeys. In “Trichinellosis” (C. E. Tanner, A. MartinezFernandez, and F. Bolas-Femandez, Eds.), pp. 293-298. C.S.I.C. Press, Madrid. DENNIS, D. T., DESPOMMIER, D. D., AND DAVIS, N. 1970. Infectivity of the newborn larva of Trichinella spiralis in the rat. Journal of Parasitology 56, 974977. DESGREZ, A., MORETTI, J. L., ROBERT, J., AND VINOT, J. M. 1977. “Abreg6 de MCdecine Nucleaire.” Masson, Paris. DESPOMMIER, D. D., ARON, L., AND TURGEON, L. 1975. Trichinella spiralis: Growth of the intracellular (muscle) larva. Experimental Parasitology 37, 108116. DESPOMMIER, D. 1976. Musculature. In “Ecological Aspects of Parasitology” (C. R. Kennedy, Ed.), pp. 269-285. North-Holland, Amsterdam. DESPOMMIER, D. D. 1990. Trichinella spiralis: The worm that would be virus. Parasitology Today 6, 193-l%. GANSM~LLER, A., ANTEUNIS, A., VENTURIELLO, S. M., BRUSCHI, F., AND BINAGHI, R. A. 1987. An-

tibody-dependent “in vitro” cytotoxicity of newborn Trichinellu spiralis larvae: Nature of the cells involved. Parasite Immunology 9, 281-292. HARLEY, J. P., AND GALLICCHIO, V. 1971. Trichinella spiralis: Migration of larvae in the rat. Experimental Parasitology 30, 11-21. HERRICK, W. W., AND JANEWAY, T. C. 1909. Demonstration of the Trichinella spiralis in the circulat-

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ing blood of man. Archives of Internal Medicine 3, 263-266. “Human Serum Albumin Millimicrosphere Kit (TCK9) Instructions.” 1984. Sorin Biomedica, Vercelli, Italy. JEFFERY, P. K., AND CORRIN, B. 1984. Structural analysis of the Respiratory Tract. In “Immunology of the Lung and Upper Respiratory Tract” (J. Bienenstock, Ed.), pp. l-27. McGraw-Hill, New York. JUNGERY, M., CLARK, N. W. T., AND PARKHOUSE, R. M. E. 1983. A major change in surface antigens during the maturation of newborn larvae of Trichinella spiralis. Molecular

and Biochemical

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ogy 17, 101-109. MARKWELL, M. A. K. 1982. A new solid-state reagent to iodinate proteins. I. Conditions for the efficient labeling of antiserum. Analytical Biochemistry 125, 427-432. ORTEGA-PIERRES, G., MCKENZIE, C. D., AND PARKHOUSE, R. M. E. 1984. Protection against Trichinella spiralis induced by a monoclonal antibody that promotes killing of newborn larvae by granulocytes. Parasite

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6, 275-284.

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SANTOLICANDRO, A. M., MANCINI, P., FAZIO, F., STRATA, G. C., AND GIUNTINI, C. 1971. The re-

gional distribution of pulmonary blood volume studied by radioactive microspheres. Journal of Nuclear Biology and Medicine 15, 10-l 1. SPENCER, H. 1977. Pulmonary parasitic diseases. In “Pathology of the Lung” (K. Spencer, Ed.), 3rd ed., pp. 327-370. Pergamon Press, Oxford. WANG, C. H., AND BELL, R. G. 1986a. Trichinella spiralis: Newborn larval migration route in rats reexamined. Experimental Parasitology 61, 7685. WANG, C. H., AND BELL, R. G. 1986b. Trichinella spiralis: Vascular recirculation and organ retention of newborn larvae in rats. Experimental Parasitology 62, 430441. WILSON, R. A. 1990. Pulmonary immune response to parasites. In “Parasites: Immunity and Pathology” (J. M. Behnke, Ed.), pp. 208-248, Taylor & Francis, London. Received 29 July 1991;accepted with revision 9 March 1992

Trichinella spiralis: modifications of the cuticle of the newborn larva during passage through the lung.

A scintigraphic method was developed to study the distribution of radioactivity after iv injection of 131I-labeled Trichinella spiralis newborn larvae...
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