Aug 20, 1984 - Extracts of Schistosoma japonicum eggs were found to exhibit hemolytic activity on ... factors in S. japonicum eggs seemed to be important in.
Vol. 46, No. 2
INFECTION AND IMMUNITY, Nov. 1984, P. 514-518
0019-9567/84/110514-05$02.00/0
Copyright © 1984, American Society for Microbiology
Hemolytic Factors in Schistosoma japonicum Eggs HIROKO
ASAHI,1* ATSUKO MORIBAYASHI,2 FUJIRO SENDO,3 AND TAKATOSHI KOBAYAKAWA1
Department of Parasitology, 1 and Laboratory of Technology,2 National Institute of Health, 2-10-35, Kamiosaki, Shinagawa-ku, Tokyo 141, and Department of Parasitology, Yamagata University School of Medicine, lida Zao,
Yamagata, 990-23,3 Japan Received 25 June 1984/Accepted 20 August 1984
Extracts of Schistosoma japonicum eggs were found to exhibit hemolytic activity on erythrocytes of various species. The hemolytic reaction took place more rapidly at 37°C than at 4°C and did not require divalent cations. The degree of hemolysis was dependent on the. concentration of the egg extracts. The hemolytic factors seemed to be lipid in nature because of being heat-stable and soluble in chloroform solvent. Further analysis by means of thin-layer chromatography showed that hemolytic activity in the extracts was due to free fatty acids. Analysis by gas chromatography revealed that fatty acids in the egg extracts consist of myristic, palmitic, palmitoleic, stearic, oleic, linoleic, linolenic, and arachidonic acids. Among them, based on the experiments with commercially available fatty acids, arachidonic acid was the most intensively hemolytic, followed by linoleic, linolenic, oleic, palmitoleic, and myristic acids in that order, whereas palmitic acid showed weak activity only in the presence of divalent cations. From the estimated calculation, it was assumed that 1 mg of protein of the egg extracts contains as much as 0.22 mg of free fatty acids.
The major pathology of schistosomiasis appears to be due to schistosoma egg-induced tissue damage, including cellmediated granulomatous inflammation in the host tissues (23). It has been suggested that in addition to the crucial role played by immunological reactions, certain substances which have the ability to directly damage host tissues and cells may play an important role in the pathogenesis of schistosomiasis, particularly in granuloma formation (3, 4,
350-mesh wire screens. The mature eggs retained on a sieve with a mesh opening of 40 FLm (350 mesh) in diameter were washed with physiological saline solution. This method yielded S. japonicum eggs almost entirely free from tissue debris of the murine intestines. About 70% of the S. japonicum eggs collected were mature and showed active flame cell movement of miracidia. S. japonicum EE were prepared as follows. Collected eggs were suspended in a 20-fold volume of 0.15 M veronalbuffered saline (pH 7.4) containing 0.15 mM CaC12 and 0.5 mM MgCl2 (VBS21) and then distrupted by sonication at 4°C. In addition, they were triturated by repeated freezethawing until no intact eggs could be seen. The clear supernatant (S. japonicum EE), obtained by centrifugation of the egg homogenates at 4°C for 30 min at 12,000 x g, was dialyzed against distilled water. S. japonicum EE and the sediment containing mainly egg shells were lyophilized and subjected to assay for LA or subsequent purifications. The protein content of the extracts was determined by the method of Lowry et al. (13). Measurement of LA. Sheep and mouse RBCs in Alsever solution were purchased from Nippon Bio-Test Laboratories Inc., Tokyo, Japan. Human RBCs (group 0, Rh-) were prepared from heparinized blood. On the day of use the RBCs were washed with 0.15 M phosphate-buffered saline (PBS [pH 7.2]), VBS2+ containing 0.1% gelatinc (GVBS2+), or 0.15 M VBS (pH 7.4) containing 10 mM EDTA and 0.1% gelatine (GVBS-EDTA). LA was measured according to the method described by Weltzien (24), with some modificatioris. Briefly, 1 volume of RBC suspension (sheep, 8 x 108 cells per ml; human or murine, 4 x 108 cells per ml), unless otherwise specified, was mixed with 1.5 volumes of test sample. In the experiment on the effect of divalent cations on the LA of S. japonicum EE, GVBS-EDTA Was used as the medium instead of GVBS2+. After 60 min at 37°C, unless otherwise specified, the optical density of the centrifuged and diluted (1:5) supernatant was read spectrophotometrically at 541 nm to measure hemoglobin release. The results were represented as the percentage of control (100%) hemolysis obtained by lysing the same number of RBCs with distilled water.
23). We found that soluble extracts of Schistosoma japonicum eggs exhibit hemolytic activity (LA) on erythrocytes (RBCs) of various species. Since the hemolytic phenomenon has been well-proven to be valuable for the study of toxic effect on cellular surfaces, and since Smith et al. (22) reported that a lysophosphatide mixture similar to those naturally found in schistosoma eggs, which are generally known to be markedly cytotoxic and irritative to cells and their membrane, has been one of the casual substances in the formation of schistosomal granuloma, the presence of the hemolytic factors in S. japonicum eggs seemed to be important in regard to pathogenesis around the eggs. The present report demonstrates the occurrence of hemolytic factors in soluble extracts from S. japonicum eggs, and the results extend to include purification and identification of the hemolytic factors, which apparently seem to be free fatty acids (FFA). MATERIALS AND METHODS Preparation of S. japonicum EE. S. japonicum egg extracts (EE) were prepared with S. japonicum eggs obtained from the intestines of white Swiss mice of the Webster strain (NIH) which had been inoculated with S. japonicum by intraperitoneal injection of 40 to 60 cercariae 7 to 9 weeks previously. The intestines were homogenized in a Waring blender, followed by digestion with 0.4% trypsin (1:250; Difco Laboratories, Detroit, Mich.) at 370C for 3 h, as described by Browne and Thomas (2). S. japonicum eggs were sedimented several times and then separated from tissue debris by being sieved through 50-, 100-, 200-, and *
Corresponding author. 514
HEMOLYTIC FACTORS IN S. JAPONICUM EGGS
VOL. 46, 1984
TABLE 1. LA of S. japonicum EE, the sediments from S. japonicum egg homogenates, and trypsin % LAa in: Prepn and RBC GVBS2+ PBS
515
anol extracts, were further evaporated and suspended in diluent buffer to be assayed for LA. Crude lipids of the sediment from S. japonicum egg homogenates were also extracted in the same manner as S.
japonicum BE.
S. japonicum EE, 480 ,.ug of protein/ml Sheep Human Mouse
69 84 86
S. japonicum EEb, 480 ,ug of protein/ml Sheep Human Mouse
50 43 78
Sediments from S. japonicum egg homogenates Sheep
94 78
2 1
Human
0.2% Trypsin Sheep Human Mouse
2 1 12
Buffer Sheep Human Mouse
2 2 8
5 2
Mean percentage of duplicated test samples. For 100% hemolysis of sheep, human, and mouse RBCs, values were 0.730, 0.770, and 0.730, a
respectively, at an optical density of 541 nm. b S. japonicum eggs were collected from mice infected with S. japonicum without being subjected to trypsin digestion.
Alternatively, the dosage which led to 50% hemolysis was graphically extrapolated from the hemolysis-concentration curves in quantitative assays. Extraction of lipids. The lyophilized S. japonicum EE was homogenized in a chloroform-methanol mixture (2:1 [vol/ vol]) to extract crude lipids which were further extracted by the method of Bligh and Dyer (1). The three fractions obtained, namely, precipitates, chloroform, tnd water-meth-
Separation and analysis of lipids. Thin-layer chromatography (TLC) of chloroform extracts of S. japonicum EE (total lipids) was carried out on a silica gel thin-layer plate (0.25 mm thick; E. Merck AG, Darmstadt, Federal Republic of Germany) with use of either chloroform-methanol-water (65:25:4 [vol/vol]) or petroleum ether-diethyl ether-acetic acid (80:30:1 [vol/vol]) as a developing solvent system. Silica gel bands were located with use of iodine vapor, 50% sulfuric acid, Dittmer reagent (5), and ninhydrin reagent. Each fraction was extracted from the silica gel plate to be assayed for LA. Gas-liquid chromatography (GLC) was performed with a Shimadzu type GC 7A apparatus equipped with a hydrogen flame ionization detector; the glass column (210 by 0.3 cm) was packed with 25% diethylene glycol succinate or 1.5% SE-30 (Applied Science Laboratories, Inc., State College, Pa.) on Chromosorb W (Shimadzu Scientific Instruments, Inc., Kyoto, Japan). Each fatty acid was methylated with diazomethane and identified by comparing the retention time with those of methylated standards. Furthermore, the peak area corresponding to each fatty acid was quantitatively calculated with a Hewlett Packard model 3385 (integrator for chromatography). Chemicals. Cholesterol and saturated fatty acids as myristic (C14:0), palmitic (C16:0), and stearic (C18:0) acids were purchased from Wako Pure Chemical Industries, Ltd., Osaka, Japan. Unsaturated fatty acids as palmitoleic (C16:l), oleic (C18:1); linoleic (C18:2), linolenic (C18:3), and arachidonic (C20:4) acids were purchased from Sigma Chemical Co., St. Louis, Mo. Cholesteryl stearate was synthesized at this laboratory. RESULTS Lysis of RBCs by S. japonicum EE. S. japonicum EE prepared with or without the trypsin digestion step and the sediment of the S. japonicum egg homogenate were tested for their ability to hemolyze RBCs of sheep, humans, and
.8001 .700
.600 E c LL,
.500.400.300
0 0
.200.100
120 240 30 60 960 final concentration of SEE, Ig protein/ml FIG. 1. Effect of graded concentrations of S. japonicum EE on hemolysis. Each test sample was incubated at 37°C for 1 h. Each bar represents the mean + standard deviation. The following concentrations of sheep RBCs as target cells were employed, with GVBS2" as 0
medium: 0.8
x
108 RBCs per ml (A), 1.6
x 108
RBCs per ml (x), and 3.2 x 108 RBCs per ml (0). OD, Optical density; SEE, S. japonicum EE.
516
ASAHI ET AL.
INFECT. IMMUN.
Sheep RBC
100-
Human RBC
90 80 o
70
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INCUBATION TIME, min. course of hemolysis of sheep and
FIG. 2. Time human RBCs by S. japonicum EE. Each reaction mixture containing RBCs and S. japonicum EE (480 ,ug of protein per ml) was incubated at 37°C ( ) or 4°C (--- -). Each point represents the mean percentage of duplicated samples. Either GVBS2+ (0) or PBS (x) was used as medium for the experiments. Controls are GVBS2+ (A) or PBS (A) alone.
mice. GVBS2+ containing 0.2% trypsin served as the control. S. japonicum EE at the concentration of 480 pLg of protein per ml exhibited LA towards sheep, human, and mouse RBCs, whereas no LA was observed in the sediment of S. japonicum egg homogenates (Table 1). S. japonicum EE prepared without trypsinization also exhibited similar LA excluding the possible effect of trypsin upon the LA of S. japonicum EE during the process of S. japonicum EE preparation. Characteristics of hemolysis by S. japonicum EE. (i) Kinetics of hemolysis by S. japonicum EE. The kinetics of hemolysis by S. japonicum EE as a function of S. japonicum EE concentration was studied. S. japonicum EE at a final concentration ranging from 0 to 960 ,ug of protein per ml was added to a sheep RBC suspension in GVBS2+ at concentrations of 0.8 x 108, 1.6 x 108, and 3.2 x 108 cells per ml. The degree of hemolysis was dependent on the concentration of S. japonicum EE, and the plot Qf released hemoglobin levels (optical density at 541 nm) versus the concentration of S. japonicum EE took the form of a sigmoid curve, except when the concentration of S. japonicum EE was less than 60 pg of protein per ml (Fig. 1). (ii) Effect of temperature and time. The reaction mixtures of sheep and human RBCs and S. japonicum EE at a concentration of 480 pLg of protein per ml were incubated at 37 or 4°C for the indicated periods of time, followed by spectrophotometric determination of hemoglobin release. A significant degree of hemolysis was observed as early as 30 min after the initiation of incubation at 37°C, whereas at 4°C, although the degree of hemolysis increased as the time of incubation elapsed, a markedly low degree of LA was observed as late as 120 min in both of the RBC mixtures employed (Fig. 2). (iii) Effect of heat treatment of S. japonicum EE. To see whether the LA of S. japonicum EE is heat-labile, S. japonicum EE that was boiled at 100°C for 5 min with subsequent cooling and sonication was subjected to the assay. The LA of S. japonicum EE at a concentration of 480
,ug of protein per ml on human and sheep RBCs remained unaffected, suggesting that heat-stable factors in S. japonicum EE could be responsible for LA. (iv) Effect of EDTA. The possible effect of EDTA upon the LA of S. japonicum EE was tested by replacing GVBS2+ with GVBS-EDTA. The LA of S. japonicum EE at a concentration of 480 ,ug of protein per ml on human and sheep RBCs was apparently unchanged, suggesting that the reaction is not dependent on the divalent cations of Mg2+ and Ca2+. Purification of hemolytic constituents from S. japonicum EE. (i) Extraction with chloroform and methanol. In an attempt to further characterize the constituents in S. japonicum EE responsible for their LA, their solubility in chloroform and methanol was studied. Among five fractions obtained from both S. japonicum EE (precipitates, methanolwater extracts, and chloroform extracts) and the sediments of S. japonicum egg homogenates (precipitates and methanol-chloroform extracts), as extracted by the method of Bligh and Dyer (1), none except the chloroform extracts of S. japonicum EE exhibited LA on sheep and human RBCs, suggesting that the hemolytic, active constituents are lipid in nature.
(ii) Separation of chloroform extracts of S. japonicum EE by TLC. Chloroform extracts obtained from S. japonicum EE were further separated by TLC with a solvent mixture of chloroform-methanol-water (65:25:4 [vol/vol]). Iodine vapor staining revealed the presence of five spots (Fig. 3, Fl through F5). Three spots (Fl, F2, and F3) and one spot (F3) were detectable with Dittmer reagent and ninhydrin reagent, respectively. Each spot eluted from the plate was assayed for its LA. Only components in spot F4 exhibited LA on sheep and human RBCs. Therefore, components in spot F4
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origin FIG. 3. TLC of chloroform extracts of S. japonicum EE. These plates were developed with a solvent mixture of chloroform-methanol-water (65:25:4 [vol/vol]). Iodine vapor (lane A), Dittmer reagent (lane B), and ninhydrin reagent (lane C) were employed as indicators. S, Chloroform extracts of S. japonicum EE.
HEMOLYTIC FACTORS IN S. JAPONICUM EGGS
VOL. 46, 1984
517
exhibited weak LA only in the presence of divalent cations. The experimental mixture of fatty acids also exhibited LA, as was the case for FFA extracted from S. japonicum EE.
top X
DISCUSSION
i
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1
2
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origin
4
F4
1
2
3 4
FIG. 4. TLC of fraction F4 isolated from chloroform extracts of S. japonicum EE. A solvent mixture of petroleum ether-diethyl ether-acetic acid (80:30:1 [vol/vol]) was used for the development. Sulfuric acid (50%) was employed for differentiation of steroids from neutral lipids. Standards: stearic acid (Cj,:0) (lane 1), methyl stearate (lane 2), cholesteryl stearate (lane 3), and cholesterol (lane 4).
were
chromatographed again by TLC with the solvent
system of petroleum ether-diethyl ether-acetic acid (80:30:1 [vol/vol]). Two spots (Fig. 4, F4-i and F4-ii) were obtained,
and each of them was tested for the LA. LA was found only in a fraction extracted from spot F4-ii. Furthermore, the comparative study between the Rf value of spot F4-ii and those of the standards stearic acid, methyl stearate, cholesteryl stearate, and cholesterol revealed that the main components in spot F4-ii exhibiting LA are FFA but not sterols (Fig. 4). Identification of FFA responsible for LA. Constituents in spot F4-ii, which were methylated, were further analyzed by GLC for identification of the varieties of acid involved. The gas chromatogram (Fig. 5) shows that the FFA from S. japonicum EE contained 3.8% myristic (C140), 38.1% palmitic (C16:0), 3.3% palmitoleic (C16:1), 19.1% stearic (Ci8:0), 22.0% oleic (C18:1), 4.8% linoleic (C18:2), 4.7% linolenic (C18:3), and 2.6% arachidonic (C204) acids, as well as two unidentified acids. From the estimated calculation performed by comparing the peak area of each of the FFA with those of reference fatty acids, the contents of which were known beforehand, it was assumed that 1.00 mg of S. japonicum EE protein contains as much as 0.22 mg of FFA. Commercially available FFA corresponding to the FFA detected in the gas chromatogram, including myristic, palmitic, palmitoleic, stearic, oleic, linoleic, linolenic, and arachidonic acids, were quantitatively tested for their LA with human RBCs. In addition, assays were performed upon cholesterol, cholesteryl stearate, and a fatty acid mixture which was experimentally prepared by mixing commercially available fatty acids at the same composition as that of FFA in S. japonicum EE. The results in Table 2 demonstrate that arachidonic acid was the most markedly hemolytic, followed by linoleic, linolenic, oleic, palmitoleic, and myristic acids in that order, whereas stearic acid, cholesterol, and cholesteryl stearate did not exhibit significant LA at a concentration of less than 100 ,ug/ml. On the other hand, palmitic acid
The present paper demonstrates that EE from S. japonicum eggs exhibited LA on RBCs of various species. Hemolysis took place more rapidly at 37°C than at 4°C and did not apparently require divalent cations. The degree of hemolysis was dependent on the concentration of S. japonicum EE. Factors responsible for LA were heat stable and found in chloroform extracts of S. japonicum EE. Analyses by means of TLC and GLC showed that the LA of S. japonicum EE was due to FFA. GLC of the FFA fraction from S. japonicum EE revealed the presence of myristic, palmitic, palmitoleic, stearic, oleic, linoleic, linolenic, and arachidonic acids. Among them, arachidonic, linoleic, linolenic, oleic, palmitoleic, palmitic, and myristic acids exhibited LA. Smith et al. (21, 22) reported schistosome eggs to be abundant in FFA. The present study demonstrates that FFA in S. japonicum EE predominantly consisted of unsaturated, long-chain FFA, some of which were found to be hemolytically active. In general, FFA from various sources are known to exhibit different biological activities depending on the chemical structure in relation to their chain length, the degree of unsaturation, and the isomerism and surface properties of the target with which they interact (8, 15, 17); from an immunological point of view, increasing dosages of polyunsaturated fatty acids have been shown to produce, successively, in vivo and in vitro immune activation and inhibition (14-16, 25) and lymphocytolysis (9, 18). Transient delayedtype hypersensitivity has been successfully induced by injection with lysozyme conjugated with several varieties of fatty acids (10), and eosinophil-chemotactic factors have been shown to be generated and released from polymorphonucle-
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rCN
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CD~CC
: rz °X , QO) _
A^ injection
10
_
CDC_
20
30
min
FIG. 5. Pattern of GLC of FFA fraction F4-ii from S. japonicum EE. The percent composition of each peak is presented in parentheses. The concentration of total FFA in S. japonicum EE was estimated as 0.22 mg/mg of protein of S. japonicum EE. *, Not identified.
518
ASAHI ET AL.
INFECT. IMMUN.
TABLE 2. LA of each of the fatty acids, cholesterol, cholesterol derivative, and fatty acid mixture Dosage in ,ug/ml for 50% hemolysis" (mM) in: Test sample
GVBS2+
PBS
Myristic acid (C14:0) Palmitic acid (C16:0) Palmitoleic acid (C16: 1) Stearic acid (C18:0) Oleic acid (C18:1) Linoleic acid (C18:2) Linolenic acid (C18:3) Arachidonic acid (C20:4) Fatty acid mixture' Cholesterol Cholesteryl stearate
80 + 2 (0.357) 86 ± 0 (0.336) 39 + 1 (0.154) >100 41 ± 1 (0.145) 19 ± 0 (0.068) 23 + 1 (0.083) 19 ± 1 (0.063) 43 1 > 100 > 100
38 ± 1 (0.170) >100 37 1 (0.146) >100 46 ± 7 (0.163) 30 ± 8 (0.107) 32 ± 5 (0.115) 18 ± 0 (0.059) 36 2 > 100 >100
4.
5. 6.
7.
100' (22 ,ug of FFA) 100' (22 ,ug of FFA)
8.
a Mean ± standard deviation of at least three experiments. b The percent composition of each fatty acid was adjusted to that of FFA from S.japonicum EE. c The concentration is expressed in micrograms of protein per milliliter.
9.
S. japonicum EE
10.
ar cells by arachidonic acid (11), which was one of the FFA detected in S. japonicium eggs as described above.
However, little is known about the possible significance of FFA derived from parasitic helminths, except for their lipid metabolism (7). Of particular interest is the observation, possibly relevant to parasitic infections, that the hemolytic substance identified as being an unsaturated FFA of oleic acid has been produced by or derived from malaria parasites which might be implicated in black-water fever cases (12). These previous studies led us to consider the possibility that FFA released from S. japonicum eggs through excretory microscopic pores in their shells (20) might exert a modulatory influence on the host immune responses, such as chemotaxis and phagocytosis, possibly relating to schistosomal granuloma formation with prominent participation of eosinophiles at the sites (19), although, at present, there is no direct evidence to suggest that the hemolytic-cytotoxic effect of FFA in S. japonictum eggs has in vivo relevance to the
pathology of shistosomiasis. Incidentally, it has to be noted that Schistosoma mansoni eggs contain lysophosphatides (22), as well as the w, antigen, which has hepatocytotoxic effects (6). In fact, we have also observed that trace quantities of lysophosphatides in S. japonicum eggs are unable to exhibit any hemolytic activity (data not shown). A functional study on the in vivo role of FFA obtained from S. japonicum eggs is presently being undertaken. ACKNOWLEDGMENTS The invaluable advice of J. Yasuda, Department of Blood Products, and S. Nakazawa, Laboratory of Technology, National Institute of Health, Tokyo, Japan, is gratefully acknowledged. LITERATURE CITED 1. Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911917. 2. Browne, H. G., and J. I. Thomas. 1953. A method for isolating pure, viable schistosome eggs from host tissues. J. Parasitol. 49:371-374. 3. Boros, D. L., and K. S. Warren. 1970. Delayed hypersensitivitytype granuloma formation and dermal reaction induced and
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elicited by a soluble factor isolated from Schistosoma mansoni eggs. J. Exp. Med. 132:488-507. Byram, J. E., M. J. Doenhoff, R. Musallam, L. H. Brink, and F. von Lichtenberg. 1979. Schistosoma mansoni infections in T-cell deprived mice, and the ameliorating effect of administering homologous chronic infection serum. II. Pathology. Am. J. Trop. Med. Hyg. 28:274-285. Dittmer, J. C., and R. L. Lester. 1964. A simple, specific spray for the detection of phospholipids on thin-layer chromatograms. J. Lipid. Res. 5:126-127. Dunne, D. W., S. Lucas, Q. Bikle, S. Pearson, L. Madgwick, J. Bain, and M. J. Doenhoff. 1981. Identification and partial purification of an antigen (wl) from Schistosoma mansoni eggs which is putatively hepatotoxic in T-cell deprived mice. Trans. R. Soc. Trop. Med. Hyg. 75:54-71. Frayha, G. J., and J. D. Smyth. 1983. Lipid metabolism in parasitic helminths. Adv. Parasitol. 22:309-387. Kanai, K., and E. Kondo. 1979. Antibacterial and cytotoxic aspects of long-chain fatty acids as cell surface events: selected topics. Jpn. J. Med. Sci. Biol. 32:135-174. Kigoshi, S., and R. Ito. 1973. High levels of free fatty acids in lymphoid cells, with special reference to their cytotoxicity. Experientia 15:1408-1420. Kojima, A., M. Sugimoto, and Y. Egashira. 1976. Immunogenicity of lysozyme derivatives lipid-conjugated to various degrees in mice treated with and without cyclophosphamide: dissociation of delayed-type hypersensitivity and helper function. Jpn. J. Med. Sci. Biol. 29:323-333. Konig, W., H. Tesch, and N. Frickhofen. 1978. Generation and release of eosinophil chemotactic factor from human polymorphonuclear neutrophils by arachidonic acid. Eur. J. Immunol. 8:434-437. Laser, H. 1948. Hemolytic system in the blood of malariainfected monkeys. Nature (London) 161:560. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. Meade, C. J., and J. Mertin. 1976. The mechanism of immunoinhibition by arachidonic and linoleic acid: effects on the lymphoid and reticuloendothelial systems. Int. Arch. Allergy Appl. Immunol. 51:2-24. Meade, C. J., and J. Mertin. 1978. Fatty acids and immunity. Adv. Lipid Res. 16:127-165. Mertin, J., and D. Hughes. 1975. Specific inhibitory action of polyunsaturated fatty acids on lymphocyte transformation induced by PHA and PPD. Int. Arch. Allergy Appl. Immunol. 48:203-210. Nieman, C. 1954. Influences of trace amounts of fatty acids on the growth of microoganisms. Bacteriol. Rev. 18:147-163. Okudaira, H., I. Takaoka, H. Okada, R. Furuse-Irie, S. Kawachi, S. Nojima, and K. Nishioka. 1970. Cytotoxic factor demonstrated in lymph node extract. J. Biochem. 68:379-394. Phillips, S. M., and D. G. Colley. 1978. Immunologic aspects of host responses to schistosomiasis: resistance, immunopathology, and eosinophil involvement. Prog. Allergy 24:49-182. Sawada, T., K. Hara, K. Takagi, Y. Nagazawa, and S. Oka. 1956. Cytochemical studies on the hepatic tissue of mice following infections with Sc histosomajaponic um. Am. J. Trop. Med.
Hyg. 5:847-859. 21. Smith, T. M., B. L. Doughty, and J. N. Brown. 1977. Fatty acid and lipid class composition of Schistosoma japonicum eggs. Comp. Biochem. Physiol. 57B:59-63. 22. Smith, T. M., H. L. Lucia, B. L. Doughty, and F. C. von Lichtenberg. 1971. The role of phospholipids in schistosome granuloma. J. Infect. Dis. 123:629-639. 23. Warren, K. S. 1973. The pathology of Schistosoma infections. Helminthological Abstracts ser. A 42:591-633. 24. Weltzien, H. U. 1973. Slow-reacting hemolytic phosphatides: Benzylated lysolecithins. Biochim. Biophys. Acta 311:6-14. 25. Weyman, C., J. Belin, A. D. Smith, and R. H. S. Thompson. 1975. Linoleic acid as an immunosuppressive agent. Lancet
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