Vol. 15, No. 1 Printed in U.S.A.
INFECTION AND IMMUNITY, Jan. 1977, p. 84-90 Copyright C) 1977 American Society for Microbiology
Effect of Trichinella spiralis Infection on Passive Cutaneous Anaphylaxis in Mice JOHN J. MUNOZ* AND R. L. COLE Rocky Mountain Laboratory, Hamilton, Montana 59840
Received for publication 28 June 1976
Infection of CFW mice with Trichinella spiralis induced a state of relative unresponsiveness to passive cutaneous anaphylaxis (PCA) induced with hen egg albumin and its corresponding antibodies. The unresponsiveness was to PCA produced either with immunoglobulin G, (IgG,) or IgE type of antibodies, but was more pronounced with the latter. As few as 25 larvae given by stomach tube 20 days before induced this resistance, although 400 larvae induced a greater resistance. When 400 to 600 larvae were fed to mice, the refractoriness of these mice to PCA was noticed 15 days later. The sera of infected mice had the ability to inhibit mainly PCA induced by IgE. This inhibitory property of sera from infected mice was more pronounced 35 days after infection than 10 months later, when only weak inhibitory activity was detected. Purified rat IgE inhibited the PCA reactions induced in both mice and rats with mouse IgE-type antibody. At high concentrations, evidence of inhibition of the IgG1-induced PCA in mice was also obtained. We believe that the relative unresponsiveness of infected mice is due to an increase in production of IgE which competitively blocks the mast cell sites for other IgE molecules.
aphylaxis (PCA) induced with either IgG, or IgE type of antibodies.
Infection with Trichinella spiralis in some strains of mice induces a state of histamine and serotonin hypersensitivity (2, 13) and increases the levels of immunoglobulin E (IgE) with specificity to trichinella antigens (3, 11). These properties are in some respects similar to those of the substance from Bordetella pertussis that we have called pertussigen (J. J. Munoz, Fed. Proc. 35:813, 1976), which also increases the susceptibility of some mouse strains to vasoactive amines and increases the levels of IgE with specificity to antigens given with it (4). A significant increase in the levels of IgE with specificty to a given antigen should induce a state of relative unresponsiveness to other antigens, because IgE fixes strongly to mast cells and high concentrations of IgE with one specificity should successfully compete with mast cell binding sites for IgE molecules with different specificities. This was demonstrated by Stanworth et al. (19, 20) when they showed that human myeloma IgE or its Fc piece blocked the Prausnitz-Kustner (P-K) reaction in humans and by Jarrett et al. (7), who showed competitive inhibition of IgE in rats. Any treatment that increases IgE production to antigens other than the one involved in hypersensitivity reactions should competitively inhibit reactions due to IgE. Therefore, we have explored the possibility that mice infected with T. spiralis might become more resistant to passive cutaneous an-
MATERIALS AND METHODS T. spiralis. These worms were kept in mice infected by stomach tube with 200 to 600 larvae. When larvae were needed, mice infected for at least 1 month were processed by a modification of the method used by Weatherly (21). Briefly, the method consists of sacrificing infected mice, removing the skin, internal organs, heads, tails, and feet, and homogenizing the carcass (mainly muscle and bone) in a Waring Blendor for 60 s in a solution (500 ml/ mouse) of pepsin (1% dried pepsin in 0.5% HCl). The homogenized suspension was incubated for 2 h at 370C with constant shaking and filtered through a double thickness of cheesecloth, and the larvae were allowed to settle for at least 15 min. The supernatant fluid was drawn off to about 2.5 cm from the bottom. The settled larvae were diluted with saline and allowed to settle again in a funnel fitted with a clear plastic tube and clamp. When the larvae had settled, they were collected in as small a volume as possible and washed by being added to the top of a funnel filled with fresh physiological saline. This process was repeated two or three times until the larval suspension was cleared of any cloudiness. The suspension was standardized by counting the larvae in a known volume as previously described (13). Mice were infected by means of a stomach tube (18-gauge animal-feeding needle, Popper and Sons, Inc.) with 0.1 to 0.2 ml containing the appropriate number of larvae. 84
VOL. 15, 1977
Mice. CFW male and female mice reared in our laboratory were used. C57BL/6J female mice were purchased from the Jackson Laboratory, Bar Harbor, Me. B. pertussis extract. B. pertussis extract (BPE) was made by a modification of the method previously described (12). Acetone-extracted cells were suspended in 1 M NaCl-0.05 M sodium pyrophosphate at pH 7.4. The suspension was left overnight at 2 to 5C with constant stirring, and then the cell debris was separated by centrifugation. The clear supernatant fluid was extensively dialyzed against distilled water and lyophilized. Mouse antibody. The reaginic type of antibody (IgE) was contained in a pool of antisera from C57BL/6J mice immunized intraperitoneally with a mixture of 50 to 125 spg of hen egg albumin (HEA) and 5 to 50 ,ug of BPE dissolved in 0.2 ml of phosphate-buffered saline. Twenty-one to 26 days later, the mice were given subcutaneously a booster dose of 5 ug of HEA in saline, and the mice were bled 7 to 9 days later. Sera with high titers of 72-h PCA antibody were pooled and titrated for their content of anti-HEA of the IgG, (2-h PCA) and IgE (72-h PCA) classes of immunoglobulins by PCA reactions performed on mice. The IgG, type of mouse anti-HEA was produced in C57BL/6J mice that had been immunized intraperitoneally with a mixture of 125 ,ug of HEA and 50 j.g of BPE in 0.2 ml of saline. Then, at weekly intervals, intraperitoneal injections of 0.5 ml of complete Freund adjuvant emulsified in saline were given. On the 4th week and weekly thereafter, intraperitoneal injections of 0.5 ml of complete Freund adjuvant containing 6 ,ug of HEA were given until marked ascites developed. The ascitic fluid was then collected, centrifuged, and kept frozen. This fluid, which contained good titers of anti-HEA in both IgG1 and IgE, was fractionated to separate IgG, from IgE by first precipitating the globulins at 50% saturation with ammonium sulfate. The precipitated globulins were dialyzed in 0.005 M phosphate buffer (pH 7.9) and passed through a diethylaminoethylcellulose column equilibrated with the same buffer. Fractions were eluted by increasing the buffer concentration to 0.02, 0.03, 0.05, and 0.5 M phosphate at pH 7.9. The IgGI was eluted in the 0.02 M fraction, and the IgE was eluted in the 0.05 M fraction. The 0.02 M fraction was dialyzed, lyophilized, and then used in the present work. This preparation did not induce 72-h PCA in mice. The serum with IgE antibody, when heated at 560C for 3 h, failed to induce PCA reactions, thus indicating that the antiHEA antibodies were mainly of the IgE class. The IgG, did not produce PCA reactions in rat skin, whereas the IgE antibody preparation did in a titer similar to that obtained in mice as reported by Ovary et al. (15). Purification of rat myeloma IgEIR, was done from myeloma ascitic fluid. The technique was that used by H. Metzger (personal communication). Briefly, it consisted of the following steps. Twenty milliliters of ascitic fluid was dialyzed against borate-NaCl buffer (222.6 g of boric acid + 168.3 g of NaCl in 18 liters of water at pH 8). Saturated
EFFECT OF T. SPIRALIS ON PCA
85
(NH4)2S04 was added to the dialyzed fluid to bring the salt concentration to 37% saturation. After standing overnight at 2 to 50C, the solution was centrifuged clear and the precipitate was discarded. To the supernatant fluid, additional saturated (NH4)2SO4 was added to bring it to 48% saturation. This mixture was allowed to stand overnight at 2 to 50C and then centrifuged. The supernatant fluid was discarded, and the precipitate was dissolved in borate-NaCl buffer and chromatographed in a Sepharose 6B column (2.5 by 83 cm) equilibrated in the same buffer. The protein peak containing IgE was rechromatographed in the same column, and the material under the rather symmetrical peak of IgE was dialyzed against 0.025 M tris(hydroxymethyl)aminomethane (Tris)-phosphate buffer, pH 8 (80 ml of 2.5 M Tris + 150 ml of 1 M NaH2PO,, 700 ml of water; the pH was adjusted to 8.0 with 10 N NaOH, and the volume was brought up to 6 liters). The dialyzed material was chromatographed on a DE-52 column (volume of 110 ml) equilibrated with Trisphosphate buffer. The IgE in this column is not retarded and comes off in the first protein peak. This fraction was considered to be pure rat IgE. The fractionation was monitored by gel diffusion tests performed with a specific anti-rat IgE serum kindly supplied by H. Metzger. The final concentration of the IgE was determined by the optical density at 280 nm (optical density/1.36 = milligrams of protein/ milliliter). Antigen. Five-times-recrystallized HEA was purchased from Nutritional Biochemicals Corp. PCA reactions. PCA reactions were performed as previously described (4).
RESULTS Effect of T. spirlis infection on PCA reaction. Mice that had been infected with 600 larvae 34 days before were used as test animals for PCA reactions. Dilutions of IgG, antibody and IgE antibody were tested in four infected mice and four normal mice by intracutaneous administration of 0.05 ml of the chosen dilutions. Two hours later, mice sensitized with IgG, were challenged intravenously with 0.2 ml of a mixture of 0.5% Evans blue + 0.5% HEA, and the diameter of the reactions was measured 30 min later. Mice sensitized with IgE antibody were similarly challenged 3 days later, and reactions were measured 30 min later. The results are given in Table 1. It is clear from these results that infected mice did not respond as well as normal mice to either IgGi- or IgE-mediated PCA. The range of concentrations of IgE used in this experiment was rather low, and we did not know from these results whether infected mice were capable of responding to PCA induced with IgE. In the following experiments we increased the range of concentrations. Effect of worm load on PCA reaction. Mice were infected with either 25, 100, or 400 larvae
86
INFECT. IMMUN.
MUNOZ AND COLE
TABLE 1. Effect of T. spiralis infection on PCA' Concn of IgG, Dilution of serum contain25
Normal
ing IgE
(Aglml)
Recipient mouse
50
100
Ob 11 15 0 10 15
1:3,000 0 0
1:2,000 6wc 8w
1:1,000
10 9
0 0 11w 0 0 T. spiralis 0 0 0 12w 0 0 infected 0 a CFW mice that had been infected with 600 T. spiralis larvae 34 days earlier were used. b Numbers are the diameter in millimeters of the area of bluing measured on the underside of the skin. Each reading represents a separate mouse. Only one site per mouse was used. c w, Weak reaction.
per mouse. Twenty and 41 days later, the IgE and IgG, PCA antibodies were titrated in these mice (Table 2). Photographs of the reactions obtained 20 days after infection are given in Fig. 1 and 2. Twenty days after infection, mice infected with only 25 larvae showed a moderate resistance to manifest reactions induced with antibodies of the IgG, or IgE class. When 100 or 400 larvae were given, however, a striking inhibition was observed for both types of antibodies. Forty-one days after infection, strong inhibition was observed after even the small dose of 25 larvae. Effect of duration of T. spiralis infection on ability of mice to give PCA reactions. This experiment was designed to determine when after infection resistance to PCA was first observed. Mice were infected with 400 larvae and at intervals thereafter were tested as above for their ability to develop PCA reactions with the two types of antibodies (Table 3). PCA responses to IgG, and IgE types of antibodies were inhibited strongly beginning 20 days after the infection, and the inhibition seemed stronger at 41 days. An unexpected finding in this experiment was that the PCA reactions induced by IgG, antibody were considerably larger in mice that had harbored the infection for only 10 days than in normal mice or those that had carried the infection for 15 days or longer. The significance of this finding is presently under investigation. Effect of serum from normal and T. spiralis-infected mice on PCA reactions. As indicated in the introduction, T. spiralis infections augment the levels of IgE in the animal, and this increase in IgE could be responsible for preventing, competitively, PCA due to IgE with specificty to other antigens. The previous experiments clearly showed that T. spiralisinfected mice were more resistant to PCA reactions than were the normal animals. It was
then important to see whether the serum of infected animals had a substance that could prevent PCA reactions from taking place. The antiserum containing IgE antibody to HEA was diluted 1:250, and the IgG, preparation was made to contain 100 ug/ml. To 0.3 ml of each antibody, 0.3 ml of diluted normal or infected mouse serum was added. Each of two mice was then given intracutaneously 0.05 ml of each mixture, and 2 or 72 h later the mice were challenged intravenously with the dye-HEA mixture. Sera from mice that had been infected for various periods of time with T. spiralis were used. The sera of infected mice inhibited the PCA induced with IgE to HEA having little effect on PCA induced with IgG, (Table 4). In addition, it is evident that the inhibitory substance was more concentrated in the sera of mice infected 35 days before bleeding than in those that had carried the infection 4 to 10 months. The sera of normal mice had no demonstrable inhibition of either type of antibody. The substance responsible for this inhibition is most likely IgE. We have demonstrated IgEtype antibodies specific for T. spiralis antigens by 72-h PCA reactions induced with these sera TABLE 2. Effect of larval load on reactivity of skin to PCA No. of larvae given 25
20a 41
100
20 41
400
20 41
0
Concn of IgG,
Days after infections
(Ag/ml)
Dilution of serum containing IgE
0 0 0
50 100 1:500 1:250 1:125 7w 14 12w 20 +d 14 12 14 11 10 + 10 0 12 0 lOw 16 9w 0 0
0 0 0 0
low 18 15 0 low 9w lOw 13
0 0 0 0
10 14 12w
0 0 0 0
25
12Wb .
0 0 0 0 9w 11w 11 12
0 +
0 0 14 16 12 10
lOw 16 19 17 18
10 10 10 11
9w 10 7w 10 0 8w 12w 0
0 6w 0 0 12 13 13 14
0 7w 0 NIe 30 24 11 13
a On day 20, the diaphragms of six mice per group were examined under the microscope for T. spiralis larvae. Mice infected with 25 larvae had only a few larvae in the diaphragm, but all six mice were positive. The diaphragms of those receiving 100 and 400 larvae showed increasing numbers of larvae in all mice. b. c See Table 1 for meaning of numbers. w, Weak reactions either ring shaped or with clear center, or with very light concentration of dye. d _ s Doubtful reaction. eND, Not done.
VOL. 15, 1977
EFFECT OF T. SPIRALIS ON PCA
87
CONCENTRATION OF IgG1ANTIBODY TO HEA 25
50
100 womw
.1
0
V
.110,
...J'
i.
z w
'
m-
-4 -
lim.^,.
'U
4( 4c 16 -i
3 0.
100
*
I'-
0
z 400
9 ,^
FIG. 1. PCA reactions performed with different concentrations (in micrograms per milliliter) of the IgG0 type of antibody to HEA in normal mice or in mice infected 20 days earlier with 25, 100, or 400 T. spiralis larvae given by stomach tube.
using as a challenge dose a saline extract from frozen, thawed, and homogenized larvae mixed with Evans blue. Unfortunately, we do not presently have a way to quantitate mouse IgE other than by PCA reactions. We will attempt in the future to develop such a test by using the technique of producing anti-mouse IgE recently developed by Prouvost-Danon et al. (16), Lang et al. (9), and S. Lehrer (personal communication). The availability of rat myeloma IgE offers a possibility of measuring the effect of this protein on IgE of mice. It is known that mouse IgE fixes to rat mast cells and that it produces PCA reactions in rats (15), and that rat IgE fixes to mouse mast cells (17). This indicates that rat IgE may also inhibit the PCA reaction. Effect of rat IgE on PCA reaction in mice. A solution of purified rat myeloma IgE containing 500 pug/ml was made in physiological saline, and then serial 10-fold dilutions were made. Equal volumes of each dilution were mixed with either a solution of IgG, antibody containing 100 gg/ml or a 1:250 dilution of serum containing IgE antibody. The final concentration of IgG 'was 50 ,ug/ml, and the final dilution of the IgE-containing serum was 1:500. Two mice were sensitized per dilution, and two mice for
each antibody received it diluted in saline only. The results are given in Table 5. The PCA due to IgG, was not significantly inhibited by 250 ,g of rat IgE per ml of the final mixture. In some experiments, however, rat IgE at the highest concentration seemed to have some inhibitory effect because the PCA reactions had a completely clear center. The reactions induced by IgE antibody were completely inhibited by as little as 10 jig of rat IgE per ml in the mixture and partially inhibited by 2 ug/ml. Similar inhibition was obtained in rat skin sensitized with mouse antibody (IgE). These experiments show that IgE may indeed be responsible for the inhibition observed. There are methods besides worm infestation that stimualte IgE formation to various antigens; one is by immunization with B. pertussis extracts (4), and another is by giving antigen mixed with large amounts of alum (18). Preliminary studies have shown that both of these methods may also render mice more resistant to PCA reaction. DISCUSSION During a study on the stimulation of IgE in mice by an extract from B. pertussis, it occurred to us that stimulation of IgE to a heterol-
88
INFECT. IMMUN.
MUNOZ AND COLE
DsL UTON OF 1gE ANTIBODY TO HEA ri2250
2n. ,
z LU
25
0 LU
m t.
0 ~ ~ ~ ~
~
7,4;~
~
'C ALI~~~~~~~~~~~~~~~~~~~~~~~~~~
:1 100 i-'i
ci~~~~~~~~~~~~~~~~~l U-~~~=X -urj tI', Z400 FIG. 2. PCA reactions performed with different concentrations of the IgE type of antibody to HEA in normal mice or in mice infected 20 days earlier with 25, 100, or 400 T. spiralis larvae given by stomach tube. TABLE 3. Effect of duration of T. spiralis infection on resistance to PCA Days after infection 10
15 20 41
Concn of IgG, (jug/1l) Osno g gm 25 lOwa.b 11w
Dilution of serum containing IgE
50
100
1:500
1:250
1:125
25 25
34 32
0 12
10 9
20 19
+C
20 12
0 0
12 20
0 0
11w
15
0 0
0 +
10 14
0 0
0 6w
0 7w
0 0
12w low
0 0
0 0
0 NDd
0 0
9w
11 12 17 10 13 Uninfected 11w 11 14 13 controlse 12w 10 18 a, bSee Table 1 for meaning of numbers. w, Weak reaction. ±, Doubtful reaction. d ND, Not done. e Groups of uninfected mice of the same age and sex as the infected mice were used for each time period, with results similar to the control group shown in this table; for that reason the results were omitted.
ogous antigen should block allergic reactions involving mast cells. One of the most effective ways of stimulating IgE in animals, and apparently humans as well, is infection with certain
parasitic worms (5-8, 15). Indirectly, some published observations have shown that parasitism in humans may make them more resistant to allergic responses, since persons infected with helminths were found to give weaker P-K reactions (1). These observations, although they do not directly incriminate parasitism as responsible for resistance to certain allergic reactions, are highly suggestive. Jarrett et al. have definitely shown that Nippostrongylus brasiliensis infections make rats more resistant to PCA reactions (7). The results presented here also conclusively show that experimental infection of mice with T. spiralis makes these animals strikingly more refractory to PCA reactions to a heterologous antigen. Thus, infected mice sensitized with anti-HEA of either the IgG1 or IgE class did not respond to doses of these antibodies that were fully effective in normal mice of the same sex and age. The refractory state appeared about 7 days after the female worms had deposited the larvae in the intestinal mucosa and the larvae had migrated to the muscle (from 15 to 20 days after oral infection with larvae [10]). It was also apparent that the refractoriness was more pronounced 41 days after infection than at 15 or 20 days. As few as 25 larvae were sufficient to induce the refractory state, but in this
VOL. 15, 1977
EFFECT OF T. SPIRALIS ON PCA
89
TABLE 4. Effect of T. spiralis-infected mouse sera obtained at different times after infection
35 days after infection 4 mo after infection
10 mo after infection
1:8
1:4
1:2
Normal
IgE-containing serum diluted 1:500 in the
50 gg of IgG,/ml in the mixture
Type of mouse serum used
mixture
1:8
1:4
1:16
1:16
1:2
12wb 17
13w 12w
15
15
12
15
0 0
0 0
0
low
0
14w
18 18
15a 14
17
15
15
17
NDC ND
ND
ND
ND
ND
ND
ND
0 17
13
13
17
0
14
13
10
0
12w 0
low 0
12w 12w
12 12
11 12
15 11
17 13
0 17w
12w llw
12w 11
llw
Antibody in saline
15 17
a
See Table 1 for meaning of numbers.
b
w, Weak reaction.
c
ND, Not done.
TABLE 5. Effect of rat IgE on PCA reactions in mice Final concn of 50 jg of IgG,/ml rat IgE (jug~ml in mixture
IgE-containing serum diluted 1:500 in mixture
14a 0 0 33 250 14 0 0 0 50 0 0 9 13 10 6wb 8 2 10 10 14 12 15 10 0.4 14 14 13 12 Saline a See Table 1 for meaning of numbers. Columns represent readings from individual mice. b w, Weak reaction.
case it took longer to manifest itself. One hundred larvae were more effective, and 400 appeared to be even better. Since each mature female worm can produce close to 1,000 larvae (10), even the small infective dose of 25 larvae has a potential to produce some 12,500 larvae (assuming that half the infecting larvae are males and half females), which is a significant worm mass. The most important factor in the production of this refractoriness was probably IgE, because sera from infected animals inhibited PCA reactions induced by IgE and purified rat IgE also inhibited these reactions in both mice and rats. It is clear from the results that inhibition in infected mice was not exclusively manifested against the IgE class but also against the IgG1 class. We do not now know whether this inhibition was due to IgE alone or whether an increase in the IgG1 in the infected mice was responsible for inhibition of IgG1-induced PCA. Since IgG, is normally present in high concentrations in the sera of mice, it is more probable that IgE in high titers may be
11
14 14
also competing with IgG1. This should be further investigated with purified IgG, and IgE fractions from mouse serum. If the inhibition observed in this work was due to an increased concentration of IgE, one should also be able to increase the resistance of mice to PCA by other methods known to increase IgE. We are presently investigating this
possibility. The implication of these findings with respect to human allergies involving the IgE and possibly the IgG class of immunoglobulins is clear. It should be possible to block mast cell receptors by greatly increasing the IgE concentration in the blood to antigens that normally are not present in the environment. If this can be done, mast cells may well become relatively unresponsive to antigens to which the person is allergic. It has already been shown that IgE myeloma protein or its Fc piece blocks the P-K reaction (19, 20). Passive transfer of IgE would probably not be practical because of its antigenicity, its limited availability, and the required frequent administration. If endogenous production of IgE were increased, most of these problems could be circumvented. ACKNOWLEDGMENTS We wish to express our appreciation to H. Metzger for supplying the rat ascitic fluid and the methods to purify the rat IgE used in this work. We also would like to thank R. K. Bergman for his help in writing this manuscript.
LITERATURE CITED 1. Bazaral, M., H. A. Orgel, and R. N. Hamburger. 1973. The influence of serum IgE levels of selected recipi-
ents, including patients with allergy, helminthiasis and tuberculosis, on the apparent P-K titre of a reaginic serum. Clin. Exp. Immunol. 14:117-125.
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2. Briggs, N. T. 1963. Hypersensitivity in murine trichinosis: some responses of trichinella-infected mice to antigen and 5-hydroxytryptophan. Ann. N.Y. Acad.
12. Munoz, J., and B. M. Hestekin. 1963. Antigens of Bor-
Sci. 113:456-466. Briggs, N. T. 1963. Immunological injury of mast cells in mice actively and passively sensitized to antigens from Trichinella spiralis. J. Infect. Dis. 113:22-32. Clausen, C. R., J. Munoz, and R. K. Bergman. 1969. Reaginic-type of antibody in mice stimulated by extracts of Bordetella pertussis. J. Immunol. 103:768777. Cueva, J., and Q. F. B. Blanca E. Navarro. 1973. IgE en alergicos y parasitados en Mexico. Alergia (Mexico City) 21:1-11. Gougerot,M. A., A. Pasticier, G. Saimot, J. P. Coulaud, A. Ricour, and M. Payet. 1975. Les IgE seriques dan les parasitoses: a propos d'une etude chez 27 Africians de louest. Bull. Soc. Pathol. Exot. 68:297303. Jarrett, E. E. E., T. S. C. Orr, and P. Riley. 1971. Inhibition of allergic reactions due to competition for mast cell sensitization sites by two reagins. Clin. Exp. Immunol. 9:585-594. Johansson, S. G. O., T. Mellbin, and B. Vahliquist. 1968.Immunoglobulin levels in Ethiopian preschool children with special reference to high concentrations of immunoglobulin E (IgND). Lancet i:1118-1121. Lang, G. M., W. Y. Lee, B. G. Carter, and A. H. Sehon. 1976. Production of goat, rat and guinea pig antisera to murine IgE. J. Immunol. 116:881-883. Larsh, J. E., Jr. 1963. Experimental trichinosis. Adv. Parasitol. 1:213-286. Mota I., E. H. Sadun, R. M. Bradshaw, and R. W. Gore. 1969. The immunological response of mice infected with Trichinella spiralis. Biological and physico-chemical distinction of two homocytotropic anti-
147:524-526. 14. Ogilvie, B. M. 1964. Reagin-like antibodies in animals immune to helminth parasites. Nature (London) 204:91-92. 15. Ovary, Z., S. S. Caiazza, and S. Kojima. 1975. PCA reactions with mouse antibodies in mice and rats. Int. Arch. Allergy Appl. Immunol. 48:16-21. 16. Prouvost-Danon, A., R. Binaghi, S. Rochas, and Y. Boussac-Aron. 1972. Immunochemical identification of mouse IgE. Immunology 23:481-491. 17. Prouvost-Danon, A., J. Wyczolkowska, R. Binaghi, and A. Abadie. 1975. Mouse and rat IgE: cross sensitization of mast cells and antigenic relationships. Immunology 29:151-162. 18. Revoltella, R., and Z. Ovary. 1969. Reaginic antibody production in different mouse strains. Immunology 17:45-54. 19. Stanworth, D. R., J. H. Humphrey, H. Bennich, and S. G. 0. Johansson. 1967. Specific inhibition of the Prausnitz-Kustner reaction by an atypical human myeloma protein. Lancet ii:330-332. 20. Stanworth, D. R., J. H. Humphrey, H. Bennich, and S. G. 0. Johansson. 1968. Inhibition of Prausnitz-Kdstner reaction by proteolytic-cleavage fragments of a human myeloma protein of immunoglobulin class E.
3. 4.
5.
6.
7.
8.
9.
10. 11.
bodies. Immunology 16:71-81.
detella pertussis. III. The protective antigen. Proc. Soc. Exp. Biol. Med. 112:799-805. 13. Munoz, J. J., F. Sogandares-Bernal, and R. K. Bergman. 1974.Histamine sensitization of mice by Trichinella spiralis infection. Proc. Soc. Exp. Biol. Med.
Lancet ii:17-18.
21. Weatherly, N. F. 1970. Increased survival of Swiss mice given sublethal infections of Trichinella spiralis. J. Parasitol. 56:748-752.
INFECTION AND IMMUNITY, Jan. 1977, p. 84-90 Copyright C) 1977 American Society for Microbiology
Effect of Trichinella spiralis Infection on Passive Cutaneous Anaphylaxis in Mice JOHN J. MUNOZ* AND R. L. COLE Rocky Mountain Laboratory, Hamilton, Montana 59840
Received for publication 28 June 1976
Infection of CFW mice with Trichinella spiralis induced a state of relative unresponsiveness to passive cutaneous anaphylaxis (PCA) induced with hen egg albumin and its corresponding antibodies. The unresponsiveness was to PCA produced either with immunoglobulin G, (IgG,) or IgE type of antibodies, but was more pronounced with the latter. As few as 25 larvae given by stomach tube 20 days before induced this resistance, although 400 larvae induced a greater resistance. When 400 to 600 larvae were fed to mice, the refractoriness of these mice to PCA was noticed 15 days later. The sera of infected mice had the ability to inhibit mainly PCA induced by IgE. This inhibitory property of sera from infected mice was more pronounced 35 days after infection than 10 months later, when only weak inhibitory activity was detected. Purified rat IgE inhibited the PCA reactions induced in both mice and rats with mouse IgE-type antibody. At high concentrations, evidence of inhibition of the IgG1-induced PCA in mice was also obtained. We believe that the relative unresponsiveness of infected mice is due to an increase in production of IgE which competitively blocks the mast cell sites for other IgE molecules.
aphylaxis (PCA) induced with either IgG, or IgE type of antibodies.
Infection with Trichinella spiralis in some strains of mice induces a state of histamine and serotonin hypersensitivity (2, 13) and increases the levels of immunoglobulin E (IgE) with specificity to trichinella antigens (3, 11). These properties are in some respects similar to those of the substance from Bordetella pertussis that we have called pertussigen (J. J. Munoz, Fed. Proc. 35:813, 1976), which also increases the susceptibility of some mouse strains to vasoactive amines and increases the levels of IgE with specificity to antigens given with it (4). A significant increase in the levels of IgE with specificty to a given antigen should induce a state of relative unresponsiveness to other antigens, because IgE fixes strongly to mast cells and high concentrations of IgE with one specificity should successfully compete with mast cell binding sites for IgE molecules with different specificities. This was demonstrated by Stanworth et al. (19, 20) when they showed that human myeloma IgE or its Fc piece blocked the Prausnitz-Kustner (P-K) reaction in humans and by Jarrett et al. (7), who showed competitive inhibition of IgE in rats. Any treatment that increases IgE production to antigens other than the one involved in hypersensitivity reactions should competitively inhibit reactions due to IgE. Therefore, we have explored the possibility that mice infected with T. spiralis might become more resistant to passive cutaneous an-
MATERIALS AND METHODS T. spiralis. These worms were kept in mice infected by stomach tube with 200 to 600 larvae. When larvae were needed, mice infected for at least 1 month were processed by a modification of the method used by Weatherly (21). Briefly, the method consists of sacrificing infected mice, removing the skin, internal organs, heads, tails, and feet, and homogenizing the carcass (mainly muscle and bone) in a Waring Blendor for 60 s in a solution (500 ml/ mouse) of pepsin (1% dried pepsin in 0.5% HCl). The homogenized suspension was incubated for 2 h at 370C with constant shaking and filtered through a double thickness of cheesecloth, and the larvae were allowed to settle for at least 15 min. The supernatant fluid was drawn off to about 2.5 cm from the bottom. The settled larvae were diluted with saline and allowed to settle again in a funnel fitted with a clear plastic tube and clamp. When the larvae had settled, they were collected in as small a volume as possible and washed by being added to the top of a funnel filled with fresh physiological saline. This process was repeated two or three times until the larval suspension was cleared of any cloudiness. The suspension was standardized by counting the larvae in a known volume as previously described (13). Mice were infected by means of a stomach tube (18-gauge animal-feeding needle, Popper and Sons, Inc.) with 0.1 to 0.2 ml containing the appropriate number of larvae. 84
VOL. 15, 1977
Mice. CFW male and female mice reared in our laboratory were used. C57BL/6J female mice were purchased from the Jackson Laboratory, Bar Harbor, Me. B. pertussis extract. B. pertussis extract (BPE) was made by a modification of the method previously described (12). Acetone-extracted cells were suspended in 1 M NaCl-0.05 M sodium pyrophosphate at pH 7.4. The suspension was left overnight at 2 to 5C with constant stirring, and then the cell debris was separated by centrifugation. The clear supernatant fluid was extensively dialyzed against distilled water and lyophilized. Mouse antibody. The reaginic type of antibody (IgE) was contained in a pool of antisera from C57BL/6J mice immunized intraperitoneally with a mixture of 50 to 125 spg of hen egg albumin (HEA) and 5 to 50 ,ug of BPE dissolved in 0.2 ml of phosphate-buffered saline. Twenty-one to 26 days later, the mice were given subcutaneously a booster dose of 5 ug of HEA in saline, and the mice were bled 7 to 9 days later. Sera with high titers of 72-h PCA antibody were pooled and titrated for their content of anti-HEA of the IgG, (2-h PCA) and IgE (72-h PCA) classes of immunoglobulins by PCA reactions performed on mice. The IgG, type of mouse anti-HEA was produced in C57BL/6J mice that had been immunized intraperitoneally with a mixture of 125 ,ug of HEA and 50 j.g of BPE in 0.2 ml of saline. Then, at weekly intervals, intraperitoneal injections of 0.5 ml of complete Freund adjuvant emulsified in saline were given. On the 4th week and weekly thereafter, intraperitoneal injections of 0.5 ml of complete Freund adjuvant containing 6 ,ug of HEA were given until marked ascites developed. The ascitic fluid was then collected, centrifuged, and kept frozen. This fluid, which contained good titers of anti-HEA in both IgG1 and IgE, was fractionated to separate IgG, from IgE by first precipitating the globulins at 50% saturation with ammonium sulfate. The precipitated globulins were dialyzed in 0.005 M phosphate buffer (pH 7.9) and passed through a diethylaminoethylcellulose column equilibrated with the same buffer. Fractions were eluted by increasing the buffer concentration to 0.02, 0.03, 0.05, and 0.5 M phosphate at pH 7.9. The IgGI was eluted in the 0.02 M fraction, and the IgE was eluted in the 0.05 M fraction. The 0.02 M fraction was dialyzed, lyophilized, and then used in the present work. This preparation did not induce 72-h PCA in mice. The serum with IgE antibody, when heated at 560C for 3 h, failed to induce PCA reactions, thus indicating that the antiHEA antibodies were mainly of the IgE class. The IgG, did not produce PCA reactions in rat skin, whereas the IgE antibody preparation did in a titer similar to that obtained in mice as reported by Ovary et al. (15). Purification of rat myeloma IgEIR, was done from myeloma ascitic fluid. The technique was that used by H. Metzger (personal communication). Briefly, it consisted of the following steps. Twenty milliliters of ascitic fluid was dialyzed against borate-NaCl buffer (222.6 g of boric acid + 168.3 g of NaCl in 18 liters of water at pH 8). Saturated
EFFECT OF T. SPIRALIS ON PCA
85
(NH4)2S04 was added to the dialyzed fluid to bring the salt concentration to 37% saturation. After standing overnight at 2 to 50C, the solution was centrifuged clear and the precipitate was discarded. To the supernatant fluid, additional saturated (NH4)2SO4 was added to bring it to 48% saturation. This mixture was allowed to stand overnight at 2 to 50C and then centrifuged. The supernatant fluid was discarded, and the precipitate was dissolved in borate-NaCl buffer and chromatographed in a Sepharose 6B column (2.5 by 83 cm) equilibrated in the same buffer. The protein peak containing IgE was rechromatographed in the same column, and the material under the rather symmetrical peak of IgE was dialyzed against 0.025 M tris(hydroxymethyl)aminomethane (Tris)-phosphate buffer, pH 8 (80 ml of 2.5 M Tris + 150 ml of 1 M NaH2PO,, 700 ml of water; the pH was adjusted to 8.0 with 10 N NaOH, and the volume was brought up to 6 liters). The dialyzed material was chromatographed on a DE-52 column (volume of 110 ml) equilibrated with Trisphosphate buffer. The IgE in this column is not retarded and comes off in the first protein peak. This fraction was considered to be pure rat IgE. The fractionation was monitored by gel diffusion tests performed with a specific anti-rat IgE serum kindly supplied by H. Metzger. The final concentration of the IgE was determined by the optical density at 280 nm (optical density/1.36 = milligrams of protein/ milliliter). Antigen. Five-times-recrystallized HEA was purchased from Nutritional Biochemicals Corp. PCA reactions. PCA reactions were performed as previously described (4).
RESULTS Effect of T. spirlis infection on PCA reaction. Mice that had been infected with 600 larvae 34 days before were used as test animals for PCA reactions. Dilutions of IgG, antibody and IgE antibody were tested in four infected mice and four normal mice by intracutaneous administration of 0.05 ml of the chosen dilutions. Two hours later, mice sensitized with IgG, were challenged intravenously with 0.2 ml of a mixture of 0.5% Evans blue + 0.5% HEA, and the diameter of the reactions was measured 30 min later. Mice sensitized with IgE antibody were similarly challenged 3 days later, and reactions were measured 30 min later. The results are given in Table 1. It is clear from these results that infected mice did not respond as well as normal mice to either IgGi- or IgE-mediated PCA. The range of concentrations of IgE used in this experiment was rather low, and we did not know from these results whether infected mice were capable of responding to PCA induced with IgE. In the following experiments we increased the range of concentrations. Effect of worm load on PCA reaction. Mice were infected with either 25, 100, or 400 larvae
86
INFECT. IMMUN.
MUNOZ AND COLE
TABLE 1. Effect of T. spiralis infection on PCA' Concn of IgG, Dilution of serum contain25
Normal
ing IgE
(Aglml)
Recipient mouse
50
100
Ob 11 15 0 10 15
1:3,000 0 0
1:2,000 6wc 8w
1:1,000
10 9
0 0 11w 0 0 T. spiralis 0 0 0 12w 0 0 infected 0 a CFW mice that had been infected with 600 T. spiralis larvae 34 days earlier were used. b Numbers are the diameter in millimeters of the area of bluing measured on the underside of the skin. Each reading represents a separate mouse. Only one site per mouse was used. c w, Weak reaction.
per mouse. Twenty and 41 days later, the IgE and IgG, PCA antibodies were titrated in these mice (Table 2). Photographs of the reactions obtained 20 days after infection are given in Fig. 1 and 2. Twenty days after infection, mice infected with only 25 larvae showed a moderate resistance to manifest reactions induced with antibodies of the IgG, or IgE class. When 100 or 400 larvae were given, however, a striking inhibition was observed for both types of antibodies. Forty-one days after infection, strong inhibition was observed after even the small dose of 25 larvae. Effect of duration of T. spiralis infection on ability of mice to give PCA reactions. This experiment was designed to determine when after infection resistance to PCA was first observed. Mice were infected with 400 larvae and at intervals thereafter were tested as above for their ability to develop PCA reactions with the two types of antibodies (Table 3). PCA responses to IgG, and IgE types of antibodies were inhibited strongly beginning 20 days after the infection, and the inhibition seemed stronger at 41 days. An unexpected finding in this experiment was that the PCA reactions induced by IgG, antibody were considerably larger in mice that had harbored the infection for only 10 days than in normal mice or those that had carried the infection for 15 days or longer. The significance of this finding is presently under investigation. Effect of serum from normal and T. spiralis-infected mice on PCA reactions. As indicated in the introduction, T. spiralis infections augment the levels of IgE in the animal, and this increase in IgE could be responsible for preventing, competitively, PCA due to IgE with specificty to other antigens. The previous experiments clearly showed that T. spiralisinfected mice were more resistant to PCA reactions than were the normal animals. It was
then important to see whether the serum of infected animals had a substance that could prevent PCA reactions from taking place. The antiserum containing IgE antibody to HEA was diluted 1:250, and the IgG, preparation was made to contain 100 ug/ml. To 0.3 ml of each antibody, 0.3 ml of diluted normal or infected mouse serum was added. Each of two mice was then given intracutaneously 0.05 ml of each mixture, and 2 or 72 h later the mice were challenged intravenously with the dye-HEA mixture. Sera from mice that had been infected for various periods of time with T. spiralis were used. The sera of infected mice inhibited the PCA induced with IgE to HEA having little effect on PCA induced with IgG, (Table 4). In addition, it is evident that the inhibitory substance was more concentrated in the sera of mice infected 35 days before bleeding than in those that had carried the infection 4 to 10 months. The sera of normal mice had no demonstrable inhibition of either type of antibody. The substance responsible for this inhibition is most likely IgE. We have demonstrated IgEtype antibodies specific for T. spiralis antigens by 72-h PCA reactions induced with these sera TABLE 2. Effect of larval load on reactivity of skin to PCA No. of larvae given 25
20a 41
100
20 41
400
20 41
0
Concn of IgG,
Days after infections
(Ag/ml)
Dilution of serum containing IgE
0 0 0
50 100 1:500 1:250 1:125 7w 14 12w 20 +d 14 12 14 11 10 + 10 0 12 0 lOw 16 9w 0 0
0 0 0 0
low 18 15 0 low 9w lOw 13
0 0 0 0
10 14 12w
0 0 0 0
25
12Wb .
0 0 0 0 9w 11w 11 12
0 +
0 0 14 16 12 10
lOw 16 19 17 18
10 10 10 11
9w 10 7w 10 0 8w 12w 0
0 6w 0 0 12 13 13 14
0 7w 0 NIe 30 24 11 13
a On day 20, the diaphragms of six mice per group were examined under the microscope for T. spiralis larvae. Mice infected with 25 larvae had only a few larvae in the diaphragm, but all six mice were positive. The diaphragms of those receiving 100 and 400 larvae showed increasing numbers of larvae in all mice. b. c See Table 1 for meaning of numbers. w, Weak reactions either ring shaped or with clear center, or with very light concentration of dye. d _ s Doubtful reaction. eND, Not done.
VOL. 15, 1977
EFFECT OF T. SPIRALIS ON PCA
87
CONCENTRATION OF IgG1ANTIBODY TO HEA 25
50
100 womw
.1
0
V
.110,
...J'
i.
z w
'
m-
-4 -
lim.^,.
'U
4( 4c 16 -i
3 0.
100
*
I'-
0
z 400
9 ,^
FIG. 1. PCA reactions performed with different concentrations (in micrograms per milliliter) of the IgG0 type of antibody to HEA in normal mice or in mice infected 20 days earlier with 25, 100, or 400 T. spiralis larvae given by stomach tube.
using as a challenge dose a saline extract from frozen, thawed, and homogenized larvae mixed with Evans blue. Unfortunately, we do not presently have a way to quantitate mouse IgE other than by PCA reactions. We will attempt in the future to develop such a test by using the technique of producing anti-mouse IgE recently developed by Prouvost-Danon et al. (16), Lang et al. (9), and S. Lehrer (personal communication). The availability of rat myeloma IgE offers a possibility of measuring the effect of this protein on IgE of mice. It is known that mouse IgE fixes to rat mast cells and that it produces PCA reactions in rats (15), and that rat IgE fixes to mouse mast cells (17). This indicates that rat IgE may also inhibit the PCA reaction. Effect of rat IgE on PCA reaction in mice. A solution of purified rat myeloma IgE containing 500 pug/ml was made in physiological saline, and then serial 10-fold dilutions were made. Equal volumes of each dilution were mixed with either a solution of IgG, antibody containing 100 gg/ml or a 1:250 dilution of serum containing IgE antibody. The final concentration of IgG 'was 50 ,ug/ml, and the final dilution of the IgE-containing serum was 1:500. Two mice were sensitized per dilution, and two mice for
each antibody received it diluted in saline only. The results are given in Table 5. The PCA due to IgG, was not significantly inhibited by 250 ,g of rat IgE per ml of the final mixture. In some experiments, however, rat IgE at the highest concentration seemed to have some inhibitory effect because the PCA reactions had a completely clear center. The reactions induced by IgE antibody were completely inhibited by as little as 10 jig of rat IgE per ml in the mixture and partially inhibited by 2 ug/ml. Similar inhibition was obtained in rat skin sensitized with mouse antibody (IgE). These experiments show that IgE may indeed be responsible for the inhibition observed. There are methods besides worm infestation that stimualte IgE formation to various antigens; one is by immunization with B. pertussis extracts (4), and another is by giving antigen mixed with large amounts of alum (18). Preliminary studies have shown that both of these methods may also render mice more resistant to PCA reaction. DISCUSSION During a study on the stimulation of IgE in mice by an extract from B. pertussis, it occurred to us that stimulation of IgE to a heterol-
88
INFECT. IMMUN.
MUNOZ AND COLE
DsL UTON OF 1gE ANTIBODY TO HEA ri2250
2n. ,
z LU
25
0 LU
m t.
0 ~ ~ ~ ~
~
7,4;~
~
'C ALI~~~~~~~~~~~~~~~~~~~~~~~~~~
:1 100 i-'i
ci~~~~~~~~~~~~~~~~~l U-~~~=X -urj tI', Z400 FIG. 2. PCA reactions performed with different concentrations of the IgE type of antibody to HEA in normal mice or in mice infected 20 days earlier with 25, 100, or 400 T. spiralis larvae given by stomach tube. TABLE 3. Effect of duration of T. spiralis infection on resistance to PCA Days after infection 10
15 20 41
Concn of IgG, (jug/1l) Osno g gm 25 lOwa.b 11w
Dilution of serum containing IgE
50
100
1:500
1:250
1:125
25 25
34 32
0 12
10 9
20 19
+C
20 12
0 0
12 20
0 0
11w
15
0 0
0 +
10 14
0 0
0 6w
0 7w
0 0
12w low
0 0
0 0
0 NDd
0 0
9w
11 12 17 10 13 Uninfected 11w 11 14 13 controlse 12w 10 18 a, bSee Table 1 for meaning of numbers. w, Weak reaction. ±, Doubtful reaction. d ND, Not done. e Groups of uninfected mice of the same age and sex as the infected mice were used for each time period, with results similar to the control group shown in this table; for that reason the results were omitted.
ogous antigen should block allergic reactions involving mast cells. One of the most effective ways of stimulating IgE in animals, and apparently humans as well, is infection with certain
parasitic worms (5-8, 15). Indirectly, some published observations have shown that parasitism in humans may make them more resistant to allergic responses, since persons infected with helminths were found to give weaker P-K reactions (1). These observations, although they do not directly incriminate parasitism as responsible for resistance to certain allergic reactions, are highly suggestive. Jarrett et al. have definitely shown that Nippostrongylus brasiliensis infections make rats more resistant to PCA reactions (7). The results presented here also conclusively show that experimental infection of mice with T. spiralis makes these animals strikingly more refractory to PCA reactions to a heterologous antigen. Thus, infected mice sensitized with anti-HEA of either the IgG1 or IgE class did not respond to doses of these antibodies that were fully effective in normal mice of the same sex and age. The refractory state appeared about 7 days after the female worms had deposited the larvae in the intestinal mucosa and the larvae had migrated to the muscle (from 15 to 20 days after oral infection with larvae [10]). It was also apparent that the refractoriness was more pronounced 41 days after infection than at 15 or 20 days. As few as 25 larvae were sufficient to induce the refractory state, but in this
VOL. 15, 1977
EFFECT OF T. SPIRALIS ON PCA
89
TABLE 4. Effect of T. spiralis-infected mouse sera obtained at different times after infection
35 days after infection 4 mo after infection
10 mo after infection
1:8
1:4
1:2
Normal
IgE-containing serum diluted 1:500 in the
50 gg of IgG,/ml in the mixture
Type of mouse serum used
mixture
1:8
1:4
1:16
1:16
1:2
12wb 17
13w 12w
15
15
12
15
0 0
0 0
0
low
0
14w
18 18
15a 14
17
15
15
17
NDC ND
ND
ND
ND
ND
ND
ND
0 17
13
13
17
0
14
13
10
0
12w 0
low 0
12w 12w
12 12
11 12
15 11
17 13
0 17w
12w llw
12w 11
llw
Antibody in saline
15 17
a
See Table 1 for meaning of numbers.
b
w, Weak reaction.
c
ND, Not done.
TABLE 5. Effect of rat IgE on PCA reactions in mice Final concn of 50 jg of IgG,/ml rat IgE (jug~ml in mixture
IgE-containing serum diluted 1:500 in mixture
14a 0 0 33 250 14 0 0 0 50 0 0 9 13 10 6wb 8 2 10 10 14 12 15 10 0.4 14 14 13 12 Saline a See Table 1 for meaning of numbers. Columns represent readings from individual mice. b w, Weak reaction.
case it took longer to manifest itself. One hundred larvae were more effective, and 400 appeared to be even better. Since each mature female worm can produce close to 1,000 larvae (10), even the small infective dose of 25 larvae has a potential to produce some 12,500 larvae (assuming that half the infecting larvae are males and half females), which is a significant worm mass. The most important factor in the production of this refractoriness was probably IgE, because sera from infected animals inhibited PCA reactions induced by IgE and purified rat IgE also inhibited these reactions in both mice and rats. It is clear from the results that inhibition in infected mice was not exclusively manifested against the IgE class but also against the IgG1 class. We do not now know whether this inhibition was due to IgE alone or whether an increase in the IgG1 in the infected mice was responsible for inhibition of IgG1-induced PCA. Since IgG, is normally present in high concentrations in the sera of mice, it is more probable that IgE in high titers may be
11
14 14
also competing with IgG1. This should be further investigated with purified IgG, and IgE fractions from mouse serum. If the inhibition observed in this work was due to an increased concentration of IgE, one should also be able to increase the resistance of mice to PCA by other methods known to increase IgE. We are presently investigating this
possibility. The implication of these findings with respect to human allergies involving the IgE and possibly the IgG class of immunoglobulins is clear. It should be possible to block mast cell receptors by greatly increasing the IgE concentration in the blood to antigens that normally are not present in the environment. If this can be done, mast cells may well become relatively unresponsive to antigens to which the person is allergic. It has already been shown that IgE myeloma protein or its Fc piece blocks the P-K reaction (19, 20). Passive transfer of IgE would probably not be practical because of its antigenicity, its limited availability, and the required frequent administration. If endogenous production of IgE were increased, most of these problems could be circumvented. ACKNOWLEDGMENTS We wish to express our appreciation to H. Metzger for supplying the rat ascitic fluid and the methods to purify the rat IgE used in this work. We also would like to thank R. K. Bergman for his help in writing this manuscript.
LITERATURE CITED 1. Bazaral, M., H. A. Orgel, and R. N. Hamburger. 1973. The influence of serum IgE levels of selected recipi-
ents, including patients with allergy, helminthiasis and tuberculosis, on the apparent P-K titre of a reaginic serum. Clin. Exp. Immunol. 14:117-125.
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INFECT. IMMUN.
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2. Briggs, N. T. 1963. Hypersensitivity in murine trichinosis: some responses of trichinella-infected mice to antigen and 5-hydroxytryptophan. Ann. N.Y. Acad.
12. Munoz, J., and B. M. Hestekin. 1963. Antigens of Bor-
Sci. 113:456-466. Briggs, N. T. 1963. Immunological injury of mast cells in mice actively and passively sensitized to antigens from Trichinella spiralis. J. Infect. Dis. 113:22-32. Clausen, C. R., J. Munoz, and R. K. Bergman. 1969. Reaginic-type of antibody in mice stimulated by extracts of Bordetella pertussis. J. Immunol. 103:768777. Cueva, J., and Q. F. B. Blanca E. Navarro. 1973. IgE en alergicos y parasitados en Mexico. Alergia (Mexico City) 21:1-11. Gougerot,M. A., A. Pasticier, G. Saimot, J. P. Coulaud, A. Ricour, and M. Payet. 1975. Les IgE seriques dan les parasitoses: a propos d'une etude chez 27 Africians de louest. Bull. Soc. Pathol. Exot. 68:297303. Jarrett, E. E. E., T. S. C. Orr, and P. Riley. 1971. Inhibition of allergic reactions due to competition for mast cell sensitization sites by two reagins. Clin. Exp. Immunol. 9:585-594. Johansson, S. G. O., T. Mellbin, and B. Vahliquist. 1968.Immunoglobulin levels in Ethiopian preschool children with special reference to high concentrations of immunoglobulin E (IgND). Lancet i:1118-1121. Lang, G. M., W. Y. Lee, B. G. Carter, and A. H. Sehon. 1976. Production of goat, rat and guinea pig antisera to murine IgE. J. Immunol. 116:881-883. Larsh, J. E., Jr. 1963. Experimental trichinosis. Adv. Parasitol. 1:213-286. Mota I., E. H. Sadun, R. M. Bradshaw, and R. W. Gore. 1969. The immunological response of mice infected with Trichinella spiralis. Biological and physico-chemical distinction of two homocytotropic anti-
147:524-526. 14. Ogilvie, B. M. 1964. Reagin-like antibodies in animals immune to helminth parasites. Nature (London) 204:91-92. 15. Ovary, Z., S. S. Caiazza, and S. Kojima. 1975. PCA reactions with mouse antibodies in mice and rats. Int. Arch. Allergy Appl. Immunol. 48:16-21. 16. Prouvost-Danon, A., R. Binaghi, S. Rochas, and Y. Boussac-Aron. 1972. Immunochemical identification of mouse IgE. Immunology 23:481-491. 17. Prouvost-Danon, A., J. Wyczolkowska, R. Binaghi, and A. Abadie. 1975. Mouse and rat IgE: cross sensitization of mast cells and antigenic relationships. Immunology 29:151-162. 18. Revoltella, R., and Z. Ovary. 1969. Reaginic antibody production in different mouse strains. Immunology 17:45-54. 19. Stanworth, D. R., J. H. Humphrey, H. Bennich, and S. G. 0. Johansson. 1967. Specific inhibition of the Prausnitz-Kustner reaction by an atypical human myeloma protein. Lancet ii:330-332. 20. Stanworth, D. R., J. H. Humphrey, H. Bennich, and S. G. 0. Johansson. 1968. Inhibition of Prausnitz-Kdstner reaction by proteolytic-cleavage fragments of a human myeloma protein of immunoglobulin class E.
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21. Weatherly, N. F. 1970. Increased survival of Swiss mice given sublethal infections of Trichinella spiralis. J. Parasitol. 56:748-752.
