3-Methylcholanthrene-induced immunosuppression in mice to Trichinella spiralis antigens.

Immunopharmacology and Immunotoxicology

ISSN: 0892-3973 (Print) 1532-2513 (Online) Journal homepage: http://www.tandfonline.com/loi/iipi20

3-Methylcholanthrene-Induced Immunosuppression in Mice to Trichinella Spiralis Antigens Brian E. Johnson, Robin G. Bell & Rodney R. Dietert To cite this article: Brian E. Johnson, Robin G. Bell & Rodney R. Dietert (1990) 3Methylcholanthrene-Induced Immunosuppression in Mice to Trichinella Spiralis Antigens, Immunopharmacology and Immunotoxicology, 12:2, 237-256 To link to this article: http://dx.doi.org/10.3109/08923979009019671

Published online: 28 Sep 2008.

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IMMUNOPHARMACOLOGY AND IMMUNOTOXICOLOGY 12(2), 237-256 (1990)

3-METHYLCHOLANTHRENE-INDUCED IMUNOSUPPRESSION IN MICE

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TO TRICHINELLA SPIRALIS ANTIGENS Brian E. Johnsonln2*, Robin G. Bell3, and Rodney R. Oietert'** 'Institute for Comparative & Environmental Toxicology 2Department of Poultry and Avian Science 3J.A. Baker Institute for Animal Health Cornell University, Ithaca, New York 14853 ABSTRACT The immunosuppressive effects of in vivo [subcutaneous) exposure to 40 or 80 mg/kg 3-methylcholanthrene (MC) were examined in aryl hydrocarbon hydroxyl ase (AHH) responsi ve C57BL/6 (86) and AHH non-responsive OBA/2 (D2) inbred strains of mice. Twenty-four hours after treatment with carcinogen or vehicle alone, animals were primed with crude L, muscle larvae antigen from 1. spiralis. Immune status was assessed in vitro after six days as antigen-specific lymphoprol iferation. The prol iferation of splenocytes from MC-treated 02 and B6 mice was significantly impaired compared to controls. To examine the cellular basis of the immunosuppression, primed splenocytes from control and MCtreated mice were separated into adherent and non-adherent fractions on Sephadex G-10 columns. When antigen-pul sed adherent cells from MC-treated 86 and 02 mice were recombined with control non-adherent cells from syngeneic and B6D2F1 mice, T-cell prol iferation was significantly reduced. This suppression was not observed with the addition of increased numbers of adherent cells. Non-adherent cells from MC-treated mice showed a decreased capacity to respond to the presence of control antigen-pulsed adherent cells from appropriate mice. These results suggest that MC treatment has a similar suppressive effect on the immune responses of both B6 and 02 mice that involves the quality of accessory cell -T-cell interactions .

23 7 Copyright 0 1990 by Marcel Dekker, Inc.

JOHNSON, BELL, AND DIETERT

238

Introduction The development and r e g u l a t i o n o f an immune response r e q u i r e s t h e e f f e c t i v e i n t e r a c t i o n o f lymphocytes w i t h processed antigen bound t o t h e surface o f macrophages (Me) o r o t h e r accessory c e l l s (1,2).

The p r e s e n t a t i o n o f compl ex antigens


involves t h e association o f a n t i g e n i c fragments w i t h major h i s t o c o m p a t i b i l i t y complex (MHC) encoded p r o t e i n s on c e l l surfaces. Equally important i s t h e p r o d u c t i o n o f cytokines by both M8s and T - c e l l s . Chemicals which modulate any o f these processes can a l t e r immunocompetence. A v a r i e t y o f chemicals are known t o modify immune processes.

These i n c l u d e t h e p o l y c y c l i c aromatic hydrocarbon carcinogens (PAHC) such as Benzo(a)pyrene (BP), 3-Methylcholanthrene (MC),

and

7,12-Dimethyl benz(a)anthracene (DMBA) ( 3 ) . I n responsive i n d i v i d u a l s , these compounds induce t h e de novo synthesis o f phase I and I 1 metabolic enzymes (4). One o f these products, t h e phase Imixed-function monooxygenase, a r y l hydrocarbon hydroxyl ase (AHH), r e s u l t s i n t h e a c t i v a t i o n o f pro-carcinogenic PAHCs t o r e a c t i v e nucleophiles ( 5 ) . It has been suggested t h a t t h i s

process may be responsible f o r t h e genotoxic e f f e c t s o f these compounds (6). The i n d u c t i o n o f AHH a c t i v i t y by PAHCs i s mediated by t h e

&I locus (7).

This locus has been i m p l i c a t e d i n immune

suppression associated w i t h exposure t o a wide v a r i e t y o f carcinogenic and non-carcinogenic PAHs. I n s i g h t i n t o t h e mechanism o f PAHC-mediated immunotoxici t y was f i r s t provided by Stjernsward (8,9) whose observations suggested a c o r r e l a t i o n between t h e c a r c i n o g e n i c i t y and immunosuppressive p o t e n t i a l o f carcinogenic PAHCs ( l O , l l , 12). Subsequently, Yamashita and Hamaoka (13) found t h a t OMBA a f f e c t e d p r i m a r i l y Me and T c e l l s , b u t had l i t t l e e f f e c t on B - c e l l s . More

.

r e c e n t l y , Blanton, e t a1 (14) provided evidence t h a t B - c e l l s were a l s o adversely a f f e c t e d by exposure t o BP. I n antigen presentation assays using i r r a d i a t e d spleen c e l l s , M8s from BP-

3-METHYLCHOLANTHRENE-INDUCED IMMUNOSUPPRESSION

239

t r e a t e d B6C3F1 mice were l e s s e f f e c t i v e s t i m u l a t o r s o f T - c e l l pro1 i f e r a t i o n (15). The present study addresses both t h e mechanism(s) o f PAHCmediated immunosuppression and t h e r o l e o f t h e & I genotype. Methylchol anthrene, one o f t h e most thoroughly c h a r a c t e r i z e d AHH


inducers and a potent carcinogen, was used t o examine t h e e f f e c t s o f acute i n v i v o exposure on antigen presenting c e l l s and T - c e l l s . Here, t h e immunosuppressive e f f e c t s o f MC-treatment are contrasted i n the archetype "AHH-inducibl el' (Ahb) C57BL/6 and "non-inducibl e"

(Ahd)

DBA/2 s t r a i n s o f mice. Materi a1 s and Methods

An ima1 s Female C57BL/6 and DBA/E mice were purchased from The Jackson Laboratory (Bar Harbor, ME). A l l mice were 4-5 weeks o l d a t t h e time o f purchase, and acclimated an a d d i t i o n a l 2-4 weeks. Mice were housed 4 per cage i n 30x18~13cm polycarbonate cages, allowed f r e e access t o water, and f e d Charles R i v e r RMH 1000 formula

& libitum.

Photoperiod was maintained a t 16 h r d a y l i g h t

and 8 h r darkness. Materi a1 s MC, T r i c a p r y l i n , Concanavalin A (Con A) and Lidocaine (Sigma

Chemical Co., S t . Louis, MO), Sephadex G-10 (Pharmacia, Inc., Piscataway, NJ), D i f f - Q u i c k (Baxter Health Care Co., McGaw Park, I L ) , twenty-four and n i n e t y - s i x w e l l t i s s u e c u l t u r e p l a t e s (Costar, Cambridge, MA) were a l l purchased from sources as indicated. T r i t i a t e d (3H-methyl) thymidine (3H-TdR) was obtained from I C N Pharmaceuticals, Inc. ( I r v i n e , CA), w h i l e a l l o t h e r chemicals were o f reagent grade and were purchased from commercial sources. FITC-conjugated mouse monoclonal IgGl a n t i - I a was purchased from Bioproducts f o r Science, Inc.,

Indianapolis, IN.

2 40

JOHNSON, BELL, AND DIETEKT


MC Treatment and Immunization Dose-response experiments showed a maximal suppression o f the plaque-forming cell responses of MC-treated mice at doses of 40-80 mg/kg MC (data not shown). On this basis, mice were given a single sc injection o f either MC (40 or 80 mg/kg), dissolved in tricapryl in, or tricapryl in alone (vehicle control). Twenty-four hours later, mice were primed by ip injection with 150 pg of crude Trichinella sDiralis L, muscle larvae (Tsp) antigen, prepared as described (16), in Freund's incomplete adjuvant. Six days after immunization the mice were killed by cervical dislocation and their spleens coll ected aseptical ly. Cell SeDarat i on Single cell suspensions of splenocytes were prepared as described (17). Ficoll -Hypaque (Pharmacia, Inc., Piscataway, NJ) gradient centrifugation was used to produce a cell fraction enriched for mononucl ear 1 eukocytes, These cell s were separated into adherent and non-adherent fractions by the method o f Jerrells, et al. (18). Briefly, sterile columns, made from 12 cc syringe barrels containing 5 ml of thoroughly washed Sephadex G-10 were prepared and rinsed with approximately 30 ml of cell culture medium (CCM) with 5% horse serum (HS). Prior to the addition of cells, the columns were warmed to 37OC in a CO, incubator. The cell suspension was added to the columns and allowed to drain into the matrix, followed by an incubation of 30 min under standard conditions (STC) of 37OC, 5% CO, and 95% humidity. Non-adherent cells were eluted from the columns with 15-20 ml of warm CCM with 5% HS. Cells were pelleted and resuspended to a concentration of 4x106 viable cells per ml in CCM with 5% HS. Prior to pulsing adherent cells with 2 ml (0.5 mg/ml) of crude Tsp muscle larvae antigen, the columns were rinsed with an additional 10-12 ml o f CCM which was discarded.



241

After an incubation of 2 hr at STC with antigen, adherent cells were removed from the columns with the addition of ice cold RPMI 1640 with 0.5% (w/v) lidocaine followed by a vigorous rinsing using a 10 ml wide-bore pipette. The matrix was further rinsed with approximately 20 ml of ice cold RPMI 1640 without HS. The adherent fraction was pelleted and resuspended in CCM with 5% HS to a concentration of 2 . 5 ~ 1 0viable ~ cells per ml. Cytospins were prepared from each fraction and differentially stained with "Diff-Quick." Non-adherent fractions consisted of 299% lymphocytes. The adherent fraction consisted o f 75-80% MBs. Cell s were counted with a hemacytometer; vi abi 1 i ty was estimated by trypan blue dye exclus on. Ant iqen Presentation This portion of the assay was mod fied from that previously described by Wassom, et a1 ., (19). In brief, 4x105 non-adherent cells in 100 pl were added to the wells of a 96 well, flatbottomed microtiter plate (Costar, 205 Broadway Ave., Cambridge, MA). In addition, 2 . 5 ~ 1 0or ~ 2 . 5 ~ 1 0antigen ~ pulsed adherent cells, in a volume of 100 pl , were added to each well. Plates were then incubated at STC for 96 hr. Following the incubation period, cultures were pulsed with 3H-TdR (1.5 pCi/well) and harvested with a microharvesterTM (Bellco Glass, Inc., Vineland, NJ) 18-24 hr later. Cvtoki ne Production A si ngl e-cell suspension of spl enocytes was prepared as described. The cells were resuspended to a concentration of 8x106 viable cells/ml in CCM with 5% HS. One-half ml volumes of cells were distributed to the wells of a 24 well polystyrene tissue culture plate along with 0.5 ml of Con A, at 5 or 10 pg/ml or crude Tsp L, muscle larvae antigen at 100 or 200 pg/ml. The

242


plates were incubated for 24 hr a t STC and the cell culture fluid harvested. Samples were assayed for activity the same day.


Cvtokine Assay Cytokine activity was assessed by the method of Gi l l i s, d.,(20) and used the IL-2 dependent, cloned murine cytotoxic 1cell line CTLL-2 (ATCC TIB 214; 21). Briefly, c e l l s were washed twice in RPMI 1640 with 5% HS and adjusted t o a concentration of 2x10, viable cells/ml in CCM with 5% HS. Cells were counted and viability determined as before. A 100 pl of cell suspension was added t o each well o f a 96 well, flat-bottom, tissue culture plate. One-hundredpl aliquots of culture fluid t o be assayed for cytokine activity were run in tr ipl i cat e a t a final dilution of 1:2. Following an incubation period of 48 hr a t STC, the cultures were pulsed with 0.5 pCi of 3H-TdR per well and harvested with a microharvesterTM(Be11co G1 ass, Inc. , Vi nel and, NJ) 18-24 hr 1ater Preliminary experiments with MC, conducted and analyzed as described by Gillis, e t a l . , (20), failed t o reveal the presence of inhibitory substances in culture fluids produced as described above. For this reason, samples were run in t r i p l i c a t e as 1:2 dilutions. Approximate units of activity were calculated by extrapolation t o a standard curve run on each plate of each assay. The lymphokine standard used in these assays was produced from Con A (10 ug/ml) stimulated rat splenocytes cultured a t 1x106 cells/ml for 24 hr a t STC in RPMI 1640 supplemented with insulin, 10 ug/ml; transferrin, 32 ug/ml; ZnCl,, 1.2 ng/ml; bovine albumin, 32 mg/ml; HS, 0.1%; phorbol 12-myristate-13-acetate, 5 ng/ml This material was concentrated by vacuum dialysis a t 4 O C and extensively dialyzed against Dul becco's phosphate buffered saline. Probi t analysis revealed approximately 400 ED,, Units of activity per ml.

.

.

3-METHYLCHOLANTHRENE-INDUCEDIMMUNOSUPPRESSION

243


Detection of Ia bv Flow cvtometrv Two-million spleen cells were dispensed into 5 ml polystyrene tubes (Fa1 con) and pel 1 eted. Cell s were washed once with FPBS (PBS with 1% FBS; 0.05% NaN,), pelleted and resuspended in 50 pl of a 1:20 dilution of FITC-conjugated primary antibody and incubated 20 min. at 4OC. The cells were washed twice more in FPBS and resuspended in 1 ml of fixative (FPBS with 1% formaldehyde). A Becton Dickinson Electronics Laboratory (Mountain View, CA) FACS 440 equipped with a Spectra Physics argon-ion laser was used for cytometric analysis. The laser was operated at 150 mW and 10,240 cells from each sample were evaluated. Forward, low-angle scatter measurements were made on incident light scattered through a angle of 2O-12' and used a 488/10 nm bandpass filter to block fluorescence. Fluorescence determinations were made through a 530/30 nm bandpass filter. Statistical Methods Data from mu1 tiple experiments were analyzed by two factor analysis, assuming a random effects model. The factors were replicate and treatment. The remaining data were analyzed by one factor analysis of variance, where differences among elements were ascertained using the protected LSD method. The nominal 5% probability of rejecting the null hypothesis when true was used throughout. All statistical procedures were as described by Snedecor and Cochran (22). Results Figures 1 and 2 show the results of assays involving the recombination of non-adherent cell fractions with antigen-pulsed G-10 adherent cells (APC), predominantly M8s, from control and MC-treated mice. The assays were replicated a minimun of three times with each strain of mice. Due to variations in the levels

2 44


Antigen Presentation by Adherent Splenocytes from Control & MC Treated DBA/2 mice


I ,

2.5X103 control M8 treated D2 T-cells

.

2.5X103 treated M8 control D2 T-cells

.

2.5X103 treated M8 control F1 T-cells

. 2.5X104 control M8 treated D2 T-cells 2.5X104 treated M8 control D2 T-cells 2.5X104 treated MU control F1 T-cells I

+

I

+ +

+

*

+ + l . . . . . l w . . . l . . . . . l . . . : : l : . : : k

!5

Splenocytes from MC- and vehicle-treated DBA/2 mice were separated by adherence on Sephadex G-10. Two concentrations of antigen-pul sed macrophages from MC-treated mice (MC-APC) were recombined with antigen primed T-cells from vehicle control, B6D2F1 and syngeneic mice. In addition, non-adherent T-cell fractions from MC-exposed mice were combined with antigen-pul sed macrophages from control, vehicle-treated mice. The antigenspecific proliferation of primed T-cells was measured as H-TdR uptake and the response is shown as a percent of control; i.e. as a percent of the response generated with antigen-pul sed accessory cells from vehicle-treated control mice. Asterisks indicate experimental groups in which the responses associated with cell s from MC-treated mice were significantly different from those using cells from vehicle-treated mice. (~$0.05).


245

Antigen Presentation by Adherent Splenocytes from Control & MC Treated C57BL/6 Mice


-

2.5X103 control M8 treated B6 T-cells 2.5X103 treated M8 control B6 T-cells

-

2.5X103 treated M8 control F1 T-cells

+ +

-\\\\\\\\\\\\\\y* b

+

-\\\\\\\\\

*

l . . . . . l . . . . . , . . . . . l . . . . + I . . . . . I . . . . . I . . . . . I . . . ’

FIG. 2 Spl enocytes from MC- and vehicle-treated C57BL/6 mice were separated and recombined as described in the text and fig. 1 legend. Asterisks indicate experimental groups in which the responses associated with cell s from MC-treated mice were significantly different from those using cells from vehicletreated mice (ps0.05). of 3H-TdR incorporation between assays the results were expressed as the arc sine of the ratio of incorporation by MC-treated vs. untreated control cultures and analyzed as a randomized blocks design. Incorporation of 3H-TdR by nonadherent cells or APC alone ranged from 300 to 1000 CPM depending upon the number used and the experiment. The ability of APC from MC-treated 86 mice to stimulate the proliferation of either B6D2F1 or syngeneic T-cells from vehicleexposed control mice was unaltered when 2. 5x104 adherent cell s


2 46


were used. However, 2 . 5 ~ 1 0M8s ~ from MC-treated mice showed a significantly decreased ability to stimulate the proliferation of control T-cells from either B6D2F1 (in one experiment 1,138 f 173 vs. 2,595 f 362 CPM; p=0.0084) or syngeneic mice (1,303 f 136 vs. 2,854 f 476 CPM; p=0.026). Non-adherent cells from MC-treated B6 mice also demonstrated a significantly decreased capacity to prol iferate, relative to control T-cell fractions, when stimulated with APC from vehicle-treated mice. However, the depression o f the lymphoprol iferative response was significant only when 2 . 5 ~ 1 0 ~ APC were used. A similar picture was observed in D2 mice. Adherent cells from MC-treated mice showed an impaired ability to stimulate control T-cells from syngeneic and F1 mice. In a typical experiment, pooled nonadherent cells from untreated mice were mixed with 2 . 5 ~ 1 0or ~ 2 . 5 ~ 1 0APC ~ from MC-treated or untreated individuals. This resulted in incorporations of 1742 f 214 and 3115 f 194 CPM respectively when 2 . 5 ~ 1 0APC ~ were used to present antigen (ps0.0001) and 10,513 f 2736 and 14,346 f 1181 CPM with 2 . 5 ~ 1 0APC ~ (p=0.25). Also, syngeneic non-adherent cells from MC-treated mice showed a significant reduction in prol iferation when adherent cells from control animals were used to present antigen. The results of flow cytometric assays of splenic adherent cell fractions are listed in Table 1. Adherent cells from MC-treated D2 mice showed significantly increased forward 1 ight scatter compared to controls, while the mean log of channel fluorescence (labeling intensity) of these cells was slightly, but not significantly, decreased. The proportion of labeled cells from MC-treated D2 mice was significantly less than that of controls. Lastly, the ratio of immunofluorescence and scatter, which can serve acceptably as a parameter representative of surface antigen density (23), showed a highly significant difference (piO.0001) between cells from control and treated D2

Scatter

88.84*f 5.97 57.30 f 4.10 61.48 f 2.30

Mc

C57BL/6J control

MC 111.14 f 1.30

110.22 f 1.10

117.82 f 3.28

121.56 f 5.38

Intensity

Log FITC

23.47 f 1.34

26.59 f 2.14

29.47*f 1.27

40.12 f 3.31

Cell s

X Labeled

f 0.200

1.818 f 0.055

1.997

1.330*f 0.027

1.487 f 0.018

FITC/FLA Ratio

*

Entry i s significantly different from respective control ( ~ $ 0 . 0 5 ) .

allice were treated S.C. with MC dissolved in tricaprylin a t 40 mg/kg, 7 days prior t o assay. Controls were treated S.C. with tricaprylin alone. Each value represents the mean f SEM for a minimum of 6 mice per group.

81.73 f 2.49

control

DBA/2J

Treatmenta Forward Light

Strain

The expression o f c l a s s I1 MHC antigens by splenic adherent cells from control and MC-treated DBA/L and C57BL/6 nice.

Table I


z

0

W

v)

cn

m

.cr a

rd

C

cn

2 0

zz

W

0

B

n

0 C

z

W

I

2 B

5FI

z

r3

z

3-

r

0

2

c)

r

r3 2 4

M

z

I

w


248


strain mice. In contrast, no significant differences were apparent for any of these parameters in 86 mice. ~ cytokine production in response to The results of j . vitrQ mitogen and antigen are illustrated in Figures 3 and 4. A number of cytokines, including interleukins, interferons, monokines and prostaglandins are known to be produced by Con A-stimulated splenocyte cultures. As such, no attempt was made to discriminate the contribution of individual cytokines in these assays. No differences were observed in the Con A stimulated cytokine activity by splenocytes from DBA/2 mice. Cells from 86 mice, treated with 40 or 80 mg/kg MC, showed significantly less cytokine activity in response to Con A (5 pg/ml). The same trend was present at 10 pg/ml, but the difference was not significant. In contrast with antigen specific cytokine production, treatment of both D2 and 86 strains with 40 mg/kg MC resulted in a significant depression of cytokine activity produced by primed splenocytes stimulated with either 100 or 200 pg/ml of crude Tsp antigen

.

Discussion In these experiments, factors which have been shown to play an integral role in the development of immune responses were contrasted in "AHH-induci bl e" (Ahb) 86 and "AHH-non-induci bl ell (Ahd) D2 strains o f mice. In Ahb individuals, with high levels o f the cytosolic Ah receptor (the putative protein product of the &I locus), AHH activity is rapidly induced (24,25). Although noninducible (Ahd) strains possess some constitutive AHH activity (17,26) and very low levels of the Ah receptor (27), they respond poorly to PAHs. Mice which have the AHH-inducible phenotype have been shown to be more susceptible to the immunosuppressive effects of a number of carcinogenic as well as non-carcinogenic polycycl ic, heterocyclic and halogenated hydrocarbons (7). On this basis, the Ah locus has been implicated in the induction of

249


Cytokine Production by Splenocytes from C57BL/6J mice


lo

T

tt

0 Vehicle controls Iz9 Mice treated with 40 rng/kg MC CiGi4 Mice treated with 80 rng/kg MC

8.0

6.0

4.0

0

8

$ -.2 "u"

2.0

0.0 Control

Con-A

5w

Con-A 1opg

TED loo'pg

TSP

200'pg

FIG. 3

Cytokine production by antigen-primed splenocytes from MCand vehicl e-treated C57BL/6 mice. Splenocytes were prepared as described i n the t e x t and stimulated i n vitro w i t h e i t h e r crude T . spiralis antigen o r t h e mitogen Con A. Sample a c t i v i t y i s represented as the s p e c i f i c H-TdR incorporation by t h e 11-2 dependent c e l l l i n e CTLL-2. Asterisks denote groups i n which responses were s i g n i f i c a n t l y d i f f e r e n t than those o f v e h i c l e t r e a t e d c o n t r o l s ( ~ $ 0 . 0 5 ) . U n i t s o f cytokine a c t i v i t y are approximate and were derived by l i n e a r e x t r a p o l a t i o n t o a doseresponse curve generated w i t h a cytokine standard prepared i n house. immunosuppression i n association w i t h exposure t o some o f these compounds (28,29). These observations have been confirmed i n &Icongenic s t r a i n s o f mice which are thought t o d i f f e r o n l y a t t h e Ah locus (30). From the r e s u l t s o f the antigen presentation assays, i t i s evident t h a t a s i n g l e i n v i v Q exposure t o MC can i n t e r f e r e w i t h the a b i l i t y o f antigen-pulsed accessory c e l l s t o s t i m u l a t e


2 50

Cytokine Production by Splenocytes from DBA/2J mice. 10

C

.G -cI

0 Vehicle controla ISY Mice treated with 40 rng/kg MC. Mice treated with 80 rng/kg MC.

8.0

8

b T

6.0


Fk 8:s

0

=a

35 4.0

l Y 4 4

TLl I =

"0 8 E 0 a n

&?

8

Trichinella spiralis: immunosuppression in challenge infections of Swiss mice.

Specificity of affinity-purified Trichinella spiralis antigens.

Immunity to Trichinella spiralis infection in vitamin A-deficient mice.

Serglycin proteoglycans limit enteropathy in Trichinella spiralis-infected mice.

Studies of macrophage function during Trichinella spiralis infection in mice.

Selective immunodepression in mice by Trichinella spiralis extracts and infections.

Eosinophils and resistance to Trichinella spiralis.

Recovery of Trichinella spiralis larvae.

Trichinella spiralis: the in situ localization of muscle larva antigens recognized by humans.

Experimental Trichinella spiralis infection in sheep.

Immunosuppression of in vivo and in vitro lymphocyte responses in swine induced by Trichinella spiralis or excretory-secretory antigens of the parasite.

Proteolytic enzymes from Trichinella spiralis larvae.

Helminthotoxic responses of intestinal eosinophils to Trichinella spiralis newborn larvae.

The Use of Recombinant 31 kDa Antigens of Trichinella spiralis for Serodiagnosis of Experimental Trichinellosis.

Early serodiagnosis of trichinellosis by ELISA using excretory-secretory antigens of Trichinella spiralis adult worms.

Characterization of Trichinella spiralis antigens sharing an immunodominant, carbohydrate-associated determinant distinct from phosphorylcholine.

Matrix metalloproteinase (MMP)-2 and MMP-9 as inflammation markers of Trichinella spiralis and Trichinella pseudospiralis infections in mice.

T and B cells in the transfer of immunity against Trichinella spiralis in mice.

Trichinella spiralis: low vaccine potential of glutathione S-transferase against infections in mice.

Effect of Trichinella spiralis infection on passive cutaneous anaphylaxis in mice.

Trichinella spiralis: dose dependence and kinetics of the mucosal immune response in mice.

Activation and Recruitment of Regulatory T Cells via Chemokine Receptor Activation in Trichinella spiralis-Infected Mice.

Trichinella spiralis: anaphylactic antibody formation and susceptibility in strains of inbred mice.

Protective immunity against Trichinella spiralis infection induced by TsNd vaccine in mice.