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The Journal of Nutrition

Nutrition and Disease

Dietary Plasma Protein Supplements Prevent

the Release of Mucosal Proinflammatory

Mediators in Intestinal Inflammation in Rats

1,2

Anna Pe ´rez-Bosque,3* Lluı ¨sa Miro ´ ,3 Javier Polo,4 Louis Russell,5 Joy Campbell,5 Eric Weaver,6

Joe Crenshaw,5 and Miquel Moreto ´ 3

3Department of Physiology, Faculty of Pharmacy and the Nutrition and Food Safety Research Institute of the University of Barcelona,

Barcelona 08028, Spain; 4APC Europe S.A., Granollers 08403, Spain; 5APC Inc, Ankeny, IA 50021; and 6Proliant Health and Biologicals,

Ankeny, IA 50021

Abstract

Spray-dried plasma (SDP) is a complex mixture of active proteins that modulates the immune response of gut-associated

lymphoid tissue. We examined whether SDP and Ig concentrate (IC)supplementation could modulate cytokine expression

and inflammatory mediators in rats challenged with Staphylococcus aureus enterotoxin B (SEB). Wistar-Lewis rats were

fed diets supplemented with SDP (8% wt:wt), IC (1.5% wt:wt), or milk proteins (control diet) from weaning (d 21) to d 34

after birth. On d 32 and 35, the rats were given SEB (0.5 mg/kg; intraperitoneal). Six hours after the second SEB dose,

jejunal mucosa and Peyer’s patches (PP) from the small intestine were collected. The cytokines interferon-g (IFNg ), tumor

necrosis factor-a (TNFa), interleukin (IL)-6, IL-10, transforming growth factor- b (TGF b), and leukotrienne B4 (LTB4) were

analyzed using commercial kits. SEB increased the release of proinflammatory mediators (IFNg , TNFa, IL-6, and LTB4) in

PP (P , 0.05) and in the mucosa (P , 0.05). In both tissues, SDPprevented the increase in IFNg , IL-6, and LTB4 induced by

SEB (P , 0.05). IC reduced the expression of TNFa and LTB4 in PP and mucosa (P , 0.05). SDP supplementation

increased IL-10 and mature TGF b concentrations in intestinal mucosa from both inflamed and noninflamed rats. Both SDP

and IC increased the mature:total TGF b ratio (all P , 0.05). Both supplements were effective at preventing the SEB-

induced increase in proinflammatory:antiinflammatory cytokine ratios in PP and mucosa and in serum. The preventive

effects of plasma supplements on intestinal inflammation involve modulation of intestinal cytokines, characterized by anincreased expression of antiinflammatory cytokines. J. Nutr. 140: 25–30, 2010.

Introduction

The gastrointestinal tract provides a protective interface be-tween the luminal compartment, containing large amounts of microbes and antigens derived from food, and its internal milieu.The intestinal mucosa controls the penetration of luminalantigens and the generation of immunologic responses in thegut and dysregulation of these processes causes intestinalinflammation. Even in the absence of inflammatory stimuli, thehealthy intestine is in a proinflammatory state that is character-

ized by the generation of proinflammatory cytokines (1).Because the host’s immune responses can be modulated by

diet (2), the dietary approach offers a therapeutic potential inconditions associated with gut-barrier dysfunction and inflam-matory response. Dietary supplementation with spray-dried

plasma (SDP)7 improves the intestinal homeostasis reducing thebasal activation of the immune system (3,4). Furthermore, SDPfeed can reduce intestinal inflammation in unchallenged weanedpigs, as indicated by decreased intraepithelial lymphocytes andlamina propria cell density in the large intestine (5).

Recent information about the mechanism of action of SDPhas been obtained in a rat model of mild intestinal inflammation.The model is based on the systemic administration of entero-toxin B of Staphylococcus aureus (SEB) (4). This activates themucosal immune system affecting both organized and diffusegut-associated lymphoid tissue (GALT). Plasma supplementpreparations, either SDP (full SDP) or Ig concentrate (IC), canprevent SEB-induced increases in some GALT populations, suchas the activated T helper cells present in Peyer’s patches (PP) (4).They can also partly prevent the SEB-induced increase inparacellular flux across the epithelium, due to the reduced

1 Supported by grant no. RDITCRD06-1-001 from the Generalitat de Catalunya,

Spain.2 Author disclosures: A. Perez-Bosque, L. Miro, and M. Moreto, no conflicts of

interest. J. Polo is employed by APC-Europe; L. Russell, J. Campbell, and J.

Crenshaw are employed by APC Inc, Ankeny. E. Weaver is employed by Proliant

Health and Biologicals, Ankeny.

* To whom correspondence should be addressed. E-mail: [email protected].

7 Abbreviations used: GALT, gut-associated lymphoid tissue; GAPDH, glyceral-

dehyde phosphate dehydrogenase; IC, Ig concentrate; IFNg , interferon-g ; IL,

interleukin; iNOS, inducible nitric oxide synthase; LTB4, leukotriene B4; PP,

Peyer’s patches; SDP, spray-dried plasma; SEB, Staphylococcus aureus enter-

otoxin B; TGF b, transforming growth factor- b; TNFa, tumor necrosis factor-a.

0022-3166/08 $8.00 ã 2010 American Society for Nutrition.

Manuscript received July 3, 2009. Initial review completed July 27, 2009. Revision accepted October 29, 2009. 25First published online November 18, 2009; doi:10.3945/jn.109.112466.

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expression of tight-junction proteins (6), and the SEB-inducedreduction in D-glucose maximal transport without affectingthe passive component (7). Studies examining mucosal diffuseGALT populations showed that an SEB challenge increased thecytotoxic lymphocyte populations of T-gd cells and natural killercells and the number of activated intraepithelial lymphocytes(8). These results indicate that plasma protein supplements canmodulate the degree of activation of diffuse GALT.

Because mucosal cytokines are important in the regulation of mucosal function, we examined the effects of SDP and IC

supplements on indicators of intestinal inflammation, measuringthe expression of proinflammatory cytokines [interferon (IFNg ),tumor necrosis factor-a (TNFa), interleukin (IL)-6] and inflam-matory mediators [leukotrienne B4 (LTB4) and inducible oxidenitric synthase (iNOS)]. A second objective was to studywhether plasma protein supplementation affects the expressionof antiinflammatory cytokines such as IL-10 and transforminggrowth factor- b (TGF b). Our results support the view thatplasma supplements modulate the balance of proinflammatoryand antiinflammatory cytokines in the intestinal mucosa.

Materials and Methods

Animals, diets, and the SEB model. Male Wistar Lewis rats weresupplied by Harlan Ibe ´rica and kept under stable temperature and

humidity conditions with a 12-h-light/-dark cycle. All protocols used in

this study were approved by the University of Barcelona’s Ethics

Committee for Animal Experimentation and by the Catalan Govern-ment. In accordance with our institution’s guidelines, the number of

animals used in our studies was kept as low as possible at 7–8 rats per

group. At d 21, rats were weaned and distributed randomly to the

different experimental groups and fed the experimental diets until d 35.

Pelleted diets were formulated to meet the NRC requirements (9) for

laboratory animals (Table 1). They were isoenergetic, isonitrogenous, and

had the same lysine content. SDP is a feed ingredient obtained bycentrifuging the RBC of hygienically collected blood from pigs (10). IC

was obtained by purifying plasma Ig (11) providing a similar amount of

immunoglobulins as SDP with a much lower inclusion level in the diet.Both ingredients were spray-dried to obtain a stable powder productcontaining active Ig. Maintenance of the native structure was checked by

immunoelectrophoresis and Western blotting. Activity was verified by an

ELISA that specifically recognized Escherichia coli (12). Diets supple-

mented with SDP or IC were formulated to have a similar IgG content (10

g/kg of diet). The effective daily intake of dietary supplements between d

21 and the end of the experiment (d 35) ranged from 480 to 800 mg/d forSDAP and from 90 to 150 mg/d for IgC. Both SDP and IC were derived

from the same batch of fresh porcine blood (APC Europe S.A.).

Intestinal inflammation was induced by intraperitoneal administration

of 0.5 mg/kg SEB (Toxin Technology) dissolved in PBS on d 32 and 35.

Unchallenged rats received PBS (1 mL/kg). Rats were anesthetized

intraperitoneally with ketamine and xylazine (100 and 10 mg/kg,

respectively) 6 h after the second dose of SEB and serum and tissues

were harvested and stored at 2808C until processing for further analysis.

Rats were killed by cervical dislocation.

Study protocols. We used 2 different protocols. For protocol 1, the

groups studied were: the control group (not challenged and fed the

control diet) and groups treated with SEB and fed either the control diet

(SEB group) or a diet supplemented with SDP (SEB-SDP group) or IC(SEB-IC group). The objective of this protocol was to assess whether the

antiinflammatory effects of SDP and IC supplementation described

previously (4,6,8) involves modulation of the cytokine profile and

proinflammatory mediators during intestinal inflammation. To this end,

the concentrations of IFNg , TNFa, IL-6, and IL-10 were determined in

PP, intestinal mucosa, and serum. The concentration of LTB4 was

determined in PP and in the intestinal mucosa and the expression of iNOS was analyzed in the jejunal mucosa. For protocol 2, in view of the

results obtained with protocol 1, we wanted to assess whether SDP and

IC were also able to modulate the expression of antiinflammatory

cytokines in unchallenged rats. The studies were performed on 6experimental groups. Three groups were not treated with SEB and fed

either the control diet or a diet supplemented with SDP or IC (control,

SDP, and IC groups, respectively). The other 3 groups were challengedvia SEB and fed 1 of the same 3 diets (groups SEB, SEB-SDP, and SEB-IC,

respectively). The variables measured were the concentration of IL-10and TGF b in samples of the jejunal mucosa.

Determination of cytokines and LTB 4 . Blood was collected by cardiac

puncture from all rats at the end of the experimental period to obtain

serum. Excised PP from small intestine and samples of jejunal mucosa

were homogenized in lysis buffer containing 0.1 mmol/L EDTA, 0.1

mmol/L EGTA, 1% v:v Triton X-100, and 2% v:v of protease inhibitorcocktail (Sigma). The homogenate was centrifuged at 48C at 14,000 3 g

for 10 min. Cytokines and proinflammatory mediators were determined

in the supernatant and in serum. IFNg , TNFa, IL-6, and IL-10 were

measured by Cytometric Bead Array (Becton Dickinson). TGF b contentwas determined in the supernatant by ELISA (Promega). LTB4 was

measured by competitive ELISA (Assay Designs). All assays wereperformed following the manufacturer’s instructions.

RNA isolation and analysis. RNA extraction and RT were carried out

as described previously (13). For real-time PCR determinations, we used

a template of cDNA in a 20-mL reaction containing 0.2 mmol/L of eachprimer and 23 SyBr GreenER SuperMix (Invitrogen). The primers used

were: iNOS forward, 59-CGG TTC ACA GTC TTG GTG AAA G-39;

iNOS reverse, 59-GCT CTG ACG CGG GAA GCC ATG ACC T-39;

fragment size 76 bp; glyceraldehyde phosphate dehydrogenase(GAPDH) forward, 59-AGG TCG GTG TGA ACG GAT TTG-39;

GAPDH reverse, 59-TGT AGA CCA TGT AGT TGC GGT CA-39;

fragment size 122 bp. Real-time PCR was performed on an ABI Prism

7000 Sequence Detection system (Applied Biosystems). The PCR

conditions were as described elsewhere (13). Samples were tested induplicate and the average values were used for the quantification, which

was carried out using the 22 DDCT method (14). To confirm PCR

amplification of the intended product, representative samples wereanalyzed by electrophoresis on a 2% agarose gel. Product fidelity was

confirmed by melt-curve analysis.

Statistical analysis. All data are expressed as mean 6 SEM of 7–8 rats.

For protocol 1, 2 1-way ANOVA were performed to analyze differences

in proinflammatory cytokines and mediators, and proinflammatory:

antiinflammatory cytokine ratios between the control and SEB groups. A

second ANOVA followed by the Bonferroni post hoc test was performed

to test for significant differences in the same variables in the 3 challenged

groups (SEB, SEB-SDP, SEB-IC). For protocol 2, a 2-way ANOVA wascarried out followed by theBonferroni post hoc test to analyze the effects

of diet and SEB challenge on IL-10 and TGF b concentration as well as

TABLE 1 Composition of experimental diets

Ingredient, % Control SDP IC

SDP1 – 8 –

IC2 – – 1.50

Corn starch 19.93 30.88 22.47

Skim milk 53.07 34.05 49.02

Sugar 10 10 10

Soybean oil 7 7 7

Cellulose 5 5 5

AIN-93 GM3 3.5 3.5 3.5

AIN-93 VX3 1 1 1

DL-methionine 0.25 0.32 0.26

Choline bitartrate 0.25 0.25 0.25

1 AP-820. APC-Europe.2 APC-Europe.3

Provided by Harlan Iberica.

26 Perez-Bosque et al.

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the interaction factors (diet3

SEB challenge). Correlation coefficientswere calculated by Pearson parametric methods, using SPSS-14.0software. Differences were considered significant at P , 0.05.

Results

Proinflammatory profile in intestinal mucosa. SEB in-creased the concentrations of TNFa and IL-6 by 40–60% inboth mucosa and PP (P , 0.05; Fig. 1A,C ). Enterotoxinadministration doubled the mucosal concentration of IFNg and LTB4 and also increased its amount in PP by 60% (P, 0.01;Fig. 1B,D). Furthermore, SEB administration increased mucosaliNOS expression by 22% (P , 0.05; Table 2). In serum, SEB

increased TNFa by 92% (P,

0.01) and IFNg by 47% (P =0.063) but did not affect the IL-6 concentration (Table 3).In the jejunum, dietary supplementation with SDP did not

alter the SEB effects on TNFa but reduced the increased IFNg

expression by ~25% in both PP and mucosa (P , 0.05; Fig. 1B).The IL-6 and LTB4 concentrations in PP and mucosa of the SEB-SDP group were less than in the SEB group (P , 0.05, Fig. 1C ;P , 0.01, Fig. 1D, respectively) and the enterotoxin-inducedincrease of iNOS expression in intestinal mucosa (P , 0.05;Table 2). SDP also reduced the increase in the serum concen-trations of TNFa and IFNg induced by SEB (P , 0.05; Table 3).The IC supplement prevented the SEB-induced increase of IFNg ,TNFa, and LTB4 in the intestinal mucosa and the expression of TNFa and LTB4 in PP (P , 0.05; Fig. 1). Dietary IC

supplementation also reduced the increased iNOS expressionafter the SEB challenge (P , 0.05; Table 2) but did not modifythe SEB-induced increase of serum proinflammatory cytokines.

Antiinflammatory cytokine profile in intestinal mucosa. Ourresults showed that administration of the enterotoxin did notaffect the mucosal expression of IL-10 or mature TGF b, althoughthe synthesis of total TGF b was reduced (Table 4). However, SDPsupplementation increased IL-10 and mature TGF b concentra-tions in the intestinal mucosa in unchallenged rats (Table 4). Theresults for IL-10 obtained from the SEB-treated groups weresimilar to those obtained using protocol 1. SDP supplementationalso increased the ratio of mature TGF b:total TGF b (Table 4).

The effects of the IC supplement showed a similar pattern, with

intermediate IL-10 values between those of SDP-supplementedrats and control rats. IC also increased the mature TGF b:totalTGF b ratio in the intestinal mucosa (P , 0.05) in bothunchallenged and challenged animals (Table 4).

Proinflammatory cytokine to antiinflammatory cytokine

balance. We calculated the proinflammatory:antiinflammatorycytokine ratios (IFNg :IL-10, IL-6:IL-10, and TNFa:IL-10) in thejejunal mucosa, PP, and serum. The results showed that SEBincreased all 3 ratios in the jejunal mucosa (Fig. 2A) and inserum (Fig. 2C ) and 2 of them in PP (Fig. 2B). The 2 dietarysupplements prevented the increase in proinflammatory:antiin-flammatory ratios induced by SEB in mucosa, PP, and serum.Moreover, the mucosal TNFa:IL-10 ratios correlated well withmucosal iNOS expression (r, 0.607; P , 0.001).

Discussion

GALT is responsible for the immunological response at themucosal interface. It mediates tolerance to nonpathogenicantigens, such as food antigens, and initiates an effectiveimmune reaction against pathogens to maintain immunologicalhomeostasis (15). These effects are mediated by the immune cellpopulations present in organized and diffuse GALT, whichproduce a wide range of cytokine and mediator substances thatcan perturb the barrier and defense function of the intestinal

epithelium.

FIGURE 1 Concentrations of TNFa (A), IFNg (B ), IL-6 (C ), LTB4 (D ), and IL-10 (E ) in jejunal mucosa and PP from control, SEB, SEB-SDP, and

SEB-IC rats. Results are means 6 SEM, n = 7–8. *Different from control, P , 0.05. Means for rats challenged with SEB without a common letter

differ, P , 0.05.

TABLE 2 Levels of mRNA of iNOS in jejunal mucosa fromcontrol, SEB, SEB-SDP, and SEB-IC rats1,2

iNOS (arbitrary units)

Control 1.00 6 0.04

SEB 1.22 6 0.04a*

SEB-SDP 1.05 6 0.05b

SEB-IC 1.03 6 0.06b

1 Results are mean 6 SEM, n = 7–8. *Different from control, P , 0.05. Means for rats

challenged with SEB without a common letter differ, P , 0.05.2

Levels of mRNA were normalized to GAPDH.

Antiinflammatory effects of plasma supplements 27

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Previous studies using a rat model of intestinal inflammationinduced by the systemic administration of SEB show that dietarysupplementation with SDP or IC can reduce GALT stimulation.SEB is a potent activator of the intestinal immune system thatcan trigger a high percentage of T cells, especially in the smallintestine (16). Our results from rats challenged with SEB showedthat the enterotoxin induced a mild mucosal immune activation,involving Tgd lymphocytes and activated CD4 in PP, and Tgdand activated lymphocytes in both lamina propria and intraep-ithelial compartments (4,8). Activated mast cells and eosinophils

were also stimulated (17). Stimulation of the mucosal immunesystem by SEB results in increased water content in feces,reduced epithelial tightness, and impaired nutrient transport(4,6,7). It is well known that SEB induces a rapid increase incirculating cytokines, such as IL-2, IL-6, and IFNg (18). Inaddition, cytokines reduce the expression of tight-junctionproteins, hence altering mucosal permeability (19), and decreasethe expression of the a-subunit of the Na/K ATPase. Thus, theyaffect nutrient and electrolyte transport across the epithelium(7,20). Because most of the effects of GALT activation in the ratSEB model are reduced or prevented by dietary SDP or IC (17),we studied the involvement of mucosal cytokines as candidatesthat may mediate the effects of dietary plasma supplements.

The analysis of cytokine release induced by SEB administra-tion was conducted in the immune compartments that areresponsible for 3 different functions: in PP, because they are thesite of induction of mucosal immune responses; in the intestinalmucosa, because it is the effector that controls luminal interac-tions; and finally in the serum, which serves as an indicator of

systemic activation. SEB increased proinflammatory cytokines inboth mucosal and PP compartments. This was consistent withthe previous observation of GALT stimulation by SEB at PP andmesenteric lymph nodes (the inductor site) (4) and at laminapropria and intraepithelial lymphocytes (the effector site) (8). Inaddition, the mucosal GALT stimulation induced by SEB drivesthe mucosal immune response toward a Th1 type (cellularimmunity) response, because IFNg and TNFa enhance activa-tion and proliferation of Th1 cells (21,22). SEB also increasediNOS expression at the mucosal level. Because this effect was

well correlated with the TNFa:IL10 ratio, we suggest that thechanges in iNOS were the result of the imbalance of proinflammatory:antiinflammatory cytokine ratios.

Cytokines determine the nature of the immune response inintestinal inflammation by inducing the release of inflammatorymediators such as reactive oxygen species, nitric oxide, leuko-trienes, platelet-activating factor, and prostaglandins (23). Leu-kotrienes are a family of lipid mediators that play a key role inthe pathogenesis of inflammation. For example, LTB4 is one of the most potent chemoattractants and activators of neutrophilsand other cells such as macrophages and T-cells and is involved ininflammatory diseases (24). Moreover, IL-6 and TNFa amplifythe immune response by enhancing the proliferation of T cells,

thus promoting leukocyte infiltration and facilitating cell-cellsignaling. The present study shows that dietary plasma supple-mentation modulates the expression of proinflammatory cyto-kines (TNFa, IFNg , and IL-6) and other inflammatory mediators(LTB4, iNOS), and antiinflammatory IL-10 and TGF b.

Although there were some differences in cytokine expressionbetween SDP and IC groups of unchallenged rats, the 2ingredients had similar effects on inflammation. Dietary supple-mentation of IC and SDP has similar effects on intestinalinflammation and both can reduce the balance from proinflam-matory to antiinflammatory cytokines that characterizes the SEBcondition. This is in agreement with studies evaluating IC andSDP on the growth of young pigs (25). The effects of plasma

supplements on cytokine release are consistent with the preventiveeffects observed in activated CD4 and Tgd subsets in PP and onactivated lymphocytes and cytotoxic populations in laminapropria in rats challenged with SEB (4,8). Proinflammatorycytokines induce iNOS expression and contribute to alteredintestinal integrity in inflammatory intestinal disorders. Therefore,

TABLE 3 Concentrations of TNFa, IFNg , IL-6, and IL-10 inserum from control, SEB, SEB-SDP, and SEB-IC rats1

Control SEB SEB-SDP SEB-IC

ng/L

TNFa 113 6 19 217 6 17*a 136 6 29b 168 6 17ab

IFNg 20.1 6 3.7 29.5 6 1.9a 20.1 6 3.3b 23.9 6 2.6ab

IL-6 64.1 6 9.6 74.1 6 5.5 62.0 6 9.3 71.3 6 8.7

IL-10 116 6 19 98 6 11a 172 6 30b 142 6 20ab

1 Results are mean 6 SEM, n = 7–8. *Different from control, P , 0.05. Means for ratschallenged with SEB without a common letter differ, P , 0.05.

TABLE 4 Antiinflammatory cytokine concentration in mucosa of jejunum from control, SDP, IC, SEB,SEB-SDP, and SEB-IC rats1

Diet

Not

challenged Challenged Diet Challenge

Diet 3 SEB

challenge

IL-10, ng/g tissue Control 0.63 6 0.02a

0.64 6 0.03a

SDP 0.75 6 0.04b 0.86 6 0.07b

IC 0.68 6 0.03a 0.72 6 0.08a ,0.001 NS2 NS

Mature TGF b, pg/mg protein Control 6.43 6 1.22a 5.92 6 0.68a

SDP 9.96 6 1.59b 7.43 6 1.77b

IC 7.76 6 0.96ab 6.89 6 0.75ab 0.031 NS NS

Total TGF b, pg/mg protein Control 93.7 6 6.8 71.7 6 9.7

SDP 98.6 6 16.9 65.2 6 6.8

IC 81.7 6 13.2 67.4 6 4.7 NS 0.045 NS

Mature TGF b:total TGF b ratio Control 0.067 6 0.009a 0.072 6 0.006a

SDP 0.106 6 0.012b 0.120 6 0.015b

IC 0.102 6 0.013b 0.107 6 0.013b 0.022 NS NS

1 Results are mean 6 SEM, n = 7–8. Within a challenge group, means with superscripts without a common letter differ, P , 0.05.2

NS, Not significant, P $ 0.05.


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the increase in IFNg and TNFa in the present study may explainthe reduction in intestinal barrier function observed in previousexperiments (6), because both cytokines can reduce the epithelialpermeability by internalizing tight junction proteins (26).Results also suggest that increased intestinal permeability afterSEB administration can be mediated by macrophage activationand NO synthesis. Proinflammatory cytokines also have severaleffects on transport functions. For example, IFNg down-regulates Na+-coupled D-glucose transport in T84 cells (27)

and TNFa reduces fructose uptake in rabbit intestine (28) andmay therefore be responsible for the effects on glucose transportobserved previously (7). Taken together, the reduction inmucosal expression of TNFa, IFNg , and IL-6, and the loweriNOS activity (hence decreasing NO production) and LTB4expression (consistent with lower mast cell proliferation) allpoint to a reduction in the mucosal chemical mediators that areresponsible for intestinal inflammation with dietary supplemen-tation of SDP or IC. The observation that dietary supplemen-tation with plasma proteins increases the mucosal expression of IL-10 and mature TGF b suggests the involvement of theseantiinflammatory cytokines in the control of the production of proinflammatory cytokines. These effects contribute to intesti-

nal homeostasis, because both IL-10 and TGF b are important in

the control of T cells and of innate immune cell-mediatedintestinal pathology. They are also both important in theacquisition of regulatory function by peripheral T cells, asrecently reviewed by Coombes and Maloy (29).

In view of the role of IL-10 in the amelioration of intestinalinflammation (30), it is worth noting that SDP can increasemucosal IL-10 in either the presence or absence of an SEBchallenge. Also interesting are the effects of SDP and IC onTGF b, a cytokine that plays an important role in the homeo-stasis of intestinal epithelial cells, contributing to the mainte-

nance of the intestinal barrier (31). However, IL-10 secretionwas not modified by SEB, perhaps because the pattern of response of this cytokine to the enterotoxin stimulation isdifferent from that of proinflammatory cytokines, as shown byAssenmacher et al. (32). Therefore, plasma supplements caneither directly or indirectly [e.g. by changing the luminalbacterial profile as suggested by Martı ´n-Oru ´ e et al. (33)]regulate the expression of mucosal antiinflammatory mediators.In turn, this will reduce SEB-evoked immune cell activation andthe concomitant epithelial dysfunction, which confirms theresults reported by Lu et al. (34) using an in vitro coculturemodel. The observation that the effects of SEB on the release of systemic proinflammatory cytokine are small is in agreement

with previous results in the spleen (4) and indicates that SEB haslittle effect on the peripheral immune system. This supports theview that the increased proinflammatory cytokine concentrationin serum in rats challenged with SEB reflects the activation of themucosal immune system rather than the activation of peripheralcompartments of the immune system. The increase in IL-10concentration in serum due to dietary plasma supplementationcould also explain the reduction in the cytotoxic lymphocytepopulation (such as NK cells) observed in the spleen (4).

Intestinal inflammation has been shown to negatively affectgrowth in young animals and disruption of gut barrier functionis thought to have a range of health consequences in humans,including exacerbation of inflammatory bowel disease (35). A

supplementary protein source, such as plasma protein or IC,may play an important role in limiting the consequences of intestinal inflammation, potentially at a much lower cost thanother methods. Recent findings in a mouse model of colitis (36)showed that both SDP and IC can partially prevent the structuraland functional changes observed in the colon during thedevelopment of the disease, which highlights the role of diet inthe treatment of inflammatory bowel disease.

Acknowledgments

We thank M. Bajaj-Elliott and W. Dhaliwal for advice on PCRdeterminations. We also thank Monica Maijo ´ for invaluabletechnical assistance. M.M., J.P., L.R., Joy C., E.W., Joe C., and

A.P-B. designed research; A.P-B. and L.M. conducted research; J.P. provided essential materials; A.P-B. and M.M. analyzed thedata and wrote the paper; M.M., J.P., L.R., Joy C., E.W., Joe C.,and A.P-B. discussed the results. All authors have read andapproved the final manuscript.

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