Food allergy/hypersensitivity: Antigenicity or timing?

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Immunobiology 214 (2009) 269–278

Food allergy/hypersensitivity: Antigenicity or timing?

Patrıcia Olaya Paschoala,b,�, Sylvia M.N. Camposb, Monique M.B. Pedruzzib,Valeria Garridob, Monica Bissoa, Danielle M.F. Antunesb, Alberto F. Nobregac,Gerlinde Teixeirab

aPrograma de Pos-Graduacao em Patologia, Faculdade de Medicina, Universidade Federal Fluminense, BrazilbLaboratorio do Grupo de Imunologia Gastrintestinal, Departamento de Imunobiologia, Instituto de Biologia,

Universidade Federal Fluminense, BrazilcUniversidade Federal do Rio de Janeiro

Received 30 October 2007; received in revised form 25 August 2008; accepted 15 September 2008

Abstract

Mechanisms involved in the induction of oral tolerance (OT) or systemic immunization through the oral rout arestill poorly understood. In our previous studies, we have shown that when normal mice eat peanuts they becometolerant, with no gut alterations. Conversely, if immunized with peanut proteins prior to a challenge diet (CD)containing peanuts they develop chronic inflammation of the gut. Our aim is to evaluate the consequences of theintroduction of a novel protein in the diet of animals presenting antigen-specific gut inflammation. Adult, femaleC57BL/6J mice were divided in control (C) and experimental (E) groups. C1–C3 received peanut proteinimmunization, animals of the control groups C4 were sham immunized, and control group C5 received ovalbumin(OVA) immunization. The experimental group was immunized with peanut protein extract. Before initial exposure to a30-day peanut containing CD, the experimental group was divided into 5 groups (E1–E5). OVA feeding began 7 daysprior CD (E1) on day 0 (E2), 7 (E3), 14 (E4) and 21 (E5) during CD. Our results show that oral exposure to a novelprotein (OVA) in the absence of gut inflammation (E1) leads to low levels of systemic antibody (Ab) titers, comparableto tolerant animals. Conversely, as off initial induction of inflammation, groups submitted to OVA (OT) protocoldevelop increasingly higher systemic Ab titers similar to animals of the immune control group. In conclusion, ourprotocol indicates that timing is more important than the antigenicity when a novel protein is offered, in the diet.r 2008 Elsevier GmbH. All rights reserved.

Keywords: Food allergy; Ovalbumin; Oral tolerance; Peanuts

Introduction

For many years the term allergy was used asa synonym for IgE-mediated hypersensitivity, thus

associated to severe potentially life-threatening anaphy-lactic reactions. However, clinical observations haveshown that not all allergies have this as the underliningmechanism. A force task of the European Academy ofAllergy and Clinical Immunology published in 2001 arevised nomenclature for allergies (Johansson et al.,2001). This force task proposed that allergies arehypersensitivity reactions initiated by immunologicmechanisms and that adverse reactions to food should

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0171-2985/$ - see front matter r 2008 Elsevier GmbH. All rights reserved.

doi:10.1016/j.imbio.2008.09.007

�Corresponding author at: Instituto de Biologia, Alameda Barros

Terra s/n, Centro, Niteroi, 24020-150, Rio de Janeiro, Brazil.

Tel.: 55 21 26292313.

E-mail address: [email protected] (P.O. Paschoal).


be called food hypersensitivity. When immunologicmechanisms have been demonstrated, the appropriateterm is food allergy, and, if the role of IgE is highlighted,the term is IgE-mediated food allergy. All otherreactions, previously referred to as ‘‘food intolerance’’,should be referred to as nonallergic food hypersensitiv-ity as previously suggested by Bruijnzeel-Koomen et al.(1995) and Ortolani et al. (1999).

In 1946, Merill Chase showed that oral administra-tion of a contact-sensitizing agent (2,4-dinitrochloro-benzene) does not lead to sensitization, but ratherprevents the animal from eliciting an immune responseupon subsequent subcutaneous injections or cutaneouschallenge. The specific induction of these regulatedresponses by administration of antigen through thegastrointestinal tract is known as oral tolerance (OT)(Mayer and Shao, 2004). Thus, food allergy representsan abrogation of normal OT to that specific foodstuff(Scott and Sampson, 2006).

For efficient strategies aimed at an active cure of foodallergies, a better understanding of the pathogenicmechanisms is strongly needed (Sampsom et al., 2005).In contrast to current thought that food allergy isprimarily a Th2-driven process, there is accumulatingdata from the literature indicating that food allergiesmay also be a result of a mixed Th1Th2 immuneresponse.

The mucosal immune system utilizes a very complexand tightly regulated system of suppression andcontrolled inflammation to distinguish between innoc-uous and potentially harmful antigens of the intestinallumen. Although the exact mechanisms governing thisregulation remains to be elucidated, evidence exists forthe involvement of a variety of cell types, includingCD4+, CD8+ T cell, regulatory T cells, B cells, anddendritic cells (Mayer, 2005). When both the ability toremain tolerant to the abundance of dietary antigensand prevention of luminal bacterial flora to harm thehost fail, disease may install (Kraus et al., 2004a).

OT induction is a key feature of intestinal immunity,generating systemic low responsiveness to ingestedantigens (Worbs et al., 2006). Mechanisms governingtolerance induction have been well characterized inselected mouse strains. Similar studies in man arelacking, although there is evidence that tolerance canbe induced. The ability to induce OT in humans wasreported by Husby et al., although the type of tolerancegenerated to a neo-antigen, keyhole limpet hemocyanin(KLH), was slightly different from that seen in murinemodels. Tolerance for delayed-type hypersensitivity(DTH) responses but not antibody (Ab) responses wasseen (Husby et al., 1994; Kraus et al., 2004b). In diseasestates, tolerance is altered and this may account for thepresence of mucosal inflammation.

In food hypersensitivity there is evidence that aller-gens may be handled differently and this may play a role

in disease expression (Mayer and Shao, 2004). It hasbeen clearly demonstrated that an upregulated Th1immune response and subsequent overproduction ofTh1 cytokines may lead to autoimmune diseases such as,inflammatory bowel disease, autoimmune arthritis,experimental autoimmune encephalomyelitis, and type-I diabetes (Kuchroo et al., 1995; Neurath et al., 2002).Thus, there is an intense interest in understanding theregulation of the immune response, with the goal ofdeveloping therapeutic strategies. In cases of multiplefood allergies, restricted diets may result in unbalancednutrition and pose a considerable hardship to the familyof a food-allergic child (Nowak-Wegrzyn, 2003).

In previous studies of our group, we have shown thatcrude peanut protein extract is immunogenic both inmice and rats. All animals responded with high levels ofsystemic antibodies (po0.001) as compared to salinecontrols. No differences were observed when comparinganimals within strains nor when comparing youngadults (2–4 months) with older animals (12–16 months).Submitting animals to a peanut containing challengediet (CD) induces important alterations of the gutmucosal architecture, typical of chronic inflammation inimmunized but not in normal animals (Teixeira, 2003;Teixeira et al., 2008a, in press). In this study, we showthe consequences of the administration of a non relatedprotein in the presence of a chronic antigen-specific gutinflammation.

Methods

Animals

C57BL/6J female mice, 2–3 months old, wereobtained by the local Animal Resources Center (Uni-versidade Federal Fluminense). These animals wererandomly divided into 2 groups: control and experi-mental. These were subsequently subdivided into threecontrol groups (n ¼ 7) and 5 experimental groups(n ¼ 14). All animals were individually marked, to beable to perform paired statistical analysis. This researchwas approved by the animal ethics committee of theSchool of Medicine of the Fluminense Federal Uni-versity.

Immunization protocols

The immunization protocol utilized in this study was:100 mg specific protein (peanut protein extract orovalbumin (OVA)) plus 1mg of adjuvant [Al(OH)3], ina final volume of 200 ml by subcutaneous route (sc) forthe primary immunization and 100 mg of specific proteinwithout adjuvant, performed 21 days later, for thebooster immunization.

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For normal controls the animals were sham immu-nized with 1mg of adjuvant [Al(OH)3], in a final volumeof 200 ml of saline by sc for the primary immunization

and saline without adjuvant, performed 21 days later,for the booster immunization.

All animals of the experimental groups (E1–E5) andcontrol groups C1–C3 received peanut protein immuni-zation, animals of the control groups C4 were shamimmunized, and control group C5 received OVAimmunization.

Tolerization to OVA or peanuts

To induce OT, animals received to drink a sweetened20% egg white solution (diluted in saline v/v with 5% ofsaccharine) or to eat a diet containing exclusively peanutseeds for 7 days ad libitum prior to the immunizationprotocol described above with the respective proteins.(Teixeira, 2003; Teixeira et al., 2008a, in press).

Bleeding

Animals were bled 200 ml from the retrorbital plexusprior to manipulation, after each immunization andduring the inflammation induction protocol. The serumwas collected and stored at �20 1C until analyses.

Induction of the antigen-specific inflammatory gut

reaction

Two weeks after booster immunization with peanutprotein extract, experimental animals were submitted toa CD with peanuts in natura, during a 30-day period asdescribed by Teixeira (2003), Teixeira et al. (2008a) inpress.

Animals of control group immunized to peanuts (C1)continued to receive commercial chow, (C2) whichhad been submitted to peanuts OT protocol and (C3)immunized to peanuts received peanuts for a 30-dayperiod.

Timing of OVA feeding in mice with antigen-specific

inflammation of the gut

To determine the consequences of gut inflammationon the induction of OT, the 5 experimental animalsgroups received OVA to drink at different weeks ofexposure to the peanut containing CD. The first group(E1) received OVA to drink from day 7 through day 0(week 1) of initiation of peanut containing CD. Thesecond group (E2) received OVA to drink as of day 0 ofthe CD (week 1). The third group (E3), fourth (E4), andthe fifth groups (E5) received OVA to drink as of the7th, 14th, and 21st days after the beginning of the CD(weeks 2, 3, and 4), respectively (Table 1).

Determination of Ab levels

An enzyme-linked immunosorbent assay (ELISA)was performed to detect total IgG. Briefly, 96-wellmicro plates (Alfa, Brazil) were coated with 10 mg ofpeanut protein extract or OVA in 0.1M PBS bufferovernight at 4 1C per well. After a thorough wash thewells were blocked with PBS-gelatin before incubationof the serum samples for 3 h at RT. Detection wasperformed with goat anti-mouse IgG HRP (1:5000,Sigma) and OPD (o-phenylenediamine, Sigma). Thereaction was interrupted with 0.1M sulphuric acid andread at 492 nm in an ELISA micro plate reader (Anthos2010, Germany). The results were expressed as arbitrary

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Table 1. Timing of oral tolerance induction to Ovalbumin in the course of gut inflammation due to the exposure of a challenge diet

containing the specific antigen to which animals had been previously immunized with peanut protein extract

Challenge diet (weeks) Recovery (weeks)

�1 1 2 3 4 1 2 3 4 5 6 7

Group E1 OVAayb

yyc

Group E2 OVA y

yy

Group E3 OVA y

yy

Group E4 OVA y

yy

Group E5 OVA y

yy

aOVA 1 week of free access to sweetened egg white solution.by time of killing after induction of gut inflammation by the removal of normal mouse chow and introduction of challenge diet.

cyy time of killing after recovery of gut inflammation by the removal of challenge diet and introduction of normal mouse chow.

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units of ELISA corresponding to the area under thedilution curve of each serum.

Histomorphology

As depicted in Table 1, animals of the experimentalgroups were euthanized at different times of CDexposure (1, 2, 3, and 4 weeks) and recovery period(1, 3, 4, 5, 6, and 7 weeks) to collect intestinal segments.Animals of the control groups were euthanized atthe end of their respective experimental protocols diet),C1 – immune without gut inflammation (immunizedwith peanut protein without 4 weeks of CD); C2 –tolerant (OT to peanut protocol with 4 weeks of CD);C3 – immune (immunized with peanut protein with 4weeks of CD); C4 – normal (sham immunized withoutexposure to CD).

Intestinal segments of all animals were fixed with 10%buffered formaldehyde and stained with hematoxylin-eosin (HE) and analyzed to quantify gut alterations ofthe histological architecture. For the purpose of thisstudy, we examined the histological parameters from theduodenum analyzing the general aspects of the tissuesuch as integrity of the intestinal structure, number ofvilli per field, edema, congestion and leukocyte infiltrateand establishing the ratios between villi height/widthand intestinal epithelial cells/intraepithelial leukocytes(IEC/IEL).

Statistical analysis

Statistical analysis was performed by using Fisher’stest, ANOVA, and Tukey0s post test to determine theminimum significance difference (MSD) using Graph-Pad InStat program by GraphPad Software Incs.

Results

Immunogenicity and induction of OT to peanuts innatura

Confirming prior data total protein peanut extract isimmunogenic. All immunized animals responded withsignificantly higher levels of systemic antibodies(po0.001) when compared to saline controls. Whenanimals had free access to the seeds in the diet for 7consecutive days prior to immunization a significantreduction of specific Ab titers were observed comparedto immunized group (po0.001) (Fig. 1).

Induction of antigen-specific inflammation of the gut

To quantify the evolution of gut inflammation,induced by a peanut containing CD for a 4 week period

half of the animals of groups E1, E2, E3, and E4 waseuthanized after 1, 2, 3, and 4 weeks of exposure,respectively. The rest of the animals were killed 1, 3, 4, 5,6, and 7 weeks of recovery (removal of peanuts andreintroduction of normal mouse chow) as depicted inTable 1.

Macroscopic analysis of the intestine

The macroscopic analysis of the peritoneum pertain-ing to animals of the experimental group showed a palecoloring of organs in general and revealed a frailconsistency of the intestinal tissue as of the third weekof CD in contrast to the control group. Animals of therecovery group also showed macroscopic improvementas of the third week (data not shown).

Microscopic analysis of the intestine

The first microscopic parameter of the duodenumevaluated was the villi height/villi width (vh/vw) ratio(Fig. 2). Immune animals of the group that had notreceived CD (C1) presented a normal vh/vw ratio(2.7970.63) as did the groups which received CD forone (E2) (2.0570.13) and 2 weeks (E3) (2.8370.75). Asof the third week of CD significant alterations becomeevident. The most obvious architectural alterations wereobserved in the last experimental group E5 (1.3170.18),where significant shortening of the villi accompanied byenlargement of the vw was seen leading to a verysignificant difference (po0.01) when compared to thegroup without CD (C1) and tolerant control groups(C2). After removal of CD all groups presented recoveryof the intestinal villi with significant changes as of thethird week (Fig. 2).

The second parameter evaluated was the numberof the IEC in relation to the number of IEL per villi.

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0

2

4

6

8

10

12

Zero

Arb

itrar

y U

nits

of E

LIS

A

ExperimentalTolerantControl

Booster Challenge diet

Fig. 1. Total IgG anti-peanuts-Ab titers of tolerant, experi-

mental and control animals before any manipulation (zero),

after booster immunization (booster) and after CD (peanuts).

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The total number of cells decreases as the inflammatoryprocess is installed. A positive correlation can beestablished between this cell count and histomorphologyvh (data not shown). As the inflammatory processaggravates, during the 4 weeks of CD exposure, theIEC/IEL ratio decreases from 0.6070.07 (E2) to0.3770.11 (E5). After the removal of the CD anormalization of the vh and cell count was seen,although these ratios do not reach normal levels forboth parameters within the 7-week period observed. Theimprovement was slower for the cell count than for thearchitecture of the villi (Figs. 3 and 4).

In Fig. 4, we demonstrate the differences between thedifferent experimental groups. Representative examplesof histological findings are shown in the microscopicanalysis. In agreement with the macroscopy Fig. 4Ashows a preserved intestinal structure of control animals

which had been immunized but had not received theCD. In Fig. 4B the mucosa of a tolerant animaldemonstrates a similar architectural structure comparedto the control group. On the other hand, figures C, D, E,and F, pertaining to groups E1–E4 killed after differenttimes of CD exposure 1–4 weeks, respectively, demon-strates increasing alterations typical of chronic inflam-matory process, such as: villi shortening, villi widening,edema, congestion, and leukocyte infiltration in duode-num.

Timing of OVA feeding in mice with antigen-specific

inflammation of the gut

The experimental groups (E1–E5) that were submittedto a diet containing sweetened OVA ad libitum during 7

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050

100150200250300350400450

0

Time of challenge diet(weeks)

Mea

sure

in µ

m

Villi heightVilli width

0

1

2

3

4

5

6

Vill

i hei

ght w

idth

ratio

1 2 3 4 1 3 4 5 6 7

Time of recovery (weeks).0

Time of challenge diet(weeks)

1 2 3 4 1 3 4 5 6 7

Time of recovery (weeks).

Fig. 2. Mean measures showing evolution of vh and vw (left panel) and the ratio established between these measures (right panel)

during exposure to CD and recovery period of C57Bl/6J experimental mice (n ¼ 7).

0

50

100

150

200

250

300

0

Weeks of challenge diet

Num

ber o

f cel

ls p

er v

illi

IECIEL

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

IEC

/IEL

ratio

1 2 3 4 1 3 4 5 6 7

Weeks of recovery0

Weeks of challenge diet

1 2 3 4 1 3 4 5 6 7

Weeks of recovery

Fig. 3. Mean intestinal epithelial cell (IEC) and intra epithelial leukocytes (IEL) count during induction of gut inflammation due to

exposure to CD containing specific antigen and during recovery after reintroduction of conventional mouse chow (left panel).

Establishment of the IEC/IEL ratio during CD and recovery period (right panel) (n ¼ 6).



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Fig. 4. Histomorphology (HE) of small intestine – (A) Control animal. (B) Tolerant animal. (C) Animal with 1 week of gut

inflammation induction. (D) Animal with 2 weeks of gut inflammation induction. (E) Animal with 3 weeks of gut inflammation

induction. (F) Animal with 4 weeks of gut inflammation induction. Progressive alterations such as presence of edema, leukocyte

infiltrate, modification of normal architecture of villi can be observed in panels C, D, E, and F.

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

E1Control groupsExperimental groups

Arb

itrar

y U

nits

of E

LIS

A

E2 E3 E4 E5 Tolerant Immune

Fig. 5. (}) Individual and (–) mean anti-OVA IgG, after free ingestion of sweetened OVA in the diet, prior (E1) and during (E2–E5)

the induction of an antigen-specific gut inflammation due to a peanut containing CD, compared to normal and immune control

groups (n ¼ 7).

P.O. Paschoal et al. / Immunobiology 214 (2009) 269–278274


days at different timings of the peanut containing CDpresented increasing anti-OVA specific Ab titers. Theanti-OVA Ab titers after oral exposure to sweetened eggwhite and prior to systemic immunization of the 5groups showed that animals of E1 (0.5770.14), E2(1.0670.41) and E3 (0.7170.27) present low mean Abtiters, respectively) not significantly different fromtolerant controls. Groups E4 (1.5970.49) and E5(2.5471.07) present anti-OVA Ab titers compatible toprimary systemic immunization and are significantlydifferent from groups E1–E3 (po0.01) (Fig. 5).

Although all groups presented a significant elevationof total Ab titers after systemic immunization animals ofgroup E1 (1.3270.468) presented the lowest elevation.The Ab titers of group E1 differ significantly from

immune control animals (po0.001) but not fromtolerant animals (p40.5) thus being considered tolerant.These animals also presented a significant differencefrom all other experimental groups. On the otherhand groups E2 (4.1170.62), E3 (4.3570.38), E4(3.3670.28), and E5 (5.4770.73) present significantdifferences when compared to tolerant group but not toImmunized group thus being considered systemicallysensitized. When comparing these latter groups (E2–E5)a scatter in the individual Ab titers can be seen (Fig. 6).

Fig. 7 depicts the mean elevation of Ab titers betweenoral administration of OVA and systemic immunizationindicating that all animals that receive OVA to drink asof the introduction of the peanut containing CD becomesystemically sensitized to OVA.

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0.00

1.00

2.00

3.00

4.00

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6.00

7.00

8.00

E1

Control groupsExperimental groups

Arb

itrar

y U

nits

of E

LIS

A

Tolerant ImmuneE2 E3 E4 E5

Fig. 6. (}) Individual and (–) mean anti-OVA IgG, after OVA booster in groups that had free access to sweetened OVA in the diet,

prior (E1) and during (E2–E5) the induction of an antigen-specific gut inflammation due to a peanut containing CD. (n ¼ 7)

compared to normal and immune control groups.

0

1

2

3

4

5

6

Oral exposure

Control GroupsExperimental Groups

Arb

itrar

y U

nits

Of

ELI

SA E1

E2E3

E4

E5

TolerantImmune

Booster Primary Booster

Fig. 7. Comparison of mean anti-OVA IgG titers of control and experimental groups. Experimental groups: anti-OVA Ab titers

after Ova feeding over a 7-day period – (E1) began 7 days prior to peanut containing CD (E2) on day 0, (E3) on day 7, (E4) on day

14 and (E5) on day 21 of CD and after the booster inoculation of OVA. Control groups: Ab titers after primary and booster

inoculation.



Discussion

Food allergy is a disease that affects approximately6% of young children and 3.7% of the adult populationin the USA (Scott and Sampson, 2006). Recognition offood allergy is largely based on symptoms reported bypatients or parents. While IgE-mediated food allergy isrelatively easy to diagnose, identification of non-IgE-mediated food allergy relies on suggestive symptoms,frequently mimicked by various other adverse reactionsto foods and other diseases. (Engelmann et al., 2008).

Systemic introduction of an antigen whether byinjection (subcutaneous or intramuscular) or injury,may lead to local infiltration of inflammatory cells andspecific immunoglobulin production (Strid et al., 2005).In contrast, antigens introduced at mucosal surfacesgenerally induce active immune tolerance to the antigens(Mayer and Shao, 2004). In this work, we confirm thatpeanut antigens elicit qualitatively distinct immuneresponses based on their initial rout of entry and onprior immune experience of the animal to the respectiveantigen.

As we have previously described when mice and ratsare systemically immunized to peanut protein extractand then exposed exclusively to a diet based on rawpeanuts they develop inflammation of the gut. Thefeatures of this model are prominent mononuclearleukocyte infiltrate and lamina propria edema and largernumbers of goblets cells inducing architectural changessuch as, villous atrophy and crypt hyperplasia. Clini-cally, these animals present significant weight loss whichcan be explained by the histological findings of the gut,which in turn may be the cause of lower food intake(Teixeira et al., 2008b), and a lower absorption rate(Teixeira et al., 2008a, in press). These are in agreementto the typical signs seen in human celiac disease (glutenenteropathy), and which are observed in other food-induced enteropathies. We have also described thatwhen immunized animals are given the choice, theyavoid the allergenic foodstuff eating the other compo-nents of the diet and consequently do not develop gutalterations (Teixeira et al., 2008b).

Foods contain substances that can both control thephysiological functions of the body and modulate theimmune responses (Kaminogawa and Nanno, 2004).Although both OT and allergic responses have beenestablished both in humans and in animal models thesedepend on complex interactions involving the geneticbackground, the type, timing and dose of Ag and isthought to be a consequence of the introduction ofproteins in the gut in physiologic conditions (Vaz andPordeus, 2005; Faria and Weiner, 2005; Teixeira et al.,2008a, b in press).

Furthermore, dealing specifically with it has beenshown that the allergic reactions to foods may provokecharacteristic responses in the skin, gastrointestinal and

respiratory tract, regardless of the immunopathogenicmechanism responsible for the reaction (Sampsom et al.,2005). As such allergic responses require complexinteractions between the protein and the immunesystem, which are notoriously difficult to predict (Hubyet al., 2000).

There has been considerable recent broadening ofbasic concepts of intestinal food allergy, in particularthe importance of non-IgE-mediated mechanisms. Thetraditional emphasis on IgE-mediated allergy nowappears inappropriate in light of current studies of thebasic mechanisms of OT to dietary antigen and ofincreasing recognition of the requirement for earlyinfectious challenge in the prevention of allergicsensitization (Chehade, 2007).

This major change in emphasis has been elicited bothby basic scientific studies and by the recognition of novelpatterns of food allergic diseases within the pediatricpopulation. A rapid increase in food-allergic sensitiza-tion has been noted in the last decade in special, twopreviously rare phenomena: multiple food allergies andsensitization of exclusively breast-fed infants to antigenseaten by the mother have become commonplace(Murch, 2000).

In our study, we propose that prior systemicimmunization associated to the port of entry of thespecific antigen is a strong cause of induction of chronicinflammatory reactions of the gut. Although othersensitizing mechanisms that simulate the clinical settingsuch as skin sensitization (Strid et al., 2005) or oralsensitization associated to cholera toxin (Mayer, 2005)could have been used, we chose to sensitize animalsusing the subcutaneous rout in order to quantify theexact amount of antigen that was given.

In our results, the group of animals which receivedOVA before (E1) the initiation of the 4-week peanut CDpresented specific Ab titers similar to the tolerant group.Animals which received free access to OVA during thefirst and second week of the CD (E2 and E3) presentedspecific Ab titers similar to the primary immunization ofimmune control animals. As of the third week of CD, weshow that the specific Ab titers of the groups thatreceived OVA during this period, groups E4 and E5,were similar booster level of the immune control group.After booster immunization group E1 continued topresent low Ab titers in contrast to all other experi-mental groups (E2–E5) that received OVA to drink as ofday 1 of CD exposure. These latter groups presented Abtiters equivalent or higher to immune control groupconfirming their immunological status.

Thus, oral administration of OVA in mice sensitizedto peanuts before the induction of chronic inflammationdue to the exposure to a CD containing the specificantigen resulted in the development of OT. In contrast,as of the exposure to the CD which induces significantinflammatory changes to the gut, the introduction of the



novel protein led to a shift from the induction of OT tosystemic immunization.

Although we have not determined the profile of thecells that infiltrate the gut during CD we could arguethat these are cells with an inflammatory profile whichhave been elicited during systemic immunization andthat migrate to the gut upon exposure to the specificantigen inducing a shift from a tolerogenic to animmunizing environment in the gut.

In contrast to current thought that peanut allergy isprimarily a Th2-driven process, there is accumulatingdata from the literature indicating that this food allergymay be a result of a mixed Th1Th2 immune response.The absence of systemic anaphylactic reactions, thepresence of a mononuclear infiltrate with few mast cellsand eosinophils in our murine model corroborates to theidea that this is a mixed Th1Th2 modulation of peanutallergy. Finally, the verification of similar inflammatoryresponses in the gut of mice with different geneticbackgrounds (Balb/c and C57BL/B6) provides aninteresting method for the study of this mixed immuneresponse to peanut proteins. The animals that weresubmitted to a chronic gut inflammatory process showedmore heterogeneity in the individual immune responseswhen compared to systemic immunization. This may bedue to the fact that the animals were free to determinethe amount of daily intake.

The intrinsic potential of protein allergens to inducesensitization will be manifest only in susceptibleindividuals, and then only if the allergen is encounteredin sufficient quantities and via a relevant route ofexposure (Huby et al., 2000). In our model this fact canbe seen in the animals that are immunized (thussusceptible) but not challenged in the diet in which theintroduction of a novel protein leads to OT due tonormal gut environment.

Preliminary results indicate that during the recoveryperiod of the chronic inflammation of the gut aconcomitant recovery of the ability to induce OT is shownarguing that the deregulation of the normal physiology istransitory depending on local exposure to the allergen.

In conclusion the induction of OT or systemicimmunization to a novel protein depends on the localenvironment of the gut. Taking these results to theclinical setting we could suggest that children with gutalterations should not be exposed to novel proteins as ameasure to avoid multiple food allergies.

References

Bruijnzeel-Koomen, C., Ortolani, C., Aas, K., Bindslev-

Jensen, C., Bjorksten, B., Moneret-Vautrin, D., Wuthrich,B., 1995. Adverse reactions to food. European academy of

allergology and clinical immunology subcommittee. Allergy

50, 623–635.

Chehade, M., 2007. IgE and non-IgE-mediated food allergy:

treatment in 2007. Curr. Opin. Allergy Clin. Immunol. 7,

264–268.

Engelmann, P.A., Beyer, K., Wesley Burks, A., Lack, G.,

Liacouras, C.A., Hourihane, J.O., Sampson, H.A., Soderg-

ren, E., 2008. New visions for food allergy: an iPAC

summary and future trends. Pediatr. Allergy Immunol.

(Suppl. 19), 26–39.

Faria, A., Weiner, H., 2005. Oral tolerance. Immunol. Rev.

206, 232–259.

Huby, R., Dearman, R., Kimber, I., 2000. Why are some

proteins allergens? Toxicol. Sci. 55, 235–246.

Husby, S., Mestecky, J., Moldoveanu, Z., Holland, S., Elson,

C., 1994. Oral tolerance in humans. T cell but not B

cell tolerance after antigen feeding. J. Immunol. 152,

4663–4670.

Johansson, S.G., Hourihane, J.O., Bousquet, J., Bruijnzeel-

Koomen, C., Dreborg, S., Haahtela, T., Kowalski, M.L.,

Mygind, N., Ring, J., van Cauwenberge, P., van Hage-

Hamsten, M., Wuthrich, B., 2001. EAACI (the European

academy of allergology and cinical immunology) nomen-

clature task force. A revised nomenclature for allergy. An

EAACI position statement from the EAACI nomenclature

task force. Allergy 56, 813–824.

Kaminogawa, S., Nanno, M., 2004. Modulation of immune

functions by foods. Evid-Based Complement. Altern. Med.

1, 241–250.

Kraus, T.A., Toy, L., Chan, L., Childs, J., Cheifetz, A.,

Mayer, L., 2004a. Failure to induce oral tolerance in

Cronh’s and ulcerative colitis patients: possible genetic risk.

Ann. NY Acad. Sci. 20, 225–238.

Kraus, TA., Toy, L., Chan, L., Childs, J., Mayer, L., 2004b.

Failure to induce oral tolerance to a soluble protein in

patients with inflammatory bowel disease. Gastoenterology

126, 1771–1778.

Kuchroo, V., Das, M., Brown, J., Ranger, A., 1995. B7-1 and

B7-2 costimulatory molecules activate differentially the

Th1/Th2 developmental pathways: application to autoim-

mune disease therapy. Cell 80, 707–718.

Mayer, L., 2005. Mucosal immunity. Immunol. Rev. 206, 5–21.

Mayer, L., Shao, L., 2004. Therapeutic potencial of oral

tolerance. Nat. Rev. 4, 1–14.

Murch, S., 2000. The immunologic basis for intestinal food

allergy. Curr. Opin. Gastroenterol. 16, 552–557.

Neurath, M., Finotto, S., Glimcher, L., 2002. The role of

Th1/Th2 polarization in mucosal immunity. Nat. Med. 8,

567–573.

Nowak-Wegrzyn, A., 2003. Future approaches to food allergy.

Pediatrics 111, 1672–1680.

Ortolani, C., Bruijnzeel-Koomen, C., Bengtsson, U., Bindslev-

Jensen, C., Bjorksten, B., Høst, A., Ispano, M., Jarish, R.,

Madsen, C., Nekam, K., Paganelli, R., Poulsen, L.K.,

Wuthrich, B., 1999. Controversial aspects of adverse

reactions to food. European academy of allergology and

clinical immunology (EAACI) reactions to food subcom-

mittee. Allergy 54, 27–45.

Sampsom, J., Van Berkel, L., Van Helvoort, J., Unger, W.,

Jansen, W., Thepen, T., Mebius, R., Verbeek, S., Kraal, G.,

2005. FcgRIIB regulates nasal and oral tolerance: a role for

dendritic cells. J. Immunol. 174, 5279–5287.

ARTICLE IN PRESSP.O. Paschoal et al. / Immunobiology 214 (2009) 269–278 277


Scott, H., Sampson, H., 2006. Food allergy. J. Allergy Clin.

Immunol. 117, 470–475.

Strid, J., Hourihane, J., Kimber, I., Callard, R., Strobel, S.,

2005. Epicutaneous exposure to peanut protein prevents

oral tolerance and enhances allergic sensitization. Clin.

Exp. Allergy 35, 757–766.

Teixeira, G., 2003. Um Modelo Murino de InflamacaoIntestinal Cronica Tolerancia e Imunizacao Oral: antigeni-

cidade versus Temporalidade. Universidade Federal Flu-

minense, Niteroi, p. 213.Teixeira, G., Antunes, D., Castro Jr., A., Campos, S.,

Pedruzzi, M., Paschoal, P., Costa, J., Araujo, V.,

Andrade, L., Moreno, S., Caldas, L., Cardoso, G.,

Nobrega, A., 2008a. Induction of an antigen specific gut

inflammatory reaction in mice and rats: a model for

human inflammatory bowel disease. Braz. Arch. Biol.

Technol. (in press).

Teixeira, G., Paschoal, P., Oliveira, V., Pedruzzi, M., Campos,

S., Andrade, L., Nobrega, A., 2008b. Diet selection in

immunologically manipulated mice. Immunobiology 213,

1–12.

Vaz, N., Pordeus, V., 2005. Visita a imunologia. Arq. Bras.

Cardiol. 85, 350–362.

Worbs, T., Bode, U., Yan, S., Hoffmann, M., Hintzen, G.,

Bernhardt, G., Foster, R., Pabst, O., 2006. Oral tolerance

originates in the intestinal immune system and relies on

antigen carriage by dendritic cells. J. Exp. Med. 203,

519–527.


Food allergy/hypersensitivity: Antigenicity or timing?

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