Food allergy, the pathogenesis of which is still unknown, has become a serious public health issue, as its prevalence has increased over the last two decades and around 10% of children are affected [1
]. Probiotics are live microorganisms that confer a health benefit on the host when administered in adequate amounts [2
]. Currently, the Food Allergy and Anaphylaxis Guidelines of the European Academy of Allergy and Clinical Immunology state “there is no evidence to recommend prebiotics or probiotics or other dietary supplements based on particular nutrients to prevent food allergy” [3
]. The number of reports on both basic experiments and clinical trials designed to clarify the effects of probiotics or gut microbiota on allergic disorders has been increasing [4
]. For instance, Clostridium butyricum
significantly ameliorates intestinal anaphylaxis symptoms in mice with food allergy [13
]. Pre- and postnatal Lactobacillus reuteri
supplementation decreases allergen responsiveness in infancy [14
]. Although these probiotics (C. butyricum
and L. reuteri
) have been widely used for their expected health benefits, evidence for their effectiveness against food allergy is still insufficient.
The intestinal barrier, which acts as both a mechanical and a microbial barrier, plays an important role in the development of immune tolerance to prevent allergens passing through the intestinal epithelia from the external environment [15
]. Thus, increased intestinal permeability is thought to be associated with the pathogenesis of food allergy. Recently, it has been reported that the intestinal microbiota contributes to the organization of epithelial barrier function, and changes the bacterial community linked to intestinal permeability and chronic gastrointestinal disease, especially food allergy [17
The aim of the present study was to explore the mechanism responsible for changes in the morphology and function of the intestinal barrier using a juvenile–young adult rat model of food sensitization, focusing on the contribution of intestinal microbiota and the effects of probiotics (C. butyricum
and L. reuteri
) and antibiotics. In this context, previous fragmentary reports have indicated that in ovalbumin (OVA)-sensitized rats, intestinal permeability is increased in association with damage to mucosal tight junctions (TJs) and expression of molecules involved in TJ regulation [19
], or that probiotics decrease Th2 responses and improve intestinal barrier function [20
]. However, the mechanisms underlying the relationship between intestinal epithelial barrier function and regulation of gut microbiota in the context of food allergy have not been precisely clarified, and further accumulation of scientific data is needed. With this aim, we have been studying the use of different types of probiotics and antibiotic treatments in OVA-sensitized rats. Here, we conducted a comprehensive investigation of serum-IgE, gut permeability, ultrastructural features, previously unreported TJ-associated proteins (e.g., claudin-1, -3, -5, and -7), Th2 cytokines, and fecal microbiota, and evaluated the relationships among them. As the results, the TJs of OVA-sensitized rats were disrupted, whereas rats that received probiotics were only mildly affected. The sensitized rats also showed a reduction in both TJ-related proteins expression and localization. Clostridiaceae were increased by probiotics administration, especially Alkaliphilus
, relative to the ovalbumin-sensitized group. Thus, gut microbiota appears to play a role in regulating TJ proteins leading to barrier function.
2. Materials and Methods
2.1. Animal Handling and Study Design
Four-week-old male Brown Norway SPF rats were obtained from Charles River Laboratories (Tokyo, Japan). They were housed under specific pathogen-free conditions at 23 (±3) °C with a 12 h light/dark cycle and a relative humidity of 30%–70% during experiments, and were provided with conventional food and water ad libitum. Protocols for all animal studies were approved by the Institutional Animal Experiment Committee of Jichi Medical University (Approval Number: 17082-01), in accordance with the Institutional Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science, and Technology. Rats were divided into an antigen sensitized group and a control group. In the sensitized group, 1 mg of OVA (Worthington Biochemical Corp, Lakewood, NJ, USA) in 1 mL of PBS was administrated intragastrically daily for 48 days without the use of an adjuvant, and the control group was administered 1 mL of PBS in the same way. Each group was further subdivided into one receiving antibiotics for ablation of intestinal bacteria and one receiving probiotics (C. butyricum
and L. reuteri
). On day 49, all OVA-sensitized rats were then orally challenged with OVA (100 mg) solution in 1 mL of PBS [19
]. On day 50, the intestine and blood were collected from every rat (n
= 5–7 rats per group: PBS only; 6, PBS + antibiotics (Abx); 5, PBS + C. butyricum
(CB); 5, PBS + L. reuteri
(LR); 5, OVA only; 7, OVA + Abx; 7, OVA + CB; 7, OVA + LR; 6) (Figure 1
2.2. Ablation of Intestinal Flora by Antibiotic Treatment
Antibiotic treatment was performed in accordance with a previous report with some modification [21
]. Briefly, amphotericin-B (Fuji Pharma, Tokyo, Japan) was administered by gavage at 1 mg/kg every 12 hours before the start of the experiment. From day zero, water flasks freely available to the rats were supplemented with 1 g/L ampicillin (Astellas Pharma, Tokyo, Japan), and an antibiotic cocktail consisting of 50 mg/kg vancomycin (Shionogi Pharma, Tokyo, Japan), 100 mg/kg kanamycin (Meiji Seika Pharma, Tokyo, Japan), 100 mg/kg metronidazole (Shionogi Pharma, Tokyo, Japan), and 1 mg/kg amphotericin-B was administered by antibiotic gavage every 12 hours. A gavage volume of 10 mL/kg body weight was delivered via a gastric tube without prior sedation. The antibiotic cocktail was prepared freshly every day, and ampicillin and water were renewed every seventh day (Figure 1
2.3. Probiotic Treatment
Probiotic treatment groups comprising an antigen sensitized group and a control group were further divided into three subgroups. The probiotics (C. butyricum
[MIYAIRI 588®, Miyarisan Pharmaceutical Co., Ltd., Tokyo, Japan] at 1 × 108
CFU/mL, L. reuteri
[DSM 17938, Bio Gaia Japan Co., Ltd., Stockholm, Sweden] at 1 × 109
CFU/mL, respectively; 5–7 rats per group) were administrated respectively by daily gavage with 1 mg OVA in 1 mL PBS for seven weeks (49 days) in the sensitized group. The control group received the same concentrations and dosages of probiotics in 1 mL PBS in the same way (Figure 1
2.4. Measurement of Serum OVA-IgE
Blood was collected from the jugular vein on days 0, 14, 28, and 50 from the start of the experiment. Each sample was allowed to clot for 1 h at room temperature and was then centrifuged at 2000× g (15 min, 4 °C); all sera were stored at −20 °C. The serum OVA-specific IgE was assayed by ELISA in accordance with the manufacturer’s instructions (Cusabio Technology LLC, Houston, TX, USA). The final OD value was detected at 450 nm wavelength using a microplate reader (Benchmark Plus, Microplate Reader, Bio-Rad, USA). We prepared a standard curve by plotting standard concentration on the X-axis and absorbance on the Y-axis, and used it to calculate the IgE concentration of each sample from its absorbance.
2.5. Evaluation of Intestinal Permeability
Intestinal permeability was determined by measuring the lactulose/mannitol ratio in urine samples in each group (days 14, 28, and 50). After a 24 h fast, rats were administrated 100 mg of lactulose and 50 mg of mannitol (dissolved in 1 mL distilled water) orally. The percentage absorption of these sugars was determined from the amount of excreted lactulose and mannitol measured during the first six hours after ingestion, using an EnzyChrom intestinal permeability assay kit (BioAssay Systems, Hayward, CA, USA) in accordance with the manufacturer’s instructions. Any increase in this ratio indicated increased intestinal permeability, as lactulose is only absorbed though intercellular spaces.
2.6. Hematoxylin and Eosin (HE) Staining
On day 50, intestinal samples were collected during deep anesthesia by intraperitoneal injection of Nembutal (Dainippon Sumitomo, Tokyo, Japan). For HE staining, specimens of the jejunum were fixed with 4% paraformaldehyde in a 50 mM phosphate buffer (Wako, Osaka, Japan), pH 7.4, for 24 h at 4 °C and stained with HE after dehydration, embedding, and slicing. The structure and morphological changes were observed and analyzed using a microscope (Olympus, Tokyo, Japan). Villus length was determined by measuring the distance from the crypt base to the villus tip using ImageJ software (Version 1.50, National Institutes of Health, Bethesda, MD, USA). The degree of inflammation was evaluated using an intestinal inflammation scoring system based on the following parameters: (1) inflammatory cell infiltration, (2) damage to the surface epithelium, and (3) irregular villous and crypt loss [22
]. We also counted eosinophil infiltration in the lamina propria of the jejunal mucosa. Five to seven animals from each experimental group were evaluated, and a minimum of 15 well-oriented villi from each section were measured and observed by microscopy.
2.7. Transmission Electron Microscopy
Under deep anesthesia, small pieces (about 1.5 mm × 1.5 mm × 2 mm) of the jejunum were rapidly excised and fixed with 2.5% glutaraldehyde in a 0.1 M phosphate buffer, pH 7.4, for 2 h at 4 °C. The specimens were then post-fixed with 1% osmium tetroxide in a 0.1 M phosphate buffer for 1.5 h at 4 °C. The pieces of the jejunum were then dehydrated in a graded ethanol series, transferred to propylene oxide, embedded in epoxy resin (Quetol 812; Nisshin EM Co., Tokyo, Japan), and polymerized for 48 h at 60 °C. The specimen blocks were cut into ultrathin sections with an ultramicrotome (UCT; Leica Microsystems, Waltzer, Germany), stained with uranyl acetate and lead citrate, after which the structural and morphological changes in epithelial cells and tight junctions (TJs) were examined using a transmission electron microscope (HT7700; Hitachi, Tokyo, Japan). The apical junction length and width of the TJ and adherens junction of jejunum epithelial cells were measured using ImageJ software (Version 1.50, National Institutes of Health, Bethesda, MD, USA). Representative data were obtained from 10–15 measurements per sample (n = 5–7 per samples per group).
2.8. Immunofluorescence Staining
The fixed jejunal samples were immersed in 30% sucrose in 50 mM PBS for two days at 4 °C, embedded in Tissue-Tek OCT compound (Sakura FineTechnical, Tokyo, Japan), and frozen on dry ice. Cryosections (thickness: 4 μm) were obtained using a cryostat (CM3000; Leica Microsystems, Wetzlar, Germany) and blocked with 2% normal goat serum (Vector Laboratories, Burlingame, CA, USA) for 30 min at 30 °C. Tissues were incubated in antibody retrieval buffer with 0.01 M citrate, pH 6.0, for 10 min at 90 °C, and then with primary antibodies against the TJ proteins claudin-1 (Cat#717800), claudin-2 (Cat#51600), claudin-7 (Cat#349100), and zonula occludens (ZO-1) (Cat#617300) [Thermo Fisher Scientific (Waltham, MA, USA)], claudin-3 (SAB4200607), and claudin-5 (SAB4200537) [Sigma-Aldrich (St. Louis, MO, USA)] at 4 °C overnight. After being washed with PBS, the fixed jejunal tissues were incubated with the secondary antibody Alexa-Fluor-488-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min at 30 °C. Nuclei were visualized in Vectashield Hardset Mounting medium with DAPI (Vector Laboratories, CA, USA). Imaging was performed using a confocal laser microscope, FluoViewTM FV1000 (Olympus, Tokyo, Japan), equipped with ×20 and ×40 objective lenses.
2.9. Real-Time PCR
After the rats were sacrificed on day 50, 25–40 mg of jejunal tissue was collected and ground in liquid nitrogen, and then transferred to a 1.5 mL EP tube. The total RNA (n
= 5–7 rats per group) was extracted using TRIzol Reagent (Cat#15596018, Invitrogen, Carlsbad, CA, USA) and reverse-transcribed to cDNA using a Superscript® VILOTM
kit (Invitrogen, Carlsbad, CA, USA). Primers for TJ proteins and cytokines were designed using the Primer-Blast software package (National Center for Biotechnology Information, Rockville, Bethesda, MD, USA) based on the mRNA sequences in GenBank (National Center for Biotechnology Information, Bethesda, MD USA). These primer-sequences are listed in Table 1
. Real-time PCR was performed using ABI 7500 Fast Real-time PCR (ABI 7500; Applied Biosystems, Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) and samples were run in triplicate. The reaction conditions were as follows: 50 °C for 2 min, 95 °C for 2 min for the holding step, followed by 40 cycles of 95 °C for 3 s and 60 °C for 30 s. As an internal control, β-actin was used for standardization of the transcript results, and relative gene expression levels were calculated by the (2−ΔΔCT
) method. We confirmed the relative quantitative PCR (2−ΔΔCT
) values between OVA sensitization rats and non-sensitization rats. We adopted PBS only (control) samples instead of non-sensitization samples because there were no remarkable differences between them.
2.10. Fecal DNA Isolation and 16S rRNA Sequence Analysis
On day 50, feces were collected in a sterile tube filled with 1 mL PBS and then immediately frozen at −80 °C. For isolation of DNA, 100–300 mg of fecal material was ground with silica beads and extracted with a QIAamp DNA stool mini kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s instructions. PCR products for the V4 region of the 16S rRNA gene were amplified with region-specific primers that included the Illumina flowcell adapter (Illumina, San Diego, CA, USA) sequences and 12-base barcodes on the reverse primer. PCR production of the bacterial DNA template was quantified using Invitrogen’s PicoGreen. Taxonomic classification of 16S rRNA targeting amplicon reads and the eight most abundant bacterial sequences was performed using Illumina 16S Metagenomics workflow in the Miseq Reporter software curated by the GreenGene taxonomic database (https://basespace.illumina.com/analyses/
). Alpha diversity was calculated based on the Shannon index for richness and evenness of bacterial sequences at rarefraction depth reads of the operational taxonomic unit sample.
2.11. Statistical Analysis
All group comparisons, except the microbiota analysis, were performed using analysis of variance (ANOVA) with the Tukey–Kramer method for multiple comparison after normality test. The microbiota analysis was performed using permutational multivariate analysis of variance (PERMANOVA) using PRIMER version 7 software (PRIMER-e, New Zealand). Box and whisker plots were created using GraphPad Prism 7 software (GraphPad Software, USA). Differences at p < 0.05 were considered to be statistically significant.
In the present study, using juvenile–young adult rats with food sensitization as a model, we found that not only antigen (OVA) sensitization, but also antibiotic treatment induced leaky gut, causing allergen absorption and food sensitization, presumably through significant expression of key TJ proteins. Moreover, administration of probiotics prevented this increase in intestinal permeability, presumably through an increase in Clostridiaceae, as well as a significant influence on the expression of TJ proteins.
One of the functions of the epithelial barrier is to prevent macromolecular antigens and other harmful substances from being absorbed [23
]. It is reported that intestinal permeability may increase when the intestinal mucosal barrier function is damaged during allergy, and that such epithelial barrier dysfunction leads to excessive transport of macromolecular substances, which are absorbed into the deep tissues and have the potential to induce antigen-related intestinal inflammation or hypersensitivity [25
]. The results of our lactulose/mannitol assay showed that intestinal permeability was increased after OVA sensitization, and this was further confirmed by electron microscopy (shortened and widened intercellular TJs of intestinal villus epithelial cells). These changes in OVA-sensitized rats were not prevented by administration of antibiotics, but were prevented by treatment with probiotics, as further confirmed by electron microscopy. These findings are in line with a previous report indicating that TJ and adherens junction microstructure is markedly disrupted and widened after OVA sensitization and treatment with antibiotics [29
TJs play an important role in the maintenance of intestinal permeability, which is considered to determine selective cellular absorption [30
]. TJs comprise multiple proteins forming a functional complex, and the major function of most TJ proteins is to maintain the integrity of the epithelial barrier. Transmembrane proteins, such as occludin, claudins, and junctional adhesion molecules, are structural proteins arranged in a linear manner, whereas the cytoplasmic adhesion proteins, ZO-1, -2, and -3, form a supporting cytoskeletal structure [32
]. These proteins play a pivotal role in the regulation of TJ permeability and intestinal barrier function [33
]. Our real-time PCR study showed that OVA down-regulated the expression of ZO-1; occludin; and claudin-1, -3, -5, -7, -8, -9, and -15. These findings are in line with a previous similar study of OVA-sensitized rats, which demonstrated significant down-regulation of the TJ-mediating proteins ZO-1 and claudin-8 and -15 [19
]. These results suggest that OVA induces damage to the intestinal barrier, and that TJ permeability is related to the expression and regulation of these proteins. Although a previous report has indicated that elevated expression of claudin-2 in epithelial cells plays an important role in epithelial barrier dysfunction as well as the pathogenesis of intestinal antigen-specific hypersensitivity [39
], we found no significant increase of claudin-2 expression in the present study. This may have been partly owing to the experimental design, as another previous report that adopted a similar experimental design also demonstrated no significant differences in the claudin-2 level between an antigen sensitization group and a control group [19
]. On the other hand, treatment with C. butyricum
and L. reuteri
up-regulated the expression of ZO-1; occludin; and claudin-1, -3, -5, -7, -8, -9, and -15. Similar beneficial effects of probiotics on the association between intestinal permeability and TJ protein expression have been reported. In Bet v1 pollen-sensitized mice, a mixture of Lactobacillus
strains up-regulated the expression of occludin and the TJ molecule ZO-1, and improved the function of the gut epithelial barrier [29
]. In rats treated with dextran sulfate sodium, which has been widely used as a model for inducing acute and chronic colitis, a probiotic mixture of strain VSL#3 was shown to protect against increased intestinal permeability by up-regulating of expression of occludin, ZO-1, and claudins 1–5 [40
Using an OVA-sensitized rat model, we further detected downregulation of the Th2-associated cytokines IL-4 and IL-13, and upregulation of the Th1-associated cytokine INF-γ in the groups administered probiotics, suggesting that probiotics induced an immunoregulatory response. These findings are consistent with previous studies showing that probiotic administration or supplementation exerted beneficial effects by improvement of the Th1/Th2 balance in a mouse model of food allergy [13
]. Regarding the mechanism of tolerance acquisition in infancy, it involves a complex combination of physiological and immunological regulation. One of the factors involved is food antigen degradation in the lumen as the digestive function develops. Others include prevention of the absorption of high-antigenic substances through the development of gut barrier function and secretory IgA production. Organization of the gut epithelium makes it a tight, efficient barrier with filtering properties against the entry of allergens. Secretory IgA-based immune complexes promote the induction of non-inflammatory cytokines, ensuring low reactivity against transported antigens. In addition, regulatory T cells and clonal deletion of antigen-specific T cells, as well as anergy induction, are crucial factors in tolerance [41
]. Our study suggests that probiotics could be advantageous in terms of tolerance because neither gut permeability nor the serum-specific IgE level were increased, and expression of mRNA for IL-4, IL-13, and TNF-α was decreased in the jejunum.
The microbiota of OVA-sensitized rats was significantly more diverse than that of each of the probiotic treatment groups and control (PBS only) group. This is compatible with previous clinical reports that demonstrated that the microbiota of children with food allergy was significantly more diverse than that of healthy controls [42
]. Clostridiaceae were also significantly enriched by treatment with C. butyricum
and L. reuteri
. It was possible to rule out any contamination because the rats were housed in individual ventilation cages to keep them separated from the other rats and any possible exposures, including that via air. Furthermore, we paid careful attention to all technical procedures, agents, and equipment (probiotic solutions or gavage tubes were kept in different places) to avoid contamination.
It is reported that a combination of specific immunotherapy with C. butyricum
significantly enforces the therapeutic effect against food allergen-related inflammation in mouse intestine [12
]. Stefka et al. reported that Clostridium
regulates innate lymphoid cell function to alter gut epithelial permeability and reduce allergen uptake into the systemic circulation [10
]. C. butyricum
can produce butyrate, which is not only the main energy source for enterocyte regeneration [45
], but also an important immunomodulatory molecule in the intestine. Butyrate can stimulate the rearrangement of TJ proteins of epithelial cells and improve gut barrier integrity [46
were significantly increased in the OVA sensitized with L. reuteri
administration group, and the latter was also significantly increased in the OVA sensitized with C. butyricum
administration group in comparison with the OVA only group, respectively. Recently, a reduced Akkermansia muciniphila
level in the gut microbiota of children with allergic asthma has been reported [47
]. Another study demonstrated that A. muciniphila
significantly increased the expression of occludin and claudin-4. Both A. muciniphila
and extracellular vesicles increased the expression of ZO-2 in the cell line. The researchers concluded that A. muciniphila
and its extracellular vesicles might both increase the integrity of the intestinal barrier and reduce inflammation [48
]. A. muciniphila
is a mucus-colonizing member of the gut microbiota that has evolved to specialize in the degradation and utilization of host mucus. Mucus degradation and fermentation by A. muciniphila
are known to result in the production of acetate [49
]. L. reuteri
also produces lactate and acetate, which become directly available to coexisting butyrogenic bacteria, which normally produce butyrate through carbohydrate fermentation or amino acids degradation pathways within the same mucosal niche. Aside from stimulation of the rearrangement of TJ proteins in epithelial cells and improvement of gut barrier integrity, butyrate can stimulate mucus production from epithelial cells [46
]. Therefore, one of the reasons for the increase of Akkermansia
revealed by the fecal microbiota analysis of the L. reuteri
administration group could have been thickening of the mucus layer, leading to enhancement of gut barrier integrity in a way. There is a “coexistence cycle” associated with Akkermansia
, so to speak, and the acceleration of mucus production by butyrate may lead to an increase in the number of Akkermansia
. Similar effects are expected for C. butyricum
, which produces butyrate, although the fecal microbiota analysis revealed no significant differences. On another note, there is little information about gut Alkaliphilus
. Alkaliphilus transvaalensis
is reportedly connected to chitin degradation with production of high short chain fatty acids (SCFAs; acetate, propionate, and butyrate) [50
]. Thus, although speculative, Alkaliphilus
may be integrated into the gut microbiota ecosystem where many microorganism networks are formed, and produce SCFAs.
In our present study, Oscillospira
was significantly enriched in the OVA only group, and reduced in both the C. butyricum
-treated and L. reuteri
-treated OVA groups. A previous report has revealed that dietary intervention with extensively hydrolyzed casein formula supplemented with L. rhamnosus
GG accelerates tolerance acquisition in infants with cow’s milk allergy. Fecal samples from infants with cow’s milk allergy demonstrated that Oscillospira
was reduced in infants that became tolerant, but significantly enriched in those that remained allergic [44
]. Therefore, from the viewpoint of Oscillospira
, it seemed that both C. butyricum
and L. reuteri
played a beneficial role in our study.
At the species level, the probiotics we administered were not detected at all by a fecal microbiota analysis (data not shown). Therefore, it is considered that these probiotics did not colonize the gut. They might have passed through the gut within one day and fallen below the detection sensitivity limit. A previous study has also reported that administered C. butyricum
grew in, but did not colonize the rat intestine because intestinal C. butyricum
cells disappeared (ELISA) three days after administration [51
]. Further investigation will be needed in order to clarify why none of the administered probiotics were detected.
We found that OVA sensitization alone changed the composition of gut microbiota in comparison with controls, in terms of the relative abundance of bacterial phyla. Similarly, Andreassen et al. recently reported that allergen immunization in a food allergy model induced profound changes in the composition of the gut microbiome [52
]. This is considered to indicate that the gut mucosal immune system can affect the composition of gut commensal bacteria. In other words, there is a bidirectional interaction between gut microbiota formation and the mucosal immune system. The gut microbiota signatures could have been affected by OVA sensitization through complex ecosystem effects.
Because the jejunum, where most food absorption occurs in the digestive tract, plays an important role in epithelial barrier function in food allergy, we focused on the jejunum in the present study. However, microbiota composition and immune cell composition vary throughout the intestine, and these regional variations will influence the interactions between them. Therefore, further investigation of different segments of the gastrointestinal tract (ileum, colon, and so on) is needed. We acknowledge that the sample size used in this study was small, and this is the major limitation of our study. Furthermore, despite the use of inbred rodents and standardized maintenance, handling, and exposure to the animals in the same groups, there is considerable inter-individual variation in the immune responses. A previous study demonstrated that some of the immunized experimental animals appeared to be non-responders or low responders to immunization owing to inter-individual variation, even though the animals were brought up in the same environment and treated in the same manner [52
]. Therefore, our results might have been affected by non-immune response bias.
A recent ground-breaking clinical trial demonstrated that the combination of probiotics (L. rhamnosus
) and peanut oral immunotherapy provided a long-lasting clinical benefit and persistent suppression of the allergic immune response in children with peanut allergy [53
]. In that study, the microbial composition of stool samples was not analyzed in order to examine the effects of the combination therapy, and the synergistic action of probiotics with oral immunotherapy was not assessed; however, the findings suggest that probiotics may be able to enhance the tolerance-inducing capacity of oral immunotherapy. Further basic experiments and clinical trials are needed in order to address the important and as-yet-unanswered question of whether or not probiotics provide a significant benefit for the prevention or treatment of food allergy.