Sublingual Omp16-driven redirection of the allergic intestinal response in a pre-clinical model of food allergy.

BACKGROUND: IgE-mediated food allergy remains a significant and growing worldwide problem. Sublingual immunotherapy (SLIT) shows an excellent safety profile for food allergy, but the clinical efficacy needs to be improved. This study assessed the effects of the Toll-like receptor 4 agonist outer membrane protein (Omp) 16 from Brucella abortus combined with cow´s milk proteins (CMP) through the sublingual route to modulate cow's milk allergy in an experimental model. METHODS: Mice sensitized with cholera toxin and CMP were orally challenged with the allergen to elicit hypersensitivity reactions. Then, mice were treated with a very low amount of CMP along with Omp16 as a mucosal adjuvant, and finally, animals were re-exposed to CMP. Systemic and mucosal immune parameters were assessed in vivo and in vitro. RESULTS: We found that the sublingual administration of Omp16 + CMP induced a buccal Th1 immune response that modulated the intestinal allergic response with the suppression of symptoms, reduction of IgE and IL-5, and up-regulation of IgG2a and IFN-γ. The adoptive transfer of submandibular IFN-γ-producing α4ß7+ CD4+ and CD8+ cells conferred protection against allergic sensitization. The use of Omp16 + CMP promoted enhanced protection compared to CMP alone. CONCLUSION: In conclusion, Omp16 represents a promising mucosal adjuvant that can be used to improve the clinical and immune efficacy of SLIT for food allergy.

Many Patients With Irritable Bowel Syndrome Have Atypical Food Allergies Not Associated With Immunoglobulin E.

BACKGROUND & AIMS: Confocal laser endomicroscopy (CLE) is a technique that permits real-time detection and quantification of changes in intestinal tissues and cells, including increases in intraepithelial lymphocytes and fluid extravasation through epithelial leaks. Using CLE analysis of patients with irritable bowel syndrome (IBS), we found that more than half have responses to specific food components. Exclusion of the defined food led to long-term symptom relief. We used the results of CLE to detect reactions to food in a larger patient population and analyzed duodenal biopsy samples and fluid from patients to investigate mechanisms of these reactions. METHODS: In a prospective study, 155 patients with IBS received 4 challenges with each of 4 common food components via the endoscope, followed by CLE, at a tertiary medical center. Classical food allergies were excluded by negative results from immunoglobulin E serology analysis and skin tests for common food antigens. Duodenal biopsy samples and fluid were collected 2 weeks before and immediately after CLE and were analyzed by histology, immunohistochemistry, reverse transcription polymerase chain reaction, and immunoblots. Results from patients who had a response to food during CLE (CLE+) were compared with results from patients who did not have a reaction during CLE (CLE-) or healthy individuals (controls). RESULTS: Of the 108 patients who completed the study, 76 were CLE+ (70%), and 46 of these (61%) reacted to wheat. CLE+ patients had a 4-fold increase in prevalence of atopic disorders compared with controls (P = .001). Numbers of intraepithelial lymphocytes were significantly higher in duodenal biopsy samples from CLE+ vs CLE- patients or controls (P = .001). Expression of claudin-2 increased from crypt to villus tip (P < .001) and was up-regulated in CLE+ patients compared with CLE- patients or controls (P = .023). Levels of occludin were lower in duodenal biopsy samples from CLE+ patients vs controls (P = .022) and were lowest in villus tips (P < .001). Levels of messenger RNAs encoding inflammatory cytokines were unchanged in duodenal tissues after CLE challenge, but eosinophil degranulation increased, and levels of eosinophilic cationic protein were higher in duodenal fluid from CLE+ patients than controls (P = .03). CONCLUSIONS: In a CLE analysis of patients with IBS, we found that more than 50% of patients could have nonclassical food allergy, with immediate disruption of the intestinal barrier upon exposure to food antigens. Duodenal tissues from patients with responses to food components during CLE had immediate increases in expression of claudin-2 and decreases in occludin. CLE+ patients also had increased eosinophil degranulation, indicating an atypical food allergy characterized by eosinophil activation.

Oral exposure to the free amino acid glycine inhibits the acute allergic response in a model of cow's milk allergy in mice.

The conditionally essential amino acid glycine functions as inhibitory neurotransmitter in the mammalian central nervous system. Moreover, it has been shown to act as an anti-inflammatory compound in animal models of ischemic perfusion, post-operative inflammation, periodontal disease, arthritis and obesity. Glycine acts by binding to a glycine-gated chloride channel, which has been demonstrated on neurons and immune cells, including macrophages, polymorphonuclear neutrophils and lymphocytes. The present study aims to evaluate the effect of glycine on allergy development in a cow's milk allergy model. To this end, C3H/HeOuJ female mice were supplemented with glycine by oral gavage (50 or 100 mg/mouse) 4 hours prior to sensitization with cow's milk whey protein, using cholera toxin as adjuvant. Acute allergic skin responses and anaphylaxis were assessed after intradermal allergen challenge in the ears. Mouse mast cell protease-1 (mMCP-1) and whey specific IgE levels were detected in blood collected 30 minutes after an oral allergen challenge. Jejunum was dissected and evaluated for the presence of mMCP-1-positive cells by immunohistochemistry. Intake of glycine significantly inhibited allergy development in a concentration dependent manner as indicated by a reduction in; acute allergic skin response, anaphylaxis, serum mMCP-1 and serum levels of whey specific IgE. In addition, in-vitro experiments using rat basophilic leukemia cells (RBL), showed that free glycine inhibited cytokine release but not cellular degranulation. These findings support the hypothesis that the onset of cow's milk allergy is prevented by the oral intake of the amino acid glycine. An adequate intake of glycine might be important in the improvement of tolerance against whey allergy or protection against (whey-induced) allergy development.

Interleukin-13 suppresses interleukin-10 via inhibiting A20 in peripheral B cells of patients with food allergy.

The regulatory B cells (Breg) are important in the body immunity. The differentiation process of Breg is not fully understood yet. Ubiquitin A20 has immune regulatory functions. This study aims to investigate the role of A20 in the regulation of interleukin (IL)-10 in B cells. In this study, B cells were isolated from the peripheral blood samples of healthy subjects and patients with food allergy (FA). The B cells were analyzed by flow cytometry, real time RT-PCR, Western blotting and chromatin immunoprecipitation. We observed that the frequency of Breg and the levels of A20 in B cells were markedly lower in FA patients than in healthy controls. In vitro deletion of A20 compromised the expression of IL-10. B cells in FA patients showed higher levels of histone deacetylase (HDAC)-11 than in healthy subjects. Exposure to IL-13 in the culture induced high levels of HDAC11 in B cells. IL-13 also repressed the expression of A20 in B cells, in which HDAC11 played a critical role via inducing the chromatin remoldeling at the IL-10 promoter locus. Mice with A20-deficient B cells are prone to FA. In summary, ubiquitin A20 can increase the IL-10 expression in B cells, which can be affected by the IL-13-induced HDAC11. To inhibit HDAC11 may have therapeutic potential for FA.

Mesenteric IL-10-producing CD5+ regulatory B cells suppress cow's milk casein-induced allergic responses in mice.

Food allergy is a hypersensitive immune reaction to food proteins. We have previously demonstrated the presence of IL-10-producing CD5(+) B cells and suggested their potential role in regulating cow's milk casein allergy in humans and IgE-mediated anaphylaxis in mice. In this study, we determined whether IL-10-producing CD5(+) regulatory B cells control casein-induced food allergic responses in mice and, if so, the underlying mechanisms. The induction of oral tolerance (OT) by casein suppressed casein-induced allergic responses including the decrease of body temperature, symptom score, diarrhea, recruitment of mast cells and eosinophils into jejunum, and other biological parameters in mice. Notably, the population of IL-10-producing CD5(+) B cells was increased in mesenteric lymph node (MLN), but not in spleen or peritoneal cavity (PeC) in OT mice. The adoptive transfer of CD5(+) B cells from MLN, but not those from spleen and PeC, suppressed the casein-induced allergic responses in an allergen-specific and IL-10-dependent manner. The inhibitory effect of IL-10-producing CD5(+) B cells on casein-induced allergic response was dependent on Foxp3(+) regulatory T cells. Taken together, mesenteric IL-10-producing regulatory B cells control food allergy via Foxp3(+) regulatory T cells and could potentially act as a therapeutic regulator for food allergy.

Histopathologic findings in children diagnosed with cow's milk protein allergy.

BACKGROUND: Cow's milk protein allergy is the most common cause of food allergy. The challenge test, either open or doubled-blind with a placebo control, is regarded as the criterion standard. Endoscopy and histologic findings are considered a method that can aid in the diagnosis of this entity. AIMS: The aim of this study was to describe the histopathologic findings in children suspected of cow's milk protein allergy that were seen at our hospital. MATERIAL AND METHODS: A descriptive, observational study was conducted on 116 children clinically suspected of presenting with cow's milk protein allergy that were seen at the Department of Gastroenterology and Nutrition of the Instituto Nacional de Pediatría. Upper endoscopy and rectosigmoidoscopy with biopsies were performed and the findings were described. RESULTS: Of the 116 patients, 64 (55.17%) were girls and 52 (44.83%) were boys. The rectum was the site with the greatest presence of eosinophils per field in both groups, followed by the duodenum. In general, more than 15 eosinophils were found in 46% of the patients. CONCLUSIONS: Between 40 and 45% of the cases had the histologic criterion of more than 15 to 20 eosinophils per field and the rectosigmoid colon was the most affected site. Therefore, panendoscopy and rectosigmoidoscopy with biopsy and eosinophil count are suggested.

The degree of whey hydrolysis does not uniformly affect in vitro basophil and T cell responses of cow's milk-allergic patients.

BACKGROUND: Several studies investigated whether hydrolysed proteins can induce tolerance to cow's milk (CM) in children at risk of developing CM allergy. Due to methodological problems and inconsistent findings, the evidence for a tolerogenic effect is limited. A major problem is that different hydrolysates may give different outcomes due to variations in their production and composition. OBJECTIVE: The aim of the study was to investigate the effect of the degree of hydrolysis on the allergenicity and immunogenicity of whey hydrolysates. METHODS: The hydrolysis of whey was stopped at different time-points between 1 and 60 min. In 18 CM allergic patients, the allergenicity of the hydrolysates was determined by immunoblot and the basophil activation test. To test immunogenicity, CM-specific T cell lines were generated. RESULTS: In most patients, increasing time of hydrolysis decreased IgE recognition and basophil activation. However, in five patients, hydrolysed proteins induced more basophil activation than non-hydrolysed proteins. The immunoblot data indicated that these patients recognized either a 25- to 30-kDa degradation product of casein or a 10-kDa degradation product of whey. Although T cell activation was decreased in all patients over time, half of them still showed a positive response to the proteins after 60 min of hydrolysis. CONCLUSION: Increasing the time of hydrolysis reduces both allergenicity and immunogenicity of whey hydrolysates in most but not all patients. This indicates that not the degree of hydrolysis is decisive but the presence and stability of IgE and T cell epitopes in the hydrolysate recognized by individual patients.

Association of allergen-specific regulatory T cells with the onset of clinical tolerance to milk protein.

BACKGROUND: About 70% of children with milk allergy tolerate extensively heated milk (HM) products and outgrow their allergy earlier than those who react to HM. OBJECTIVE: To test the hypothesis that HM-tolerant children have a higher precursor frequency of adaptive allergen-specific regulatory T (Treg) cells. METHODS: Allergic, HM-tolerant, outgrown, or control subjects were defined by oral food challenge. PBMCs were cultured with purified caseins and controls for 7 days, and proliferating CD25(+)CD27(+) Treg cells were identified by flow cytometry. Proliferating cells were also characterized for their expression of FoxP3, CTLA 4, CD45RO, and CD127. Allergen-specific Treg cell origin and function were assessed by depletion of CD25(hi) cells before culture. RESULTS: There was a higher percentage (median [25th% to 75th%], 16.85% [7.1-31.7]) of proliferating allergen-specific CD25(+)CD27(+) T cells from cultures of HM-tolerant subjects (n = 18) than subjects with allergy (n = 8; 4.91% [2.6-7.5]; P < .01). Control subjects with no history of milk allergy (n = 7) also had low percentages of these cells (2.9% [2.4-6.0]), whereas outgrown subjects (n = 7) had intermediate percentages (9.0% [2.7-16.4]). There were no significant differences between the patient groups in the frequency of polyclonal Treg cells or allergen-specific effector T cells. Allergen-specific Treg cells were found to be FoxP3(+)CD25(hi)CD27(+), cytotoxic T lymphocyte-associated antigen 4(+), CD45RO(+)CD127(-) and were derived from circulating CD25(hi) T cells. Depletion of the CD25(hi) cells before in vitro culture significantly enhanced allergen-specific effector T-cell expansion. CONCLUSION: A higher frequency of milk allergen-specific Treg cells correlates with a phenotype of mild clinical disease and favorable prognosis.

Hypogammaglobulinaemia secondary to cow-milk allergy in children under 2 years of age.

Symptomatic hypogammaglobulinaemia in children younger than 2 years of age was studied to rule out a primary immunodeficiency. Thirty-four patients were referred to the Immunology Service to study the hypogammaglobulinaemia-associated clinical picture. Food allergy was documented in 10 patients by personal and familial history, presence of specific immunoglobulin E (IgE) and elevated total serum IgE levels. Coeliac disease and human immunodeficiency virus infection were also ruled out. Protein loss through stools was assessed by clearance of alpha1-antitrypsin (AAT). Serum immunoglobulin levels were determined by nephelometry and functional antibodies were studied by enzyme-linked immunosorbent assay. The cellular immune response was assessed by in vitro lymphocyte proliferation in response to mitogens and cell subsets were analysed by flow cytometry. In five patients of the 10 patients we suspected a protein loss through the mucosa. Four of these five patients showed an increased AAT and the other showed an extensive cutaneous lesion. Immunological studies revealed normal antibody function, in vitro lymphoproliferative responses and cell numbers in four of the 5 patients. One patient showed abnormally low numbers of CD4(+) T cells as well as a defective proliferative response to mitogens. After diagnosis of cow milk allergy, milk was replaced with infant milk formula containing hydrolysed proteins. Recovery of immunoglobulin values and clinical resolution were achieved. Hypogammaglobulinaemia during early childhood in some children may be secondary to cow milk allergy, and immunoglobulins and cells may leak through the inflamed mucosa. Resolution of symptoms as well as normalization of immunoglobulin values may be easily achieved by avoidance of the offending allergen.

Interaction among human leucocyte antigen-peptide-T cell receptor complexes in cow's milk allergy: the significance of human leucocyte antigen and T cell receptor-complementarity determining region 3 loops.

BACKGROUND: Allergic individuals respond to only a few specific antigens, therefore allergic diseases are characterized by antigen specificity. Clarification of the mechanism of antigen specificity will lead to progress in the therapy of allergic diseases. OBJECTIVES: The purpose of this study is to determine the specific association among T cell epitopes, antigen-presenting molecules and T cell receptor (TCR), and to determine the TCR usage in the pathogenesis of allergies using antigen-specific T cell clones (TCCs). The results can clarify the mechanism of the antigen specificity of allergic diseases, and provide new therapeutic possibilities using analogue peptides. METHODS: Short-term T cell clones specific to beta-lactoglobulin (BLG) were established from peripheral blood mononuclear cells (PBMCs) collected from five patients allergic to cow's milk. We then identified the T cell epitopes and antigen-presenting molecules, and examined TCR usage. We also determined the sequence of the TCR-complementarity-determining region 3 (CDR3). RESULTS: Six TCCs established from the five patients recognized three different peptides, and BLGp97-117 was recognized by four of the six TCCs. BLGp101-112 (KYLLFCMENSAE) was the core sequence in the fragment. Sequence analysis of TCR by the RT-PCR method revealed a marked heterogeneity in TCR usage, and similar amino acid sequences were recognized in the CDR3 region. Four of the six TCCs recognized BLG in association with human leucocyte antigen (HLA)-DRB1*0405 as antigen-presenting molecules. CONCLUSION: We proposed the motif of the interaction between the HLA-DRB1*0405 allele and antigen peptide, and suggested that HLA-DRB1*0405 is an immunoregulatory gene product for T cell responses to BLG.

Fecal tumor necrosis factor alpha, eosinophil cationic protein and IgE levels in infants with cow's milk allergy and gastrointestinal manifestations.

Infants with atopic eczema exhibit a specific fecal protein pattern after oral challenge with cow's milk, characterized by an increase in both eosinophil cationic protein (ECP) and tumor necrosis factor (TNF)alpha. The aim of our study was to determine the pattern of these proteins in allergic infants with intestinal manifestations. TNFalpha, ECP and immunoglobulin E (IgE) were measured in stools from 13 infants with intestinal symptoms and 10 healthy infants. The allergic infants underwent two stool collections, one before a cow's milk challenge and the other after the challenge, either at the onset of clinical manifestations (n=6) or 15 days after the challenge if no clinical manifestations occurred (n=7). Baseline TNFalpha, ECP and IgE levels were low in all infants. The concentration of TNFalpha increased after the challenge in infants positive to challenge (p<0.05) but not in those negative to challenge. ECP and IgE levels remained low after the challenge in all the allergic infants. These data confirm that fecal TNFalpha and ECP levels indicate various reaction types of food allergy and that different immunologic disturbances lead to atopic eczema or intestinal symptoms during food allergy. Fecal protein pattern can thus be a useful tool in diagnosing food allergy in infants with intestinal manifestations.

Effect of terfenadine on TNF alpha release from peripheral blood mononuclear cells during cow's milk allergy.

BACKGROUND: In infants with cow's milk allergy and intestinal symptoms, peripheral blood mononuclear cells stimulated in vitro with cow's milk proteins, secrete large amounts of the proinflammatory cytokine TNF alpha thus altering intestinal barrier capacity. Terfenadine, an antihistaminic drug, inhibits the release of several inflammatory mediators, including histamine, prostaglandins and leukotrienes. OBJECTIVES: To test the potential ability of terfenadine to inhibit TNF alpha secretion by mononuclear cells from infants with cow's milk allergy. METHODS: Mononuclear cells from infants allergic to cow's milk proteins were stimulated in vitro for 6 days by a mixture of milk proteins (beta-lactoglobulin, alpha-lactalbumin and casein) with or without terfenadine (0.1-1 microM) and culture supernatants were assayed for TNF alpha by enzyme immunoassay. The effect of culture supernatants on intestinal barrier capacity was evaluated by measuring the electrical resistance (index of integrity) of filter-grown HT29-19 A intestinal cells in Ussing chambers. RESULTS: During active cow's milk allergy, mononuclear cells stimulated with cow's milk proteins secreted large amounts of TNF alpha which significantly reduced the electrical resistance of HT29-19 A intestinal cells. There was a dose-dependent decrease in TNF alpha secretion in the presence of terfenadine, with a maximal inhibition of 62% of this secretion at 1 microM. Accordingly, terfenadine-treated mononuclear cells supernatants did not alter the electrical resistance of intestinal HT29.19 A cells. CONCLUSION: These results indicate that in infants with intestinal dysfunction due to cow's milk allergy, terfenadine is a potent inhibitor of the TNF alpha secretion induced by sensitizing milk protein antigens. This inhibition prevents the degradation of intestinal function as measured in an intestinal cell line, in vitro.

In vitro hydrolysis of gliadin and casein peptides: secondary defect in coeliac disease shown by organ culture.

BACKGROUND: Small-intestinal organ culture was used as an in vitro model of coeliac disease, studying biopsy specimens from patients with coeliac disease, cow's milk allergy, and controls. METHODS: Organ culture incubations were done using the pure gliadin peptide B3144 (amino acid sequences 3-56 of alpha-type gliadins) and a control peptide from casein (amino acid sequences 152-193 of alpha s1-casein). The importance of using negative controls was stressed by non-specific tissue damage. By reversed-phase high-performance liquid chromatography of organ culture supernatants, 27 specimens were further investigated. RESULTS: There was good retrieval of peptide calibration peaks after culture. Qualitative and quantitative evaluation of chromatography runs showed degradation of at least 29% of B3144 and 37% of Cas-P. Normal mucosa (controls and coeliac patients on a gluten-free diet) was able to hydrolyse peptide fractions completely, whereas incubation with damaged mucosa (coeliac disease, cow's milk allergy) left initial peptides. CONCLUSION: It is concluded, using a pure single gliadin peptide, that deficient peptide hydrolysis in coeliac disease was a secondary event.

Jejunal response to beta-lactoglobulin in infants with cow's milk allergy.

Weaning is a transient period of life during which maternal proteins are replaced by foreign proteins. Concomitantly, in early postnatal life, both digestive and immune systems undergo a maturation process. Allergy to cow's milk protein may develop in human infants during weaning, determining digestive, respiratory, cutaneous or systemic symptoms. We studied the intestinal response to bovine milk beta-lactoglobulin (beta-LG) in infants with cow's milk allergy, first during the active phase, and then during the symptom-free stage. During the active phase, transepithelial transport of the beta-LG across the intestinal epithelial layer stimulated the sensitized subepithelial immune cells. This stimulation induced a rise in short-circuit current suggestive of an electrogenic chloride secretion and impaired protein handling by the epithelium. These findings underline the dual role of the epithelial layer in intestinal function: on one hand, it takes an active part in allowing dietary antigens to stimulate the submucosal system, and on the other hand becomes the target for mediators involved in food allergy.

Mononuclear cells from infants allergic to cow's milk secrete tumor necrosis factor alpha, altering intestinal function.

BACKGROUND/AIMS: Intestinal dysfunction observed during cow's milk allergy (CMA) is incompletely understood, and neither the effector cells nor the mediators responsible have been clearly identified. This study was undertaken to better characterize the implication of mononuclear cells in food allergy. METHODS: Peripheral blood mononuclear cells (PBMC) from infants with CMA were cultured in the presence of cow's milk proteins (CMP), and the release of tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), and interleukin 4 and 6 was measured. The effect of culture supernatants was tested on HT29 cl.19A intestinal cell monolayers mounted in Ussing chambers. RESULTS: When stimulated by CMP, PBMC from infants with CMA released more TNF-alpha than those from control infants (429 +/- 92 vs. 205 +/- 34 pg/mL). Culture supernatants did not directly stimulate electrogenic chloride secretion by HT29 cl.19A cells, but epithelial barrier capacity was altered as shown by the significant decrease in electrical resistance (85 +/- 17 vs. 135 +/- 14 omega.cm2 in controls) and the increases in intact horseradish peroxidase, [14C]mannitol, and 22Na+ fluxes. These effects were reversed when culture supernatants were neutralized with anti-TNF-alpha antibodies. Recombinant human-TNF-alpha altered the HT29 cl.19A epithelial barrier capacity, and its effect was highly potentiated by IFN-gamma. CONCLUSIONS: These results indicate that during CMA, the high level of TNF-alpha released by mononuclear cells after milk protein challenge acts synergistically with IFN-gamma to increase the intestinal permeability.