Pierre-Henri Benhamou

Epicutaneous immunotherapy on intact skin using a new delivery system in a murine model of allergy

Background Allergen‐specific immunotherapy, subcutaneous immunotherapy (SCIT) or oral, has been used for almost a century to redirect inappropriate immune responses in atopic patients. A new mode of administration through the intact skin [epicutaneous immunotherapy (EPIT)], using an original epicutaneous delivery system, may represent an alternative to these classical methods.

Epicutaneous Immunotherapy Results in Rapid Allergen Uptake by Dendritic Cells through Intact Skin and Downregulates the Allergen-Specific Response in Sensitized Mice

Epicutaneous immunotherapy onto intact skin has proved to be an efficient and safe alternative treatment of allergy in an animal model with various allergens and in children for cow’s milk allergy. The aim of this study was to analyze the different steps of the immunological handling of the allergen when deposited on intact skin using an epicutaneous delivery system and its immune consequences in sensitized BALB/c mice. As expected, when applied on intact skin, OVA exhibits neither a passive passage through the skin nor any detectable systemic delivery. The current study demonstrates that, after a prolonged application on intact skin, OVA is taken up by dendritic cells in the superficial layers of the stratum corneum and transported, after internalization, to the draining lymph nodes, with variations according to the previous level of sensitization of the mice. When OVA is applied with the epicutaneous delivery system repeatedly, specific local and systemic responses are down-modulated in association with the induction of regulatory T cells. Besides providing new insights into skin function in the presence of allergens, this study indicates that the skin might have a tolerogenic role, at least when kept intact.

Epicutaneous Immunotherapy Using a New Epicutaneous Delivery System in Mice Sensitized to Peanuts

Background: Peanut allergy is a life-threatening condition for which new efficient and safe treatment is expected. We evaluated epicutaneous immunotherapy (EPIT) as a new alternative treatment for peanut allergy in sensitized mice. Methods: Sixty BALB/c mice were sensitized by gavages with peanut protein extract (PPE) mixed with cholera toxin. An epicutaneous delivery system, coated with 100 µg PPE (Viaskin®, DBV Technologies, Paris, France), was applied to intact skin every week during 48 h (EPIT; n = 20). This group was compared with sensitized mice treated with subcutaneous immunotherapy (SCIT; n = 20), untreated sensitized mice (sham, n = 20), and naive mice (naive; n = 20). After the 8-week treatment, a histamine release test, airway hyperreactivity measurement by plethysmography, and a resistance-compliance measurement after the challenge were performed. Blood and bronchoalveolar lavage were sampled for serology, cytokines, and cytology. Results: Specific IgE (sIgE) increased after sensitization in the EPIT (0.26 µg/ml) and SCIT (0.21 µg/ml) groups and decreased after treatment (0.09 µg/ml, p < 0.001 and 0.06 µg/ml, p < 0.001, respectively). The IgG1/IgG2a ratio decreased in the EPIT and SCIT groups versus the sham group (3.7; p < 0.001 and 2.7; p < 0.01 and 15.1, respectively). At the higher metacholine concentration, enhanced pause values were lower in the EPIT and SCIT groups than in the sham group (7.29, 6.74, and 10.99, p < 0.01, respectively), and did not differ from that of the naive group (5.06). Resistance-compliance was reversed in the treated groups versus the sham group (p < 0.001). IL-4, IL-5, IL-13, eotaxin, and eosinophils were reduced in the BAL of the EPIT and SCIT groups versus the sham group (p < 0.001). Conclusion: In peanut-sensitized mice, based on biological and physiological responses, EPIT is as efficient as subcutaneous treatment which is the reference method in immunotherapy.

The 13carbon urea breath test for the noninvasive detection of Helicobacter pylori in children: comparison with culture and determination of minimum analysis requirements.

BACKGROUND The purpose of the study was to determine the accuracy of the labelled 13carbon urea breath test for the diagnosis of Helicobacter pylori in children and to simplify the 13carbon urea breath test in identifying the most discriminating sampling time. METHODS H. pylori was searched for in 100 children aged 10.5+/-4.5 years by histology, bacteriological counts, and culture on antral biopsies together with serology and 13carbon urea breath test. Breath samples were obtained before ingestion (T0) of 75 mg urea-13C and every 10 minutes after until T60. 13CO2 excess ratio was measured by isotope ratio mass spectrometry, and values expressed as delta per mil over baseline enrichment (delta 13CO2). The arithmetic mean (Mdelta 13CO2) of T20 to T60 values was calculated and the test considered positive with Mdelta 3CO2 higher than Mdelta 13CO2 + 3 SD as determined in noninfected children. RESULTS Mdelta 13CO2 of noninfected children as assessed by culture was 1.4+/-0.6 per mil, determining a positive cut-off value of 3.44 per mil. Mdelta 13CO2 was correlated in 11 children with biopsy bacteriological counts. Both culture and 13carbon urea breath test were positive in 38 of 100 children, without any discordance. Plotting 13carbon urea breath test results at each sampling time versus Mdelta 13CO2 showed weaker correlations at T20, T30, T50, and T60, than at T40. The two-sample method at T0 and T30, T40, T50, had high sensitivity and specificity. Single-sample analysis obtained at T40 gave a comparable sensitivity and a slightly reduced specificity. CONCLUSION 13carbon urea breath test is sensitive and specific in children. Two samples collected at T0 and T40 provide the most discriminating procedure.

Epicutaneous Immunotherapy (EPIT) Blocks the Allergic Esophago-Gastro-Enteropathy Induced by Sustained Oral Exposure to Peanuts in Sensitized Mice

Background Food allergy may affect the gastrointestinal tract and eosinophilia is often associated with allergic gastrointestinal disorders. Allergy to peanuts is a life-threatening condition and effective and safe treatments still need to be developed. The present study aimed to evaluate the effects of sustained oral exposure to peanuts on the esophageal and jejunal mucosa in sensitized mice. We also evaluated the effects of desensitization with epicutaneous immunotherapy (EPIT) on these processes. Methods Mice were sensitized by gavages with whole peanut protein extract (PPE) given with cholera toxin. Sensitized mice were subsequently exposed to peanuts via a specific regimen and were then analysed for eosinophilia in the esophagus and gut. We also assessed mRNA expression in the esophagus, antibody levels, and peripheral T-cell response. The effects of EPIT were tested when intercalated with sensitization and sustained oral peanut exposure. Results Sustained oral exposure to peanuts in sensitized mice led to severe esophageal eosinophilia and intestinal villus sub-atrophia, i.e. significantly increased influx of eosinophils into the esophageal mucosa (136 eosinophils/mm2) and reduced villus/crypt ratios (1.6±0.15). In the sera, specific IgE levels significantly increased as did secretion of Th2 cytokines by peanut-reactivated splenocytes. EPIT of sensitized mice significantly reduced Th2 immunological response (IgE response and splenocyte secretion of Th2 cytokines) as well as esophageal eosinophilia (50 eosinophils/mm2, p<0.05), mRNA expression of Th2 cytokines in tissue - eotaxin (p<0.05), IL-5 (p<0.05), and IL-13 (p<0.05) -, GATA-3 (p<0.05), and intestinal villus sub-atrophia (2.3±0.15). EPIT also increased specific IgG2a (p<0.05) and mRNA expression of Foxp3 (p<0.05) in the esophageal mucosa. Conclusions Gastro-intestinal lesions induced by sustained oral exposure in sensitized mice are efficaciously treated by allergen specific EPIT.

A Dose Determination Study of Polyethylene Glycol 4000 in Constipated Children: Factors Influencing the Maintenance Dose

Objectives: To determine the doses of polyethylene glycol (PEG) 4000 without additional salts allowing normal bowel habits in childhood functional constipation. Methods: This multicenter noncomparative study allocated children to 4 groups: 6-12 months, 13 months-3 years, 4-7 years, and 8-15 years. Constipation was defined as <1 stool/d for more than 1 month in children aged 6-12 months and <3 stools/w for more than 3 months in older children. Children randomly received either a nominal or a double starting dose. Treatment scheduled for 3 months could be adapted. Data were collected daily by the parents and rated at each visit by the investigator. Results: In the 96 children included, the median (interquartile) effective daily doses were by groups; 3.75 (2.50-5.00) g, 6.00 (4.00-7.43) g, 11.71 (7.00-16.00) g, and 16.00 (16.00-24.00) g, respectively, i.e., around 0.50 g/day/kg with a potential increment of the maintenance dose with higher initial dosages. More children had a final dosage identical to the initial one when started on the nominal dose (73%) than with the double one (42%, P < 0.003). More than 90% of children recovered normal bowel habits. Fecal soiling ceased in >60% of children with this symptom at enrolment. Fecal mass in the rectum and abdominal pain were markedly reduced and appetite improved. Conclusions: A daily dose of PEG 4000 around 0.50 g/day/kg in children aged 6 months to 15 years is effective in more than 90% of constipated children and 60% of those with fecal soiling.

The regulatory T cells induction by epicutaneous immunotherapy is sustained and mediates long‐term protection from eosinophilic disorders in peanut‐sensitized mice

Allergen‐specific immunotherapy favours immune deviation from a Th2 to a Th1 response and increases the number of regulatory T cells (Tregs). Epicutaneous immunotherapy (EPIT) of sensitized mice decreases the clinical and the allergen‐specific Th2 responses and increases local and peripheral Foxp3+ Tregs.

Stimulation by recombinant human growth hormone of growth and development of remaining bowel after subtotal ileojejunectomy in rats.

Summary The impact of human recombinant growth hormone (GH) after massive small bowel resection was studied in 38 weaning female Wistar rats (65 + 5 days old; 193 + 26 g). Animals underwent a 80% small bowel resection, leaving in place similar lengths of jejunum and ileum. Animals were assigned to four groups: group A (n = 9), small bowel resection only; group B (n = 10), resection and treatment with 0.2 GH units (GHU) s.c. every other day; group C (n = 9), resection and 0.4 GHU; and group D (n = 10), laparotomy without intestinal resection. Twenty-eight days later, weight gain (percentage of initial weight) was 1 + 3 in group A, 12 + 8 in group B, 12 + 9 in group C, and 16 + 7 in group D; p < 0.001, groups B-D. Time to recover initial weight was 26.2 + 3.3 days in group A; 11.7 + 5.4 days in group B (p < 0.001); and 16 + 6.1 days in group C (p < 0.001 vs. A). The size of the intestinal remnant after the rats were killed was 1.3 + 0.6 cm (13 + 10% of initial length) in A; 5.15 + 2.4 cm (37 + 18%) in B (p < 0.01); 4.2 + 2.3 cm (33 + 20%) in C (p < 0.01 vs. A); and 4.4 + 3.5 cm (5.8 + 4.9%) in D (p < 0.001 vs A). Villus height and diameter, average number of mitosis per field, and muscular layer and wall thickness were greater in groups A, B, and C than in group D (p < 0.001). Villus height and muscular thickness of the jejunal part were increased in group C compared with A (p < 0.05). In conclusion, GH improves the postoperative intestinal adaptation process in terms of both weight gain and small bowel lengthening.

Intact skin and not stripped skin is crucial for the safety and efficacy of peanut epicutaneous immunotherapy (EPIT) in mice

BackgroundEpicutaneous immunotherapy (EPIT) on intact skin with an epicutaneous delivery system has already been used in preclinical and clinical studies. In epicutaneous vaccination and immunotherapy, the stripping of skin before application of the allergen is suggested to facilitate the passage of allergen through immune cells.ObjectivesThe aim of this study was to compare the immunological response induced by EPIT performed on intact and stripped skin in a mouse model of peanut allergy.MethodsAfter oral sensitization with peanut and cholera toxin, BALB/c mice were epicutaneously treated using an epicutaneous delivery system (Viaskin® (DBV Technologies, Paris) applied either on intact skin or on stripped skin. Following EPIT, mice received an exclusive oral peanut regimen, aimed at triggering esophageal and jejunal lesions. We assessed eosinophil infiltration by histology, mRNA expression in the esophagus, antibody levels and peripheral T-cell response.ResultsEPIT on intact skin significantly reduced Th2 immunological response (IgE response and splenocyte secretion of Th2 cytokines) as well as esophageal eosinophilia (2.7 ± 0.9, compared to Sham 19.9 ± 1.5, p < 0.01), mRNA expression of Th2 cytokines in tissue and intestinal villus sub-atrophia (2.9 ± 0.2 vs Sham, 2.1 ± 0.2, p < 0.05). By contrast, EPIT on stripped skin reinforced Th2 systemic immunological response as well as eosinophil infiltration (26.8 ± 15.1), mRNA expression of Th2 cytokines and duodenal villus/crypt-ratio (2.4 ± 0.3).ConclusionsEpicutaneous allergen-specific immunotherapy needs the integrity of superficial layers of the stratum corneum to warranty safety of treatment and to induce a tolerogenic profile of the immune response.

Epicutaneous immunotherapy induces gastrointestinal LAP+ regulatory T cells and prevents food-induced anaphylaxis

Background: The attempt to induce oral tolerance as a treatment for food allergy has been hampered by a lack of sustained clinical protection. Immunotherapy by nonoral routes, such as the skin, may be more effective for the development of maintained tolerance to food allergens. Objective: We sought to determine the efficacy and mechanism of tolerance induced by epicutaneous immunotherapy (EPIT) in a model of food‐induced anaphylaxis. Methods: C3H/HeJ mice were sensitized to ovalbumin (OVA) orally or through the skin and treated with EPIT using OVA‐Viaskin patches or oral immunotherapy using OVA. Mice were orally challenged with OVA to induce anaphylaxis. Antigen‐specific regulatory T (Treg)‐cell induction was assessed by flow cytometry using a transgenic T‐cell transfer model. Results: By using an adjuvant‐free model of food allergy generated by epicutaneous sensitization and reactions triggered by oral allergen challenge, we found that EPIT induced sustained protection against anaphylaxis. We show that the gastrointestinal tract is deficient in de novo generation of Treg cells in allergic mice. This defect was tissue‐specific, and epicutaneous application of antigen generated a population of gastrointestinal‐homing LAP+Foxp3− Treg cells. The mechanism of protection was found to be a novel pathway of direct TGF‐&bgr;–dependent Treg‐cell suppression of mast cell activation, in the absence of modulation of T‐ or B‐cell responses. Conclusions: Our data highlight the immune communication between skin and gastrointestinal tract, and identifies novel mechanisms by which epicutaneous tolerance can suppress food‐induced anaphylaxis. GRAPHICAL ABSTRACT Figure. No caption available.