Extensively hydrolysed infant formulas: Need for aligned definition of peptide size characteristics and standardisation of analytical methods.

Letter by Nutten et al. on article by Levin et al. (Levin ME, Blackhurst DM, Kirstein F, Kok D, van der Watt GF, Marais AD. Residual allergenicity of amino acid-based and extensively hydrolysed cow's milk formulas. S Afr Med J 2017;107(9):763-767. S Afr Med J 2017;107(3):258-263. https://doi.org/10.7196/SAMJ.2017.v107i9.12137); and response by Levin et al.

Long-term reduction in food allergy susceptibility in mice by combining breastfeeding-induced tolerance and TGF-ß-enriched formula after weaning.

BACKGROUND: Oral tolerance induction in early life is a promising approach for food allergy prevention. Its success requires the identification of factors necessary for its persistence. OBJECTIVES: We aimed to assess in mice duration of allergy prevention by breastfeeding-induced oral tolerance and whether oral TGF-ß supplementation after weaning would prolong it. METHODS: We quantified ovalbumin (OVA) and OVA-specific immunoglobulin levels by ELISA in milk from the EDEN birth cohort. As OVA-specific Ig was found in all samples, we assessed whether OVA-immunized mice exposed to OVA during lactation could prevent allergic diarrhoea in their 6- and 13-week-old progeny. In some experiments, a TGF-ß-enriched formula was given after weaning. RESULTS: At 6 weeks, only 13% and 34% of mice breastfed by OVA-exposed mothers exhibited diarrhoea after six and seven OVA challenges vs. 44% and 72% in mice breastfed by naïve mothers (P = 0.02 and 0.01). Protection was associated with decreased levels of MMCP1 and OVA-specific IgE (P < 0.0001). At 13 weeks, although OVA-specific IgE remained low (P = 0.001), diarrhoea occurrence increased to 32% and 46% after six and seven OVA challenges in mice breastfed by OVA-exposed mothers. MMCP1 levels were not significantly inhibited. Supplementation with TGF-ß after weaning induced a strong protection in 13-week-old mice breastfed by OVA-exposed mothers compared with mice breastfed by naive mothers (0%, 13% and 32% of diarrhoea at the fifth, sixth and seventh challenges vs. 17, 42 and 78%; P = 0.05, 0.0043 and 0.0017). MMCP1 levels decreased by half compared with control mice (P = 0.02). Prolonged protection was only observed in mice rendered tolerant by breastfeeding and was associated with an improved gut barrier. CONCLUSIONS: In mice, prevention of food allergy by breastfeeding-induced tolerance is of limited duration. Nutritional intervention by TGF-ß supplementation after weaning could prolong beneficial effects of breast milk on food allergy prevention.

Beneficial effect of Lactococcus lactis NCC 2287 in a murine model of eosinophilic esophagitis.

BACKGROUND: Eosinophilic esophagitis (EoE) is a severe inflammatory disease of the esophagus which is characterized histologically by an eosinophilic infiltration into the esophageal tissue. The efficacy of probiotics in the context of atopic diseases has been well investigated but, to date, there has been no study which has evaluated probiotic effects on EoE inflammation. This study sought to identify a probiotic which improves esophageal inflammation in experimental EoE. METHODS: Two candidate probiotics, Lactococcus lactis NCC 2287 and Bifidobacterium lactis NCC 2818, were tested in a murine model of EoE elicited by epicutaneous sensitization with Aspergillus fumigatus protein extract. Administration of bacterial strains in drinking water was used, respectively, as a preventive or treatment measure, or continuously throughout the study. Inflammatory parameters were assessed in the esophagus, skin, and lungs after allergen challenge. RESULTS: In this EoE model, supplementation with L. lactis NCC 2287 significantly decreased esophageal and bronchoalveolar eosinophilia but only when given as a therapeutic treatment. No significant effect on eosinophilia was observed when NCC 2287 was given as a preventive or a continuous intervention. NCC 2287 supplementation had no significant effect on immunoglobulin levels, skin symptom scores, or on transepidermal water loss. Supplementation with another probiotic, B. lactis NCC 2818, had no significant effect on esophageal eosinophilia. CONCLUSION: We identified a L. lactis strain, able to attenuate esophageal eosinophilic inflammation in a preclinical model of EoE. This effect is strain specific and depends on the timing and duration of bacterial supplementation. Confirmation of these observations in human clinical trials is warranted.

Oral immunotherapy with low allergenic hydrolysed egg in egg allergic children.

BACKGROUND: A major drawback of oral immunotherapy for food allergy is the possibility of severe side-effects. We assessed both safety and efficacy of a low allergenic hydrolysed egg (HydE) preparation used in a double-blind placebo-controlled randomized study in egg allergic children. METHODS: In a pilot multicentre study, 29 egg allergic patients (aged 1-5.5 years) were administered daily for 6 months 9 g HydE or placebo in a blinded, randomized manner. Safety was verified by oral food challenge to assess tolerance towards HydE at the start and efficacy by an open oral food challenge (OFC, primary outcome) at the end. Additionally, changes in basophil activation and specific IgE and IgG4 were assessed. RESULTS: All egg allergic patients randomized to HydE (n = 15) tolerated the full dose at day 1 and received the maintenance dose from the start at home. No statistically significant difference was observed on the final OFC (36% and 21% had a negative OFC in the treatment and placebo groups, respectively). Specific IgG4 levels increased, while both CD203c+ and CD63+ basophils decreased significantly more over time in the treatment than in the placebo group. CONCLUSIONS: HydE can be regarded as a safe, low allergenic product to use in children allergic to egg. Although not significant, HydE given for 6 months increased numerically the proportion of patients becoming tolerant to egg. HydE induced a modulation of the immune response towards better tolerance. A longer treatment period and/or a higher dose may improve the clinical outcome and should be evaluated.

Hydrolysed egg displays strong decrease in allergenicity and is well tolerated by egg-allergic patients.

Food allergies are believed to be on the rise, and currently, management relies on the avoidance of the food. Hen's egg allergy is after cow's milk allergy the most common food allergy; eggs are used in many food products and thus difficult to avoid. A technological process using a combination of enzymatic hydrolysis and heat treatment was designed to produce modified hen's egg with reduced allergenic potential. Biochemical (SDS-PAGE, Size exclusion chromatography and LC-MS/MS) and immunological (ELISA, immunoblot, RBL-assays, animal model) analysis showed a clear decrease in intact proteins as well as a strong decrease of allergenicity. In a clinical study, 22 of the 24 patients with a confirmed egg allergy who underwent a double-blind food challenge with the hydrolysed egg remained completely free of symptoms. Hydrolysed egg products may be beneficial as low-allergenic foods for egg-allergic patients to extent their diet.

Immune-modulatory effect of probiotic Bifidobacterium lactis NCC2818 in individuals suffering from seasonal allergic rhinitis to grass pollen: an exploratory, randomized, placebo-controlled clinical trial.

BACKGROUND/OBJECTIVES: Probiotics are defined as 'living micro-organisms that when administered in adequate amounts confer a health benefit to the host'. Different probiotic strains have been investigated for beneficial effects on allergic disorders. The purpose of the current study was to evaluate the effect of orally administering the probiotic Nestlé culture collection (NCC)2818 Bifidobacterium lactis strain on immune parameters and nasal symptom scores in subjects suffering from seasonal allergic rhinitis (SAR). SUBJECTS/METHODS: The study was a double-blinded, parallel, randomized placebo-controlled trial conducted during the peak of the pollen season. Adult subjects with clinical history of SAR and positive skin prick test to grass pollen were recruited. The subjects received B. lactis NCC2818 or placebo for 8 weeks and completed symptom questionnaires every week. Whole blood was collected at baseline (V1), 4 weeks (V2) and 8 weeks (V3) to measure immune parameters. RESULTS: Concentrations of Th-2 cytokines, secreted by stimulated blood lymphocytes, were significantly lower in the probiotic group compared with the placebo group at V3 (interleukin (IL)-5, P=0.016; IL-13, P=0.005). Total nasal symptom scores were significantly lower in the second month of the study (weeks 5-8) in the probiotic group compared with the placebo group (P=0.03). Also, percentages of activated CD63 expressing basophils were significantly lower in the probiotic group at V2 (P=0.02). CONCLUSIONS: Oral administration of the probiotic NCC2818 mitigates immune parameters and allergic symptoms during seasonal exposure. These promising results warrant that B. lactis NCC2818 be investigated further in large-scale trials for management of respiratory allergy.

Effect of Lactobacillus paracasei ST11 on a nasal provocation test with grass pollen in allergic rhinitis.

BACKGROUND: Probiotics have been associated with prevention and improvement of symptoms in atopic diseases such as atopic dermatitis. However, few studies exist that document their efficacy for upper airways allergies such as allergic rhinitis. OBJECTIVE: To investigate the effect of short-term oral administration of Lactobacillus paracasei ST11 on a nasal provocation test (NPT) with grass pollen. METHODS: Thirty-one adult volunteers with allergic rhinitis were enrolled in a randomized, double-blind, placebo-controlled study, based on two 4-week cross-over periods of product consumption (ST11-fermented milk vs. placebo), separated by a wash-out period of 6-8 weeks. Objective and subjective clinical parameters of NPT as well as systemic and nasal immunological parameters were compared between the two treatment periods (registration number: NCT 011 50 253). RESULTS: Subjects that received ST11-fermented milk had lower nasal congestion than subjects under placebo (visual analogical scale; P<0.05). Nasal pruritus followed the same trend. However, no significant change in combined nasal reaction threshold was observed between the two periods. IL-5 secretion by peripheral blood mononuclear cells and serum allergen-specific IgG4 were significantly lower in ST11-fermented milk group compared to placebo group. IL-8 and IL-10 secretion followed the same trend. CONCLUSION AND CLINICAL RELEVANCE: Short-term treatment with ST11-fermented milk before NPT significantly improved a clinical marker of NPT (subjective nasal congestion) and down-regulated systemic immune markers (IL-5 from peripheral blood mononuclear cells and serum IgG4). These data strongly suggest that probiotics may down modulate key parameters of allergic rhinitis and warrant future evaluation in seasonal trials.

Antibiotic administration early in life impairs specific humoral responses to an oral antigen and increases intestinal mast cell numbers and mediator concentrations.

In this study, we assessed the effect of administering the antibiotic amoxicillin to rat pups on the immune response to orally fed ovalbumin (OVA). We first established that amoxicillin administration durably altered the gut microbiota of these animals. In parallel, we observed that the induction of the specific humoral response to ovalbumin was impaired when it occurred during antibiotic administration to the rat pups. We also examined the consequences of those observations on further allergic reactions. Amoxicillin administration had no significant impact on subsequent sensitization to OVA, as nonexacerbated systemic allergic responses were induced in antibiotic-treated animals. However, increased rat mast cell protease II levels and higher mast cell numbers were detected in their small intestines, independently of the antigen administration. Globally, our data suggest that antibiotic administration early in life negatively affects the specific immune response to a luminal antigen when it is first introduced during antibiotic administration. The increased mast cell numbers and mediator concentrations in the intestinal mucosae of the antibiotic-treated animals may testify to the early stages of an altered immune system homeostasis.

Attenuation of colon inflammation through activators of the retinoid X receptor (RXR)/peroxisome proliferator-activated receptor gamma (PPARgamma) heterodimer. A basis for new therapeutic strategies.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is highly expressed in the colon mucosa and its activation has been reported to protect against colitis. We studied the involvement of PPARgamma and its heterodimeric partner, the retinoid X receptor (RXR) in intestinal inflammatory responses. PPARgamma(1/)- and RXRalpha(1/)- mice both displayed a significantly enhanced susceptibility to 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis compared with their wild-type littermates. A role for the RXR/PPARgamma heterodimer in the protection against colon inflammation was explored by the use of selective RXR and PPARgamma agonists. TNBS-induced colitis was significantly reduced by the administration of both PPARgamma and RXR agonists. This beneficial effect was reflected by increased survival rates, an improvement of macroscopic and histologic scores, a decrease in tumor necrosis factor alpha and interleukin 1beta mRNA levels, a diminished myeloperoxidase concentration, and reduction of nuclear factor kappaB DNA binding activity, c-Jun NH(2)-terminal kinase, and p38 activities in the colon. When coadministered, a significant synergistic effect of PPARgamma and RXR ligands was observed. In combination, these data demonstrate that activation of the RXR/PPARgamma heterodimer protects against colon inflammation and suggest that combination therapy with both RXR and PPARgamma ligands might hold promise in the clinic due to their synergistic effects.

Role of the high affinity immunoglobulin E receptor in bacterial translocation and intestinal inflammation.

A role for immunoglobulin E and its high affinity receptor (Fc epsilon RI) in the control of bacterial pathogenicity and intestinal inflammation has been suggested, but relevant animal models are lacking. Here we compare transgenic mice expressing a humanized Fc epsilon RI (hFc epsilon RI), with a cell distribution similar to that in humans, to Fc epsilon RI-deficient animals. In hFc epsilon RI transgenic mice, levels of colonic interleukin 4 were higher, the composition of fecal flora was greatly modified, and bacterial translocation towards mesenteric lymph nodes was increased. In hFc epsilon RI transgenic mice, 2,4,6-tri-nitrobenzenesulfonic acid (TNBS)-induced colitis was also more pronounced, whereas Fc epsilon RI-deficient animals were protected from colitis, demonstrating that Fc epsilon RI can affect the onset of intestinal inflammation.

[Peroxisome proliferator-activated receptor (PPAR) gamma: a new target for the treatment of inflammatory bowel disease]. / Le récepteur activé par les proliférateurs des peroxysomes (PPAR) gamma: une nouvelle cible thérapeutique pour le traitement des maladies inflammatoires chroniques de l'intestin.

UNLABELLED: The peroxisome proliferator-activated receptor (PPAR) gamma is highly expressed in the colon mucosa. In vitro, it regulates inflammation. AIM: To evaluate the anti-inflammatory functions of PPARgamma agonist during a trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice. METHODS: Colitis was induced in Balb/c mice after intra-rectal administration of TNBS. The intensity of inflammation was assessed 2 and 5 days after colitis induction by macroscopic and histologic scores and by the quantification of colon myeloperoxidase (MPO), IL-1B and TNFalpha mRNA concentrations. The therapeutic role of PPARgamma agonist given by oral gavage was assessed in preventive and treatment modes. RESULTS: TNBS induced severe macroscopic and histologic lesions, with high mucosal MPO, IL-1B and TNFalpha mRNA concentrations. PPARgamma agonist given preventively or in treatment mode allowed a significant decrease of macroscopic and histologic scores through a normalization of MPO, IL-1B and TNFalpha mRNA concentrations. CONCLUSION: PPARgamma agonist decreases the intensity of TNBS induced colitis through normalization of IL-1B and TNFalpha expression. PPARgamma agonists may be proposed as new therapeutic agents in inflammatory bowel diseases.

Schistosoma mansoni schistosomula reduce E-selectin and VCAM-1 expression in TNF-alpha-stimulated lung microvascular endothelial cells by interfering with the NF-kappaB pathway.

The recruitment of immune cells into the lungs is a key step in protection against murine schistosomiasis. In this phenomenon, pulmonary (micro)vascular endothelial cells (EC) probably play a central role, by expressing specific adhesion molecules on their surface. Recently, we have shown that Schistosoma mansoni schistosomula, the parasitic stage which resides in the lungs, could activate microvascular EC to acquire an anti-inflammatory phenotype. In the present study, we tested the hypothesis that schistosomula could also regulate the expression of adhesion molecules in vitro by human lung microvascular EC (HMVEC-l) in the present of the pro-inflammatory cytokine TNF-alpha. We found that lipophilic substance(s) present in the excretory/secretory products from schistosomula selectively reduce the TNF-alpha-induced synthesis of E-selectin and VCAM-1 mRNA and proteins without affecting ICAM-1. This inhibitory effect appears to be mediated by a cyclic AMP/protein kinase A (cAMP/PKA) pathway that probably interferes with the NF-kappaB pathway induced by TNF-alpha at the level of the E-selectin promoter, whereas a cAMP-independent pathway appears to operate in VCAM-1 down-modulation. Finally, schistosomula also significantly reduce the VLA-4/VCAM-1-dependent adherence of leukocytes to TNF-alpha-stimulated HMVEC-l. We speculate that this mechanism could represent a new stratagem that parasites may use to escape the immune system by controlling leukocyte recruitment to the lungs.

Infection of mice lacking interleukin-7 (IL-7) reveals an unexpected role for IL-7 in the development of the parasite Schistosoma mansoni.

A single intradermal administration of recombinant interleukin-7 (IL-7) has been shown to aggravate the course of murine schistosomiasis, to favor the development of Th2-associated antibodies specific for the parasite, and to alter migration kinetics and/or migratory route of the parasite within its vertebrate host. Here we show that after infection of IL-7-deficient mice with Schistosoma mansoni, the predominant parasite-specific humoral response follows a Th1 pattern, and the development of the parasite is greatly impaired. In IL-7-deficient mice, increased numbers of larvae reach the lungs and fewer larvae reach the liver, compared to control mice. In the absence of IL-7, female worms show an altered fecundity, leading to decreased numbers of eggs trapped in the tissues and to an amelioration of the pathology of the infected host. The most striking observation is the blockade of parasite growth in an IL-7-defective environment, leading to dwarf male and female worms. The results of this study have important implications for the role of IL-7 in the host-parasite relationship and show how parasites can disable or evade the host immune response.

Schistosoma mansoni activates host microvascular endothelial cells to acquire an anti-inflammatory phenotype.

Since endothelial cells (ECs) play a key role in immune defense mechanisms and in immunopathology, we investigated whether the intravascular helminth parasite Schistosoma mansoni could interact with and activate resting ECs in vitro. Microscopic analysis revealed that the lung-stage schistosomula specifically attached to microvascular ECs. This adherence was associated to active cellular processes involving actin filament formation. Since variation of permeability of cultured capillary brain ECs is a good marker for endothelial activation, the transendothelial passage of a low-molecular-weight molecule (inulin) on monolayers of bovine brain capillary ECs (BBCEC) was measured in response to parasites. Schistosomula induced a dramatic decrease in transendothelial permeability, a characteristic marker for the generation of an anti-inflammatory phenotype to ECs. This paracellular barrier enhancing effect on endothelial monolayers was due to a soluble substance(s) (below 1 kDa in size) secreted from S. mansoni schistosomula and not by mechanisms associated to adherence between parasites and ECs. The reinforcement of the endothelial barrier function was accompanied by an elevation of intracellular concentration of cyclic AMP (cAMP). The use of specific kinase inhibitors confirms that schistosomula activate ECs through a cAMP/protein kinase A pathway that leads to an increased phosphorylation of the myosin light-chain kinase. These combined findings suggest that the secretory/excretory products from schistosomula possess anti-inflammatory factor(s) that signal host microvascular endothelium. The immunological consequences of such activation are discussed.

Selectin and Lewis(x) are required as co-receptors in antibody-dependent cell-mediated cytotoxicity of human eosinophils to Schistosoma mansoni schistosomula.

Killing of Schistosoma mansoni larvae by human eosinophils via antibody-dependent cell-mediated cytotoxicity (ADCC) mechanisms requires adherence between effector cells and parasite targets. The role of adhesion molecules in this mechanism was investigated using blocking monoclonal antibodies (mAb) and soluble ligands. We show that, along with the Mac-1 alpha chain, interactions between selectins and LewisX-related structures, both expressed by eosinophils and parasite targets, play a critical part in the antibody-dependent cytotoxic function of eosinophils. To further elucidate the interactions between adhesion molecules and eosinophil Fc receptors, ADCC was performed with IgG1 or IgA mAb. We found that mAb directed against Mac-1 alpha chain or against LewisX could significantly inhibit the IgG1-, but not IgA cytotoxicity. This result might be explained, at least in part, by the inhibitory effect of these mAb on the release by eosinophils of eosinophil cationic protein, one of the major mediators involved in target killing. Taken together, these results suggest novel interactions between Fc receptors and selectins and LewisX-related structures which might act as co-receptors for eosinophil-mediated cytotoxicity.

Dermal endothelial cells and keratinocytes produce IL-7 in vivo after human Schistosoma mansoni percutaneous infection.

The parasite Schistosoma mansoni infects its definitive mammalian host through an obligatory cutaneous penetration. In this work, we studied early immune response following migration of larvae through human skin, the first immunocompetent organ encountered by the parasite. For this purpose we used an experimental model of severe combined immunodeficient mice engrafted with human skin and injected with autologous PBL. Six days after percutaneous infection, we observed an infiltration of lymphocytes within the human skin, predominantly composed of CD4+ T cells. Moreover, among the cytokines potentially present in the infected skin, immunohistochemistry analysis revealed an in vivo expression of IL-7 in the epidermal layers and strikingly at the level of vascular endothelium. Using an in vitro coculture system, we showed that the S. mansoni larvae directly trigger IL-7 production by human dermal microvascular endothelial cells but not by keratinocytes. Finally, measurements of IL-7 concentrations in plasma of 187 S. mansoni-infected individuals showed that the youngest, which are also the most infected, displayed the highest IL-7 levels. Together, these findings describe dermal endothelial cells as a novel source of IL-7, a cytokine particularly important in schistosomiasis.