Direct effects of fermented cow's milk product with Lactobacillus paracasei CBA L74 on human enterocytes

Cow's milk fermented with Lactobacillus paracasei CBA L74 (FM-CBAL74) exerts a preventive effect against infectious diseases in children. We evaluated if this effect is at least in part related to a direct modulation of non-immune and immune defence mechanisms in human enterocytes. Human enterocytes (Caco-2) were stimulated for 48 h with FM-CBAL74 at different concentrations. Cell growth was assessed by colorimetric assay; cell differentiation (assessed by lactase expression), tight junction proteins (zonula occludens1 and occludin), mucin 2, and toll-like receptor (TRL) pathways were analysed by real-time PCR; innate immunity peptide synthesis, beta-defensin-2 (HBD-2) and cathelicidin (LL-37) were evaluated by ELISA. Mucus layer thickness was analysed by histochemistry. FMCBA L74 stimulated cell growth and differentiation, tight junction proteins and mucin 2 expression, and mucus layer thickness in a dose-dependent fashion. A significant stimulation of HBD-2 and LL-37 synthesis, associated with a modulation of TLR pathway, was also observed. FM-CBAL74 regulates non-immune and immune defence mechanisms through a direct interaction with the enterocytes. These effects could be involved in the preventive action against infectious diseases demonstrated by this fermented product in children

Prognostic factors facilitating multiple food allergies and atopiv march occurrence in children with Non-IgE mediated gastrointestinal Food Allergy: results of two years follow up of the NIGEFA project

Objectives and Study: Non-IgE mediated gastrointestinal food allergies (non-IgE-GIFA) are an increasing problem in pediatric gastroenterology clinical practice. These conditions include food protein-induced: enterocolitis syndrome (FPIES), enteropathy (FPE), allergic proctocolitis (FPIAP), and motility disorders (FPIMD). The NIGEFA project is focused on the investigation of main clinical features, prognostic factors (presence atopic dermatitis (AD), multiple food allergies, diagnostic delay, and familial history of allergy), and natural history (atopic march (AM) prevalence and timing of immune tolerance acquisition). Methods: Prospective observational study evaluating children with non-IgE-GIFA diagnosed according to standard criteria observed at a tertiary center for pediatric gastroenterology and allergy (both sexes, aged <36 m, follow up 12 m after diagnosis). Main anamnestic, demographic, and clinical data were collected from all enrolled patients. Immune tolerance acquisition was evaluated by the result of oral food challenge. Results: A total of 100 patients were enrolled: 58% male, mean age at diagnosis (SD) 8.5(8.8) m. Non-IgE-GIFA conditions were: FPE (44%), FPIES (11%), FPIAP (18%), FPIMD (27%). Mean diagnostic delay was 5.3 (7.4) m. Multiple non-IgE-GIFA were observed in 47% at baseline. Familial history of allergy was observed in 64% of subjects. Presence of AD before the onset of non-IgE-GIFA was observed in 40% of subjects. The overall rate of immune tolerance acquisition at 12 m was 27%, with a higher rate in FPIAP (44%) compared with FPIMD (29.6%), FPE (22.7%) and FPIES (9.1%) subjects (p<0.05). The rate of immune tolerance acquisition at 12 m was significantly lower in children with familial history of allergy (-48%, estimated risk ratio (RR)0.52 (95% CI 0.28 to 0.99, p<0.05)) and in those with multiple non-IgE-GIFA (-61%, RR at 12 m 0.39 (95% CI 0.18 to 0.85, p<0.05)). At 12 m follow up, the rate of subjects presenting AM was 24% with no difference among the 4 disease groups. The occurrence of AM was significantly higher in subjects with multiple (38%) vs. mono non-IgE-GIFA (11%) (p<.001) at baseline, with an estimated RR of 3.38 (95% CI 1.47 to 7.81, p<0.01) at 12 m. Moreover, for every 1-month of diagnostic delay there was an increase of 1.04 RR(95% CI 1.01 to 1.07) of AM occurrence at 12 m. No associations with other potential predictors (sex, familial allergy risk, AD before the onset of GIFA, type of non-IgE-GIFA) were found. Conclusions: These data shed lights on prognostic factors and natural history of non-IgE-GIFA suggesting the importance of early diagnosis in preventing the occurrence of AM occurrence in these patients. Contac

Hepatic Mitochondrial Dysfunction and Immune Response in a Murine Model of Peanut Allergy.

BACKGROUND: Evidence suggests a relevant role for liver and mitochondrial dysfunction in allergic disease. However, the role of hepatic mitochondrial function in food allergy is largely unknown. We aimed to investigate hepatic mitochondrial dysfunction in a murine model of peanut allergy. METHODS: Three-week-old C3H/HeOuJ mice were sensitized by the oral route with peanut-extract (PNT). We investigated: 1. the occurrence of effective sensitization to PNT by analysing acute allergic skin response, anaphylactic symptoms score, body temperature, serum mucosal mast cell protease-1 (mMCP-1) and anti-PNT immunoglobulin E (IgE) levels; 2. hepatic involvement by analysing interleukin (IL)-4, IL-5, IL-13, IL-10 and IFN-γ mRNA expression; 3. hepatic mitochondrial oxidation rates and efficiency by polarography, and hydrogen peroxide (H₂O₂) yield, aconitase and superoxide dysmutase activities by spectrophotometry. RESULTS: Sensitization to PNT was demonstrated by acute allergic skin response, anaphylactic symptoms score, body temperature decrease, serum mMCP-1 and anti-peanut IgE levels. Liver involvement was demonstrated by a significant increase of hepatic Th2 cytokines (IL-4, IL-5 and IL-13) mRNA expression. Mitochondrial dysfunction was demonstrated by lower state 3 respiration rate in the presence of succinate, decreased fatty acid oxidation in the presence of palmitoyl-carnitine, increased yield of ROS proven by the inactivation of aconitase enzyme and higher H₂O₂ mitochondrial release. CONCLUSIONS: We provide evidence of hepatic mitochondrial dysfunction in a murine model of peanut allergy. These data could open the way to the identification of new mitochondrial targets for innovative preventive and therapeutic strategies against food allergy

Lactobacillus rhamnosus GG-supplemented formula expands butyrate-producing bacterial strains in food allergic infants.

Dietary intervention with extensively hydrolyzed casein formula supplemented with Lactobacillus rhamnosus GG (EHCF+LGG) accelerates tolerance acquisition in infants with cow's milk allergy (CMA). We examined whether this effect is attributable, at least in part, to an influence on the gut microbiota. Fecal samples from healthy controls (n=20) and from CMA infants (n=19) before and after treatment with EHCF with (n=12) and without (n=7) supplementation with LGG were compared by 16S rRNA-based operational taxonomic unit clustering and oligotyping. Differential feature selection and generalized linear model fitting revealed that the CMA infants have a diverse gut microbial community structure dominated by Lachnospiraceae (20.5±9.7%) and Ruminococcaceae (16.2±9.1%). Blautia, Roseburia and Coprococcus were significantly enriched following treatment with EHCF and LGG, but only one genus, Oscillospira, was significantly different between infants that became tolerant and those that remained allergic. However, most tolerant infants showed a significant increase in fecal butyrate levels, and those taxa that were significantly enriched in these samples, Blautia and Roseburia, exhibited specific strain-level demarcations between tolerant and allergic infants. Our data suggest that EHCF+LGG promotes tolerance in infants with CMA, in part, by influencing the strain-level bacterial community structure of the infant gut

Proteomic and immunological characterization of a new food allergen from hazelnut (Corylus avellana).

Hazelnuts (Corylus avellana) are one of the most common sources of life-long IgE-mediated food allergies. In this study, we investigated the IgE-reactivity pattern of children with hazelnut allergy (N=15) from Regione Campania, located in Southern Italy, and addressed proteomic strategies for characterizing IgE-binding proteins. For all of the patients (15/15), the predominant IgE-reactive component was a minor ~55kDa protein not previously described. Similar to the hazelnut 11S globulin Cor a 9 allergen, the immunoreactive protein consisted of two subunits linked via a disulfide bridge. In contrast to Cor a 9, only the 20.7kDa alkaline subunit exhibited IgE-affinity. The immunogenic subunit was purified by a two-step chromatographic procedure, but peptide mass fingerprinting was unsuccessful in identifying it, due to the incompleteness of the annotated hazelnut genome. Several tryptic peptides were de novo sequenced by tandem mass spectrometry and showed a high degree of homology with the 11S globulin storage proteins from other seeds, some of which have already been reported as food allergens. The structural characterization suggests that the new putative allergen is a divergent isoform of the hazelnut 11S globulin. These results provide a new platform for developing innovative diagnostic and therapeutic intervention plans. BIOLOGICAL SIGNIFICANCE:Over the years, at least five proteins have been reported as potential food hazelnut allergens. The predominance of specific allergens appears to be strictly related to the geographical origin of the allergic subjects. The complex patterns of the IgE-reactivity of hazelnut storage proteins result in a poor diagnostic and prognostic accuracy. In the perspective of a component-resolved "molecular approach" to the hazelnut allergy we investigated the immune-reactivity patterns to hazelnuts of 15 patients (14 in the pediatric age range) from Region Campania, located in Southern Italy. For all the patients the predominant IgE-reactive component was a minor ~55kDa protein not previously annotated in either protein or genomic databases. The putative allergen was isolated, partially characterized by MS/MS de novo sequencing and appears to be an isoallergen of the hazelnut 11S globulin Cor a 9. Like this latter, the immunoreactive protein consisted of two subunits linked via a disulfide. In contrast to Cor a 9, only the 20.7kDa alkaline subunit exhibited IgE-affinity, in analogy to 11S allergens from other seeds (pistachio, cashew, soybean). We believe that the application of combined immunochemical and proteomic strategies to characterize the new food allergen could be of interest for the readers of Journal of Proteomics. In addition, the results of this study have functional worth in providing a new platform to plan innovative diagnostic and therapeutic intervention approaches to treat hazelnut allergy