Rosita Aitoro

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.

Immunoregulatory Pathways Are Active in the Small Intestinal Mucosa of Patients with Potential Celiac Disease

OBJECTIVES:Potential celiac disease (CD) relates to subjects with a normal small intestinal mucosa who are at increased risk of developing CD as indicated by positive CD-associated serology. The objective of this study was to investigate in the small intestinal mucosa of such patients the state of immunological activation with special emphasis on immunoregulatory circuits.METHODS:Duodenal biopsies from active CD (n=48), potential CD (n=58), and control patients (n=45) were studied. RNA expression for interferon γ (IFNγ) and interleukin-10 (IL-10) were quantified by real-time quantitative PCR. The percentage of CD4+CD25+Foxp3+ T regulatory cells (Foxp3+Tregs) was determinated by flow cytometry and the number of Foxp3+ and IL-15+ cells by immunohistochemistry. Furthermore, we analyzed the suppressive function of CD4+CD25+ T cells, isolated from potential CD biopsy samples, as well as the effect of IL-15, on autologous peripheral blood responder CD4+CD25– T cells.RESULTS:In potential CD patients with Marsh 1 lesion, IFNγ-RNA expression was significantly less than in active, but enhanced if compared with potential CD patients with Marsh 0 lesion and with controls (P<0.001). The number of IL-15+ cells in subjects with potential CD was increased in comparison with controls (P<0.05), but lower than active CD (P<0.01). IL-10-RNA expression was upregulated in Marsh 0 potential CD patients if compared with those with Marsh 1 lesion (P<0.01) and controls (P<0.001), whereas there were no differences with active CD. The ratio IL-10/IFNγ reached the highest value in Marsh 0 potential CD compared with the other groups (P<0.05). The percentage of Foxp3+Tregs was also higher in potential CD compared with controls (P<0.05), although it was lower than in active CD (P<0.01). In co-culture assay, intestinal CD4+CD25+ T cells from potential CD patients exerted suppressive effects on T responder cells, and their activity was not impaired by IL-15.CONCLUSIONS:Potential CD patients show a low grade of inflammation that likely could be due to active regulatory mechanisms preventing the progression toward a mucosal damage.

Bugs for atopy: the Lactobacillus rhamnosus GG strategy for food allergy prevention and treatment in children

Food allergy (FA) is a major health issue for children living in Western countries. At this time the only proven treatment for FA is elimination of offender antigen from the diet. It is becoming clear that the development of gut microbiota exerts a profound influence on immune system maturation and tolerance acquisition. Increasing evidence suggests that perturbations in gut microbiota composition of infants are implicated in the pathogenesis of FA. These findings have unveiled new strategies to prevent and treat FA using probiotics bacteria or bacterial substance to limit T-helper (Th)/Th2 bias, which changes during the disease course. Selected probiotics administered during infancy may have a role in the prevention and treatment of FA. Lactobacillus rhamnosus GG (LGG) is the most studied probiotic in this field. Administration of LGG in early life have a role in FA prevention. Preliminary evidence shows that LGG accelerates oral tolerance acquisition in cows milk allergic infants. We are understanding the mechanisms elicited by LGG and metabolites in influencing food allergen sensitization. A deeper definition of these mechanisms is opening the way to new immunotherapeutics for children affected by FA that can efficiently limit the disease burden.

Gut Microbiota as a Target for Preventive and Therapeutic Intervention against Food Allergy

The gut microbiota plays a pivotal role in immune system development and function. Modification in the gut microbiota composition (dysbiosis) early in life is a critical factor affecting the development of food allergy. Many environmental factors including caesarean delivery, lack of breast milk, drugs, antiseptic agents, and a low-fiber/high-fat diet can induce gut microbiota dysbiosis, and have been associated with the occurrence of food allergy. New technologies and experimental tools have provided information regarding the importance of select bacteria on immune tolerance mechanisms. Short-chain fatty acids are crucial metabolic products of gut microbiota responsible for many protective effects against food allergy. These compounds are involved in epigenetic regulation of the immune system. These evidences provide a foundation for developing innovative strategies to prevent and treat food allergy. Here, we present an overview on the potential role of gut microbiota as the target of intervention against food allergy.

Extensively hydrolyzed casein formula alone or with L. rhamnosus GG reduces β‐lactoglobulin sensitization in mice

Extensively hydrolyzed casein formula (EHCF) has been proposed for the prevention and is commonly used for the treatment of cows milk allergy (CMA). The addition of the probiotic Lactobacillus rhamnosus GG (LGG) to EHCF may induce faster acquisition of tolerance to cows milk. The mechanisms underlying this effect are largely unexplored. We investigated the effects of EHCF alone or in combination with LGG on β‐lactoglobulin (BLG) sensitization in mice.

Food Allergies: Novel Mechanisms and Therapeutic Perspectives

Childhood food allergy (FA) rates have rapidly increased with significant direct medical costs for the health care system and even larger costs for the families with a food-allergic child. The possible causes of food allergy become the target of intense scrutiny in recent years. Increasing evidence underline the importance in early life of gut microbiome in the development of allergic diseases. There are a range of factors in the modern environment that may be associated with changes to both the gut microbiome and risk of FA, such as mode of delivery, antibiotic exposure, infant feeding practices, farming environment, and country of origin. Knowledge of the relationship between early life gut microbiome and allergic diseases may facilitate development of novel preventive and treatment strategies. Based on our current knowledge, there are no currently available approved therapies for food allergy. More studies are needed to evaluate the safety and efficacy of allergen-specific and allergen-nonspecific approaches, as well as combination approaches.

In the Intestinal Mucosa of Children With Potential Celiac Disease IL-21 and IL-17A are Less Expressed than in the Active Disease

Objectives:Potential celiac disease (CD) patients are at an increased risk to developing CD as indicated by positive CD-associated serology. We investigated in duodenal mucosa of such patients the presence of both IL-21 and IL-17A and the role of gliadin peptides and IL-15 in their expression.Methods:Duodenal biopsies from 76 active CD, 90 potential CD, and 58 control patients were analyzed for IL-21 and/or IL-17A production by quantitative real-time PCR, immunohistochemistry, flow cytometry, and ELISA. The presence of IL-21 receptor was investigated by western blot. Potential CD duodenal fragments were cultured with gliadin peptides (PTG) and/or IL-15 and the expression/production of IL-21 and IL-17A assessed by quantitative real-time PCR and by immunohistochemistry.Results:In potential CD, IL-21 was lower than in active CD, in terms of RNA expression (P<0.01), density of lamina propria (LP) IL-21+ cells (P<0.05), and protein secretion (P<0.05). Also, IL-21R was weakly detectable in potential CD. Several LP cell types produced IL-21 in CD. In potential CD, CD4+IL-21+ cells increased after PMA-ionomycin stimulation and co-produced IFN-γ but not IL-17A. After 24 hours of culture stimulation with PTG, IL-21-producing cells increased but not the ones producing IL-17A. This increase was further enhanced by the addition of IL-15 to culture medium.Conclusions:In potential CD, IL-21 is less expressed than in active CD; however, IL-21-producing cells are present and prone to respond after specific stimuli. This suggests a key role of IL-21 in the progression of mucosal damage in CD.

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.

Intestinal Production of Anti-Tissue Transglutaminase 2 Antibodies in Patients with Diagnosis Other Than Celiac Disease

It has been hypothesized that gluten-dependent production of anti-tissue-transglutaminase 2 (anti-TG2) antibodies may occur only at an intestinal level. We have investigated intestinal production of anti-TG2 antibodies in 136 patients with normal serum levels of anti-TG2 antibodies and normal duodenal mucosa. Intestinal deposits of anti-TG2 antibodies were evaluated by immunofluorescence and anti-TG2 antibodies released in organ culture supernatants measured by ELISA. Intestinal antibody libraries were obtained from 10 subjects. Immunohistochemistry for CD25+, CD3+, and TCR-γδ+ was assessed in subjects with positive (n = 32) and negative (n = 31) intestinal anti-TG2 antibodies. Globally 33/136 (24%) seronegative patients produced anti-TG2 autoantibodies at an intestinal level. Antibody libraries analysis confirmed the anti-TG2 antibodies mucosal production in all (n = 8) positive subjects. Lamina propria CD25+ cell count was significantly (p < 0.05) higher in patients with intestinal anti-TG2. Moreover, 13/32 (41%) of them showed high TCR-γδ+/CD3+ ratios. Intestinal anti-TG2 antibody production does not show absolute specificity for CD. It is seen more often in association with inflamed mucosa. Further investigations are necessary to prove the possible role of dietary gluten.

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

Cows 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.