Isabel Polanco

The HLA-DQ2 genotype selects for early intestinal microbiota composition in infants at high risk of developing coeliac disease

Objective Intestinal dysbiosis has been associated with coeliac disease (CD), but whether the alterations are cause or consequence of the disease is unknown. This study investigated whether the human leukocyte antigen (HLA)-DQ2 genotype is an independent factor influencing the early gut microbiota composition of healthy infants at family risk of CD. Design As part of a larger prospective study, a subset (n=22) of exclusively breastfed and vaginally delivered infants with either high genetic risk (HLA-DQ2 carriers) or low genetic risk (non-HLA-DQ2/8 carriers) of developing CD were selected from a cohort of healthy infants with at least one first-degree relative with CD. Infant faecal microbiota was analysed by 16S rRNA gene pyrosequencing and real time quantitative PCR. Results Infants with a high genetic risk had significantly higher proportions of Firmicutes and Proteobacteria and lower proportions of Actinobacteria compared with low-risk infants. At genus level, high-risk infants had significantly less Bifidobacterium and unclassified Bifidobacteriaceae proportions and more Corynebacterium, Gemella, Clostridium sensu stricto, unclassified Clostridiaceae, unclassified Enterobacteriaceae and Raoultella proportions. Quantitative real time PCR also revealed lower numbers of Bifidobacterium species in infants with high genetic risk than in those with low genetic risk. In high-risk infants negative correlations were identified between Bifidobacterium species and several genera of Proteobacteria (Escherichia/Shigella) and Firmicutes (Clostridium). Conclusions The genotype of infants at family risk of developing CD, carrying the HLA-DQ2 haplotypes, influences the early gut microbiota composition. This finding suggests that a specific disease-biased host genotype may also select for the first gut colonisers and could contribute to determining disease risk.

Influence of Milk-Feeding Type and Genetic Risk of Developing Coeliac Disease on Intestinal Microbiota of Infants: The PROFICEL Study

Interactions between environmental factors and predisposing genes could be involved in the development of coeliac disease (CD). This study has assessed whether milk-feeding type and HLA-genotype influence the intestinal microbiota composition of infants with a family history of CD. The study included 164 healthy newborns, with at least one first-degree relative with CD, classified according to their HLA-DQ genotype by PCR-SSP DQB1 and DQA1 typing. Faecal microbiota was analysed by quantitative PCR at 7 days, and at 1 and 4 months of age. Significant interactions between milk-feeding type and HLA-DQ genotype on bacterial numbers were not detected by applying a linear mixed-model analysis for repeated measures. In the whole population, breast-feeding promoted colonization of C. leptum group, B. longum and B. breve, while formula-feeding promoted that of Bacteroides fragilis group, C. coccoides-E. rectale group, E. coli and B. lactis. Moreover, increased numbers of B. fragilis group and Staphylococcus spp., and reduced numbers of Bifidobacterium spp. and B. longum were detected in infants with increased genetic risk of developing CD. Analyses within subgroups of either breast-fed or formula-fed infants indicated that in both cases increased risk of CD was associated with lower numbers of B. longum and/or Bifidobacterium spp. In addition, in breast-fed infants the increased genetic risk of developing CD was associated with increased C. leptum group numbers, while in formula-fed infants it was associated with increased Staphylococcus and B. fragilis group numbers. Overall, milk-feeding type in conjunction with HLA-DQ genotype play a role in establishing infants gut microbiota; moreover, breast-feeding reduced the genotype-related differences in microbiota composition, which could partly explain the protective role attributed to breast milk in this disorder.

Influence of Environmental and Genetic Factors Linked to Celiac Disease Risk on Infant Gut Colonization by Bacteroides Species

ABSTRACT Celiac disease (CD) is an immune-mediated enteropathy involving genetic and environmental factors whose interaction might influence disease risk. The aim of this study was to determine the effects of milk-feeding practices and the HLA-DQ genotype on intestinal colonization of Bacteroides species in infants at risk of CD development. This study included 75 full-term newborns with at least one first-degree relative suffering from CD. Infants were classified according to milk-feeding practice (breast-feeding or formula feeding) and HLA-DQ genotype (high or low genetic risk). Stools were analyzed at 7 days, 1 month, and 4 months by PCR and denaturing gradient gel electrophoresis (DGGE). The Bacteroides species diversity index was higher in formula-fed infants than in breast-fed infants. Breast-fed infants showed a higher prevalence of Bacteroides uniformis at 1 and 4 months of age, while formula-fed infants had a higher prevalence of B. intestinalis at all sampling times, of B. caccae at 7 days and 4 months, and of B. plebeius at 4 months. Infants with high genetic risk showed a higher prevalence of B. vulgatus, while those with low genetic risk showed a higher prevalence of B. ovatus, B. plebeius, and B. uniformis. Among breast-fed infants, the prevalence of B. uniformis was higher in those with low genetic risk than in those with high genetic risk. Among formula-fed infants, the prevalence of B. ovatus and B. plebeius was increased in those with low genetic risk, while the prevalence of B. vulgatus was higher in those with high genetic risk. The results indicate that both the type of milk feeding and the HLA-DQ genotype influence the colonization process of Bacteroides species, and possibly the disease risk.

Influence of early environmental factors on lymphocyte subsets and gut microbiota in infants at risk of celiac disease; the PROFICEL study.

INTRODUCTIONnIt is known that the HLA genotype can explain about a 40% of the genetic risk of celiac disease (CD), thus, other genetic predisposing factors as well as factors that subtly modulate T cell activation and differentiation need to be studied. This includes environmental factors that are currently believed to impact on the immune system and gut microbiota development.nnnAIMnTo assess the associations between early environmental factors (EEF), lymphocyte subsets, and intestinal microbiota composition in infants at familial risk for CD.nnnSTUDY DESIGNnProspective observational study.nnnSUBJECTSnFifty-five 4 month-old infants with at least a first-degree relative suffering CD. Infants were classified according to type of delivery, mothers antibiotic intake during pregnancy and during labor, milk-feeding practices, early infections and antibiotic intake, rotavirus vaccine administration, and allergy incidence within the first 18 months of life.nnnMETHODSnLymphocyte subsets and gut microbiota composition were studied at the age of 4 months.nnnRESULTSnFormula feeding and infants infections were associated with higher CD3+, CD4+, CD4+CD38+, CD4+CD28+ and CD3+CD4+CD45RO+ counts (P0.01). Infant s infections were also associated with higher CD4+CD25+, CD4+HLA-DR+ and NK cell counts (P0.01). Cesarean delivery and rotavirus vaccine administration were associated with lower percentage of CD4+CD25+ cells. Infants antibiotic intake was associated and correlated with lower counts of Bifidobacterium longum and higher counts of Bacteroides fragilis group.nnnCONCLUSIONSnInfant s infections and antibiotic intake in the first 4 months of life are the EEF more strongly and/or frequently associated to lymphocyte subpopulations and microbiota composition, respectively, in infants at risk of CD.

Dietary assessment in children and adolescents: issues and recommendations.

The assessment of dietary intake in children and adolescents is of great interest for different purposes. The characteristics of each developmental stage and associated cognitive abilities are two factors that influence the ability of children to provide valid and reliable information on food consumption. The ability to remember, limitations of vocabulary or the ability to identify different foods are some of the relevant aspects. In addition, often parents or caregivers provide surrogate information and their degree of knowledge depends on the time they spend with the child and on whether they share meals. As children grow they become more independent and increasingly spend more time away from their parents. Children also have limitations to recognize food models and photographs and associate them with different amounts of food consumed. On the other hand, often children and adolescents perceive long interviews or self-administered questionnaires they as long and boring. The use of new technologies is contributing to the development of new tools adapting dietary assessment the methods to the cognitive abilities of children, introducing gaming environments and narrative structures that attract their interest and improve the quality of information they report..

Influence of breastfeeding versus formula feeding on lymphocyte subsets in infants at risk of coeliac disease: the PROFICEL study.

PurposeIn addition to genetic risk, environmental factors might influence coeliac disease (CD) development. We sought to assess the effect of the interaction between milk-feeding practices and the HLA-DQ genotype on peripheral lymphocyte subsets and their activation markers in infants at familial risk for CD.Methods170 newborns were classified in 3 different genetic risk groups (high risk, HR; intermediate risk, IR; and low risk, LR) after DQB1 and DQA1 typing. Lymphocyte subsets were studied at the age of 4xa0months by flow cytometry analysis.Results79 infants were receiving exclusive breastfeeding (BF) and 91 partial breastfeeding or formula feeding (FF). Regarding genetic risk, 40 infants were classified in HR group, 75 in IR group and 55 in LR group. Two-way ANOVA did not show significant interactions between the type of milk feeding and genetic risk group on the lymphocyte subsets analysed. One-way ANOVA for milk-feeding practice alone showed that the percentage of CD4xa0+xa0CD25+ cells was significantly higher in BF group than in FF group (BF, 10.92xa0±xa02.71; FF, 9.94xa0±xa02.96; pxa0=xa00.026), and absolute counts of CD4xa0+xa0CD38+ cells were significantly higher in FF group than in BF group (FF, 2,881.23xa0±xa0973.48; BF, 2,557.95xa0±xa0977.06; pxa0=xa00.038). One-way ANOVA for genetic risk alone showed that absolute counts of NK cells were significantly higher in IR group than HR and LR groups (IR, 539.24xa0±xa0340.63; HR, 405.01xa0±xa0239.53; LR, 419.86xa0±xa0262.85; pxa0=xa00.028).ConclusionLymphocyte subset profiles in the early stages of life could be modulated by milk-feeding practices and genetic risk separately. Breastfeeding might have a positive immunomodulatory effect on lymphocyte subsets in infants at risk of CD.

Th17-Related Genes and Celiac Disease Susceptibility

Th17 cells are known to be involved in several autoimmune or inflammatory diseases. In celiac disease (CD), recent studies suggest an implication of those cells in disease pathogenesis. We aimed at studying the role of genes relevant for the Th17 immune response in CD susceptibility. A total of 101 single nucleotide polymorphisms (SNPs), mainly selected to cover most of the variability present in 16 Th17-related genes (IL23R, RORC, IL6R, IL17A, IL17F, CCR6, IL6, JAK2, TNFSF15, IL23A, IL22, STAT3, TBX21, SOCS3, IL12RB1 and IL17RA), were genotyped in 735 CD patients and 549 ethnically matched healthy controls. Case-control comparisons for each SNP and for the haplotypes resulting from the SNPs studied in each gene were performed using chi-square tests. Gene-gene interactions were also evaluated following different methodological approaches. No significant results emerged after performing the appropriate statistical corrections. Our results seem to discard a relevant role of Th17 cells on CD risk.

Influence of early environmental factors on lymphocyte subsets and gut microbiota in infants at risk of celiac disease; the PROFICEL study | Influencia de factores ambientales tempranos sobre las subpoblaciones de linfocitos y la microbiota intestinal de niños con riesgo de desarrollar enfermedad celíaca; el estudio PROFICEL

INTRODUCTIONnIt is known that the HLA genotype can explain about a 40% of the genetic risk of celiac disease (CD), thus, other genetic predisposing factors as well as factors that subtly modulate T cell activation and differentiation need to be studied. This includes environmental factors that are currently believed to impact on the immune system and gut microbiota development.nnnAIMnTo assess the associations between early environmental factors (EEF), lymphocyte subsets, and intestinal microbiota composition in infants at familial risk for CD.nnnSTUDY DESIGNnProspective observational study.nnnSUBJECTSnFifty-five 4 month-old infants with at least a first-degree relative suffering CD. Infants were classified according to type of delivery, mothers antibiotic intake during pregnancy and during labor, milk-feeding practices, early infections and antibiotic intake, rotavirus vaccine administration, and allergy incidence within the first 18 months of life.nnnMETHODSnLymphocyte subsets and gut microbiota composition were studied at the age of 4 months.nnnRESULTSnFormula feeding and infants infections were associated with higher CD3+, CD4+, CD4+CD38+, CD4+CD28+ and CD3+CD4+CD45RO+ counts (P0.01). Infant s infections were also associated with higher CD4+CD25+, CD4+HLA-DR+ and NK cell counts (P0.01). Cesarean delivery and rotavirus vaccine administration were associated with lower percentage of CD4+CD25+ cells. Infants antibiotic intake was associated and correlated with lower counts of Bifidobacterium longum and higher counts of Bacteroides fragilis group.nnnCONCLUSIONSnInfant s infections and antibiotic intake in the first 4 months of life are the EEF more strongly and/or frequently associated to lymphocyte subpopulations and microbiota composition, respectively, in infants at risk of CD.

Increased prevalence of pathogenic bacteria in the gut microbiota of infants at risk of developing celiac disease: The PROFICEL study

ABSTRACT Celiac disease (CD) is an immune-mediated enteropathy involving genetic and environmental factors, whose interaction influences disease risk. The intestinal microbiota, including viruses and bacteria, could play a role in the pathological process leading to gluten intolerance. In this study, we investigated the prevalence of pathogens in the intestinal microbiota of infants at familial risk of developing CD. We included 127 full-term newborns with at least one first-degree relative with CD. Infants were classified according to milk-feeding practice (breastfeeding or formula feeding) and HLA-DQ genotype (low, intermediate or high genetic risk). The prevalence of pathogenic bacteria and viruses was assessed in the faeces of the infants at 7 days, 1 month and 4 months of age. The prevalence of Clostridium perfringens was higher in formula-fed infants than in breast-fed over the study period, and that of C. difficile at 4 months. Among breastfed infants, a higher prevalence of enterotoxigenic E. coli (ETEC) was found in infants with the highest genetic risk compared either to those with a low or intermediate risk. Among formula-fed infants, a higher prevalence of ETEC was also found in infants with a high genetic risk compared to those of intermediate risk. Our results show that specific factors, such as formula feeding and the HLA-DQ2 genotype, previously linked to a higher risk of developing CD, influence the presence of pathogenic bacteria differently in the intestinal microbiota in early life. Further studies are warranted to establish whether these associations are related to CD onset later in life.

Influence of early environmental factors on peripheral lymphocyte subsets and gut microbiota in infants at risk for celiac disease

Genetic risk linked to the HLA (Human Leucocyte Antigen) system is not enough to explain the incidence of celiac disease (CD) and other genetic and environmental factors have been suggested to play a role . The immune system and microbiota are not fully developed at birth and single antigen encounter in the intestine is a key process to achieve full development . Environmental factors through this or other route are believed to impact on the immune system and microbiota as well. We sought to assess the effect of several early environmental factors on lymphocyte subsets and microbiota composition in infants at familial risk for CD. For this purpose, 55 infants with a first degree relative suffering CD were recruited before birth. Information on early environmental factors was prospectively collected including type of delivery, mother’s antibiotic intake during pregnancy, mother’s antibiotic administration during labour, milkfeeding practices, infections and antibiotic intake in the first 4 months of life and rotavirus vaccine administration. Lymphocyte subsets in peripheral blood and gut microbiota composition in faeces samples were studied at the age of 4 months by flow cytometry analysis and qPCR respectively. Linear regression analysis showed that, the absolute counts of total lymphocytes, CD3 + , CD4 + , CD4 +CD38+ , CD4+CD28+ , were positively associated with formula-fed infants and infant’s infections in the first 4 months of age. The percentage of CD4 +CD25 + was positively associated with vaginal delivery and antibiotic use by mothers during pregnancy, and negatively with rotavirus vaccine, and their absolute counts were associated positively with infant’s infections. The absolute counts of CD3+CD4+CD45RO + were positively associated with formula-feeding and infant’s infections, and negatively with infant’s antibiotic intake. CD4 +HLA-DR + cell counts were positively associated with infant’s infections. The percentages and absolute counts of NK cells were positively associated with infant’s infections and antibiotic administration in mothers during labor. Regarding microbiota, infant’s antibiotic intake is associated with higher counts of Bacteroides spp. and lower counts of B. longum. Cesarean delivery is associated with higher counts of B. angulatum and lower counts of B. catenulatum. B. angulatum counts were also positively associated with infant’s antibiotic administration in the first 4 months of life and negatively with formula feeding and antibiotic administration during pregnancy. In conclusion, the balance between the effects of the preand post-natal factors on lymphocyte subsets and microbiota composition might modify the risk for future development of immune-related diseases such as celiac disease.