Amalia Capilla

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.

Bioengineering of functional human induced pluripotent stem cell-derived intestinal grafts

Patients with short bowel syndrome lack sufficient functional intestine to sustain themselves with enteral intake alone. Transplantable vascularized bioengineered intestine could restore nutrient absorption. Here we report the engineering of humanized intestinal grafts by repopulating decellularized rat intestinal matrix with human induced pluripotent stem cell-derived intestinal epithelium and human endothelium. After 28 days of in vitro culture, hiPSC-derived progenitor cells differentiate into a monolayer of polarized intestinal epithelium. Human endothelial cells seeded via native vasculature restore perfusability. Ex vivo isolated perfusion testing confirms transfer of glucose and medium-chain fatty acids from lumen to venous effluent. Four weeks after transplantation to RNU rats, grafts show survival and maturation of regenerated epithelium. Systemic venous sampling and positron emission tomography confirm uptake of glucose and fatty acids in vivo. Bioengineering intestine on vascularized native scaffolds could bridge the gap between cell/tissue-scale regeneration and whole organ-scale technology needed to treat intestinal failure patients.There is a need for humanised grafts to treat patients with intestinal failure. Here, the authors generate intestinal grafts by recellularizing native intestinal matrix with human induced pluripotent stem cell-derived epithelium and human endothelium, and show nutrient absorption after transplantation in rats.

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.

Oblique scanning laser microscopy for simultaneously volumetric structural and molecular imaging using only one raster scan

Multi-modal three dimensional (3D) optical imaging combining both structural sensitivity and molecular specificity is highly desirable in biomedical research. In this paper, we present a method termed oblique scanning laser microscopy (OSLM) to combine optical coherence tomography (OCT), for simultaneously volumetric structural and molecular imaging with cellular resolution in all three dimensions. Conventional 3D laser scanning fluorescence microscopy requires repeated optical sectioning to create z-stacks in depth. Here, the use of an obliquely scanning laser eliminates the z-stacking process, then allows highly efficient 3D OCT and fluorescence imaging by using only one raster scan. The current setup provides ~3.6 × 4.2 × 6.5 μm resolution in fluorescence imaging, ~7 × 7 × 3.5 μm in OCT in three dimensions, and the current speed of imaging is up to 100 frames per second (fps) over a volume about 0.8 × 1 × 0.5 mm3. We demonstrate several mechanisms for molecular imaging, including intrinsically expressed GFP fluorescence, autofluorescence from Flavin proteins, and exogenous antibody-conjugated dyes. We also demonstrate potential applications in imaging human intestinal organoids (HIOs), colon mucosa, and retina.

Modeling APC mutagenesis and familial adenomatous polyposis using human iPS cells

Mutations in the gene Adenomatous Polyposis Coli or APC appear in most sporadic cases of colorectal cancer and it is the most frequent mutation causing hereditary Familial Adenomatous Polyposis. The detailed molecular mechanism by which APC mutations predispose to the development of colorectal cancer is not completely understood. This is in part due to the lack of accessibility to appropriate models that recapitulate the early events associated with APC mediated intestinal transformation. We have established a novel platform utilizing human induced Pluripotent Stem cells or iPSC from normal or FAP-specific APC mutant individuals and evaluated the effect of the mutation in the cells before and after differentiation into intestinal organoids. In order to minimize genetic background effects, we also established an isogenic platform using TALEN-mediated gene editing. Comparison of normal and APC mutant iPSC revealed a significant defect in cell identity and polarity due to the presence of APC in heterozygosity as well as chromosomal aberrations including abnormal anaphases and centrosome numbers. Importantly, upon specification into intestinal progeny, APC heterozygosity was responsible for a major change in the transcriptional identity of the cells with dysregulation of key signaling pathways, including metabolic reprogramming, abnormal lipid metabolism and intestinal-specific cadherin expression. In conclusion, we have developed a novel iPSC/intestinal model of APC mutagenesis and provide strong evidence that APC in heterozygosity imparts a clear phenotypic and molecular defect, affecting basic cellular functions and integrity, providing novel insights in the earlier events of APC-mediated tumorigenesis.

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.

Peripheral lymphocyte subsets in infants at risk for celiac disease. Effect of milk feeding practices and HLA genotype. The PROFICEL study

T. Pozo, A. Capilla, A. Marcos, Y. Sanz, G. De Palma, I. Polanco, M. D. Garcı́a-Novo, G. Castillejo, C. Ribes-Koninckx, V. Varea, J. A. Garrote, C. Calvo, F. Palau and E. Nova Grupo de Inmunonutricion, Instituto del Frı́o-ICTAN, CSIC, Madrid, Spain, Instituto de Biomedicina de Valencia (CSIC), Valencia, Spain, Ecofisiologı́a Microbioana, Instituto de Agroquı́mica y Tecnologı́a de Alimentos (CSIC), Valencia, Spain, Gastroenterologı́a, Nutrición y Hepatologı́a Pediátrica, Hospital Universitario Sant Joan de Deu and Institut Dexeus, Barcelona, Spain, Servicio de Gastroenterologia y Nutrición Pediátrica, Hospital Universitario La Paz, Madrid, Spain, Unidad de Gastroenterologı́a Pediátrica, Hospital Universitario Sant Joan de Reus, Tarragona, Spain, Unidad de Gastroenterologia, Hospital Infantil Universitario La Fe, Valencia, Spain, Research Unit and Paediatrics Service, Hospital Clı́nico Universitario, Valladolid, Spain and Unidad de Gastroenterologia, Hospital Universitario Infantil Niño Jesús, Madrid, Spain

Gut colonisation process of newborns and breast-fed babies at risk of developing coeliac disease

G. De Palma, I. Nadal, A. Capilla, E. Nova, A. Marcos, T. Pozo, V. Varea, I. Polanco, G. Castillejo, C. Ribes-Coninckx, J. A. Garrote, C. Calvo, M. D. Garcia-Novo, A. López, M. L. Cilleruelo, F. Palau and Y. Sanz Instituto de Agroquı́mica y Tecnologı́a de Alimentos (CSIC), P.O. Box 73, 46100 Burjassot-Valencia, Spain, Instituto de Biomedicina de Valencia (CSIC), Valencia, Spain, Instituto del Frı́o-ICTAN (CSIC), Madrid, Spain, Gastroenterologı́a, Nutrición y Hepatologı́a Pediátrica, Hospital Universitario Sant Joan de Deu and Institut Dexeus, Barcelona, Spain, Servicio de Gastroenterologia y Nutrición Pediátrica, Hospital Universitario La Paz, Madrid, Spain, Unidad de Gastroenterologı́a Pediátrica, Hospital Universitario Sant Joan de Reus, Tarragona, Spain, Unidad de Gastroenterologia, Hospital Infantil Universitario La Fe, Valencia, Spain, Research Unit and Paediatrics Service, Hospital Clı́nico Universitario, Valladolid, Spain, Unidad de Gastroenterologia, Hospital Universitario Infantil Niño Jesús, Madrid, Spain and Servicio de Pediatrı́a, Hospital Severo Ochoa, Leganés, Madrid, Spain