Gestational cholestyramine treatment protects adult offspring of ApoE-deficient mice against maternal-hypercholesterolemia-induced atherosclerosis.

AIM: Perinatal hypercholesterolemia exacerbates the development of atherosclerotic plaques in adult offspring. Here, we aimed to study the effect of maternal treatment with cholestyramine, a lipid-lowering drug, on atherosclerosis development in adult offspring of hypercholesterolemic ApoE-deficient (ApoE-/-) mice. METHODS: ApoE-/- mice were treated with 3% cholestyramine (CTY) during gestation (G). After weaning, offspring (CTY-G) were fed control diet until sacrificed at 25weeks of age. Atherosclerosis development in the aortic root of offspring was assessed after oil-red-o staining, along with some of predefined atherosclerosis regulators such as LDL and HDL by high-performance liquid chromatography (HPLC), and bile acids (BA) and trimethylamine N-oxide (TMAO) by liquid chromatography-mass spectrometry (LC-MS/MS). RESULTS: In pregnant dams, cholestyramine treatment resulted in significantly lower plasma total- and LDL-cholesterol as well as gallbladder total BA levels. In offspring, both males and females born to treated dams displayed reduced atherosclerotic plaques areas along with less lipid deposition in the aortic root. No significant change in plasma total cholesterol or triglycerides was measured in offspring, but CTY-G males had increased HDL-cholesterol and decreased apolipoproteins B100 to A-I ratio. This latter group also showed reduced gallbladder total and specifically tauro-conjugated bile acid pools, whereas for CTY-G females, hydrophilic plasma tauro-conjugated BA pool was significantly higher. They also benefited from lower plasma TMAO. CONCLUSION: Prenatal cholestyramine treatment reduces atherosclerosis development in adult offspring of ApoE-/- mice along with modulating the plaques' composition as well as some related biomarkers such as HDL-C, bile acids and TMAO.

Human Breast Milk miRNAs: Their Diversity and Potential for Preventive Strategies in Nutritional Therapy.

The endogenous miRNAs of breast milk are the products of more than 1000 nonprotein-coding genes, giving rise to mature small regulatory molecules of 19-25 nucleotides. They are incorporated in macromolecular complexes, loaded on Argonaute proteins, sequestrated in exosomes and lipid complexes, or present in exfoliated cells of epithelial, endothelial, or immune origins. Their expression is dependent on the stage of lactation; however, their detection depends on progress in RNA sequencing and the reappraisal of the definition of small RNAs. Some miRNAs from plants are detected in breast milk, opening the possibility of the stimulation of immune cells from the allergy repertoire. Each miRNA harbors a seeding sequence, which targets mRNAs, gene promoters, or long noncoding RNAs. Their activities depend on their bioavailability. Efficient doses of miRNAs are estimated to be roughly 100 molecules in the cytoplasm of target cells from in vitro and in vivo experiments. Each miRNA is included in networks of stimulation/inhibition/sequestration, driving the expression of cellular phenotypes. Three types of stress applied during lactation to manipulate miRNA supply were explored using rodent offspring: a foster mother, a cafeteria diet, and early weaning. This review presents the main mature miRNAs described from current mothers' cohorts and their bioavailability in experimental models as well as studies assessing the potential of miR-26 or miR-320 miRNA families to alter offspring phenotypes.

Oral Delivery of miR-320-3p with Lipidic Aminoglycoside Derivatives at Mid-Lactation Alters miR-320-3p Endogenous Levels in the Gut and Brain of Adult Rats According to Early or Regular Weaning.

To investigate if the artificial delivery of microRNAs naturally present in the breastmilk can impact the gut and brain of young rats according to weaning. Animals from a new transgenic rat line expressing the green-fluorescent protein in the endocrine lineage (cholecystokinin expressing cells) received a single oral bolus of miR-320-3p or miR-375-3p embedded in DiOleyl-Succinyl-Paromomycin (DOSP) on D-12. The pups were weaned early (D-15), or regularly (D-30). The expression of relevant miRNA, mRNAs, chromatin complexes, and duodenal cell density were assessed at 8 h post-inoculation and on D-45. The miR-320-3p/DOSP induced immediate effects on H3K4me3 chromatin complexes with polr3d promoter (p < 0.05). On regular weaning, on D-45, miR-320-3p and 375-3p were found to be downregulated in the stomach and upregulated in the hypothalamus (p < 0.001), whereas miR-320-3p was upregulated in the duodenum. After early weaning, miR-320-3p and miR-375-3p were downregulated in the stomach and the duodenum, but upregulated in the hypothalamus and the hippocampus. Combination of miR-320-3p/DOSP with early weaning enhanced miR-320-3p and chromogranin A expression in the duodenum. In the female brain stem, miR-320-3p, miR-504, and miR-16-5p levels were all upregulated. Investigating the oral miRNA-320-3p loads in the duodenal cell lineage paved the way for designing new therapeutics to avoid unexpected long-term impacts on the brain.

Early weaning leads to disruption of homeostatic and hedonic eating behaviors and modulates serotonin (5HT) and dopamine (DA) systems in male adult rats.

Early weaning is associated with disruption of eating behavior. However, little is known about the mechanisms behind it. 5HT and DA systems are key regulators of homeostatic and hedonic eating behaviors, respectively. Thus, this study aims to evaluate the effects of early weaning on feeding behavior and 5HT and DA systems. For this, rats were submitted to regular (PND30) or early weaning (PND15) and between PND250 and PND300 were evaluated food intake of standard diet in response to 4 h food deprivation, during the 24 h period and per phase of the circadian cycle, in addition to the palatable food intake. Additionally, body mass and mRNA expression of 5HT1B, 5HT2C, SERT, DRD1 and DRD2 were evaluated in the hypothalamus and brainstem. The results demonstrate that early weaning promoted an increase in standard food intake in response to a 4 h food deprivation in the 24 h period and in the dark phase of the circadian cycle, in addition to an increased palatable food intake. No differences in body mass between regular or early weaning were observed. In the hypothalamus, increased mRNA expression of SERT and DRD1 was observed, but decreased 5HT1B mRNA expression. In the brainstem, the expression of 5HT1B, SERT, 5HT2C, DRD1 and DRD2 was increased in early weaned rats. In a nutshell, the stress promoted by early weaning has programmed the animals to be hyperphagic and to increase their palatable food intake, which was associated with modulation of 5HT and DA systems.

Oral Delivery of miRNA With Lipidic Aminoglycoside Derivatives in the Breastfed Rat.

CONTEXT: Specific targeting of endogenous miRNAs which are involved in epigenetics, may help understanding homeostasis with therapeutic benefits. We use new biologically inspired vehicles consisting of lipoaminoglycosides to deliver in vivo mir-320-3p, a known human breast milk exosomal miRNA, or its antagomiR. MATERIALS AND METHODS: Four lipoaminoglycosides were screened for cytotoxicity and their biophysical properties. 1-h breast-restricted rats received single-oral treatment of either the lipoaminoglycoside Dioleyl-Succinyl Paromomycin (DOSP) complexed with miRNA or antagomiR, or of control medium at the light on (ZeitGeber Time: ZT-0H) or off (ZT-12H). Glycemia, triglycerides, cholesterol, free-fatty acid were assayed at 0, 4, 8, and 12 h post-treatment. In the stomach, small intestine, liver, plasma, adipose tissue, plexus choroid, and cortex, relevant miRNA with precursors and mRNA (polr3d, hspb6, c-myc, stat1, clock, bmal1, per1, npas2, sirt1-6, and cyclinD1) were quantified by q-PCR. Expression of POLR3D and HSPB6 proteins were analyzed in stomach and liver by Western blot. Immunoprecipitations with anti-AGO1 and 2 were performed on nuclear and cytoplasmic fractions of gastric cells along with detection of miRNA-320-3p in nucleoli. Chromatin ImmunoPrecipitation with anti-Trimethyl-histone-3-Lys-4 and Lys-27 detecting the polr3d promoter and miR-320-3p, were performed for all groups. RESULTS: Selected DOSP (diameter: 80-200 nm) did not alter gastric extracellular vesicle secretion a few hours after intake. The miR-320-3p was mainly found in gastric or small intestinal cells, reaching the blood and liver in low amount. We have found significant up-regulation of polr3d mRNA (ANOVA, p < 0.0001) at ZT-20H for the miR-320-3p-supplemented group and a higher expression of POLR3D for antagomiR group (ANOVA, p < 0.05). We had a low accumulation of miR-320-3p at ZT-20H in nucleoli, without stat1 evolution. Delivering a high amount of miRNA or antagomiR disrupts RNA-Induced Silencing Complexes in cytoplasm triggering some transfer of extracellular molecules into nuclei with alteration of immune complexes on the polr3d promoter (with a higher amount found in the K4 histone-3-me3 immune complexes at ZT-20H). CONCLUSION: Extracellular miRNAs embedded in DOSP have a rapid impact on RNAi and on nuclear chromatin complexes depending on the daily rhythm. An integrative view of the impact of extracellular miRNA on physiology will improve assaying epigenetic manipulations following nutritional stress.

Effectiveness of an antenatal maternal supplementation with prebiotics for preventing atopic dermatitis in high-risk children (the PREGRALL study): protocol for a randomised controlled trial.

INTRODUCTION: Atopic dermatitis (AD) is a chronic inflammatory disease affecting 10%-15% of children in Europe. There is a need for new primary preventive therapeutic strategies in at-risk populations. Recent research has indicated that atopic diseases are associated with a disrupted gut microbial 'balance' in early life raising the possibility that interventions which yield optimal patterns of microflora could improve host's health. Prebiotics, sugars with immunomodulatory properties that stimulate the diversity of the digestive microbiota, are ideal candidates for such research. So far, most clinical trials have focused on improving infant gut colonisation postnatally. However, prenatal life is a crucial period during which different tolerance mechanisms are put in place. We aim to determine whether antenatal prebiotics supplementation prevents AD in high-risk children. METHODS AND ANALYSIS: This is a randomised, multicentre, double-blind, trial to evaluate the effectiveness of antenatal prebiotic maternal supplementation (galacto-oligosaccharide/inulin) in pregnant women versus placebo on the occurrence of AD at 1 year of age in at-risk children (defined as having a maternal history of atopic disease). Participating women will be randomised to daily ingestion of a prebiotics or placebo (maltodextrin) from 20 weeks' gestation until delivery. The primary outcome is the prevalence of AD at 1 year of age, using the version of the UK Working Party Diagnostic Criteria optimised for preventive studies. Key secondary endpoints are AD severity, quality of life and prebiotics tolerance. The target sample size is 376 women (188 patients per group) which will provide 80% power to detect a 33% reduction of the risk of AD in the verum group (α=0.05). The primary analysis will be based on the intention-to-treat principle. ETHICS AND DISSEMINATION: Results will be presented in peer-reviewed journals and at international conferences. Ethics approval for the study was obtained from the institutional ethical review board of 'Comité de Protection des Personnes Sud Ouest-Outre-Mer III' of the University Hospital Centre of Bordeaux (2017/13). TRIAL REGISTRATION NUMBER: NCT03183440; Pre-results.

Exosomes and miRNA-Loaded Biomimetic Nanovehicles, a Focus on Their Potentials Preventing Type-2 Diabetes Linked to Metabolic Syndrome.

Exosomes are small membrane vesicles of 30-150 nm, members of the extracellular vesicle family and secreted by various cell types. Different studies describe specific microRNA (miRNA) with altered expression in serum and/or plasma of patients suffering from diabetes or metabolic syndrome. Diabetic cardiomyocyte-derived exosomes loaded with miRNAs like miR-320-3p (or 320a) have been shown regulating angiogenesis on endothelial cell cultures. Insufficient myocardial angiogenesis is the major manifestation of diabetes-caused ischemic cardiovascular disease. Studies on transfer of functional microRNAs between mouse dendritic cells via exosomes have shown that some miRNAs (miR-320-3p, 29b-3p, 7a-5p) are distributed in immature and mature exosomes. Among these miRNAs, miR-320-3p is better known in epigenetics for silencing polr3d gene by binding to its promoter in Human Embryonic Kidney-293 cells. Moreover, quantitative and stoichiometric analysis of the microRNA content of exosomes highlights the lack of reliable natural source of such particles loaded with miRNA opening the need for tailoring exosomes or nanoparticles delivering efficiently miRNA intimately linked to immunity, metabolism and epigenetics in target cells. However, loading of extracellular mature miRNA into recipient cells comes with a cost by at least impeding dynamic localization of miRNAs in nucleoli or inefficient miRNA delivery due to rapid recycling by exonucleases. All these works are calling for the design of new biomimetic vehicles and in vivo assessment of miRNA functionality when delivered by natural or biomimetic nanoparticles in order to control metabolic diseases from infancy to adulthood.

Maternal protein restriction during gestation and lactation in the rat results in increased brain levels of kynurenine and kynurenic acid in their adult offspring.

Early malnutrition is a risk factor for depression and schizophrenia. Since the offspring of malnourished dams exhibit increased brain levels of serotonin (5-HT), a tryptophan-derived neurotransmitter involved in the pathophysiology of these mental disorders, it is believed that the deleterious effects of early malnutrition on brain function are due in large part to altered serotoninergic neurotransmission resulting from impaired tryptophan (Trp) metabolism. However, tryptophan is also metabolized through the kynurenine (KYN) pathway yielding several neuroactive compounds including kynurenic (KA), quinolinic (QA) and xanthurenic (XA) acids. Nevertheless, the impact of perinatal malnutrition on brain kynurenine pathway metabolism has not been examined to date. Here, we used ultra-performance liquid chromatography-tandem mass spectrometry for the simultaneous quantification of tryptophan and a set of seven compounds spanning its metabolism through the serotonin and kynurenine pathways, in the brain of embryos and adult offspring of rat dams fed a protein-restricted (PR) diet. Protein-restricted embryos showed reduced brain levels of Trp, serotonin and KA, but not of KYN, XA, or QA. In contrast, PR adult rats exhibited enhanced levels of Trp in the brainstem and cortex along with increased concentrations of 5-HT, kynurenine and XA. The levels of XA and KA were also increased in the hippocampus of adult PR rats. These results show that early protein deficiency induces selective and long-lasting changes in brain kynurenine metabolism. Given the regulatory role of KYN pathway metabolites on brain development and function, these changes might contribute to the risk of developing psychiatric disorders induced by early malnutrition.

Evidence-Based Clinical Use of Nanoscale Extracellular Vesicles in Nanomedicine.

Recent research has demonstrated that all body fluids assessed contain substantial amounts of vesicles that range in size from 30 to 1000 nm and that are surrounded by phospholipid membranes containing different membrane microdomains such as lipid rafts and caveolae. The most prominent representatives of these so-called extracellular vesicles (EVs) are nanosized exosomes (70-150 nm), which are derivatives of the endosomal system, and microvesicles (100-1000 nm), which are produced by outward budding of the plasma membrane. Nanosized EVs are released by almost all cell types and mediate targeted intercellular communication under physiological and pathophysiological conditions. Containing cell-type-specific signatures, EVs have been proposed as biomarkers in a variety of diseases. Furthermore, according to their physical functions, EVs of selected cell types have been used as therapeutic agents in immune therapy, vaccination trials, regenerative medicine, and drug delivery. Undoubtedly, the rapidly emerging field of basic and applied EV research will significantly influence the biomedicinal landscape in the future. In this Perspective, we, a network of European scientists from clinical, academic, and industry settings collaborating through the H2020 European Cooperation in Science and Technology (COST) program European Network on Microvesicles and Exosomes in Health and Disease (ME-HAD), demonstrate the high potential of nanosized EVs for both diagnostic and therapeutic (i.e., theranostic) areas of nanomedicine.

[The new paradigm of the developmental origin of health and diseases (DOHaD)--Epigenetics and environment: evidence and missing links]. / Le nouveau paradigme de l'origine développementale de la santé et des maladies (DOHaD)--Épigénétique, environnement: preuves et chaînons manquants.

According to the new paradigm of the Developpemental Origins of Health and Disease (DOHaD), the environmental factors to which an individual is exposed throughout his life can leave an epigenetic footprint on the genome. A crucial period is the early development, where the epigenome is particularly sensitive to the effects of the environment, and during which the individual builds up his health capital that will enable him to respond more or less well to the vagaries of life. The research challenge is to decipher the modes of action and the epigenetic mechanisms put into play by environmental factors that lead to increased disease susceptibility or resilience. The challenge for health is to translate these scientific discoveries into action through, among others, the establishment of preventive recommendations to slow down the growing incidence of non communicable diseases.

[Epigenetics in transgenerational responses to environmental impacts: from facts and gaps]. / Épigénétique et réponses transgénérationnelles aux impacts de l'environnement--Des faits aux lacunes.

The existence of non-genetic and non-cultural mechanisms that transfer information on the memory of parental exposures to various environments, determining the reactivity of the following generations to their environments during their life, are of growing interest. Yet fundamental questions remain about the nature, the roles and relative importance of epigenetic marks and processes, non-coding RNAs, or other mechanisms, and their persistence over generations. A model incorporating the various transmission systems, their cross-talks and windows of susceptibility to the environment as a function of sex/gender of parent and offspring, has yet to be built.

[The sensory and nutritional experience of parents guide offsprings' feeding behavior]. / L'expérience sensorielle et nutritionnelle des parents et leur état métabolique orientent le comportement alimentaire de leur descendance.

Eating behavior is strongly regulated by intrinsic physiological factors and largely influenced by the individual and cultural environments. Excessive food intake and sedentary lifestyle are the main reasons for the global epidemic of obesity. The influence of family background on eating habits makes no doubt but the fact that the nutritional, metabolic and hormonal status of the parents before conception, and of the mother during gestation and lactation, may influence the child's future eating behavior is an innovative concept that opens the way for preventive policies. In the last decades, research on human cohorts and animal models have targeted biological mechanisms (neuroanatomical, epigenetic) that give some clues on how eating behavior can be formatted by early nutrition and related sensory experience.

Maternal and fetal tryptophan metabolism in gestating rats: effects of intrauterine growth restriction.

L-Tryptophan (L-Trp) is a precursor for serotonin (5-HT) and nicotinamide adenine dinucleotide (NAD) synthesis. Both 5-HT and NAD may impact energy metabolism during gestation given that recent studies have demonstrated that increased 5-HT production is crucial for increasing maternal insulin secretion, and that sirtuin, an NAD(+)-dependent protein deacetylase, regulates endocrine signaling. Infants born with intrauterine growth restriction (IUGR) are at a higher risk of metabolic disease once they reach adulthood. IUGR is associated with altered maternal-fetal amino acid transfer. Whether IUGR affects L-Trp metabolism in mother and fetus has not been fully elucidated. Recently, we developed an analytical method using stable isotope-labeled L-Trp to explore the metabolism of L-Trp and its main metabolites, L-kynurenine (L-Kyn), 5-HT and quinolinic acid (QA). In this study, dams submitted to dietary protein restriction throughout gestation received intravenous infusions of stable isotope-labeled (15)N2-L-Trp to determine whether L-Trp metabolism is affected by IUGR. Samples were obtained from maternal, fetal and umbilical vein plasma, as well as the amniotic fluid (AF), placenta and liver of the mother and the fetus after isotope infusion. We observed evidence for active L-Trp transfer from mother to fetus, as well as de novo synthesis of 5-HT in the fetus. Plasma 5-HT was decreased in undernourished mothers. In IUGR fetuses, maternal-fetal L-Trp transfer remained unaffected, but conversion to QA was impaired, implying that NAD production also decreased. Whether such alterations in tryptophan metabolism during gestation have adverse consequences and contribute to the increased risk of metabolic disease in IUGR remains to be explored.

MiRNA Analysis by Quantitative PCR in Preterm Human Breast Milk Reveals Daily Fluctuations of hsa-miR-16-5p.

BACKGROUND AND AIMS: Human breast milk is an extremely dynamic fluid containing many biologically-active components which change throughout the feeding period and throughout the day. We designed a miRNA assay on minimized amounts of raw milk obtained from mothers of preterm infants. We investigated changes in miRNA expression within month 2 of lactation and then over the course of 24 hours. MATERIALS AND METHODS: Analyses were performed on pooled breast milk, made by combining samples collected at different clock times from the same mother donor, along with time series collected over 24 hours from four unsynchronized mothers. Whole milk, lipids or skim milk fractions were processed and analyzed by qPCR. We measured hsa-miR-16-5p, hsa-miR-21-5p, hsa-miR-146-5p, and hsa-let-7a, d and g (all -5p). Stability of miRNA endogenous controls was evaluated using RefFinder, a web tool integrating geNorm, Normfinder, BestKeeper and the comparative ΔΔCt method. RESULTS: MiR-21 and miR-16 were stably expressed in whole milk collected within month 2 of lactation from four mothers. Analysis of lipids and skim milk revealed that miR-146b and let-7d were better references in both fractions. Time series (5H-23H) allowed the identification of a set of three endogenous reference genes (hsa-let-7d, hsa-let-7g and miR-146b) to normalize raw quantification cycle (Cq) data. We identified a daily oscillation of miR-16-5p. PERSPECTIVES: Our assay allows exploring miRNA levels of breast milk from mother with preterm baby collected in time series over 48-72 hours.

Long-lasting effect of perinatal exposure to L-tryptophan on circadian clock of primary cell lines established from male offspring born from mothers fed on dietary protein restriction.

BACKGROUND AIMS: Maternal undernutrition programs metabolic adaptations which are ultimately detrimental to adult. L-tryptophan supplementation was given to manipulate the long-term sequelae of early-life programming by undernutrition and explore whether cultured cells retain circadian clock dysregulation. METHODS: Male rat pups from mothers fed on low protein (8%, LP) or control (18%, CP) diet were given, one hour before light off, an oral bolus of L-tryptophan (125 mg/kg) between Day-12 and Day-21 of age. Body weight, food intake, blood glucose along with the capacity of colonization of primary cells from biopsies were measured during the young (45-55 days) and adult (110-130 days) phases. Circadian clock oscillations were re-induced by a serum shock over 30 hours on near-confluent cell monolayers to follow PERIOD1 and CLOCK proteins by Fluorescent Linked ImmunoSorbent Assay (FLISA) and period1 and bmal1 mRNA by RT-PCR. Cell survival in amino acid-free conditions were used to measure circadian expression of MAP-LC3B, MAP-LC3B-FP and Survivin. RESULTS: Tryptophan supplementation did not alter body weight gain nor feeding pattern. By three-way ANOVA of blood glucose, sampling time was found significant during all phases. A significant interaction between daily bolus (Tryptophan, saline) and diets (LP, CP) were found during young (p = 0.0291) and adult (p = 0.0285) phases. In adult phase, the capacity of colonization at seeding of primary cells was twice lower for LP rats. By three-way ANOVA of PERIOD1 perinuclear/nuclear immunoreactivity during young phase, we found a significant effect of diets (p = 0.049), daily bolus (p<0.0001) and synchronizer hours (p = 0.0002). All factors were significantly interacting (p = 0.0148). MAP-LC3B, MAP-LC3B-FP and Survivin were altered according to diets in young phase. CONCLUSIONS: Sequelae of early-life undernutrition and the effects of L-tryptophan supplementation can be monitored non-invasively by circadian sampling of blood D-glucose and on the expression of PERIOD1 protein in established primary cell lines.

Non-invasive exploration of neonatal gastric epithelium by using exfoliated epithelial cells.

BACKGROUND & AIMS: In preterm infants, exfoliated gastric epithelial cells can be retrieved from aspirates sampled through the naso-gastric feeding tube. Our aims were to determine (1) whether the recovery of exfoliated cells is feasible at any time from birth through the removal of the nasogastric tube, (2) whether they can be grown in culture in vitro, and (3) whether the physiological state of exfoliated cells expressing H+/K+ -ATPases reflects that of their counterparts remaining in situ at the surface of the gastric epithelium in neonatal rat pups. METHODS: In infants, gastric fluid aspirates were collected weekly after birth or every 3 hours over 24-h periods, and related to clinical parameters (Biocollection PROG/09/18). In rat pups submitted to a single fasting/refeeding cycle, we explored circadian exfoliation with the cellular counter-parts in the gland. All samples were analyzed by confocal imaging and Enzyme-Linked Immunosorbent Assay. RESULTS: Epithelial cells were identified by microscopy using membrane-bound anti-H+/K+ ATPases antibody, assessed for nucleus integrity, and the expression of selected proteins (autophagy, circadian clock). On 34 infants, the H+/K+-ATPase-positive cells were consistently found quiescent, regardless of gestational age and feeding schedule from day-5 of life to the day of removal of the naso-gastric tube. By logistic regression analysis, we did find a positive correlation between the intensity of exfoliation (cellular loss per sample) and the postnatal age (p<0.001). The H+/K+ ATPase-positive cells established in culture retained the expression of a biomarker of progenitor status (Pouf5F1-Oct4). In rat pups, the expression pattern of Survivin in H+/K+ ATPase-positive exfoliated cells paralleled that observed in cells remaining at the surface of the gastric gland. CONCLUSIONS: Tracking parietal cells can improve clinical monitoring and understanding of the autophagic death via the phosphatidylinositol 3-kinase/Akt/survivin pathway.

Exfoliated epithelial cells: potentials to explore gastrointestinal maturation of preterm infants / Células epiteliais exfoliadas: potencial para investigar a maturação gastrointestinal em crianças pré-termo

Exfoliated epithelial cells represent valuable source of information on the physiopathological state of the mucosa. However, the interpretation of data obtained from exfoliated cells is complicated by the conditions of isolation as well as the health of the subject. Exfoliation is either: a) a natural loss of body cells implying a molecular signal related to the turnover of terminally differentiated cells and to the progressive mobilization of proliferative as well as stem cells or b) the result of manual exfoliation by applying mechanical constraints like scraping. Depending on the methodology of isolation, exfoliated epithelial cells are believed to be either in apoptosis or in anoïkis. Most studies are using microscopic examination to demonstrate the presence of typical cells along with measurements on a limited number of biomarkers. Only few studies using proteomics or transcriptomics are available and they open discussion about tissue references and normalization. The main advantage of measures realized on exfoliated epithelial cells is that they are strictly non-invasive and open the possibility to evaluate maturation of gastric and intestinal tissues in long-term experiments performed on the same animal or in translational research on samples recovered from preterm infants.

Células epiteliais exfoliadas são uma fonte de informação valiosa sobre o estado fisiológico da mucosa. Contudo, a interpretação dos dados obtidos de células exfoliadas é complicado pelas condições de isolamento e da saúde do sujeito. A exfoliação é: a) uma perda natural de células implicando um sinal molecular relacionado ao turnover terminal de células diferenciadas e à progressiva mobilização de células proliferativas e de células tronco ou b) o resultado de aplicação de fatores mecânicos como raspagem. Dependendo do método de islolamento, acredita-se que podem estar em apoptose ou em anoikis. A maior parte dos estudos tem utilizado exame microscópico para demonstrar a presença de células típicas com medições sobre um número limitado de biomarcadores. Apenas uns poucos estudos usando proteômica ou transcriptômica estão disponíveis e discutem sobre referenciais de tecidos e normalização. A principal vantagem de medidas realizadas em células epiteliais exfoliadas é que elas são estritamente não invasivas e abertas à possibilidade de avaliar a maturação de tecidos gástricos e intestinais em experimentos de longo prazo feitos no mesmo animal ou em pesquisas translacionais com amostras obtidas de crianças pré-termo.

Recovery of exfoliated cells from the gastrointestinal tract of premature infants: a new tool to perform "noninvasive biopsies?".

To gain insight into specific gene expression in the gastrointestinal (GI) tract of preterm infants, we adapted a method to isolate exfoliated epithelial cells. Gastric residual fluid aspirates (n = 89) or stool samples (n = 10) were collected from 96 neonates (gestational age, 24-36 wk). Cells were characterized by microscopic observation, cytokeratin-18 immunodetection, and expression of transcripts. The human origin of cellular DNA was confirmed by amplification of specific X and Y chromosome sequences. Isolation yielded 100-500 cells per sample for gastric aspirates (n = 8) and 10-20 cells for fecal samples (n = 5). Epithelial origin was confirmed by immunodetection of cytokeratin 18. Analyses of reverse transcribed products, using two independent methods, from 15 gastric fluid and two stool samples showed that 18S-rRNA and transcripts of beta-actin, glyceraldehyde-3-phosphate dehydrogenase (gapdh), and period1 were in quantities corresponding to at least 10 cells. On 59 aspirates, we found beta-actin transcripts (all but one), cytokeratin 18 (eight positive of eight samples), SLC26-A7-1 (13 positive of 19 samples), period2 (17 positive of 17 samples), and clock (25 positive of 26 samples). Exfoliated cells can be recovered from gastric aspirates and fecal samples and serve as a tool to investigate the impact of therapeutic and nutritional regimens on the maturation of GI functions.