Non-Invasive Exploration of Neonatal Gastric Epithelium by Using Exfoliated Epithelial Cells

Abstract

Background & AimsIn 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.MethodsIn 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.ResultsEpithelial 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.ConclusionsTracking parietal cells can improve clinical monitoring and understanding of the autophagic death via the phosphatidylinositol 3-kinase/Akt/survivin pathway.

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

“…The re-induction of PERIOD1 protein expression in our primary cells observed between 6 and 18 h was similar to the PERIOD1 reactivity described in rat brain region between 6 and 13 h [ 23 ]. By focusing on the perinuclear and nuclear localization of PERIOD1, we have been able to appreciate the level of synchronization of our cells (PERIOD1 can be detected during transit at the perinuclear and nuclear membranes locations) as well as the total nuclear intensity of expression according to previously described methods [23], [35]. A daily bolus of L-tryptophan had a profound effect on the profiles of PERIOD1 protein expression for both diets.…”

“…The general aim was to design functional assays with living cells which may be sampled in long term experiments under similar conditions as ours or with humans by non-invasive means like skin fibroblasts [26], hair follicles [51], urinary cells [52] or exfoliated cells of gastric [35], or nasal [53] epitheliums. The availability of 50 primary cell lines retaining nutritional stress-related alterations in PERIOD1 expression open the way to design functional assays on living cells on the dynamic of the circadian epigenome [54] like determining if the profile of H3K9/K14 histone acetylation [55] in fibroblasts is comparable to the one found in neurons.…”

“…For quantification, preparations were observed under a Leica fluorescent video microscope (x 40 magnification) with Metamorph software. The intensity of labeling by the primary – secondary antibodies complex was normalized by the total surface of the cellular body at the best plane of acquisition by densitometry with ImageJ 1.42. software as described [35].…”

Noninvasive molecular fingerprinting of host–microbiome interactions in neonates

“…Subsequently, exfoliated epithelial cells had been used as sentinels of in vivo exposure to nutritional regimens [55,56] or as markers of disease states, including cancer in adults [57,58] and children with inflammatory bowel disease [59]. More recently, the feasibility of identifying protein markers and amplifying genes by polymerase chain reaction was demonstrated in exfoliated gastric cells from preterm infants [52,60]. Kaeffer and colleagues studied isolated exfoliated cells obtained from gastric aspirates [52,60] and stool samples [52] obtained from preterm infants.…”

“…inflammatory bowel disease [59]. More recently, the feasibility of identifying protein markers and amplifying genes by polymerase chain reaction was demonstrated in exfoliated gastric cells from preterm infants [52,60]. Kaeffer and colleagues studied isolated exfoliated cells obtained from gastric aspirates [52,60] and stool samples [52] obtained from preterm infants.…”