title: Live colonocytes in newborn stool: surrogates for evaluation of gut physiology and disease pathogenesis.
abstract: Studies of gastrointestinal pathophysiology are not feasible by biopsies in human neonates. We examined the utility of live colonocytes in stool in studying cellular markers during early neonatal life. Expression of IgA, IgG, cluster of differentiation-45 cells (CD45), and toll-like receptors-2 and 4 (TLR2 and TLR4) were analyzed by flow cytometry. Colonocyte RNA extracts were used in quantitative real-time PCR (qRT-PCR) to examine the expression of cytokeratin-19, ribosomal protein-24, and tight-junction (Tj) protein zonula occludens-1 (ZO-1). Colonocyte yield varied between 5 × 10⁴ to 2 × 10⁶ cells/g of stool. Meconium samples yielded a highly enriched population of viable cells. Although low, all samples showed CD45-positive cells during the initial weeks of life. Starting as early as d 2, IgA expression was observed in 69% of the cells. Low to moderate expression of IgG was observed with a linear increase as the infants grew. There was an almost total lack of TLR2 staining; however, >55% of the colonocytes showed TLR4 expression. Although high levels of IgA in gut cells may serve as a natural protectant during neonatal period, increased TLR4 may provide a niche for lipopolysaccharide (LPS)-mediated epithelial damage. Use of stool colonocytes can be a valuable noninvasive approach for studying gut pathophysiology in the neonatal period.
authors.affiliation: Department of Environmental, Agricultural and Occupational Health, Center for Global Health and Development, College of Public Health, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA.
authors.forename: Dinesh S
authors.initials: DS
authors.lastname: Chandel
authors.forename: Gheorghe T
authors.initials: GT
authors.lastname: Braileanu
authors.forename: June-Home J
authors.initials: JH
authors.lastname: Chen
authors.forename: Hegang H
authors.initials: HH
authors.lastname: Chen
authors.forename: Pinaki
authors.initials: P
authors.lastname: Panigrahi
mesh_terms: D015415:Biomarkers / Q000378:metabolism*; D003106:Colon / Q000166:cytology*; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D005243:Feces / Q000166:cytology*; D005434:Flow Cytometry; D041981:Gastrointestinal Tract / Q000378:metabolism / Q000502:physiology* / Q000503:physiopathology*; D006801:Humans; D007070:Immunoglobulin A / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism; D007231:Infant, Newborn; D053539:Keratin-19 / Q000378:metabolism; D017493:Leukocyte Common Antigens / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012269:Ribosomal Proteins / Q000378:metabolism; D051195:Toll-Like Receptor 2 / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000378:metabolism; D062826:Zonula Occludens-1 Protein
chemical_list: D015415:Biomarkers; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D053539:Keratin-19; D008565:Membrane Proteins; D010750:Phosphoproteins; D012269:Ribosomal Proteins; C569042:TJP1 protein, human; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D062826:Zonula Occludens-1 Protein; D017493:Leukocyte Common Antigens