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Live colonocytes in newborn stool: surrogates for evaluation of gut physiology and disease pathogenesis
Dinesh S Chandel1, Gheorghe T Braileanu, June-Home J Chen
1Department 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.
Insights
Live colonocytes in infant stool offer a noninvasive method to study neonatal gut health. This approach tracks immune markers like IgA and TLR4, aiding in understanding early gastrointestinal pathophysiology.
Area of Science:
- Neonatal immunology
- Gastrointestinal pathophysiology
- Cellular biology
Background:
- Studying neonatal gastrointestinal pathophysiology via biopsies is invasive and often infeasible.
- Live colonocytes in stool present a potential noninvasive alternative for cellular analysis in neonates.
Purpose of the Study:
- To evaluate the utility of live colonocytes isolated from neonatal stool for studying cellular markers.
- To analyze the expression of immune and cellular markers in early neonatal life using stool-derived colonocytes.
Main Methods:
- Colonocytes were isolated from neonatal stool samples.
- Flow cytometry was used to analyze the expression of IgA, IgG, CD45, TLR2, and TLR4.
- Quantitative real-time PCR (qRT-PCR) was employed to assess the expression of cytokeratin-19, ribosomal protein-24, and ZO-1.
Main Results:
- Colonocyte yields ranged from 5 × 10⁴ to 2 × 10⁶ cells/g, with meconium providing enriched viable cells.
- CD45-positive cells were detected in all samples; IgA was present in 69% by day 2, and IgG showed a linear increase.
- TLR4 expression was observed in >55% of colonocytes, while TLR2 staining was minimal.
Conclusions:
- Stool-derived colonocytes are a viable noninvasive tool for neonatal gut research.
- High IgA may protect the neonatal gut, but increased TLR4 could indicate susceptibility to LPS-mediated damage.
- This method facilitates the study of gut pathophysiology during the critical neonatal period.
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.
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