We’d initially planned to add insulin-like growth aspect (IGF)-I and IGF-II measurements but finally didn’t pursue these analytes because rat amniotic liquid was small in quantity and because individual amniotic liquid IGF-I and IGF-II amounts reported in existing books (39) were a log-fold less than the concentrations of HGF we detected inside our amniotic liquid samples. Experimental NEC Amniotic liquid supplementation. NCT-501 Amniotic liquid elevated cell migration, proliferation, and cell success in vitro. These results had been reproduced by HGF and obstructed by anti-HGF antibody or a PI3K inhibitor. HGF transactivated many RTKs in IEC6 cells, indicating that its results extended to multiple signaling pathways. Finally, similar to amniotic fluid, recombinant HGF also reduced the frequency and severity of NEC-like injury in rat pups. Amniotic fluid supplementation protects rat pups against experimental NEC, which is mediated, at least in part, by HGF. = 5) by laparotomy and needle aspiration from the amniotic sacs. The procedure was performed on day 17C18 of pregnancy (and not later) to obtain late-gestation amniotic fluid and yet collect adequate amounts because the volume of amniotic fluid decreases with advancing gestation in rats. Endotoxin concentrations were measured by a commercially available limulus lysate assay (Pierce, Rockford, IL). NCT-501 All amniotic fluid samples were kept frozen in aliquots until the time of study, with care to avoid multiple freeze-thaw cycles. Human Amniotic Fluid We also collected amniotic fluid from 30 women who underwent cesarean deliveries at 39 wk gestation. These samples were collected at University of Texas Medical Branch, Galveston, after approval by the local Institutional Review Board. Women with multiple gestation, major fetal anomalies, diabetes LAMC2 mellitus, or chorioamnionitis were excluded. All amniotic fluid samples were stored in aliquots and conditions were monitored according to the Biospecimen Reporting for Improved Study Quality (BRISQ) guidelines (25). Amniotic Fluid Cytokine Concentrations Cytokine concentrations were measured by a magnetic bead-based multiplex assay (Bioplex array, Bio-Rad, Hercules, CA) that included interleukin (IL)-1, IL-6, transforming growth factor (TGF)-1, TGF-2, monocyte chemotactic protein-1 (MCP-1)/CC-motif ligand-2 (CCL2), macrophage inflammatory protein-1 (MIP-1)/CCL3, MIP-3/CCL20, and tumor necrosis factor (TNF). Hepatocyte growth factor (HGF) NCT-501 and epidermal growth factor NCT-501 were measured by enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN). HGF concentrations in human amniotic fluid were measured by specific immunoassays (Luminex, Austin, TX). These analytes were short listed on the basis of known gut epithelial effects and existing of data on expression in amniotic fluid (1, 15, 19, 39). We had initially planned to include insulin-like growth factor (IGF)-I and IGF-II measurements but finally decided not to pursue these analytes because rat amniotic fluid was limited in volume and because human amniotic fluid IGF-I and IGF-II levels reported in existing literature (39) were a log-fold lower than the concentrations of HGF we detected in our amniotic fluid samples. Experimental NEC Amniotic fluid supplementation. We divided 80 pups (delivered from 7 dams) into a formula-fed control group and an intervention group that received formula supplemented with 30% (vol/vol) amniotic fluid (30% dilution was chosen based on in vitro studies, where amniotic fluid-induced IEC migration/proliferation reached a plateau at 30%). HGF supplementation. We divided 58 rat pups into HGF and control groups. The two groups received a total of 18 ng recombinant HGF or an equivalent amount of bovine serum albumin divided in all feeds over a day. HGF dose was based on the estimated amount of HGF received by a term rat fetus (20C25 mlkg?1h?1 ingested amniotic fluid, average weight of 5 g, and median HGF concentration in rat amniotic fluid = 5 ng/ml). Experimental NEC. To induce NEC-like injury, rat pups were placed in.