Live colonocytes in newborn stool: surrogates for evaluation of gut physiology and disease pathogenesis.

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.

Probiotic bacteria change Escherichia coli-induced gene expression in cultured colonocytes: Implications in intestinal pathophysiology.

AIM: To investigate the change in eukaryotic gene expression profile in Caco-2 cells after infection with strains of Escherichia coli and commensal probiotic bacteria. METHODS: A 19,200 gene/expressed sequence tag gene chip was used to examine expression of genes after infection of Caco-2 cells with strains of normal flora E. coli, Lactobacillus plantarum, and a combination of the two. RESULTS: The cDNA microarray revealed up-regulation of 155 and down-regulation of 177 genes by E. coli. L. plantarum up-regulated 45 and down-regulated 36 genes. During mixed infection, 27 genes were up-regulated and 59 were down-regulated, with nullification of stimulatory/inhibitory effects on most of the genes. Expression of several new genes was noted in this group. CONCLUSION: The commensal bacterial strains used in this study induced the expression of a large number of genes in colonocyte-like cultured cells and changed the expression of several genes involved in important cellular processes such as regulation of transcription, protein biosynthesis, metabolism, cell adhesion, ubiquitination, and apoptosis. Such changes induced by the presence of probiotic bacteria may shape the physiologic and pathologic responses they trigger in the host.

A novel peroxisome proliferator--activated receptor alpha/gamma dual agonist ameliorates dyslipidemia and insulin resistance in prediabetic rhesus monkeys.

TAK-559, a newly developed non-thiazolidinedione, activates both peroxisome proliferator-activated receptors alpha and gamma. We investigated the effects of TAK-559 on dyslipidemia and insulin resistance in nonhuman primates. Five adult male obese prediabetic rhesus monkeys were studied on vehicle and after TAK-559 treatment (0.3, 1.0, 3.0 mg/kg per day) for a total of 12 weeks. No significant changes were observed in body weight and fasting plasma glucose, total plasma cholesterol, very low-density lipoprotein-triglyceride, and low-density lipoprotein cholesterol levels. TAK-559 treatment resulted in significant elevation of circulating high-density lipoprotein (HDL) cholesterol levels, consisting of an increase in large HDL particles and a decrease in small dense HDL particles. Nuclear magnetic resonance data exhibited a less atherogenic lipoprotein profile with treatment. Plasma triglyceride and apolipoprotein B-100 levels decreased, whereas apolipoprotein A-I increased during TAK-559 treatment. Hyperinsulinemia and insulin resistance (quantitative insulin sensitivity check index and homeostasis model assessment) were significantly corrected with the highest dose of 3.0 mg/kg per day in these prediabetic monkeys. In addition, no adverse effects on representative liver function parameters were observed during the study period. These results suggest that TAK-559 had beneficial effects on lipoprotein profiles and insulin sensitivity, without any side effect on body weight, which suggests that TAK-559 may provide a potentially safe approach for delaying the onset of type 2 diabetes mellitus and may reduce the risk of cardiovascular disease. The positive effects of TAK-559 in nonhuman primates have led to further clinical trials of TAK-559 in Europe and the United States.

Effect of ovarian steroids on basal and oxytocin-induced prostaglandin F2alpha secretion from pig endometrial cells.

These studies were undertaken to determine how treatment with 100 nM progesterone and/or 10 nM oestradiol-17beta acutely (3 h; Experiment 1) or chronically (72 h; Experiments 2-4) influenced basal and oxytocin (OT)-stimulated prostaglandin (PG) F(2alpha) secretion, in enriched cultures of pig endometrial luminal epithelial, glandular epithelial and stromal cells obtained on Day 16 (Experiments 1, 2 and 4) or Day 12 (Experiment 3) after oestrus. In Experiment 1, acute treatment with progesterone stimulated PGF(2alpha) secretion from each cell type on Day 16, whereas acute oestradiol treatment inhibited the stimulatory action of progesterone on PGF(2alpha) secretion only in glandular epithelial cells. In Experiment 2, OT stimulated phospholipase (PL) C activity in luminal epithelial cells on Day 16 only in the presence of chronic oestradiol treatment. For glandular epithelial cells on Day 16, OT stimulated PLC activity only in the presence of chronic treatment with steroid. In stromal cells on Day 16, OT stimulated PLC activity in the absence of steroids and the response to OT was further enhanced by oestradiol. In the absence of chronic treatment with steroid, OT did not stimulate PGF(2alpha) secretion from luminal epithelial cells, but oestradiol induced a response to OT. For glandular epithelial cells, OT-induced PGF(2alpha) secretion was not altered by steroids, whereas the stimulatory response to OT was inhibited by oestradiol or progesterone in stromal cells. For endometrial cells obtained on Day 12 after oestrus in Experiment 3, OT only stimulated PGF(2alpha) release from glandular epithelial and stromal cells. For luminal epithelial cells obtained on Day 16 after oestrus and cultured under polarizing conditions in Experiment 4, secretion of PGF(2alpha) occurred preferentially from the basolateral surface and was stimulated by OT more from the basolateral surface than from the apical surface. Oxytocin-induced PGF(2alpha) secretion from the apical surface was enhanced by chronic treatment with oestradiol, whereas that from the basolateral surface was enhanced by chronic treatment with progesterone. In summary, oestradiol enhanced OT-induced PGF(2alpha) secretion from the apical surface of luminal epithelial cells and reduced the response of stromal cells to OT, actions that may contribute to the reorientation of PGF(2alpha) from endocrine secretion (i.e. towards the uterine vasculature) to exocrine secretion (i.e. towards the uterine lumen) during pregnancy recognition in pigs.

Angiotensin II increases intracellular calcium concentration in pig endometrial stromal cells through type 1 angiotensin receptors, but does not stimulate phospholipase C activity or prostaglandin F2alpha secretion.

Although the presence of endometrial receptors for angiotensin (Ang) II has been demonstrated, a specific function for AngII in the uterus has not been identified. Cytosolic free Ca2+ concentration [Ca2+]i, phospholipase C (PLC) activity and prostaglandin (PG) F2alpha secretion in response to AngII and oxytocin (OT) were measured in pig endometrial stromal cells collected 16 days after oestrus. Treatment with 100 nM OT or AngII increased (P<0.001) [Ca2+]i in stromal cells similarly (720 +/- 34 v. 690 +/- 33 pM, respectively). Subsequent administration of OT or AngII to the same cells induced smaller [Ca2+]i increases (25% or 35% of the initial responses, respectively) that occurred only if the second exposure to the same agent took place at least 5 min after the first. When administered sequentially, OT and AngII each induced a full response within 1 min of the previous treatment, regardless of which peptide was applied first. Whereas OT increased PLC activity and PGF2alpha secretion in stromal cells (P<0.01), AngII did not increase either PLC activity or PGF2alpha secretion. Type I AngII (AT1) receptors were present on stromal cells, whereas AT2 receptors were absent. Therefore, the effect of AngII in stromal cells was mediated via AT1 receptors. That AngII increased [Ca2+]i in stromal cells, but did not increase PLC or PGF2alpha secretion, indicates that either AngII releases a pool of Ca2+ through a mechanism that is not mediated by PLC and is not involved in PGF2alpha secretion or that a mechanism for PGF2alpha production other than one involving Ca2+ may exist.