Effect of amniotic fluid on the in vitro culture of human corneal endothelial cells.

The present study was designed to evaluate the effects of human amniotic fluid (HAF) on the growth of human corneal endothelial cells (HCECs) and to establish an in vitro method for expanding HCECs. HCECs were cultured in DMEM-F12 supplemented with 20% fetal bovine serum (FBS). Confluent monolayer cultures were trypsinized and passaged using either FBS- or HAF-containing media. Cell proliferation and cell death ELISA assays were performed to determine the effect of HAF on cell growth and viability. The identity of the cells cultured in 20% HAF was determined using immunocytochemistry (ICC) and real-time reverse transcription polymerase chain reaction (RT-PCR) techniques to evaluate the expression of factors that are characteristic of HCECs, including Ki-67, Vimentin, Na+/K+-ATPase and ZO-1. HCEC primary cultures were successfully established using 20% HAF-containing medium, and these cultures demonstrated rapid cell proliferation according to the cell proliferation and death ELISA assay results. The ICC and real time RT-PCR results indicated that there was a higher expression of Na+/K+-ATPase and ZO-1 in the 20% HAF cell cultures compared with the control (20% FBS) (P < 0.05). The 20% HAF-containing medium exhibited a greater stimulatory effect on HCEC growth and could represent a potential enriched supplement for HCEC regeneration studies.

Postnatal amniotic fluid intake reduces gut inflammatory responses and necrotizing enterocolitis in preterm neonates.

Preterm neonates are susceptible to gastrointestinal disorders such as necrotizing enterocolitis (NEC). Maternal milk and colostrum protects against NEC via growth promoting, immunomodulatory, and antimicrobial factors. The fetal enteral diet amniotic fluid (AF), contains similar components, and we hypothesized that postnatal AF administration reduces inflammatory responses and NEC in preterm neonates. Preterm pigs (92% gestation) were delivered by caesarean section and fed parental nutrition (2 days) followed by enteral (2 days) porcine colostrum (COLOS, n = 7), infant formula (FORM, n = 13), or AF supplied before and after introduction of formula (AF, n = 10) in experiment 1, and supplied only during the enteral feeding period in experiment 2 (FORM, n = 16; AF, n = 14). The NEC score was reduced in both AF and COLOS pigs, relative to FORM, when AF was provided prior to full enteral feeding (9.9 and 7.7 compared with 17.3, P < 0.05). There was no effect of AF when provided only during enteral feeding. AF pigs showed decreased bacterial abundance in colon and intestinal inflammation-related genes (e.g., TNF-α, IL-1α, IL-6, NOS) were downregulated, relative to FORM pigs with NEC. Anti-inflammatory properties of AF were supported by delayed maturation and decreased TNF-α production in murine dendritic cells, as well as increased proliferation and migration, and downregulation of IL-6 expression in intestinal cells (IEC-6, IPEC-J2). Like colostrum, AF may reduce NEC development in preterm neonates by suppressing the proinflammatory responses to enteral formula feeding and gut colonization when provided before the onset of NEC.

Human amniotic fluid promotes retinal pigmented epithelial cells' trans-differentiation into rod photoreceptors and retinal ganglion cells.

To evaluate the effect of human amniotic fluid (HAF) on retinal pigmented epithelial cells growth and trans-differentiation into retinal neurons, retinal pigmented epithelium (RPE) cells were isolated from neonatal human cadaver eye globes and cultured in Dulbecco's modified Eagle's medium-F12 supplemented with 10% fetal bovine serum (FBS). Confluent monolayer cultures were trypsinized and passaged using FBS-containing or HAF-containing media. Amniotic fluid samples were received from pregnant women in the first trimester of gestation. Cell proliferation and death enzyme-linked immunosorbent assays were performed to assess the effect of HAF on RPE cell growth. Trans-differentiation into rod photoreceptors and retinal ganglion cells was also studied using immunocytochemistry and real-time polymerase chain reaction techniques. Primary cultures of RPE cells were successfully established under FBS-containing or HAF-containing media leading to rapid cell growth and proliferation. When RPE cells were moved to in vitro culture system, they began to lose their differentiation markers such as pigmentation and RPE65 marker and trans-differentiated neural-like cells followed by spheroid colonies pertaining to stem/progenitor cells were morphologically detected. Immunocytochemistry (ICC) analysis of HAF-treated cultures showed a considerable expression of Rhodopsin gene (30% Rhodopsin-positive cells) indicating trans-differentiation of RPE cells to rod photoreceptors. Real-time polymerase chain reaction revealed an HAF-dose-dependant expression of Thy-1 gene (RGC marker) and significant promoting effect of HAF on RGCs generation. The data presented here suggest that HAF possesses invaluable stimulatory effect on RPE cells growth and trans-differentiation into retinal neurons. It can be regarded as a newly introduced enriched supplement in serum-free kinds of media used in neuro-retinal regeneration studies.

Intestinal macromolecule absorption in the fetal pig after infusion of colostrum in utero.

The ability of the fetal pig intestine to absorb large proteins was investigated in utero. Six pregnant sows were anesthetized (Na pentobarbital) at 99-102 d of gestation (term = 115 +/- 2 d), and a catheter was inserted into the esophagus of two to three fetuses per sow. Via these catheters, sterile solutions (10.0 mL) of colostrum whey (CW, n = 5), milk whey (MW, n = 5), or amniotic fluid (AF, n = 4) were infused into the fetal pig stomachs every 6 h for 6-8 d starting on the day after surgery (d 0). Levels of IgG in the three fluids were 120, 0.5, and 0 mg/mL, respectively. During the first 2-3 d of infusion, plasma IgG levels rose rapidly in the CW fetuses (to 7.5 +/- 0.8 mg/mL), whereas IgG remained absent in plasma from MW and AF fetuses. Absorption of a macromolecule marker, BSA, was also higher when the marker was given with CW rather than with MW or AF. However, when all three treatment groups were given CW + BSA on the last experimental day (d 6-8), the mean BSA increment in the CW group was only 5-8% of that in the AF group, with intermediate values for the MW group. Neither at the beginning nor at the end of the experiment was macromolecule uptake in individual CW fetuses correlated with their cortisol level in plasma. The prenatal pig intestine is similar to the neonatal pig intestine in that colostrum stimulates both the macromolecule absorption and the cessation of macromolecule uptake (intestinal closure). However, fetal pigs have a lower protein absorptive capacity and a longer preclosure period than newborn pigs; this may be related to an immature structure and function and a slow enterocyte proliferation rate in the prenatal pig intestine.

Systemic and luminal influences on the perinatal development of the gut.

At birth, the mammalian gastrointestinal tract (GIT) must be able to support a shift from mainly parenteral nutrition in the fetus (via the placenta) to enteral nutrition in the neonate. In the perinatal period the GIT therefore undergoes enhanced growth as well as morphological and functional differentiation, and this maturational programme is influenced by a complex interplay of local, systemic and luminal factors. This review shows how systemic and luminal factors may influence GIT development in the perinatal period of the pig and sheep, two long-gestation species. Adrenocortical hormones play a pivotal role in the prepartum maturation of the GIT in addition to their better known effects on the development of many other tissues and body systems. More particularly, in the fetal pig and sheep, the prenatal development of gastric acid and gastrin secretion, and of GIT hydrolase activities (chymosin, pepsin, amylase, lactase, aminopeptidases) is influenced by cortisol. Additionally, glucocorticoids exert effects throughout the GIT by influencing morphological, cytological, and functional differentiation. Since the GIT epithelial cells comprise a renewing cell population there are also changes in cell kinetics. In addition to systemic factors, the presence of growth factors, hormones and nutrients from swallowed amniotic fluid (fetus) and colostrum (neonate) may influence GIT development. In utero, fetal fluid ingestion has been shown to modulate tissue growth, macromolecule and immunoglobulin transport, enterocyte differentiation, cell turnover and activity of brush-border hydrolases. These effects may be mediated via regulatory peptides (e.g. insulin-like growth factor I, gastrin-releasing peptides, insulin, epidermal growth factor, gastrin). A physiological role of luminally derived growth factors is supported by a number of unique structural and functional adaptations of the GIT in the fetus and neonate (low luminal proteolysis, intestinal macromolecule transport). Thus, in the pig and sheep, both systemic and luminal factors appear to play critical roles in GIT development in the perinatal period.

Antepartum fetal surveillance. Nonstress test, contraction stress test, and biophysical profile.

The three most common methods used to evaluate fetal well being in utero are the nonstress test, contraction stress test, and the biophysical profile. This article reviews recent literature pertaining to these methods of antepartum fetal surveillance and compares and contrasts the different modalities.

Pulmonary hemodynamics in fetal lambs during development at normal and increased oxygen tension.

During the latter third of gestation, the number of resistance vessels in the lungs of the fetal sheep increases by 10-fold even after correction for lung growth. We measured pulmonary arterial pressure and blood flow directly and calculated total pulmonary resistance (pressure divided by flow) in intrauterine fetal lambs at 93-95 days and at 136 days of gestation (term is 145-148 days). In addition, we used a hyperbaric chamber to increase oxygen tension in the fetuses and measured the effect on the pulmonary circulation. When corrected for wet weight of the lungs, pulmonary blood flow did not change with advancing gestation (139 +/- 42 to 103 +/- 45 ml.100 g-1.min-1). Pulmonary arterial pressure increased (42 +/- 5 to 49 +/- 3 mmHg); thus total pulmonary resistance increased with advancing gestation from 0.32 +/- 0.12 to 0.55 +/- 0.21 mmHg.100 g.min.ml-1. If the blood flow is corrected for dry weight of the lungs, neither pulmonary blood flow nor total pulmonary resistance changed with advancing gestation. Increasing oxygen tension increased pulmonary blood flow 10-fold in the more mature fetuses but only 0.2-fold in the less mature fetuses. At the normal low oxygen tension of the fetus, pulmonary blood flow does not increase between these two points of gestation in the fetal lamb despite the increase in vessel density in the lungs. However, during elevated oxygen tension, pulmonary blood flow does increase in proportion to the increase in vessel density.

Amino acids and human amniotic fluid increase DNA biosynthesis in pancreatic islets of adult mouse, but this effect is lost following exposure to streptozotocin.

The present study describes the effects of growth hormone (GH), amino acids, and human amniotic fluid on the function and replication of normal and SZ-treated adult mouse pancreatic islets. Thus, mouse islets were exposed in vitro to SZ or vehicle only, and maintained in culture for 7 days in RPMI 1640 containing 11.1 mM glucose and the different supplements described above. Supplementation with amino acids increased the insulin accumulation in the medium, DNA biosynthesis, and polyamine contents in the control islets. In the SZ-treated islets, amino acids increased insulin accumulation in the medium and polyamine contents, but not DNA biosynthesis. Culture of control islets in the presence of 10% human amniotic fluid increased the insulin accumulation in the medium, islet insulin content, total protein and (pro)insulin biosynthesis, and DNA biosynthesis. However, in the SZ-treated islets, the effects of human amniotic fluid were limited to an increase in insulin content and insulin accumulation in the medium. GH significantly increased insulin accumulation in the medium in control, but not SZ-treated islets. In both groups of islets, GH failed to induce a significant increase in thymidine incorporation. It is concluded that amino acids and human amniotic fluid are potent stimulators of DNA biosynthesis in adult mouse pancreatic beta cells, perhaps due to an increase in cellular polyamine contents. However, following exposure to streptozotocin, the islets are not anymore responsive to these stimulators of DNA biosynthesis.

The effect of diet supplementation and addition of zinc in vitro on the growth-supporting property of amniotic fluid in African women.

The effect of diet supplementation throughout pregnancy on third-trimester amniotic fluid growth-supporting activity (GSP) was studied in 100 African women; 32 were given zinc supplementation, 22 each animal and vegetable supplements, respectively, and 24 served as control subjects. No difference in GSP was noted in any of the four groups, the majority of fluids (65%) being noninhibitory. Zinc levels in fluids from African women were much lower than those described for other population groups at corresponding periods of gestation. Although zinc levels in liquor rose following dietary zinc supplementation, these remained lower than values described in white patients. In vitro addition of zinc (up to 153 mu moles per liter final concentration) to 17 noninhibitory African liquors resulted in these fluids becoming inhibitory.

PIP: The effect of 3 diet supplementation regimes throughout the latter part of pregnancy on the growth-supporting property (GSP) of third trimester fluids was studied in 100 African women: 32 were given zinc, 22 each animal and vegetable supplements, respectively; and 24 were controls. No significant increase of inhibitory activity was found in any of the 4 groups, the majority of fluids (overall average 65%) being noninhibitory. Zinc levels were very low as compared with Caucasian women. The effect of in vitro addition of zinc on the GSP of 17 noninhibitory fluids from African women showed 5 fluids becoming bacteriostatic and 12 becoming bactericidal after adding zinc at a final concentration of 15.3 mcmol/l; raising levels 5 and 10 times those concentrations rendered all fluids bactericidal. Zinc alone did not affect the growth of Staphylococcus aureus in control subjects without liquor.

Bacterial growth inhibition by amniotic fluid. VII. The effect of zinc supplementation on bacterial inhibitory activity of amniotic fluids from gestation of 20 weeks.

Twenty human amniotic fluids obtained from gestations of 20 weeks' duration supported bacterial growth. Nine of the 20 fluids could be made inhibitory by adjusting the phosphate to zinc ratios of the fluids to less than 200 mug per milliliter. These fluids contained the phosphate-sensitive bacterial inhibitor previously, but the fluids contained sufficient phosphate to inactivate the antibacterial system. The remaining 11 amniotic fluids did not contain the peptide component of the phosphate-sensitive bacterial inhibitor and could not be made inhibitory by adjusting the phosphate to zinc ratio to less than 200 mug per milliliter. The data obtained suggested synthesis of the peptide component may occur at a gestational age of approximately 20 weeks. The peptide may indirectly be detected in fluids by determining whether antibacterial activity is obtained when the phosphate to zinc ratio of the fluids is adjusted to less than 200 mug per milliliter.