Proteinaceous components in goat amniotic fluid enhance the expression of keratinocyte differentiation-related proteins.

ObjectiveTo investigate the effect of mitogen-activated protein kinase (MAPK) signaling pathway in epidermal terminal differentiation.MethodsThe MAPK pathways (p38, ERK1/2, JNK) were inhibited by SB203580, PD98059, and SP600125 in normal human epidermal keratinocytes (NHEKs), respectively. Western blotting assays were performed to detect expression of filaggrin and differentiation-related proteins. The mRNA expressions of differentiation-related proteins were detected by real-time quantitative PCR (qRT-PCR).ResultsInhibition of MAPK pathway by SB203580, PD98059, and SP600125 resulted in significant reduction of filaggrin expression in NHEKs. Inhibition of the p38 MAPK pathway decreased the expression of differentiation-related proteins (cytokeratin 5, cytokeratin 14, ST14, and SPRR3), Akt, and NF-κB. Inhibition of JNK also suppressed expression of cytokeratin 14, SPRR3, Akt, and NF-κB. However, inhibition of ERK1/2 merely decreased expression of SPRR3 and Akt.ConclusionMAPK pathways regulates epidermal terminal differentiation in NHEKs. The p38 signaling pathway plays an especially important role.

Potential activity of chicken amniotic fluid in epidermal development by promoting keratinocyte differentiation.

Differentiation Associated Changes in Gene Expression Profiles of Interstitial Cystitis and Control Urothelial Cells

Intrauterine environment and fetal allergic sensitization.

Amniotic Stem Cells: Potential in Regenerative Medicine

Thyroxine (T4), 3,3',5'-tri-iodothyronine (reverse T3; rT3) and di-iodothyronines (3,3'-T2 and 3',5'-T2) were measured in pig amniotic fluid (AF) and allantoic fluid (Al) between 32 and 113 days of normal pregnancy. Low but measurable quantities of T4 in AF and Al (2.1 +/- 0.3 and 3.2 +/- 0.5 nmol/l respectively) were found before the onset of fetal thyroid gland function, which indicates the maternal source of T4. The presence of rT3 (55.8 +/- 4.1 pmol/l in AF and 49.8 +/- 5.3 pmol/l in Al), 3,3'-T2 (45.5 +/- 0.6 pmol/l in AF and 49.2 +/- 9.2 pmol/l in Al) and 3',5'-T2 (20.8 +/- 2.6 pmol/l in AF and 24.0 +/- 2.2 pmol/l in Al) may be attributed to the monodeiodinase system already active in fetal pig tissues in early pregnancy, as demonstrated previously. T3 concentration was undetectable in both AF and Al. An approximately twofold increase in the levels of T4, rT3 and T2s in AF and Al at mid-gestation was observed. T4 and rT3 in AF showed a positive correlation with protein concentrations. AF rT3 concentration (but not T4) correlated with rT3 in the cord and maternal serum. The 3,3'-T2 and 3',5'-T2 in AF and Al showed parallel changes to rT3, while the rT3/3,3'-T2 and rT3/3',5'-T2 molar ratios remained constant. T4 concentrations in AF and Al were markedly lower than in corresponding maternal and fetal serum; the rT3 concentration in Al was equal to that in AF and two to four times lower than in fetal serum. In spite of differences between serum hormone patterns in the pig and human near term, iodothyronine concentrations in AF showed some similarities, mainly the following: undetectable T3, a strong correlation between rT3, T4 and AF total protein and the presence of 3,3'-T2 and 3',5'-T2 in measurable levels. Comparative data for Al, except the ones in the present study in the pig, are not available.

The objective of this study was to describe the dynamic changes in protein composition and protein abundance in amniotic and allantoic fluids from buffaloes during gestation. Amniotic and allantoic fluids were collected during the first, second and third trimesters of gestation. The foetuses were measured and weighed. Fluid samples were centrifuged at 800g for 10min and then at 10,000g for 60min at 4°C. The supernatant was collected to determine the total protein concentration. Based on total protein concentration, an aliquot (50μg) was used for in-solution tryptic digestion, and mass spectrometry analysis (nano-LC-MS/MS) was performed. A multivariate statistical analysis of the proteomic data was conducted. Across the different stages of buffalo gestation, fifty-one proteins were found in the amniotic fluid, and twenty-one were found in the allantoic fluid. A total of twelve proteins were common among the stages, and four presented significant differences (VIP score α>1). Fibronectin and alpha-1-antiproteinase were more abundant in the amniotic fluid than in the allantoic fluid. Alpha-2-macroglobulin and alpha-2-HS-glycoprotein were more abundant in the allantoic fluid than in the amniotic fluid. Alpha-2-macroglobulin participates in remodelling and growth of the uterus at beginning of the gestation (first trimester), and these findings indicate that can serve as a potential tool for the early diagnosis of pregnancy in buffaloes.

Lce1 Family Members Are Nrf2-Target Genes that Are Induced to Compensate for the Loss of Loricrin

Amniotic and allantoic fluids from experimentally infected sheep contain immunoglobulin specific for Chlamydophila abortus

Amniotic fluid is a dynamic and complex mixture that reflects the physiological status of the developing fetus. In this study, the human amniotic fluid (AF) proteome of a 16-18-week normal pregnancy was profiled and analyzed to investigate the composition and functions of this fluid. Due to the complexity of AF, we utilized three different fractionation strategies to provide greater coverage. Two types of two-dimensional LC/MS/MS as well as an LC-SDS-PAGE-LC-MS/MS platform were used. A total of 16 AF samples between gestational ages of 16 and 18 weeks from women carrying chromosomally normal fetuses were analyzed by one of the three fractionation methods followed by a common reverse phase LC-MS/MS step. Mascot and The Global Proteome Machine engines were used to search the International Protein Index human database for peptide sequence identification. The list of proteins was generated by combining the results of both engines through the PeptideProphet of Scaffold software. All identified proteins were combined to generate the AF proteome comprising 1,026 unique gene matches or 842 non-redundant proteins. This list includes most of the currently used biomarkers for pregnancy-associated pathologic conditions such as preterm delivery, intra-amniotic infection, and chromosomal anomalies of the fetus. The subcellular localization, tissue expression, functions, and networks of the AF proteome were analyzed by various bioinformatic tools. These data will contribute to the better understanding of amniotic fluid function and to the discovery of novel biomarkers for prenatal diagnosis of fetal abnormalities.

Filaggrin is an essential structural protein of the stratum corneum binding to the keratin intermediate filaments to form a dense protein-lipid matrix. However, the function of filaggrin in epidermal terminal differentiation is not completely understood. To evaluate the effects of filaggrin on normal human epidermal keratinocytes (NHEKs) and to investigate the relevant mechanisms. Short hairpin RNA (shRNA) technology was used to knock-down filaggrin in normal human epidermal keratinocytes (NHEKs). Western blot and real-time quantitative PCR (qRT-PCR) were performed to detect expression of filaggrin, differentiation-related proteins and MAPK-related proteins. Filaggrin was successfully knocked down in NHEKs (99% efficiency). We found that the lack of filaggrin significantly decreased the expression of some differentiation-related proteins, including Cytokeratin 5 protein, Cytokeratin 14 protein, ST14 protein and SPRR3 protein (P<0.05). In addition, filaggrin knock-down significantly decreased expression of p-p38, p-ERK1/2, p-JNK, p-Akt, and p-NF-κB in NHEKs. Our study shows that filaggrin regulates epidermal terminal differentiation and impairs MAPK signaling pathway in normal human epidermal keratinocytes.

Intra-amniotic infections (IAI) are one of the reasons for preterm birth. High mobility group box 1 (HMGB1) is a nuclear protein with various physiological functions, including tissue healing. Its excessive extracellular release potentiates inflammatory reaction and can revert its action from beneficial to detrimental. We infected the amniotic fluid of a pig on the 80th day of gestation with 1 × 104 colony forming units (CFUs) of E. coli O55 for 10 h, and evaluated the appearance of HMGB1, receptor for glycation endproducts (RAGE), and Toll-like receptor (TLR) 4 in the amniotic membrane and fluid. Sham-infected amniotic fluid served as a control. The expression and release of HMGB1 were evaluated by Real-Time PCR, immunofluorescence, immunohistochemistry, and ELISA. The infection downregulated HMGB1 mRNA expression in the amniotic membrane, changed the distribution of HMGB1 protein in the amniotic membrane, and increased its level in amniotic fluid. All RAGE mRNA, protein expression in the amniotic membrane, and soluble RAGE level in the amniotic fluid were downregulated. TLR4 mRNA and protein expression and soluble TLR4 were all upregulated. HMGB1 is a potential target for therapy to suppress the exaggerated inflammatory response. This controlled expression and release can, in some cases, prevent the preterm birth of vulnerable infants. Studies on suitable animal models can contribute to the development of appropriate therapy.

In this model article, we present a protocol for continuous amniotic fluid exchange in rabbits using a novel system to test the effects of growth factor-deficient, artificial amniotic fluid on bowel development. Background: Ideally, the EXTrauterine Environment for Neonatal Development (EXTEND) will provide physiologic support to the extreme premature infant. An important component of that environment is the amniotic fluid. Thus, we developed an animal model to study the growth factors found within amniotic fluid and inform design of a synthetic fluid to optimize fetal development. Methods: We designed a model of amniotic fluid exchange within the pregnant rabbit, continuously removing the natural fluid from around 2 fetuses per doe and replacing it with a physiologic electrolyte solution during the final 100 h of gestation. Two fetuses from the contralateral uterine horn were used as sham-operated controls. Thirty-eight fetuses were analyzed, 19 in each group. We analyzed the fetal growth and bowel development. Results: Ultrasound after 100 h of exchange showed equivalent fluid volumes, p = 0.63. Cultures were negative for bacterial colonization. Final fluid protein concentrations were 11.6% that of control fluid (mean 1,451 ± 224.2 vs. 12,491 ± 849.2 μg/mL). There was no significant difference in fetal growth, with experimental weights 91.4% of control weights, p = 0.07. Fetal bowel weights (90.1%, p = 0.16) and lengths (94.2%, p = 0.49) were also not significantly less compared to controls. There was no significant difference in villous height or crypt depth measurements between the groups, and absorptive capacity of the bowel was not different between groups, p = 0.44. Conclusion: This animal model allows for manipulation of the components of amniotic fluid. Marked reduction of natural amniotic fluid proteins during gestation does not appear to significantly impair fetal growth or bowel development. Further work with this model will assess the importance of amniotic fluid components for normal development to inform design of a synthetic fluid for use during EXTEND.

The objective of this study was to evaluate the biochemical indices of normal fetal kidney maturation in early pregnancy. Urea, creatinine, gamma-glutamyltransferase, and beta 2-microglobulin levels were measured on paired samples of amniotic and coelomic fluids and maternal serum collected at the time of pregnancy termination in a group of woman with pregnancies between 8 and 11 wk, or on paired samples of amniotic fluid and maternal serum collected from woman undergoing early transabdominal amniocentesis between 11 and 16 wk. Before 11 wk of gestation (n = 12), significantly lower concentrations of creatinine and beta 2-microglobulin, and higher concentration of gamma-glutamyltransferase were found in amniotic fluid compared with both maternal serum and coelomic fluid. Significant positive correlations were found between gestational age (8-16 wk) and amniotic fluid (n = 47) levels of urea (r = 0.45; p < 0.01), creatinine (r = 0.68; p < 0.001), gamma-glutamyltransferase (r = 0.71; p < 0.001), and beta 2-microglobulin (r = 0.69; p < 0.001). The maternal serum levels of the corresponding variables did not varied significantly. The abrupt increase in creatinine, gamma-glutamyltransferase, and beta 2-microglobulin amniotic fluid concentrations after 10 wk of gestation provides biochemical evidence for the maturation of the fetal renal glomerular function at a time when the reabsorption capacity of the proximal tubular cells is not established. We suggest that this important variation in the amniotic fluid composition, unrelated to any comparable changes in the maternal serum, reflects the fetal kidney development from the mesonephros to the metanephros.

Simple SummaryFetal fluid contents influence growth and development, fetal wellbeing and a protection mechanism for the fetus itself. Very little is known about the composition of fetal fluids during pregnancy in cats and about changes in contents that occur throughout gestation. In this study, initial results regarding the biochemical composition of fetal fluids are provided. A comparison with maternal serum was also performed. A complete understanding of trends can be helpful for the early detection of some pathologies during pregnancy, such as in humans and mares.Fetal fluid contents have functions in protecting fetuses and are essential for fetal development and maturation. However, little is known about the exact physiological functions of fetal fluids in fetal development, as well as the changing composition throughout the gestational period in cats. In this study, the biochemical composition of amniotic (AMN) and allantoic (ALL) fluids was investigated, as well as in the maternal serum of pregnant queens. Eighteen queens were included in this study and assigned to six different groups, D20, D25, D30, D40, D45 and D60, according to the gestational stage of fetal development. A total of 44 amniotic and 37 allantoic samples were collected. Fetal fluids contained lesser concentrations of alanine aminotransferase, albumin, cholesterol, triglycerides, creatine kinase, amylase, total protein and globulin than maternal serum. Other variables, such as aspartate aminotransferase, gamma-glutamyl transferase, bilirubin and alkaline phosphatase, were in different concentrations at specific stages of gestation when compared to maternal serum. There were no differences between fetal fluids and maternal serum for lactate dehydrogenase, urea, lipase or glucose concentrations. There were greater concentrations of creatinine in amniotic fluid than in allantoic fluid or maternal serum. Based on the results of this study, fetal fluids do not accumulate as a result of the simple filtration of maternal blood, but rather, the fetus produces many of these components as a consequence of organ development and maturation.