Assessment of radial glia in the frontal lobe of fetuses with Down syndrome.

Down syndrome (DS) occurs with triplication of human chromosome 21 and is associated with deviations in cortical development evidenced by simplified gyral appearance and reduced cortical surface area. Radial glia are neuronal and glial progenitors that also create a scaffolding structure essential for migrating neurons to reach cortical targets and therefore play a critical role in cortical development. The aim of this study was to characterise radial glial expression pattern and morphology in the frontal lobe of the developing human fetal brain with DS and age-matched controls. Secondly, we investigated whether microstructural information from in vivo magnetic resonance imaging (MRI) could reflect histological findings from human brain tissue samples. Immunohistochemistry was performed on paraffin-embedded human post-mortem brain tissue from nine fetuses and neonates with DS (15-39 gestational weeks (GW)) and nine euploid age-matched brains (18-39 GW). Radial glia markers CRYAB, HOPX, SOX2, GFAP and Vimentin were assessed in the Ventricular Zone, Subventricular Zone and Intermediate Zone. In vivo diffusion MRI was used to assess microstructure in these regions in one DS (21 GW) and one control (22 GW) fetal brain. We found a significant reduction in radial glial progenitor SOX2 and subtle deviations in radial glia expression (GFAP and Vimentin) prior to 24 GW in DS. In vivo, fetal MRI demonstrates underlying radial projections consistent with immunohistopathology. Radial glial alterations may contribute to the subsequent simplified gyral patterns and decreased cortical volumes observed in the DS brain. Recent advances in fetal MRI acquisition and analysis could provide non-invasive imaging-based biomarkers of early developmental deviations.

Cervicovaginal natural antimicrobial expression in pregnancy and association with spontaneous preterm birth.

There is much interest in the role of innate immune system proteins (antimicrobial peptides) in the inflammatory process associated with spontaneous preterm birth (sPTB). After promising pilot work, we aimed to validate the association between the antimicrobial peptides/proteins elafin and cathelicidin and sPTB. An observational cohort study of 405 women at high-risk, and 214 women at low-risk of sPTB. Protein concentrations of elafin and cathelicidin, and the enzyme human neutrophil elastase (HNE) were measured in over 1,000 cervicovaginal fluid (CVF) samples (10 to 24 weeks' gestation). Adjusted CVF cathelicidin and HNE concentrations (but not elafin) were raised in high-risk women who developed cervical shortening and who delivered prematurely and were predictive of sPTB < 37 weeks, with an area under the curve (AUC) of 0.75 (95% CI 0.68 to 0.81) for cathelicidin concentration at 14 to 15+6 weeks. Elafin concentrations were affected by gestation, body mass index and smoking. CVF elafin in early pregnancy was modestly predictive of sPTB < 34 weeks (AUC 0.63, 0.56-0.70). Alterations in innate immune response proteins in early pregnancy are predictive of sPTB. Further investigation is warranted to understand the drivers for this, and their potential to contribute towards clinically useful prediction techniques.

Host Defense Peptide Expression in Human Cervical Cells and Regulation by 1,25-Dihydroxyvitamin D3 in the Presence of Cytokines and Bacterial Endotoxin.

Host defense peptides (HDPs) in the pregnant female reproductive tract provide protection against infection. The relationship between HDPs and infection/inflammation is poorly understood. Therefore, we investigated the regulation of HDPs by 1α, 25-dihydroxyvitamin D3 (1,25-(OH)2) in the presence/absence of infectious/inflammatory agents. Endocervical epithelial cells (END1/E6E7, n = 6) were exposed to 1,25-(OH)2, calcipotriol, interleukin 1ß (IL-1ß), granulate-macrophage colony-stimulating factor (GM-GSF), and lipopolysaccharide (LPS). Elafin, human beta defensin (hBD2), cathelicidin, secretory leucocyte protease inhibitor, interleukin 8, 1,25-(OH)2 receptor, and toll-like receptor 4 (TLR4) expression was determined using quantitative polymerase chain reaction and/or enzyme-linked immunosorbent assay. Host defense peptide gene and protein expression was assessed in cervicovaginal cells/fluid, respectively, from first trimester pregnant women (n = 8-12). Interleukin 1ß induced elafin and hBD2. The 1,25-(OH)2 induced cathelicidin expression in the presence of IL-1ß and LPS. The 1,25-(OH)2 also attenuated IL-1ß-induced IL-8 expression and LPS enhancement of TLR4. Host defense peptides and TLR4 profiles in cervicovaginal cells and fluid samples from pregnant women were similar to END1/E6E7 cells. In conclusion, HDPs are differentially regulated in END1/E6E7 cells. The 1,25-(OH)2 induction of cathelicidin and suppression of IL-8 highlights a mechanism by which 1,25-(OH)2 supplementation could enhance the pregnant innate immune defenses.

Nuclear factor of activated T-cell isoform expression and regulation in human myometrium.

BACKGROUND: During pregnancy, myometrial gene and protein expression is tightly regulated to accommodate fetal growth, promote quiescence and ultimately prepare for the onset of labour. It is proposed that changes in calcium signalling, may contribute to regulating gene expression and that nuclear factor of activated T-cell (NFAT) transcription factors (isoforms c1-c4) may be involved. Currently, there is little information regarding NFAT expression and regulation in myometrium. METHODS: This study examined NFAT isoform mRNA expression in human myometrial tissue and cells from pregnant women using quantitative PCR. The effects of the Ca(2+) ionophore A23187 and in vitro stretch (25 % elongation, static strain; Flexercell FX-4000 Tension System) on NFAT expression were determined in cultured human myometrial cells. RESULTS: Human myometrial tissue and cultured cells expressed NFATc1-c4 mRNA. NFATc2 gene expression in cultured cells was increased in response to 6 h stretch (11.5 fold, P < 0.001, n = 6) and calcium ionophore (A23187, 5 µM) treatment (20.6 fold, P < 0.001, n = 6). This response to stretch was significantly reduced (90 %, P < 0.001, n = 10) in the presence of an intracellular calcium chelator, BAPTA-AM (20 µM). CONCLUSIONS: These data suggest that NFATc2 expression is regulated by intracellular calcium and in vitro stretch, and that the stretch response in human myometrial cells is dependent upon intracellular calcium signalling pathways. Our findings indicate a potentially unique role for NFATc2 in mediating stretch-induced gene expression per se and warrant further exploration in relation to the mechanisms promoting uterine smooth muscle growth in early pregnancy and/or labour.

Raised trappin2/elafin protein in cervico-vaginal fluid is a potential predictor of cervical shortening and spontaneous preterm birth.

Early spontaneous preterm birth is associated with inflammation/infection and shortening of the cervix. We hypothesised that cervico-vaginal production of trappin2/elafin (peptidase inhibitor 3) and cathelicidin antimicrobial peptide (cathelicidin), key components of the innate immune system, are altered in women who have a spontaneous preterm birth. The aim was to determine the relationship between cervico-vaginal fluid (CVF) trappin2/elafin and cathelicidin protein concentrations with cervical length in woman at risk of spontaneous preterm birth. Trappin2/elafin and cathelicidin were measured using ELISA in longitudinal CVF samples (taken between 13 to 30 weeks' gestation) from 74 asymptomatic high risk women (based on obstetric history) recruited prospectively. Thirty six women developed a short cervix (<25 mm) by 24 weeks' and 38 women did not. Women who developed a short cervix had 2.71 times higher concentrations of CVF trappin2/elafin from 14 weeks' versus those who did not (CI 1.94-3.79, p<0.0005). CVF trappin2/elafin before 24 weeks' was 1.79 times higher in women who had a spontaneous preterm birth <37 weeks' (CI: 1.05-3.05, p = 0.034). Trappin2/elafin (>200 ng/ml) measured between 14+0-14+6 weeks' of pregnancy predicted women who subsequently developed a short cervix (n = 11, ROC area = 1.00, p = 0.008) within 8 weeks. Cathelicidin was not predictive of spontaneous delivery. Vitamin D status did not correlate with CVF antimicrobial peptide concentrations. Raised CVF trappin2/elafin has potential as an early pregnancy test for prediction of cervical shortening and spontaneous preterm birth. This justifies validation in a larger cohort.

STIM and Orai isoform expression in pregnant human myometrium: a potential role in calcium signaling during pregnancy.

Store-operated calcium (Ca(2+)) entry (SOCE) can be mediated by two novel proteins, STIM/Orai. We have previously demonstrated that members of the TRPC family, putative basal and store operated calcium entry channels, are present in human myometrium and regulated by labor associated stimuli IL-1ß and mechanical stretch. Although STIM and Orai isoforms (1-3) have been reported in other smooth muscle cell types, there is little known about the expression or gestational regulation of STIM and Orai expression in human myometrium. Total RNA was isolated from lower segment human myometrial biopsies obtained at Cesarean section from women at the time of preterm no labor (PTNL), preterm labor (PTL), term non-labor (TNL), and term with labor (TL); primary cultured human uterine smooth muscle cells, and a human myometrial cell line (hTERT-HM). STIM1-2, and Orai1-3 mRNA expression was assessed by quantitative real-time PCR. All five genes were expressed in myometrial tissue and cultured cells. STIM1-2 and Orai2-3 expression was significantly lower in cultured cells compared tissue. This has implications with regard investigation of the contribution of these proteins in cultured cells. Orai2 was the most abundant Orai isoform in human myometrium. Expression of STIM1-2/Orai1-3 did not alter with the onset of labor. Orai1 mRNA expression in cultured cells was enhanced by IL-1ß treatment. This novel report of STIM1-2 and Orai1-3 mRNA expression in pregnant human myometrium and Orai1 regulation by IL-1ß indicates a potential role for these proteins in calcium signaling in human myometrium during pregnancy.