Associations between HLA class II alleles and IgE sensitization to allergens in the Qatar Biobank cohort.

Background: Allergic disorders are the consequence of IgE sensitization to allergens. Population studies have shown that certain human leukocyte antigen (HLA) alleles are associated with increased or decreased risk of developing allergy. Objective: We aimed to characterize the relationship between HLA class II allelic diversity and IgE sensitization in an understudied Arab population. Methods: We explored associations between IgE sensitization to 7 allergen mixes and mesquite (comprising 41 food or aeroallergens) and 45 common classical HLA class II alleles in a well-defined cohort of 797 individuals representing the general adult population of Qatari nationals and long-term residents. To do so, we performed HLA calling from whole genome sequencing data at 2-field resolution using 2 independent algorithms. We then applied 3 different regression models to assess either each allergen mix independently, in the context of IgE sensitization to other allergens tested, or polysensitization. Results: More than half (n = 447) of the study participants showed IgE sensitization to at least 1 allergen, most of them (n = 400) to aeroallergens (Phadiatop). We identified statistically significant negative and positive associations with 24 HLA class II alleles. These have been reported to confer risk or protection from variety of diseases; however, only a few have previously been associated with allergy in other populations. Conclusions: Our study reveals several new risk and protective genetic markers for allergen-specific IgE sensitization. This is a first and essential step toward a better understanding of the origins of allergic diseases in this understudied population.

A Novel STK4 Mutation Impairs T Cell Immunity Through Dysregulation of Cytokine-Induced Adhesion and Chemotaxis Genes.

PURPOSE: Human serine/threonine kinase 4 (STK4) deficiency is a rare, autosomal recessive genetic disorder leading to combined immunodeficiency; however, the extent to which immune signaling and host defense are impaired is unclear. We assessed the functional consequences of a novel, homozygous nonsense STK4 mutation (NM_006282.2:c.871C > T, p.Arg291*) identified in a pediatric patient by comparing his innate and adaptive cell-mediated and humoral immune responses with those of three heterozygous relatives and unrelated controls. METHODS: The genetic etiology was verified by whole genome and Sanger sequencing. STK4 gene and protein expression was measured by quantitative RT-PCR and immunoblotting, respectively. Cellular abnormalities were assessed by high-throughput RT-RCR, RNA-Seq, ELISA, and flow cytometry. Antibody responses were assessed by ELISA and phage immunoprecipitation-sequencing. RESULTS: The patient exhibited partial loss of STK4 expression and complete loss of STK4 function combined with recurrent viral and bacterial infections, notably persistent Epstein-Barr virus viremia and pulmonary tuberculosis. Cellular and molecular analyses revealed abnormal fractions of T cell subsets, plasmacytoid dendritic cells, and NK cells. The transcriptional responses of the patient's whole blood and PBMC samples indicated dysregulated interferon signaling, impaired T cell immunity, and increased T cell apoptosis as well as impaired regulation of cytokine-induced adhesion and leukocyte chemotaxis genes. Nonetheless, the patient had detectable vaccine-specific antibodies and IgG responses to various pathogens, consistent with a normal CD19 + B cell fraction, albeit with a distinctive antibody repertoire, largely driven by herpes virus antigens. CONCLUSION: Patients with STK4 deficiency can exhibit broad impairment of immune function extending beyond lymphoid cells.

Cervicovaginal microbiota and metabolome predict preterm birth risk in an ethnically diverse cohort.

The syndrome of spontaneous preterm birth (sPTB) presents a challenge to mechanistic understanding, effective risk stratification, and clinical management. Individual associations between sPTB, self-reported ethnic ancestry, vaginal microbiota, metabolome, and innate immune response are known but not fully understood, and knowledge has yet to impact clinical practice. Here, we used multi-data type integration and composite statistical models to gain insight into sPTB risk by exploring the cervicovaginal environment of an ethnically heterogenous pregnant population (n = 346 women; n = 60 sPTB < 37 weeks' gestation, including n = 27 sPTB < 34 weeks). Analysis of cervicovaginal samples (10-15+6 weeks) identified potentially novel interactions between risk of sPTB and microbiota, metabolite, and maternal host defense molecules. Statistical modeling identified a composite of metabolites (leucine, tyrosine, aspartate, lactate, betaine, acetate, and Ca2+) associated with risk of sPTB < 37 weeks (AUC 0.752). A combination of glucose, aspartate, Ca2+, Lactobacillus crispatus, and L. acidophilus relative abundance identified risk of early sPTB < 34 weeks (AUC 0.758), improved by stratification by ethnicity (AUC 0.835). Increased relative abundance of L. acidophilus appeared protective against sPTB < 34 weeks. By using cervicovaginal fluid samples, we demonstrate the potential of multi-data type integration for developing composite models toward understanding the contribution of the vaginal environment to risk of sPTB.

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.

Rationale and design of SuPPoRT: a multi-centre randomised controlled trial to compare three treatments: cervical cerclage, cervical pessary and vaginal progesterone, for the prevention of preterm birth in women who develop a short cervix.

BACKGROUND: Clinically, once a woman has been identified as being at risk of spontaneous preterm birth (sPTB) due to a short cervical length, a decision regarding prophylactic treatment must be made. Three interventions have the potential to improve outcomes: cervical cerclage (stitch), vaginal progesterone and cervical pessary. Each has been shown to have similar benefit in reduction of sPTB, but there have been no randomised control trials (RCTs) to compare them. METHODS: This open label multi-centre UK RCT trial, will evaluate whether the three interventions are equally efficacious to prevent premature birth in women who develop a short cervix (<25 mm on transvaginal ultrasound). Participants will be asymptomatic and between 14+0 and 23+6 weeks' gestation in singleton pregnancies. Eligible women will be randomised to cervical cerclage, Arabin pessary or vaginal progesterone (200 mg once daily) (n = 170 women per group). The obstetric endpoints are premature birth rate <37 weeks' of gestation (primary), 34 weeks and 30 weeks (secondary outcomes) and short-term neonatal outcomes (a composite of death and major morbidity). It will also explore whether intervention success can be predicted by pre-intervention biomarker status. DISCUSSION: Preterm birth is the leading cause of perinatal morbidity and mortality and a short cervix is a useful way of identifying those most at risk. However, best management of these women has presented a clinical conundrum for decades. Given the promise offered by cerclage, Arabin pessary and vaginal progesterone for prevention of preterm birth in individual trials, direct comparison of these prophylactic interventions is now essential to establish whether one treatment is superior. If, as we hypothesise, the three interventions are equally efficacious, this study will empower women to make a choice of treatments based on personal preference and quality of life issues also explored by the study. Our exploratory analysis into whether the response to intervention is related to the pre-intervention biomarker status further our understanding of the pathophysiology of spontaneous preterm birth and help focus future research questions. TRIAL REGISTRATION: EudraCT Number: 2015-000456-15 . Registered 11th March 2015.

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.