Circulatory Neutrophils Exhibit Enhanced Neutrophil Extracellular Trap Formation in Early Puerperium: NETs at the Nexus of Thrombosis and Immunity.

Pregnancy is associated with elevated maternal levels of cell-free DNA of neutrophil extracellular trap (NET) origin, as circulatory neutrophils exhibit increased spontaneous NET formation, mainly driven by G-CSF and finely modulated by sex hormones. The postpartum period, on the other hand, involves physiological alterations consistent with the need for protection against infections and fatal haemorrhage. Our findings indicate that all relevant serum markers of neutrophil degranulation and NET release are substantially augmented postpartum. Neutrophil pro-NETotic activity in vitro is also upregulated particularly in post-delivery neutrophils. Moreover, maternal puerperal neutrophils exhibit a strong pro-NETotic phenotype, associated with increased levels of all key players in the generation of NETs, namely citH3, MPO, NE, and ROS, compared to non-pregnant and pregnant controls. Intriguingly, post-delivery NET formation is independent of G-CSF in contrast to late gestation and complemented by the presence of TF on the NETs, alterations in the platelet activity status, and activation of the coagulation cascade, triggered by circulating microparticles. Taken together, our results reveal the highly pro-NETotic and potentially procoagulant nature of postpartum neutrophils, bridging an overt immune activation with possible harmful thrombotic incidence.

Markers of neutrophil extracellular traps predict adverse outcome in community-acquired pneumonia: secondary analysis of a randomised controlled trial.

Neutrophil extracellular traps (NETs) are a hallmark of the immune response in inflammatory diseases. However, the role of NETs in community-acquired pneumonia (CAP) is unknown. This study aims to characterise the impact of NETs on clinical outcomes in pneumonia.This is a secondary analysis of a randomised controlled, multicentre trial. Patients with CAP were randomly assigned to either 50 mg prednisone or placebo for 7 days. The primary end-point was time to clinical stability; main secondary end-points were length of hospital stay and mortality.In total, 310 patients were included in the analysis. Levels of cell-free nucleosomes as surrogate markers of NETosis were significantly increased at admission and declined over 7 days. NETs were significantly associated with reduced hazards of clinical stability and hospital discharge in multivariate adjusted analyses. Moreover, NETs were associated with a 3.8-fold increased adjusted odds ratio of 30-day mortality. Prednisone treatment modified circulatory NET levels and was associated with beneficial outcome.CAP is accompanied by pronounced NET formation. Patients with elevated serum NET markers were at higher risk for clinical instability, prolonged length of hospital stay and 30-day all-cause mortality. NETs represent a novel marker for outcome and a possible target for adjunct treatments of pneumonia.

PAD4 Deficiency Decreases Inflammation and Susceptibility to Pregnancy Loss in a Mouse Model.

Inflammation is thought to play a critical role in the pathogenesis of placentation disorders such as recurrent miscarriages, growth restriction, and preeclampsia. Recently, neutrophil extracellular traps (NETs) have emerged as a potential mechanism for promoting inflammation in both infectious and noninfectious disorders. To investigate a pathogenic role for NETs in placentation disorders, we studied a model of antiangiogenic factor-mediated pregnancy loss in wild-type (WT) mice and in mice deficient in peptidylarginine deiminase 4 (Padi4-/-) that are unable to form NETs. Overexpression of soluble fms-like tyrosine kinase 1 (sFlt-1), an antiangiogenic protein that is pathogenically linked with abnormal placentation disorders during early gestation, resulted in pregnancy loss and large accumulation of neutrophils and NETs in WT placentas. Interestingly, sFlt-1 overexpression in Padi4-/- mice resulted in dramatically lower inflammatory and thrombotic response, which was accompanied by significant reduction in pregnancy losses. Inhibition of NETosis may serve as a novel target in disorders of impaired placentation.

Development of Late-Onset Preeclampsia in Association with Road Densities as a Proxy for Traffic-Related Air Pollution.

INTRODUCTION: Previous epidemiological studies indicate an association between maternal exposure to air pollution and an increased risk of hypertensive disorders in pregnancy. We analyzed the association between the occurrence of mild/severe and early-/late-onset preeclampsia (PE) and traffic-related air pollution (TRAP). MATERIALS AND METHODS: Based on retrospective data, 50 pregnant women with PE were selected and matched with a control group of healthy pregnant women according to their age, parity, and number of fetuses. The total length of major roads around the women's home within a radius of 100, 200, 300, and 500 m and the distances from the domicile to the nearest 'first class' main road and freeway were used as a proxy indicator of TRAP. We compared a PE subgroup and control group in terms of their exposure to TRAP. RESULTS: Late-onset PE cases showed a significantly higher occurrence with density of major roads within a radius of 100-300 m compared to early onset cases (p = 0.006; 0.02; 0.04). In addition, a significantly shorter distance to the nearest 'first class' main road was observed in late-onset PE cases (p = 0.0078). CONCLUSIONS: Exposure to TRAP during pregnancy was associated with an increased risk for the development of late-onset PE.

Elevated Levels of Total Cell-Free DNA in Maternal Serum Samples Arise from the Generation of Neutrophil Extracellular Traps.

INTRODUCTION: The analysis of cell-free DNA from maternal blood samples has facilitated the noninvasive detection of fetal aneuploidies or hereditary Mendelian disorders. In this context, previous studies have indicated that the pool of cell-free DNA is greater in maternal serum than in plasma samples, necessitating optimized collection and storage protocols. As the source of this increased amount of cell-free DNA is not clear, we have now examined whether neutrophil extracellular traps (NETs) contribute to this material. MATERIAL AND METHODS: Serum samples were collected in all three trimesters of normal healthy pregnant women, and at term from cases with manifest preeclampsia. The presence of NET-derived material was demonstrated by the detection of cell-free DNA fragments complexed to neutrophil granular proteins (i.e. myeloperoxidase). RESULTS: Our data indicate that NET-derived cell-free DNA/myeloperoxidase complexes were greater in serum from normal pregnant women than in normal matching nonpregnant controls. This neutrophil chromosomal material increased incrementally throughout gestation and was most pronounced in cases with preeclampsia. DISCUSSION: By detecting increased levels of cell-free DNA/myeloperoxidase complexes in maternal serum samples, our data indicate that a significant proportion of this material is derived from the generation of NETs.

Maternal serum protein profile and immune response protein subunits as markers for non-invasive prenatal diagnosis of trisomy 21, 18, and 13.

OBJECTIVES: To use proteomics to identify and characterize proteins in maternal serum from patients at high-risk for fetal trisomy 21, trisomy 18, and trisomy 13 on the basis of ultrasound and maternal serum triple tests. METHODS: We performed a comprehensive proteomic analysis on 23 trisomy cases and 85 normal cases during the early second trimester of pregnancy. Protein profiling along with conventional sodium dodecyl sulfate polyacrylamide gel electrophoresis/Tandem mass spectrometry analysis was carried out to characterize proteins associated with each trisomy condition and later validated using Western blot. RESULTS: Protein profiling approach using surface enhanced laser desorption/ionization time-of-flight mass (SELDI-TOF/MS) spectrometry resulted in the identification of 37 unique hydrophobic proteomic features for three trisomy conditions. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by Matrix Assisted Laser Desorption Ionization - Time of Flight/Time of Flight (MALDI-TOF/TOF) and western blot, glyco proteins such as alpha-1-antitrypsin, apolipoprotein E, apolipoprotein H, and serum carrier protein transthyretin were identified as potential maternal serum markers for fetal trisomy condition. The identified proteins showed differential expression at the subunit level. CONCLUSIONS: Maternal serum protein profiling using proteomics may allow non-invasive diagnostic testing for the most common trisomies and may complement ultrasound-based methods to more accurately determine pregnancies with fetal aneuploidies.

Quantitative proteomic (iTRAQ) analysis of 1st trimester maternal plasma samples in pregnancies at risk for preeclampsia.

A current major obstacle is that no reliable screening markers exist to detect pregnancies at risk for preeclampsia. Quantitative proteomic analysis employing isobaric labelling (iTRAQ) has been suggested to be suitable for the detection of potential plasma biomarkers, a feature we recently verified in analysis of pregnancies with Down syndrome foetuses. We have now examined whether this approach could yield biomarkers to screen pregnancies at risk for preeclampsia. In our study, we used maternal plasma samples obtained at 12 weeks of gestation, six from women who subsequently developed preeclampsia and six with uncomplicated deliveries. In our analysis, we observed elevations in 10 proteins out of 64 proteins in the preeclampsia study group when compared to the healthy control group. These proteins included clusterin, fibrinogen, fibronectin, and angiotensinogen, increased levels of which are known to be associated with preeclampsia. An elevation in the immune-modulatory molecule, galectin 3 binding protein, was also noted. Our pilot study, therefore, indicates that quantitative proteomic iTRAQ analysis could be a useful tool for the detection of new preeclampsia screening markers.

Can detection of late-onset PE at triage by sflt-1 or PlGF be improved by the use of additional biomarkers?

OBJECTIVE: Accurate identification of preeclampsia (PE) at triage is essential to reduce maternal and fetal morbidity and mortality. The use of maternal blood based biomarkers may facilitate the clinician's ability to assess high risk pregnancies at triage. METHODS: A prospective cross-sectional study was performed to investigate the value of soluble fms-like tyrosine kinase-1 (sFlt-1), soluble endoglin (sEng), placental growth factor (PlGF), sP-selectin, cell-free fetal DNA and total cell-free DNA in patients with late-onset PE versus gestational age-matched controls. RESULTS: The diagnosis of late-onset PE (n = 21) at triage was significantly improved by altered levels of sFlt-1, sEng, PlGF and cffDNA as compared with controls (n = 42). Areas under the receiver operating characteristic curves [AUC, Standard error (SE)] for predicting PE were for marker measurements prior to the first stage of labor as follows: sFlt-1 0.97 (SE 0.02), sEng 0.91 (SE 0.04), PlGF 0.95 (SE 0.04), cell-free fetal DNA (DYS 14) 0.84 (SE 0.06), total cell-free DNA (glyceraldehyde 3-phosphate dehydrogenase) 0.61 (SE 0.07), sP-selectin 0.51 (SE 0.07). The discrimination could be slightly improved by using the slt1-PlGF ratio: 0.98 (SE 0.02). CONCLUSION: The sFlt-1 is a useful tool for the detection of late-onset PE at triage. This can be slightly improved using a sFlt-1/PIGF ratio. The addition of other biomarkers did not improve screening performance for late-onset PE.

Microarray screening for novel preeclampsia biomarker candidates.

INTRODUCTION: Our aim was to identify novel biomarker candidates for the near-term prediction of preeclampsia in a homogenous collective. In this study, we screened at the genome-wide level for gene expression in placental villous tissue from patients with severe preeclampsia in comparison to normal healthy pregnancies. MATERIAL AND METHODS: Total RNA was extracted from placental villous tissue from 9 preeclamptic patients and 7 normotensive controls after scheduled cesarean sections. After sample pooling, gene expression analysis was performed using six Affymetrix Human Gene 1.0 ST arrays, followed by quantitative RT-PCR and validation of selected markers in the serum of patients at the protein level. RESULTS: In total, 896 significantly differentially expressed genes were identified (p ≤ 0.05). After restricting these to molecules present in the circulation, 9 upregulated and 5 downregulated genes were selected. Four of them (ß-hCG, HTRA4, LHB1, all upregulated; and NOX4, downregulated) were validated by quantitative real-time RT-PCR. Finally, the maternal plasma protein levels of 2 of these genes (LHB and ß-hCG) were confirmed to be significantly different between preeclampsia cases and controls. DISCUSSION: We identified 14 potential new biomarker candidates for preeclampsia and validated 4 of them by quantitative RT-PCR and 2 of them with subsequent serum protein analyses. Further studies will assess the optimal marker combination for the imminent prediction of impending preeclampsia.

Determination of fetal chromosome aberrations from fetal DNA in maternal blood: has the challenge finally been met?

The analysis of cell-free fetal nucleic acids in maternal blood for prenatal diagnosis has been transformed by several recent profound technology developments. The most noteworthy of these are 'digital PCR' and 'next-generation sequencing' (NGS), which might finally deliver the long-sought goal of noninvasive detection of fetal aneuploidy. Recent data, however, indicate that NGS might even be able to offer a much more detailed appraisal of the fetal genome, including paternal and maternal inheritance of point mutations for mendelian disorders such as ß-thalassaemia. Although these developments are very exciting, in their current form they are still too complex and costly, and will need to be simplified considerably for their optimal translation to the clinic. In this regard, targeted NGS does appear to be a step in the right direction, although this should be seen in the context of ongoing progress with the isolation of fetal cells and with proteomic screening markers.

Pregnancy and reproduction in autoimmune rheumatic diseases.

Despite evidence for the important role of oestrogens in the aetiology and pathophysiology of chronic immune/inflammatory diseases, the previous view of an unequivocal beneficial effect of oestrogens on RA compared with a detrimental effect on SLE has to be reconsidered. Likewise, the long-held belief that RA remits in the majority of pregnant patients has been challenged, and shows that only half of the patients experience significant improvement when objective disease activity measurements are applied. Pregnancies in patients with SLE are mostly successful when well planned and monitored interdisciplinarily, whereas a small proportion of women with APS still have adverse pregnancy outcomes in spite of the standard treatment. New prospective studies indicate better outcomes for pregnancies in women with rare diseases such as SSc and vasculitis. Fertility problems are not uncommon in patients with rheumatic disease and need to be considered in both genders. Necessary therapy, shortly before or during the pregnancy, demands taking into account the health of both mother and fetus. Long-term effects of drugs on offspring exposed in utero or during lactation is a new area under study as well as late effects of maternal rheumatic disease on children.

Detection of increased amounts of cell-free fetal DNA with short PCR amplicons.

AIM: A digital PCR approach has recently been suggested to detect greater amounts of cell-free fetal DNA in maternal plasma than conventional real-time quantitative PCR (qPCR). Because the digital qPCR approach uses shorter PCR amplicons than the real-time qPCR assay, we investigated whether a real-time qPCR assay appropriately modified for such short amplicons would improve the detection of cell-free fetal DNA. METHOD: We developed a novel universal-template (UT) real-time qPCR assay that was specific for the DYS14 sequence on Y chromosome and had a short amplicon size of 50 bp. We examined this "short" assay with 50 maternal plasma samples and compared the results with those for a conventional real-time qPCR assay of the same locus but with a longer amplicon (84 bp). RESULTS: Qualitatively, both assays detected male cell-free fetal DNA with the same specificity and detection capability. Quantitatively, however, the new UT real-time qPCR assay for shorter amplicons detected, on average, almost 1.6-fold more cell-free fetal DNA than the conventional real-time qPCR assay with longer amplicons. CONCLUSIONS: The use of short PCR amplicons improves the detection of cell-free fetal DNA. This feature may prove useful in attempts to detect cell-free fetal DNA under conditions in which the amount of template is low, such as in samples obtained early in pregnancy.

Quantitative proteomics analysis of maternal plasma in Down syndrome pregnancies using isobaric tagging reagent (iTRAQ).

Currently no specific biomarkers exist for the screening of pregnancies at risk for Down syndrome (DS). Since a quantitative proteomic approach with isobaric labelling (iTRAQ) has recently been suggested to be highly suitable for the discovery of novel plasma biomarkers, we have now used this method to examine for potential quantitative changes in the plasma proteome of the pregnancies bearing DS fetuses in comparison to normal healthy babies. In our study, we used plasma from six women with DS pregnancies and six with uncomplicated pregnancies care were taken to match cases and controls for gestational and maternal age, as these could be a confounder. In our quantitative proteomics analysis we were able to detect 178 proteins using iTRAQ labelling in conjunction with 4800 MALDI TOF/TOF. Amongst these we observed changes in betaHCG, a known screening marker for DS, indicating that our assay was functional. We found a number of elevated proteins Ig lambda chain C region, serum amyloid P-component, amyloid beta A4, and under expressed proteins like gamma-actin and titin in DS pregnancies. These proteins are also found in the sera of patients with Alzheimer disease, which share similar pathologies of DS. Our study therefore indicates that the iTRAQ labelling approach may be indeed useful for the detection of novel biomarkers.

Noninvasive prenatal exclusion of haemoglobin Bart's using foetal DNA from maternal plasma.

OBJECTIVE: Prenatal diagnosis of alpha-thalassaemia requires invasive testing associated with a risk of miscarriage. Cell-free foetal DNA in maternal plasma presents an alternative source of foetal genetic material for noninvasive prenatal diagnosis. We aimed to exclude HbBart's noninvasively by detection of unaffected paternal alleles in maternal plasma using quantitative fluorescence PCR (QF-PCR). METHOD: Microsatellite markers (16PTEL05, 16PTEL06) within the breakpoint regions of -(SEA), -(FIL) and -(THAI) deletions were analysed using QF-PCR of maternal plasma from 30 families. In this blinded study, genotypes were confirmed using conventional PCR. Maternal plasma from two known cases of HbBart's were also analysed. RESULTS: HbBart's was excluded in 10 out of 30 (33.3%, 95% CI, 17.3-52.8%) mothers by identifying the presence of nondeleted paternally inherited fetal alleles; either only 16PTEL05 (n = 1) or only 16PTEL06 (n = 4), or both (n = 5), and confirmed through direct analysis of fetal DNA. Paternally inherited foetal alleles of 16PTEL05 and 16PTEL06 were not detected in maternal plasma of the two known HbBarts cases. False negatives were excluded with the detection of paternally inherited fetal control marker, D21S1270 in maternal plasma. CONCLUSION: We show proof-of-principle that such a test can accurately exclude HbBart's in the foetus by identifying the nondeleted paternally inherited fetal alleles in maternal plasma in one out of three pregnancies, avoiding invasive testing in these pregnancies.

Placental Protein 13 (Galectin-13) Polarizes Neutrophils Toward an Immune Regulatory Phenotype.

Termed as galectin-13, placental protein 13 (PP13) is exclusively expressed in the placenta of anthropoid primates. Research on PP13 in normal and pathologic pregnancies show alteration of PP13 concentrations in pregnancy affected by preeclampsia or gestational diabetes. Galectins are also described as potent immunomodulators, and PP13 regulates T cell function in the placenta. Therefore, this study aims to investigate the effects of PP13 on neutrophils; a cell type often ignored in pregnancy, but present in the uterus and placenta from the early stages of pregnancy. Since neutrophil function is dysregulated during pathologic pregnancies, a link between PP13 and neutrophil activity is possible. We determined that PP13 reduces the apoptosis rate in neutrophils. Also, PP13 increases the expression of PD-L1 and production of HGF, TNF-α, reactive oxygen species (ROS), and MMP-9 in these cells. This phenotype resembles one observed in permissive tumor neutrophils; able to sustain tissue and vessel growth, and inhibit T cell activation. At the same time, PP13 does not alter all neutrophil functions, i.e., extrusion of neutrophil extracellular traps, degranulation, phagocytosis, and ROS production following bacterial exposure. PP13 seems to play an essential role in regulating the activity of neutrophils in the placenta by polarizing them toward a placental-growth-permissive phenotype.

G-CSF Infusion for Stem Cell Mobilization Transiently Increases Serum Cell-Free DNA and Protease Concentrations.

G-CSF for stem cell mobilization increases circulating levels of myeloid cells at different stages of maturation. Polymorphonuclear cells (PMNs) are also mobilized in high numbers. It was previously reported that G-CSF primes PMNs toward the release of neutrophils extracellular traps (NETs). Since NETs are often involved in thrombotic events, we hypothesized that high G-CSF blood concentrations could enhance PMN priming toward NET formation in healthy hematopoietic stem cell donors, predisposing them to thrombotic events. However, we found that G-CSF does not prime PMNs toward NETs formation, but increases the serum concentration of cell-free DNA, proteases like neutrophils elastase and myeloperoxidase, and reactive oxygen species. This could possibly create an environment disposed to induce thrombotic events in the presence of additional predisposing factors.

High-throughput hacking of the methylation patterns in breast cancer by in vitro transcription and thymidine-specific cleavage mass array on MALDI-TOF silico-chip.

Over the last decade, the rapidly expanding interest in the involvement of DNA methylation in developmental mechanisms, human diseases, and malignancies has highlighted the need for an accurate, quantitative, and high-throughput assay. Existing methods are limited and are often too laborious for high-throughput analysis or inadequate for quantitative analysis of methylation. Recently, a MassCLEAVE assay has been developed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to analyze base-specific methylation patterns after bisulfite conversion. To find an efficient and more cost-effective high-throughput method for analyzing the methylation profile in breast cancer, we developed a method that allows for the simultaneous detection of multiple target CpG residues by using thymidine-specific cleavage mass array on matrix-assisted laser desorption/ionization time-of-flight silicon chips. We used this novel quantitative approach for the analysis of DNA methylation patterns of four tumor suppressor genes in 96 breast tissue samples from 48 patients with breast cancer. Each individual contributed a breast cancer specimen and corresponding adjacent normal tissue. We evaluated the accuracy of the approach and implemented critical improvements in experimental design.

Proteomic technologies for prenatal diagnostics: advances and challenges ahead.

Proteomics-based identification of biomarkers for fetal abnormalities in maternal plasma, amniotic fluid and reproductive fluids has made significant progress in the past 5 years. This is attributed mainly to advances in various technology platforms associated with mass spectrometry-based techniques. As these techniques are highly sensitive and require only small quantities of body fluids, it is hoped that they will pave the way for the development of effective noninvasive approaches, without subjecting the developing fetus to the same degree of harm as current invasive procedures (e.g., amniocentesis). It is possible that these developments will include same-day analyses, thereby permitting rapid intervention when necessary. To date, a host of body fluids, such as maternal serum and plasma, amniotic fluid, cervical fluid, vaginal fluid, urine, saliva or fetal material, such as placental trophoblast, fetal membranes or cord blood, have been used successfully in the quest to develop markers for a number of pregnancy-related pathologies. In the current review update we focus on the emergence of proteomics as a major platform technology in studying various types of fetal conditions and developing markers for pregnancy-related disorders, such fetal aneuploidy, preterm birth, preeclampsia, intra-amniotic infection and fetal stress. Should the development of these markers be successful, then it is to be envisaged that proteomic approaches will become standard of care for a number of disease conditions associated with feto-maternal health.

Potential markers of preeclampsia--a review.

Preeclampsia is a leading cause of maternal and fetal/neonatal mortality and morbidity worldwide. The early identification of patients with an increased risk for preeclampsia is therefore one of the most important goals in obstetrics. The availability of highly sensitive and specific physiologic and biochemical markers would allow not only the detection of patients at risk but also permit a close surveillance, an exact diagnosis, timely intervention (e.g. lung maturation), as well as simplified recruitment for future studies looking at therapeutic medications and additional prospective markers. Today, several markers may offer the potential to be used, most likely in a combinatory analysis, as predictors or diagnostic tools. We present here the current knowledge on the biology of preeclampsia and review several biochemical markers which may be used to monitor preeclampsia in a future, that, we hope, is not to distant from today.

Circulating cell-free DNA as a potential biomarker for minimal and mild endometriosis.

It has recently been reported that high concentrations of circulating cell-free (ccf) nucleic acids in plasma and serum could be used as biomarkers for non-invasive monitoring a wide variety of malignant and benign proliferations and inflammatory conditions. Endometriosis is one of the most common benign gynaecological proliferations with inflammatory activation in premenopausal women. Real-time multiplex polymerase chain reaction was used for synchronized quantification of the glyceraldehyde-3-phosphate dehydrogenase gene sequence in nuclear DNA (nDNA) and the ATP synthase-8 gene sequence in mitochondrial DNA (mtDNA). DNA was extracted from 500 microl serum and plasma of 19 cases with endometriosis to measure the total amount of ccf nDNA and ccf mtDNA. The concentration of ccf nDNA in plasma was significantly higher in the endometriosis group than in the control group (P = 0.046). The cut-off value selected by a receiver operating characteristic curve could provide a sensitivity of 70% and a specificity of 87% to discriminate between the minimal or mild cases and normal controls. The finding of significantly increased concentrations of ccf nDNA in plasma of patients with endometriosis suggests that ccf nDNA might be a potential biomarker for developing non-invasive diagnostic test in endometriosis.