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OBJECTIVES: To investigate the performance of an automated system for the extraction of cell-free DNA of maternal and fetal origin from stored serum samples for subsequent quantitative real-time polymerase chain reaction (PCR) analysis. METHODS: Thirty-two maternal blood samples between the early second trimester and term were obtained. Cell-free DNA was extracted from replicate stored sera using a column-based manual isolation procedure and with an automated system, the MagNA Pure(trade mark) LC Instrument. Real-time quantitative PCR for the ubiquitous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and male-specific DYS14 loci was performed. RESULTS: The extraction yields for both total and fetal DNA and the quality of the purified nucleic acids were similar for the automated system or the manual procedure. However, the number of false-negative results in samples collected early in pregnancy was reduced with the automated extraction. Furthermore, the extraction rate by the automated system was highly reproducible over time. CONCLUSIONS: We validated the use of an automated extraction system for the isolation of fetal DNA from stored serum. This procedure might be exploited in the future for high-throughput non-invasive fetal gene analysis of archived serum samples. Copyright (c) 2008 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Abstract 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.

First opened in 1964 in London, the Brook Advisory Centres (BAC) were the first centres to provide contraceptive advice and sexual counselling to unmarried people in postwar Britain. Drawing on archival materials, medical articles published by BAC members and oral history interviews with former counsellors, this paper looks at tensions present in sexual health counselling work between progressive views on young people's sexuality and moral conservatism. In so doing, this paper makes two inter-related arguments. First, I argue that BAC doctors, counsellors and social workers simultaneously tried to adopt a non-judgmental listening approach to young people's sexual needs and encouraged a model of heteronormative sexual behaviours that was class-based and racialised. Second, I argue that emotional labour was central in BAC staff's attempt to navigate and smooth these tensions. This emotional labour and the tensions within it is best illustrated by BAC's pyschosexual counselling services, which on the one hand tried to encourage youth sexual pleasure and on the other taught distinctive gendered sexual roles that contributed to pathologising teenage sexual behaviours and desire.In all, I contend that, in resorting to an emotionally orientated counselling, BAC members reconfigured for the young the new form of sexual subjectivity that had been in the making since the interwar years, that is, the fact that individuals regarded themselves as sexual beings and expressed feelings and anxieties about sex. BAC's counselling work was as much a rupture with the interwar contraceptive counselling tradition-since it operated in a new climate, stressed a non-judgmental listening approach and catered for the young-as it was a continuity of some of the values of the earlier movement., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ.)

Purpose: For patients with resectable stage IIIA(N2) non-small-cell lung cancer, neoadjuvant chemotherapy with cisplatin and docetaxel followed by surgery resulted in a 1-year event-free survival (EFS) rate of 48% in the SAKK 16/00 trial and is an accepted standard of care. We investigated the additional benefit of perioperative treatment with durvalumab., Methods: Neoadjuvant treatment consisted of three cycles of cisplatin 100 mg/m 2 and docetaxel 85 mg/m 2 once every 3 weeks followed by two doses of durvalumab 750 mg once every 2 weeks. Durvalumab was continued for 1 year after surgery. The primary end point was 1-year EFS. The hypothesis for statistical considerations was an improvement of 1-year EFS from 48% to 65%., Results: Sixty-eight patients were enrolled, 67 were included in the full analysis set. Radiographic response rate was 43% (95% CI, 31 to 56) after neoadjuvant chemotherapy and 58% (95% CI, 45 to 71) after sequential neoadjuvant immunotherapy. Fifty-five patients were resected, of which 34 (62%) achieved a major pathologic response (MPR; ≤ 10% viable tumor cells) and 10 (18%) among them a complete pathologic response. Postoperative nodal downstaging (ypN0-1) was observed in 37 patients (67%). Fifty-one (93%) resected patients had an R0 resection. There was no significant effect of pretreatment PD-L1 expression on MPR or nodal downstaging. The 1-year EFS rate was 73% (two-sided 90% CI, 63 to 82). Median EFS and overall survival were not reached after 28.6 months of median follow-up. Fifty-nine (88%) patients had an adverse event grade ≥ 3 including two fatal adverse events that were judged not to be treatment-related., Conclusion: The addition of perioperative durvalumab to neoadjuvant chemotherapy in patients with stage IIIA(N2) non-small-cell lung cancer is safe and exceeds historical data of chemotherapy alone with a high MPR and an encouraging 1-year EFS rate of 73%., Competing Interests: Sacha I. RothschildConsulting or Advisory Role: Bristol Myers Squibb, AstraZeneca, Boehringer Ingelheim, Eisai, Roche, Novartis, Merck Serono, MSD Oncology, Pfizer, Takeda, AbbVieResearch Funding: Boehringer Ingelheim, AstraZeneca, Bristol Myers Squibb, Eisai, Merck Serono, AbbVieExpert Testimony: Roche, AstraZeneca, Bristol Myers SquibbTravel, Accommodations, Expenses: Roche Pharma AG, Lilly, Bristol Myers Squibb, AstraZeneca, Merck Sharp & Dohme, Amgen Alfred ZippeliusHonoraria: BMSi, MSD, Roche, NBE Therapeutics, ACM Pharma, Hookipa Biotech, BeyondSpring PharmaceuticalsResearch Funding: Roche, NBE Therapeutics, Secarna, ACM Pharma, Hookipa Biotech, Beyondsprings Spasenija Savic PrinceHonoraria: NovartisConsulting or Advisory Role: Diaceutics Ireland Limited, Merck (Schweiz) AG, AstraZeneca AG Daniel BetticherTravel, Accommodations, Expenses: Bayer Adrienne BettiniHonoraria: MSD OncologyConsulting or Advisory Role: MSD Oncology, AstraZenecaTravel, Accommodations, Expenses: Janssen Oncology Martin FrühConsulting or Advisory Role: BMS, AstraZeneca, MSD, Takeda, Roche, LillySpeakers' Bureau: PfizerResearch Funding: BMS, AstraZeneca Markus JoergerConsulting or Advisory Role: Novartis, AstraZeneca, Basilea Pharmaceutical, Bayer, Bristol Myers Squibb, Debiopharm Group, Merck, Roche, SanofiResearch Funding: AstraZeneca, Basilea Pharmaceutical, Bayer, Bristol Myers Squibb, Daiichi Sankyo, Immunophotonics, InnoMedica, Janssen Oncology, Lilly, Merck, Novartis, Pfizer, PharmaMar, Roche, Sanofi, Takeda Laetitia A. MautiConsulting or Advisory Role: Takeda, Roche, AstraZeneca, Bristol Myers Squibb, Merck Sharp & Dohme, PfizerTravel, Accommodations, Expenses: Takeda, Bristol Myers Squibb, Merck, Roche, AstraZeneca Christian BritschgiConsulting or Advisory Role: AstraZeneca, Pfizer, Roche, Takeda, Janssen-Cilag, Boehringer IngelheimTravel, Accommodations, Expenses: AstraZeneca, Takeda Walter WederConsulting or Advisory Role: AstraZeneca, Covidien/MedtronicSpeakers' Bureau: AstraZeneca, Covidien/MedtronicTravel, Accommodations, Expenses: AstraZeneca, Covidien/Medtronic Solange PetersHonoraria: Roche, Bristol Myers Squibb, Novartis, Pfizer, MSD, AstraZeneca, Takeda, Illumina, MedscapeConsulting or Advisory Role: Roche/Genentech, Novartis, Bristol Myers Squibb, Pfizer, MSD, Amgen, AstraZeneca, Janssen, Regeneron, Merck Serono, Boehringer Ingelheim, Takeda, Lilly, AbbVie, Bayer, Biocartis, Debiopharm Group, Illumina, PharmaMar, Sanofi, Seattle Genetics, Blueprint Medicines, Daiichi Sankyo, Incyte, Bioinvent, Clovis Oncology, Vaccibody, Phosplatin TherapeuticsResearch Funding: Roche, BMS, MSD, Amgen, Lilly, AstraZeneca, Pfizer, Illumina, Merck Serono, Novartis, Biodesix, Boehringer Ingelheim, Iovance Biotherapeutics, Phosplatin TherapeuticsTravel, Accommodations, Expenses: Roche, Bristol Myers Squibb, MSD, Sanofi, IncyteUncompensated Relationships: Journal of Thoracic Oncology, ESMO, European Thoracic Oncology Platform (ETOP), Annals of Oncology Michael MarkConsulting or Advisory Role: Roche, AstraZeneca, Takeda, BMS, MSD OncologyTravel, Accommodations, Expenses: Pfizer, Roche, Takeda Richard CathomasHonoraria: Janssen-Cilag, Astellas Pharma, BMS, Debiopharm GroupConsulting or Advisory Role: Astellas Pharma, Bristol Myers Squibb, Pfizer, Roche, MSD Oncology, Janssen-Cilag, Bayer, Sanofi, IpsenTravel, Accommodations, Expenses: AstraZeneca Adrian F. OchsenbeinConsulting or Advisory Role: TolremoResearch Funding: argenxPatents, Royalties, Other Intellectual Property: Patent on the anti-CD70 antibody cusatuzumab Wolf-Dieter JanthurConsulting or Advisory Role: Roche, Takeda, MSD, Novartis Nicolas MachStock and Other Ownership Interests: MaxVAX SAResearch Funding: MaxiVaxPatents, Royalties, Other Intellectual Property: I am an inventor on patent owned by MaxiVAX SA and on patent co-owned by Geneva University Hospital and MaxiVAX SAUncompensated Relationships: MaxiVax Patrizia FroeschConsulting or Advisory Role: Pfizer, Takeda, Roche, Boehringer Ingelheim, AstraZeneca, Novartis, Bayer Pierre BohanesHonoraria: MSD, BayerTravel, Accommodations, Expenses: Janssen Corinne RusterholzEmployment: Roche Miklos PlessHonoraria: Janssen-CilagConsulting or Advisory Role: AbbVie, AstraZeneca, Boehringer Ingelheim, Bristol Myers Squibb, Eisai Europe, MSD Oncology, Novartis, Pfizer/EMD Serono, Roche, Takeda, Merck SeronoExpert Testimony: TakedaTravel, Accommodations, Expenses: Vifor Pharma, Bristol Myers Squibb, Boehringer Ingelheim, AstraZenecaNo other potential conflicts of interest were reported.

Background: KRAS mutations are found in 20-25 % of non-squamous non-small cell lung cancer (NSCLC) and therapies targeting the RAS/MEK/ERK pathway are in development. We performed a multicenter open-label phase 1B trial to determine the recommended phase 2 dose and early antitumor activity of the MEK-inhibitor binimetinib combined with cisplatin and pemetrexed., Methods: Eligible patients (pts) had stage III-IV NSCLC unsuitable for curative treatment, KRAS exon 2 or 3 (codon 12, 13 or 61) mutations, no prior systemic therapy. Pts were enrolled into part 1: 3 + 3 design with dose escalation in 2 dose levels (DL) of binimetinib and part 2: expansion cohort at the maximum tolerated dose (MTD). Pts received 4 cycles of cisplatin 75 mg/m 2 , pemetrexed 500 mg/m 2 and binimetinib 30 (DL1)/45 mg (DL2) orally twice a day (bid) d1-14 q3w followed by pemetrexed and binimetinib until progressive disease (PD) or unacceptable toxicity., Results: From May 2017 to Dec 2019, 18 pts (13 dose escalation, 5 expansion cohort) were enrolled. Median age was 60 (48-73, range). KRAS mutations were 87.5 % at codon 12. No DLT occurred in the dose escalation cohort. Median number of cycles was 2 (1-17, range). Treatment discontinuation was mainly due to PD (33 %) or pts/physicians' decision (27 %). Together with the expansion cohort, 16 pts were evaluable for safety. Most frequent treatment-related grade 3 AEs were lung infection (25 %), fatigue (19 %), anemia (19 %). Overall response rate among 9 evaluable pts receiving binimetinib at MTD (45 mg bid) was 33 % (7-70 %, 95 % CI). Median progression-free survival was 5.7 months (1.1-14.0, 95 % CI) and overall survival 6.5 months (1.8-NR, 95 % CI)., Conclusions: Pts treated with combination of cisplatin, pemetrexed and binimetinib presented no unexpected toxicity. No early signal of increased antitumor activity of binimetinib added to chemotherapy was observed in our pts population., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The Swiss Group for Clinical Cancer Research (SAKK) conducted the SAKK 35/03 randomized trial (NCT00227695) to investigate different rituximab monotherapy schedules in patients with follicular lymphoma (FL). Here, we report their long-term treatment outcome. Two-hundred and seventy FL patients were treated with 4 weekly doses of rituximab monotherapy (375 mg/m2); 165 of them, achieving at least a partial response, were randomly assigned to maintenance rituximab (375 mg/m2 every 2 months) on a short-term (4 administrations; n = 82) or a long-term (up to a maximum of 5 years; n = 83) schedule. The primary end point was event-free survival (EFS). At a median follow-up period of 10 years, median EFS was 3.4 years (95% confidence interval [CI], 2.1-5.5) in the short-term arm and 5.3 years (95% CI, 3.5-7.5) in the long-term arm. Using the prespecified log-rank test, this difference is not statistically significant (P = .39). There also was not a statistically significant difference in progression-free survival or overall survival (OS). Median OS was 11.0 years (95% CI, 11.0-NA) in the short-term arm and was not reached in the long-term arm (P = .80). The incidence of second cancers was similar in the 2 arms (9 patients after short-term maintenance and 10 patients after long-term maintenance). No major late toxicities emerged. No significant benefit of prolonged maintenance became evident with longer follow-up. Notably, in symptomatic patients in need of immediate treatment, the 10-year OS rate was 83% (95% CI, 73-89%). These findings indicate that single-agent rituximab may be a valid first-line option for symptomatic patients with advanced FL., (© 2020 by The American Society of Hematology.)

This article uses the audio recordings of sexual counselling sessions carried out by Dr Joan Malleson, a birth control activist and committed family planning doctor in the early 1950s, which are held at the Wellcome Library in London as a case study to explore the ways Malleson and the patients mobilised emotions for respectively managing sexual problems and expressing what they understood as constituting a 'good sexuality' in postwar Britain. The article contains two interrelated arguments. First, it argues that Malleson used a psychological framework to inform her clinical work. She resorted to an emotion-based therapy that linked sexual difficulties with unconscious, repressed feelings rooted in past events. In so doing, Malleson actively helped to produce a new form of sexual subjectivity where individuals were encouraged to express their feelings and emotions, breaking with the traditional culture of emotional control and restraint that characterized British society up until the fifties. Second, I argue that not only Malleson but also her patients relied on emotions. The performance of mainly negative emotions reveals what they perceived as the 'normal' and sexual 'ideal'. Sexual therapy sessions reflected the seemingly changing nature of the self towards a more emotionally aware and open one that adopted both the language of emotions and that of popular psychology to articulate his or her sexual difficulties., (© The Author(s) [2019]. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

This paper explores the influence of English female doctors on the creation of the International Planned Parenthood Federation (IPPF) and the production and circulation of contraceptive knowledge in England and, to a lesser extent in France, between 1930 and 1970. By drawing on the writings of female doctors and proceedings of international conferences as well as the archives of the British Medical Women's Federation (MWF) and Family Planning Association (FPA), on the one hand, and Mouvement Français pour le Planning Familial (MFPF), on the other, this paper explores the agency of English female doctors at the national and transnational level. I recover their pioneering work and argue that they were pivotal in legitimising family planning within medical circles. I then turn to their influence on French doctors after World War II. Not only were English medical women active and experienced agents in the family planning movement in England; they also represented a conduit of information and training crucial for French doctors. Transfer of knowledge across the channel was thus a decisive tool for implementing family planning services in France.

This special issue adopts a comparative approach to the politics of reproduction in twentieth-century France and Britain. The articles investigate the flow of information, practices and tools across national boundaries and between groups of experts, activists and laypeople. Empirically grounded in medical, news media and feminist sources, as well as ethnographic fieldwork, they reveal the practical similarities that existed between countries with officially different political regimes as well as local differences within the two countries. Taken as a whole, the special issue shows that the border between France and Britain was more porous than is typically apparent from nationally-focused studies: ideas, people and devices travelled in both directions; communication strategies were always able to evade the rule of law; contraceptive practices were surprisingly similar in both countries; and religion loomed large in debates on both sides of the channel.

This paper examines the introduction to Britain of the Gräfenberg ring, an early version of what later became known as an intrauterine device (IUD). The struggle during the interwar years to establish the value of the ring provides an opportunity for a case study of the evaluation and acceptance of a new medical device. With the professionalization of the birth control movement and the expansion of birth control clinics in interwar Britain, efforts to develop better scientific means for contraception grew rapidly. At the end of the nineteenth century, methods for controlling fertility ranged from coitus interruptus and abstinence, to diverse substances ingested or placed into the vagina, to barrier methods. The first decades of the twentieth century brought early work on chemical contraceptives as well as a number of new intrauterine devices, among them the Gräfenberg ring. Developing a cheap, reliable, and widely acceptable contraceptive became a pressing goal for activists in the voluntary birth control movement in Britain between the wars. Yet, tensions developed over the best form of contraception to prescribe. By situating the Gräfenberg ring within the context of the debates and competition among British medical and birth control professionals, this paper reveals broader issues of power relationships and expertise in the assessment of a new medical technology., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Purpose: Rituximab maintenance therapy has been shown to improve progression-free survival in patients with follicular lymphoma; however, the optimal duration of maintenance treatment remains unknown., Patients and Methods: Two hundred seventy patients with untreated, relapsed, stable, or chemotherapy-resistant follicular lymphoma were treated with four doses of rituximab monotherapy in weekly intervals (375 mg/m(2)). Patients achieving at least a partial response were randomly assigned to receive maintenance therapy with one infusion of rituximab every 2 months, either on a short-term schedule (four administrations) or a long-term schedule (maximum of 5 years or until disease progression or unacceptable toxicity). The primary end point was event-free survival (EFS). Progression-free survival, overall survival (OS), and toxicity were secondary end points. Comparisons between the two arms were performed using the log-rank test for survival end points., Results: One hundred sixty-five patients were randomly assigned to the short-term (n = 82) or long-term (n = 83) maintenance arms. Because of the low event rate, the final analysis was performed after 95 events had occurred, which was before the targeted event number of 99 had been reached. At a median follow-up period of 6.4 years, the median EFS was 3.4 years (95% CI, 2.1 to 5.3) in the short-term arm and 5.3 years (95% CI, 3.5 to not available) in the long-term arm (P = .14). Patients in the long-term arm experienced more adverse effects than did those in the short-term arm, with 76% v 50% of patients with at least one adverse event (P < .001), five versus one patient with grade 3 and 4 infections, and three versus zero patients discontinuing treatment because of unacceptable toxicity, respectively. There was no difference in OS between the two groups., Conclusion: Long-term rituximab maintenance therapy does not improve EFS, which was the primary end point of this trial, or OS, and was associated with increased toxicity., Competing Interests: Authors' disclosures of potential conflicts of interest are found in the article online at www.jco.org. Author contributions are found at the end of this article., (© 2015 by American Society of Clinical Oncology.)

Purpose: Our main objective was to prospectively determine the prognostic value of [(18)F]fluorodeoxyglucose positron emission tomography/computed tomography (PET/CT) after two cycles of rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone given every 14 days (R-CHOP-14) under standardized treatment and PET evaluation criteria., Patients and Methods: Patients with any stage of diffuse large B-cell lymphoma were treated with six cycles of R-CHOP-14 followed by two cycles of rituximab. PET/CT examinations were performed at baseline, after two cycles (and after four cycles if the patient was PET-positive after two cycles), and at the end of treatment. PET/CT examinations were evaluated locally and by central review. The primary end point was event-free survival at 2 years (2-year EFS)., Results: Median age of the 138 evaluable patients was 58.5 years with a WHO performance status of 0, 1, or 2 in 56%, 36%, or 8% of the patients, respectively. By local assessment, 83 PET/CT scans (60%) were reported as positive and 55 (40%) as negative after two cycles of R-CHOP-14. Two-year EFS was significantly shorter for PET-positive compared with PET-negative patients (48% v 74%; P = .004). Overall survival at 2 years was not significantly different, with 88% for PET-positive versus 91% for PET-negative patients (P = .46). By using central review and the Deauville criteria, 2-year EFS was 41% versus 76% (P < .001) for patients who had interim PET/CT scans after two cycles of R-CHOP-14 and 24% versus 72% (P < .001) for patients who had PET/CT scans at the end of treatment., Conclusion: Our results confirmed that an interim PET/CT scan has limited prognostic value in patients with diffuse large B-cell lymphoma homogeneously treated with six cycles of R-CHOP-14 in a large prospective trial. At this point, interim PET/CT scanning is not ready for clinical use to guide treatment decisions in individual patients., (© 2015 by American Society of Clinical Oncology.)

Human pregnancy is accompanied by a mild systemic inflammatory response, which includes the activation of monocytes circulating in maternal blood. This response is exaggerated in preeclampsia, a placental-dependent disorder specific to human pregnancies. We and others showed that placental syncytiotrophoblast membrane microparticles (STBM) generated in vitro from normal placentas stimulated peripheral blood monocytes, which suggest a contribution of STBM to the systemic maternal inflammation. Here, we analyzed the inflammatory potential of STBM prepared from preeclamptic placentas on primary monocytes and investigated the mode of action in vitro. STBM generated in vitro by placental villous explants of normal or preeclamptic placentas were co-incubated with human peripheral blood monocytes. In some cases, inhibitors of specific cellular functions or signaling pathways were used. The analysis of the monocytic response was performed by flow cytometry, enzyme-linked immunoassays, real-time PCR, and fluorescence microscopy. STBM derived from preeclamptic placentas up-regulated the cell surface expression of CD54, and stimulated the secretion of the pro-inflammatory interleukin (IL)-6 and IL-8 in a similar, dose-dependent manner as did STBM prepared from normal placentas. STBM bound to the cell surface of monocytes, but phagocytosis was not necessary for activation. STBM-induced cytokine secretion was impaired in the presence of inhibitors of toll-like receptor (TLR) signaling or when nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation was blocked. Our results suggest that the inflammatory reaction in monocytes may be initiated by the interaction of STBM with TLRs, which in turn signal through NF-κB to mediate the transcription of genes coding for pro-inflammatory factors.

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., (© 2012 John Wiley & Sons, Ltd.)

Preeclampsia is a common disorder of the second half of pregnancy that complicates 2% to 7% of all pregnancies worldwide and remains a major cause of maternal and fetal morbidity and mortality. Although the origin of the disease is still elusive, population-based studies have suggested that it might implicate genetic, immunologic, or physiologic factors. On the other hand, there is no doubt that the placenta plays an important role in its development. In preeclampsia, the shedding of placenta debris, such as syncytiotrophoblast microparticles (STBMs) and DNA and messenger RNA molecules, into the maternal peripheral blood is increased. The analysis of this material may give new insight into placentation and the underlying etiology of this disorder, as well as yield new tracks of research for the understanding of the molecular mechanisms, leading to the generation of the clinical symptoms.

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.

Erythroblast cell differentiation involves self-controlled and limited nuclear proteolysis prior nucleus loss. Early evidence suggests that apoptotic-like pathways are activated during this process. The chromatin of developing erythroblasts becomes fragmented in vivo, however, the exact mechanisms and molecules involved remain elusive. In this study, erythroblasts were differentiated in culture from CD34-enriched umbilical cord blood progenitor cells and the characteristics of DNA fragmentation were examined. This analysis shows that the chromatin of differentiating erythroblasts is cleaved into discrete fragments of 50-200 kb. This process most likely involves one or several endonucleases as we detect in vivo double strand DNA cleavage. However, major players of the apoptotic DNA degradation, caspase activated DNase and apoptosis inducing factor, are not activated in these cells. Therefore, our data suggests that erythroblast chromatin degradation may involve enzymes distinct form those active in apoptotic cells.

Human pregnancy is a metabolic and immune challenge for the mother who has to accommodate in her womb a semi-allogeneic fetus whose energy needs increase tremendously with gestation. Recent compelling research has suggested that proper inflammatory changes and oxidative balance are a requisite for successful pregnancy. The placenta is an integral component of this inflammatory response as it actively produces a variety of cytokines and immunomodulatory hormones. In preeclampsia, a life-threatening disorder of pregnancy that is characterized by widespread damage and dysfunction of the maternal endothelium, placental oxidative stress and aberrant cytokine expression induces an exaggerated maternal systemic inflammatory response to pregnancy.

The transcription factor PAX5 is a critical regulator of B-cell commitment and development. Although normally not expressed in myeloid progenitors, PAX5 has recently been shown to be frequently expressed in myeloid malignancies and to suppress expression of myeloid differentiation genes, compatible with an effect on the differentiation or maintenance of myeloid progenitors. However, previous studies in which PAX5 was ectopically expressed in normal myeloid progenitors in vivo and in vitro provided conflicting results as to the effect of PAX5 on myeloid development. Herein, we demonstrate that on ectopic expression of PAX5 in bone marrow multipotent stem/progenitor cells, cells with a biphenotypic B220(+)GR-1/MAC-1(+) phenotype are produced. These remain cytokine-dependent, but unlike control-transduced cells they sustain long-term generation of myeloid progenitors in vitro and remain capable of myeloid differentiation. Notably, PAX5(+)B220(+)GR-1/MAC-1(+) myeloid progenitors coexpress, at the single-cell level, myeloid genes and otherwise B-cell-specific PAX5 target genes. These findings establish that ectopic expression of PAX5 introduces extensive self-renewal properties in otherwise short-lived myeloid progenitors. Along with the established ectopic expression of PAX5 in acute myeloid leukemia, this motivates a careful investigation of the potential involvement of ectopic PAX5 expression in myeloid and biphenotypic leukemias.

Apoptosis plays an important role in red blood cell development, notably by regulating the fate of early erythroid progenitors. We show here that, by contrast, mature erythroblasts are resistant to apoptosis. Treatment of these cells with several apoptosis-inducing agents failed to trigger caspase activation and oligonucleosomal DNA fragmentation. Interestingly, we find that cytochrome c levels are dramatically reduced even though the cells contain mitochondria. Supplementation of cytosolic extracts from mature erythroblasts with cytochrome c, however, did not rescue caspase activation. This was not due to the presence of inhibitors of apoptosis, as these proteins were also missing in these cells. We also show that cytochrome c depletion is a normal event during erythroblast differentiation, which follows transient, developmentally induced caspase activation and correlates with the loss of response to cytokine withdrawal or drug-induced apoptosis. Our data therefore suggest that erythroblasts acquire resistance to apoptosis during maturation through the developmentally induced depletion of cytochrome c and other crucial regulators of the apoptotic machinery.

Objectives: Cell-free fetal RNA from placental origin is present in the peripheral blood of pregnant women. Therefore, its qualitative analysis might provide insights into the physiological condition of the placenta. Here, we examine whether oxidative stress affects the integrity of placentally derived mRNA in vitro., Methods: Placental explants were cultured under normal or oxidative conditions, and the levels of placental and syncytiotrophoblast microparticle associated glyceraldehyde-3-phosphate dehydrogenase 3' versus 5' mRNA fragments were analyzed by real-time reverse-transcriptase polymerase chain reaction., Result: The relative ratio of 3' to 5' mRNA fragments associated with placental microparticles was significantly altered upon culture under oxidative stress., Conclusions: Our data suggest that oxidative stress reduces the levels of full-length, particle-associated glyceraldehyde-3-phosphate dehydrogenase mRNA transcripts released by the placenta. Therefore, analysis of the microparticle-coupled mRNA integrity in pregnant women might prove useful to diagnose disorders such as preeclampsia, where placental oxidative stress is involved., (Copyright (c) 2007 S. Karger AG, Basel.)

Recent studies have provided new insight into aberrations in the immunological interplay between mother and fetus and their potential role in the development of recurrent fetal loss and preeclampsia. The action of anti-phospholipid antibodies in recurrent fetal loss is now proposed to involve the complement system, neutrophil activation and the production of TNFalpha by immune bystander cells. A clear involvement of the immune system is emerging in preeclampsia, involving mainly the innate arm, especially neutrophils. The activation of peripheral neutrophils by placentally released inflammatory debris triggers the induction of neutrophil extracellular traps (NETs), which may lead to an occlusion of the intervillous space, thereby further promoting a condition of placental hypoxia. It has, hence, been suggested that new therapeutic strategies be developed, including the possible use of TNFalpha antagonists in cases of recurrent miscarriage. These strategies need to be addressed with caution due to the possible induction of fetal congenital abnormalities.

Recent studies have suggested that pre-eclampsia may result from endothelial cell damage and overt immune activity triggered by the elevated release of syncytiotrophoblast-derived micro-particles (STBM). In this context, STBM have been reported to inhibit lymphocyte proliferation and induce Jurkat T cell apoptosis. In this study, STBM were prepared by three different in vitro methods (mechanical dissection, villous explant culture, and placental perfusion) and their functional properties were tested on T lymphocytes enriched from peripheral blood samples. Mechanically- and villous explant-derived STBM significantly inhibited activation, proliferation and cytokine release by T lymphocytes, while placental perfusion-derived STBM significantly induced T cell proliferation and a slight increase in IFNgamma release. None of the STBM preparations caused T cell apoptosis. Therefore, STBM prepared by different methods in vitro exhibit different effects on circulating T cells, a feature that will have to be taken into account when considering their potential role in normal pregnancy and pre-eclampsia.

Objective: The purpose of this study was to analyze the potential of placental-conditioned medium to activate endothelial cells in vitro and to identify the placental factors that mediate this effect., Study Design: Placental-conditioned medium was generated by the culturing of normal term placental villous explants for up to 48 hours. Human umbilical vein endothelial cells were exposed to the conditioned media, and cellular proliferation, viability, and activation were investigated., Results: The proliferation of endothelial cells that were exposed to 20% placental-conditioned medium was reduced by 25%, but their survival was not compromised. Conditioned medium also up-regulated the expression of E-selectin and stimulated the release of soluble intercellular adhesion molecule-1 and the secretion of interleukin-6. Treatment with interleukin-1 receptor antagonist, but not with an anti-tumor necrosis factor-alpha neutralizing antibody, blocked the release of soluble intercellular adhesion molecule-1 and interleukin-6., Conclusion: Placentally derived interleukin-1 may be 1 of the potential mediators of the maternal inflammatory response that is observed in late pregnancy.

Background: Analysis of fetal DNA in maternal plasma has recently been introduced as a new method for noninvasive prenatal diagnosis, particularly for the analysis of fetal genetic traits, which are absent from the maternal genome, e.g., RHD or Y-chromosome-specific sequences. To date, the analysis of other fetal genetic traits has been more problematic because of the overwhelming presence of maternal DNA sequences in the circulation. We examined whether different biochemical properties can be discerned between fetal and maternal circulatory DNA., Methods: Plasma DNA was examined by agarose gel electrophoresis. The fractions of fetal and maternal DNA in size-fractionated fragments were assayed by real-time PCR. The determination of paternally and maternally inherited fetal genetic traits was examined by use of highly polymorphic chromosome-21-specific microsatellite markers., Results: Size fractionation of circulatory DNA indicated that the major portion of cell-free fetal DNA had an approximate molecular size of <0.3 kb, whereas maternally derived sequences were, on average, considerably larger than 1 kb. Analysis of size-fractionated DNA (

Recent developments of surrogate assays for human hematopoietic stem cells (HSC) have facilitated efforts at improving HSC gene transfer efficiency. Through the use of xenograft transplantation models, such as nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, successful oncoretroviral gene transfer to transplantable hematopoietic cells has been achieved. However, because of the low frequency and/or homing efficiency of SCID repopulating cells (SRC) in bone marrow (BM), studies have primarily focused on cord blood (CB). The recently developed extended (> 60 days) long-term culture-initiating cell (ELTC-IC) assay detects an infrequent and highly quiescent candidate stem cell population in BM as well as CB of the CD34(+)CD38(-) phenotype. Although these characteristics suggest that ELTC-IC and SRC might be closely related, attempts to oncoretrovirally transduce ELTC-IC have been unsuccessful. Here, recently developed conditions (high concentrations of SCF + FL + Tpo in serum-free medium) supporting expansion of BM CD34(+)CD38(-) 12 week ELTC-IC promoted efficient oncoretroviral transduction of BM and CB ELTC-IC. Although SRC can be transduced with oncoretroviral vectors, this is frequently associated with loss of reconstituting activity, posing a problem for development of clinical HSC gene therapy. However, previous attempts at expanding transduced HSC posttransduction resulted in compromised rather than improved gene marking. Utilizing conditions promoting cell divisions and transduction of ELTC-IC we show that although 5 days of ex vivo culture is sufficient to obtain maximum gene transfer efficiency to SRC, extension of the expansion period to 12 days significantly enhances multilineage reconstitution activity of transduced SRC, supporting the feasibility of improving gene marking through ex vivo expansion.

Interleukin-2 (IL-2) responsiveness of T lymphocytes is controlled through transcription of the IL-2 receptor (IL-2R) alpha subunit by antigen and by IL-2 itself. IL-2 induces IL-2Ralpha transcription via an IL-2-responsive enhancer (IL-2rE), whose activity depends on the cooperative binding of IL-2-induced STAT5 to two sites and of constitutively active Elf-1 to a third one. Here we describe the changes in IL-2rE chromatin that occur in normal T lymphocytes upon activation of IL-2Ralpha expression. In cells induced to transiently express IL-2Ralpha with concanavalin A (which mimics antigen), none of the IL-2rE sites is occupied despite the presence of Elf-1 and STAT1, which bind to the IL-2rE in vitro. The two STAT binding sites are occupied rapidly upon IL-2 stimulation, concomitantly with STAT5 activation. Occupation of the Elf-1 binding site is delayed, although Elf-1 concentration and binding activity are not modified by IL-2. Digestion of T-cell chromatin with DNase I and micrococcal nuclease shows that IL-2 induces the appearance of nuclease-hypersensitive sites flanking the IL-2rE. Thus IL-2, in addition to activating STAT5, appears to regulate IL-2Ralpha transcription by making IL-2Ralpha chromatin accessible to transcription factors.