Search

While adoptive cell therapies have revolutionized cancer immunotherapy, current autologous chimeric antigen receptor (CAR) T cell manufacturing face challenges in scaling to meet patient demands. CAR T cell production still largely rely on fed-batch, manual, open processes that lack environmental monitoring and control, whereas most perfusion-based, automated, closed-system bioreactors currently suffer from large footprints and working volumes, thus hindering process development and scaling-out. Here, we present a means of conducting anti-CD19 CAR T cell culture-on-a-chip. We show that T cells can be activated, transduced, and expanded to densities exceeding 150 million cells/mL in a two-milliliter perfusion-capable microfluidic bioreactor, thus enabling the production of CAR T cells at clinical dose levels in a small footprint. Key functional attributes such as exhaustion phenotype and cytolytic function were comparable to T cells generated in a gas-permeable well. The process intensification and online analytics offered by the microbioreactor could facilitate high-throughput process optimization studies, as well as enable efficient scale-out of cell therapy manufacturing, while providing insights into the growth and metabolic state of the CAR T cells duringex vivoculture.

The specific cytokines that regulate pediatric acute respiratory distress syndrome (PARDS) pathophysiology remains unclear. Here, we evaluated the respiratory cytokine profile in PARDS to identify the molecular signatures associated with severe disease. A multiplex suspension immunoassay was used to profile 45 cytokines, chemokines and growth factors. Cytokine concentrations were compared between severe and non-severe PARDS, and correlated with oxygenation index (OI). Partial least squares regression modelling and regression coefficient plots were used to identify a composite of key mediators that differentially segregated severe from non-severe disease. The mean (standard deviation) age and OI of this cohort was 5.2 (4.9) years and 17.8 (11.3), respectively. Early PARDS patients with severe disease exhibited a cytokine signature that was up-regulated for IL-12p70, IL-17A, MCP-1, IL-4, IL-1β, IL-6, MIP-1β, SCF, EGF and HGF. In particular, pro-inflammatory cytokines (IL-6, MCP-1, IP-10, IL-17A, IL-12p70) positively correlated with OI early in the disease. Whereas late PARDS was characterized by a differential lung cytokine signature consisting of both up-regulated (IL-8, IL-12p70, VEGF-D, IL-4, GM-CSF) and down-regulated (IL-1β, EGF, Eotaxin, IL-1RA, and PDGF-BB) profiles segregating non-severe and severe groups. This cytokine signature was associated with increased transcription, T cell activation and proliferation as well as activation of mitogen-activated protein kinase pathway that underpin PARDS severity.

A critical initial stage of skeletal morphogenesis involves formation of highly compact aggregates of mesenchymal cells, known as mesenchymal condensations, appearing as regularly-spaced pattern of spots. Conventional computational models to understand their patterning have been based on chemotaxis, haptotaxis, and reaction-diffusion equations. In this work, we investigate the mesenchymal condensations from a different perspective, namely topological defects within liquid crystal-like pattern. Using bone marrow-derived mesenchymal stromal cells (bm-MSCs), we observed emergence of cellular swirls in confluent in-vitro cultures, followed by appearance of mesenchymal condensations at the centers of the selfassembled swirls. Specifically, the condensations appeared at the ‘comet-like’ (+1/2) and ‘spiral-shaped’ (+1) topological defect sites within the swirl pattern. Next, with the rationale that cellular swirls precede skeletal morphogenesis, and supported with the qualitative observation that swirl pattern-features are donor-specific, we probed the correlation between swirl pattern and the chondrogenic differentiation outcome of bm-MSCs. Towards this, we first generated and imaged cellular swirls systematically across 5 donors by controlling seeding density, culture vessel geometry, and culture duration. We observed that the swirl pattern features quantified as variance of coherency correlated strongly with the cartilage matrix proteins, sulfated glycosaminoglycan and collagen-II, quantified from the standard in-vitro chondrogenic differentiation assay. Our work shows that swirl-pattern quantification provides a novel and powerful tool to predict efficacy of bm-MSCs for in-vitro cartilage regeneration.Significance StatementMesenchymal condensation is a critical stage in the formation of bone and cartilage, where the mesenchymal cells form high density cell clusters that are regularly spaced. In this work, we inspect the patterning of these condensations in-vitro from a novel perspective. We first show that at high density, bone-marrow-derived mesenchymal stromal cells (bm-MSCs) self-assemble to form cellular swirls resembling the vortices in a turbulent flow. This is followed by cell aggregations at the centers of the vortices, which show correspondence to mesenchymal condensations. Interestingly, we observed that the swirl pattern made by bm-MSCs isolated from human donors, varies from individual to individual and correlates with their propensity to differentiate into cartilage. This suggests that swirl pattern quantification via image analysis can be used to predict differentiation outcome, in context of regenerative cell therapy.

Women with endometriosis have a profound association with autoimmunity. An excess of autoantigens in the peritoneal cavity resulting from retrograde menstruation could lead to inflammation and pathologic autoimmunity. Using a native-conformation protein array, proteome-wide analysis of autoantibodies (AAbs) against 1623 proteins were profiled in peritoneal fluids (PF) of 25 women with endometriosis and 25 endometriosis-negative women. 46% of endometriotic women have five or more AAbs. Diverse cognate autoantigens were identified and corresponding AAbs against proteins involved in implantation, B-cell activation/development, and aberrant migration and mitogenicity. AAbs recognizing tumour suppressor protein p53 were the most frequent at 35% and were targeted against native and citrullinated p53 forms. Further, unsupervised hierarchical clustering and integrative pathway analysis, we observed clusters of endometriosis-associated infertile women with 60% positive for two or more AAbs which are involved in PDGF, TGF-β, RAC1/PAK1/p38/MMP2 signaling, LAT2/NTAL/LAB-mediated calcium mobilisation and integrin-mediated cell adhesion. Together, our data identifies peritoneal autoimmunity in a significant subset of women with endometriosis, with diverse impact on infertility and disease pathophysiology.

Background: Poor ovarian responders (POR) are women undergoing in-vitro fertilization who respond poorly to ovarian stimulation, resulting in the retrieval of lower number of oocytes, and subsequently lower pregnancy rates. The follicular fluid (FF) provides a crucial microenvironment for the proper development of follicles and oocytes through tightly controlled metabolism and cell signaling. Conversely, dysregulated FF metabolism and cytokine production in POR could impart or reflect detrimental effects on oocytes. Androgens such as dehydroepiandrosterone (DHEA) have been proposed to alter the POR follicular microenvironment and induce metabolic changes but its utility in improving pregnancy rates remain uncertain, partially due to the unknown impact DHEA imposes on the FF metabolome and cytokine profiles. Methods: FF samples were collected from 52 POR patients who underwent IVF with DHEA supplementation (DHEA+) and without (DHEA-). The FF samples were split for profiling analyses using untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomics and a large-scale multiplex suspension immunoassay covering 65 cytokines, chemokines and growth factors. Multivariate statistical modelling by partial least squares-discriminant regression (PLSR) analysis was performed for revealing metabolome-scale differences. Further, differential metabolite analysis between the two groups was performed by PLSR β-coefficient regression analysis and Student’s t-test. Results: Untargeted metabolomics identified 118 FF metabolites of diverse chemistries and concentrations which spanned three orders of magnitude. They include metabolic products highly associated with ovarian function – amino acids for regulating pH and osmolarity, lipids such fatty acids and cholesterols for oocyte maturation, and glucocorticoids for ovarian steroidogenesis. The FF metabolomic profiles of DHEA+ and DHEA- groups were notably distinct as classified by multivariate partial least squares regression analysis. Specifically, glycerophosphocholine, linoleic acid, progesterone, and valine were significantly lower in DHEA+ relative to DHEA-. Using the large-scale cytokine profiling assay, we observed significantly lower MCP1, IFNγ, LIF and VEGF-D levels in DHEA+ relative to DHEA. Conclusions: Collectively, our large-scale study characterized a diversity of metabolites and cytokines in the FF of POR individuals and identified several of these compounds associated DHEA supplementation.

To study differences in cytokine expression profiles between women with ongoing pregnancy and those experiencing spontaneous miscarriage, among women who presented with threatened miscarriage before week 16 of gestation.Prospective cohort study.Academic hospital.In this prospective cohort study, 155 pregnant women, comprising normal pregnant women recruited from antenatal clinics (n = 97) and women with threatened miscarriage recruited from an emergency walk-in clinic (n = 58).None.Sixty-five serum cytokines quantified using multiplex immunoassay correlated with miscarriage outcomes.Among women presenting with threatened miscarriage, those who eventually miscarried had significantly lower levels of interleukin (IL)-2, IL-12p70, IL-17A, B-cell-activating factor, B lymphocyte chemoattractant, basic nerve growth factor, interferon-γ, tumor necrosis factor-related apoptosis-inducing ligand, thymic stromal lymphopoietin, and tumor necrosis factor-α and higher levels of vascular endothelial growth factor A, IL-21, and stromal cell-derived factor 1α than those with ongoing pregnancy. Comparisons between normal pregnancies and women with threatened miscarriage who eventually miscarried revealed significant differences across 7 cytokines: B-cell-activating factor; B lymphocyte chemoattractant; basic nerve growth factor; IL-17A; fractalkine/CX3CL1; vascular endothelial growth factor A; and CCL22. Vascular endothelial growth factor A exhibited a negative correlation with the progesterone level (r = -0.270). The cluster of significant cytokines alludes to T cell proliferation, B-cell proliferation, natural killer cell-mediated cytotoxicity, and apoptosis as important pathways that determine pregnancy outcomes. Bioinformatic analysis further revealed alteration of the suppressor of cytokine signaling proteins family of Janus kinase-signal transducer and activator of transcription signaling axis by cytokines as a plausible key molecular mechanism in spontaneous miscarriage.This study demonstrates that the regulated balance between the proinflammatory and anti-inflammatory pathways is crucial to maintaining pregnancy. A better understanding of the cytokines associated with immunomodulatory effects may lead to novel targets for the prediction and treatment of spontaneous miscarriage.

Metabolism is the collection of biochemical reactions enabled by chemically diverse metabolites, which facilitate different physiological processes to exchange substances and synthesize energy in diverse living organisms. Metabolomics has emerged as a cutting-edge method to qualify and quantify the metabolites in different biological matrixes, and it has the extraordinary capacity to interrogate the biological significance that underlies metabolic modification and modulation. Liquid chromatography combined with mass spectrometry (LC/MS), as a robust platform for metabolomics analysis, has increased in popularity over the past 10 years due to its excellent sensitivity, throughput, and versatility. However, metabolomics investigation currently provides us with only phenotype data without revealing the biochemical functions and associated mechanisms. This limitation indeed weakens the core value of metabolomics data in a broad spectrum of the life sciences. In recent years, the scientific community has actively explored the functional features of metabolomics and translated this cutting-edge approach to be used to solve key multifaceted questions, such as disease pathogenesis, the therapeutic discovery of drugs, nutritional issues, agricultural problems, environmental toxicology, and microbial evolution. Here, we are the first to briefly review the history and applicable progression of LC/MS-based metabolomics, with an emphasis on the applications of metabolic phenotyping. Furthermore, we specifically highlight the next era of LC/MS-based metabolomics to target functional metabolomes, through which we can answer phenotype-related questions to elucidate biochemical functions and associated mechanisms implicated in dysregulated metabolism. Finally, we propose many strategies to enhance the research capacity of functional metabolomics by enabling the combination of contemporary omics technologies and cutting-edge biochemical techniques. The main purpose of this review is to improve the understanding of LC/MS-based metabolomics, extending beyond the conventional metabolic phenotype toward biochemical functions and associated mechanisms, to enhance research capability and to enlarge the applicable scope of functional metabolomics in small-molecule metabolism in different living organisms.

Nucleic acids-based molecular diagnostic tools incorporating the CRISPR/Cas system are being developed as rapid and sensitive methods for pathogen detection. However, most CRISPR/Cas-based diagnostics lack quantitative detection ability. Here, we report Warm-Start RApid DIgital Crispr Approach (WS-RADICA), which uses commercially available digital chips for the rapid, sensitive, and quantitative detection of nucleic acids. WS-RADICA detected as little as 1 copy/μl SARS-CoV-2 RNA in 40 min (qualitative detection) or 60 min (quantitative detection). WS-RADICA can be easily adapted to various digital devices: two digital devices were evaluated for both DNA and RNA quantification, with linear dynamic ranges of 0.8-12777 copies/µL for DNA and 1.2-18391 copies/µL for RNA (both R2 values > 0.99). Moreover, WS-RADICA had greater sensitivity and inhibitor tolerance than a bulk RT-LAMP-Cas12b reaction and similar performance to RT-qPCR and RT-dPCR. Given its speed, sensitivity, quantification capability, and inhibitor tolerance, WS-RADICA shows great promise for a variety of applications requiring nucleic acid quantification.

Despite numerous advances in in vitro fertilization (IVF) techniques since its first success in 1978, almost half of the patients treated remain childless. The multifactorial nature of IVF treatment means that success is dependent on variables, including the quality of oocytes. Therefore, new technologies are needed to objectively and quantitatively examine how each oocyte can be selected or optimized to achieve for the best possible outcomes for patients. Here, we report an optimized digital polymerase chain reaction (dPCR) for direct absolute quantification of nucleic acids within 3.5 h without the need for sample extraction or purification. Using individual oocytes, the developed method demonstrated absolute quantification with a linear dynamic range of 0.65 – 33 copies/µL (r2=0.999), high accuracy and excellent reproducibility of Gapdh (0.72-16.95 copies/oocyte), Hprt1 (1.05-19.05 copies/oocyte) and ATPase 6, (5.55-32358.15 copies/oocyte) in ovaries even from the same mouse. Finally, dPCR was used to validate extracellular microRNAs from oocytes incubated with a toxic unsaturated very-long chained ceramide. This study therefore shows the feasibility of dPCR for the rapid and sensitive absolute quantification of DNA/RNA and extracellular miRNA for the study of oocytes.

Flaviviral infections result in a wide spectrum of clinical outcomes, ranging from asymptomatic infection to severe disease. Although the correlates of severe disease have been explored1-4, the pathophysiology that differentiates symptomatic from asymptomatic infection remains undefined. To understand the molecular underpinnings of symptomatic infection, the blood transcriptomic and metabolomic profiles of individuals were examined before and after inoculation with the live yellow fever viral vaccine (YF17D). It was found that individuals with adaptive endoplasmic reticulum (ER) stress and reduced tricarboxylic acid cycle activity at baseline showed increased susceptibility to symptomatic outcome. YF17D infection in these individuals induced maladaptive ER stress, triggering downstream proinflammatory responses that correlated with symptomatic outcome. The findings of the present study thus suggest that the ER stress response and immunometabolism underpin symptomatic yellow fever and possibly even other flaviviral infections. Modulating either ER stress or metabolism could be exploited for prophylaxis against symptomatic flaviviral infection outcome.

Aim: In complex biological matrixes, many sphingolipids are present with multiple reaction monitoring traces or lack of standard for verification, potentially leading to inaccurate identification and quantitation. Results/methodology: Based on these retention times of available standards, we devised a retention time bracketing approach to identify and predict sphingolipids of the same homologous series. Excellent concordance of predicted and observed retention times (

Accelerated aerobic glycolysis is a distinctive metabolic property of cancer cells that confers dependency on glucose for survival. However, the selective therapeutic targeting of this vulnerability has yielded mixed results owing to the different sensitivities of each cancer type to glucose removal and lack of insight into the underlying mechanisms of glucose deprivation-induced cell death.Here, we screened multiple cell lines to determine their sensitivities to glucose deprivation and found that the cell lines most sensitive to glucose deprivation failed to activate AMPK, a major regulator of metabolic adaptation, resulting in metabolic catastrophe.Notably, glucose deprivation-induced AMPK dysregulation and rapid cell death were observed only in cancer cell lines with high expression of cystine/glutamate antiporter xCT. While this phenomenon was prevented by pharmacological or genetic inhibition of xCT, overexpression of xCT sensitized resistant cancer cells to glucose deprivation. We found that cystine uptake and glutamate export through xCT under glucose deprivation contributes to rapid NADPH depletion, leading to the collapse of the redox system, which subsequently inactivates AMPK by inhibitory oxidation and phosphorylation. This AMPK dysregulation caused a failure of metabolic switch to fatty acid oxidation upon glucose deprivation, resulting in mitochondrial dysfunction and cell death.Taken together, these findings suggest a novel cross-talk between the metabolic and signal transduction and reveal a metabolic vulnerability in xCT-high expressing cancer cells to glucose deprivation and provide a rationale for targeting glucose metabolism in these cancer cells.

Zika virus (ZIKV) is an Aedes mosquito-borne flavivirus that causes debilitating congenital and developmental disorders. No licensed ZIKV vaccine or treatment exist, due largely to an incomplete understanding of ZIKV pathogenesis. In this study, we used a panel of ZIKV strains, including a pair that differ by just a single amino acid substitution (phenylalanine to leucine substitution, M-F37L) in the membrane (M) protein, and coupled these with unbiased whole genome gene expression studies to demarcate the border between attenuated and pathogenic infection. We identified infection-induced metabolic dysregulation as a minimal set of host alterations that differentiated attenuated from pathogenic ZIKV strains. Glycolytic rewiring reduced availability of tricarboxylic acid (TCA) cycle substrates, resulting in impaired oxidative phosphorylation and mitochondrial dysfunction that triggered inflammation and apoptosis in pathogenic but not attenuated ZIKV strains. Exogenous supplementation of pyruvate and glutamine that feed directly into the TCA cycle, reduced inflammatory and apoptotic responses induced by wild-type ZIKV infection. Critically, pyruvate supplementation to ZIKV-infected dams prevented gross fetal developmental abnormalities and rescued fetal growth. Our findings thus pinpoint dysregulated metabolism as an underpinning of ZIKV pathogenicity and that pyruvate supplementation to expectant women could be explored as a prophylaxis for congenital Zika syndrome.

Rapid diagnostics of adventitious agents in biopharmaceutical/cell manufacturing release testing and the fight against viral infection have become critical. Quantitative real-time PCR and CRISPR-based methods rapidly detect DNA/RNA in 1 h but suffer from inter-site variability. Absolute quantification of DNA/RNA by methods such as digital PCR reduce this variability but are currently too slow for wider application. Here, we report a RApid DIgital Crispr Approach (RADICA) for absolute quantification of nucleic acids in 40-60 min. Using SARS-CoV-2 as a proof-of-concept target, RADICA allows for absolute quantification with a linear dynamic range of 0.6-2027 copies/µL (R2 value > 0.99), high accuracy and low variability, no cross-reactivity to similar targets, and high tolerance to human background DNA. RADICA's versatility is validated against other targets such as Epstein-Barr virus (EBV) from human B cells and patients' serum. RADICA can accurately detect and absolutely quantify EBV DNA with similar dynamic range of 0.5-2100 copies/µL (R2 value > 0.98) in 1 h without thermal cycling, providing a 4-fold faster alternative to digital PCR-based detection. RADICA therefore enables rapid and sensitive absolute quantification of nucleic acids which can be widely applied across clinical, research, and biomanufacturing areas.

What are the potential biomarkers for peritoneal endometriosis in peritoneal fluid and serum?Case-control studies composed of independent discovery and validation sets were conducted. In the discovery set, untargeted liquid chromatography-mass spectrometry (LC-MS/MS) metabolomics, multivariable and univariable analyses were conducted to generate global metabolomic profiles of peritoneal fluid for endometriosis and to identify potential metabolites that could distinguish peritoneal endometriosis (n = 10) from controls (n = 31). The identified metabolites from the discovery set were validated in independent peritoneal fluid (n =19 peritoneal endometriosis and n = 20 controls) and serum samples (n = 16 peritoneal endometriosis and n = 19 controls) using targeted metabolomics. The area under the receiver-operating characteristics curve (AUC) analysis was used to evaluate the diagnostic performance of peritoneal endometriosis metabolites.In the discovery set, peritoneal fluid phosphatidylcholine (34:3) and phenylalanyl-isoleucine were significantly increased in peritoneal endometriosis groups compared with control groups, with AUC 0.77 (95% CI 0.61 to 0.92; P = 0.018) and AUC 0.98 (95% CI 0.95 to 1.02; P0.001), respectively. In the validation set, phenylalanyl-isoleucine retained discriminatory performance to distinguish peritoneal endometriosis from controls in both peritoneal fluid (AUC 0.77, 95% CI 0.61 to 0.92; P = 0.006) and serum samples (AUC 0.81, 95% CI 0.64 to 0.99; P = 0.004), with notably stronger discrimination between peritoneal endometriosis and controls in proliferative phase.Our preliminary results propose phenylalanyl-isoleucine as a potential biomarker of peritoneal endometriosis, which may be used as a minimally invasive diagnostic biomarker of peritoneal endometriosis.

Poor ovarian responders (POR) are women undergoing in-vitro fertilization who respond poorly to ovarian stimulation, resulting in the retrieval of lower number of oocytes, and subsequently lower pregnancy rates. The follicular fluid (FF) provides a crucial microenvironment for the proper development of follicles and oocytes. Conversely, dysregulated FF metabolome and cytokinome could have detrimental effects on oocytes in POR. Androgens such as dehydroepiandrosterone (DHEA) have been proposed to alter the POR follicular microenvironment but its effects on the FF metabolome and cytokine profiles is unknown. In this study, untargeted LC-MS/MS metabolomics was performed on FF of POR patients with DHEA supplementation (DHEA+) and without (DHEA-) in a randomized clinical trial (N=52). Untargeted metabolomics identified 118 FF metabolites of diverse chemistries, which included lipids, steroids, amino acids, hormones, among others. FF metabolomes were different between DHEA+ and DHEA- groups. Specifically, glycerophosphocholine, linoleic acid, progesterone, and valine were significantly lower in DHEA+ relative to DHEA-. Among cytokines, MCP1, IFNγ, LIF and VEGF-D were significantly lower in DHEA+ relative to DHEA. Collectively, our data suggest a role of DHEA on these metabolic and cytokines pathways, and these FF metabolites could be used to guide future studies in DHEA supplementation regimen.

Quantitative real-time PCR and CRISPR-based methods detect SARS-CoV-2 in 1 hour but do not allow for the absolute quantification of virus particles, which could reduce inter-lab variability and accelerate research. The 4-hour reaction time of the existing digital PCR-based method for absolute virus quantification is too long for widespread application. We report a RApid DIgital Crispr Approach (RADICA) for the absolute quantification of SARS-CoV-2 DNA and Epstein–Barr virus DNA in human samples that yields results within 1 hour. For validation, we compared RADICA to digital PCR for quantifying synthetic SARS-CoV-2 DNA and Epstein–Barr viral DNA. RADICA allows absolute quantification of DNA with a dynamic range from 0.6 to 2027 copies/µL (R2 value > 0.98), without cross-reactivity on similar virus or human background DNA. Thus, RADICA can accurately detect and quantify nucleic acid in 1h without thermal cycling, providing a 4-fold faster alternative to digital PCR-based virus detection.

ObjectiveTo identify potential serum biomarkers in women with peritoneal endometriosis (PE) by first looking at its source in the peritoneal fluid (PF).DesignCase-control pilot studies, comprising independent discovery and validation sets.SettingKK Women’s and Children’s Hospital, Singapore.Patient(s)Women with laparoscopically confirmed PE and absence of endometriosis (control).Intervention(s)None.Main Outcome Measure(s)In the discovery set, we used untargeted liquid chromatography-mass spectrometry (LC-MS/MS) metabolomics, multivariable and univariable analyses to generate global metabolomic profiles of PF for endometriosis and to identify potential metabolites that could distinguish PE (n=10) from controls (n=31). Using targeted metabolomics, we validated the identified metabolites in PF and sera of cases (n=16 PE) and controls (n=19). We performed the area under the receiver-operating characteristics curve (AUC) analysis to evaluate the diagnostic performance of PE metabolites.Result(s)In the discovery set, PF phosphatidylcholine (34:3) and phenylalanyl-isoleucine were significantly increased in PE than controls groups, with AUC 0.77 (95% confidence interval 0.61-0.92; p=0.018) and AUC 0.98 (0.95-1.02; pp=0.006) and serum samples (AUC 0.81; 0.64-0.99; p=0.004).Conclusion(s)Our preliminary results propose phenylalanyl-isoleucine as a potential biomarker of PE, which may be used as a minimally-invasive diagnostic biomarker of PE.

Nucleic acids-based molecular diagnostic tools incorporating the CRISPR/Cas system are being developed as rapid and sensitive methods for pathogen detection. However, most CRISPR/Cas-based diagnostics lack quantitative detection ability. Here, we report Warm-Start RApid DIgital Crispr Approach (WS-RADICA) for the rapid, sensitive, and quantitative detection of nucleic acids. WS-RADICA detected as little as 1 copy/μl SARS-CoV-2 RNA in 40 min (qualitative detection) or 60 min (quantitative detection). WS-RADICA can be easily adapted to various digital devices: two digital chips were evaluated for both DNA and RNA quantification, with linear dynamic ranges of 0.8-12777 copies/μL for DNA and 1.2-18391 copies/μL for RNA (both R

Endometriosis is a common inflammatory gynecological disorder which causes pelvic scarring, pain, and infertility, characterized by the implantation of endometrial-like lesions outside the uterus. The peritoneum, ovaries, and deep soft tissues are the commonly involved sites, and endometriotic lesions can be classified into three subphenotypes: superficial peritoneal endometriosis (PE), ovarian endometrioma (OE), and deep infiltrating endometriosis (DIE). In 132 women diagnosed laparoscopically with and without endometriosis (n = 73, 59 respectively), and stratified into PE, OE, and DIE, peritoneal fluids (PF) were characterized for 48 cytokines by using multiplex immunoassays. Partial-least-squares-regression analysis revealed distinct subphenotype cytokine signatures—a six-cytokine signature distinguishing PE from OE, a seven-cytokine signature distinguishing OE from DIE, and a six-cytokine-signature distinguishing PE from DIE—each associated with different patterns of biological processes, signaling events, and immunology. These signatures describe endometriosis better than disease stages (p < 0.0001). Pathway analysis revealed the association of ERK1 and 2, AKT, MAPK, and STAT4 linked to angiogenesis, cell proliferation, migration, and inflammation in the subphenotypes. These data shed new insights on the pathophysiology of endometriosis subphenotypes, with the potential to exploit the cytokine signatures to stratify endometriosis patients for targeted therapies and biomarker discovery.

Study question Does an early proliferative phase endometrial biopsy harvested during ovarian stimulation harbour information predictive of the outcome following fresh embryo transfer (ET) in that same cycle? Summary answer Transcriptome analysis of the whole-tissue endometrium did not reveal significant differential gene expression (DGE) in relation to the outcome; however, the secretome profile of isolated, cultured and in vitro decidualized endometrial stromal cells (EnSCs) varied significantly between patients who had a live birth compared to those with an implantation failure following fresh ET in the same cycle as the biopsy. What is known already In the majority of endometrial receptivity research protocols, biopsies are harvested during the window of implantation (WOI). This, however, precludes ET in that same cycle, which is preferable as the endometrium has been shown to adapt over time. Endometrial biopsies taken during ovarian stimulation have been reported not to harm the chances of implantation, and in such biopsies DGE has been observed between women who achieve pregnancy versus those who do not. The impact of the endometrial proliferative phase on human embryo implantation remains unclear, but deserves further attention, especially since in luteal phase endometrial biopsies, a transcriptional signature predictive for repeated implantation failure has been associated with reduced cell proliferation, possibly indicating proliferative phase involvement. Isolation, culture and in vitro decidualization (IVD) of EnSCs is a frequently applied basic research technique to assess endometrial functioning, and a disordered EnSC secretome has previously been linked with failed implantation. Study design, size, duration This study was nested in a randomized controlled trial (RCT) investigating the effect of endometrial scratching during the early follicular phase of ovarian stimulation on clinical pregnancy rates after IVF/ICSI. Of the 96 endometrial biopsies available, after eliminating those without fresh ET and after extensive matching in order to minimize the risk of potential confounding, 18 samples were retained to study two clinical groups: nine biopsies of patients with a live birth versus nine biopsies of patients with an implantation failure, both following fresh ET performed in the same cycle as the biopsy. We studied the proliferative endometrium by analysing its transcriptome and by isolating, culturing and decidualizing EnSCs in vitro. We applied this latter technique for the first time on proliferative endometrial biopsies obtained during ovarian stimulation for in-cycle outcome prediction, in an attempt to overcome inter-cycle variability. Participants/materials, setting, methods RNA-sequencing was performed for 18 individual whole-tissue endometrial biopsies on an Illumina HiSeq1500 machine. DGE was analysed three times using different approaches (DESeq2, EdgeR and the Wilcoxon rank-sum test, all in R). EnSC isolation and IVD was performed (for 2 and 4 days) for a subset of nine samples, after which media from undifferentiated and decidualized cultures were harvested, stored at −80°C and later assayed for 45 cytokines using a multiplex suspension bead immunoassay. The analysis was performed by partial least squares regression modelling. Main results and the role of chance After correction for multiple hypothesis testing, DGE analysis revealed no significant differences between endometrial samples from patients who had a live birth and those with an implantation failure following fresh ET. However secretome analysis after EnSC isolation and culture, showed two distinct clusters that clearly corresponded to the two clinical groups. Upon IVD, the secretome profiles shifted from that of undifferentiated cells but the difference between the two clinical groups remained yet were muted, suggesting convergence of cytokine profiles after decidualization. Limitations, reasons for caution Caution is warranted due to the limited sample size of the study and the in vitro nature of the EnSC experiment. Validation on a larger scale is necessary, however, hard to fulfil given the very limited availability of in-cycle proliferative endometrial biopsies outside a RCT setting. Wider implications of the findings These data support the hypothesis that the endometrium should be assessed not only during the WOI and that certain endometrial dysfunctionalities can probably be detected early in a cycle by making use of the proliferative phase. This insight opens new horizons for the development of endometrial tests, whether diagnostic or predictive of IVF/ICSI treatment outcome. Study funding/competing interest(s) This study was supported by Fonds Wetenschappelijk Onderzoek (FWO, Flanders, Belgium, 11M9415N, 1 524 417N), Wetenschappelijk Fonds Willy Gepts (WFWG G160, Universitair Ziekenhuis Brussel, Belgium) and the National Medicine Research Council (NMRC/CG/M003/2017, Singapore). There are no conflicts of interests. Trial registration number NCT02061228.

Endometriosis is a disease characterized by regurgitated lesions which are invasive and migratory, embedding at ectopic, extra-uterine locations. Extracellular glucosylceramides (GlcCers), bioactive sphingolipids potentiating signals for cell migration, are found in elevated levels in endometriosis; however underlying mechanisms that result in cellular migration are poorly defined. Here, we demonstrated that internalized GlcCer induced migratory activity in immortalized human endometrial stromal cells (HESCs), with highest potency observed in long-chain GlcCer. Long-chain ceramide (Cer) similarly induced cellular migration and mass spectrometry results revealed that the migratory behavior was contributed through glycosylation of ceramides. Cells treated with GlcCer synthase inhibitor, or RNAi-mediated knockdown of glucosylceramide synthase (GCS), the enzyme catalyzing GlcCer production attenuated cell motility. Mechanistic studies showed that GlcCer acts through stromal cell-derived factor-1 alpha and its receptor, CXC chemokine receptor 4 (SDF-1α-CXCR4) signaling axis and is dependent on phosphorylation of LYN kinase at Tyr396, and dephosphorylation of Tyr507. Migration was prominently attenuated in cells exposed to CXCR4 antagonist, AMD3100, yet can be rescued with diprotin A, which prevents the degradation of SDF-1α. Furthermore, blocking of LYN kinase activity in the presence of SDF-1α and GlcCer reduced HESC migration, suggesting that LYN acts downstream of GlcCer-SDF-1α-CXCR4 axis as part of its intracellular signal transduction. Our results reveal a novel role of long-chain GlcCer and the dialog between GlcCer, LYNpTyr396 and SDF-1α-CXCR4 in inducing HESC migration. This finding may improve our understanding how endometriotic lesions invade to their ectopic sites, and the possibility of using GlcCer to modulate the SDF-1α-CXCR4-LYNpTyr396 axis in endometriosis.

The prognosis of dengue remains a challenge in the early, objective triage of patients with dengue fever of differing severity. Circulating immuno-modulating proteins have brought new possibilities as prognostic markers of severe dengue (SD). This systematic review is devoted to understanding the potential utility of blood-based cytokines and chemokines as prognostication markers of SD based on the current literature. PubMed and Embase were searched. Of 794 candidate articles, 685 abstracts were screened against our exclusion/inclusion criteria and 25 (3.6 %) studies met the quality assessments. A total of 18 studies were retrospective observational and 2 were prospective cohort studies. Elevated IL-10, up to day 7 of fever onset, stood out as a candidate prognostic marker for SD using the 1997 and 2009 World Health Organization (WHO) case definitions. IFNγ was another potential prognostic marker of SD (1997 WHO case definition), but its levels varied between studies. Significant heterogeneity in methodologies and patient cohorts prevent ready application of IL-10 and IFNγ as prognostic markers to other dengue populations. Our results suggest that the current non-randomized studies are delivering inconsistent messages and higher-quality studies, with consistent methodologies and validation in independent patient cohorts, are needed to delineate confounding variables. Major gaps identified were full accounting and transparency of sampling days, dengue virus type, infection status and age group.

Neutralizing antibodies (nAbs) are a critical component for protection against dengue virus (DENV) infection, but little is known about the immune mechanisms governing their induction and whether such mechanisms can be harnessed for vaccine development. In this study, we profiled the early immune responses to flaviviruses in human peripheral blood mononuclear cells and screened a panel of toll-like receptor (TLR) agonists that stimulate the same immune signatures. Monocyte/macrophage-driven inflammatory responses and interferon responses were characteristics of flavivirus infection and associated with induction of nAbs in humans immunized with the yellow fever vaccine YF-17D. The signatures were best reproduced by the combination of TLR agonists Pam3CSK4 and PolyI:C (PP). Immunization of both mice and macaques with a poorly immunogenic recombinant DENV-2 envelope domain III (EDIII) induced more consistent nAb and CD4+ T-cell responses with PP compared to alum plus monophosphoryl lipid A. Induction of nAbs by PP required interferon-mediated signals in macrophages in mice. However, EDIII + PP vaccination only provided partial protection against viral challenge. These results provide insights into mechanisms underlying nAb induction and a basis for further improving antigen/adjuvant combinations for dengue vaccine development., Dengue vaccine: adjuvants for induction of neutralizing antibodies Neutralizing antibodies (nAb) are associated with protection from symptomatic dengue virus infection and current dengue vaccine development aims to elicit a strong nAb response. However, immune mechanisms underlying nAb development in response to dengue infection or vaccination are not completely understood. In this study, led by Jianzhu Chen from the Interdisciplinary Research Group in Infectious Diseases in Singapore, researchers identify a combination of two TLR agonists that elicits better nAb responses than currently used adjuvants. Comparison of transcriptomes of human PBMCs infected with flaviviruses identifies an immune signature that is associated with induction of nAbs and a similar immune signature can be elicited with a combination of two TLR agonists. Vaccination of mice or non-human primates using these two TLR agonists as adjuvant elicits a strong nAb response. However, this vaccination regimen could not confer protection in the animals, suggesting that further improvements of this vaccine candidate will be necessary.

Flaviviral infections result in a wide spectrum of clinical outcomes, ranging from asymptomatic infection to severe disease. Although the correlates of severe disease have been explored

Successful vascularization is essential in wound healing, the histo-integration of biomaterials, and other aspects of regenerative medicine. We developed a functional in vitro assay to dissect the complex processes directing angiogenesis during wound healing, whereby vascular cell spheroids were induced to sprout in the presence of classically (M1) or alternatively (M2) activated macrophages. This simulated a microenvironment, in which sprouting cells were exposed to the inflammatory or proliferation phases of wound healing, respectively. We showed that M1 macrophages induced single-cell migration of endothelial cells and pericytes. In contrast, M2 macrophages augmented endothelial sprouting, suggesting that vascular cells infiltrate the wound bed during the inflammatory phase and extensive angiogenesis is initiated upon a switch to a predominance of M2. Interestingly, M1 and M2 shared a pro-angiogenic secretome, whereas pro-inflammatory cytokines were solely secreted by M1. These results suggested that acute inflammatory factors act as key inducers of vascular cell infiltration and as key negative regulators of angiogenesis, whereas pro-angiogenic factors are present throughout early wound healing. This points to inflammatory factors as key targets to modulate angiogenesis. The here-established wound healing assay represents a useful tool to investigate the effect of biomaterials and factors on angiogenesis during wound healing.

Neutralizing antibodies (nAbs) are a critical component for protection against dengue virus (DENV) infection, but little is known about the immune mechanisms governing their induction and whether such mechanisms can be harnessed for vaccine development. In this study, we profiled the early immune responses to flaviviruses in human peripheral blood mononuclear cells and screened a panel of toll-like receptor (TLR) agonists that stimulate the same immune signatures. Monocyte/macrophage-driven inflammatory responses and interferon responses were characteristics of flavivirus infection and associated with induction of nAbs in humans immunized with the yellow fever vaccine YF-17D. The signatures were best reproduced by the combination of TLR agonists Pam

During gestation the developing human fetus is exposed to a diverse range of potentially immune-stimulatory molecules including semi-allogeneic antigens from maternal cells, substances from ingested amniotic fluid, food antigens, and microbes. Yet the capacity of the fetal immune system, including antigen-presenting cells, to detect and respond to such stimuli remains unclear. In particular, dendritic cells, which are crucial for effective immunity and tolerance, remain poorly characterized in the developing fetus. Here we show that subsets of antigen-presenting cells can be identified in fetal tissues and are related to adult populations of antigen-presenting cells. Similar to adult dendritic cells, fetal dendritic cells migrate to lymph nodes and respond to toll-like receptor ligation; however, they differ markedly in their response to allogeneic antigens, strongly promoting regulatory T-cell induction and inhibiting T-cell tumour-necrosis factor-α production through arginase-2 activity. Our results reveal a previously unappreciated role of dendritic cells within the developing fetus and indicate that they mediate homeostatic immune-suppressive responses during gestation.

Objective To characterize the peritoneal fluid (PF) sphingolipid profile in endometriosis-associated infertility (EAI), and to assess the plausible functional role(s) of ceramides in oocyte maturation potential. Design Retrospective case-control study and in vitro mouse oocyte study. Setting University-affiliated hospital and university laboratory. Subjects Twenty-seven infertile patients diagnosed with endometriosis and 20 infertile patients who did not have endometriosis; BALB/c female mice. Intervention(s) None. Main Outcome Measure(s) PF sphingolipid concentrations. Number of metaphase II (MII) mouse oocytes. Result(s) Liquid chromatography–tandem mass spectrometry revealed 11 significantly elevated PF sphingolipids in infertile women with severe endometriosis compared with infertile women without endometriosis (change >50%, false discovery rate ≤10%). Logistic regression analysis identified three very-long-chain ceramides potentially associated with EAI. Functional studies revealed that very-long-chain ceramides may compromise or induce murine MII oocyte maturation. The oocyte maturation effects induced by the very long-chain ceramides were triggered by alterations in mitochondrial superoxide production in a concentration-dependent manner. Scavenging of mitochondrial superoxide reversed the maturation effects of C24:0 ceramide. Conclusion(s) EAI is associated with accumulation of PF very-long-chain ceramides. Mouse studies demonstrated how ceramides affect MII oocyte maturation, mediating through mitochondrial superoxide. These results provide an opportunity for direct functional readout of pathophysiology in EAI, and future therapies targeted at this sphingolipid metabolism may be harnessed for improved oocyte maturation.

In cycling human endometrium, menstruation is followed by rapid estrogen-dependent growth. Upon ovulation, progesterone and rising cellular cAMP levels activate the transcription factor Forkhead box O1 (FOXO1) in endometrial stromal cells (EnSCs), leading to cell cycle exit and differentiation into decidual cells that control embryo implantation. Here we show that FOXO1 also causes acute senescence of a subpopulation of decidualizing EnSCs in an IL-8 dependent manner. Selective depletion or enrichment of this subpopulation revealed that decidual senescence drives the transient inflammatory response associated with endometrial receptivity. Further, senescent cells prevent differentiation of endometrial mesenchymal stem cells in decidualizing cultures. As the cycle progresses, IL-15 activated uterine natural killer (uNK) cells selectively target and clear senescent decidual cells through granule exocytosis. Our findings reveal that acute decidual senescence governs endometrial rejuvenation and remodeling at embryo implantation, and suggest a critical role for uNK cells in maintaining homeostasis in cycling endometrium.

The endometrial perivascular microenvironment is rich in mesenchymal stem-like cells that express type 1 integral membrane protein Sushi domain containing 2 (SUSD2) but the role of these cells in the decidual transformation of this tissue in pregnancy is unknown. We used an antibody directed against SUSD2 (W5C5) to isolate perivascular (W5C5+) and nonperivascular (W5C5−) fibroblasts from mid-luteal biopsies. We show that SUSD2 expression, and hence the ratio of W5C5+:W5C5− cells, changes in culture depending on cell-cell contact and activation of the Notch signaling pathway. RNA sequencing revealed that cultures derived from W5C5+ progenitor cells remain phenotypically distinct by the enrichment of novel and established endometrial perivascular signature genes. In an undifferentiated state, W5C5+-derived cells produced lower levels of various chemokines and inflammatory modulators when compared with their W5C5− counterparts. This divergence in secretomes was switched and became more pronounced upon decidualization, which transformed perivascular W5C5+ cells into the dominant source of a range of chemokines and cytokines, including leukemia inhibitory factor and chemokine (C-C motif) ligand 7. Our findings suggest that the decidual response is spatially organized at the embryo-maternal interface with differentiating perivascular cells establishing distinct cytokine and chemokine profiles that could potentially direct trophoblast toward maternal vessels and govern local immune responses in pregnancy.