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Monoclonal IgG antibodies constitute the fastest growing class of therapeutics. Thus, there is an intense interest to design more potent antibody formats, where long plasma half-life is a commercially competitive differentiator affecting dosing, frequency of administration and thereby potentially patient compliance. Here, we report on an Fc-engineered variant with three amino acid substitutions Q311R/M428E/N434W (REW), that enhances plasma half-life and mucosal distribution, as well as allows for needle-free delivery across respiratory epithelial barriers in human FcRn transgenic mice. In addition, the Fc-engineered variant improves on-target complement-mediated killing of cancer cells as well as both gram-positive and gram-negative bacteria. Hence, this versatile Fc technology should be broadly applicable in antibody design aiming for long-acting prophylactic or therapeutic interventions. Antibody based biologics are a rapidly growing class of therapeutics with interest to enhance their performance, distribution, longevity and effectivity. Here, authors report the engineering of human IgG Fc to enhance plasma half-life, mucosal distribution and killing of cancer cells and bacteria.

Exposure to maternal stress during pregnancy can have adverse effects on the fetus, which has potential long-term effects on offspring's development and health. We investigated the kinetics and metabolism of the hormones and amino acids: cortisol, cortisone, tryptophan and serotonin in the term placenta in an ex vivo human placental perfusion model. The placentas used in the experiments were donated from families participating in the Maternal Stress and Placental Function project with a known maternal stress background. Cortisol, cortisone, tryptophan and serotonin were added simultaneously to the maternal side in the 6 hour ex vivo term human recirculating placental perfusion model, in four experimental set-ups: without inhibitors, with carbenoxolone -that inhibits cortisol metabolism into cortisone, with fluoxetine that inhibits the serotonin transporter, and with PCPA that inhibits metabolism of tryptophan into serotonin. The concentration of cortisol and cortisone, and tryptophan and serotonin were quantified using UPLC and HPLC-MS respectively. Cortisol was rapidly metabolized into cortisone in the placenta, to a somewhat lesser degree when adding the inhibitor carbenoxolone, resulting in higher fetal exposure to cortisol. Serotonin was also rapidly metabolized in the placenta. When adding fluoxetine a peak of fetal serotonin levels was seen in the first hour of the perfusion. No effect was seen of the maternal stress levels on placental transport kinetics in this study. Inhibiting the metabolism of cortisol in the placenta increased fetal exposure to cortisol as expected. Unexpectedly we saw an increased fetal exposure to serotonin when inhibiting the serotonin transporter, which may be related to the increased serotonin concentration on the maternal side of the placenta. No effect on placental kinetics were evident on maternal stress levels during the pregnancy as the majority of participating mothers had normal stress levels.

Prenatal stress affects the health of the pregnant woman and the fetus. Cortisol blood levels are elevated in pregnancy, and fetal exposure to cortisol is regulated by the placenta enzyme 11β-HSD2. A decrease in enzyme activity allows more maternal cortisol to pass through the placental barrier. Combining the fetal and maternal cortisol to cortisone ratio into the adjusted fetal cortisol exposure (AFCE) represents the activity of the enzyme 11β-HSD2 in the placenta. To investigate the effect of prenatal maternal stress on the ratio of cortisol and cortisone in maternal and fetal blood at birth in a normal population. Maternal self-reported stress was assessed at one time-point, as late in the pregnancy as convenient for the participant, using the Depression Anxiety Stress Scales (DASS-42), Pregnancy Related Anxiety (PRA), and Major Life Events during pregnancy. The study included 273 participants from Copenhagen University Hospital. Maternal and umbilical cord blood was sampled directly after birth and cortisol and cortisone concentrations were quantified using UPLC chromatography. Data were analyzed in a five-step regression model with addition of possible confounders. The primary outcome was AFCE, and plasma concentrations of maternal and fetal cortisol and cortisone were secondary outcomes. Significant associations were seen for the primary outcome AFCE and the plasma concentrations of maternal cortisol and fetal cortisone with exposure to Pregnancy Related Anxiety (PRA), though the associations were reduced when adjusting for birth related variables, especially delivery mode. The weight of the placenta affected the associations of exposures on AFCE, but not plasma concentrations of cortisol and cortisone in mother and fetus. Moreover, the study demonstrated the importance of delivery mode and birth strain on cortisol levels right after delivery. Our main finding was associations between PRA and AFCE, which shows the effect of maternal stress on placental cortisol metabolism.

During placental malaria, Plasmodium falciparum infected erythrocytes sequester in the placenta, causing health problems for both the mother and fetus. The specific adherence is mediated by the VAR2CSA protein, which binds to placental chondroitin sulfate (CS) on chondroitin sulfate proteoglycans (CSPGs) in the placental syncytium. However, the identity of the CSPG core protein and the cellular impact of the interaction have remain elusive. In this study we identified the specific CSPG core protein to which the CS is attached, and characterized its exact placental location. VAR2CSA pull-down experiments using placental extracts from whole placenta or syncytiotrophoblast microvillous cell membranes showed three distinct CSPGs available for VAR2CSA adherence. Further examination of these three CSPGs by immunofluorescence and proximity ligation assays showed that syndecan-1 is the main receptor for VAR2CSA mediated placental adherence. We further show that the commonly used placental choriocarcinoma cell line, BeWo, express a different set of proteoglycans than those present on placental syncytiotrophoblast and may not be the most biologically relevant model to study placental malaria. Syncytial fusion of the BeWo cells, triggered by forskolin treatment, caused an increased expression of placental CS-modified syndecan-1. In line with this, we show that rVAR2 binding to placental CS impairs syndecan-1-related Src signaling in forskolin treated BeWo cells, but not in untreated cells.

The Danish part of the large European Human biomonitoring pilot project Demonstration of a study to Coordinate and Perform Human biomonitoring on a European Scale (DEMOCOPHES) investigated the urine, hair and blood concentrations of 66 different environmental chemicals in a group of 145 Danish school children aged 6-11 years and their mothers from rural and urban areas in autumn 2011. Some - but not all - results were published; however, the concurrence of the chemicals has not been assessed. The measured concentrations of polybrominated diphenyl ethers (PBDEs) and glyphosate is assessed to complete the investigation of all 66 chemicals in DEMOCOPHES. The concentrations of PBDEs were measured in plasma samples of 143 mothers and 116 children. Glyphosate was measured in a subsample of 27 urine samples. Previously assessed chemicals were polychlorinated biphenyls (PCBs), and polyfluoroalkyl substances (PFASs) analyzed in blood samples, mercury analyzed in hair, and phthalate metabolites, parabens, phenols, cadmium, paracetamol and cotinine analyzed in urine samples. Differences in concentrations between mothers and children were assessed, and the associations between the concentrations of the different environmental chemicals. investigated by correlation analysis. PBDE47 was found in relatively high levels compared with previous Danish results in both mothers and children, with a significantly higher level in the children compared to their mothers. Glyphosate in concentrations around 1 ng/mL was detected in all 27 samples. The analyzed environmental exposures seem to follow a pattern where chemicals within the same classes are strongly correlated and where children and mothers are exposed to the same chemicals. The correlations between the measured environmental chemicals indicate that a specific exposure pattern may exist, where people who are highly exposed to one class of environmental chemicals also may be highly exposed to certain other classes. As some of the compounds were measured in higher levels in children compared to mothers, increased focus also on the exposure in young children is recommended. For more detailed investigation of specific exposure sources more studies with increased power and detailed questionnaires should be developed.

Background Polybrominated diphenyl ethers (PBDEs) have been widely used as flame retardants in consumer products. PBDEs may affect thyroid hormone homeostasis, which can result in irreversible damage of cognitive performance, motor skills and altered behaviour. Thus, in utero exposure is of very high concern due to critical windows in fetal development. Methods A human ex vivo placenta perfusion system was used to study the kinetics and extent of the placental transfer of BDE-47, BDE-99 and BDE-209 during four-hour perfusions. The PBDEs were added to the maternal circulation and monitored in the maternal and fetal compartments. In addition, the perfused cotyledon, the surrounding placental tissue as well as pre-perfusion placental tissue and umbilical cord plasma were also analysed. The PBDE analysis included Soxhlet extraction, clean-up by adsorption chromatography and GC-MS analysis. Results and Discussion Placental transfer of BDE-47 was faster and more extensive than for BDE-99. The fetal-maternal ratios (FM-ratio) after four hours of perfusion were 0.47 and 0.25 for BDE-47 and BDE-99, respectively, while the indicative permeability coefficient (IPC) measured after 60 minutes of perfusion was 0.26 h -1 and 0.10 h -1 , respectively. The transport of BDE-209 seemed to be limited. These differences between the congeners may be related to the degree of bromination. Significant accumulation was observed for all congeners in the perfused cotyledon as well as in the surrounding placental tissue. Conclusion The transport of BDE-47 and BDE-99 indicates in utero exposure to these congeners. Although the transport of BDE-209 was limited, however, possible metabolic debromination may lead to products which are both more toxic and transportable. Our study demonstrates fetal exposure to PBDEs, which should be included in risk assessment of PBDE exposure of women of child-bearing age.

Background Placental malaria occurs when Plasmodium falciparum infected erythrocytes sequester in the placenta. Placental parasite isolates bind to chondroitin sulphate A (CSA) by expression of VAR2CSA on the surface of infected erythrocytes, but may sequester by other VAR2CSA mediated mechanisms, such as binding to immunoglobulins. Furthermore, other parasite antigens have been associated with placental malaria. These findings have important implications for placental malaria vaccine design. The objective of this study was to adapt and describe a biologically relevant model of parasite adhesion in intact placental tissue. Results The ex vivo placental perfusion model was modified to study adhesion of infected erythrocytes binding to CSA, endothelial protein C receptor (EPCR) or a transgenic parasite where P. falciparum erythrocyte membrane protein 1 expression had been shut down. Infected erythrocytes expressing VAR2CSA accumulated in perfused placental tissue whereas the EPCR binding and the transgenic parasite did not. Soluble CSA and antibodies specific against VAR2CSA inhibited binding of infected erythrocytes. Conclusion The ex vivo model provides a novel way of studying receptor-ligand interactions and antibody mediated inhibition of binding in placental malaria.

This study reports for the 1st time the incidence and interobserver variation of morphologic findings in a series of 34 term placentas from pregnancies with normal outcome used for perfusion studies. Histologic evaluation of placental tissue is challenging, especially when it comes to defining “normal tissue” versus “pathologic lesions.” A scoring system for registration of abnormal morphologic findings was developed. Light microscopic examination was performed independently by 2 pathologists, and interobserver variation was analyzed. Findings in normal and perfused tissue were compared and selected findings were tested against success parameters from the perfusions. Finally, the criteria for frequent lesions with fair to poor interobserver variation in the nonperfused tissue were revised and reanalyzed. In the perfused tissue, the perfusion artefact “trophoblastic vacuolization,” which is believed to represent dilated transtrophoblastic channels, was reproducible and significantly correlated to the perfusion marker “fetal leakage.” In longer perfusions, microscopy of the perfused cotyledon revealed bacteria in the fetal vessels. This finding led to an adjustment in the perfusion protocol with addition of antibiotics to the medium. In the “normal” tissue, certain lesions were very frequent and showed only fair or poor interobserver agreement. Revised minimum criteria for these lesions were defined and found reproducible. This study has emphasized the value of pathologic examination as a supplement in placental perfusion models. Examination of the perfused cotyledon for trophoblastic vacuolization is recommended as an additional quality marker in perfusion models. The study also underlines the need for exact definitions of abnormality in frequent placental lesions.

Objectives To investigate the effects of cognitively demanding work tasks and office noise on heart rate variability (HRV), cardiovascular responses and electromyography (EMG) activity in the trapezius muscles. Methods Ten female volunteers were exposed to simulated open-plan office noise for 35 min (Leq 65 dBA), while engaged in cognitively demanding tasks. Task performance, self-rated stress and energy, affective state, perceived exertion in the shoulders and in the head, EMG in the left and right trapezius muscle, blood pressure, heart period length, HRV, and salivary cortisol were measured. Results Cognitively demanding work tasks were associated with changes in HRV, systolic blood pressure and EMG that reflects increased sympathetic activity in the autonomic nervous system. No effect of noise was observed, except for a higher rating of perceived exertion in the head and, contrary to expectations, a 4% lower diastolic blood pressure in the noise conditions. Conclusions Psychophysiological measures reflected the mental load imposed by cognitive work tasks. Short-term exposure to office noise resulted in increased ratings of perceived exertion in the head, but not in physiological stress reactions.

•Comparability of placental malaria vaccine potency assays was investigated.•Results from different binding inhibition assays cannot be compared directly.•Sera should be tested in a dilution series.•The level of parasite binding avidity affects the antibody inhibition. Malaria in pregnancy is associated with significant morbidity in pregnant women and their offspring. Plasmodium falciparum infected erythrocytes (IE) express VAR2CSA that mediates binding to chondroitin sulphate A (CSA) in the placenta. Two VAR2CSA-based vaccines for placental malaria are in clinical development. The purpose of this study was to evaluate the robustness and comparability of binding inhibition assays used in the clinical development of placental malaria vaccines. The ability of sera from animals immunised with different VAR2CSA constructs to inhibit IE binding to CSA was investigated in three in vitro assays using 96-well plates, petri dishes, capillary flow and an ex vivo placental perfusion assay. The inter-assay variation was not uniform between assays and ranged from above ten-fold in the flow assay to two-fold in the perfusion assay. The intra-assay variation was highest in the petri dish assay. A positive correlation between IE binding avidity and the level of binding after antibody inhibition in the petri dish assay indicate that high avidity IE binding is more difficult to inhibit. The highest binding inhibition sensitivity was found in the 96-well and petri dish assays compared to the flow and perfusion assays where binding inhibition required higher antibody titers. The inhibitory capacity of antibodies is not easily translated between assays and the high sensitivity of the 96-well and petri dish assays stresses the need for comparing serial dilutions of serum. Furthermore, IE binding avidity must be in the same range when comparing data from different days. There was an overall concordance in the capacity of antibody-mediated inhibition, when comparing the in vitro assays with the perfusion assay, which more closely represents in vivo conditions. Importantly the ID1-ID2a protein in a liposomal formulation, currently in a phase I trial, effectively induced antibodies that inhibited IE adhesion in placental tissue.