A comprehensive battery of flow cytometric immunoassays for the in vitro testing of chemical effects in human blood cells

BackgroundThere is a growing need for immunological assays to test toxic and modulatory effects of chemicals. The assays should be easy to use, reproducible and superior to cell line-based assays. We have therefore developed a comprehensive portfolio of assays based on primary human blood cells that are suitable for testing chemical effects.MethodsThe flow cytometry-based assays were designed to target a wide range of human peripheral blood mononuclear cells and whole blood, including T cells, NK cells, B cells, basophils and innate-like T cells such as γδT, MAIT and NKT cells. We have selected a set of activation markers for each immune cell, e.g: CD154 (T cells), CD137, CD107a (NK cells), CD63 (basophils), CD69, CD83 (B cells), CD69, IFN-γ (MAIT cells) and we selected cell specific stimuli: aCD3 antibodies (T cells); E. coli and cytokines IL-12/15/18 (MAIT cells); CpG ODN2006, R848 or aCD40 antibodies (B cells), fMLP or aFcϵR1 (basophils) or K562 cells (NK cells).ResultsBy selecting immune cell-specific markers and cell-specific stimuli, we were able to induce particular immune responses from the targeted immune cells. For example, the response to stimulation with anti-CD3 antibodies was in 36.8% of CD107a+CD8+ cells. Cytokine stimulation induced the production of IFN-γ in 30% of MAIT cells. After stimulation with E. coli, around 50% of MAIT cells produced TNF. About 40% of basophils responded to aFcƐR1 stimulation. Similar activation ranges were achieved in K562-stimulated NK cells.ConclusionOur test portfolio covers the most relevant immune cells present in human blood, providing a solid basis for in vitro toxicity and immunomodulatory testing of chemicals. By using human blood, the natural composition of cells found in the blood can be determined and the effects of chemicals can be detected at the cellular level

Role of female sex hormones, estradiol and progesterone, in mast cell behavior

Female sex hormones have long been suspected to have an effect on mast cell (MC) behavior. This assumption is based on the expression of hormone receptors in MCs as well as on the fact that many MC-related pathophysiological alterations have a different prevalence in females than in males. Further, serum IgE levels are much higher in allergic female mice compared to male mice. Ovariectomized rats developed less airway inflammation compared to sham controls. Following estrogen replacement ovariectomized rats re-established airway inflammation levels’ found in intact females. In humans, a much higher asthma prevalence was found in women at reproductive age as compared to men. Serum levels of estradiol and progesterone have been directly correlated with the clinical and functional features of asthma. Around 30–40% of women who have asthma experienced worsening of their symptoms during the perimenstrual phase, the so-called perimenstrual asthma. Postmenopausal women receiving hormone replacement therapy have an increased risk of new onset of asthma. Beside, estrus cycle dependent changes on female sex hormones are related to changes on MC number in mouse uterine tissue and estradiol and progesterone were shown to induce uterine MC maturation and degranulation. We will discuss here the currently available information concerning the role of these female sex hormones on MC behavior

Human Breast Milk: From Food to Active Immune Response With Disease Protection in Infants and Mothers

Breastfeeding is associated with long-term wellbeing including low risks of infectious diseases and non-communicable diseases such as asthma, cancer, autoimmune diseases and obesity during childhood. In recent years, important advances have been made in understanding the human breast milk (HBM) composition. Breast milk components such as, non-immune and immune cells and bioactive molecules, namely, cytokines/chemokines, lipids, hormones, and enzymes reportedly play many roles in breastfed newborns and in mothers, by diseases protection and shaping the immune system of the newborn. Bioactive components in HBM are also involved in tolerance and appropriate inflammatory response of breastfed infants if necessary. This review summarizes the current literature on the relationship between mother and her infant through breast milk with regard to disease protection. We will shed some light on the mechanisms underlying the roles of breast milk components in the maintenance of health of both child and mother

Urticaria in Pregnancy and Lactation

Chronic urticaria (CU) is a mast cell-driven chronic inflammatory disease with a female predominance. Since CU affects mostly females in reproductive age, pregnancy is an important aspect to consider in the context of this disease. Sex hormones affect mast cell (MC) biology, and the hormonal changes that come with pregnancy can modulate the course of chronic inflammatory conditions, and they often do. Also, pregnancy-associated changes in the immune system, including local adaptation of innate and adaptive immune responses and skewing of adaptive immunity toward a Th2/Treg profile have been linked to changes in the course of inflammatory diseases. As of now, little is known about the effects of pregnancy on CU and the outcomes of pregnancy in CU patients. Also, there are no real-life studies to show the safety of urticaria medications during pregnancy. The recent PREG-CU study provided the first insights on this and showed that CU improves during pregnancy in half of the patients, whereas it worsens in one-third; and two of five CU patients experience flare-ups of their CU during pregnancy. The international EAACI/GA²LEN/EuroGuiDerm/APAAACI guideline for urticaria recommends adopting the samemanagement strategy in pregnant and lactating CU patients; starting treatment with standard doses of second-generation (non-sedative) H1 antihistamines, to increase the dose up to 4-folds in case of no response, and to add omalizumab in antihistamine-refractory patients; but also emphasizes the lack of evidence-based information on the safety and efficacy of urticaria treatments during pregnancy. The PREG-CU study assessed treatments and their outcomes during pregnancy. Here, we review the reported effects of sex hormones and pregnancy-specific immunological changes on urticaria, we discuss the impact of pregnancy on urticaria, and we provide information and guidance on the management of urticaria during pregnancy and lactation

Pro-inflammatory Diet Pictured in Children With Atopic Dermatitis or Food Allergy:Nutritional Data of the LiNA Cohort

Background: Lifestyle and environmental factors are known to contribute to allergic disease development, especially very early in life. However, the link between diet composition and allergic outcomes remains unclear. Methods: In the present population-based cohort study we evaluated the dietary intake of 10-year-old children and analyses were performed with particular focus on atopic dermatitis or food allergy, allergic diseases known to be affected by dietary allergens. Dietary intake was assessed via semi-quantitative food frequency questionnaires. Based on these data, individual nutrient intake as well as children’s Dietary Inflammatory Index (C-DII™) scores were calculated. Information about atopic manifestations during the first 10 years of life and confounding factors were obtained from standardized questionnaires during pregnancy and annually thereafter. Results: Analyses from confounder-adjusted logistic regression models (n = 211) revealed that having atopic outcomes was associated with having a pro-inflammatory pattern at the age of 10 years: OR = 2.22 (95% CI: 1.14–4.31) for children with atopic dermatitis and OR = 3.82 (95% CI: 1.47–9.93) for children with food allergy in the first 10 years of life. Conclusion: A pro-inflammatory dietary pattern might worsen the atopic outcome and reduce the buffering capacity of the individual against harmful environmental exposures or triggers. For pediatricians it is recommended to test for the individual tolerance of allergenic foods and to increase the nutrient density of tolerable food items to avoid undesirable effects of eating a pro-inflammatory diet.</p

Pro-inflammatory Diet Pictured in Children With Atopic Dermatitis or Food Allergy: Nutritional Data of the LiNA Cohort

Background: Lifestyle and environmental factors are known to contribute to allergic disease development, especially very early in life. However, the link between diet composition and allergic outcomes remains unclear. Methods: In the present population-based cohort study we evaluated the dietary intake of 10-year-old children and analyses were performed with particular focus on atopic dermatitis or food allergy, allergic diseases known to be affected by dietary allergens. Dietary intake was assessed via semi-quantitative food frequency questionnaires. Based on these data, individual nutrient intake as well as children’s Dietary Inflammatory Index (C-DIITM) scores were calculated. Information about atopic manifestations during the first 10 years of life and confounding factors were obtained from standardized questionnaires during pregnancy and annually thereafter. Results: Analyses from confounder-adjusted logistic regression models (n = 211) revealed that having atopic outcomes was associated with having a pro-inflammatory pattern at the age of 10 years: OR = 2.22 (95% CI: 1.14–4.31) for children with atopic dermatitis and OR = 3.82 (95% CI: 1.47–9.93) for children with food allergy in the first 10 years of life Conclusion: A pro-inflammatory dietary pattern might worsen the atopic outcome and reduce the buffering capacity of the individual against harmful environmental exposures or triggers. For pediatricians it is recommended to test for the individual tolerance of allergenic foods and to increase the nutrient density of tolerable food items to avoid undesirable effects of eating a pro-inflammatory diet

p45 NF-E2 regulates syncytiotrophoblast differentiation by post-translational GCM1 modifications in human intrauterine growth restriction

AbstractPlacental insufficiency jeopardizes prenatal development, potentially leading to intrauterine growth restriction (IUGR) and stillbirth. Surviving fetuses are at an increased risk for chronic diseases later in life. IUGR is closely linked with altered trophoblast and placental differentiation. However, due to a paucity of mechanistic insights, suitable biomarkers and specific therapies for IUGR are lacking. The transcription factor p45 NF-E2 (nuclear factor erythroid derived 2) has been recently found to regulate trophoblast differentiation in mice. The absence of p45 NF-E2 in trophoblast cells causes IUGR and placental insufficiency in mice, but mechanistic insights are incomplete and the relevance of p45 NF-E2 for human syncytiotrophoblast differentiation remains unknown. Here we show that p45 NF-E2 negatively regulates human syncytiotrophoblast differentiation and is associated with IUGR in humans. Expression of p45 NF-E2 is reduced in human placentae complicated with IUGR compared with healthy controls. Reduced p45 NF-E2 expression is associated with increased syncytiotrophoblast differentiation, enhanced glial cells missing-1 (GCM1) acetylation and GCM1 desumoylation in IUGR placentae. Induction of syncytiotrophoblast differentiation in BeWo and primary villous trophoblast cells with 8-bromo-adenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) reduces p45 NF-E2 expression. Of note, p45 NF-E2 knockdown is sufficient to increase syncytiotrophoblast differentiation and GCM1 expression. Loss of p45 NF-E2 using either approach resulted in CBP-mediated GCM1 acetylation and SENP-mediated GCM1 desumoylation, demonstrating that p45 NF-E2 regulates post-translational modifications of GCM1. Functionally, reduced p45 NF-E2 expression is associated with increased cell death and caspase-3 activation in vitro and in placental tissues samples. Overexpression of p45 NF-E2 is sufficient to repress GCM1 expression, acetylation and desumoylation, even in 8-Br-cAMP exposed BeWo cells. These results suggest that p45 NF-E2 negatively regulates differentiation and apoptosis activation of human syncytiotrophoblast by modulating GCM1 acetylation and sumoylation. These studies identify a new pathomechanism related to IUGR in humans and thus provide new impetus for future studies aiming to identify new biomarkers and/or therapies of IUGR.</jats:p

Paraoxonase 2 protein is spatially expressed in the human placenta and selectively reduced in labour

Humans parturition involves interaction of hormonal, neurological, mechanical stretch and inflammatory pathways and the placenta plays a crucial role. The paraoxonases (PONs 1–3) protect against oxidative damage and lipid peroxidation, modulation of endoplasmic reticulum stress and regulation of apoptosis. Nothing is known about the role of PON2 in the placenta and labour. Since PON2 plays a role in oxidative stress and inflammation, both features of labour, we hypothesised that placental PON2 expression would alter during labour. PON2 was examined in placentas obtained from women who delivered by cesarean section and were not in labour and compared to the equivalent zone of placentas obtained from women who delivered vaginally following an uncomplicated labour. Samples were obtained from 12 sites within each placenta: 4 equally spaced apart pieces were sampled from the inner, middle and outer placental regions. PON2 expression was investigated by Western blotting and real time PCR. Two PON2 forms, one at 62 kDa and one at 43 kDa were found in all samples. No difference in protein expression of either isoform was found between the three sites in either the labour or non-labour group. At the middle site there was a highly significant decrease in PON2 expression in the labour group when compared to the non-labour group for both the 62 kDa form (p = 0.02) and the 43 kDa form (p = 0.006). No spatial differences were found within placentas at the mRNA level in either labour or non-labour. There was, paradoxically, an increase in PON2 mRNA in the labour group at the middle site only. This is the first report to describe changes in PON2 in the placenta in labour. The physiological and pathological significance of these remains to be elucidated but since PON2 is anti-inflammatory further studies are warranted to understand its role

Preeclampsia and Blood Pressure Trajectory during Pregnancy in Relation to Vitamin D Status

Every tenth pregnancy is affected by hypertension, one of the most common complications and leading causes of maternal death worldwide. Hypertensive disorders in pregnancy include pregnancy-induced hypertension and preeclampsia. The pathophysiology of the development of hypertension in pregnancy is unknown, but studies suggest an association with vitamin D status, measured as 25-hydroxyvitamin D (25(OH)D). The aim of this study was to investigate the association between gestational 25(OH)D concentration and preeclampsia, pregnancy-induced hypertension and blood pressure trajectory. This cohort study included 2000 women. Blood was collected at the first (T1) and third (T3) trimester (mean gestational weeks 10.8 and 33.4). Blood pressure at gestational weeks 10, 25, 32 and 37 as well as symptoms of preeclampsia and pregnancy-induced hypertension were retrieved from medical records. Serum 25(OH)D concentrations (LC-MS/MS) in T1 was not significantly associated with preeclampsia. However, both 25(OH)D in T3 and change in 25(OH)D from T1 to T3 were significantly and negatively associated with preeclampsia. Women with a change in 25(OH)D concentration of ≥30 nmol/L had an odds ratio of 0.22 (p = 0.002) for preeclampsia. T1 25(OH)D was positively related to T1 systolic (β = 0.03, p = 0.022) and T1 diastolic blood pressure (β = 0.02, p = 0.016), and to systolic (β = 0.02, p = 0.02) blood pressure trajectory during pregnancy, in adjusted analyses. There was no association between 25(OH)D and pregnancy-induced hypertension in adjusted analysis. In conclusion, an increase in 25(OH)D concentration during pregnancy of at least 30 nmol/L, regardless of vitamin D status in T1, was associated with a lower odds ratio for preeclampsia. Vitamin D status was significantly and positively associated with T1 blood pressure and gestational systolic blood pressure trajectory but not with pregnancy-induced hypertension

Longitudinal associations of DNA methylation and sleep in children : a meta-analysis

Publisher Copyright: © 2022, The Author(s).Background: Sleep is important for healthy functioning in children. Numerous genetic and environmental factors, from conception onwards, may influence this phenotype. Epigenetic mechanisms such as DNA methylation have been proposed to underlie variation in sleep or may be an early-life marker of sleep disturbances. We examined if DNA methylation at birth or in school age is associated with parent-reported and actigraphy-estimated sleep outcomes in children. Methods: We meta-analysed epigenome-wide association study results. DNA methylation was measured from cord blood at birth in 11 cohorts and from peripheral blood in children (4–13 years) in 8 cohorts. Outcomes included parent-reported sleep duration, sleep initiation and fragmentation problems, and actigraphy-estimated sleep duration, sleep onset latency and wake-after-sleep-onset duration. Results: We found no associations between DNA methylation at birth and parent-reported sleep duration (n = 3658), initiation problems (n = 2504), or fragmentation (n = 1681) (p values above cut-off 4.0 × 10–8). Lower methylation at cg24815001 and cg02753354 at birth was associated with longer actigraphy-estimated sleep duration (p = 3.31 × 10–8, n = 577) and sleep onset latency (p = 8.8 × 10–9, n = 580), respectively. DNA methylation in childhood was not cross-sectionally associated with any sleep outcomes (n = 716–2539). Conclusion: DNA methylation, at birth or in childhood, was not associated with parent-reported sleep. Associations observed with objectively measured sleep outcomes could be studied further if additional data sets become available.Peer reviewe