Neonatal stress exposure and DNA methylation of stress-related and neurodevelopmentally relevant genes: An exploratory study.

BACKGROUND: Stress exposure during Neonatal Intensive Care Unit (NICU) stay may have long-lasting effects on neurodevelopmental outcomes in extremely preterm infants. Altered DNA methylation of stress-related and neurodevelopmentally relevant genes may be an underlying mechanism. AIMS: This exploratory study aimed to investigate the association between neonatal stress exposure and DNA methylation in these genes at two different time points: early during the NICU stay (7-14 days after birth) and later, at discharge from the NICU. SUBJECTS: We included 45 extremely preterm infants in this prospective cohort study, gestational age 24-30 weeks. OUTCOME MEASURES: We collected fecal samples at days 7-14 (n = 44) and discharge (n = 28) and determined DNA methylation status in predefined regions of NR3C1, SLC6A4, HSD11B2, OPRM1, SLC7A5, SLC1A2, IGF2, NNAT, BDNF and GABRA6 using pyrosequencing. Because of low DNA concentrations in some fecal samples, we could do so in 25-50 % of collected samples. We prospectively quantified daily neonatal stress exposure using the Neonatal Infant Stressor Scale (NISS) and explored associations between cumulative NISS scores and average DNA methylation status. RESULTS: Rates of methylation of most genes were not statistically different between day 7-14 and discharge, except for OPRM1. We found moderately high and mostly negative correlation coefficients upon discharge with the cumulative NISS for the NR3C1, SLC6A4, SLC1A2, IGF2, BDNF and OPRM1 genes, albeit not statistically significant. CONCLUSIONS: Findings suggest that expression of stress-related and neurodevelopmentally relevant genes may be differently regulated following higher neonatal stress exposure. Larger studies should challenge the findings of this study and ideally test the effects on gene expression.

Hypermethylation of CTDSPL2 prior to necrotizing enterocolitis onset.

Background: Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in preterm infants. Epigenetic changes in DNA methylation may be present prior to NEC onset. Methods: 24 preterm infants with NEC and 45 matched controls were included. Human DNA was isolated from stool samples and methylation of CTDSPL2, HERC1, NXPE3 and PTGDR was measured using pyrosequencing. Results: CTDSPL2 displayed a higher DNA methylation of 51% compared with 17% in controls, prior to NEC onset (p = 0.047). Discussion: Noninvasive measurement of methylation in stool allows for comparison with healthy preterm controls. This potentially allows future biomarker or risk predictor use. The effect of CTDSPL2 hypermethylation on gene expression remains unclear.

What is this article about? Necrotizing enterocolitis (NEC) is a common emergency condition affecting the gastrointestinal system of preterm infants. Epigenetic changes in DNA methylation may be present in infants before the onset of NEC. DNA methylation is a natural process that can help turn genes on or off, thereby affecting their function. This study focused on measuring the amount of DNA methylation in certain genes in preterm infants who developed NEC. What were the results? This study included 24 preterm infants with NEC and 45 matched healthy controls. The researchers isolated human DNA from stool samples, and the amount of DNA methylation of four specific genes was measured. They found that one of the genes, CTDSPL2, had significantly higher DNA methylation in infants who later developed NEC than in healthy infants. What do the results of the study mean? In this study, researchers found that CTDSPL2 showed a higher level of DNA methylation in stool samples of infants who later developed NEC. The effect of this change remains unclear, but may affect the way cells grow and respond to injury or infection, which could contribute to the development of NEC. Measuring DNA methylation in stool samples provides a noninvasive method for identifying DNA methylation changes in preterm infants. Comparing the amount of DNA methylation in healthy infants with that in preterm infants at risk of NEC may help predict the risk of developing NEC.

Methylation of the serotonin reuptake transporter gene and non-motor symptoms in dystonia patients.

BACKGROUND: Dystonia is a rare movement disorder, in which patients suffer from involuntary twisting movements or abnormal posturing. Next to these motor symptoms, patients have a high prevalence of psychiatric comorbidity, suggesting a role for serotonin in its pathophysiology. This study investigates the percentage of DNA methylation of the gene encoding for the serotonin reuptake transporter (SLC6A4) in dystonia patients and the associations between methylation levels and presence and severity of psychiatric symptoms. METHODS: Patients with cervical dystonia (n = 49), myoclonus dystonia (n = 41) and dopa-responsive dystonia (DRD) (n = 27) and a group of healthy controls (n = 56) were included. Psychiatric comorbidity was evaluated with validated questionnaires. Methylation levels of 20 CpG sites situated 69 to 213 base pairs upstream of the start codon of SLC6A4 were investigated. Methylation in dystonia patients was compared to healthy controls, correcting for age, and correlated with psychiatric comorbidity. RESULTS: Bootstrapped quantile regression analysis showed that being a dystonia patient compared to a healthy control significantly explains the methylation level at two CpG sites (CpG 24: pseudo-R2 = 0.05, p = 0.04, CpG 32: pseudo-R2 = 0.14, p = 0.03). Subgroup analysis revealed that being a DRD patient significantly explained a part of the variance of methylation levels at two CpG sites (CpG 21: pseudo-R2 = 0.03, p = 0.00, CpG 24: pseudo-R2 = 0.06, p = 0.03). Regression analysis showed that methylation level at CpG 38 significantly explained a small proportion of the variance of severity score for anxiety (R2 = 0.07, p = 0.04) and having a diagnosis of depression (Nagelkerke R2: 0.11, p = 0.00). Genotype of the 5-HTTLPR polymorphism had no additional effect on these associations. CONCLUSIONS: This study showed an association between percentage of methylation at several specific sites of the promoter region of SLCA64 and (dopa-responsive) dystonia patients compared to healthy controls. Furthermore, methylation levels were associated with severity of anxiety and presence of a depressive disorder in the dystonia group. This study suggests alterations in the serotonergic metabolism in dystonia patients, and its relation with the non-motor symptoms.

Differential Placental DNA Methylation of NR3C1 in Extremely Preterm Infants With Poorer Neurological Functioning.

Background: Understanding underlying mechanisms of neurodevelopmental impairment following preterm birth may enhance opportunities for targeted interventions. We aimed to assess whether placental DNA methylation of selected genes affected early neurological functioning in preterm infants. Methods: We included 43 infants, with gestational age <30 weeks and/or birth weight <1,000 g and placental samples at birth. We selected genes based on their associations with several prenatal conditions that may be related to poor neurodevelopmental outcomes. We determined DNA methylation using pyrosequencing, and neurological functioning at 3 months post-term using Prechtl's General Movement Assessment, including the Motor Optimality Score-Revised (MOS-R). Results: Twenty-four infants had atypical MOS-R, 19 infants had near-optimal MOS-R. We identified differences in average methylation of NR3C1 (encoding for the glucocorticoid receptor) [3.3% (95%-CI: 2.4%-3.9%) for near-optimal vs. 2.3% (95%-CI: 1.7%-3.0%), p = 0.008 for atypical], and at three of the five individual CpG-sites. For EPO, SLC6A3, TLR4, VEGFA, LEP and HSD11B2 we found no differences between the groups. Conclusion: Hypomethylation of NR3C1 in placental tissue is associated with poorer neurological functioning at 3 months post-term in extremely preterm infants. Alleviating stress during pregnancy and its impact on preterm infants and their neurodevelopmental outcomes should be further investigated.

Perinatal exposure to fluoxetine and maternal adversity affect myelin-related gene expression and epigenetic regulation in the corticolimbic circuit of juvenile rats.

Many pregnant women experience symptoms of depression, and are often treated with selective serotonin reuptake inhibitor (SSRI) antidepressants, such as fluoxetine. In utero exposure to SSRIs and maternal depressive symptoms is associated with sex-specific effects on the brain and behavior. However, knowledge about the neurobiological mechanisms underlying these sex differences is limited. In addition, most animal research into developmental SSRI exposure neglects the influence of maternal adversity. Therefore, we used a rat model relevant to depression to investigate the molecular effects of perinatal fluoxetine exposure in male and female juvenile offspring. We performed RNA sequencing and targeted DNA methylation analyses on the prefrontal cortex and basolateral amygdala; key regions of the corticolimbic circuit. Perinatal fluoxetine enhanced myelin-related gene expression in the prefrontal cortex, while inhibiting it in the basolateral amygdala. SSRI exposure and maternal adversity interacted to affect expression of genes such as myelin-associated glycoprotein (Mag) and myelin basic protein (Mbp). We speculate that altered myelination reflects altered brain maturation. In addition, these effects are stronger in males than in females, resembling known behavioral outcomes. Finally, Mag and Mbp expression correlated with DNA methylation, highlighting epigenetic regulation as a potential mechanism for developmental fluoxetine-induced changes in myelination.

Altered neurodevelopmental DNA methylation status after fetal growth restriction with brain-sparing.

It is under debate how preferential perfusion of the brain (brain-sparing) in fetal growth restriction (FGR) relates to long-term neurodevelopmental outcome. Epigenetic modification of neurotrophic genes by altered fetal oxygenation may be involved. To explore this theory, we performed a follow-up study of 21 FGR children, in whom we prospectively measured the prenatal cerebroplacental ratio (CPR) with Doppler sonography. At 4 years of age, we tested their neurodevelopmental outcome using the Wechsler Preschool and Primary Scale of Intelligence, the Child Behavior Checklist, and the Behavior Rating Inventory of Executive Function. In addition, we collected their buccal DNA to determine the methylation status at predefined genetic regions within the genes hypoxia-inducible factor-1 alpha (HIF1A), vascular endothelial growth factor A (VEGFA), erythropoietin (EPO), EPO-receptor (EPOR), brain-derived neurotrophic factor (BDNF), and neurotrophic tyrosine kinase, receptor, type 2 (NTRK2) by pyrosequencing. We found that FGR children with fetal brain-sparing (CPR <1, n = 8) demonstrated a trend (0.05 < p < 0.1) toward hypermethylation of HIF1A and VEGFA at their hypoxia-response element (HRE) compared with FGR children without fetal brain-sparing. Moreover, in cases with fetal brain-sparing, we observed statistically significant hypermethylation at a binding site for cyclic adenosine monophophate response element binding protein (CREB) of BDNF promoter exon 4 and hypomethylation at an HRE located within the NTRK2 promoter (both p <0.05). Hypermethylation of VEGFA was associated with a poorer Performance Intelligence Quotient, while hypermethylation of BDNF was associated with better inhibitory self-control (both p <0.05). These results led us to formulate the hypothesis that early oxygen-dependent epigenetic alterations due to hemodynamic alterations in FGR may be associated with altered neurodevelopmental outcome in later life. We recommend further studies to test this hypothesis.

DNA Methylation of TLR4, VEGFA, and DEFA5 Is Associated With Necrotizing Enterocolitis in Preterm Infants.

Background: Epigenetic changes, such as DNA methylation, may contribute to an increased susceptibility for developing necrotizing enterocolitis (NEC) in preterm infants. We assessed DNA methylation in five NEC-associated genes, selected from literature: EPO, VEGFA, ENOS, DEFA5, and TLR4 in infants with NEC and controls. Methods: Observational cohort study including 24 preterm infants who developed NEC (≥Bell Stage IIA) and 45 matched controls. DNA was isolated from stool samples and methylation measured using pyrosequencing. We investigated differences in methylation prior to NEC compared with controls. Next, in NEC infants, we investigated methylation patterns long before, a short time before NEC onset, and after NEC. Results: Prior to NEC, only TLR4 CpG 2 methylation was increased in NEC infants (median = 75.4%, IQR = 71.3-83.8%) versus controls (median = 69.0%, IQR = 64.5-77.4%, p = 0.025). In NEC infants, VEGFA CpG 3 methylation was 0.8% long before NEC, increasing to 1.8% a short time before NEC and 2.0% after NEC (p = 0.011; p = 0.021, respectively). A similar pattern was found in DEFA5 CpG 1, which increased from 75.4 to 81.4% and remained 85.3% (p = 0.027; p = 0.019, respectively). These changes were not present for EPO, ENOS, and TLR4. Conclusion: Epigenetic changes of TLR4, VEGFA, and DEFA5 are present in NEC infants and can differ in relation to the time of NEC onset. Differences in DNA methylation of TLR4, VEGFA, and DEFA5 may influence gene expression and increase the risk for developing NEC. This study also demonstrates the use of human DNA extraction from stool samples as a novel non-invasive method for exploring the bowel of preterm infants and which can also be used for necrotizing enterocolitis patients.

Antidepressant treatment is associated with epigenetic alterations of Homer1 promoter in a mouse model of chronic depression.

BACKGROUND: Understanding the neurobiology of depression and the mechanism of action of therapeutic measures is currently a research priority. We have shown that the expression of the synaptic protein Homer1a correlates with depression-like behavior and its induction is a common mechanism of action of different antidepressant treatments. However, the mechanism of Homer1a regulation is still unknown. METHODS: We combined the chronic despair mouse model (CDM) of chronic depression with different antidepressant treatments. Depression-like behavior was characterized by forced swim and tail suspension tests, and via automatic measurement of sucrose preference in IntelliCage. The Homer1 mRNA expression and promoter DNA methylation were analyzed in cortex and peripheral blood by qRT-PCR and pyrosequencing. RESULTS: CDM mice show decreased Homer1a and Homer1b/c mRNA expression in cortex and blood samples, while chronic treatment with imipramine and fluoxetine or acute ketamine application increases their level only in the cortex. The quantitative analyses of the methylation of 7 CpG sites, located on the Homer1 promoter region containing several CRE binding sites, show a significant increase in DNA methylation in the cortex of CDM mice. In contrast, antidepressant treatments reduce the methylation level. LIMITATIONS: Homer1 expression and promotor methylation were not analyzed in different blood cell types. Other CpG sites of Homer1 promoter should be investigated in future studies. Our experimental approach does not distinguish between methylation and hydroxymethylation. CONCLUSIONS: We demonstrate that stress-induced depression-like behavior and antidepressant treatments are associated with epigenetic alterations of Homer1 promoter, providing new insights into the mechanism of antidepressant treatment.

Mid-gestation low-dose LPS administration results in female-specific excessive weight gain upon a western style diet in mouse offspring.

Gestational complications, including preeclampsia and gestational diabetes, have long-term adverse consequences for offspring's metabolic and cardiovascular health. A low-grade systemic inflammatory response is likely mediating this. Here, we examine the consequences of LPS-induced gestational inflammation on offspring's health in adulthood. LPS was administered to pregnant C57Bl/6J mice on gestational day 10.5. Maternal plasma metabolomics showed oxidative stress, remaining for at least 5 days after LPS administration, likely mediating the consequences for the offspring. From weaning on, all offspring was fed a control diet; from 12 to 24 weeks of age, half of the offspring received a western-style diet (WSD). The combination of LPS-exposure and WSD resulted in hyperphagia and increased body weight and body fat mass in the female offspring. This was accompanied by changes in glucose tolerance, leptin and insulin levels and gene expression in liver and adipose tissue. In the hypothalamus, expression of genes involved in food intake regulation was slightly changed. We speculate that altered food intake behaviour is a result of dysregulation of hypothalamic signalling. Our results add to understanding of how maternal inflammation can mediate long-term health consequences for the offspring. This is relevant to many gestational complications with a pro-inflammatory reaction in place.

Sex and Exposure to Postnatal Chlorpyrifos Influence the Epigenetics of Feeding-Related Genes in a Transgenic APOE Mouse Model: Long-Term Implications on Body Weight after a High-Fat Diet.

Developmental exposure to toxicants and diet can interact with an individual's genetics and produce long-lasting metabolic adaptations. The different isoforms of the apolipoprotein E (APOE) are an important source of variability in metabolic disorders and influence the response to the pesticide chlorpyrifos (CPF). We aimed to study the epigenetic regulation on feeding control genes and the influence of postnatal CPF exposure, APOE genotype, and sex, and how these modifications impact on the metabolic response to a high-fat diet (HFD). Both male and female apoE3- and apoE4-TR mice were exposed to CPF on postnatal days 10-15. The DNA methylation pattern of proopiomelanocortin, neuropeptide Y, leptin receptor, and insulin-like growth factor 2 was studied in the hypothalamus. At adulthood, the mice were given a HFD for eight weeks. The results highlight the importance of sex in the epigenetic regulation and the implication of CPF treatment and APOE genotype. The body weight progression exhibited sex-dimorphic differences, apoE4-TR males being the most susceptible to the effects induced by CPF and HFD. Overall, these results underscore the pivotal role of sex, APOE genotype, and developmental exposure to CPF on subsequent metabolic disturbances later in life and show that sex is a key variable in epigenetic regulation.

Gestational oxidative stress protects against adult obesity and insulin resistance.

Pregnancy complications such as preeclampsia cause increased fetal oxidative stress and fetal growth restriction, and associate with a higher incidence of adult metabolic syndrome. However, the pathophysiological contribution of oxidative stress per se is experimentally difficult to discern and has not been investigated. This study determined, if increased intrauterine oxidative stress (IUOx) affects adiposity, glucose and cholesterol metabolism in adult Ldlr-/-xSod2+/+ offspring from crossing male Ldlr-/-xSod2+/+ mice with Ldlr-/-xSod2 +/- dams (IUOx) or Ldlr-/-xSod2 +/- males with Ldlr-/-xSod2+/+ dams (control). At 12 weeks of age mice received Western diet for an additional 12 weeks. Adult male IUOx offspring displayed lower body weight and reduced adiposity associated with improved glucose tolerance compared to controls. Reduced weight gain in IUOx was conceivably due to increased energy dissipation in white adipose tissue conveyed by higher expression of Ucp1 and an accompanying decrease in DNA methylation in the Ucp1 enhancer region. Female offspring did not show comparable phenotypes. These results demonstrate that fetal oxidative stress protects against the obesogenic effects of Western diet in adulthood by programming energy dissipation in white adipose tissue at the level of Ucp1.

Differential placental DNA methylation of VEGFA and LEP in small-for-gestational age fetuses with an abnormal cerebroplacental ratio.

BACKGROUND: In Fetal Growth Restriction 'fetal programming' may take place via DNA methylation, which has implications for short-term and long-term health outcomes. Small-for-gestational age fetuses are considered fetal growth restricted, characterized by brain-sparing when fetal Doppler hemodynamics are abnormal, expressed as a cerebroplacental ratio (CPR) <1. We aimed to determine whether brain-sparing is associated with altered DNA methylation of selected genes. METHODS: We compared DNA methylation of six genes in 41 small-for-gestational age placentas with a normal or abnormal CPR. We selected EPO, HIF1A, VEGFA, LEP, PHLDA2, and DHCR24 for their role in angiogenesis, immunomodulation, and placental and fetal growth. DNA methylation was analyzed by pyrosequencing. RESULTS: Growth restricted fetuses with an abnormal CPR showed hypermethylation of the VEGFA gene at one CpG (VEGFA-309, p = .001) and an overall hypomethylation of the LEP gene, being significant at two CpGs (LEP-123, p = .049; LEP-51, p = .020). No differences in methylation were observed for the other genes. CONCLUSIONS: VEGFA and LEP genes are differentially methylated in placentas of small-for-gestational age fetuses with brain-sparing. Hypermethylation of VEGFA-309 in abnormal CPR-placentas could indicate successful compensatory mechanisms. Methylation of LEP-51 is known to suppress LEP expression. Hypomethylation in small-for-gestational age placentas with abnormal CPR may result in hyperleptinemia and predispose to leptin-resistance later in life.

Human sFLT1 Leads to Severe Changes in Placental Differentiation and Vascularization in a Transgenic hsFLT1/rtTA FGR Mouse Model.

The anti-angiogenic soluble fms-like tyrosine kinase 1 (sFLT1) is one of the candidates in the progression of preeclampsia, often associated with fetal growth restriction (FGR). Therapeutic agents against preeclampsia with/without FGR, as well as adequate transgenic sFLT1 mouse models for testing such agents, are still missing. Much is known about sFLT1-mediated endothelial dysfunction in several tissues; however, the influence of sFLT1 on placental and fetal development is currently unknown. We hypothesize that sFLT1 is involved in the progression of FGR by influencing placental differentiation and vascularization and is a prime candidate for interventional strategies. Therefore, we generated transgenic inducible human sFLT1/reverse tetracycline-controlled transactivator (hsFLT1/rtTA) mice, in which hsFLT1 is ubiquitously overexpressed during pregnancy in dams and according to the genetics in hsFLT1/rtTA homozygous and heterozygous fetuses. Induction of hsFLT1 led to elevated hsFLT1 levels in the serum of dams and on mRNA level in all placentas and hetero-/homozygous fetuses, resulting in FGR in all fetuses at term. The strongest effects in respect to FGR were observed in the hsFLT1/rtTA homozygous fetuses, which exhibited the highest hsFLT1 levels. Only fetal hsFLT1 expression led to impaired placental morphology characterized by reduced placental efficiency, enlarged maternal sinusoids, reduced fetal capillaries, and impaired labyrinthine differentiation, associated with increased apoptosis. Besides impaired placental vascularization, the expression of several transporter systems, such as glucose transporter 1 and 3 (Glut-1; Glut-3); amino acid transporters, solute carrier family 38, member one and two (Slc38a1; Slc38a2); and most severely the fatty acid translocase Cd36 and fatty acid binding protein 3 (Fabp3) was reduced upon hsFLT1 expression, associated with an accumulation of phospholipids in the maternal serum. Moreover, the Vegf pathway showed alterations, resulting in reduced Vegf, Vegfb, and Plgf protein levels and increased Bad and Caspase 9 mRNA levels. We suggest that hsFLT1 exerts an inhibitory influence on placental vascularization by reducing Vegf signaling, which leads to apoptosis in fetal vessels, impairing placental differentiation, and the nutrient exchange function of the labyrinth. These effects were more pronounced when both the dam and the fetus expressed hsFLT1 and ultimately result in FGR and resemble the preeclamptic phenotype in humans.

From blood to lung tissue: effect of cigarette smoke on DNA methylation and lung function.

BACKGROUND: Genetic and environmental factors play a role in the development of COPD. The epigenome, and more specifically DNA methylation, is recognized as important link between these factors. We postulate that DNA methylation is one of the routes by which cigarette smoke influences the development of COPD. In this study, we aim to identify CpG-sites that are associated with cigarette smoke exposure and lung function levels in whole blood and validate these CpG-sites in lung tissue. METHODS: The association between pack years and DNA methylation was studied genome-wide in 658 current smokers with >5 pack years using robust linear regression analysis. Using mediation analysis, we subsequently selected the CpG-sites that were also associated with lung function levels. Significant CpG-sites were validated in lung tissue with pyrosequencing and expression quantitative trait methylation (eQTM) analysis was performed to investigate the association between DNA methylation and gene expression. RESULTS: 15 CpG-sites were significantly associated with pack years and 10 of these were additionally associated with lung function levels. We validated 5 CpG-sites in lung tissue and found several associations between DNA methylation and gene expression. CONCLUSION: This study is the first to validate a panel of CpG-sites that are associated with cigarette smoking and lung function levels in whole blood in the tissue of interest: lung tissue.

The fetal programming effect of prenatal smoking on Igf1r and Igf1 methylation is organ- and sex-specific.

The impact of prenatal smoke exposure (PSE) on DNA methylation has been demonstrated in blood samples from children of smoking mothers, but evidence for sex-dependent smoke-induced effects is limited. As the identified differentially methylated genes can be associated with developmental processes, and insulin-like growth factors (IGFs) play a critical role in prenatal tissue growth, we hypothesized that PSE induces fetal programming of Igf1r and Igf1. Using a mouse model of smoking during pregnancy, we show that PSE alters promoter methylation of Igf1r and Igf1 and deregulates their gene expression in lung and liver of fetal (E17.5) and neonatal (D3) mouse offspring. By further comparing female versus male, lung versus liver, or fetal versus neonatal time point, our results demonstrate that CpG site-specific aberrant methylation patterns sex-dependently vary per organ and time point. Moreover, PSE reduces gene expression of Igf1r and Igf1, dependent on organ, sex, and offspring's age. Our results indicate that PSE may be a source of organ-specific rather than general systemic fetal programming. This is exemplified here by gene promoter methylation and mRNA levels of Igf1r and Igf1, together with a sex- and organ-specific naturally established correlation of both parameters that is affected by prenatal smoke exposure. Moreover, the comparison of fetuses with neonates suggests a CpG site-dependent reversibility/persistence of PSE-induced differential methylation patterns.

Early micronutrient supplementation protects against early stress-induced cognitive impairments.

Early-life stress (ES) impairs cognition later in life. Because ES prevention is problematic, intervention is needed, yet the mechanisms that underlie ES remain largely unknown. So far, the role of early nutrition in brain programming has been largely ignored. Here, we demonstrate that essential 1-carbon metabolism-associated micronutrients (1-CMAMs; i.e., methionine and B vitamins) early in life are crucial in programming later cognition by ES. ES was induced in male C57Bl/6 mice from postnatal d (P)2-9. 1-CMAM levels were measured centrally and peripherally by using liquid chromatography-mass spectroscopy. Next, we supplemented the maternal diet with 1-CMAM only during the ES period and studied cognitive, neuroendocrine, neurogenic, transcriptional, and epigenetic changes in adult offspring. We demonstrate that ES specifically reduces methionine in offspring plasma and brain. Of note, dietary 1-CMAM enrichment during P2-9 restored methionine levels and rescued ES-induced adult cognitive impairments. Beneficial effects of this early dietary enrichment were associated with prevention of the ES-induced rise in corticosterone and adrenal gland hypertrophy did not involve changes in maternal care, hippocampal volume, neurogenesis, or global/Nr3c1-specific DNA methylation. In summary, nutrition is important in brain programming by ES. A short, early supplementation with essential micronutrients can already prevent lasting effects of ES. This concept opens new avenues for nutritional intervention.-Naninck, E. F. G., Oosterink, J. E., Yam, K.-Y., de Vries, L. P., Schierbeek, H., van Goudoever, J. B., Verkaik-Schakel, R.-N., Plantinga, J. A., Plosch, T., Lucassen, P. J., Korosi, A. Early micronutrient supplementation protects against early stress-induced cognitive impairments.

Aging impairs long-term hematopoietic regeneration after autologous stem cell transplantation.

Most of our knowledge of the effects of aging on the hematopoietic system comes from studies in animal models. In this study, to explore potential effects of aging on human hematopoietic stem and progenitor cells (HSPCs), we evaluated CD34(+) cells derived from young (<35 years) and old (>60 years) adult bone marrow with respect to phenotype and in vitro function. We observed an increased frequency of phenotypically defined stem and progenitor cells with age, but no distinct differences with respect to in vitro functional capacity. Given that regeneration of peripheral blood counts can serve as a functional readout of HSPCs, we compared various peripheral blood parameters between younger patients (≤50 years; n = 64) and older patients (≥60 years; n = 55) after autologous stem cell transplantation. Patient age did not affect the number of apheresis cycles or the amount of CD34(+) cells harvested. Parameters for short-term regeneration did not differ significantly between the younger and older patients; however, complete recovery of all 3 blood lineages at 1 year after transplantation was strongly affected by advanced age, occurring in only 29% of the older patients, compared with 56% of the younger patients (P = .009). Collectively, these data suggest that aging has only limited effects on CD34(+) HSPCs under steady-state conditions, but can be important under consitions of chemotoxic and replicative stress.

A single center analysis of nucleophosmin in acute myeloid leukemia: value of combining immunohistochemistry with molecular mutation analysis.

Mutations of nucleophosmin 1 are frequently found in acute myeloid leukemia and lead to aberrant cytoplasmic accumulation of nucleophosmin protein. Immunohistochemical staining is therefore recommended as the technique of choice in front-line screening. In this study, we assessed the sensitivity and specificity of immunohistochemistry on formalin-fixed bone marrow biopsies compared with gold standard molecular analysis to predict nucleophosmin 1 mutation status in 119 patients with acute myeloid leukemia. Discrepant cases were further characterized by gene expression analyses and fluorescence in situ hybridization. A large overlap between both methods was observed. Nevertheless, nine patients demonstrated discordant results at initial screening. Five cases demonstrated nuclear staining of nucleophosmin 1 by immunohistochemistry, but a nucleophosmin 1 mutation by molecular analysis. In two cases this could be attributed to technical issues and in three cases minor subpopulations of myeloblasts had not been discovered initially. All tested cases exhibited the characteristic nucleophosmin-mutated gene expression pattern. Four cases had cytoplasmic nucleophosmin 1 staining and a nucleophosmin-mutated gene expression pattern without a detectable nucleophosmin 1 mutation. In two of these cases we found the chromosomal translocation t(3;5)(q25;q35) encoding the NPM-MLF1 fusion protein. In the other discrepant cases the aberrant cytoplasmic nucleophosmin staining and gene expression could not be explained. In total six patients (5%) had true discordant results between immunohistochemistry and mutation analysis. We conclude that cytoplasmic nucleophosmin localization is not always caused by a conventional nucleophosmin 1 mutation and that in the screening for nucleophosmin 1 abnormalities, most information will be obtained by combining immunohistochemistry with molecular analysis.