Immunoprofiling Correlates of Protection Against SHIV Infection in Adjuvanted HIV-1 Pox-Protein Vaccinated Rhesus Macaques.

Human immunodeficiency virus type 1 (HIV-1) infection remains a major public health threat due to its incurable nature and the lack of a highly efficacious vaccine. The RV144 vaccine trial is the only clinical study to date that demonstrated significant but modest decrease in HIV infection risk. To improve HIV-1 vaccine immunogenicity and efficacy, we recently evaluated pox-protein vaccination using a next generation liposome-based adjuvant, Army Liposomal Formulation adsorbed to aluminum (ALFA), in rhesus monkeys and observed 90% efficacy against limiting dose mucosal SHIV challenge in male animals. Here, we analyzed binding antibody responses, as assessed by Fc array profiling using a broad range of HIV-1 envelope antigens and Fc features, to explore the mechanisms of ALFA-mediated protection by employing machine learning and Cox proportional hazards regression analyses. We found that Fcγ receptor 2a-related binding antibody responses were augmented by ALFA relative to aluminium hydroxide, and these responses were associated with reduced risk of infection in male animals. Our results highlight the application of systems serology to provide mechanistic insights to vaccine-elicited protection and support evidence that antibody effector responses protect against HIV-1 infection.

From Cell Death to Regeneration: Rebuilding After Injury.

The ability to regrow lost or damaged tissues is widespread, but highly variable among animals. Understanding this variation remains a challenge in regeneration biology. Numerous studies from Hydra to mouse have shown that apoptosis acts as a potent and necessary mechanism in regeneration. Much is known about the involvement of apoptosis during normal development in regulating the number and type of cells in the body. In the context of regeneration, apoptosis also regulates cell number and proliferation in tissue remodeling. Apoptosis acts both early in the process to stimulate regeneration and later to regulate regenerative patterning. Multiple studies indicate that apoptosis acts as a signal to stimulate proliferation within the regenerative tissues, producing the cells needed for full regeneration. The conservation of apoptosis as a regenerative mechanism demonstrated across species highlights its importance and motivates the continued investigation of this important facet of programmed cell death. This review summarizes what is known about the roles of apoptosis during regeneration, and compares regenerative apoptosis with the mechanisms and function of apoptosis in development. Defining the complexity of regenerative apoptosis will contribute to new knowledge and perspectives for understanding mechanisms of apoptosis induction and regulation.

Studying In Vivo Retinal Progenitor Cell Proliferation in Xenopus laevis.

The efficient generation and maintenance of retinal progenitor cells (RPCs) are key goals needed for developing strategies for productive eye repair. Although vertebrate eye development and retinogenesis are well characterized, the mechanisms that can initiate RPC proliferation following injury-induced regrowth and repair remain unknown. This is partly because endogenous RPC proliferation typically occurs during embryogenesis while studies of retinal regeneration have largely utilized adult (or mature) models. We found that embryos of the African clawed frog, Xenopus laevis, successfully regrew functional eyes after ablation. The initiation of regrowth induced a robust RPC proliferative response with a concomitant delay of the endogenous RPC differentiation program. During eye regrowth, overall embryonic development proceeded normally. Here, we provide a protocol to study regrowth-dependent RPC proliferation in vivo. This system represents a robust and low-cost strategy to rapidly define fundamental mechanisms that regulate regrowth-initiated RPC proliferation, which will facilitate progress in identifying promising strategies for productive eye repair.

DNA methylation of NR3c1 in infancy: Associations between maternal caregiving and infant sex.

Caregivers play a critical role in scaffolding infant stress reactivity and regulation, but the mechanisms by which this scaffolding occurs is unclear. Animal models strongly suggest that epigenetic processes, such as DNA methylation, are sensitive to caregiving behaviors and, in turn, offspring stress reactivity. We examined the direct effects of caregiving behaviors on DNA methylation in infants and infant stress reactivity. Infants and mothers (N = 128) were assessed during a free play when infants were 5 months old. Maternal responsiveness and appropriate touch were coded. and infant buccal epithelial cells were sampled to assess for DNA methylation of the glucocorticoid receptor gene, NR3c1 exon 1F. Infant cortisol reactivity was assessed in response to the still-face paradigm. Greater levels of maternal responsiveness and appropriate touch were related to less DNA methylation of specific regions in NR3c1 exon 1F, but only for females. There was no association with maternal responsiveness and appropriate touch or DNA methylation of NR3c1 exon 1F on prestress cortisol or cortisol reactivity. Our results are discussed in relation to programming models that implicate maternal care as an important factor in programing infant stress reactivity.

Los cuidadores juegan un papel esencial en el andamiaje de la reactividad y regulación del estrés infantil pero los mecanismos por medio de los cuales aparece este andamiaje no están claros. Los modelos animales fuertemente sugieren que los procesos epigenéticos, tales como la metilación del ADN, son sensibles a los comportamientos de prestaciones de cuidado y por consiguiente a la reactividad al estrés por parte de los hijos. Examinamos los efectos directos que los comportamientos de prestaciones de cuidado tienen sobre la metilación de ADN en infantes y, por consiguiente, la reactividad del estrés infantil. Los infantes y sus madres (N = 128) fueron evaluados durante una sesión de juego libre cuando los infantes tenían 5 meses de edad. Se codificó la sensibilidad materna y la apropiada forma de tocar y se obtuvo muestra de las células epiteliales bucales del infante para analizar la metilación de ADN del gen receptor glucocorticoide, NR3c1, exón 1F. Se evaluó la reactividad del infante al cortisol como respuesta al paradigma de la cara quieta. Niveles mayores de sensibilidad materna y apropiada forma de tocar se relacionaron con menos metilación de ADN de regiones específicas en NR3c1 exón 1F, aunque sólo en las niñas. No se presentó ninguna asociación con la sensibilidad materna y la apropiada forma de tocar, o metilación de ADN de NR3c1 exón 1F en el cortisol pre-estrés o la reactividad del cortisol. Nuestros resultados se discuten en relación con modelos de programación que implican cuidado materno como un importante factor en la programación de la reactividad del estrés del infante.

Les personnes prenant soin des enfants jouent un rôle critique dans l'échafaudage de la réaction au stress du nourrisson et la régulation mais les mécanismes selon lesquels cet échafaudage se bâtit ne sont pas clairs. Les modèles animaux suggèrent fortement que des processus épigénétiques, comme la méthylation de l'ADN, sont sensibles au comportements de qui prend soin d'eux et en conséquence déclenchent un réaction au stress. Nous avons examiné les effets directs des comportements soignants sur la méthylation de l'ADN chez les bébés, en ensuite sur la réaction au stress du nourrisson. Des nourrissons et leurs mères (N = 128) ont été évalués au moyen d'un jeu libre quand les bébés avaient 5 mois d'âge. La réaction maternelle et le toucher approprié ont été codés et des cellules épithéliales buccales du bébé ont été prélevées afin d'évaluer la méthylation de l'ADN du gène récepteur glucocorticoïde, le NR3c1 exon 1F. La réaction du cortisol du bébé a été évaluée en réponse au paradigme du visage immuable. Des niveaux plus élevés de réaction maternelle et de toucher approprié étaient liés à une méthylation de l'ADN des régions spécifiques de NR3c1 exon 1F moindre, mais seulement chez les filles. On n'a trouvé aucun lien avec la réaction maternelle et le toucher approprié ou de méthylation NR3c1 exon 1F de l'ADN sur le cortisol pré-test ou de réaction du cortisol. Nos résultats sont discutés en relation aux modèles de programme qui impliquent que le soin maternel en tant que facteur important dans la programmation de la réaction au stress du bébé.

Using the Xenopus Developmental Eye Regrowth System to Distinguish the Role of Developmental Versus Regenerative Mechanisms.

A longstanding challenge in regeneration biology is to understand the role of developmental mechanisms in restoring lost or damaged tissues and organs. As these body structures were built during embryogenesis, it is not surprising that a number of developmental mechanisms are also active during regeneration. However, it remains unclear whether developmental mechanisms act similarly or differently during regeneration as compared to development. Since regeneration is studied in the context of mature, differentiated tissues, it is difficult to evaluate comparative studies with developmental processes due to the latter's highly proliferative environment. We have taken a more direct approach to study regeneration in a developmental context (regrowth). Xenopus laevis, the African clawed frog, is a well-established model for both embryology and regeneration studies, especially for the eye. Xenopus eye development is well-defined. Xenopus is also an established model for retinal and lens regeneration studies. Previously, we demonstrated that Xenopus tailbud embryo can successfully regrow a functional eye that is morphologically indistinguishable from an age-matched control eye. In this study, we assessed the temporal regulation of retinal differentiation and patterning restoration during eye regrowth. Our findings showed that during regrowth, cellular patterning and retinal layer formation was delayed by approximately 1 day but was restored by 3 days when compared to eye development. An assessment of the differentiation of ganglion cells, photoreceptor cells, and Müller glia indicated that the retinal birth order generated during regrowth was consistent with that observed for eye development. Thus, retina differentiation and patterning during regrowth is similar to endogenous eye development. We used this eye regrowth model to assess the role of known mechanisms in development versus regrowth. Loss-of-function studies showed that Pax6 was required for both eye development and regrowth whereas apoptosis was only required for regrowth. Together, these results revealed that the mechanisms required for both development and regrowth can be distinguished from regrowth-specific ones. Our study highlights this developmental model of eye regrowth as a robust platform to systematically and efficiently define the molecular mechanisms that are required for regeneration versus development.

Hydroxymethylation is uniquely distributed within term placenta, and is associated with gene expression.

The conversion of cytosine to 5-methylcystosine (5mC) is an important regulator of gene expression. 5mC may be enzymatically converted to 5-hydroxymethylcytosine (5hmC), with a potentially distinct regulatory function. We sought to investigate these cytosine modifications and their effect on gene expression by parallel processing of genomic DNA using bisulfite and oxidative bisulfite conversion in conjunction with RNA sequencing. Although values of 5hmC across the placental genome were generally low, we identified ∼21,000 loci with consistently elevated levels of 5-hydroxymethycytosine. Absence of 5hmC was observed in CpG islands and, to a greater extent, in non-CpG island-associated regions. 5hmC was enriched within poised enhancers, and depleted within active enhancers, as defined by H3K27ac and H3K4me1 measurements. 5hmC and 5mC were significantly elevated in transcriptionally silent genes when compared with actively transcribed genes. 5hmC was positively associated with transcription in actively transcribed genes only. Our data suggest that dynamic cytosine regulation, associated with transcription, provides the most complete epigenomic landscape of the human placenta, and will be useful for future studies of the placental epigenome.-Green, B. B., Houseman, E. A., Johnson, K. C., Guerin, D. J., Armstrong, D. A., Christensen, B. C., Marsit, C. J. Hydroxymethylation is uniquely distributed within term placenta, and is associated with gene expression.

The Contributions of Maternal Sensitivity and Maternal Depressive Symptoms to Epigenetic Processes and Neuroendocrine Functioning.

This study tested whether maternal responsiveness may buffer the child to the effects of maternal depressive symptoms on DNA methylation of NR3C1, 11ß-HSD2, and neuroendocrine functioning. DNA was derived from buccal epithelial cells and prestress cortisol was obtained from the saliva of 128 infants. Mothers with depressive symptoms who were more responsive and who engaged in more appropriate touch during face-to-face play had infants with less DNA methylation of NR3C1 and 11ß-HSD2 compared to mothers with depressive symptoms who were also insensitive. The combination of exposure to maternal depressive symptoms and maternal sensitivity was related to the highest prestress cortisol levels, whereas exposure to maternal depressive symptoms and maternal insensitivity was related to the lowest prestress cortisol levels.

Maternal smoking during pregnancy is associated with mitochondrial DNA methylation.

Maternal smoking during pregnancy (MSDP) has detrimental effects on fetal development and on the health of the offspring into adulthood. Energy homeostasis through ATP production via the mitochondria (mt) plays a key role during pregnancy. This study aimed to determine if MSDP resulted in differences in DNA methylation to the placental mitochondrial chromosome at the transcription and replication control region, the D-Loop, and if these differences were also present in an alternate neonatal tissue (foreskin) in an independent birth cohort. We investigated mtDNA methylation by bisulfite-pyrosequencing in two sections of the D-Loop control region and in long interspersed nuclear element-1 (LINE-1) genomic sequences in placenta from 96 mother-newborn pairs that were enrolled in a Rhode Island birth cohort along with foreskin samples from 62 infants from a Kentucky birth cohort. In both placenta and foreskin, mtDNA methylation in the light chain D-Loop region 1 was positively associated with MSDP in placenta (difference+2.73%) (P=0.001) and foreskin (difference+1.22%) (P=0.08). Additionally, in foreskin, a second segment of the D-Loop-heavy chain region 1 showed a small but significant change in methylation with MSDP (+0.4%, P=0.04). No methylation changes were noted in either tissue at the LINE-1 repetitive element. We identified a similar pattern of epigenetic effect to mitochondria arising in cells from different primordial lineages and in different populations, associated with MSDP. These robust and consistent results build evidence that MSDP may impact mt D-Loop methylation, as one mechanism through which this exposure affects newborn health.

Placental epigenetic patterning of glucocorticoid response genes is associated with infant neurodevelopment.

AIM: To determine associations between methylation of NR3C1, HSD11B2, FKBP5 and ADCYAP1R1 and newborn neurobehavioral outcomes. METHODS: In 537 newborns, placental methylation was quantified using bisulfite pyrosequencing. Profiles of neurobehavior were derived via the Neonatal Intensive Care Unit Network Neurobehavioral Scales. Using exploratory factor analysis, the relationships between methylation factor scores and neurobehavioral profiles were examined. RESULTS: Increased scores of the factor characterized by NR3C1 methylation were associated with membership in a reactive, poorly regulated profile (odds ratio: 1.47; 95% CI: 1.00-2.18), while increased scores of the factor characterized by HSD11B2 methylation reduced this risk. CONCLUSION: These results suggest that coordinated regulation of these genes influences neurobehavior and demonstrates the importance of examining these alterations in a harmonized fashion.

Expression of imprinted genes in placenta is associated with infant neurobehavioral development.

Genomic imprinting disorders often exhibit delayed neurobehavioral development, suggesting this unique mechanism of epigenetic regulation plays a role in mental and neurological health. While major errors in imprinting have been linked to adverse health outcomes, there has been little research conducted on how moderate variability in imprinted gene expression within a population contributes to differences in neurobehavioral outcomes, particularly at birth. Here, we profiled the expression of 108 known and putative imprinted genes in human placenta samples from 615 infants assessed by the Neonatal Intensive Care Unit (NICU) Network Neurobehavioral Scales (NNNS). Data reduction identified 10 genes (DLX5, DHCR24, VTRNA2-1, PHLDA2, NPAP1, FAM50B, GNAS-AS1, PAX8-AS1, SHANK2, and COPG2IT1) whose expression could distinguish between newborn neurobehavioral profiles derived from the NNNS. Clustering infants based on the expression pattern of these genes identified 2 groups of infants characterized by reduced quality of movement, increased signs of asymmetrical and non-optimal reflexes, and increased odds of demonstrating increased signs of physiologic stress and abstinence. Overall, these results suggest that common variation in placental imprinted gene expression is linked to suboptimal performance on scales of neurological functioning as well as with increased signs of physiologic stress, highlighting the central importance of the control of expression of these genes in the placenta for neurobehavioral development.

Prenatal predictors of infant self-regulation: the contributions of placental DNA methylation of NR3C1 and neuroendocrine activity.

We examined whether placental DNA methylation of the glucocorticoid receptor gene, NR3C1 was associated with self-regulation and neuroendocrine responses to a social stressor in infancy. Placenta samples were obtained at birth and mothers and their infants (n = 128) participated in the still-face paradigm when infants were 5 months old. Infant self-regulation following the still-face episode was coded and pre-stress cortisol and cortisol reactivity was assessed in response to the still-face paradigm. A factor analysis of NR3C1 CpG sites revealed two factors: one for CpG sites 1-4 and the other for sites 5-13. DNA methylation of the factor comprising NR3C1 CpG sites 5-13 was related to greater cortisol reactivity and infant self-regulation, but cortisol reactivity was not associated with infant self-regulation. The results reveal that prenatal epigenetic processes may explain part of the development of infant self-regulation.

The Role of Placental 11-Beta Hydroxysteroid Dehydrogenase Type 1 and Type 2 Methylation on Gene Expression and Infant Birth Weight.

Maternal stress has been linked to infant birth weight outcomes, which itself may be associated with health later in life. The placenta acts as a master regulator for the fetal environment, mediating intrauterine exposures to stress through the activity of genes regulating glucocorticoids, including the 11beta-hydroxysteroid dehydrogenase (HSD11B) type 1 and 2 genes, and so we hypothesized that variation in these genes will be associated with infant birth weight. We investigated DNA methylation levels at six sites across the two genes, as well as mRNA expression for each, and the relationship to infant birth weight. Logistic regressions correcting for potential confounding factors revealed a significant association between methylation at a single CpG site within HSD11B1 and being born large for gestational age. In addition, our analysis identified correlations between methylation and gene expression, including sex-specific transcriptional regulation of HSD11B2. Our work is one of the first comprehensive views of DNA methylation and expression in the placenta for both HSD11B types 1 and 2, linking epigenetic alterations with the regulation of fetal stress and birth weight outcomes.