Benjamin B. Green

MicroRNA molecular profiling from matched tumor and bio-fluids in bladder cancer

BackgroundMicroRNAs have been identified as potential cancer biomarkers due to their presence and stability in many body fluids including urine and plasma, but the relationship of the pattern of expression of these messengers across various biological media has not been addressed and could provide important information in order to translate these biomarkers for epidemiologic or clinical use.MethodsWe analyzed microRNA of matched FFPE-tumor tissue, plasma, urine exosomes (n = 16) and WBCs (n = 11) from patients with bladder cancer, using Nanostring miRNA assays and droplet digital PCR for validation. Pearson correlations were used to compare expression between media.ResultsNumerous microRNAs were detected and overlapping from specific bio-specimen sources. MiR-4454 and miR-21 overexpression was found in three sources: tumor, WBCs and urine. Additionally, miR-15b-5p, miR-126-3p, miR-93-5p, and miR-150-5p were common to tumor/WBCs, while miR-720/3007a, miR-205, miR-200c-3p and miR-29b-3p common to tumor/urine. Significant associations were noted between the log-adjusted average miRNA counts in tumor vs. WBCs (r = 0.418 p < 0.001), and tumor vs. urine (r = 0.38 p < 0.001). No association was seen tumor vs. plasma exosome miRs (r = 0.07 p = 0.06).ConclusionsMicroRNA profiling from matched samples in patients shows a significant number of microRNAs up regulated in bladder tumors are identifiable in urine exosomes and WBCs of the same patient, but not in blood plasma. This study demonstrated varying relationships between miRNA detected in biological media from the same patient, and serves to inform the potential of urine-based microRNAs as biomarkers for bladder cancer and potentially other malignancies.

Epigenome-wide assessment of DNA methylation in the placenta and arsenic exposure in the New Hampshire Birth Cohort Study (USA)

Background: Arsenic is one of the most commonly encountered environmental toxicants, and research from model systems has suggested that one mode of its toxic activity may be through alterations in DNA methylation. In utero exposure to arsenic can affect fetal, newborn, and infant health, resulting in a range of phenotypic outcomes. Objectives: This study examined variation in placental DNA methylation and its relationship to arsenic exposure in 343 individuals enrolled in the New Hampshire Birth Cohort Study. Methods: Linear regression models using a reference-free correction to account for cellular composition were employed to determine CpG loci affected by arsenic levels. Results: Total arsenic measured in maternal urine during the second trimester was not associated with methylation in the placenta, whereas arsenic levels quantified through maternal toenail collected at birth were associated with methylation at a single CpG locus (p = 4.1 × 10–8). Placenta arsenic levels were associated with 163 differentially methylated loci (false discovery rate < 0.05), with 11 probes within the LYRM2 gene reaching genome-wide significance (p < 10–8). Measurement of LYRM2 mRNA levels indicated that methylation was weakly to moderately correlated with expression (r = 0.15, p < 0.06). In addition, we identified pathways suggesting changes in placental cell subpopulation proportions associated with arsenic exposure. Conclusions: These data demonstrate the potential for arsenic, even at levels commonly experienced in a U.S. population, to have effects on the DNA methylation status of specific genes in the placenta and thus supports a potentially novel mechanism for arsenic to affect long-term children’s health. Citation: Green BB, Karagas MR, Punshon T, Jackson BP, Robbins DJ, Houseman EA, Marsit CJ. 2016. Epigenome-wide assessment of DNA methylation in the placenta and arsenic exposure in the New Hampshire Birth Cohort Study (USA). Environ Health Perspect 124:1253–1260; http://dx.doi.org/10.1289/ehp.1510437

Placental expression profile of imprinted genes impacts birth weight

The importance of imprinted genes in regulating feto-placental development has been long established. However, a comprehensive assessment of the role of placental imprinted gene expression on fetal growth has yet to be conducted. In this study, we examined the association between the placental expression of 108 established and putative imprinted genes and birth weight in 677 term pregnancies, oversampled for small for gestational age (SGA) and large for gestational age (LGA) infants. Using adjusted multinomial regression analyses, a 2-fold increase in the expression of 9 imprinted genes was positively associated with LGA status: BLCAP [odds ratio (OR) = 3.78, 95% confidence interval (CI): 1.83, 7.82], DLK1 [OR = 1.63, 95% CI: 1.27, 2.09], H19 [OR = 2.79, 95% CI: 1.77, 4.42], IGF2 [OR = 1.43, 95% CI:1.31, 2.40], MEG3 [OR = 1.42, 95% CI: 1.19, 1.71], MEST [OR = 4.78, 95% CI: 2.64, 8.65], NNAT [OR = 1.40, 95% CI: 1.05, 1.86], NDN [OR = 2.52, 95% CI: 1.72, 3.68], and PLAGL1 [OR = 1.85, 95% CI: 1.40, 2.44]. For SGA status, a 2-fold increase in MEST expression was associated with decreased risk [OR = 0.31, 95% CI: 0.17, 0.58], while a 2-fold increase in NNAT expression was associated with increased risk [OR = 1.52, 95% CI: 1.1, 2.1]. Following a factor analysis, all genes significantly associated with SGA or LGA status loaded onto 2 of the 8 gene-sets underlying the variability in the dataset. Our comprehensive placental profiling of imprinted genes in a large birth cohort supports the importance of these genes for fetal growth. Given that abnormal birth weight is implicated in numerous diseases and developmental abnormalities, the expression pattern of placental imprinted genes has the potential to be developed as a novel biomarker for postnatal health outcomes.

Select Prenatal Environmental Exposures and Subsequent Alterations of Gene-Specific and Repetitive Element DNA Methylation in Fetal Tissues

Strong evidence implicates maternal environmental exposures in contributing to adverse outcomes during pregnancy and later in life through the developmental origins of health and disease hypothesis. Recent research suggests these effects are mediated through the improper regulation of DNA methylation in offspring tissues, specifically placental tissue, which plays a critical role in fetal development. This article reviews the relevant literature relating DNA methylation in multiple tissues at or near delivery to several prenatal environmental toxicants and stressors, including cigarette smoke, endocrine disruptors, heavy metals, as well as maternal diet. These human studies expand upon previously reported outcomes in animal model interventions and include effects on both imprinted and non-imprinted genes. We have also noted some of the strengths and limitations in the approaches used, and consider the appropriate interpretation of these findings in terms of their effect size and their relationship to differential gene expression and potential health outcomes. The studies suggest an important role of DNA methylation in mediating the effects of the intrauterine environment on children’s health and a need for additional research to better clarify the role of this epigenetic mechanism as well as others.

Between-cow variation in dermal fibroblast response to lipopolysaccharide reflected in resolution of inflammation during Escherichia coli mastitis

Effective response to mammary gland infection depends on efficient early innate immune response. The desired response would be one that is sufficient to clear the infection with a rapid return to the production of high-quality milk and limited tissue damage. In this study, 43 early lactation cows were ranked based on the ability of their fibroblasts to produce IL-8 in response to Escherichia coli lipopolysaccharide. Subsequently, the effect of a low or high response phenotype on the response to E. coli mastitis was determined. Untreated fibroblasts produced no detectable IL-8, whereas the range of IL-8 production in response to LPS (100 ng/mL) was approximately 7-fold between the lowest and highest responding cultures. Similar patterns of between-cow variation were observed in fibroblast production of IL-8 and IL-6 in response to IL-1β and Pam2CSK4 (a synthetic diacylated lipopeptide ligand). Four low and 4 high responder cows were challenged in late lactation with intramammary infusion of E. coli. All cows developed clinical mastitis in the challenged quarters and all cows cleared the infection within 8 d. However, somatic cell count began to decline earlier in the low responder group, and milk BSA concentration (an indicator of tissue damage) was also lower in low responders compared with high responders. Milk production from the challenged quarter was markedly depressed in both groups, but returned toward prechallenge values earlier in low responder cows. Dermal fibroblast cells appear predictive of a cows response to mastitis. In this study, the low responder phenotype was sufficient to contain an E. coli infection with a more rapid return to the production of high quality milk.

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

ABSTRACT 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.

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.

Age dependent changes in the LPS induced transcriptome of bovine dermal fibroblasts occurs without major changes in the methylome

BackgroundBy comparing fibroblasts collected from animals at 5-months or 16-months of age we have previously found that the cultures from older animals produce much more IL-8 in response to lipopolysaccharide (LPS) stimulation. We now expand this finding by examining whole transcriptome differences in the LPS response between cultures from the same animals at different ages, and also investigate the contribution of DNA methylation to the epigenetic basis for the age-dependent increases in responsiveness.ResultsAge-dependent differences in IL-8 production by fibroblasts in response to LPS exposure for 24 h were abolished by pretreatment of cultures with a DNA demethylation agent, 5-aza-2′deoxycytidine (AZA). RNA-Seq analysis of fibroblasts collected from the same individuals at either 5 or 16 months of age and exposed in parallel to LPS for 0, 2, and 8 h revealed a robust response to LPS that was much greater in the cultures from older animals. Pro-inflammatory genes including IL-8, IL-6, TNF-α, and CCL20 (among many other immune associated genes), were more highly expressed (FDR < 0.05) in the 16-month old cultures following LPS exposure. Methylated CpG island recovery assay sequencing (MIRA-Seq) revealed numerous methylation peaks spread across the genome, combined with an overall hypomethylation of gene promoter regions, and a remarkable similarity, except for 20 regions along the genome, between the fibroblasts collected at the two ages from the same animals.ConclusionsThe fibroblast pro-inflammatory response to LPS increases dramatically from 5 to 16 months of age within individual animals. A better understanding of the mechanisms underlying this process could illuminate the physiological processes by which the innate immune response develops and possibly individual variation in innate immune response arises. In addition, although relatively unchanged by age, our data presents a general overview of the bovine fibroblast methylome as a guide for future studies in cattle epigenetics utilizing this cell type.

The use of dermal fibroblasts as a predictive tool of the toll-like receptor 4 response pathway and its development in Holstein heifers

The innate immune system comprises the hosts first line of defense against invading pathogens, and variation in the magnitude of this response between animals has been shown to affect susceptibility to mastitis. The toll-like receptor (TLR) family of proteins initiates the response to invading bacteria, specifically with TLR4 recognizing lipopolysaccharide (LPS) of gram-negative microbes. The underlying genetic variation in the TLR4 pathway leading to differential response is not well understood; therefore, the objective of this work was to determine the efficacy in which the response to LPS by dermal fibroblasts could be used to predict the actual systemic response of that animal to an intravenous endotoxin challenge. To accomplish this, dermal fibroblasts were isolated from 15 Holstein heifers at 5, 11, and 16 mo of age and exposed to either LPS or IL-1β; then, the production of IL-8 in medium was quantified by ELISA. Animals were ranked based upon the magnitude of the fibroblast IL-8 response, and 8 heifers were selected [4 low responders (LR) and 4 high responders (HR)] for challenge with an intravenous bolus dose (0.5 μg/kg of body weight) of LPS. Overall, between-animal variation in fibroblast IL-8 production following LPS or IL-1β was high, indicating appreciable differences in the TLR4 pathway of the animals. Ranking of the fibroblast responses was consistent across the 3 sampling times for each animal; however, the absolute response increased, and the age at which the fibroblasts were obtained was consistent with the potential for age-related changes in cell function to affect immune function processes. Following systemic LPS challenge, HR heifers had higher plasma concentrations of tumor necrosis factor-α and IL-8 than LR heifers. However, LR heifers had a stronger febrile response than HR heifers. The use of dermal fibroblasts under laboratory conditions appears to represent a practical model for predicting the innate immune response in vivo and could act as an important tool in mapping genetic differences of the TLR4 pathway.

Cow-to-cow variation in fibroblast response to a toll-like receptor 2/6 agonist and its relation to mastitis caused by intramammary challenge with Staphylococcus aureus.

Staphylococcus aureus is a common cause of chronic mammary gland infections in dairy cattle. However, the inflammatory response and duration of infection following pathogen exposure is variable between individual animals. To investigate interanimal differences in immune response, dermal fibroblast cultures were established from skin biopsies collected from 50 early lactation Holstein cows. The fibroblasts ability to produce IL-8 in response to a 24-h treatment with a synthetic toll-like receptor 2/6 agonist (Pam2CSK4) was used to assign a response phenotype to the animals. Five high-responding and 5 low-responding animals were then selected for an intramammary challenge with S. aureus to evaluate differences in the inflammatory response, chronicity of infection, and development of antibodies to the pathogen. All animals exhibited clinical symptoms of mastitis at 24h postchallenge. Animals previously classified as high responders experienced a greater inflammatory response characterized by elevated levels of milk somatic cell count, IL-8, and BSA following the challenge compared with low responders. In addition, antibodies toward the challenge strain of S. aureus reached higher levels in whey from the challenged gland of high responders compared with low responders. Despite the antibody response, all 5 high responders were chronically infected for the 6-wk duration of the study, whereas 2 of the low responders cleared the infection, although 1 of these did become reinfected. The observed differences between animals classified as low and high responders based on their fibroblast responsiveness suggests that this cell type can be used to further examine the causes of interanimal variation in response to mammary infection.