Pilot examination of independent and interactive effects of maltreatment and neighborhood risk on child attachment.

Elucidating the influence of microsystem and exosystem factors on development is an important goal of developmental psychopathology. This study examined the effects of maltreatment and neighborhood risk on child-caregiver attachment. Maltreatment records, neighborhood risk indices, and Strange Situation data were collected from a diverse sample of 170 four-year-old children and their caregivers. Relative contributions of maltreatment, neighborhood risk, and their interaction on attachment insecurity and disorganization were explored via latent moderation. Maltreated children demonstrated higher rates of insecure attachment, but not attachment disorganization, independent of neighborhood risk. Controlling for maltreatment, preliminary results suggested no effects of neighborhood risk on attachment. Findings support prior research that has identified maltreatment as a salient risk to the formation of secure attachment relationships. However, results add heterogeneity to the limited research investigating effects of neighborhood on attachment. Overall, this study highlights the importance of examining multilevel ecological risk in relation to attachment relationship development.

Transient growth factor expression via mRNA in lipid nanoparticles promotes hepatocyte cell therapy to treat murine liver diseases.

Primary human hepatocyte (PHH) transplantation is a promising alternative to liver transplantation, whereby liver function could be restored by partial repopulation of the diseased organ with healthy cells. However, currently PHH engraftment efficiency is low and benefits are not maintained long-term. Here we refine two mouse models of human chronic and acute liver diseases to recapitulate compromised hepatocyte proliferation observed in nearly all human liver diseases by overexpression of p21 in hepatocytes. In these clinically relevant contexts, we demonstrate that transient, yet robust expression of human hepatocyte growth factor and epidermal growth factor in the liver via nucleoside-modified mRNA in lipid nanoparticles, whose safety was validated with mRNA-based COVID-19 vaccines, drastically improves PHH engraftment, reduces disease burden, and improves overall liver function. This novel strategy may overcome the critical barriers to clinical translation of cell therapies with primary or stem cell-derived hepatocytes for the treatment of liver diseases.

Infant Feeding Practices and Metal Concentrations in Children's Blood.

This cohort study assesses the association between 4 infant feeding practices and concentrations of 8 nonessential and 7 essential metals in red blood cells.

VEGFA mRNA-LNP promotes biliary epithelial cell-to-hepatocyte conversion in acute and chronic liver diseases and reverses steatosis and fibrosis.

The liver is known for its remarkable regenerative ability through proliferation of hepatocytes. Yet, during chronic injury or severe hepatocyte death, proliferation of hepatocytes is exhausted. To overcome this hurdle, we propose vascular-endothelial-growth-factor A (VEGFA) as a therapeutic means to accelerate biliary epithelial-cell (BEC)-to-hepatocyte conversion. Investigation in zebrafish establishes that blocking VEGF receptors abrogates BEC-driven liver repair, while VEGFA overexpression promotes it. Delivery of VEGFA via nonintegrative and safe nucleoside-modified mRNA encapsulated into lipid nanoparticles (mRNA-LNPs) in acutely or chronically injured mouse livers induces robust BEC-to-hepatocyte conversion and elimination of steatosis and fibrosis. In human and murine diseased livers, we further identified VEGFA-receptor KDR-expressing BECs associated with KDR-expressing cell-derived hepatocytes. This work defines KDR-expressing cells, most likely being BECs, as facultative progenitors. This study reveals unexpected therapeutic benefits of VEGFA delivered via nucleoside-modified mRNA-LNP, whose safety is widely validated with COVID-19 vaccines, for harnessing BEC-driven repair to potentially treat liver diseases.

Growth Hormone Accelerates Recovery From Acetaminophen-Induced Murine Liver Injury.

Background and Aims: Acetaminophen (APAP) overdose is the leading cause of acute liver failure, with one available treatment, N-acetyl cysteine (NAC). Yet, NAC effectiveness diminishes about ten hours after APAP overdose, urging for therapeutic alternatives. This study addresses this need by deciphering a mechanism of sexual dimorphism in APAP-induced liver injury, and leveraging it to accelerate liver recovery via growth hormone (GH) treatment. GH secretory patterns, pulsatile in males and near-continuous in females, determine the sex bias in many liver metabolic functions. Here, we aim to establish GH as a novel therapy to treat APAP hepatotoxicity. Approach and Results: Our results demonstrate sex-dependent APAP toxicity, with females showing reduced liver cell death and faster recovery than males. Single-cell RNA sequencing analyses reveal that female hepatocytes have significantly greater levels of GH receptor expression and GH pathway activation compared to males. In harnessing this female-specific advantage, we demonstrate that a single injection of recombinant human GH protein accelerates liver recovery, promotes survival in males following sub-lethal dose of APAP, and is superior to standard-of-care NAC. Alternatively, slow-release delivery of human GH via the safe nonintegrative lipid nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP), a technology validated by widely used COVID-19 vaccines, rescues males from APAP-induced death that otherwise occurred in control mRNA-LNP-treated mice. Conclusions: Our study demonstrates a sexually dimorphic liver repair advantage in females following APAP overdose, leveraged by establishing GH as an alternative treatment, delivered either as recombinant protein or mRNA-LNP, to potentially prevent liver failure and liver transplant in APAP-overdosed patients.

VEGFA mRNA-LNP promotes biliary epithelial cell-to-hepatocyte conversion in acute and chronic liver diseases and reverses steatosis and fibrosis.

The liver is known for its remarkable regenerative ability through proliferation of hepatocytes. Yet, during chronic injury or severe hepatocyte death, proliferation of hepatocytes is exhausted. To overcome this hurdle, we propose vascular-endothelial-growth-factor A (VEGFA) as a therapeutic means to accelerate biliary epithelial cell (BEC)-to-hepatocyte conversion. Investigation in zebrafish establishes that blocking VEGF receptors abrogates BEC-driven liver repair, while VEGFA overexpression promotes it. Delivery of VEGFA via non-integrative and safe nucleoside-modified mRNA encapsulated into lipid-nanoparticles (mRNA-LNP) in acutely or chronically injured mouse livers induces robust BEC-to-hepatocyte conversion and reversion of steatosis and fibrosis. In human and murine diseased livers, we further identified VEGFA-receptor KDR-expressing BECs associated with KDR-expressing cell-derived hepatocytes. This defines KDR-expressing cells, most likely being BECs, as facultative progenitors. This study reveals novel therapeutic benefits of VEGFA delivered via nucleoside-modified mRNA-LNP, whose safety is widely validated with COVID-19 vaccines, for harnessing BEC-driven repair to potentially treat liver diseases. Highlights: Complementary mouse and zebrafish models of liver injury demonstrate the therapeutic impact of VEGFA-KDR axis activation to harness BEC-driven liver regeneration.VEGFA mRNA LNPs restore two key features of the chronic liver disease in humans such as steatosis and fibrosis.Identification in human cirrhotic ESLD livers of KDR-expressing BECs adjacent to clusters of KDR+ hepatocytes suggesting their BEC origin.KDR-expressing BECs may represent facultative adult progenitor cells, a unique BEC population that has yet been uncovered.

The prenatal environment and its influence on maternal and child mitochondrial DNA copy number and methylation: A review of the literature.

Mitochondrial DNA (mtDNA) is sensitive to environmental stressors and associated with human health. We reviewed epidemiological literature examining associations between prenatal environmental, dietary, and social exposures and alterations in maternal/child mtDNA copy number (mtDNAcn) and mtDNA methylation. Evidence exists that prenatal maternal exposures are associated with alterations in mtDNAcn for air pollution, chemicals (e.g. metals), cigarette smoke, human immunodeficiency virus (HIV) infection and treatment. Evidence for their associations with mtDNA methylation was limited. Given its potential implications as a disease pathway biomarker, studies with sufficient biological specificity should examine the long-term implications of prenatal and early-life mtDNA alterations in response to prenatal exposures.

Associations of Prenatal First Trimester Essential and Nonessential Metal Mixtures with Body Size and Adiposity in Childhood.

BACKGROUND: Prenatal nonessential metals may contribute to postnatal adiposity, whereas essential metals may have metabolic benefits. We evaluated joint and individual associations between prenatal metals and childhood adiposity. METHODS: We measured concentrations of six nonessential (arsenic, barium, cadmium, cesium, lead, and mercury) and four essential (magnesium, manganese, selenium, and zinc) metals in first trimester maternal blood from a prebirth cohort. We collected anthropometric measures in early childhood, mid-childhood, and early adolescence including subscapular+tricep skinfold thickness (mm) (N = 715-859), waist circumference (cm) (N = 717-882), and body mass index (BMI) (z-score) (N = 716-875). We measured adiposity in mid-childhood and early adolescence using bone densitometry total- and trunk- fat mass index (kg/m 2 ) (N = 511-599). We estimated associations using adjusted quantile g-computation and linear regression. RESULTS: The nonessential metal mixture was associated with higher total (ß = 0.07, 95% CI = 0.01, 0.12) and trunk fat mass index (ß = 0.12, CI = 0.02, 0.22), waist circumference (ß = 0.01, CI = 0.00, 0.01), and BMI (ß = 0.24, CI = 0.07, 0.41) in mid-childhood, and total fat mass index (ß = 0.07, CI = 0.01, 0.14), and BMI (ß = 0.19, CI = 0.02, 0.37) in early adolescence. The essential metal mixture was associated with lower early adolescence total-(ß = -0.11, CI = -0.17, -0.04) and trunk- fat mass index (ß = -0.13, CI = -0.21, -0.05), subscapular+tricep skinfold thickness (ß = -0.02, CI = -0.03, -0.00), waist circumference (ß = -0.003, CI = -0.01, -0.00), and BMI (ß = -0.16, CI = -0.28, -0.04). Cadmium and cesium were individually associated with childhood adiposity at different timepoints. CONCLUSIONS: Prenatal first-trimester essential metals were associated with lower childhood adiposity, whereas nonessential metals were associated with higher adiposity into adolescence.

Associations Between Prenatal Blood Metals and Vitamins and Cord Blood Peptide Hormone Concentrations.

Background: Nonessential metals have endocrine disrupting properties, interfere with cellular processes, generate reactive oxygen and deplete antioxidants, while essential metals and vitamins act as antioxidants. The extent to which prenatal metals and vitamins are associated with cord blood hormones involved in maternal and fetal metabolic and growth processes is unknown. Methods: We measured six nonessential (arsenic, barium, cadmium, cesium, lead, mercury) and four essential (magnesium, manganese, selenium, zinc) metals and trace elements, and two vitamins (B12 and folate) in first trimester blood from participants in the longitudinal pre-birth Project Viva cohort, who were recruited between 1999-2002 in eastern Massachusetts. We measured adiponectin, C-peptide, IGF-1, IGF-2, IGFBP-3, insulin, and leptin concentrations in cord blood (~n=695). We used covariate-adjusted quantile g-computation for mixtures and linear regression for individual exposures to estimate associations with cord blood peptide hormones. Results: The essential metal mixture (magnesium, manganese, selenium, zinc) was associated with higher IGF-1 (ß=3.20 ng/ml per quartile, 95% CI: 0.39, 6.01), IGF-2 (ß=10.93 ng/ml, 95% CI: 0.08, 21.79), and leptin (ß=1.03 ng/ml, 95% CI: 0.25, 1.80). Magnesium was associated with higher leptin (ß=2.90 ng/ml, 95% CI: 0.89, 4.91), while B12 was associated with lower adiponectin, IGF-2, and leptin, but higher C-peptide. Other individual nonessential metals were associated with cord blood hormones. Conclusions: Our findings suggest that some prenatal metals and vitamins are associated with cord blood hormones, which may influence growth and development.

Health Effects of Pesticide Exposure in Latin American and the Caribbean Populations: A Scoping Review.

BACKGROUND: Multiple epidemiological studies have shown that exposure to pesticides is associated with adverse health outcomes. However, the literature on pesticide-related health effects in the Latin American and the Caribbean (LAC) region, an area of intensive agricultural and residential pesticide use, is sparse. We conducted a scoping review to describe the current state of research on the health effects of pesticide exposure in LAC populations with the goal of identifying knowledge gaps and research capacity building needs. METHODS: We searched PubMed and SciELO for epidemiological studies on pesticide exposure and human health in LAC populations published between January 2007 and December 2021. We identified 233 publications from 16 countries that met our inclusion criteria and grouped them by health outcome (genotoxicity, neurobehavioral outcomes, placental outcomes and teratogenicity, cancer, thyroid function, reproductive outcomes, birth outcomes and child growth, and others). RESULTS: Most published studies were conducted in Brazil (37%, n=88) and Mexico (20%, n=46), were cross-sectional in design (72%, n=167), and focused on farmworkers (45%, n=105) or children (21%, n=48). The most frequently studied health effects included genotoxicity (24%, n=62) and neurobehavioral outcomes (21%, n=54), and organophosphate (OP) pesticides were the most frequently examined (26%, n=81). Forty-seven percent (n=112) of the studies relied only on indirect pesticide exposure assessment methods. Exposure to OP pesticides, carbamates, or to multiple pesticide classes was consistently associated with markers of genotoxicity and adverse neurobehavioral outcomes, particularly among children and farmworkers. DISCUSSION: Our scoping review provides some evidence that exposure to pesticides may adversely impact the health of LAC populations, but methodological limitations and inconsistencies undermine the strength of the conclusions. It is critical to increase capacity building, integrate research initiatives, and conduct more rigorous epidemiological studies in the region to address these limitations, better inform public health surveillance systems, and maximize the impact of research on public policies. https://doi.org/10.1289/EHP9934.

Comparison of DNA methylation measurements from EPIC BeadChip and SeqCap targeted bisulphite sequencing in PON1 and nine additional candidate genes.

Epigenome-wide association studies (EWAS) are widely implemented in epidemiology, and the Illumina HumanMethylationEPIC BeadChip (EPIC) DNA microarray is the most-used technology. Recently, next-generation sequencing (NGS)-based methods, which assess DNA methylation at single-base resolution, have become more affordable and technically feasible. While the content of microarray technology is fixed, NGS-based approaches, such as the Roche Nimblegen, SeqCap Epi Enrichment System (SeqCap), offer the flexibility of targeting most CpGs in a gene. With the current usage of microarrays and emerging NGS-based technologies, it is important to establish whether data generated from the two platforms are comparable. We harnessed 112 samples from the Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS) birth cohort study and compared DNA methylation between the EPIC microarray and SeqCap for PON1 and nine additional candidate genes, by evaluating epigenomic coverage and correlations. We conducted multivariable linear regression and principal component analyses to assess the ability of the EPIC array and SeqCap to detect biological differences in gene methylation by the PON1-108 single nucleotide polymorphism. We found an overall high concordance (r = 0.84) between SeqCap and EPIC DNA methylation, among highly methylated and minimally methylated regions. However, substantial disagreement was present between the two methods in moderately methylated regions, with SeqCap measurements exhibiting greater within-site variation. Additionally, SeqCap did not capture PON1 SNP associated differences in DNA methylation that were evident with the EPIC array. Our findings indicate that microarrays perform well for analysing DNA methylation in large cohort studies but with limited coverage.

c-Maf: The magic wand that turns on LSEC fate.

In this issue of Cell Stem Cell, Gómez-Salinero et al. (2022) identify c-Maf as a driver for murine liver sinusoidal endothelial cell (LSEC) fate and function during liver development, homeostasis, and repair. Similarly, c-Maf defines human LSECs, and its overexpression specializes generic HUVECs into functional induced-LSECs, potentiating regenerative therapeutics.

Biodigester Cookstove Interventions and Child Diarrhea in Semirural Nepal: A Causal Analysis of Daily Observations.

BACKGROUND: Hundreds of thousands of biodigesters have been constructed in Nepal. These household-level systems use human and animal waste to produce clean-burning biogas used for cooking, which can reduce household air pollution from woodburning cookstoves and prevent respiratory illnesses. The biodigesters, typically operated by female caregivers, require the handling of animal waste, which may increase domestic fecal contamination, exposure to diarrheal pathogens, and the risk of enteric infections, especially among young children. OBJECTIVE: We estimated the effect of daily reported biogas cookstove use on incident diarrhea among children <5y old in the Kavrepalanchok District of Nepal. Secondarily, we assessed effect measure modification and statistical interaction of individual- and household-level covariates (child sex, child age, birth order, exclusive breastfeeding, proof of vaccination, roof type, sanitation, drinking water treatment, food insecurity) as well as recent 14-d acute lower respiratory infection (ALRI) and season. METHODS: We analyzed 300,133 person-days for 539 children in an observational prospective cohort study to estimate the average effect of biogas stove use on incident diarrhea using cross-validated targeted maximum likelihood estimation (CV-TMLE). RESULTS: Households reported using biogas cookstoves in the past 3 d for 23% of observed person-days. The adjusted relative risk of diarrhea for children exposed to biogas cookstove use was 1.31 (95% confidence interval (CI): 1.00, 1.71) compared to unexposed children. The estimated effect of biogas stove use on diarrhea was stronger among breastfed children (2.09; 95% CI: 1.35, 3.25) than for nonbreastfed children and stronger during the dry season (2.03; 95% CI: 1.17, 3.53) than in the wet season. Among children exposed to biogas cookstove use, those with a recent ALRI had the highest mean risk of diarrhea, estimated at 4.53 events (95% CI: 1.03, 8.04) per 1,000 person-days. DISCUSSION: This analysis provides new evidence that child diarrhea may be an unintended health risk of biogas cookstove use. Additional studies are needed to identify exposure pathways of fecal pathogen contamination associated with biodigesters to improve the safety of these widely distributed public health interventions. https://doi.org/10.1289/EHP9468.

Dietary intake and household exposures as predictors of urinary concentrations of high molecular weight phthalates and bisphenol A in a cohort of adolescents.

BACKGROUND: Phthalates and bisphenol A (BPA) are endocrine disrupting chemicals used in consumer products, building materials, and food processing and packaging materials. They are associated with adverse health outcomes, especially when exposure occurs during heightened windows of susceptibility. OBJECTIVE: We evaluated the relationship between housing and dietary characteristics and the concentration of several high-molecular-weight (HMW) phthalate metabolites and BPA in a cohort of Latina adolescents. METHODS: We collected information on recent food consumption and housing characteristics and quantified the concentration of HMW phthalate and BPA metabolites in urine collected at two different time points. We used generalized estimating equations (GEE) to assess predictors of each metabolite. RESULTS: No significant associations were observed between housing and dietary characteristics and metabolites of di(2-ethylhexyl) phthalate (DEHP) or BPA. In contrast, higher urinary monobenzyl phthalate (MBzP) concentration was associated with living in a home with vinyl or linoleum flooring (66.7% change, p-value <0.01), while higher urinary mono(3-carboxypropyl) phthalate (MCPP) concentration was associated with recent consumption of coffee (47.2% change, p-value <0.01), and fast food (30.3% change, p-value <0.05). SIGNIFICANCE: These findings may be useful in targeting interventions that reduce phthalate uptake in young adults.

Prospective Associations of Early Pregnancy Metal Mixtures with Mitochondria DNA Copy Number and Telomere Length in Maternal and Cord Blood.

BACKGROUND: Metal exposure during pregnancy influences maternal and child health. Oxidative stress and inflammation may mediate adverse effects of heavy metals, whereas essential metals may act as antioxidants. Mitochondrial DNA is a prime target for metal-induced oxidative damage. Telomere dysfunction is attributed to imbalances between reactive oxidant species and antioxidants. OBJECTIVES: We evaluated individual and joint associations of prenatal metals with mitochondrial DNA copy number (mtDNAcn) and telomere length (TL) in maternal and cord blood as biomarkers of inflammation and oxidative stress. METHODS: We measured six nonessential metals (arsenic, barium, cadmium, cesium, lead, mercury) and four essential metals (magnesium, manganese, selenium, zinc) in first-trimester maternal red blood cells in Project Viva, a U.S. prebirth cohort. We measured relative mtDNAcn (n=898) and TL (n=893) in second-trimester maternal blood and mtDNAcn (n=419) and TL (n=408) in cord blood. We used multivariable linear regression and quantile g-computation to estimate associations between prenatal metals and the biomarkers. We used generalized additive models and Bayesian kernel machine regression to examine nonlinearity and interactions. RESULTS: A 2-fold increase in maternal magnesium was associated with lower maternal [ß=-0.07, 95% confidence interval (CI): -0.10, -0.01] and cord blood (ß=-0.08, 95% CI: -0.20, -0.01) mtDNAcn. Lead was associated with higher maternal mtDNAcn (ß=0.04, 95% CI: 0.01, 0.06). Selenium was associated with longer cord blood TL (ß=0.30, 95% CI: 0.01 0.50). An association was observed between the nonessential metal mixture and higher maternal mtDNAcn (ß=0.04, 95% CI: 0.01, 0.07). There was a nonlinear relationship between cord blood mtDNAcn and magnesium; maternal mtDNAcn and barium, lead, and mercury; and maternal TL and barium. DISCUSSION: Maternal exposure to metals such as lead, magnesium, and selenium was associated with mtDNAcn and TL in maternal second trimester and cord blood. Future work will evaluate whether these biomarkers are associated with child health. https://doi.org/10.1289/EHP9294.

Transient yet Robust Expression of Proteins in the Mouse Liver via Intravenous Injection of Lipid Nanoparticle-encapsulated Nucleoside-modified mRNA.

With the recent availability of the SARS-CoV-2 mRNA-based vaccines, public attention has been drawn to this new technology and how it may be applied to other indications. Temporal activation of key hepatic regenerative pathways can induce liver regeneration, overcoming the lack of donor organs for liver transplantation and ineffectiveness of alternative treatments. Recombinant protein therapies and genetic therapies that target these pathways require frequent and repeated injections or, when integrated into the genome, may lead to deleterious effects. In contrast, nucleoside-modified mRNA encapsulated in lipid nanoparticles (mRNA-LNP) are non-integrative and induce transient yet robust expression of proteins that could serve as an ideal therapeutic tool to treat specific liver diseases. For instance, our recent publication in Nature Communications used mRNA-LNP to express hepatic mitogens, hepatocyte growth factor, and epidermal growth factor to induce liver regeneration following both acute and chronic liver injuries. Initial testing with firefly luciferase mRNA-LNP transfection and in vivo imaging confirmed specific hepatotropic delivery. In this protocol, we describe in detail the necessary steps to deliver mRNA-LNP to the murine liver and, following intravenous injection of eGFP mRNA-LNP, verify transfection efficiency using flow cytometry and liver cell specificity using immunofluorescence analyses. This procedure presents an unprecedented tool that can be customized with mRNA-LNP encoding any protein of interest to be expressed by virtually all hepatocytes, ~70% endothelial cells, and ~40% Kupffer cells for promoting liver function and/or regeneration. Graphic abstract: Experimental Design of mRNA-LNP IV Injection and Analysis of Liver Cell Specificity and Efficiency of Transfection (Created with BioRender.com).

Murine liver repair via transient activation of regenerative pathways in hepatocytes using lipid nanoparticle-complexed nucleoside-modified mRNA.

Induction of intrinsic liver regeneration is an unmet need that can be achieved by temporally activating key hepatocyte regenerative pathways. Here, we establish an efficient, safe, non-integrative method to transiently express hepatocyte-growth-factor (HGF) and epidermal-growth-factor (EGF) in hepatocytes via nucleoside-modified, lipid-nanoparticle-encapsulated mRNA (mRNA-LNP) delivery in mice. We confirm specific hepatotropism of mRNA-LNP via intravenous injection of firefly luciferase encoding mRNA-LNP, with protein expression lasting about 3 days. In the liver, virtually all hepatocytes are transfected along with a subpopulation of endothelial and Kupffer cells. In homeostasis, HGF mRNA-LNP efficiently induce hepatocyte proliferation. In a chronic liver injury mouse model recapitulating non-alcoholic fatty liver disease, injections of both HGF and EGF mRNA-LNP sharply reverse steatosis and accelerate restoration of liver function. Likewise, HGF and EGF mRNA-LNP accelerate liver regeneration after acetaminophen-induced acute liver injury with rapid return to baseline ALT levels. This study introduces mRNA-LNP as a potentially translatable safe therapeutic intervention to harness liver regeneration via controlled expression of endogenous mitogens in vivo.