Effects of Developmental Lead and Phthalate Exposures on DNA Methylation in Adult Mouse Blood, Brain, and Liver: A Focus on Genomic Imprinting by Tissue and Sex.

BACKGROUND: Maternal exposure to environmental chemicals can cause adverse health effects in offspring. Mounting evidence supports that these effects are influenced, at least in part, by epigenetic modifications. It is unknown whether epigenetic changes in surrogate tissues such as the blood are reflective of similar changes in target tissues such as cortex or liver. OBJECTIVE: We examined tissue- and sex-specific changes in DNA methylation (DNAm) associated with human-relevant lead (Pb) and di(2-ethylhexyl) phthalate (DEHP) exposure during perinatal development in cerebral cortex, blood, and liver. METHODS: Female mice were exposed to human relevant doses of either Pb (32 ppm) via drinking water or DEHP (5mg/kg-day) via chow for 2 weeks prior to mating through offspring weaning. Whole genome bisulfite sequencing (WGBS) was utilized to examine DNAm changes in offspring cortex, blood, and liver at 5 months of age. Metilene and methylSig were used to identify differentially methylated regions (DMRs). Annotatr and ChIP-enrich were used for genomic annotations and gene set enrichment tests of DMRs, respectively. RESULTS: The cortex contained the majority of DMRs associated with Pb (66%) and DEHP (57%) exposure. The cortex also contained the greatest degree of overlap in DMR signatures between sexes (n=13 and 8 DMRs with Pb and DEHP exposure, respectively) and exposure types (n=55 and 39 DMRs in males and females, respectively). In all tissues, detected DMRs were preferentially found at genomic regions associated with gene expression regulation (e.g., CpG islands and shores, 5' UTRs, promoters, and exons). An analysis of GO terms associated with DMR-containing genes identified imprinted genes to be impacted by both Pb and DEHP exposure. Of these, Gnas and Grb10 contained DMRs across tissues, sexes, and exposures, with some signatures replicated between target and surrogate tissues. DMRs were enriched in the imprinting control regions (ICRs) of Gnas and Grb10, and we again observed a replication of DMR signatures between blood and target tissues. Specifically, we observed hypermethylation of the Grb10 ICR in both blood and liver of Pb-exposed male animals. CONCLUSIONS: These data provide preliminary evidence that imprinted genes may be viable candidates in the search for epigenetic biomarkers of toxicant exposure in target tissues. Additional research is needed on allele- and developmental stage-specific effects, as well as whether other imprinted genes provide additional examples of this relationship. https://doi.org/10.1289/EHP14074.

Characteristics and Consequences of Violent Victimization in Sexual and Gender Minority Communities: An Analysis of the 2017-2021 National Crime Victimization Survey.

Purpose: This article investigates rates of violent victimization, subsequent help-seeking, and health-related consequences within sexual and gender minority (SGM) communities. Methods: Aggregate data from the 2017-2021 National Crime Victimization Survey were examined to determine nationally representative estimates of rates and distributions of violent victimization, help-seeking, and socioemotional consequences within those 16 years of age and older. Due to sample size, most analyses aggregated sexual orientation and gender identity to allow comparison of SGM persons to non-SGM persons and examine differences within the SGM population. Results: Persons who identified as lesbian, gay, or bisexual experienced violent victimization at rates two to six times higher than straight persons. Transgender persons were victimized more than three times as often than cisgender persons. SGM persons experienced higher rates of all types of violent victimization than non-SGM persons regardless of victim-offender relationship. There were differences by victim demographic characteristics, including sex, race and Hispanic origin, age, marital status, and household income. A higher proportion of SGM victims reported only problems with work/school or problems both at work/school and with family/friends. Finally, higher proportions of SGM victims reported socioemotional consequences when they were female, older, or experienced serious violent crime. Conclusion: The findings in this study continue to highlight high levels of violence experienced by SGM persons and disproportionate socioemotional consequences. There is an evident need to develop targeted interventions and provide services to address the consequences of victimization among this population. The analyses demonstrate the necessity of continued research to better understand the impact of violence on SGM communities.

piOxi database: a web resource of germline and somatic tissue piRNAs identified by chemical oxidation.

PIWI-interacting RNAs (piRNAs) are a class of small non-coding RNAs that are highly expressed and extensively studied from the germline. piRNAs associate with PIWI proteins to maintain DNA methylation for transposon silencing and transcriptional gene regulation for genomic stability. Mature germline piRNAs have distinct characteristics including a 24- to 32-nucleotide length and a 2'-O-methylation signature at the 3' end. Although recent studies have identified piRNAs in somatic tissues, they remain poorly characterized. For example, we recently demonstrated notable expression of piRNA in the murine soma, and while overall expression was lower than that of the germline, unique characteristics suggested tissue-specific functions of this class. While currently available databases commonly use length and association with PIWI proteins to identify piRNA, few have included a chemical oxidation method that detects piRNA based on its 3' modification. This method leads to reproducible and rigorous data processing when coupled with next-generation sequencing and bioinformatics analysis. Here, we introduce piOxi DB, a user-friendly web resource that provides a comprehensive analysis of piRNA, generated exclusively through sodium periodate treatment of small RNA. The current version of piOxi DB includes 435 749 germline and 9828 somatic piRNA sequences robustly identified from M. musculus, M. fascicularis and H. sapiens. The database provides species- and tissue-specific data that are further analyzed according to chromosome location and correspondence to gene and repetitive elements. piOxi DB is an informative tool to assist broad research applications in the fields of RNA biology, cancer biology, environmental toxicology and beyond. Database URL:  https://pioxidb.dcmb.med.umich.edu/.

Effects of Developmental Lead and Phthalate Exposures on DNA Methylation in Adult Mouse Blood, Brain, and Liver Identifies Tissue- and Sex-Specific Changes with Implications for Genomic Imprinting.

Background: Maternal exposure to environmental chemicals can cause adverse health effects in offspring. Mounting evidence supports that these effects are influenced, at least in part, by epigenetic modifications. Objective: We examined tissue- and sex-specific changes in DNA methylation (DNAm) associated with human-relevant lead (Pb) and di(2-ethylhexyl) phthalate (DEHP) exposure during perinatal development in cerebral cortex, blood, and liver. Methods: Female mice were exposed to human relevant doses of either Pb (32ppm) via drinking water or DEHP (5 mg/kg-day) via chow for two weeks prior to mating through offspring weaning. Whole genome bisulfite sequencing (WGBS) was utilized to examine DNAm changes in offspring cortex, blood, and liver at 5 months of age. Metilene and methylSig were used to identify differentially methylated regions (DMRs). Annotatr and Chipenrich were used for genomic annotations and geneset enrichment tests of DMRs, respectively. Results: The cortex contained the majority of DMRs associated with Pb (69%) and DEHP (58%) exposure. The cortex also contained the greatest degree of overlap in DMR signatures between sexes (n = 17 and 14 DMRs with Pb and DEHP exposure, respectively) and exposure types (n = 79 and 47 DMRs in males and females, respectively). In all tissues, detected DMRs were preferentially found at genomic regions associated with gene expression regulation (e.g., CpG islands and shores, 5' UTRs, promoters, and exons). An analysis of GO terms associated with DMR-containing genes identified imprinted genes to be impacted by both Pb and DEHP exposure. Of these, Gnas and Grb10 contained DMRs across tissues, sexes, and exposures. DMRs were enriched in the imprinting control regions (ICRs) of Gnas and Grb10, with 15 and 17 ICR-located DMRs across cortex, blood, and liver in each gene, respectively. The ICRs were also the location of DMRs replicated across target and surrogate tissues, suggesting epigenetic changes these regions may be potentially viable biomarkers. Conclusions: We observed Pb- and DEHP-specific DNAm changes in cortex, blood, and liver, and the greatest degree of overlap in DMR signatures was seen between exposures followed by sex and tissue type. DNAm at imprinted control regions was altered by both Pb and DEHP, highlighting the susceptibility of genomic imprinting to these exposures during the perinatal window of development.

Developmental exposures to common environmental contaminants, DEHP and lead, alter adult brain and blood hydroxymethylation in mice.

Introduction: The developing epigenome changes rapidly, potentially making it more sensitive to toxicant exposures. DNA modifications, including methylation and hydroxymethylation, are important parts of the epigenome that may be affected by environmental exposures. However, most studies do not differentiate between these two DNA modifications, possibly masking significant effects. Methods: To investigate the relationship between DNA hydroxymethylation and developmental exposure to common contaminants, a collaborative, NIEHS-sponsored consortium, TaRGET II, initiated longitudinal mouse studies of developmental exposure to human-relevant levels of the phthalate plasticizer di(2-ethylhexyl) phthalate (DEHP), and the metal lead (Pb). Exposures to 25 mg DEHP/kg of food (approximately 5 mg DEHP/kg body weight) or 32 ppm Pb-acetate in drinking water were administered to nulliparous adult female mice. Exposure began 2 weeks before breeding and continued throughout pregnancy and lactation, until offspring were 21 days old. At 5 months, perinatally exposed offspring blood and cortex tissue were collected, for a total of 25 male mice and 17 female mice (n = 5-7 per tissue and exposure). DNA was extracted and hydroxymethylation was measured using hydroxymethylated DNA immunoprecipitation sequencing (hMeDIP-seq). Differential peak and pathway analysis was conducted comparing across exposure groups, tissue types, and animal sex, using an FDR cutoff of 0.15. Results: DEHP-exposed females had two genomic regions with lower hydroxymethylation in blood and no differences in cortex hydroxymethylation. For DEHP-exposed males, ten regions in blood (six higher and four lower) and 246 regions (242 higher and four lower) and four pathways in cortex were identified. Pb-exposed females had no statistically significant differences in blood or cortex hydroxymethylation compared to controls. Pb-exposed males, however, had 385 regions (all higher) and six pathways altered in cortex, but no differential hydroxymethylation was identified in blood. Discussion: Overall, perinatal exposure to human-relevant levels of two common toxicants showed differences in adult DNA hydroxymethylation that was specific to sex, exposure type, and tissue, but male cortex was most susceptible to hydroxymethylation differences by exposure. Future assessments should focus on understanding if these findings indicate potential biomarkers of exposure or are related to functional long-term health effects.

Are social interactions preferentially attended in real-world scenes? Evidence from change blindness.

In change detection paradigms, changes to social or animate aspects of a scene are detected better and faster compared with non-social or inanimate aspects. While previous studies have focused on how changes to individual faces/bodies are detected, it is possible that individuals presented within a social interaction may be further prioritised, as the accurate interpretation of social interactions may convey a competitive advantage. Over three experiments, we explored change detection to complex real-world scenes, in which changes either occurred by the removal of (a) an individual on their own, (b) an individual who was interacting with others, or (c) an object. In Experiment 1 (N = 50), we measured change detection for non-interacting individuals versus objects. In Experiment 2 (N = 49), we measured change detection for interacting individuals versus objects. Finally, in Experiment 3 (N = 85), we measured change detection for non-interacting versus interacting individuals. We also ran an inverted version of each task to determine whether differences were driven by low-level visual features. In Experiments 1 and 2, we found that changes to non-interacting and interacting individuals were detected better and more quickly than changes to objects. We also found inversion effects for both non-interaction and interaction changes, whereby they were detected more quickly when upright compared with inverted. No such inversion effect was seen for objects. This suggests that the high-level, social content of the images was driving the faster change detection for social versus object targets. Finally, we found that changes to individuals in non-interactions were detected faster than those presented within an interaction. Our results replicate the social advantage often found in change detection paradigms. However, we find that changes to individuals presented within social interaction configurations do not appear to be more quickly and easily detected than those in non-interacting configurations.

PIWI-Interacting RNA (piRNA) and Epigenetic Editing in Environmental Health Sciences.

PURPOSE OF REVIEW: The epigenome modulates gene expression in response to environmental stimuli. Modifications to the epigenome are potentially reversible, making them a promising therapeutic approach to mitigate environmental exposure effects on human health. This review details currently available genome and epigenome editing technologies and highlights ncRNA, including piRNA, as potential tools for targeted epigenome editing. RECENT FINDINGS: Zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease (CRISPR/Cas) research has significantly advanced genome editing technology, with broad promise in genetic research and targeted therapies. Initial epigenome-directed therapies relied on global modification and suffered from limited specificity. Adapted from current genome editing tools, zinc finger protein (ZFP), TALE, and CRISPR/nuclease-deactivated Cas (dCas) systems now confer locus-specific epigenome editing, with promising applicability in the field of environmental health sciences. However, high incidence of off-target effects and time taken for screening limit their use. FUTURE DEVELOPMENT: ncRNA serve as a versatile biomarker with well-characterized regulatory mechanisms that can easily be adapted to edit the epigenome. For instance, the transposon silencing mechanism of germline PIWI-interacting RNAs (piRNA) could be engineered to specifically methylate a given gene, overcoming pitfalls of current global modifiers. Future developments in epigenome editing technologies will inform risk assessment through mechanistic investigation and serve as potential modes of intervention to mitigate environmentally induced adverse health outcomes later in life.

Toxicoepigenetics and Environmental Health: Challenges and Opportunities.

The rapidly growing field of toxicoepigenetics seeks to understand how toxicant exposures interact with the epigenome to influence disease risk. Toxicoepigenetics is a promising field of environmental health research, as integrating epigenetics into the field of toxicology will enable a more thorough evaluation of toxicant-induced disease mechanisms as well as the elucidation of the role of the epigenome as a biomarker of exposure and disease and possible mediator of exposure effects. Likewise, toxicoepigenetics will enhance our knowledge of how environmental exposures, lifestyle factors, and diet interact to influence health. Ultimately, an understanding of how the environment impacts the epigenome to cause disease may inform risk assessment, permit noninvasive biomonitoring, and provide potential opportunities for therapeutic intervention. However, the translation of research from this exciting field into benefits for human and animal health presents several challenges and opportunities. Here, we describe four significant areas in which we see opportunity to transform the field and improve human health by reducing the disease burden caused by environmental exposures. These include (1) research into the mechanistic role for epigenetic change in environment-induced disease, (2) understanding key factors influencing vulnerability to the adverse effects of environmental exposures, (3) identifying appropriate biomarkers of environmental exposures and their associated diseases, and (4) determining whether the adverse effects of environment on the epigenome and human health are reversible through pharmacologic, dietary, or behavioral interventions. We then highlight several initiatives currently underway to address these challenges.

From little things, big things grow: An exploratory analysis of the national cost of peripheral intravenous catheter insertion in Australian adult emergency care.

OBJECTIVE: To estimate the total economic impact of peripheral intravenous catheter (PIVC) or cannula insertion and use in adult Australian EDs, including those cannulas that remain unused for therapeutic purposes. METHODS: Searches on Australian government websites were conducted to find rates of insertion, complications and cost of cannula; following this, gaps in national data sets were filled with MEDLINE and PubMed searches to estimate the total cost of cannula use in Australian EDs. Once the data were collected, totals were combined to establish an estimated cost for the listed categories. RESULTS: The estimated cost of cannulation in Australia may be up to A$594 million per year, including the cost of insertion (equipment and staff), cost of complications such as Staphylococcus aureus bacteraemia and phlebitis, and patient-centred costs (lost patient productivity, infiltration, occlusion and dislodgement). Approximately A$305.9 million is attributed to unused cannulas and approximately 11 790 days of clinician time is spent annually inserting cannula that remains idle. CONCLUSION: The figures developed in the present study represent an important educational opportunity to encourage thoughtful consideration of all interventions, no matter how small. ED cannula insertion represents a large economic and health cost to Australia's health system, many of which remain unused. There are no national data sets that record complications associated with PIVCs and we highlight the urgent need for improved data.

A medium composition containing normal resting glucose that supports differentiation of primary human airway cells.

Primary cells isolated from the human respiratory tract are the state-of-the-art for in vitro airway epithelial cell research. Airway cell isolates require media that support expansion of cells in a basal state to maintain the capacity for differentiation as well as proper cellular function. By contrast, airway cell differentiation at an air-liquid interface (ALI) requires a distinct medium formulation that typically contains high levels of glucose. Here, we expanded and differentiated human basal cells isolated from the nasal and conducting airway to a mature mucociliary epithelial cell layer at ALI using a medium formulation containing normal resting glucose levels. Of note, bronchial epithelial cells expanded and differentiated in normal resting glucose medium showed insulin-stimulated glucose uptake which was inhibited by high glucose concentrations. Normal glucose containing ALI also enabled differentiation of nasal and tracheal cells that showed comparable electrophysiological profiles when assessed for cystic fibrosis transmembrane conductance regulator (CFTR) function and that remained responsive for up to 7 weeks in culture. These data demonstrate that normal glucose containing medium supports differentiation of primary nasal and lung epithelial cells at ALI, is well suited for metabolic studies, and avoids pitfalls associated with exposure to high glucose.

Genome-wide CRISPR/Cas9 deletion screen defines mitochondrial gene essentiality and identifies routes for tumour cell viability in hypoxia.

Mitochondria are typically essential for the viability of eukaryotic cells, and utilize oxygen and nutrients (e.g. glucose) to perform key metabolic functions that maintain energetic homeostasis and support proliferation. Here we provide a comprehensive functional annotation of mitochondrial genes that are essential for the viability of a large panel (625) of tumour cell lines. We perform genome-wide CRISPR/Cas9 deletion screening in normoxia-glucose, hypoxia-glucose and normoxia-galactose conditions, and identify both unique and overlapping genes whose loss influences tumour cell viability under these different metabolic conditions. We discover that loss of certain oxidative phosphorylation (OXPHOS) genes (e.g. SDHC) improves tumour cell growth in hypoxia-glucose, but reduces growth in normoxia, indicating a metabolic switch in OXPHOS gene function. Moreover, compared to normoxia-glucose, loss of genes involved in energy-consuming processes that are energetically demanding, such as translation and actin polymerization, improve cell viability under both hypoxia-glucose and normoxia-galactose. Collectively, our study defines mitochondrial gene essentiality in tumour cells, highlighting that essentiality is dependent on the metabolic environment, and identifies routes for regulating tumour cell viability in hypoxia.

MEMS Deformable Mirrors for Space-Based High-Contrast Imaging.

Micro-Electro-Mechanical Systems (MEMS) Deformable Mirrors (DMs) enable precise wavefront control for optical systems. This technology can be used to meet the extreme wavefront control requirements for high contrast imaging of exoplanets with coronagraph instruments. MEMS DM technology is being demonstrated and developed in preparation for future exoplanet high contrast imaging space telescopes, including the Wide Field Infrared Survey Telescope (WFIRST) mission which supported the development of a 2040 actuator MEMS DM. In this paper, we discuss ground testing results and several projects which demonstrate the operation of MEMS DMs in the space environment. The missions include the Planet Imaging Concept Testbed Using a Recoverable Experiment (PICTURE) sounding rocket (launched 2011), the Planet Imaging Coronagraphic Technology Using a Reconfigurable Experimental Base (PICTURE-B) sounding rocket (launched 2015), the Planetary Imaging Concept Testbed Using a Recoverable Experiment - Coronagraph (PICTURE-C) high altitude balloon (expected launch 2019), the High Contrast Imaging Balloon System (HiCIBaS) high altitude balloon (launched 2018), and the Deformable Mirror Demonstration Mission (DeMi) CubeSat mission (expected launch late 2019). We summarize results from the previously flown missions and objectives for the missions that are next on the pad. PICTURE had technical difficulties with the sounding rocket telemetry system. PICTURE-B demonstrated functionality at >100 km altitude after the payload experienced 12-g RMS (Vehicle Level 2) test and sounding rocket launch loads. The PICTURE-C balloon aims to demonstrate 10 - 7 contrast using a vector vortex coronagraph, image plane wavefront sensor, and a 952 actuator MEMS DM. The HiClBaS flight experienced a DM cabling issue, but the 37-segment hexagonal piston-tip-tilt DM is operational post-flight. The DeMi mission aims to demonstrate wavefront control to a precision of less than 100 nm RMS in space with a 140 actuator MEMS DM.

Angiogenic and Arthritogenic Properties of the Soluble Form of CD13.

Aminopeptidase N/CD13 is expressed by fibroblast-like synoviocytes (FLS) and monocytes (MNs) in inflamed human synovial tissue (ST). This study examined the role of soluble CD13 (sCD13) in angiogenesis, MN migration, phosphorylation of signaling molecules, and induction of arthritis. The contribution of sCD13 was examined in angiogenesis and MN migration using sCD13 and CD13-depleted rheumatoid arthritis (RA) synovial fluids (SFs). An enzymatically inactive mutant CD13 and intact wild-type (WT) CD13 were used to determine whether its enzymatic activity contributes to the arthritis-related functions. CD13-induced phosphorylation of signaling molecules was determined by Western blotting. The effect of sCD13 on cytokine secretion from RA ST and RA FLS was evaluated. sCD13 was injected into C57BL/6 mouse knees to assess its arthritogenicity. sCD13 induced angiogenesis and was a potent chemoattractant for MNs and U937 cells. Inhibitors of Erk1/2, Src, NF-κB, Jnk, and pertussis toxin, a G protein-coupled receptor inhibitor, decreased sCD13-stimulated chemotaxis. CD13-depleted RA SF induced significantly less MN migration than sham-depleted SF, and addition of mutant or WT CD13 to CD13-depleted RA SF equally restored MN migration. sCD13 and recombinant WT or mutant CD13 had similar effects on signaling molecule phosphorylation, indicating that the enzymatic activity of CD13 had no role in these functions. CD13 increased the expression of proinflammatory cytokines by RA FLS, and a CD13 neutralizing Ab inhibited cytokine secretion from RA ST organ culture. Mouse knee joints injected with CD13 exhibited increased circumference and proinflammatory mediator expression. These data support the concept that sCD13 plays a pivotal role in RA and acute inflammatory arthritis.

Nursing care at end of life: a UK-based survey of the deaths of people living in care settings for people with intellectual disability.

BACKGROUND: People with intellectual disability are believed to be at risk of receiving poor end-of-life care. Nurses, given their advocacy role and duty to provide compassionate end-of-life care, have the potential to change this situation but research regarding this aspect of their role is limited. AIMS: This paper thus seeks to answer the question 'How and when are nurses involved in providing care at end of life for people with intellectual disability?' METHODS: A total of 38 intellectual disability care providers in the UK providing support to 13,568 people with intellectual disability were surveyed. Data regarding 247 deaths within this population were gathered in two stages and subsequently entered into SPSSX for analysis. RESULTS: Findings revealed that the majority of deaths occurred between the ages of 50 and 69 years, the most commonly reported cause of death being respiratory problems. Both community and hospital-based nurses were involved in supporting individuals during their final 3 months of life, and sometimes more than one type of nurse provided support to individuals. Generally nursing care was rated positively, although room for improvement was also identified. CONCLUSIONS: Nurses are involved in supporting people with intellectual disability at end of life and appropriate education is required to undertake this role. This may require change in curricula and subsequent research to determine the impact of such change on nursing support to this population.

Violence-Related Disparities Experienced by Black Youth and Young Adults: Opportunities for Prevention.

INTRODUCTION: The purpose of this study is to characterize violence-related disparities experienced by young blacks in the U.S. Reducing violence experienced by blacks, particularly youth, who are at substantially higher risk, is essential to improving the health of blacks in the U.S. METHODS: Data from four independent data sets for youth and adults were analyzed to examine rates of homicide, assault, injury from a physical fight, bullying victimization, and missing school because of safety concerns for non-Hispanic blacks and whites aged 10-34 years between 2010 and 2015. Disparities in adverse childhood experiences (e.g., exposure to violence and household challenges) and physical/mental health outcomes in adulthood were examined. Data were analyzed in 2017. RESULTS: Black adolescents and young adults are at higher risk for the most physically harmful forms of violence (e.g., homicides, fights with injuries, aggravated assaults) compared with whites. In addition, black adults reported exposure to a higher number of adverse childhood experiences than whites. These adverse childhood experiences were positively associated with increased odds of self-reported coronary heart disease, fair or poor physical health, experiencing frequent mental distress, heavy drinking, and current smoking. CONCLUSIONS: Disproportionate exposure to violence for blacks may contribute to disparities in physical injury and long-term mental and physical health. Understanding the violence experiences of this age group and the social contexts surrounding these experiences can help improve health for blacks in the U.S. Communities can benefit from the existing evidence about policies and programs that effectively reduce violence and its health and social consequences.

Hypoglycemia in a Patient With a Polyhormonal Pancreatic Neuroendocrine Tumor With Evidence of Endocrine Progenitors.

A 55-year-old woman with a large polyhormonal neuroendocrine tumor with unusual pathology is described. The patient presented with intermittent neuroglycopenic symptoms between more protracted asymptomatic periods occurring over the preceding 4 years. During a diagnostic 72-hour inpatient fast, she exhibited hypoglycemia at 70 hours after initiation. On computed tomography scan, a 6-cm mass was identified at the pancreatic head. The patient underwent a pylorus-preserving pancreaticoduodenectomy, and pathology was positive for cells staining for pancreatic polypeptide, insulin, and occasional double hormone (insulin plus pancreatic polypeptide)-positive cells. In addition, the tumor exhibited broad staining for ALDH1A3, a new marker of endocrine progenitors. This case serves to highlight the clinical and pathologic variability of insulin-producing tumors and raises the potential for cells in these tumors to exhibit hormone interconversion and progenitor-like states.

Localization, Shedding, Regulation and Function of Aminopeptidase N/CD13 on Fibroblast like Synoviocytes.

Aminopeptidase N/CD13 is highly expressed by fibroblast like synoviocytes (FLS) and may play a role in rheumatoid arthritis (RA). CD13 was previously detected in human synovial fluid where it was significantly increased in RA compared to osteoarthritis. In this study we found that CD13 in biological fluids (plasma, synovial fluid, FLS culture supernatant) is present as both a soluble molecule and on extracellular vesicles, including exosomes, as assessed by differential ultracentrifugation and density gradient separation. Having determined CD13 could be released as a soluble molecule from FLS, we examined potential mechanisms by which CD13 might be shed from the FLS membrane. The use of protease inhibitors revealed that CD13 is cleaved from the FLS surface by metalloproteinases. siRNA treatment of FLS revealed one of those proteases to be MMP14. We determined that pro-inflammatory cytokines (TNFα, IFNγ, IL-17) upregulated CD13 mRNA in FLS, which may contribute to the increased CD13 in RA synovium and synovial fluid. Inhibition of CD13 function by either inhibitors of enzymatic activity or anti-CD13 antibodies resulted in decreased growth and diminished migration of FLS. This suggests that CD13 may be involved in the pathogenic hyperplasia of RA FLS. This data expands potential roles for CD13 in the pathogenesis of RA.

Joint-specific DNA methylation and transcriptome signatures in rheumatoid arthritis identify distinct pathogenic processes.

Stratifying patients on the basis of molecular signatures could facilitate development of therapeutics that target pathways specific to a particular disease or tissue location. Previous studies suggest that pathogenesis of rheumatoid arthritis (RA) is similar in all affected joints. Here we show that distinct DNA methylation and transcriptome signatures not only discriminate RA fibroblast-like synoviocytes (FLS) from osteoarthritis FLS, but also distinguish RA FLS isolated from knees and hips. Using genome-wide methods, we show differences between RA knee and hip FLS in the methylation of genes encoding biological pathways, such as IL-6 signalling via JAK-STAT pathway. Furthermore, differentially expressed genes are identified between knee and hip FLS using RNA-sequencing. Double-evidenced genes that are both differentially methylated and expressed include multiple HOX genes. Joint-specific DNA signatures suggest that RA disease mechanisms might vary from joint to joint, thus potentially explaining some of the diversity of drug responses in RA patients.

Inflammatory properties of inhibitor of DNA binding 1 secreted by synovial fibroblasts in rheumatoid arthritis.

BACKGROUND: Inhibitor of DNA binding 1 (Id1) is a nuclear protein containing a basic helix-loop-helix (bHLH) domain that regulates cell growth by selective binding and prevention of gene transcription. Sources of Id1 production in rheumatoid arthritis synovial tissue (RA ST) and its range of functional effects in RA remain to be clarified. METHODS: We analyzed Id1 produced from synovial fibroblasts and endothelial cells (ECs) with histology and real-time polymerase chain reaction (RT-PCR). Fibroblast supernatants subjected to differential centrifugation to isolate and purify exosomes were measured for Id1 by enzyme-linked immunosorbent assay (ELISA). Western blotting of Id1-stimulated ECs was performed to determine the kinetics of intracellular protein phosphorylation. EC intracellular signaling pathways induced by Id1 were subsequently targeted with silencing RNA (siRNA) for angiogenesis inhibition. RESULTS: By PCR and histologic analysis, we found that the primary source of Id1 in STs is from activated fibroblasts that correlate with inflammatory scores in human RA ST and in joints from K/BxN serum-induced mice. Normal (NL) and RA synovial fibroblasts increase Id1 production with stimulation by transforming growth factor beta (TGF-ß). Most of the Id1 released by RA synovial fibroblasts is contained within exosomes. Endothelial progenitor cells (EPCs) and human dermal microvascular ECs (HMVECs) activate the Jnk signaling pathway in response to Id1, and Jnk siRNA reverses Id1-induced HMVEC vessel formation in Matrigel plugs in vivo. CONCLUSIONS: Id1 is a pleotropic molecule affecting angiogenesis, vasculogenesis, and fibrosis. Our data shows that Id1 is not only an important nuclear protein, but also can be released from fibroblasts via exosomes. The ability of extracellular Id1 to activate signaling pathways expands the role of Id1 in the orchestration of tissue inflammation.