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Vitellogenin (VTG), an egg yolk precursor, is abnormally produced by male and juvenile oviparous species after exposure to estrogens. Plasma VTG in loggerhead sea turtles (Caretta caretta) helped us understand their reproductive maturation and investigate it as a biomarker of contaminant exposure. The presence of VTG was screened in plasma from 404 loggerheads from the northwestern Atlantic Ocean using a freshwater turtle antibody in western blots. The concentrations of VTG were semiquantified using band intensities calibrated to results from a loggerhead antibody enzyme‐linked immunoassay. The detection and concentrations of VTG were in (from highest to lowest): nesting females, in‐water adult females, subadult females, smaller females, unknown sex, and males. Loggerheads from this region begin vitellogenesis at ≅77 cm straight carapace length. We classified VTG expression as abnormal in nine male or juvenile turtles. Organochlorine contaminant (OC) concentrations were measured in blood and/or fat biopsies of some turtles. One abnormal VTG female had the second highest fat polychlorinated biphenyl (PCB) and 4,4′‐dichlorodiphenyldichloroethylene concentrations compared among 43 VTG‐negative juveniles. The nine VTG‐abnormal turtles had average blood PCB concentrations 8.5% higher, but not significantly different, than 46 VTG‐negative juveniles (p = 0.453). In turtles less than 77 cm, blood PCB concentrations were significantly, but weakly, correlated with semiquantified VTG concentrations (tau = 0.1, p = 0.004). Greater blood OC concentrations were found in adult females than in males, which motivated the creation of a conceptual model of OC, VTG, and hormone concentrations across a reproductive cycle. A decision tree is also provided incorporating VTG as a sexing tool. Abnormal VTG expression cannot conclusively be linked to endocrine disruption caused by these OC concentrations. Studies should further investigate causes of abnormal VTG expression in wild sea turtles. Environ Toxicol Chem 2023;42:1309–1325. © 2023 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. [ABSTRACT FROM AUTHOR]

Objective We have previously shown, in a cohort of untreated rheumatoid arthritis (RA) patients, that the suppressive function of Treg cells is defective. However, other studies in cohorts of patients with established RA have shown that Treg cell function is normal. We hypothesized that treatment may restore Treg cell function and lead to reduced disease activity. The aim of this study was to investigate whether treatment with methotrexate (MTX) can result in epigenetic changes that lead to restoration of the Treg cell suppressive function in RA. Methods Peripheral blood samples from RA patients were assessed using 3H-thymidine incorporation to measure Treg cell suppression of T cell proliferation, and by enzyme-linked immunosorbent assay to determine Treg cell suppression of interferon-γ production. CTLA-4 and FoxP3 expression was measured by flow cytometry and quantitative polymerase chain reaction (qPCR) in Treg cells from healthy individuals and RA patients. CD4+ T cells isolated from healthy individuals were cultured with interleukin-2 (IL-2), IL-6, and tumor necrosis factor α in the presence or absence of MTX, and FoxP3 expression was determined using qPCR and flow cytometry. Methylation of the FOXP3 upstream enhancer was analyzed by bisulfite sequencing PCR. Results Defective Treg cell function was observed only in RA patients who had not been treated with MTX, whereas Treg cells from MTX-exposed RA patients had restored suppressive function. This restored suppression was associated with increased expression of FoxP3 and CTLA-4 in Treg cells. Bisulfite sequencing PCR of Treg cells cultured in MTX revealed a significant reduction in methylation of the FOXP3 upstream enhancer. Conclusion This study identifies a novel mechanism of action of MTX, in which treatment of RA patients with MTX restores defective Treg cell function through demethylation of the FOXP3 locus, leading to a subsequent increase in FoxP3 and CTLA-4 expression. [ABSTRACT FROM AUTHOR]

Treg-cell function is compromised in rheumatoid arthritis (RA). As the master regulator of Treg cells, FOXP3 controls development and suppressive function. Stable Treg-cell FOXP3 expression is epigenetically regulated; constitutive expression requires a demethylated Treg-specific demethylated region. Here, we hypothesised that methylation of the FOXP3 locus is altered in Treg cells of established RA patients. Methylation analysis of key regulatory regions in the FOXP3 locus was performed on Treg cells from RA patients and healthy controls. The FOXP3 Treg-specific demethylated region and proximal promoter displayed comparable methylation profiles in RA and healthy-donor Treg cells. We identified a novel differentially methylated region (DMR) upstream of the FOXP3 promoter, with enhancer activity sensitive to methylation-induced silencing. In RA Treg cells we observed significantly reduced DMR methylation and lower DNA methyltransferase (DNMT1/3A) expression compared with healthy Treg cells. Furthermore, DMR methylation negatively correlated with FOXP3 mRNA expression, and Treg cells isolated from rheumatoid factor negative RA patients were found to express significantly higher levels of FOXP3 than Treg cells from RhF-positive patients, with an associated decrease in DMR methylation. In conclusion, the novel DMR is involved in the regulation of Treg-cell FOXP3 expression, but this regulation is lost post-transcriptionally in RA Treg cells. [ABSTRACT FROM AUTHOR]

Objective Functionally impaired Treg cells expressing abnormally low levels of CTLA-4 have been well documented in rheumatoid arthritis (RA). However, the molecular defect underlying this reduced expression is unknown. The aims of this study were to assess the role of DNA methylation in regulating CTLA-4 expression in Treg cells isolated from RA patients and to elucidate the mechanism of their reduced suppressor function. Methods CTLA-4 expression in Treg cells from RA patients and healthy controls was measured by quantitative polymerase chain reaction (PCR) and flow cytometry. Methylation of the CTLA-4 gene promoter was analyzed by bisulfite-specific PCR, followed by sequencing. Methylation-dependent transcriptional activity of the CTLA-4 gene promoter was measured by luciferase assay, and NF-AT binding to the CTLA-4 gene promoter was determined by chromatin immunoprecipitation. The role of CTLA-4 expression in controlling Teff cells was analyzed using an autologous mixed lymphocyte reaction. Results Down-regulation of CTLA-4 expression in Treg cells from RA patients was caused by methylation of a previously unidentified NF-AT binding site within the CTLA-4 gene promoter. As a consequence, Treg cells were unable to induce expression and activation of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO), which in turn resulted in a failure to activate the immunomodulatory kynurenine pathway. Conclusion We show for the first time that epigenetic modifications contribute to defective Treg cell function in RA through an inability to activate the IDO pathway. Therefore, this study sets a precedent for investigating potential therapeutic strategies aimed at reinforcing the IDO pathway in RA patients. [ABSTRACT FROM AUTHOR]

Objective To determine whether selective blockade of tumor necrosis factor receptor I (TNFRI) affects spontaneous proinflammatory cytokine and chemokine production in ex vivo-cultured human rheumatoid arthritis synovial membrane mononuclear cells (MNCs) and to compare this response to that of TNF ligand blockade using etanercept. Methods A bispecific, single variable-domain antibody (anti-TNFRI moiety plus an albumin binding moiety [TNFRI-AlbudAb]) was used to selectively block TNFRI. Inhibition of TNFα-mediated responses in cell lines expressing TNFRI/II confirmed TNFRI-AlbudAb potency, human rhabdomyosarcoma cell line KYM-1D4 cytotoxicity, and human umbilical vein endothelial cell (HUVEC) vascular cell adhesion molecule 1 (VCAM-1) upregulation. Eighteen RA synovial membrane MNC suspensions were cultured for 2 days or 5 days, either alone or in the presence of TNFRI-AlbudAb, control-AlbudAb, or etanercept. Proinflammatory cytokines and chemokines in culture supernatants were measured by enzyme-linked immunosorbent assays. A mixed-effects statistical analysis model was used to assess the extent of TNFRI selective blockade, where the results were expressed as the percentage change with 95% confidence intervals (95% CIs). Results TNFRI-AlbudAb inhibited TNFα-induced KYM-1D4 cell cytotoxicity (50% inhibition concentration [IC50] 4 n M) and HUVEC VCAM-1 up-regulation (IC50 12 n M) in a dose-dependent manner. In ex vivo-cultured RA synovial membrane MNCs, selective blockade of TNFRI inhibited the production of proinflammatory cytokines and chemokines to levels similar to those obtained with TNF ligand blockade, without inducing cellular toxicity. Changes in cytokine levels were as follows: −23.5% (95% CI −12.4, −33.2 [ P = 0.004]) for granulocyte-macrophage colony-stimulating factor, -33.4% (95% CI -20.6, -44.2 [ P ≤ 0.0001]) for interleukin-10 (IL-10), -17.6% (95% CI 3.2, -34.2 [ P = 0.0880]) for IL-1β, and -19.0% (95% CI -3.4, -32.1 [ P = 0.0207]) for IL-6. Changes in chemokine levels were as follows: -34.2% (-14.4, -49.4 [ P = 0.0030]) for IL-8, -56.6% (-30.7, -72.9 [ P = 0.0011]) for RANTES, and -24.9% (2, -44.8 [ P = 0.0656]) for monocyte chemotactic protein 1. Conclusion In ex vivo-cultured RA synovial membrane MNCs, although a limited role of TNFRII cannot be ruled out, TNFRI signaling was found to be the dominant pathway leading to proinflammatory cytokine and chemokine production. Thus, selective blockade of TNFRI could potentially be therapeutically beneficial over TNF ligand blockade by retaining the beneficial TNFRII signaling. [ABSTRACT FROM AUTHOR]

Aqueous suspensions of fullerene aggregates (aqua-nC60) were used to investigate the movement of carbon-based nanomaterials in a marine water column and to determine their effects on different life stages of a marine teleost. Fullerene aggregates formed precipitates as a result of mixing in natural seawater, and levels of aqua-nC60 were significantly increased in bottom waters after 24 h. Exposure of Fundulus heteroclitus embryos, larvae, and adults to increasing concentrations of aqua-nC60 resulted in very little mortality, and no median lethal concentrations could be calculated at ⩽ 10 mg/L. Aggregates of aqua-nC60 did adhere to the chorion but did not affect development of the embryos or their hatching success. Movements of aqua-nC60 through the chorion and into the embryo tended to increase with higher exposure levels; however, the concentrations were extremely low and did not differ significantly. Larvae exposed to increasing concentrations of aqua-nC60 exhibited a significant dose-dependent increase in total glutathione (GSH). This was accompanied by a decreasing trend in lipid peroxidation (LPO), but LPO was not statistically different between treatments. Adult F. heteroclitus exposed to increasing concentrations of aqua-nC60 exhibited an increase in total GSH in liver tissue but not in the gill. No significant effects on LPO were observed in either tissue. Thus, we conclude that aqua-nC60 affects the oxidative stress response of F. heteroclitus and that increased antioxidant defenses provide some physiological tolerance for these materials. Environmental factors influencing uptake, metabolism, and physiological response following exposure, however, need further investigation. [ABSTRACT FROM AUTHOR]

Monitoring toxic organochlorine (OC) compounds is an important aspect in wildlife studies, especially in protected species such as sea turtles. The goal of this study was to determine whether blood OC concentrations can predict those in adipose tissue of sea turtles. Blood offers many benefits for monitoring OCs. It can be collected nondestructively from live turtles and can be sampled repeatedly for continuous monitoring. Organochlorine concentrations in blood may better represent the exposure levels of target tissues, but blood concentrations may fluctuate more than those in fatty tissues following recent dietary exposure or lipid mobilization. Paired fat and blood samples were collected from 44 live, juvenile loggerhead sea turtles and 10 juvenile Kemp's ridley sea turtle carcasses. Organochlorines were analyzed using gas chromatography with electron capture detection and mass spectrometry. Lipid-normalized OC concentrations measured in the blood significantly correlated to levels found in the fat samples of both species. This result suggests that sea turtle blood is a suitable alternative to fatty tissues for measuring OCs because blood concentrations reasonably represent those observed in the paired fat samples. However, blood OC concentrations calculated on a wet-mass basis were significantly and inversely correlated to lipid content in the fat samples. Therefore, caution should be used when monitoring spatial or temporal trends, as OC levels may increase in the blood following mobilization of fat stores, such as during long migrations, breeding, or disease events. [ABSTRACT FROM AUTHOR]

Temperature cycling between -4.5±1.0 °C and 13.0±2.0 °C in the presence of added water caused significant breakage of granitic detritus. Because cycling with only adsorbed water present also caused breakage, fragmentation is attributed to both the combined action of ice and adsorbed water and to the latter acting alone. Breakage evidently resulted from the gradual tensile opening of pre-existing cracks, weakening then splitting grains. Surviving unbroken grains show evidence of a fatigue effect. Quartz split dominantly along pre-existing subplanar microfractures whereas feldspar and biotite split along crystal cleavages. Degree of breakage and product size distribution depend on the crystalline nature of the parent material, its previous history, and the nature and duration of the breakage process. With the first two factors the same, size distributions from adsorbed water breakage alone and from that due to adsorbed water plus ice differ slightly. Both contrast strongly with those of simulated fluviatile breakage. Whereas the latter preferentially produced 2–20 μim particles (probably debris of inter-particle collisions), static breakage split coarser grains wherever major weaknesses occurred, producing less selective product size distributions with greater proportions of loess-sized material (about 20–60 μm). Characteristic inflections in the size distribution curves of our experimentally produced debris are also shown by samples from the sola of some frost-affected soils. Partially healed microfractures in plutonic quartz are normally spaced at about 1–10 μm—approaching the downward asymptotic comminution limit for brittle solids (about 1 μm). Surficial physical processes are capable of reducing only a small proportion of plutonic quartz to this size before its storage in sediments. [ABSTRACT FROM AUTHOR]

PURPOSE. To identify accurate descriptive terms for risk for violence and risk for suicide and to provide operational definitions for these terms METHODS. The Delphi technique, with two rounds, was used to differentiate the operational definitions that represent risk for suicide from those that represent risk for violence. The expert panel consisted of 23 healthcare professionals with a minimum of a master's degree. In addition to the expert panel, a control group (N = 11) participated to assess content validity. FINDINGS. Thirty-six definitions were agreed upon for suicide, 39 for violence. CONCLUSIONS. These definitions represent the basic distinctions between the two behavioral manifestations. [ABSTRACT FROM AUTHOR]

A high incidence of violence occurs in contemporary society, which is reflected in the violence exhibited by psychiatric patients. To validate the defining characteristics of potential for violence, 125 registered nurses were asked to rate the relevance of defining characteristics of the diagnosis. A diagnostic content validity index was used to ascertain which defining characteristics the nurses believed to be the most critical in the identification of violence potential. Nine major and 20 minor characteristics were identified by these nurses. [ABSTRACT FROM AUTHOR]

Objective: To investigate the mechanism that leads to the spontaneous production of tumor necrosis factor alpha (TNFalpha) in rheumatoid arthritis (RA) synovial tissue., Methods: Normal blood monocytes were cocultured either with fixed activated T cells generated from normal blood or RA synovial T cells purified from synovium. TNFalpha production was measured in supernatants from these cocultures following blockade of the transcription factor nuclear factor kappaB (NF-kappaB) using adenoviral transfer of the inhibitor of NF-kappaB kinase alpha into the responding monocytes, or blockade of phosphatidylinositol 3-kinase (PI 3-kinase) using the inhibitory drugs wortmannin or LY294002. TNFalpha production was measured by enzyme-linked immunosorbent assay., Results: TNFalpha production in synovial tissue from patients with RA but not osteoarthritis was found to be T cell dependent. The RA synovial joint T cells resembled normal T cells that had been activated for 8 days using a cocktail of cytokines. These T cells, designated Tck (cytokine-activated T cells), and RA synovial T cells both induced TNFalpha production in resting monocytes in a cell-contact-dependent manner, which was abrogated by blockage of the transcription factor NF-kappaB but augmented if PI 3-kinase was inhibited. Normal blood T cells activated conventionally via the T cell receptor with crosslinked anti-CD3 antibody resulted in TNFalpha production from monocytes; this was unaffected by NF-kappaB blockade, but was inhibited in the presence of PI 3-kinase-blocking drugs., Conclusion: These data provide strong evidence for the importance of T cells in inducing TNFalpha in chronic inflammatory rheumatoid tissue, and give insight into the mechanism whereby these T cells are activated in vivo. Furthermore, they indicate that production of TNFalpha in pathologic tissue is regulated differently from physiologic antigen-dependent TNFalpha production, which raises the possibility that selective inhibitors of TNFalpha in disease may be developed.