Using primary organotypic mouse midbrain cultures to examine developmental neurotoxicity of silver nanoparticles across two genetic strains.

Micromass culture systems have been developed as three-dimensional organotypic in vitro alternatives to test developmental toxicity. We have optimized a murine-based embryonic midbrain micromass system in two genetic strains to evaluate neurodevelopmental effects of gold-cored silver nanoparticles (AgNPs) of differing sizes and coatings-20 nm AgCitrate, 110 nm AgCitrate, and 110 nm AgPVP. AgNPs are increasingly used in consumer, commercial, and medical products for their antimicrobial properties and observations of Ag in adult and fetal brain following in vivo exposures to AgNPs have led to concerns about the potential for AgNPs to elicit adverse effects on neurodevelopment and neurological function. Cytotoxicity was assessed at three time points of development by both nominal dose and by dosimetric dose. Ag dosimetry was assessed in cultures and the gold core component of the AgNPs was used as a tracer for determination of uptake of intact AgNPs and silver dissolution from particles in the culture system. Results by both nominal and dosimetric dose show cell death increased significantly in a dose-dependent manner at later time points (days 15 and 22 in vitro) that coincide with differentiation stages of development in both strains. When assessed by dosimetric dose, cultures were more sensitive to smaller particles, despite less uptake of Ag in smaller particles in both strains.

Clinical Monitoring of Well-Appearing Infants Born to Mothers With Chorioamnionitis.

BACKGROUND: The risk of early-onset sepsis is low in well-appearing late-preterm and term infants even in the setting of chorioamnionitis. The empirical antibiotic strategies for chorioamnionitis-exposed infants that are recommended by national guidelines result in antibiotic exposure for numerous well-appearing, uninfected infants. We aimed to reduce unnecessary antibiotic use in chorioamnionitis-exposed infants through the implementation of a treatment approach that focused on clinical presentation to determine the need for antibiotics. METHODS: Within a quality-improvement framework, a new treatment approach was implemented in March 2015. Well-appearing late-preterm and term infants who were exposed to chorioamnionitis were clinically monitored for at least 24 hours in a level II nursery; those who remained well appearing received no laboratory testing or antibiotics and were transferred to the level I nursery or discharged from the hospital. Newborns who became symptomatic were further evaluated and/or treated with antibiotics. Antibiotic use, laboratory testing, culture results, and clinical outcomes were collected. RESULTS: Among 277 well-appearing, chorioamnionitis-exposed infants, 32 (11.6%) received antibiotics during the first 15 months of the quality-improvement initiative. No cases of culture result-positive early-onset sepsis occurred. No infant required intubation or inotropic support. Only 48 of 277 (17%) patients had sepsis laboratory testing. The implementation of the new approach was associated with a 55% reduction (95% confidence interval 40%-65%) in antibiotic exposure across all infants ≥34 weeks' gestation born at our hospital. CONCLUSIONS: A management approach using clinical presentation to determine the need for antibiotics in chorioamnionitis-exposed infants was successful in reducing antibiotic exposure and was not associated with any clinically relevant delays in care or adverse outcomes.

In vitro to in vivo benchmark dose comparisons to inform risk assessment of quantum dot nanomaterials.

Engineered nanomaterials are currently under review for their potential toxicity; however, their use in consumer/commercial products has continued to outpace risk assessments. In vitro methods may be utilized as tools to improve the efficiency of risk assessment approaches. We propose a framework to compare relationships between previously published in vitro and in vivo toxicity assessments of cadmium-selenium containing quantum dots (QDs) using benchmark dose (BMD) and dosimetric assessment methods. Although data were limited this approach was useful for identifying sensitive assays and strains. In vitro studies assessed effects of QDs in three pulmonary cell types across two mouse strains. Significant dose-response effects were modeled and a standardized method of BMD analysis was performed as a function of both exposure dose and dosimetric dose. In vivo studies assessed pulmonary effects of QD exposure across eight mouse strains. BMD analysis served as a basis for relative comparison with in vitro studies. We found consistent responses in common endpoints between in vitro and in vivo studies. Strain sensitivity was consistent between in vitro and in vivo studies, showing A/J mice more sensitive to QDs. Cell types were found to differentially take up QDs. Dosimetric adjustments identified similar sensitivity among cell types. Thus, BMD analysis can be used as an effective tool to compare the sensitivity of different strains, cell types, and assays to QDs. These methods allow for in vitro assays to be used to predict in vivo responses, improve the efficiency of in vivo studies, and allow for prioritization of nanomaterial assessments. This article is categorized under: Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine.

Characterization of 3D embryonic C57BL/6 and A/J mouse midbrain micromass in vitro culture systems for developmental neurotoxicity testing.

In vitro micromass culture systems have been proposed as an alternative method for developmental toxicity assessment to reduce the need for resource-intensive in vivo toxicity testing. In this study, a three-dimensional in vitro embryonic mouse midbrain culture system is characterized in two mouse strains to facilitate gene x environment considerations. Gestational day (GD) 11 C57BL/6 or GD 12 A/J mouse midbrain cells were isolated and cultured in high-density micromass format for 22days in vitro (DIV). Hematoxylin intensity and protein content revealed that neuronal differentiation increases linearly over time in both C57BL/6 and A/J cultures. Protein expression showed time-dependent proliferation markers (PCNA) increased significantly between DIV 4-6 compared to DIV 1. Early and late differentiation markers (e.g. ß-tubulin III and NMDAɛ1) were expressed between DIV 6-8 and DIV 8-15, respectively. Immunohistochemistry and protein expression results for proliferation and differentiation markers were concordant. Protein expression patterns for the two mouse strain micromass systems were similar. This study characterizes a novel method for investigating early neurogenesis and may be used to characterize neurodevelopmental toxicity in vitro. Our findings show how the use of different mouse strains in neurodevelopmental studies may extend test systems for gene and environment interaction studies.

Occupational exposure limit for silver nanoparticles: considerations on the derivation of a general health-based value.

With the increased production and widespread commercial use of silver nanoparticles (AgNPs), human and environmental exposures to silver nanoparticles are inevitably increasing. In particular, persons manufacturing and handling silver nanoparticles and silver nanoparticle containing products are at risk of exposure, potentially resulting in health hazards. While silver dusts, consisting of micro-sized particles and soluble compounds have established occupational exposure limits (OELs), silver nanoparticles exhibit different physicochemical properties from bulk materials. Therefore, we assessed silver nanoparticle exposure and related health hazards in order to determine whether an additional OEL may be needed. Dosimetric evaluations in our study identified the liver as the most sensitive target organ following inhalation exposure, and as such serves as the critical target organ for setting an occupational exposure standard for airborne silver nanoparticles. This study proposes an OEL of 0.19 µg/m(3) for silver nanoparticles derived from benchmark concentrations (BMCs) from subchronic rat inhalation toxicity assessments and the human equivalent concentration (HEC) with kinetic considerations and additional uncertainty factors. It is anticipated that this level will protect workers from potential health hazards, including lung, liver, and skin damage.

Urinary microRNAs as potential biomarkers of pesticide exposure.

MicroRNAs (miRNAs) are post-transcriptional regulators that silence messenger RNAs. Because miRNAs are stable at room temperature and long-lived, they have been proposed as molecular biomarkers to monitor disease and exposure status. While urinary miRNAs have been used clinically as potential diagnostic markers for kidney and bladder cancers and other diseases, their utility in non-clinical settings has yet to be fully developed. Our goal was to investigate the potential for urinary miRNAs to act as biomarkers of pesticide exposure and early biological response by identifying the miRNAs present in urine from 27 parent/child, farmworker/non-farmworker pairs (16FW/11NFW) collected during two agricultural seasons (thinning and post-harvest) and characterizing the between- and within-individual variability of these miRNA epigenetic regulators. MiRNAs were isolated from archived urine samples and identified using PCR arrays. Comparisons were made between age, households, season, and occupation. Of 384 miRNAs investigated, 297 (77%) were detectable in at least one sample. Seven miRNAs were detected in at least 50% of the samples, and one miRNA was present in 96% of the samples. Principal components and hierarchical clustering analyses indicate significant differences in miRNA profiles between farmworker and non-farmworker adults as well as between seasons. Six miRNAs were observed to be positively associated with farmworkers status during the post-harvest season. Expression of five of these miRNA trended towards a positive dose response relationship with organophosphate pesticide metabolites in farmworkers. These results suggest that miRNAs may be novel biomarkers of pesticide exposure and early biological response.

Sewer Gas: An Indoor Air Source of PCE to Consider During Vapor Intrusion Investigations.

The United States Environmental Protection Agency (USEPA) is finalizing its vapor intrusion guidelines. One of the important issues related to vapor intrusion is background concentrations of volatile organic chemicals (VOCs) in indoor air, typically attributed to consumer products and building materials. Background concentrations can exist even in the absence of vapor intrusion and are an important consideration when conducting site assessments. In addition, the development of accurate conceptual models that depict pathways for vapor entry into buildings is important during vapor intrusion site assessments. Sewer gas, either as a contributor to background concentrations or as part of the site conceptual model, is not routinely evaluated during vapor intrusion site assessments. The research described herein identifies an instance where vapors emanating directly from a sanitary sewer pipe within a residence were determined to be a source of tetrachloroethylene (PCE) detected in indoor air. Concentrations of PCE in the bathroom range from 2.1 to 190 ug/m3 and exceed typical indoor air concentrations by orders of magnitude resulting in human health risk classified as an "Imminent Hazard" condition. The results suggest that infiltration of sewer gas resulted in PCE concentrations in indoor air that were nearly two-orders of magnitude higher as compared to when infiltration of sewer gas was not known to be occurring. This previously understudied pathway whereby sewers serve as sources of PCE (and potentially other VOC) vapors is highlighted. Implications for vapor intrusion investigations are also discussed.

Modulation of mTOR effector phosphoproteins in blood basophils from allergic patients.

The mammalian target of rapamycin (mTOR) pathway contributes to various immunoinflammatory processes. Yet, its potential involvement in basophil responses in allergy remains unclear. In this pilot study, we quantified two key mTOR effector phosphoproteins, the eukaryotic initiation factor 4E (peIF4E) and S6 ribosomal protein (pS6rp), in blood basophils from nut allergy patients (NA, N = 16) and healthy controls (HC, N = 13). Without stimulation in vitro, basophil peIF4E levels were higher in NA than HC subjects (P = 0.014). Stimulation with nut (offending) but not chicken / rice (non-offending) extract increased basophil peIF4E and pS6rp levels (+32%, P = 0.018, and +98%, P = 0.0026, respectively) in NA but not HC subjects, concomitant with increased surface levels of CD203c and CD63, both known to reflect basophil activation. Pre-treatment with the mTOR inhibitor rapamycin decreased pS6rp and CD203c responses in nut extract-stimulated basophils in NA subjects. Thus, basophil responses to offending allergens are associated with modulation of mTOR effector phosphoproteins.

Use of Specific IgE and Skin Prick Test to Determine Clinical Reaction Severity.

AIMS: To determine whether specific IgE and skin prick test correlate better in predicting reaction severity during a double-blinded placebo controlled food challenge (DBPCFC) for egg, milk, and multiple tree nut allergens. STUDY DESIGN: Prospective study. PLACE AND DURATION OF STUDY: Department of Pediatrics, Stanford University School of Medicine, August 2009 and ongoing. METHODOLOGY: We examined the reaction severity of twenty-four subjects to nine possible food allergens: milk, egg, almond, cashew, hazelnut, peanut, sesame, pecan and walnut. Specific IgE and SPT were performed before each DBPCFC. DBPCFC results were classified into mild (1), moderate (2), or severe (3) reactions using a modified Bock's criteria. RESULTS: Twenty four subjects underwent a total of 80 DBPCFC. Eighty percent of all DBPCFCs resulted in a positive reaction. A majority, 71%, were classified as mild. No reactions occurred with a SPT of zero mm while three reactions occurred with a negative specific IgE. All reactions were reversible with medication. CONCLUSION: These data suggest that SPT and specific IgE levels are not associated with reaction severity (p<0.64 and 0.27, respectively). We also found that combining specific IgE and SPT improved specificity but did not help to achieve clinically useful sensitivity. For instance, an SPT > 5mm had a sensitivity of 91% and specificity of 50%. Combining SPT > 5mm and IgE > 7 resulted in a reduced sensitivity of 64%. Unexpectedly, a history of anaphylaxis 70% (n=17) was not predictive of anaphylaxis on challenge 4% (n=2).