Acellular oxidative potential assay for screening of amorphous silica nanoparticles.

Silica nanoparticles (SiNPs) are used in a wide range of consumer products, engineering and medical applications, with likelihood of human exposure and potential health concerns. It is essential to generate toxicity information on SiNP forms and associated physicochemical determinants to conduct risk assessment on these new materials. To address this knowledge gap, we screened a panel of custom synthesized, well-characterized amorphous SiNPs pristine and surface-modified (-C3-COOH, -C11-COOH, -NH2, -PEG) of 5 different sizes: (15, 30, 50, 75, 100 nm) for their oxidative potential using an acellular assay. The assay is based on oxidation of dithiothreitol (DTT) by reactive oxygen species and can serve as a surrogate test for oxidative stress. These materials were characterized for size distribution, aggregation, crystallinity, surface area, surface modification, surface charge and metal content. Tests for association between oxidative potential of SiNPs and their physicochemical properties were carried out using analysis of variance and correlation analyses. These test results suggest that the size of amorphous SiNPs influenced their oxidative potential irrespective of the surface modification, with 15 nm exhibiting relatively higher oxidative potential compared to the other sizes. Furthermore, SiNP surface area, surface modification and agglomeration in solution also appeared to affect oxidative potential of these SiNPs. These findings indicate that physicochemical properties are critical in influencing the oxidative behaviour of amorphous SiNPs, with potential to trigger cellular oxidative stress and thus toxicity, when exposed. This information advances our understanding of potential toxicities of these amorphous SiNPs and supports risk assessment efforts and the design of safer forms of silica nanomaterials.

Quantification of surface functional groups on silica nanoparticles: comparison of thermogravimetric analysis and quantitative NMR.

Thermogravimetric analysis (TGA) coupled with evolved gas analysis-FT-IR has been examined as a potential method to study the functional group content for surface modified silica nanoparticles. A comparison with a quantitative solution NMR method based on analysis of groups released after dissolution of the silica matrix is used to provide benchmark data for comparison and to assess the utility and limitations of TGA. This study focused primarily on commercially available silicas and tested whether it was possible to use a correction based on bare silica to account for the significant mass loss that occurs due to condensation of surface hydroxyl groups and loss of matrix-entrapped components at temperatures above ∼200 °C. Although this approach has been used successfully in the literature for in-house prepared samples, it was problematic for commercial silicas prepared by the Stöber method. For these materials the agreement between estimates from qNMR and TGA mass loss was poor in many cases. However much better agreement was observed for samples for which the mass loss above 200 °C is relatively low, such as non-porous silica, or samples for which the mass fraction of functional group is large (e.g., high molecule weight groups or multilayers). FT-IR was useful in identifying the likely structure of the components lost from the surface at various temperatures and in some cases provided evidence of contaminants in the sample. Nevertheless, in other cases correlation of thermograms and FT-IR with NMR data was necessary, particularly for samples where multi-step modification of the silica surface results in incomplete functionalization that gives a mixture of products. Overall the results indicate that TGA provides reliable results for silicas of low porosity or those for which the functional group accounts for a significant fraction of the total sample mass. It is also suitable as a supplementary or screening technique to indicate the presence of coatings or covalent surface modification, prior to applying other techniques or for routine analyses where sensitivity is not critical.

Therapeutic potential of Pak1 inhibition for pain associated with cutaneous burn injury.

Painful burn injuries are among the most debilitating form of trauma, globally ranking in the top 15 leading causes of chronic disease burden. Despite its prevalence, however, chronic pain after burn injury is under-studied. We previously demonstrated the contribution of the Rac1-signaling pathway in several models of neuropathic pain, including burn injury. However, Rac1 belongs to a class of GTPases with low therapeutic utility due to their complex intracellular dynamics. To further understand the mechanistic underpinnings of burn-induced neuropathic pain, we performed a longitudinal study to address the hypothesis that inhibition of the downstream effector of Rac1, Pak1, will improve pain outcome following a second-degree burn injury. Substantial evidence has identified Pak1 as promising a clinical target in cognitive dysfunction and is required for dendritic spine dysgenesis associated with many neurological diseases. In our burn injury model, mice exhibited significant tactile allodynia and heat hyperalgesia and dendritic spine dysgenesis in the dorsal horn. Activity-dependent expression of c-fos also increased in dorsal horn neurons, an indicator of elevated central nociceptive activity. To inhibit Pak1, we repurposed an FDA-approved inhibitor, romidepsin. Treatment with romidepsin decreased dendritic spine dysgenesis, reduced c-fos expression, and rescued pain thresholds. Drug discontinuation resulted in a relapse of cellular correlates of pain and in lower pain thresholds in behavioral tests. Taken together, our findings identify Pak1 signaling as a potential molecular target for therapeutic intervention in traumatic burn-induced neuropathic pain.

Dendritic spine remodeling following early and late Rac1 inhibition after spinal cord injury: evidence for a pain biomarker.

Neuropathic pain is a significant complication following spinal cord injury (SCI) with few effective treatments. Drug development for neuropathic pain often fails because preclinical studies do not always translate well to clinical conditions. Identification of biological characteristics predictive of disease state or drug responsiveness could facilitate more effective clinical translation. Emerging evidence indicates a strong correlation between dendritic spine dysgenesis and neuropathic pain. Because dendritic spines are located on dorsal horn neurons within the spinal cord nociceptive system, dendritic spine remodeling provides a unique opportunity to understand sensory dysfunction after SCI. In this study, we provide support for the postulate that dendritic spine profiles can serve as biomarkers for neuropathic pain. We show that dendritic spine profiles after SCI change to a dysgenic state that is characteristic of neuropathic pain in a Rac1-dependent manner. Suppression of the dysgenic state through inhibition of Rac1 activity is accompanied by attenuation of neuropathic pain. Both dendritic spine dysgenesis and neuropathic pain return when inhibition of Rac1 activity is lifted. These findings suggest the utility of dendritic spines as structural biomarkers for neuropathic pain.

Advancing Risk Analysis for Nanoscale Materials: Report from an International Workshop on the Role of Alternative Testing Strategies for Advancement.

The Society for Risk Analysis (SRA) has a history of bringing thought leadership to topics of emerging risk. In September 2014, the SRA Emerging Nanoscale Materials Specialty Group convened an international workshop to examine the use of alternative testing strategies (ATS) for manufactured nanomaterials (NM) from a risk analysis perspective. Experts in NM environmental health and safety, human health, ecotoxicology, regulatory compliance, risk analysis, and ATS evaluated and discussed the state of the science for in vitro and other alternatives to traditional toxicology testing for NM. Based on this review, experts recommended immediate and near-term actions that would advance ATS use in NM risk assessment. Three focal areas-human health, ecological health, and exposure considerations-shaped deliberations about information needs, priorities, and the next steps required to increase confidence in and use of ATS in NM risk assessment. The deliberations revealed that ATS are now being used for screening, and that, in the near term, ATS could be developed for use in read-across or categorization decision making within certain regulatory frameworks. Participants recognized that leadership is required from within the scientific community to address basic challenges, including standardizing materials, protocols, techniques and reporting, and designing experiments relevant to real-world conditions, as well as coordination and sharing of large-scale collaborations and data. Experts agreed that it will be critical to include experimental parameters that can support the development of adverse outcome pathways. Numerous other insightful ideas for investment in ATS emerged throughout the discussions and are further highlighted in this article.

ProxTom lymphatic vessel reporter mice reveal Prox1 expression in the adrenal medulla, megakaryocytes, and platelets.

Lymphatic vessels (LVs) are important structures for antigen presentation, for lipid metabolism, and as conduits for tumor metastases, but they have been difficult to visualize in vivo. Prox1 is a transcription factor that is necessary for lymphangiogenesis in ontogeny and the maintenance of LVs. To visualize LVs in the lymph node of a living mouse in real time, we made the ProxTom transgenic mouse in a C57BL/6 background using red fluorescent LVs that are suitable for in vivo imaging. The ProxTom transgene contained all Prox1 regulatory sequences and was faithfully expressed in LVs coincident with endogenous Prox1 expression. The progenies of a ProxTom × Hec6stGFP cross were imaged using two-photon laser scanning microscopy, allowing the simultaneous visualization of LVs and high endothelial venules in a lymph node of a living mouse for the first time. We confirmed the expression of Prox1 in the adult liver, lens, and dentate gyrus. These intensely fluorescent mice revealed the expression of Prox1 in three novel sites: the neuroendocrine cells of the adrenal medulla, megakaryocytes, and platelets. The novel sites identified herein suggest previously unknown roles for Prox1. The faithful expression of the fluorescent reporter in ProxTom LVs indicates that these mice have potential utility in the study of diseases as diverse as lymphedema, filariasis, transplant rejection, obesity, and tumor metastasis.

Conditional RAC1 knockout in motor neurons restores H-reflex rate-dependent depression after spinal cord injury.

A major complication with spinal cord injury (SCI) is the development of spasticity, a clinical symptom of hyperexcitability within the spinal H-reflex pathway. We have previously demonstrated a common structural motif of dendritic spine dysgenesis associated with hyperexcitability disorders after injury or disease insults to the CNS. Here, we used an adeno-associated viral (AAV)-mediated Cre-Lox system to knockout Rac1 protein expression in motor neurons after SCI. Three weeks after AAV9-Cre delivery into the soleus/gastrocnemius of Rac1-"floxed" adult mice to retrogradely infect spinal alpha-motor neurons, we observed significant restoration of RDD and reduced H-reflex excitability in SCI animals. Additionally, viral-mediated Rac1 knockdown reduced presence of dendritic spine dysgenesis on motor neurons. In control SCI animals without Rac1 knockout, we continued to observe abnormal dendritic spine morphology associated with hyperexcitability disorder, including an increase in mature, mushroom dendritic spines, and an increase in overall spine length and spine head size. Taken together, our results demonstrate that viral-mediated disruption of Rac1 expression in ventral horn motor neurons can mitigate dendritic spine morphological correlates of neuronal hyperexcitability, and reverse hyperreflexia associated with spasticity after SCI. Finally, our findings provide evidence of a putative mechanistic relationship between motor neuron dendritic spine dysgenesis and SCI-induced spasticity.

Depletion of CD4(+)CD25(+) T cells exacerbates experimental autoimmune encephalomyelitis induced by mouse, but not rat, antigens.

A key question in the field of autoimmunity concerns the fact that experimental disease is generally induced more easily with closely related, but not completely identical, tissue-restricted antigens. Here, the possibility that naturally occurring regulatory T cells (Tregs) for self-antigens are more potent than those for related antigens was investigated. The self-antigen specificity of naturally occurring Tregs was tested in experimental autoimmune encephalomyelitis (EAE) induced with mouse (self) or closely related (rat) myelin oligodendrocyte glycoproteins (MOGs). Surprisingly, Treg depletion increased EAE severity in mice immunized with mouse, but not rat, MOG. This increase was associated with increased T-cell activation and infiltration of the central nervous system, as well as increased interleukin (IL)-17 production and a higher ratio of interferon-gamma- to IL-10-producing cells. These data suggest that Tregs are specific for self-antigen and do not "cross-protect" against autoimmunity even when disease is induced with closely related foreign antigens.

Spinal cord motor neuron plasticity accompanies second-degree burn injury and chronic pain.

Burn injuries and associated complications present a major public health challenge. Many burn patients develop clinically intractable complications, including pain and other sensory disorders. Recent evidence has shown that dendritic spine neuropathology in spinal cord sensory and motor neurons accompanies central nervous system (CNS) or peripheral nervous system (PNS) trauma and disease. However, no research has investigated similar dendritic spine neuropathologies following a cutaneous thermal burn injury. In this retrospective investigation, we analyzed dendritic spine morphology and localization in alpha-motor neurons innervating a burn-injured area of the body (hind paw). To identify a molecular regulator of these dendritic spine changes, we further profiled motor neuron dendritic spines in adult mice treated with romidepsin, a clinically approved Pak1-inhibitor, or vehicle control at two postburn time points: Day 6 immediately after treatment, or Day 10 following drug withdrawal. In control treated mice, we observed an overall increase in dendritic spine density, including structurally mature spines with mushroom-shaped morphology. Pak1-inhibitor treatment reduced injury-induced changes to similar levels observed in animals without burn injury. The effectiveness of the Pak1-inhibitor was durable, since normalized dendritic spine profiles remained as long as 4 days despite drug withdrawal. This study is the first report of evidence demonstrating that a second-degree burn injury significantly affects motor neuron structure within the spinal cord. Furthermore, our results support the opportunity to study dendritic spine dysgenesis as a novel avenue to clarify the complexities of neurological disease following traumatic injury.

Physicochemical Properties Can Be Key Determinants of Mesoporous Silica Nanoparticle Potency in Vitro.

Nanoforms of mesoporous silica (mSiNPs) are increasingly applied in medicine, imaging, energy storage, catalysis, biosensors, and bioremediation. The impact of their physicochemical properties on health and the environment remain to be elucidated. In this work, newly synthesized mesoporous silica (sizes: 25, 70, 100, 170, and 600 nm; surface functionalization: pristine, C3-, and C11-COOH moieties) were assessed for cytotoxicity and induction of inflammatory responses in vitro (A549, THP-1, J774A.1 cells). All toxicity end points were integrated to obtain simple descriptors of biological potencies of these mSiNPs. The findings indicate that mSiNPs are less bioactive than the nonporous reference SiNP used in this study. The C3-COOH-modified mSiNPs were generally less cytotoxic than their pristine and C11-modified counterparts in the nanorange (≤100 nm). Carboxyl-modified mSiNPs affected inflammatory marker release across all sizes with cell-type specificity, suggesting a potential for immunomodulatory effects. Surface area, size, extent of agglomeration, ζ-potential, and surface modification appeared to be important determinants of cytotoxicity of mSiNPs based on association tests. Pathway analysis identified particle and cell-type-specific alteration of cellular pathways and functions by mSiNPs. The integration of exposure-related biological responses of multiple cell lines to mSiNPs allowed for a comprehensive evaluation of the impact of physicochemical factors on their toxicity characteristics. The integrated multilevel toxicity assessment approach can be valuable as a hazard screening tool for safety evaluations of emerging nanomaterials for regulatory purpose.

Differential cytotoxic and inflammatory potency of amorphous silicon dioxide nanoparticles of similar size in multiple cell lines.

The likelihood of environmental and health impacts of silicon dioxide nanoparticles (SiNPs) has risen, due to their increased use in products and applications. The biological potency of a set of similarly-sized amorphous SiNPs was investigated in a variety of cells to examine the influence of physico-chemical and biological factors on their toxicity. Cellular LDH and ATP, BrdU incorporation, resazurin reduction and cytokine release were measured in human epithelial A549, human THP-1 and mouse J774A.1 macrophage cells exposed for 24 h to suspensions of 5-15, 10-20 and 12 nm SiNPs and reference particles. The SiNPs were characterized in dry state and in suspension to determine their physico-chemical properties. The dose-response data were simplified into particle potency estimates to facilitate the comparison of multiple endpoints of biological effects in cells. Mouse macrophages were the most sensitive to SiNP exposures. Cytotoxicity of the individual cell lines was correlated while the cytokine responses differed, supported by cell type-specific differences in inflammation-associated pathways. SiNP (12 nm), the most cytotoxic and inflammogenic nanoparticle had the highest surface acidity, dry-state agglomerate size, the lowest trace metal and organics content, the smallest surface area and agglomerate size in suspension. Particle surface acidity appeared to be the most significant determinant of the overall biological activity of this set of nanoparticles. Combined with the nanoparticle characterization, integration of the biological potency estimates enabled a comprehensive determination of the cellular reactivity of the SiNPs. The approach shows promise as a useful tool for first-tier screening of SiNP toxicity.

Characterization of in vitro genotoxic, cytotoxic and transcriptomic responses following exposures to amorphous silica of different sizes.

The objectives of the present study were to investigate the underlying mechanisms of genetic and cellular toxicity induced by silica nanoparticles (SiNPs) and determine if such toxicity is influenced by particle size. Commercially available amorphous SiNPs (12 nm, 5-10 nm, and 10-15 nm) and micrometer sized (SiP2 µm) silica were characterised for size, chemical composition, and aggregation state. Mouse lung epithelial (FE1) cells derived from Muta™Mouse were exposed to various concentrations (12.5, 25, 50, 100 µg/ml) of SiNPs and SiP2 µm. Cellular viability, clonogenic potential, oxidative stress, micronucleus formation, and mutant frequency were measured at different post-exposure time points. Cellular internalization of particles was assessed using nanoscale hyperspectral microscopy. Biological pathway and functional perturbations were assessed using DNA microarrays. Detailed characterization of particles confirmed their size, purity, and uniform dispersion in the exposure medium. Decreased cellular viability was observed acutely at 24h at concentrations higher than 25 µg/ml for all particle types, with SiNPs being the most sensitive; loss of viability was surface area dependent at the lowest concentration tested. However, only SiNP12 showed poor long-term survival. A size-dependent increase in micronucleus formation was also observed for SiNPs. In contrast to the viability results, SiP2 µm exhibited the highest potential to induce oxidative stress compared to the SiNPs at all tested concentrations. Gene ontology and biological pathway analysis revealed significant changes in the expression of genes implicated in lysosomal functions in SiNP12-treated cells, which appear closely associated with higher SiNP12 internalization and lysosomal rearrangements in the cytoplasm of these cells. These results suggest that SiNPs induce cellular and genetic toxicity in a size-dependent manner and that the observed toxicity may be the results of higher particle internalization of smaller SiNP and subsequent lysosomal overload.

Lymphotoxin plays a crucial role in the development and function of nasal-associated lymphoid tissue through regulation of chemokines and peripheral node addressin.

The mechanism of nasal-associated lymphoid tissue (NALT) development is incompletely understood with regard to the roles of cytokines, chemokines, and vascular addressins. Development of the wild-type NALT continued in the immediate postnatal period with gradual increases in cellularity, compartmentalization into T- and B-cell zones, and expression of lymphotoxin (LT)-alpha, LT-beta, and lymphoid chemokines (CCL21, CCL19, CXCL13). High endothelial venules (HEVs) developed that expressed GlyCAM-1, HEC-6ST [an enzyme crucial for expression of luminal peripheral node addressin (PNAd)], and PNAd itself. LT-beta(-/-) and LT-alpha(-/-) NALTs had fewer cells than those of wild-type mice, reduced (LT-beta(-/-)) or absent (LT-alpha(-/-)) lymphoid chemokines, and no T- and B-cell compartmentalization. LT-beta(-/-) HEVs expressed only abluminal PNAd and no HEC-6ST or GlyCAM-1. LT-alpha(-/-) HEVs had no PNAd, HEC-6ST, or GlyCAM-1. Because intranasal immunization gives rise to vaginal IgA, immunization of LT-beta(-/-) mice, which retain cervical lymph nodes, might generate such a response. Intranasal immunization with ovalbumin and cholera toxin revealed lower cytokine levels in the LT-alpha(-/-) and LT-beta(-/-) NALTs, and undetectable vaginal IgA. In contrast, splenic cytokines and serum IgG titers, although reduced, were detectable. These data indicate that LT-alpha(3) and LT-alpha(1)beta(2) cooperatively contribute to NALT development and function through regulation of lymphoid chemokines and adhesion molecules; they are the first to implicate LT-alpha(1)beta(2) in GlyCAM-1 regulation in NALT HEV development.