Qualitative and Quantitative Neuropathology Approaches Using Magnetic Resonance Microscopy (Diffusion Tensor Imaging) and Stereology in a Hexachlorophene Model of Myelinopathy in Sprague-Dawley Rats.

It is well established that hexachlorophene, which is used as an antibacterial agent, causes intramyelinic edema in humans and animal models. The hexachlorophene myelinopathy model, in which male Sprague-Dawley rats received 25 to 30 mg/kg hexachlorophene by gavage for up to 5 days, provided an opportunity to compare traditional neuropathology evaluations with magnetic resonance microscopy (MRM) findings. In addition, stereology assessments of 3 neuroanatomical sites were compared to quantitative measurements of similar structures by MRM. There were positive correlations between hematoxylin and eosin and luxol fast blue stains and MRM for identifying intramyelinic edema in the cingulum of corpus callosum, optic chiasm, anterior commissure (aca), lateral olfactory tracts, pyramidal tracts (py), and white matter tracts in the cerebellum. Stereology assessments were focused on the aca, longitudinal fasciculus of the pons, and py and demonstrated differences between control and treated rats, as was observed using MRM. The added value of MRM assessments was the ability to acquire qualitative 3-dimensional (3-D) images and obtain quantitative measurements of intramyelinic edema in 26 neuroanatomical sites in the intact brain. Also, diffusion tensor imaging (fractional anisotropy [FA]) indicated that there were changes in the cytoarchitecture of the white matter as detected by decreases in the FA in the treated compared to the control rats. This study demonstrates creative strategies that are possible using qualitative and quantitative assessments of potential white matter neurotoxicants in nonclinical toxicity studies. Our results lead us to the conclusion that volumetric analysis by MRM and stereology adds significant value to the standard 2-D microscopic evaluations.

Evaluating the Impact of the U.S. National Toxicology Program: A Case Study on Hexavalent Chromium.

BACKGROUND: Evaluating the impact of federally funded research with a broad, methodical, and objective approach is important to ensure that public funds advance the mission of federal agencies. OBJECTIVES: We aimed to develop a methodical approach that would yield a broad assessment of National Toxicology Program's (NTP's) effectiveness across multiple sectors and demonstrate the utility of the approach through a case study. METHODS: A conceptual model was developed with defined activities, outputs (products), and outcomes (proximal, intermediate, distal) and applied retrospectively to NTP's research on hexavalent chromium (CrVI). Proximal outcomes were measured by counting views of and requests for NTP's products by external stakeholders. Intermediate outcomes were measured by bibliometric analysis. Distal outcomes were assessed through Web and LexisNexis searches for documents related to legislation or regulation changes. RESULTS: The approach identified awareness of NTP's work on CrVI by external stakeholders (proximal outcome) and citations of NTP's research in scientific publications, reports, congressional testimonies, and legal and policy documents (intermediate outcome). NTP's research was key to the nation's first-ever drinking water standard for CrVI adopted by California in 2014 (distal outcome). By applying this approach to a case study, the utility and limitations of the approach were identified, including challenges to evaluating the outcomes of a research program. CONCLUSIONS: This study identified a broad and objective approach for assessing NTP's effectiveness, including methodological needs for more thorough and efficient impact assessments in the future. Citation: Xie Y, Holmgren S, Andrews DMK, Wolfe MS. 2017. Evaluating the impact of the U.S. National Toxicology Program: a case study on hexavalent chromium. Environ Health Perspect 125:181-188; http://dx.doi.org/10.1289/EHP21.

Cyclic tensile strain enhances osteogenesis and angiogenesis in mesenchymal stem cells from osteoporotic donors.

We have shown that the uniaxial cyclic tensile strain of magnitude 10% promotes and enhances osteogenesis of human mesenchymal stem cells (hMSC) and human adipose-derived stem cells (hASC) from normal, nonosteoporotic donors. In the present study, MSC from osteoporotic donors were analyzed for changes in mRNA expression in response to 10% uniaxial tensile strain to identify potential mechanisms underlying the use of this mechanical loading paradigm for prevention and treatment of osteoporosis. Human MSC isolated from three female, postmenopausal osteoporotic donors were analyzed for their responses to mechanical loading using microarray analysis of over 47,000 gene probes. Human MSC were seeded in three-dimensional collagen type I constructs to mimic the organic extracellular matrix of bone and 10% uniaxial cyclic tensile strain was applied to promote osteogenesis. Seventy-nine genes were shown to be regulated within hMSC from osteoporotic donors in response to 10% cyclic tensile strain. Upregulation of six genes were further confirmed with real-time RT-PCR: jun D proto-oncogene (JUND) and plasminogen activator, urokinase receptor (PLAUR), two genes identified as potential key molecules from network analysis; phosphoinositide-3-kinase, catalytic, delta polypeptide (PIK3CD) and wingless-type MMTV integration site family, member 5B (WNT5B), two genes with known importance in bone biology; and, PDZ and LIM domain 4 (PDLIM4) and vascular endothelial growth factor A (VEGFA), two genes that we have previously shown are significantly regulated in hASC in response to this mechanical stimulus. Function analysis indicated that 10% cyclic tensile strain induced expression of genes associated with cell movement, cell proliferation, and tissue development, including development in musculoskeletal and cardiovascular systems. Our results demonstrate that hMSC from aged, osteoporotic donors are capable of enhanced osteogenic differentiation in response to 10% cyclic tensile strain with significant increases in the expression of genes associated with enhanced cell proliferation, musculoskeletal development, and angiogenesis. Surprisingly, cyclic tensile strain of magnitude 10% not only enhanced osteogenesis in hMSC from osteoporotic donors, but also enhanced expression of angiogenic factors. Better understanding and methodologies to promote osteogenesis in hMSC from elderly, osteoporotic donors may greatly facilitate achieving long-term success in bone regeneration and functional bone tissue engineering for this ever-growing patient population.

A high concentration of genistein down-regulates activin A, Smad3 and other TGF-ß pathway genes in human uterine leiomyoma cells.

Previously, we found that high doses of genistein show an inhibitory effect on uterine leiomyoma (UtLM) cell proliferation. In this study, using microarray analysis and Ingenuity Pathways Analysis™, we identified genes (up- or down-regulated, ≥ 1.5 fold, P ≤ 0.001), functions and signaling pathways that were altered following treatment with an inhibitory concentration of genistein (50 µg/ml) in UtLM cells. Downregulation of TGF-ß signaling pathway genes, activin A, activin B, Smad3, TGF-ß2 and genes related to cell cycle regulation, with the exception of the upregulation of the CDK inhibitor P15, were identified and validated by real- time RT-PCR studies. Western blot analysis further demonstrated decreased protein expression of activin A and Smad3 in genistein-treated UtLM cells. Moreover, we found that activin A stimulated the growth of UtLM cells, and the inhibitory effect of genistein was partially abrogated in the presence of activin A. Overexpression of activin A and Smad3 were found in tissue samples of leiomyoma compared to matched myometrium, supporting the contribution of activin A and Smad3 in promoting the growth of UtLM cells. Taken together, these results suggest that downregulation of activin A and Smad3, both members of the TGF-ß pathway, may offer a mechanistic explanation for the inhibitory effect of a high-dose of genistein on UtLM cells, and might be potential therapeutic targets for treatment of clinical cases of uterine leiomyomas.

Microarray analysis of human adipose-derived stem cells in three-dimensional collagen culture: osteogenesis inhibits bone morphogenic protein and Wnt signaling pathways, and cyclic tensile strain causes upregulation of proinflammatory cytokine regulators and angiogenic factors.

Human adipose-derived stem cells (hASC) have shown great potential for bone tissue engineering. However, the molecular mechanisms underlying this potential are not yet known, in particular the separate and combined effects of three-dimensional (3D) culture and mechanical loading on hASC osteogenesis. Mechanical stimuli play a pivotal role in bone formation, remodeling, and fracture repair. To further understand hASC osteogenic differentiation and response to mechanical stimuli, gene expression profiles of proliferating or osteogenically induced hASC in 3D collagen I culture in the presence and absence of 10% uniaxial cyclic tensile strain were examined using microarray analysis. About 847 genes and 95 canonical pathways were affected during osteogenesis of hASC in 3D culture. Pathway analysis indicated the potential roles of Wnt/ß-catenin signaling, bone morphogenic protein (BMP) signaling, platelet-derived growth factor (PDGF) signaling, and insulin-like growth factor 1 (IGF-1) signaling in hASC during osteogenic differentiation. Application of 10% uniaxial cyclic tensile strain suggested synergistic effects of strain with osteogenic differentiation media on hASC osteogenesis as indicated by significantly increased calcium accretion of hASC. There was no significant further alteration in the four major pathways (Wnt/ß-catenin, BMP, PDGF, and IGF-1). However, 184 transcripts were affected by 10% cyclic tensile strain. Function and network analysis of these transcripts suggested that 10% cyclic tensile strain may play a role during hASC osteogenic differentiation by upregulating two crucial factors in bone regeneration: (1) proinflammatory cytokine regulators interleukin 1 receptor antagonist and suppressor of cytokine signaling 3; (2) known angiogenic inductors fibroblast growth factor 2, matrix metalloproteinase 2, and vascular endothelial growth factor A. This is the first study to investigate the effects of both 3D culture and mechanical load on hASC osteogenic differentiation. A complete microarray analysis investigating both the separate effect of soluble osteogenic inductive factors and the combined effects of chemical and mechanical stimulation was performed on hASC undergoing osteogenic differentiation. We have identified specific genes and pathways associated with mechanical response and osteogenic potential of hASC, thus providing significant information toward improved understanding of our use of hASC for functional bone tissue engineering applications.

Genome-wide analysis of loss of heterozygosity and copy number amplification in uterine leiomyomas using the 100K single nucleotide polymorphism array.

PURPOSE: Uterine leiomyomas (fibroids) are benign smooth muscle tumors commonly found among reproductive-aged women. Though benign, these tumors are the leading indication for hysterectomies in the United States and cause significant morbidity. Despite the importance of this tumor in women's health, relatively little is known about the molecular etiology. METHODS: In this study, we used the Affymetrix 100K single nucleotide polymorphism (SNP) chip to assess whether the pattern and frequency of genome-wide loss of heterozygosity (LOH) and copy number amplifications is associated with clinical heterogeneity. RESULTS: Thirty-seven tumors with varying sizes and histology from eleven patients were analyzed. LOH was observed in 4/37 tumors (10.8%) and significantly associated with large-sized tumors (p<0.0014). Two tumors revealed hemizygosity on chromosome 7q, a region that has been consistently reported to have LOH. Additionally, we detected one novel region of LOH, 16p13.11 in one tumor (2.7%). Copy number amplifications were observed on all chromosomes; however, most were low-level amplifications and only detected in a single tumor. One region of amplification at 3p26.3 was detected in four tumors. CONCLUSIONS: Despite the use of a high-density SNP platform, our results suggest that genome-wide LOH and copy number amplifications are infrequent events and generally do not determine clinical and histologic characteristics of this disease.

Double-stranded RNA-activated protein kinase regulates early innate immune responses during respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is the most common cause of childhood viral bronchiolitis and lung injury. Inflammatory responses significantly contribute to lung pathologies during RSV infections and bronchiolitis but the exact mechanisms have not been completely defined. The double-stranded RNA-activated protein kinase (PKR) functions to inhibit viral replication and participates in several signaling pathways associated with innate inflammatory immune responses. Using a functionally defective PKR (PKR(-/-)) mouse model, we investigated the role of this kinase in early events of RSV-induced inflammation. Our data showed that bronchoalveolar lavage (BAL) fluid from infected PKR(-/-) mice had significantly lower levels of several innate inflammatory cytokines and chemokines. Histological examinations revealed that there was less lung injury in infected PKR(-/-) mice as compared to the wild type. A genome-wide analysis showed that several early antiviral and immune regulatory genes were affected by PKR activation. These data suggest that PKR is a signaling molecule for immune responses during RSV infections.