Catherine P. Starnes

Particle size distributions by transmission electron microscopy: an interlaboratory comparison case study.

This paper reports an interlaboratory comparison that evaluated a protocol for measuring and analysing the particle size distribution of discrete, metallic, spheroidal nanoparticles using transmission electron microscopy (TEM). The study was focused on automated image capture and automated particle analysis. NIST RM8012 gold nanoparticles (30 nm nominal diameter) were measured for area-equivalent diameter distributions by eight laboratories. Statistical analysis was used to (1) assess the data quality without using size distribution reference models, (2) determine reference model parameters for different size distribution reference models and non-linear regression fitting methods and (3) assess the measurement uncertainty of a size distribution parameter by using its coefficient of variation. The interlaboratory area-equivalent diameter mean, 27.6 nm ± 2.4 nm (computed based on a normal distribution), was quite similar to the area-equivalent diameter, 27.6 nm, assigned to NIST RM8012. The lognormal reference model was the preferred choice for these particle size distributions as, for all laboratories, its parameters had lower relative standard errors (RSEs) than the other size distribution reference models tested (normal, Weibull and Rosin-Rammler-Bennett). The RSEs for the fitted standard deviations were two orders of magnitude higher than those for the fitted means, suggesting that most of the parameter estimate errors were associated with estimating the breadth of the distributions. The coefficients of variation for the interlaboratory statistics also confirmed the lognormal reference model as the preferred choice. From quasi-linear plots, the typical range for good fits between the model and cumulative number-based distributions was 1.9 fitted standard deviations less than the mean to 2.3 fitted standard deviations above the mean. Automated image capture, automated particle analysis and statistical evaluation of the data and fitting coefficients provide a framework for assessing nanoparticle size distributions using TEM for image acquisition.

Regulation of thrombospondin-1 expression in alternatively activated macrophages and adipocytes: role of cellular cross talk and omega-3 fatty acids.

Thrombospondin-1 (TSP-1) expression in human adipose positively correlates with body mass index and may contribute to adipose dysfunction by activating transforming growth factor-β and/or inhibiting angiogenesis. Our objective was to determine how TSP-1 is regulated in adipocytes and polarized macrophages using a coculture system and to determine whether fatty acids, including the ω-3 fatty acid docosahexaenoic acid (DHA), regulate TSP-1 expression. Coculture of M1, M2a or M2c macrophages with adipocytes induced TSP-1 gene expression in adipocytes (from 2.4- to 4.2-fold, P<.05), and adipocyte coculture induced TSP-1 gene expression in M1 and M2c macrophages (M1: 8.6-fold, M2c: 26-fold; P<.05). TSP-1 protein levels in the shared media of adipocytes and M2c cells were also strongly induced by coculture (>10-fold, P<.05). DHA treatment during the coculture of adipocytes and M2c macrophages potently inhibited the M2c macrophage TSP-1 mRNA level (97% inhibition, P<.05). Adipocyte coculture induced interleukin (IL)-10 expression in M2c macrophages (10.1-fold, P<.05), and this increase in IL-10 mRNA expression was almost completely blocked with DHA treatment (96% inhibition, P<.05); thus, IL-10 expression closely paralleled TSP-1 expression. Since IL-10 has been shown to regulate TSP-1 in other cell types, we reduced IL-10 expression with siRNA in the M2c cells and found that this caused TSP-1 to be reduced in response to adipocyte coculture by 60% (P<.05), suggesting that IL-10 regulates TSP-1 expression in M2c macrophages. These results suggest that supplementation with dietary ω-3 fatty acids could potentially be beneficial to adipose tissue in obesity by reducing TSP-1 and fibrosis.

Distinct transcriptomic responses of Caenorhabditis elegans to pristine and sulfidized silver nanoparticles.

Manufactured nanoparticles (MNP) rapidly undergo aging processes once released from products. Silver sulfide (Ag2S) is the major transformation product formed during the wastewater treatment process for Ag-MNP. We examined toxicogenomic responses of pristine Ag-MNP, sulfidized Ag-MNP (sAg-MNP), and AgNO3 to a model soil organism, Caenorhabditis elegans. Transcriptomic profiling of nematodes which were exposed at the EC30 for reproduction for AgNO3, Ag-MNP, and sAg-MNP resulted in 571 differentially expressed genes. We independently verified expression of 4 genes (numr-1, rol-8, col-158, and grl-20) using qRT-PCR. Only 11% of differentially expressed genes were common among the three treatments. Gene ontology enrichment analysis also revealed that Ag-MNP and sAg-MNP had distinct toxicity mechanisms and did not share any of the biological processes. The processes most affected by Ag-MNP relate to metabolism, while those processes most affected by sAg-MNP relate to molting and the cuticle, and the most impacted processes for AgNO3 exposed nematodes was stress related. Additionally, as observed from qRT-PCR and mutant experiments, the responses to sAg-MNP were distinct from AgNO3 while some of the effects of pristine MNP were similar to AgNO3, suggesting that effects from Ag-MNP is partially due to dissolved silver ions.

DHA reduces the atrophy-associated Fn14 protein in differentiated myotubes during coculture with macrophages.

Macrophages are an important component of muscle where they are involved in complex processes such as repair, regeneration and hypertrophy. We recently reported that macrophage numbers increase in the muscle of obese patients, suggesting that muscle-resident macrophages could be involved in the development of muscle insulin resistance that is associated with obesity. Coculture of activated macrophages with human muscle cells impairs insulin signaling and induces atrophy signaling pathways in the human muscle cells; this is exacerbated by the addition of palmitic acid. In this study, we tested the hypothesis that docosahexaenoic acid (DHA), a polyunsaturated fatty acid that has anti-inflammatory properties, would have the opposite effect of palmitic acid on muscle-macrophage cocultures. Surprisingly, DHA did not stimulate insulin signaling in human muscle myotubes that were cocultured with fibroblasts or macrophages. However, DHA inhibited Fn14, the TNF-like weak inducer of apoptosis receptor that increases the expression of the muscle-specific ubiquitin ligase MuRF-1 (muscle ring-finger protein-1). DHA treatment also increased the apparent molecular mass of MuRF-1 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, suggesting that DHA causes MuRF-1 to be posttranslationally modified. In conclusion, these results suggest that DHA may have a beneficial effect on muscle mass in humans by inhibiting the induction of Fn14 by infiltrating macrophages.

Outcomes for Students Receiving School-Based Physical Therapy as Measured by the School Function Assessment.

Purpose: To describe School Function Assessment (SFA) outcomes after 6 months of school-based physical therapy and the effects of age and gross motor function on outcomes. Methods: Within 28 states, 109 physical therapists and 296 of their students with disabilities, ages 5 to 12 years, participated. After training, therapists completed 10 SFA scales on students near the beginning and end of the school year. Results: Criterion scores for many students remained stable (46%-59%) or improved (37%-51%) with the most students improving in Participation and Maintaining/Changing Positions. Students aged 5 to 7 years showed greater change than 8- to 12-year-olds on 5 scales. Students with higher gross motor function (Gross Motor Function Classification System levels I vs IV/V and II/III vs IV/V) showed greater change on 9 scales. Conclusions: Positive SFA change was recorded in students receiving school-based physical therapy; however, the SFA is less sensitive for older students and those with lower functional movement.

Physical impairment in patients with idiopathic inflammatory myopathies is associated with the American College of Rheumatology functional status measure

The goals of this study were to assess the predictive value of chart-abstracted American College of Rheumatology functional status (ACR-FS) with patient-reported ACR-FS and to relate it with measures of muscle function in a single-institution cohort of patients with idiopathic inflammatory myopathies (IIMs). Demographic and clinical data of 102 patients with IIMs regularly followed in the Rheumatology and Neurology Clinics at the University of Kentucky Medical Center between 2006 and 2012 were obtained through retrospective chart review. Clinical and functional status evaluation, muscle performance testing, and body composition measures were performed on a subset of 21 patients. ACR-FS was obtained by both chart abstraction and direct patient report. Spearman’s correlations were used to examine the relationship of ACR-FS derived from chart abstraction with direct patient report, as well as the relationship of measures of physical function and body composition with ACR-FS. ACR-FS derived from chart abstraction was significantly correlated with ACR-FS derived from direct patient report (ρ = 0.78, p < 0.001). ACR-FS derived from chart abstraction was also significantly correlated with patient-reported physical function (ρ = −0.71, p < 0.001) and physical activity (ρ = −0.58, p < 0.05), manual muscle testing (ρ = −0.66, p < 0.01), and skeletal muscle endurance as measured by the functional index-2 test (shoulder flexion ρ = −0.62, p < 0.01; hip flexion ρ = −0.65, p < 0.0; heel lift ρ = −0.67, p < 0.01; and toe lift ρ = −0.68, p < 0.01). The ACR-FS is a simple measure of disability that can be used in chart abstraction studies involving IIM patients. We have demonstrated that ACR-FS correlates well with muscle performance tests of strength and endurance.

Regulation of Small Ubiquitin-Like Modifier-1, Nuclear Receptor Coreceptor, Histone Deacetylase 3, and Peroxisome Proliferator-Activated Receptor-γ in Human Adipose Tissue

BACKGROUND This study investigated the regulation of peroxisome proliferator-activated receptor-γ (PPARγ), the histone deacetylase 3 (HDAC3)-nuclear receptor coreceptor (NCoR) complex (a corepressor of transcription used by PPARγ), and small ubiquitin-like modifier-1 (SUMO-1) (a posttranslational modifier of PPARγ) in human adipose tissue and both adipocyte and macrophage cell lines. The objective was to determine whether there were alterations in the human adipose tissue gene expression levels of PPARγ, HDAC3, NCoR, and SUMO-1 associated either with obesity or with treatment of impaired glucose tolerance (IGT) subjects with insulin-sensitizing medications. METHODS We obtained subcutaneous adipose tissue biopsies from 86 subjects with a wide range of body mass index (BMI) and insulin sensitivity (S(I)). Additionally, adipose tissue biopsies were obtained from a randomized subgroup of IGT subjects before and after 10 weeks of treatment with either pioglitazone or metformin. RESULTS The adipose mRNA levels of PPARγ, NCoR, HDAC3, and SUMO-1 correlated strongly with each other (P<0.0001); however, SUMO-1, NCoR, and HDAC3 gene expression were not significantly associated with BMI or S(I). Pioglitazone increased SUMO-1 expression by 23% (P<0.002) in adipose tissue and an adipocyte cell line (P<0.05), but not in macrophages. Small interfering RNA (siRNA)-mediated knockdown of SUMO-1 decreased PPARγ, HDAC3, and NCoR in THP-1 cells and increased tumor necrosis factor-α (TNF-α) induction in response to lipopolysaccharide (LPS). CONCLUSIONS These results suggest that the coordinate regulation of SUMO-1, PPARγ1/2, HDAC3, and NCoR may be more tightly controlled in macrophages than in adipocytes in human adipose and that these modulators of PPARγ activity may be particularly important in the negative regulation of macrophage-mediated adipose inflammation by pioglitazone.

THU0450 Relationship between the American College of Rheumatology (ACR) Classification Criteria of Functional Status and Clinical Predictors of Disability in Inflammatory Myopathies (IIM)

Background Many patients with IIMs exibit chronic muscle weakness and functional disability despite treatment. In polymyositis/ dermatomyositis (PM/DM), male sex, higher prednisone dosage and older age have been associated with muscle weakness and functional disability.[1,2] Sporadic inclusion body myositis (sIBM) itself is associated with major end-stage disability.[3] The ACR functional status (ACRFS) criteria have been used as a core measure of the consequences of impairment in IIM.[4] However, its association with known or suspected risk factors of disability warrants further investigation. Objectives To determine predictors of current/ worst ever ACRFS with known or suspected risk factors of disability and muscle weakness in patients with IIM. Methods Data were obtained from chart reviews of IIM and overlap myositis (OM) cases seen in the Rheumatology and Neurology Clinics at the University of Kentucky from May/06 until July/12. Current and worst ever ACRFS, demographic and clinical characteristics were abstracted from medical records. One-way ANOVA and Fisher’s exact/ Chi-square tests were used to compare groups on continuous/ categorical variables, respectively. Ordinal logistic regression was applied to estimate the effects of IIM type, age, sex and disease duration from diagnosis on current and worst ever ACRFS. Results 90 patients with IIM and OM were included: 38 PM, 29 DM, 12 IBM and 11 OM. When patient ages were divided into tertiles, sIBM patients were significantly older (p= 0.03). Females predominated in the PM, DM and OM groups. In sIBM, females were slightly outnumbered. Mean duration of disease from diagnosis was highest in sIBM (73 mo) compared to PM (39 mo), DM (18 mo) and OM (35 mo) (p=0.01). sIBM was more likely to be associated with presence of some degree of disability at the last time of assessment (91%), compared to all other groups combined (73%) (p=0.001). In the multivariable analysis, poorer current ACRFS and worst ever ACRFS were independently associated with higher age, and higher age and longer disease duration, respectively, controlling for all other variables. Conclusions The demographic and clinical characteristics of our cohort are consistent with previous reports. As expected, sIBM was associated with increased odds of disability. In the multivariate analysis, higher age and disease duration were identified as independent risk factors for disability. References Bronner IM, Van Der Meulen MF, De Visser M et al. Long-term outcome in polymyositis and dermatomyositis. Annals of the rheumatic diseases 65(11), 1456-1461 (2006). Clarke AE, Bloch DA, Medsger TA, Jr., Oddis CV. A longitudinal study of functional disability in a national cohort of patients with polymyositis/dermatomyositis. Arthritis and rheumatism 38(9), 1218-1224 (1995). Cox FM, Titulaer MJ, Sont JK, Wintzen AR, Verschuuren JJ, Badrising UA. A 12-year follow-up in sporadic inclusion body myositis: an end stage with major disabilities. Brain : a journal of neurology 134(Pt 11), 3167-3175 (2011). Stucki G, Stoll T, Bruhlmann P, Michel BA. Construct validation of the ACR 1991 revised criteria for global functional status in rheumatoid arthritis. Clinical and experimental rheumatology 13(3), 349-352 (1995). Acknowledgements This study was supported by the Arthritis Foundation, the Center for Clinical and Translational Science (CCTS) at the University of Kentucky, the University of Kentucky College of Medicine Clinical Scholars Program and Research Data Capture (REDCap). Disclosure of Interest None Declared

THU0449 Utility of Ultrasound (US) in Assessing Skeletal Muscle Architecture in Idiopathic Inflammatory Myopathies (IIM)

Background IIM are systemic autoimmune diseases characterized by chronic inflammation in skeletal muscle leading to proximal muscle weakness. In rheumatoid arthritis, muscle weakness has been linked to specific changes in muscle architecture that are measureable using ultrasound (US).1 However, data regarding the contribution of muscle architecture to muscle weakness in IIM is lacking. US is a non-invasive, relatively inexpensive and validated method of assessing skeletal muscle architectural parameters [i.e. anatomic cross sectional area (ACSA), fascicle length (Lf), pennation angle (θ) and muscle thickness].2,3 US determination of Lf and θ allows calculation of the physiological cross sectional area of muscle, which is a better predictor of intrinsic muscle force compared to ACSA or volume.4 Objectives To test the utility of US in determining skeletal muscle architecture in patients with IIM. To investigate associations between rectus femoris muscle ACSA (RFACSA), muscle peak torque generation, and other clinical outcomes in patients with IIM. Methods Clinical data were obtained from chart reviews of IIM cases seen in the Rheumatology clinic at the University of Kentucky from May/2006 until Jan/2013. Participant body composition (DXA), mid-thigh bilateral RFACSA and right vastus lateralis fascicle length(VLLf) were measured with the knee extended and muscle relaxed. Right knee extensor and elbow flexor muscle-specific peak torques were measured using a Biodex dynamometer. Data were analyzed using descriptive statistics and Spearman rank correlation coefficient. Results 12 patients with IIM and overlap myositis (OM) were included: 2 polymyositis (PM), 4 dermatomyositis (DM), 5 sporadic inclusion body myositis (sIBM) and 1 OM. In 1 PM and 3 sIBM patients, muscle architecture was so disrupted that VLLf could not be measured. Right and left mean RFACSA were correlated (p=0.02). Mean RFACSA was inversely correlated with total body and thigh fat (p<0.05), and positively correlated with trunk lean mass and elbow flexor maximal voluntary isometric contraction (MVIC) (p<0.05). However, RFACSA did not correlate well with knee extensor MVIC. VLLf was measured in 3 DM patients (mean=7.92cm, SD=2.03) and 1 PM patient (6.79cm), which is in the described range.2 Conclusions RFACSA was associated with body composition. It is possible that in IIM, quadriceps muscle is weaker than predicted by RFACSA or lean body mass. In severe cases of IIM, in particular sIBM, substantial disruption of muscle architecture could be detected by US. References Matschke V, Murphy P, Lemmey AB, Maddison P, Thom JM. Skeletal muscle properties in rheumatoid arthritis patients. Medicine and science in sports and exercise 2010;42:2149-55. Chleboun GS, France AR, Crill MT, Braddock HK, Howell JN. In vivo measurement of fascicle length and pennation angle of the human biceps femoris muscle. Cells, tissues, organs 2001;169:401-9. Herbert RD, Gandevia SC. Changes in pennation with joint angle and muscle torque: in vivo measurements in human brachialis muscle. J Physiol 1995;484 ( Pt 2):523-32. Narici MV, Landoni L, Minetti AE. Assessment of human knee extensor muscles stress from in vivo physiological cross-sectional area and strength measurements. Eur J Appl Physiol Occup Physiol 1992;65:438-44. Acknowledgements This study was supported by the Arthritis Foundation, the Center for Clinical and Translational Science (CCTS) at the University of Kentucky, the University of Kentucky College of Medicine Clinical Scholars Program and Research Data Capture (REDCap). Disclosure of Interest None Declared