ROLE OF HLA-B35 AND ER STRESS IN SCLERODERMA PATIENTS WITH PULMONARY ARTERIAL HYPERTENSION

Pulmonary arterial hypertension (PAH), a common complication of limited cutaneous systemic sclerosis (lcSSc), is associated with alterations of markers of inflammation and vascular damage. Endoplasmic reticulum (ER) stress and unfolded protein response (UPR) have been implicated in various diseases. The presence of the HLA-B35 allele, Human antigen class I, has emerged as an important risk factor for the development of PAH in patients with lcSSc, however the mechanisms underlying this association have not been fully elucidated. We have recently reported that the presence of HLA-B35 contributes to human dermal microvascular endothelial cell (HDMEC) dysfunction by significantly increasing production of endothelin-1 (ET-1) and significantly decreasing endothelial NO synthase (eNOS). Furthermore, HLA-B35 greatly upregulated expression of chaperones, including heat shock proteins (HSPs) HSP70 (HSPA1A and HSPA1B) and HSP40 (DNAJB1 and DNAJB9), suggesting that HLA-B35 induces the ER stress and UPR in ECs and this mechanism can mediate the induction of ET-1 in patients with PAH. The goal of this study was to better understand the role of HLA-B35-induced ER stress/UPR in the development/progression of PAH disease in lcSSc patients. First we focused on the molecular mechanisms of ET-1 induction by HLA-B35. ER stress inducer, Thapsigargin (TG) and HLA-B35 induced ET-1 expression with similar potency in HDMECs. HLA-B35 or ER stress activated the PERK/eIF2\u3b1/ATF4 branch of the UPR and modestly increased the spliced variant of X-box binding protein (XBP1), but did not affect the Activating Transcription Factor -6 (ATF6) pathways. Depletion of ATF4 decreased basal expression levels of ET-1 mRNA and protein, and completely prevented upregulation of ET-1 by HLA-B35/ER stress. Additional experiments have demonstrated that the JNK and NF-\uf06bB pathways are also required for ET-1 upregulation by HLA-B35/ER stress. Formation of the ATF4/c-JUN complex, but not the ATF4/NF-\uf06bB complex was also increased. The functional role of c-JUN in responses to HLA-B35/ER stress was further confirmed in ET-1 promoter assays. This study identified ATF4 as a novel activator of the ET-1 gene. Then we focus on whether markers of ER stress/UPR were present in PBMCs from lcSSc-PAH patients and if the presence of HLA-B35 contributes to activation of the immune cells. Several ER stress/UPR genes, including Immunoglobulin-heavy-chain binding protein (BiP), ATF4 and ATF6 and a spliced form of XBP1 were upregulated in lcSSc PBMCs, with the highest levels in patients with PAH. Also selected HSP genes, particularly DNAJB1, and IFN-related genes were found at significantly elevated levels in PBMCs from lcSSc patients, while IRF4 was significantly decreased. There was a positive correlation between DNAJB1 and severity of PAH disease (PAP) (r = 0.56, p<0.05) and between ER stress markers and IL-6 levels (r = 0.53, p< 0.0001) in lcSSc PBMCs. When we stratified all PBMC samples based on the presence of the HLA-B35 allele, we could observe that HLA-B35 positive individuals showed higher levels of selected ER stress markers when compared to HLA-B35 negative individuals. Furthermore, patients carrying HLA-B35 antigen expressed higher levels of IL-6, a key inflammatory cytokine associated with development of PAH. This study demonstrates association between select ER stress/UPR markers and lcSSc-PAH suggesting that ER stress/UPR may contribute to the altered function of circulating immune cells in lcSSc. All these associations were enhanced by the presence of HLA-B35. In conclusion, we hypothesize that HLA-B35 may play a role in EC dysfunction inducing ET1 via ER stress/UPR. Also activation of ER stress/UPR, in combination with presence of HLA-B35, might drive the inflammatory process in lcSSc-PAH

Guided Goal Setting: A Feasible Obesity Prevention Behavior Change Strategy for Low-Income Parents with Young Children

To reduce the risk of pediatric obesity, behaviorally-focused parental education is needed. This study examined the feasibility of guided goal setting in a pediatric obesity prevention intervention for low-income parents of young children. Parents from Head Start participated in a six-week nutrition, activity, and parenting intervention that included guided goal setting (N = 47). At each session, data were collected on goal selection, effort, and attainment. Pre- and postintervention data were collected on nutrition, activity, and child feeding behaviors (n = 24). A subset of parents completed in-depth interviews (n = 20). Parents reported a high level of goal effort (88%) and achievement (79%) and a preference for goal personalization and a list of goals from which to choose. In general, parents did not find the goal contract or weekly tracking as motivating as goal personalization. Pre- and postintervention assessment showed significant changes in the dietary energy density (p = .008) and vegetable behavior (p = .04) subscales with a marginal change in the snacking subscale (p = .08). Guided goal setting was a feasible behavioral strategy for the parents in this study since parents engaged in the process and demonstrated changes in serving more vegetables and fewer energy-dense foods

Evidence to Support the Use of the Retrospective Pretest method to Measure Dietary and Physical Activity Behavior and Self-Efficacy in Adolescents

This study compared the retrospective pretest-posttest method to the traditional prospective pretest-posttest method assessing adolescents’ dietary and physical activity, self-efficacy and behaviors. Participants were 7th and 8th grade students at a rural K-8th grade elementary school in Northern California (n=188). All participants completed an evaluation instrument (traditional pretest), followed by a 9-lesson dietary and physical activity intervention. Upon completion of the intervention, participants completed a second and identical evaluation instrument which served as a traditional posttest. The following day, participants completed another evaluation tool, this time formatted as a retrospective pretest. Analysis included sample t tests comparing the means of each method. Participants (n = 154) with a mean age of 13 ± .7 years old were included in the analyses (52% female). Paired sample t tests reported non-significant differences between the two methods for dietary behavior and dietary self-efficacy, yet significant differences were found for physical activity behavior (p < .05) and physical activity self-efficacy (p < .01). We conclude that the retrospective pretest-posttest method was as good a measure of dietary self-efficacy and behavior as the traditional prospective pretest-posttest method and may be better at attenuating response-shift bias when assessing physical activity self-efficacy and behavior

Evidence to Support the Use of the Retrospective Pretest method to Measure Dietary and Physical Activity Behavior and Self-Efficacy in Adolescents

This study compared the retrospective pretest-posttest method to the traditional prospective pretest-posttest method assessing adolescents’ dietary and physical activity, self-efficacy and behaviors. Participants were 7th and 8th grade students at a rural K-8th grade elementary school in Northern California (n=188). All participants completed an evaluation instrument (traditional pretest), followed by a 9-lesson dietary and physical activity intervention. Upon completion of the intervention, participants completed a second and identical evaluation instrument which served as a traditional posttest. The following day, participants completed another evaluation tool, this time formatted as a retrospective pretest. Analysis included sample t tests comparing the means of each method. Participants (n = 154) with a mean age of 13 ± .7 years old were included in the analyses (52% female). Paired sample t tests reported non-significant differences between the two methods for dietary behavior and dietary self-efficacy, yet significant differences were found for physical activity behavior (p < .05) and physical activity self-efficacy (p < .01). We conclude that the retrospective pretest-posttest method was as good a measure of dietary self-efficacy and behavior as the traditional prospective pretest-posttest method and may be better at attenuating response-shift bias when assessing physical activity self-efficacy and behavior

Assessment of the immune landscapes of advanced ovarian cancer in an optimized in vivo model

BackgroundOvarian cancer (OC) is typically diagnosed late, associated with high rates of metastasis and the onset of ascites during late stage disease. Understanding the tumor microenvironment and how it impacts the efficacy of current treatments, including immunotherapies, needs effective in vivo models that are fully characterized. In particular, understanding the role of immune cells within the tumor and ascitic fluid could provide important insights into why OC fails to respond to immunotherapies. In this work, we comprehensively described the immune cell infiltrates in tumor nodules and the ascitic fluid within an optimized preclinical model of advanced ovarian cancer.MethodsGreen Fluorescent Protein (GFP)-ID8 OC cells were injected intraperitoneally into C57BL/6 mice and the development of advanced stage OC monitored. Nine weeks after tumor injection, mice were sacrificed and tumor nodules analyzed to identify specific immune infiltrates by immunohistochemistry. Ascites, developed in tumor bearing mice over a 10-week period, was characterized by mass cytometry (CyTOF) to qualitatively and quantitatively assess the distribution of the immune cell subsets, and their relationship to ascites from ovarian cancer patients.ResultsTumor nodules in the peritoneal cavity proved to be enriched in T cells, antigen presenting cells and macrophages, demonstrating an active immune environment and cell-mediated immunity. Assessment of the immune landscape in the ascites showed the predominance of CD8+, CD4+, B–, and memory T cells, among others, and the coexistance of different immune cell types within the same tumor microenvironment.ConclusionsWe performed, for the first time, a multiparametric analysis of the ascitic fluid and specifically identify immune cell populations in the peritoneal cavity of mice with advanced OC. Data obtained highlights the impact of CytOF as a diagnostic tool for this malignancy, with the opportunity to concomitantly identify novel targets, and define personalized therapeutic options

The Black Identity, Hair Product Use, and Breast Cancer Scale

Introduction Across the African Diaspora, hair is synonymous with identity. As such, Black women use a variety of hair products, which often contain more endocrine-disrupting chemicals than products used by women of other races. An emerging body of research is linking chemicals in hair products to breast cancer, but there is no validated instrument that measures constructs related to hair, identity, and breast health. The objective of this study was to develop and validate the Black Identity, Hair Product Use, and Breast Cancer Scale (BHBS) in a diverse sample of Black women to measure the social and cultural constructs associated with Black women’s hair product use and perceived breast cancer risk. Methods Participants completed a 27-item scale that queried perceptions of identity, hair products, and breast cancer risk. Principal Component Analyses (PCA) were conducted to establish the underlying component structures, and confirmatory factor analysis (CFA) was used to determine model fit. Results Participants (n = 185) were African American (73%), African, and Caribbean Black women (27%) aged 29 to 64. PCA yielded two components that accounted for 61% of total variance. Five items measuring sociocultural perspectives about hair and identity loaded on subscale 1 and accounted for 32% of total variance (α = 0.82; 95% CI = 0.77–0.86). Six items assessing perceived breast cancer risk related to hair product use loaded on subscale 2 and accounted for 29% of total variance (α = 0.82 (95% CI = 0.74–0.86). CFA confirmed the two-component structure (Root Mean Square Error of Approximation = 0.03; Comparative Fit Index = 0.91; Tucker Lewis Index = 0.88). Conclusions The BHBS is a valid measure of social and cultural constructs associated with Black women’s hair product use and perceived breast cancer risk. This scale is useful for studies that assess cultural norms in the context of breast cancer risk for Black women

PO-435 Photoactivation of nanoparticles delivered by mesenchymal stem cells induces osteosarcoma cell death in in vitro 3D co-culture models

Introduction Osteosarcoma (OS) is a rare and aggressive tumour that mainly affects long bones of adolescents. Currently, OS patients are treated with a combination of multi-agent chemotherapy and surgery. However, 30% of patients do not respond to standard treatment. Therefore, innovative therapeutic agents are needed. Mesenchymal stem cells (MSCs) display a specific tumour-tropism and have been previously used in successful preclinical studies to deliver several therapeutic agents. Furthermore, the safety of genetically engineered MSCs was demonstrasted in ongoing clinical trial. The goal of the present study was to test in vitro whether MSCs could uptake photoactivable nanoparticles (NPs) and induce cell death of OS cells upon photoactivation. Material and methods Ptl@PMMA NPs were produced by adding tetrasulfonate aluminium phthalocyanine (Ptl) to an aqueous solution of positively charged poly-methylmethacrylate (PMMA) nanoparticles. The photosensitizer Ptl is activated in near-infrared light allowing a deep tissue penetration. Human MSC lines, isolated from the bone marrow of multiple donors, were loaded with Ptl@PMMA NPs. The MSCs' ability to internalise and retain NPs, along with their migratory properties, were tested. Cell death upon photoactivation (PDT) was evaluated in vitro, on a monolayer co-culture of MSCs and OS cells and in 3D multicellular spheroids, generated via cell suspension in ultralow attachment plates Results and discussions MSCs showed an internalisation rate of Plt@PMMA>95%, which did not alter cell viability and migratory capacity. When Ptl@PMMA-MSCs were co-cultured with a human OS cell line (SaOS-2) in monolayers, they efficiently triggered cell death upon PDT. In particular, AnnexinV/PI and CalceinAM/EthD staining showed 70% of cell death in the co-culture system. These results were also validated by a metabolic assay. Interestingly, in a 3D co-culture of the OS cell line MG63 and Ptl@PMMA-MSCs, we observed a marked reduction of the viability ( Conclusion For the first time, we demonstrated that photoactivation of MSCs loaded with Ptl@PMMA NPs can successfully induce OS cell death in a three-dimensional OS model. These results encourage further in vivo evaluation to demonstrate the specific targeting of Plt@PMMA loaded MSCs to the tumour stroma and the efficacy of PDT treatmen

Large-Scale Spray Releases: Additional Aerosol Test Results

One of the events postulated in the hazard analysis for the Waste Treatment and Immobilization Plant (WTP) and other U.S. Department of Energy (DOE) nuclear facilities is a breach in process piping that produces aerosols with droplet sizes in the respirable range. The current approach for predicting the size and concentration of aerosols produced in a spray leak event involves extrapolating from correlations reported in the literature. These correlations are based on results obtained from small engineered spray nozzles using pure liquids that behave as a Newtonian fluid. The narrow ranges of physical properties on which the correlations are based do not cover the wide range of slurries and viscous materials that will be processed in the WTP and in processing facilities across the DOE complex. To expand the data set upon which the WTP accident and safety analyses were based, an aerosol spray leak testing program was conducted by Pacific Northwest National Laboratory (PNNL). PNNL’s test program addressed two key technical areas to improve the WTP methodology (Larson and Allen 2010). The first technical area was to quantify the role of slurry particles in small breaches where slurry particles may plug the hole and prevent high-pressure sprays. The results from an effort to address this first technical area can be found in Mahoney et al. (2012a). The second technical area was to determine aerosol droplet size distribution and total droplet volume from prototypic breaches and fluids, including sprays from larger breaches and sprays of slurries for which literature data are mostly absent. To address the second technical area, the testing program collected aerosol generation data at two scales, commonly referred to as small-scale and large-scale testing. The small-scale testing and resultant data are described in Mahoney et al. (2012b), and the large-scale testing and resultant data are presented in Schonewill et al. (2012). In tests at both scales, simulants were used to mimic the relevant physical properties projected for actual WTP process streams

Comparing Two Waist-to-Height Ratio Measurements with Cardiometabolic Risk Factors among Youth with Diabetes

Background: Waist circumference (WC) is commonly measured by either the World Health Organization (WHO) or National Health and Nutrition Examination Survey (NHANES) protocol. Objective: Compare the associations of WHO vs. NHANES WC-to-height ratio (WHtR) protocols with cardiometabolic risk factors (CMRFs) in a sample of youth with diabetes. Methods: For youth (10–19 years old with type 1 [N=3082] or type 2 [N=533] diabetes) in the SEARCH for Diabetes in Youth Study, measurements were obtained of WC (by two protocols), weight, height, fasting lipids (total cholesterol, triglycerides, HDL cholesterol, Non-HDL cholesterol) and blood pressures. Associations of CMRFs with WHO and NHANES WHtR were modeled stratified by body mass index (BMI) percentiles for age/sex: lower BMI (&lt;85th BMI percentile; N=2071) vs. higher BMI (≥85th percentile; N=1594). Results: Among lower-BMI participants, both NHANES and WHO WHtR were associated (p&lt;0.005) with all CMRFs except blood pressure. Among higher-BMI participants, both NHANES and WHO WHtR were associated (p&lt;0.05) with all CMRFs. WHO WHtR was more strongly associated (p&lt;0.05) than NHANES WHtR with triglycerides, non-HDL cholesterol, and systolic blood pressure in lower-BMI participants. Among high-BMI participants, WHO WHtR was more strongly associated (p&lt;0.05) than NHANES WHtR with triglycerides and systolic blood pressure. Conclusion: Among youth with diabetes, WHtR calculated from either WC protocol captures cardiometabolic risk. The WHO WC protocol may be preferable to NHANES WC