Identifying RNA Biomarkers and Molecular Pathways Involved in Multiple Subtypes of Uveitis.

PURPOSE: Uveitis is a heterogeneous collection of diseases. We tested the hypothesis that despite the diversity of uveitides, there could be common mechanisms shared by multiple subtypes, and that evidence of these common mechanisms may be detected as gene expression profiles in whole blood. DESIGN: Cohort study. METHODS: Ninety subjects with uveitis including axial spondyloarthritis (n = 17), sarcoidosis (n = 13), inflammatory bowel disease (n = 12), tubulointerstitial nephritis with uveitis (n = 10), or idiopathic uveitis (n = 38) as well as 18 healthy controls were enrolled, predominantly at Oregon Health & Science University. RNA-Seq data generated from peripheral, whole blood identified 19,859 unique transcripts. We analyzed gene expression pathways via Kyoto Encyclopedia of Genes and Genomes and Gene Ontology (GO). We validated our list of upregulated genes by comparison to a previously published study on peripheral blood gene expression among 50 subjects with diverse forms of uveitis. RESULTS: Both the Kyoto Encyclopedia of Genes and Genomes and GO analysis identified multiple shared pathways or GO terms with a P value of <.0001. Almost all pathways related to the immune response and/or response to an infection. A total of 119 individual transcripts were upregulated by at least 1.5-fold and false discovery rate <.05, and 61 were downregulated by similar criteria. Comparing mRNA from our study with a false discovery rate <.05 and the prior report, we identified 10 common gene transcripts: ICAM1, IL15RA, IL15, IRF1, IL10RB, GSK3A, TYK2, MEF2A, MEF2B, and MEF2D. CONCLUSIONS: Many forms of uveitis share overlapping mechanisms. These data support the concept that a single therapeutic approach could benefit diverse forms of this disease.

Preliminary Report on Interleukin-22, GM-CSF, and IL-17F in the Pathogenesis of Acute Anterior Uveitis.

Purpose:Anterior uveitis is the most common anatomic subset of uveitis. We developed a novel multi-parametric flow cytometry panel to identify immune dysregulation signatures in HLA B27-associated acute anterior uveitis (AAU) and axial spondyloarthritis (AxSpA).Methods: We used fluorescence activated cell sorting to characterize T cell cytokine expression in stimulated T cell subsets from patients with AAU (n = 4) compared to healthy controls (n = 14) or subjects with AxSpA (n = 6).Results: Positive findings among subjects with AAU included a statistically significant increase in stimulated granulocyte-macrophage colony stimulating factor (GM-CSF), IL-17, and IL-22 synthesized by CD8 cells, a trend for stimulated ILC (innate lymphoid cells)-3 cells to synthesize more IL-22 (p = .07), and stimulated MAIT (mucosa associated innate lymphoid cells)-like cells that express the T cell receptor V alpha 7.2 to express IL-17A, IL-17F, and IL-22 in a greater percentage of cells relative to controls. IL-17F, GM- CSF, and IL-22 represent potentially novel targets in AAU.Conclusion: Our report is arguably the first to implicate IL-17F or ILC-3 and MAIT cells in the pathogenesis of AAU.Abbreviations AAU: acute anterior uveitis; AxSpA: axial spondyloarthritis; BASDAI: Bath ankylosing spondylitis disease activity index; CCR: chemokine receptor; DMSO: dimethylsulfoxide; EULAR:European League Against Rheumatism; FACS: fluorescence activated cell sorter; FBS: fetal bovine serum; FSC: orward light scatter; GM-CSF: granulocyte-macrophage colony stimulating factor; HC: healthy control; ILC: innate lymphoid cell; KIR: killer immunoglobulin receptor; MAIT: mucosal associated immune T cell; ND: not detected; NK: natural killer cell; OHSU-Oregon Health & Science University; PBMC: peripheral blood mononuclear cell; SSC: side light scatter; TCR: T cell receptor.

Revising the Diagnosis of Idiopathic Uveitis by Peripheral Blood Transcriptomics.

PURPOSE: To test the hypothesis that idiopathic uveitis can be categorized into subtypes based on gene expression from blood. DESIGN: Case control study. METHODS: We applied RNA-Seq to peripheral blood from patients with uveitis associated with 1 of 4 systemic diseases, including axial spondyloarthritis (n = 17), sarcoidosis (n = 13), inflammatory bowel disease (n = 12), tubulo-interstitial nephritis with uveitis (n = 10), or idiopathic uveitis (n = 38) as well as 18 healthy control subjects evaluated predominantly at Oregon Health and Science University. A high-dimensional negative binomial regression model implemented in the edgeR R package compared each disease group with the control subjects. The 20 most distinctive genes for each diagnosis were extracted. Of 80 genes, there were 75 unique genes. A classification algorithm was developed by fitting a gradient boosting tree with 5-fold cross-validation. Messenger RNA from subjects with idiopathic uveitis were analyzed to see if any fit clinically and by gene expression pattern with one of the diagnosable entities. RESULTS: For uveitis associated with a diagnosable systemic disease, gene expression profiling achieved an overall accuracy of 85% (balanced average of sensitivity plus specificity, P < .001). Although most patients with idiopathic uveitis presumably have none of these 4 associated systemic diseases, gene expression profiles helped to reclassify 11 of 38 subjects. CONCLUSIONS: Peripheral blood gene expression profiling is a potential adjunct in accurate differential diagnosis of the cause of uveitis. Validation of these results and characterization of the gene expression profile from additional discrete diagnoses could enhance the value of these observations.

Social anxiety and self-compassion in adolescents.

INTRODUCTION: Social anxiety disorder is a prevalent mental illness with a young onset age. Preliminary evidence suggested that low self-compassion may contribute to adult social anxiety, but research with youth has lagged far behind. This study investigated the relationship between selfcompassion and social anxiety in adolescents. It also examined the mediating role of three cognitive mechanisms: fear of negative evaluation, self-focused attention, and cognitive avoidance. METHODS: A total of 316 adolescents (age 14-18, 54% male) recruited in Scotland, UK, completed 7 questionnaires. RESULTS: Self-compassion was inversely correlated with social anxiety with a large effect size (r = -.551). This was partially mediated by fear of negative evaluation and cognitive avoidance, but not self-focused attention. Selfcompassion also predicted social anxiety above depression and anxiety symptoms. CONCLUSIONS: Our findings suggested that self-compassion could be an important factor in the development of social anxiety, and hence therapeutic techniques targeting self-compassion could potentially be beneficial in preventing or treating adolescent social anxiety.

Evaluation of risk communication in a mammography patient decision aid.

OBJECTIVES: We characterized patients' comprehension, memory, and impressions of risk communication messages in a patient decision aid (PtDA), Mammopad, and clarified perceived importance of numeric risk information in medical decision making. METHODS: Participants were 75 women in their forties with average risk factors for breast cancer. We used mixed methods, comprising a risk estimation problem administered within a pretest-posttest design, and semi-structured qualitative interviews with a subsample of 21 women. RESULTS: Participants' positive predictive value estimates of screening mammography improved after using Mammopad. Although risk information was only briefly memorable, through content analysis, we identified themes describing why participants value quantitative risk information, and obstacles to understanding. We describe ways the most complicated graphic was incompletely comprehended. CONCLUSIONS: Comprehension of risk information following Mammopad use could be improved. Patients valued receiving numeric statistical information, particularly in pictograph format. Obstacles to understanding risk information, including potential for confusion between statistics, should be identified and mitigated in PtDA design. PRACTICE IMPLICATIONS: Using simple pictographs accompanied by text, PtDAs may enhance a shared decision-making discussion. PtDA designers and providers should be aware of benefits and limitations of graphical risk presentations. Incorporating comprehension checks could help identify and correct misapprehensions of graphically presented statistics.

Impact of a decision aid on reducing uncertainty: pilot study of women in their 40s and screening mammography.

BACKGROUND: In 2009 the United States Preventive Services Task Force updated its breast cancer screening guidelines to recommend that average-risk women obtain a screening mammogram every two years starting at age 50 instead of annually starting at age 40. Inconsistencies in data regarding the benefit versus risk of routine screening for women less than 50-years-of-age led to a second recommendation - that women in their forties engage in a shared decision making process with their provider to make an individualized choice about screening mammography that was right for them. In response, a web-based interactive mammography screening decision aid was developed and evaluated. METHODS: The decision aid was developed using an agile, iterative process. It was further honed based on feedback from clinical and technical subject matter experts. A convenience sample of 51 age- and risk-appropriate women was recruited to pilot the aid. Pre-post decisional conflict and screening choice was assessed. RESULTS: Women reported a significant reduction in overall decisional conflict after using the decision aid (Z = -5.3, p < 0.001). These participants also reported statistically significant reductions in each of the decisional conflict subscales: feeling uncertain (Z = -4.7, p < 0.001), feeling uninformed (Z = -5.2, p < 0.001), feeling unclear about values (Z = -5.0, p < 0.001), and feeling unsupported (Z = -4.0, p < 0.001). However, a woman's intention to obtain a screening mammogram in the next 1-2 years was not significantly changed (Wilcoxon signed-rank Z = -1.508, p = 0.132). CONCLUSION: This mammography screening decision aid brings value to patient care not by impacting what a woman chooses but by lending clarity to why or how she chooses it.

Mammography Decision Aid Reduces Decisional Conflict for Women in Their Forties Considering Screening.

BACKGROUND: Clinical guidelines recommend a personalized approach to mammography screening for women in their forties; however, methods to do so are lacking. An evidence-based mammography screening decision aid was developed as an electronic mobile application and evaluated in a before-after study. METHODS: The decision aid (Mammopad) included modules on breast cancer, mammography, risk assessment, and priority setting about screening. Women aged 40-49 years who were patients of rural primary care clinics, had no major risk factors for breast cancer, and no mammography during the previous year were invited to use the decision aid. Twenty women participated in pretesting of the decision aid and 75 additional women completed the before-after study. The primary outcome was decisional conflict measured before and after using Mammopad. Secondary outcomes included decision self-efficacy and intention to begin or continue mammography screening. Differences comparing measures before versus after use were determined using Wilcoxon signed rank tests. RESULTS: After using Mammopad, women reported reduced decisional conflict based on mean Decisional Conflict Scale scores overall (46.33 versus 8.33; Z = -7.225; p < 0.001) and on all subscales (p < 0.001). Women also reported increased mean Decision Self-Efficacy Scale scores (79.67 versus 95.73; Z = 6.816, p < 0.001). Although 19% of women changed their screening intentions, this was not statistically significant. CONCLUSIONS: Women reported less conflict about their decisions for mammography screening, and felt more confident to make decisions after using Mammopad. This approach may help guide women through the decision making process to determine personalized screening choices that are appropriate for them.

Life in a landfill slum, children's health, and the Millennium Development Goals.

People living in slums can be considered left behind with regard to national successes in achieving Millennium Development Goals (MDGs). The objective of this study was to evaluate the living and working conditions of waste pickers and their children in a landfill slum located in the largest city in eastern Indonesia. A total of 113 people from the landfill slum and 1184 people from the general population participated in face-to-face interviews. Municipal solid waste (MSW) was analyzed for metals, metalloids and fecal indicator bacteria. Ambient air quality including particulate matter was measured in the landfill. Households in the landfill slum were 5.73 (p=0.04) times more likely to be below the international poverty line (MDG 1: Poverty) and 15.6 times (p<0.01) more likely to have no one in the household possessing a primary education (MDG 2: Universal Education), and 107 times (p<0.01) more likely not to have improved sanitation facilities (MDG 7: Environmental Sustainability) when compared to the general population. Diarrhea is one of the leading causes of death in children under five in Indonesia. Young children living in the landfill slum were 2.87 times (p=0.02) more likely to develop diarrhea than their general population counterparts. Other survey results and environmental measurements suggest that landfill slum children have additional adverse health effects (e.g. infections and poisoning). Poverty underlies several MDG issues that directly or indirectly affect child health. Therefore, eradicating extreme poverty will continue to be the most critical challenge for the MDGs beyond 2015.

Childhood acute respiratory infections and household environment in an Eastern Indonesian urban setting.

This pilot study evaluated the potential effect of household environmental factors such as income, maternal characteristics, and indoor air pollution on children's respiratory status in an Eastern Indonesian community. Household data were collected from cross-sectional (n = 461 participants) and preliminary childhood case-control surveys (pneumonia cases = 31 diagnosed within three months at a local health clinic; controls = 30). Particulate matter (PM2.5 and PM10) was measured in living rooms, kitchens, children's bedrooms, and outside areas in close proximity once during the case-control household interviews (55 homes) and once per hour from 6 a.m. to midnight in 11 homes. The household survey showed that children were 1.98 times (p = 0.02) more likely to have coughing symptoms indicating respiratory infection, if mothers were not the primary caregivers. More children exhibited coughing if they were not exclusively breastfed (OR = 2.18; p = 0.06) or there was a possibility that their mothers were exposed to environmental tobacco smoke during pregnancy (OR = 2.05; p = 0.08). This study suggests that household incomes and mother's education have an indirect effect on childhood pneumonia and respiratory illness. The concentrations of PM2.5 and PM10 ranged from 0.5 to 35.7 µg/m3 and 7.7 to 575.7 µg/m3, respectively, based on grab samples. PM was significantly different between the case and control groups (p < 0.01). The study also suggests that ambient air may dilute indoor pollution, but also introduces pollution into the home from the community environment. Effective intervention programs need to be developed that consider multiple direct and indirect risk factors to protect children.

Characterization of ceftazidime resistance mechanisms in clinical isolates of Burkholderia pseudomallei from Australia.

Burkholderia pseudomallei is a gram-negative bacterium that causes the serious human disease, melioidosis. There is no vaccine against melioidosis and it can be fatal if not treated with a specific antibiotic regimen, which typically includes the third-generation cephalosporin, ceftazidime (CAZ). There have been several resistance mechanisms described for B. pseudomallei, of which the best described are amino acid changes that alter substrate specificity in the highly conserved class A ß-lactamase, PenA. In the current study, we sequenced penA from isolates sequentially derived from two melioidosis patients with wild-type (1.5 µg/mL) and, subsequently, resistant (16 or ≥256 µg/mL) CAZ phenotypes. We identified two single-nucleotide polymorphisms (SNPs) that directly increased CAZ hydrolysis. One SNP caused an amino acid substitution (C69Y) near the active site of PenA, whereas a second novel SNP was found within the penA promoter region. In both instances, the CAZ resistance phenotype corresponded directly with the SNP genotype. Interestingly, these SNPs appeared after infection and under selection from CAZ chemotherapy. Through heterologous cloning and expression, and subsequent allelic exchange in the native bacterium, we confirmed the role of penA in generating both low-level and high-level CAZ resistance in these clinical isolates. Similar to previous studies, the amino acid substitution altered substrate specificity to other ß-lactams, suggesting a potential fitness cost associated with this mutation, a finding that could be exploited to improve therapeutic outcomes in patients harboring CAZ resistant B. pseudomallei. Our study is the first to functionally characterize CAZ resistance in clinical isolates of B. pseudomallei and to provide proven and clinically relevant signatures for monitoring the development of antibiotic resistance in this important pathogen.

Dominance of multidrug resistant CC271 clones in macrolide-resistant streptococcus pneumoniae in Arizona.

BACKGROUND: Rates of resistance to macrolide antibiotics in Streptococcus pneumoniae are rising around the world due to the spread of mobile genetic elements harboring mef(E) and erm(B) genes and post-vaccine clonal expansion of strains that carry them. RESULTS: Characterization of 592 clinical isolates collected in Arizona over a 10 year period shows 23.6% are macrolide resistant. The largest portion of the macrolide-resistant population, 52%, is dual mef(E)/erm(B)-positive. All dual-positive isolates are multidrug-resistant clonal lineages of Taiwan19F-14, mostly multilocus sequence type 320, carrying the recently described transposon Tn2010. The remainder of the macrolide resistant S. pneumoniae collection includes 31% mef(E)-positive, and 9% erm(B)-positive strains. CONCLUSIONS: The dual-positive, multidrug-resistant S. pneumoniae clones have likely expanded by switching to non-vaccine serotypes after the heptavalent pneumococcal conjugate vaccine release, and their success limits therapy options. This upsurge could have a considerable clinical impact in Arizona.

Multidrug-Resistant Staphylococcus aureus in US Meat and Poultry.

We characterized the prevalence, antibiotic susceptibility profiles, and genotypes of Staphylococcus aureus among US meat and poultry samples (n = 136). S. aureus contaminated 47% of samples, and multidrug resistance was common among isolates (52%). S. aureus genotypes and resistance profiles differed significantly among sample types, suggesting food animal-specific contamination.