Mass cytometry analysis reveals altered immune profiles in patients with coronary artery disease.

Objective: The importance of inflammation in atherosclerosis is well accepted, but the role of the adaptive immune system is not yet fully understood. To further explore this, we assessed the circulating immune cell profile of patients with coronary artery disease (CAD) to identify discriminatory features by mass cytometry. Methods: Mass cytometry was performed on patient samples from the BioHEART-CT study, gated to detect 82 distinct cell subsets. CT coronary angiograms were analysed to categorise patients as having CAD (CAD+) or having normal coronary arteries (CAD-). Results: The discovery cohort included 117 patients (mean age 61 ± 12 years, 49% female); 79 patients (68%) were CAD+. Mass cytometry identified changes in 15 T-cell subsets, with higher numbers of proliferating, highly differentiated and cytotoxic cells and decreases in naïve T cells. Five T-regulatory subsets were related to an age and gender-independent increase in the odds of CAD incidence when expressing CCR2 (OR 1.12), CCR4 (OR 1.08), CD38 and CD45RO (OR 1.13), HLA-DR (OR 1.06) and Ki67 (OR 1.22). Markers of proliferation and differentiation were also increased within B cells, while plasmacytoid dendritic cells were decreased. This combination of changes was assessed using SVM models in discovery and validation cohorts (area under the curve = 0.74 for both), confirming the robust nature of the immune signature detected. Conclusion: We identified differences within immune subpopulations of CAD+ patients which are indicative of a systemic immune response to coronary atherosclerosis. This immune signature needs further study via incorporation into risk scoring tools for the precision diagnosis of CAD.

Lipidomics Profiling and Risk of Coronary Artery Disease in the BioHEART-CT Discovery Cohort.

The current coronary artery disease (CAD) risk scores for predicting future cardiovascular events rely on well-recognized traditional cardiovascular risk factors derived from a population level but often fail individuals, with up to 25% of first-time heart attack patients having no risk factors. Non-invasive imaging technology can directly measure coronary artery plaque burden. With an advanced lipidomic measurement methodology, for the first time, we aim to identify lipidomic biomarkers to enable intervention before cardiovascular events. With 994 participants from BioHEART-CT Discovery Cohort, we collected clinical data and performed high-performance liquid chromatography with mass spectrometry to determine concentrations of 683 plasma lipid species. Statin-naive participants were selected based on subclinical CAD (sCAD) categories as the analytical cohort (n = 580), with sCAD+ (n = 243) compared to sCAD- (n = 337). Through a machine learning approach, we built a lipid risk score (LRS) and compared the performance of the existing Framingham Risk Score (FRS) in predicting sCAD+. We obtained individual classifiability scores and determined Body Mass Index (BMI) as the modifying variable. FRS and LRS models achieved similar areas under the receiver operating characteristic curve (AUC) in predicting the validation cohort. LRS enhanced the prediction of sCAD+ in the healthy-weight group (BMI < 25 kg/m2), where FRS performed poorly and identified individuals at risk that FRS missed. Lipid features have strong potential as biomarkers to predict CAD plaque burden and can identify residual risk not captured by traditional risk factors/scores. LRS compliments FRS in prediction and has the most significant benefit in healthy-weight individuals.

Overcoming cohort heterogeneity for the prediction of subclinical cardiovascular disease risk.

Cardiovascular disease remains a leading cause of mortality with an estimated half a billion people affected in 2019. However, detecting signals between specific pathophysiology and coronary plaque phenotypes using complex multi-omic discovery datasets remains challenging due to the diversity of individuals and their risk factors. Given the complex cohort heterogeneity present in those with coronary artery disease (CAD), we illustrate several different methods, both knowledge-guided and data-driven approaches, for identifying subcohorts of individuals with subclinical CAD and distinct metabolomic signatures. We then demonstrate that utilizing these subcohorts can improve the prediction of subclinical CAD and can facilitate the discovery of novel biomarkers of subclinical disease. Analyses acknowledging cohort heterogeneity through identifying and utilizing these subcohorts may be able to advance our understanding of CVD and provide more effective preventative treatments to reduce the burden of this disease in individuals and in society as a whole.

Third-party CMV- and EBV-specific T-cells for first viral reactivation after allogeneic stem cell transplant.

Virus-specific T-cells (VSTs) from third-party donors mediate short- and long-term antiviral effects in allogeneic hematopoietic stem cell transplant (HSCT) recipients with relapsed or refractory viral infections. We investigated early administration of third-party VSTs, together with antiviral therapy in patients requiring treatment for first cytomegalovirus (CMV) or Epstein-Barr virus (EBV) infection. Thirty HSCT patients were treated with 1 to 4 VST infusions (2 × 107 cells/m2; CMV n=27, EBV n=3) at a median of 4 days after initiation of antiviral treatment. The overall viral response rate was 100%, with a complete response (CR) rate of 94%. Of the 28 patients who achieved a CR, 23 remained virus PCR negative (n=9) or below quantitation limit (n=14) for the duration of follow-up. Four patients had brief episodes of quantifiable reactivation not requiring additional therapy, and one required a second infusion after initial CR, remaining PCR negative thereafter. All 3 patients treated for EBV post-transplant lymphoproliferative disorder achieved sustained CR. Rates of aGVHD and cGVHD after infusion were 13% and 23%, respectively. There were no serious infusion-related adverse events. VST infusion was associated with rapid recovery of CD8+CD45RA-CD62L- and a slower recovery of CD4+CD45RA-CD62L- effector memory T-cells; CMV-specific T-cells comprised up to 13% of CD8+ cells. At 1 year post-transplant, non-relapse mortality was 10%, cumulative incidence of relapse was 7%, overall survival was 88% and 25 of 27 patients had ECOG status of 0 or 1. Early administration of third-party VSTs in conjunction with antiviral treatment appears safe and leads to excellent viral control and clinical outcomes. Registered on Australian New Zealand Clinical Trials Registry as #ACTRN12618000343202.

treekoR: identifying cellular-to-phenotype associations by elucidating hierarchical relationships in high-dimensional cytometry data.

High-throughput single-cell technologies hold the promise of discovering novel cellular relationships with disease. However, analytical workflows constructed for these technologies to associate cell proportions with disease often employ unsupervised clustering techniques that overlook the valuable hierarchical structures that have been used to define cell types. We present treekoR, a framework that empirically recapitulates these structures, facilitating multiple quantifications and comparisons of cell type proportions. Our results from twelve case studies reinforce the importance of quantifying proportions relative to parent populations in the analyses of cytometry data - as failing to do so can lead to missing important biological insights.

Metabolic Signatures in Coronary Artery Disease: Results from the BioHEART-CT Study.

Despite effective prevention programs targeting cardiovascular risk factors, coronary artery disease (CAD) remains the leading cause of death. Novel biomarkers are needed for improved risk stratification and primary prevention. To assess for independent associations between plasma metabolites and specific CAD plaque phenotypes we performed liquid chromatography mass-spectrometry on plasma from 1002 patients in the BioHEART-CT study. Four metabolites were examined as candidate biomarkers. Dimethylguanidino valerate (DMGV) was associated with presence and amount of CAD (OR) 1.41 (95% Confidence Interval [CI] 1.12-1.79, p = 0.004), calcified plaque, and obstructive CAD (p < 0.05 for both). The association with amount of plaque remained after adjustment for traditional risk factors, ß-coefficient 0.17 (95% CI 0.02-0.32, p = 0.026). Glutamate was associated with the presence of non-calcified plaque, OR 1.48 (95% CI 1.09-2.01, p = 0.011). Phenylalanine was associated with amount of CAD, ß-coefficient 0.33 (95% CI 0.04-0.62, p = 0.025), amount of calcified plaque, (ß-coefficient 0.88, 95% CI 0.23-1.53, p = 0.008), and obstructive CAD, OR 1.84 (95% CI 1.01-3.31, p = 0.046). Trimethylamine N-oxide was negatively associated non-calcified plaque OR 0.72 (95% CI 0.53-0.97, p = 0.029) and the association remained when adjusted for traditional risk factors. In targeted metabolomic analyses including 53 known metabolites and controlling for a 5% false discovery rate, DMGV was strongly associated with the presence of calcified plaque, OR 1.59 (95% CI 1.26-2.01, p = 0.006), obstructive CAD, OR 2.33 (95% CI 1.59-3.43, p = 0.0009), and amount of CAD, ß-coefficient 0.3 (95% CI 0.14-0.45, p = 0.014). In multivariate analyses the lipid and nucleotide metabolic pathways were both associated with the presence of CAD, after adjustment for traditional risk factors. We report novel associations between CAD plaque phenotypes and four metabolites previously associated with CAD. We also identified two metabolic pathways strongly associated with CAD, independent of traditional risk factors. These pathways warrant further investigation at both a biomarker and mechanistic level.

Bioinformatics Analysis of Chicken miRNAs Associated with Monocyte to Macrophage Differentiation and Subsequent IFNγ Stimulated Activation.

BACKGROUND: The goal of this project was to characterize the molecular and cellular roles of various gene targets regulated by miRNAs identified in differentiating and stimulating avian macrophages. Once a monocyte arrives to a site of infection, local signals induce a redistribution of resources into a macrophage phenotype. This may involve upregulating pathogen pattern recognizing receptors and increasing the efficiency of lysosomal biogenesis, while simultaneously recycling components involved in circulatory migration and leukocyte extravasation. a monocyte tooled with chemokine surface receptors and an internal cytoskeletal structure geared towards mobility may efficiently sense, react, and migrate toward a site of infection. METHODS: Peripheral blood derived monocytes were purified and cultured from young chickens. RNA sequencing was performed on both peripheral blood monocytes during differentiation into macrophages and on mature macrophages following stimulation with interferon gamma. A set of microRNAs were identified and investigated using bioinformatics methods to ascertain their potential role in avian macrophage biology. RESULTS: Among a number of miRNAs that are found to be expressed in avian macrophages, we focused on eight specific miRNAs (miR-1618, miR-1586, miR-1633, miR-1627, miR-1646, miR-1649, miR-1610, miR-1647) associated with macrophage differentiation and activation. Expression profiles of microRNAs were characterized during differentiation and activation. Candidate miRNA targets were implicated in processes including Wnt signaling, ubiquitination, PPAR mediated macrophage function, vesicle mediated cytokine trafficking, and WD40 domain protein functions. CONCLUSION: A global theme for macrophage function that may be modulated by microRNAs is the comprehensive redistribution of the cell's protein repertoire. This redistribution involves two processes: 1) the degradation and recycling of unneeded cytoplasmic and membrane components and 2) the mobilization of newly synthesized cellular components via vesicular trafficking. Generally, it appears that macrophages need to closely regulate gene expression for differentiation to be able to activate successfully in response to a pathogen. This is a process in which miRNAs participate by affecting several pathways critical for both, differentiation and activation.

Impact from point-of-care devices on emergency department patient processing times compared with central laboratory testing of blood samples: a randomised controlled trial and cost-effectiveness analysis.

OBJECTIVE: To determine if time to disposition decisions for emergency department (ED) patients can be reduced when blood tests are processed using point-of-care (POC) devices and to conduct a cost-effectiveness analysis of POC compared with laboratory testing. METHODS: This randomised trial enrolled adults suspected of an acute coronary syndrome or presenting with conditions considered to only require blood tests available by POC. Participants were randomised to have blood tests processed by POC or laboratory. Outcomes measured were time to disposition decision and ED length-of-stay (LOS). The cost-effectiveness analysis calculated the total and mean costs per ED presentation, as well as total and mean benefits in time saved to disposition decision. RESULTS: There were 410 POC participants and 401 controls. The mean times to a disposition decision for POC versus controls were 3.24 and 3.50 h respectively, a difference of 7.6% (95% CI 0.4% to 14.3%, p=0.04), and 4.32 and 4.52 h respectively for ED LOS, a difference of 4.4% (95% CI -2.7% to 11.0%, p=0.21). Improved processing time was greatest for participants enrolled by senior staff with a reduction in time to disposition decision of 19.1% (95% CI 7.3% to 29.4%, p<0.01) and ED LOS of 15.6% (95% CI 4.9% to 25.2%, p=0.01). Mean pathology costs were $12 higher in the POC group (95% CI $7 to $18) and the incremental cost-effectiveness ratio was $113 per hour saved in time to disposition decision for POC compared with standard laboratory testing. CONCLUSIONS: Small improvements in disposition decision time were achieved with POC testing for a moderate increase in cost. Greatest benefit may be achieved when POC is targeted to senior medical staff.

Causes of constraints to patient flow in emergency departments: a comparison between staff perceptions and findings from the Patient Flow Study.

OBJECTIVES: The objectives of the study were: (i) to survey staff perceptions of causes of delay in patients' journeys through the ED; (ii) to identify and analyse key constraints to patient flow using real-time diagnostic/decision support software (Patient Flow Study); and (iii) to assess the correlation between staff perceptions and data from the Patient Flow Study. METHODS: ED and non-ED staff were surveyed prior to the Patient Flow Study. The survey involved ranking the likely reasons for delay at three set points after triage (160, 320 and 480 min). Real-time data on delay in patients' journeys through the ED were collected over a period of 5 weeks. The correlation between staff perceptions and study data at the three time points was calculated using Spearman's rank correlation coefficient. Subgroup analysis was performed on the basis of staff position, years of experience at St George and whether they had previously attended training on constraints to flow. RESULTS: A total of 68 staff responded to the questionnaire (response rate 42%). During the study period, 4555 ED attendances (97% of all presentations) were analysed for causes of constraint. Strong correlation between staff perceptions and real-time data was only found among some subgroups at the point 160 min from triage. CONCLUSIONS: Overall, staff perceptions regarding causes of constraint to patient flow do not correlate well with data obtained from real-time analysis.

Deck the halls with rows of trolleys...emergency departments are busiest over the Christmas holiday period.

OBJECTIVES: To assess changes in emergency department (ED) activity and visits to EDs that could have been managed by general practitioners (GP-type visits) in the Christmas and New Year holiday period compared with the rest of the year. DESIGN AND SETTING: Retrospective descriptive and analytical comparison of New South Wales ED visits in the holiday period and the rest of the year; data were obtained from the NSW Emergency Department Data Collection database for the period 2001 to early 2006. More detailed information in 2005-2006 allowed GP-type visits to be assessed in this period only. MAIN OUTCOME MEASURES: The change in the number and percentage of weekly ED visits during the holiday period. RESULTS: Between 2001 and 2006, average weekly counts of ED visits increased by 9% (95% CI, 7%-11%) during the holiday period. The holiday increase was largely accounted for by visits that were less urgent, and for patients who were not admitted, did not arrive by ambulance, had a shorter treatment time and arrived between 08:00 and midnight. In 2005-2006, average weekly counts of GP-type visits increased by 21% (95% CI, 15%-28%) compared with 8% (95% CI, 4%-12%) for ED visits overall. However, GP-type visits accounted for only 39% of the additional holiday visits. GP-type visits increased mainly for adults and more in urban than rural areas. CONCLUSIONS: The Christmas and New Year period is the busiest time of year for NSW EDs. However, only some of the additional holiday visits can be attributed to GP-type visits. Improving access to GPs, but also to broader hospital and community-based health care services over the holiday period, should be considered for managing the excess demand.

Acute symptomatic pulmonary embolism associated with long haul air travel to Sydney.

The association between air travel and pulmonary embolism (PE) is recognized, but the absolute risk has not been quantified. Due to its geographical isolation, more than 50% of international travelers arrive at Sydney airport after a flight of'at least 9 hours. Patients who become acutely unwell upon disembarkation are taken to one of two hospitals. In this study we reviewed the presentations at these two hospitals. A retrospective analysis of all patients presenting to the emergency departments (ED) directly from Sydney International Airport with symptomatic PE over a three-year period was undertaken. Over 36 months, 17 patients were admitted from Sydney International Airport to the ED with acute PE. All patients had flown for at least 9 hours. Seven patients had risk factors for thromboambolic disease. During the period of our review 6.58 million passengers arrived in Sydney on flights of at least 9 hours in duration, representing an incidence of 2.57 per million. In conclusion, the incidence of acutely symptomatic PE in association with long distance air travel is low and the majority of patients survive following hospital presentation.

The impact of trauma case management on patient outcomes.

BACKGROUND: Previous investigations demonstrate that nursing case management in the acute care setting improves patient outcomes. However, these findings provide limited information specific to trauma patients. METHOD: The effect of trauma case management (TCM) was measured using practice-specific variables such as in-hospital complications, missed injury rates, and length of stay. Other measures included staff satisfaction and use of allied health services. Data from 148 patients with an Injury Severity Score < 16 in the 5 months after the introduction of TCM were compared with 327 patients from the previous 12 months. RESULTS: Results demonstrated a trend toward reduced length of stay overall, more so in the older and more severely injured. TCM greatly improved missed injury detection rates (p < 0.0015) and coordinated allied health use more efficiently (p < 0.0001). Staff surveys exhibited a perceived dramatic improvement in the effectiveness of patient care (p < 0.0001). CONCLUSION: The introduction of TCM improved the efficiency and effectiveness of trauma patient care in our institution.