PAXgene Tubes Research Articles

RNA profiles to distinguish multiple sclerosis (MS) and neuromyelitis optica (NMO) in peripheral whole blood (S9.003)

RNA profiles to distinguish multiple sclerosis (MS) and neuromyelitis optica (NMO) in peripheral whole blood (S9.003)

Abstract 1040: Evaluation of blood collection tube’s performance for analysis of liquid biopsies by flow and mass cytometry

Abstract The detection of liquid biopsies, including circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) have demonstrated promise as non-invasive assays for monitoring response to therapy for patients with cancer. While ctDNA cannot predict transcriptomic and proteomic changes in response to therapy, we can measure direct cell surface antigen and gene expression changes in CTCs. In pediatric settings, blood samples are often collected from multi-institutional trials. Therefore, to translate liquid biopsies into routine practice, it is important to explore variables most likely to impact the functionality of CTCs including time to process the sample, prospective cryopreservation, blood collection tube and fixative reagents. Cell stabilizer tubes such as Streck Cell-Free DNA, CellSave, CellRescue, or PAXgene ccfDNA have been designed to prevent cell lysis and release of genomic DNA into the plasma, often observed in standard EDTA tubes. In pediatrics, we are often limited by the volume of blood we can draw for research. To this end, we have established an innovative pipeline to collect ctDNA, CTCs, and matched germline DNA from a single blood sample. Blood samples were processed for plasma extraction and CTC isolation after 4h, 24h, and 48h. We isolated rare CTCs from whole blood by combining peripheral blood cell depletion with antigen-based CTC identification. We then evaluated CTC recovery rate and cell membrane integrity (cell-membrane permeabilization) across all blood collection tubes by flow or mass cytometry. Additionally, we integrated whole transcriptome analysis of RNA transcripts in individually sorted CTCs. In this study, we detected similar levels of cell-free DNA, germline DNA and ctDNA in healthy donors and patient samples collected in the DNA stabilizer tubes up to 48h after blood collection. Surprisingly, frequent clotting was observed in the cell layer of samples collected in PAXgene tubes only 24h after blood collection, preventing further analysis of CTC quality in these tubes. At 4h, EDTA tubes and stabilizer tubes seem to perform similarly in preserving CTC integrity. However, at later timepoints, CTC recovery rate, RNA quality and cell membrane integrity were significantly higher in CellRescue tubes. Finally, we showed that CTC count, and RNA quality were slightly lower in patient samples enumerated after cryopreservation compared to the paired-samples processed the same day in both EDTA and cell-stabilizing tubes. In conclusion, we validated the CellRescue tubes as the best candidate to reduce cell lysis and protect against cell membrane permeabilization and mRNA degradation for up to 48 hours, allowing sample collection from multi-institutional studies. To the best of our knowledge, this study is the first to compare the key steps in handling blood samples and how this affects transcriptomic-based assay performance. Citation Format: Anne-Florence Blandin, Sarah Finstuen-Magro, Samuel Abbou, Kelly Klega, Alejandra E. Aguilar, Mohammad Tanhaemami, Maria Ana Isabel C. De Los Santos, Joadly Duplan, Brian Crompton. Evaluation of blood collection tube’s performance for analysis of liquid biopsies by flow and mass cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1040.

Abstract WP223: Identification Of Cerebral Amyloid Angiopathy With A Leukocyte Gene Expression Profile

Introduction: Cerebral amyloid angiopathy (CAA) is a cerebral small vessel disease featuring beta-amyloid deposits within the cerebral vasculature. It is a major cause of cognitive decline and intracerebral hemorrhage in the elderly. This study evaluated whether gene expression profiles in peripheral blood can differentiate patients with CAA from vascular risk factor and healthy controls. Methods: In 27 patients with CAA blood cell gene expression was compared to 55 controls. Total RNA was isolated from PAXgene tubes and measured by RNA sequencing. Differentially expressed genes between CAA and controls were identified by ANOVA adjusting for age and sex. Functional pathway analysis identified pathways associated with CAA. A prediction model to distinguish CAA from controls was developed using linear discriminant analysis with feature selection by forward selection. Model performance was evaluated by 10-fold leave-one-out cross validation. Results: 686 differentially expressed genes were identified (p<0.05, fold change >|1.2|), of interest ADAM15, CAMK1D, CAP1 and TGFB1 . Canonical pathway analysis identified cell movement of phagocytes, activation of phagocytes, CREB, degranulation of leukocytes, immune response of cells, inflammatory response, and IL-23 signaling. A 24 gene panel differentiated patients with CAA from controls with >95% sensitivity and specificity. The identified genes reveal differences in immune system regulation in patients with CAA compared to vascular risk factor and healthy control patients. Differences identified include a potential shift in beta-amyloid uptake by phagocytes ( TGFB1, CREB, CAMK1D ), an increase in vascular extracellular matrix disruption ( ADAM15, CAP1 ), and a possible alteration in amyloid precursor processing ( BRI3BP, SORCS3 ). Conclusion: Differences in peripheral leukocyte gene expression are present in patients with CAA compared to control patients. These relate to differences in immune activation and signaling associated with CAA. The differences in blood cell gene expression shows promise to distinguish CAA from controls, though further evaluation in larger cohorts is required to further evaluate potential diagnostic utility of this gene expression profile.

Abstract WP242: Transcriptomic Changes In Peripheral Blood Leukocytes Associated With The Cerebral Amyloid Angiopathy Small Vessel Disease Score

Introduction: Cerebral amyloid angiopathy (CAA) is a cerebrovascular disease characterized by beta-amyloid deposition within cerebral vessels. Clinical markers of CAA severity include recurrent ICH, cognitive decline, and imaging features of CAA including microhemorrhage, dilated perivascular spaces, cortical superficial siderosis, and white matter hyperintensities. CAA is an important cause of cognitive decline and intracerebral hemorrhage in the elderly, leading to the need for CAA treatments. The contribution of the immune system to CAA severity is not well understood. This study sought to evaluate changes in peripheral leukocyte gene expression associated with CAA severity. Methods: In 22 patients with CAA peripheral blood was collected into PAXgene tubes. RNA was isolated, and sequencing performed. Differentially expressed genes based on CAA small vessel disease (CAA-SVD) score as a marker of CAA severity were identified using ANOVA and functional pathway analysis. The relationship between leukocyte gene expression and CAA severity were assessed. Results: ANOVA was used to identify genes that were associated with CAA-SVD score (p≤0.05, partial correlation coefficient ≥;|0.5|). HDAC11, IL23A, TRAIL, TRAILR1 , TRAILR2 , and ICAM-1 were associated with CAA severity (CAA-SVD score). Canonical pathway analysis identified included induction of T lymphocytes, binding of antigen presentation cells, IL-12 signaling in macrophages/monocytes, activation of phagocytes, tight junction signaling, and activation of antigen presenting cells to be associated with CAA severity (p≤0.05). Conclusion: An association between the peripheral immune system and CAA severity was identified. Changes in neutrophil, monocyte, and Th17 cell gene expression in peripheral blood was associated with CAA severity. Further evaluation of immune system changes associated with CAA severity is needed to understand contribution to cerebral small vessel disease, cognitive decline, and potential roles as treatment targets or risk stratification markers in CAA.

Evaluation of the host immune response assay SeptiCyte RAPID for potential triage of COVID-19 patients

Tools for the evaluation of COVID-19 severity would help clinicians with triage decisions, especially the decision whether to admit to ICU. The aim of this study was to evaluate SeptiCyte RAPID, a host immune response assay (Immunexpress, Seattle USA) as a triaging tool for COVID-19 patients requiring hospitalization and potentially ICU care. SeptiCyte RAPID employs a host gene expression signature consisting of the ratio of expression levels of two immune related mRNAs, PLA2G7 and PLAC8, measured from whole blood samples. Blood samples from 146 adult SARS-CoV-2 (+) patients were collected within 48 h of hospital admission in PAXgene blood RNA tubes at Hospital del Mar, Barcelona, Spain, between July 28th and December 1st, 2020. Data on demographics, vital signs, clinical chemistry parameters, radiology, interventions, and SeptiCyte RAPID were collected and analyzed with bioinformatics methods. The performance of SeptiCyte RAPID for COVID-19 severity assessment and ICU admission was evaluated, relative to the comparator of retrospective clinical assessment by the Hospital del Mar clinical care team. In conclusion, SeptiCyte RAPID was able to stratify COVID-19 cases according to clinical severity: critical vs. mild (AUC = 0.93, p < 0.0001), critical vs. moderate (AUC = 0.77, p = 0.002), severe vs. mild (AUC = 0.85, p = 0.0003), severe vs. moderate (AUC = 0.63, p = 0.05). This discrimination was significantly better (by AUC or p-value) than could be achieved by CRP, lactate, creatine, IL-6, or D-dimer. Some of the critical or severe cases had “early” blood draws (before ICU admission; n = 33). For these cases, when compared to moderate and mild cases not in ICU (n = 37), SeptiCyte RAPID had AUC = 0.78 (p = 0.00012). In conclusion, SeptiCyte RAPID was able to stratify COVID-19 cases according to clinical severity as defined by the WHO COVID-19 Clinical Management Living Guidance of January 25th, 2021. Measurements taken early (before a patient is considered for ICU admission) suggest that high SeptiScores could aid in predicting the need for later ICU admission.

Transcriptomic Analysis of Whole Blood in Admixed Latinx Alzheimer Disease Cohorts

AbstractBackgroundIdentifying the genes and biological pathways involved in Alzheimer disease (AD) is critical in the effort to develop effective therapies. Significant work has identified genetic variants conferring risk and protection for AD in individuals of diverse ancestries, but identification of downstream functional effects including modulation of gene regulation is lacking, particularly in individuals of diverse ancestries. Therefore, to explore transcriptional changes between clinically diagnosed AD and cognitively intact age‐matched controls, herein we analyzed RNA sequencing data from peripheral blood collected from individuals of admixed genetic backgrounds.MethodTotal RNA was extracted from peripheral whole blood stored in PAXGene tubes from 47 Cubans (22 AD and 25 controls), 85 Peruvians (41 AD and 44 controls), and 168 Puerto Ricans (88 AD and 80 controls). PolyA selected mRNA was sequenced to more than 40 million paired end read per sample on the Illumina NovaSeq 6000. The bioinformatic pipeline included mapping to the human reference genome (GRCh38), gene quantifications against the GENCODE v35 annotation set, and differential expression was calculated using DESeq2 with sex, age at blood draw, and count of APOEe4 alleles as covariates. Functional categorization was performed by gene set enrichment of gene ontology and KEGG pathways.ResultAcross the cohorts, a total of 358 protein‐coding genes (FDR ≤ 0.05, Fold change ≥ 1.25) were differentially expressed with 238 down‐regulated and 120 up‐regulated in AD relative to controls. Despite the few genes overlapping between ethnicities, pathway analysis revealed common pathways including up‐regulation of genes involved in inflammation and RNA processing and down‐regulation of genes involved in cellular detoxification and lipid transport, among others. Interestingly, while few specific genes overlap in differential expression overlap with a published set of genes from non‐Hispanic Europeans and African Americans (Griswold et al, 2018), the pathways identified are similar.ConclusionOur analysis reveals a signature of gene expression that implicates increased inflammation and decreased cellular detoxification based on gene expression analysis in admixed Latinx AD. Convergence of pathways across these and African American and European cohorts supports the idea of distinct genes but similar underlying pathological processes contributing to AD across individuals of diverse ancestries.

Modulation of gene expression profile following consumption of high-dairy products in subjects with hyperinsulinemia

Background and aimDysregulation of gene expression is associated to a higher risk of type 2 diabetes (T2D). Further, research indicates that dairy consumption may potentially affect gene expression.The aim of this study was to examine if genes and pathways associated with T2D are differentially changed in subjects with hyperinsulinemia after high dairy (HD) diet. Methods and resultsTen obese patients with hyperinsulinemia who consumed HD (4 servings/day according to the Canadian Food Guide (2007)) for six weeks participated in this study. Before and after HD consumption, fasting blood samples were collected. Blood was taken in PAX-gene tubes and RNA was extracted and analyzed using Clariom S microarrays. Results indicated that 236 genes (137 up-regulated and 99 down-regulated; fold change (FC) ≥ ±1.2; p < 0.05) were expressed differentially between before and after HD intake. Genes related to pathways associated with insulin signaling and inflammation, such as olfactory receptor activity, G-protein-coupled receptors (GPCR), phosphatidylinositol-3-OHKinase (PI3K)/AKT2 (PI3K-AKT2), Ras signaling, Mitogen-Activated Protein Kinase (MAPK) were altered following HD. ConclusionOverall, results suggest a potential protective effect of HD intake in individuals at risk of T2D through modification of gene expression profiles. Registration number for clinical studiesNCT02961179.

PSXI-3 Decreased Circulating Concentrations of CD21 and CD19 in Canines with Heart Murmur and Associated Valvular Cardiovascular Disorder

Abstract Myxomatous mitral valve disease (MMVD) is a commonly diagnosed canine heart disease characterized by a thickening of the mitral valve. While it affects dogs of all genetic backgrounds, the breeds most commonly affected include small to medium dogs. The underlying causes of MMVD are not clear but pathological changes include alterations in the extracellular matrix and collagen, and activation of matrix metalloproteinases (MMPs), leading to subsequent myocardial remodeling. Inflammatory signaling pathways are hypothesized to influence the progression of the valve lesion contributing to valvular regurgitation. One of the clinical characteristics of MMVD in dogs is heart murmur. We assessed the circulating concentrations of inflammatory markers in dogs that were clinically diagnosed by a veterinarian as having MMVD with at least grade 4 heart murmur. RNA was extracted from blood samples collected in PAXgene tubes from dogs with heart murmurs (n=6; 4 dogs had grade 5/6 and 2 dogs had 4/6 heart murmur; 9.3-15.11 yr) and control dogs (n=6; 9.4-15.10 yr) with no history of cardiovascular problems. Gene expression was assessed using NanoString nCounter and analysis performed using Nanostring’s nSolver software. There was a significant decrease in the concentrations of CD21 (-1.88 fold) and CD19 (-1.84 fold) in MMVD compared with controls (both p&amp;lt; 0.05). CD21 and CD19 are parts of CD21/CD19/CD81 complex that act as a co-receptor to the B cell receptor. A decrease in CD19 and CD21 indicates a possible disruption in the homeostasis of B-cell activation and may contribute to chronic inflammation. In addition, pro-inflammatory cytokines/chemokines and receptors that were increased in MMVD include CCL5 (1.74), Lymphotoxin β (1.49), Interferon-γ (1.35), CCL3 (1.28), CCL4 (1.28), IL-18R1 (1.27) and MMP-9 (1.27, all NS). Our results indicate the importance of targeting the B-lymphocyte-associated signaling pathway and selected cytokines for restoring immune homeostasis and reducing inflammation in MMVD.

A cluster of type II interferon-regulated genes associates with disease activity in patients with systemic lupus erythematosus

Upregulation of interferon-regulated genes (IRGs), denoted IFN signature, in peripheral blood has been used as an indirect measure of IFN pathway activation in patients with systemic lupus erythematosus (SLE). However, it has not been determined, which IFN signatures that optimally reflect clinical disease activity.In this study, we determined an IFN signature based on the expression of 128 IRGs in whole blood from 34 SLE patients in a cross-sectional (CS) study, 11 with active lupus nephritis followed longitudinally (LS) and 15 healthy controls. Blood samples were collected in PAXgene tubes and RNA was extracted and purified using a PAXgene blood RNA kit (Qiagen). Gene expression was measured using the NanoString nCounter Gene Expression platform.The CS SLE patients with higher disease activity displayed thrice as many upregulated IRGs (n = 46) as the rest. These IRGs clustered in three groups, consisting of IRGs known to be predominantly stimulated by type I (gene cluster K1) and type II (gene clusters K2 and 3) IFNs. SLEDAI-2K scores associated with the K2 and K3 gene scores (β = 0.372 and β = 0.419, both p < 0.015) but not with K1. In the longitudinal study, the mean SLEDAI-2K score decreased after an average follow-up of 360 days (β = −2.08, P = 5.09 × 10−12). The mean K1, K2 and K3 gene scores did not change over time, however longitudinal changes in SLEDAI-2K and K3 scores were associated (β = 0.814, p = 0.007).This study validates the presence of type I IRG subsets that do not associate with disease activity in SLE patients. The novel finding in this study is the association between a type II IRG subset and disease activity. Both findings may have significant implications for choosing IRGs defining clinically relevant IFN signatures.

MicroRNA sequencing in patients with coronary artery disease - considerations for use as biomarker for thrombotic risk.

MicroRNAs (miRNAs) are small RNAs integral in the regulation of gene expression. Analysis of circulating miRNA levels may identify patients with coronary artery disease (CAD) at risk for recurrent myocardial infarction (MI) after percutaneous coronary interventions (PCIs). Subjects with CAD were selected from the GENCATH cardiac catheterization biobank. Subjects with recurrent MI after PCI were compared with those without recurrent MI during follow‐up in the initial (n = 48) and replication cohort (n = 67). Next generation MiRNA sequencing was performed on plasma samples and whole blood samples fixed with PAXGENE tubes upon collection. Overall, 164 miRNAs derived from whole blood were differentially expressed in the replication cohort between subjects with and without recurrent MI events (p < 0.05), with 69 remaining significant after false‐discovery rate (FDR) correction. None of the miRNAs in plasma was significantly different by FDR among subjects with and without MI. Overall, correlation between direction of effects between plasma and whole blood assays was variable, and only two miRNAs were concordant and significant in both. Associations of miRNA with vascular disease, MI, and thrombosis were further explored. MiRNA profiling has potential as the future biomarker for disease prognosis and treatment response marker in secondary treatment of patients with CAD after PCI. Whole blood may be the preferred sample source as compared to plasma.

A Cost Effective Liquid Biopsy Technology Using Donor Derived Cell Free Dnato Diagnose Rejection in Thoracic Organ Transplant Recipients

<h3>Purpose</h3> Solid organ transplant recipients are periodically reviewed to assess the health of transplanted organ. Surgical biopsy is considered as gold standard to monitor rejections as histological changes associated with rejection can be identified by direct microscopy or after immunohistochemical staining with rejection specific biomarker, C4d. However, a number of inconsistencies exist while interpreting the sections as criteria for quantitative scoring of endothelial enlargement and C4d-positivity in patients without clinical evidence of rejection and C4d-negativity in patients with clinical evidence of rejection is not established. Besides the above repeated biopsies can expose the organs to risk of scarring, which in turn can affect its optimal function. <h3>Methods</h3> To mitigate these inconsistencies and risks we have developed a cost effective liquid biopsy technology to diagnose allograft rejection. We first screen an array of single-nucleotide polymorphisms (SNPs) in donor and recipient genomic DNA to discriminate and identify donor specific SNPs. As these SNPs are representative of donor derived cell free DNA (dd-cfDNA), quantification of them by real time PCR in total cell free DNA (total cfDNA) fraction of recipients can be considered as indicator for allograft health. By using this technology we performed a pilot study on 12 heart or lung allograft recipients. 9ml of whole blood collected in PAXGENE tubes from the patients before biopsy were processed to extract total cfDNA and quantify the abundance of dd-cfDNA in the total cfDNA fraction by real time qPCR. <h3>Results</h3> Data analysis indicated presence of dd-cfDNA in 2/12 samples, which correlated with histopathological investigations that identified minimal acute rejection in one patient and cell mediated rejection in the other. One more patient was identified to be undergoing minimal cellular rejection one month post transplant. However, we could not detect dd-cfDNA signal in the plasma. Based on these data, our liquid biopsy technology is able to diagnose rejection in 66.6% (2/3) of patients with a negative prediction at 100% (9/9). <h3>Conclusion</h3> A cost effective Liquid Biopsy Technology using dd-cfDNA quantification by real time qPCR which has excellent correlation with conventional biopsies. Large randomised control studies need to be conducted to further validate this.

EP158: The utility of transcriptomics to increase diagnostic yield in cases of rare genetic disease

Rare diseases affect less than 1 in 200,000 individuals by definition, however, collectively are common, with approximately 30 million individuals in the United States impacted. Frequently individuals experience an arduous journey towards a rare disease diagnosis, often termed a “diagnostic odyssey”. Exome sequencing (ES) has become standard of care for patients with suspected rare genetic disease, with a diagnostic yield of 30%. Here we report on preliminary results of the largest study to date integrating transcriptome sequencing (RNAseq) with ES to increase diagnostic yield for rare disease patients. A cohort of 406 patients who were consented into the Mayo Clinic Diagnostic Odyssey study were selected for study. All patients had received clinical or research exome sequencing which failed to result in a definitive diagnosis. RNA was extracted from whole blood collected in PAXgene tubes, and sequencing libraries prepared using a hybridization capture protocol with the TruSeq RNA Access Library Prep Kit (Illumina, San Diego, CA). Paired-end 101-basepair reads were sequenced using Illumina instruments. Resulting RNA sequences were analyzed for fusion using two algorithms, LeafCutterMD and Fraser, with results integrated for review. Allele specific expression was determined using AnevaDot. Gene expression was analyzed for outlier expression patterns in individual patients compared to the entire cohort using Outrider. Fusion analysis was performed using an internal workflow previously described. The major advantages RNAseq in addition to ES are the high-throughput functional characterization of DNA variation and the detection of aberrations not identifiable by ES sequencing alone. Recent work has shown a 7.5-35% yield using RNAseq in unsolved rare disease cases, dependent on tissue and methodology. In this study RNAseq was performed on blood from 406 individuals with suspected rare genetic disease for whom clinical ES was non-diagnostic. The transcriptome data was analyzed to interrogate four different effects: genes with outlier expression, aberrant RNA-splicing, gene fusions, and allele specific expression. Splicing analysis utilized two complementary algorithms, FRASER and LeafcutterMD, to identify modified intron usage, shifted splice donor and acceptor sites, intron retention events, and creation of cryptic exons. All data were studied for ultra-rare modifications with potential disease ramifications, using the remaining patient cohort as a control RNASeq population. Our preliminary results include gene fusion events, splice site variants with confirmed splicing alterations, and significantly altered outlier gene expression which were integral to the patient’s genetic diagnosis. RNA sequencing is complementary to DNA sequencing in the search for genetic diagnoses in Mendelian disease. When analyzing RNA data, care must be taken to evaluate all forms of RNA mediated events to obtain a thorough representation of the underlying genetics. Here we discuss a 406 patient cohort study using RNA and DNA sequencing to profile patient seen at the Mayo Clinic. Complete results of this study will provide valuable information regarding the clinical utility of RNAseq in tandem with ES for identifying a genetic diagnosis for rare disease patients.

TIMP-1 expression in coronary thrombi associate with myocardial injury in ST-elevation myocardial infarction patients.

Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are considered important both in atherosclerosis and remodeling after acute myocardial infarction (AMI). We aimed to study genetic expression and presence of MMP-2, MMP-9, TIMP-1, TIMP-2 and the extracellular MMP-inducer (EMMPRIN) in coronary thrombi. Circulating levels and genetic expression in circulating leukocytes were also assessed, and relations to degree of myocardial injury measured by troponin T and time from symptom to PCI were explored. Expression of cell markers were also analyzed, indicating relations to cell types. Intracoronary thrombi were aspirated from 33 patients with ST-elevation myocardial infarction (STEMI). Blood samples with Pax-gene tubes were drawn at end of PCI and the next day. RNA was isolated from thrombi and leukocytes, and genes were relatively quantified by RT-PCR. Each thrombus was preserved for histology and immunohistochemistry analyzes. Genes coding for the five markers were present in 84-100% of thrombi and immunohistochemically stained in 96-100%. Expression of TIMP-1 in thrombi and in leukocytes correlated significantly to peak troponin T ( r = 0.393 P = 0.026, r = 0.469 P = 0.006, respectively). No significant correlations between genes expressed in thrombi and time from symptom to PCI were observed. TIMP-1 was connected mainly to monocytes/macrophages in the thrombi. MMP-2, MMP-9, TIMP-1, TIMP-2 and EMMPRIN were highly expressed in human coronary thrombi. The correlation between troponin T and the expression of TIMP-1 both in thrombi and in leukocytes at time of PCI indicates that TIMP-1 plays a role in myocardial damage early post-MI.

Gene network expression of whole blood leukocytes in dairy cows with different milk yield at dry-off.

Dairy cows at dry-off undergo several management and physiological changes, resulting in alterations in plasma biomarkers of inflammation, oxidative stress, and immune system. High milk yield at the end of lactation exacerbates these responses. The underlying mechanism of these changes has yet to be elucidated. We hypothesized altered leukocyte gene expression after dry-off and different responses in cows with different milk yield. Thirteen Holstein dairy cows were sampled at the turn of dry-off to investigated whole blood leukocyte gene expression and were grouped according to the average milk yield during the last week of lactation: low (< 15 kg/d) and high milk yield (> 15 kg/d). Blood samples were collected in PAXgene tubes (Preanalytix, Hombrechtikon, Switzerland) at -7, 7, and 34 days from dry-off (DFD) to measure mRNA abundance of 37 genes. Normalized gene abundance data were subjected to MIXED model ANOVA (SAS Institute Inc., Cary, NC). Compared with -7 DFD, at 7 DFD RNA abundance of lipoxygenase genes (ALOX5, ALOX15) and myeloperoxidase (MPO) increased, and that of the antioxidant gene (SOD2) decreased. Meanwhile, genes related to recognition and immune mediation (CD16, MYD88, TLR2), migration and cell adhesion (CX3CR1, ITGAL, ITGB2, TLN1), and the antimicrobial gene MMP9 were downregulated at 7 or 34 DFD, whereas the antimicrobial IDO1 gene was upregulated. Compared with low-producing cows, cows with high milk yield at dry-off cows had upregulated expression of the pro-inflammatory cytokines IL8 and IL18 and a greater reduction in transcript abundance of the toll-like receptor (TLR) recognition-related gene TLR2. Overall, the dry-off confirmed to be a phase of intense changes, triggering an inflammatory response and somewhat suppressing leukocyte immune function. In cows with high milk yield during the week before dry-off, the inflammatory response was exacerbated.

CircSKA3 acts as a sponge of miR-6796-5p to be associated with outcomes of ischemic stroke by regulating matrix metalloproteinase 9 expression.

This study was undertaken to screen the circular RNAs (circRNAs) influencing matrix metalloproteinase 9 (MMP9) through the competing endogenous RNA (ceRNA) network and evaluate the prognostic value of these circRNAs for acute ischemic stroke. A total of 220 ischemic stroke patients and 62healthy subjects were included in this study. RNA was isolated from blood collected in PAXgene tubes. Illumina sequencing, quantitative real-time polymerase chain reaction (qRT-PCR) validation, and luciferase reporter assay were explored to construct and verify the existence of a circRNA-microRNA (miRNA)-matrix metalloproteinase-9 (MMP9) network. The 215 ischemic stroke patients were recruited in a prognostic cohort. They were prospectively followed up for 3months after stroke onset, and a poor functional outcome was defined as a major disability or death. After Illumina sequencing, six circRNAs were predicted to bind miRNAs and then regulate MMP9messenger RNA (mRNA). qRT-PCR showed that only circSKA3 was significantly increased in ischemic stroke patients compared to healthy controls and positively associated with MMP9mRNA expression. Luciferase reporter assay further verified a direct interaction between circSKA3, MMP9, and hsa-miR-6796-5p. Patients in the top tertile of circSKA3had a 2.672-fold (p<0.05) risk of poor functional outcome, compared with those in the bottom tertile (p for trend = 0.016). The outcome was predicted by circSKA3 with area under the receiver operating characteristic curve at 0.614 (p=0.004). circSKA3 functioned as a ceRNA for hsa-miR-6796-5p to aggravate the progression of ischemic stroke via targeting MMP9. Baseline circSKA3 was positively associated with poor outcomes of ischemic stroke. circSKA3may be a potential biomarker or therapeutic target in ischemic stroke.

Plaque and remodeling markers in coronary thrombi

Abstract Background Matrix Metalloproteinases (MMPs) and their inhibitors are considered to be of importance in development of atherosclerotic coronary artery disease. MMP-9 has been associated with unstable atherosclerotic plaques and rupture, as well as left ventricular remodeling after myocardial infarction (MI), whereas MMP-2 seems to be more related to progression of stable plaques. MMP activity is modulated by Tissue Inhibitors of Metalloproteinases (TIMPs). TIMP-1 has been associated with cardiac remodeling post MI, and TIMP-2 has been discussed to inhibit plaque development and destabilization. The extracellular MMP Inducer, EMMPRIN, stimulates both MMPs and TIMPs, and has been found upregulated on the surface of monocytes in patients with acute MI, and associated with MMP-9 activity. Purpose To study whether genes encoding MMP-2, MMP-9, TIMP-1, TIMP-2 and EMMPRIN are expressed in coronary thrombi and in circulating leukocytes from STEMI patients, and whether these are related to the degree of myocardial injury measured by troponin T, and time from symptoms to PCI. Methods Intracoronary thrombi were aspirated from 33 patients with ST-elevation myocardial infarction (STEMI) treated with primary PCI. The thrombi were snap-frozen in RNA-later solution for gene expression analyses. Peripheral blood samples with Pax-gene tubes were drawn at end of the PCI procedure. RNA was isolated from the thrombi and leukocytes, and the actual genes relatively quantified by RT PCR. Peak troponin T was collected from clinical records. Results Genes coding for the five different markers were present in 84–100% of the thrombi. Median peak troponin T was 3434 m/L. The expression of TIMP-1 in the thrombi correlated significantly to peak troponin T (r=0.393, p=0.026), and dividing peak troponin T values into quartiles, the median value of TIMP-1 mRNA in Q4 was 2.5-fold higher compared to Q1–3 (p=0.107). Peak troponin T also correlated to the expression of TIMP-1 in circulating leukocytes (r=0.469, p=0.006), and in Q4 of troponin T, the median value was 1.6-fold higher compared to Q1–3 (p=0.056). There were no significant correlations between the other measured genes and troponin T, and also no associations of any genes expressed in the thrombi or in circulating leukocytes to time from symptom to PCI (median 152 min). Conclusion Genes coding for MMP-2, MMP-9, TIMP-1, TIMP-2 and EMMPRIN were highly expressed in human coronary thrombi. The positive correlation between peak troponin T and the expression of TIMP-1 both in thrombi and in circulating leukocytes at time of PCI in patients with STEMI, may indicate that the role of TIMP-1 is important in cardiac remodeling immediately post-MI. Funding Acknowledgement Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Stein Erik Hagens Foundation for Clinical Heart Research

Trypanosome SL-RNA detection in blood and cerebrospinal fluid to demonstrate active gambiense human African trypanosomiasis infection.

BackgroundSpliced Leader (SL) trypanosome RNA is detectable only in the presence of live trypanosomes, is abundant and the Trypanozoon subgenus has a unique sequence. As previously shown in blood from Guinean human African trypanosomiasis (HAT) patients, SL-RNA is an accurate target for diagnosis. Detection of SL-RNA in the cerebrospinal fluid (CSF) has never been attempted. In a large group of Congolese gambiense HAT patients, the present study aims i) to confirm the sensitivity of SL-RNA detection in the blood and; ii) to assess the diagnostic performance of SL-RNA compared to trypanosome detection in CSF.Methodology/Principal findingsBlood and CSF from 97 confirmed gambiense HAT patients from the Democratic Republic of Congo were collected using PAXgene blood RNA Tubes. Before RNA extraction, specimens were supplemented with internal extraction control RNA to monitor the extraction, which was performed with a PAXgene Blood RNA Kit. SL-RNA qPCR was carried out with and without reverse transcriptase to monitor DNA contamination. In blood, 92/97 (94.8%) HAT patients tested SL-RNA positive, which was significantly more than combined trypanosome detection in lymph and blood (78/97 positive, 80.4%, p = 0.001). Of 96 CSF RNA specimens, 65 (67.7%) were SL-RNA positive, but there was no significant difference between sensitivity of SL-RNA and trypanosome detection in CSF. The contribution of DNA to the Cq values was negligible. In CSF with normal cell counts, a fraction of SL-RNA might have been lost during extraction as indicated by higher internal extraction control Cq values.Conclusions/SignificanceDetection of SL-RNA in blood and CSF allows sensitive demonstration of active gambiense HAT infection, even if trypanosomes remain undetectable in blood or lymph. As this condition often occurs in treatment failures, SL-RNA detection in blood and CSF for early detection of relapses after treatment deserves further investigation.Trial registrationThis study was an integral part of the diagnostic trial "New Diagnostic Tools for Elimination of Sleeping Sickness and Clinical Trials: Early tests of Cure" (DiTECT-HAT-WP4, ClinicalTrials.gov Identifier: NCT03112655).

Dietary Polyphenol and Methylsulfonylmethane Supplementation Improves Immune, DAMP Signaling, and Inflammatory Responses During Recovery From All-Out Running Efforts.

Nutritional ingredients with defined mechanisms of action can be useful in the recovery of the body from the physical demands of a habitual training plan. The purpose of this study was to determine the effect of dietary supplementation with optimized curcumin, pomegranate ellagitannins, and MSM (R + MSM) on immune-associated mRNA during early recovery (i.e., up to 8 h post-exercise) following all-out running efforts (5-km, 10-km, and 21.1-km). Subjects (N = 14) were randomized to either a supplement (R + MSM) or a control group using an open label design. The study was completed over a period of 31-day prior to a scheduled half-marathon race. Venous blood samples were collected into PAXgene tubes at baseline, subsequent samples were collected at 2, 4, and 8 h after each running effort. A 574-plex mRNA Immunology Array (NanoString) was measured for each sample and ROSALIND® Advanced Analysis Software was used to examined changes in 31 annotated immune response pathways and specific mRNA changes. The greatest change in immune pathways occurred at 2 h (GSS > 3) followed by 4 h (GSS 2–3) and 8 h (GSS 1–2). R + MSM was associated with an increase in innate immunity (CAMP, LTF, TIRAP, CR1, IL1R1, CXCR1, PDCDILG2, and GNLY) and a blunted/smaller increase in damage-associated molecular pattern (DAMP) signaling/inflammation (TLR4, TLR5, S100A8, S100A9, and IFP35). We also found changes in immune-associated mRNA that have not been previously linked to exercise recovery (SOCS1, SOCS2, MME, CECAM6, MX1, IL-1R2, KLRD1, KLRK1, and LAMP3). Collectively these results demonstrate that supplementation with a combination of optimized curcumin, pomegranate ellagitannins, and methylsulfonylmethane resulted in changes that may improve biological recovery from all-out running efforts.

High-Density Blood Transcriptomics Reveals Precision Immune Signatures of SARS-CoV-2 Infection in Hospitalized Individuals.

The immune response to COVID-19 infection is variable. How COVID-19 influences clinical outcomes in hospitalized patients needs to be understood through readily obtainable biological materials, such as blood. We hypothesized that a high-density analysis of host (and pathogen) blood RNA in hospitalized patients with SARS-CoV-2 would provide mechanistic insights into the heterogeneity of response amongst COVID-19 patients when combined with advanced multidimensional bioinformatics for RNA. We enrolled 36 hospitalized COVID-19 patients (11 died) and 15 controls, collecting 74 blood PAXgene RNA tubes at multiple timepoints, one early and in 23 patients after treatment with various therapies. Total RNAseq was performed at high-density, with >160 million paired-end, 150 base pair reads per sample, representing the most sequenced bases per sample for any publicly deposited blood PAXgene tube study. There are 770 genes significantly altered in the blood of COVID-19 patients associated with antiviral defense, mitotic cell cycle, type I interferon signaling, and severe viral infections. Immune genes activated include those associated with neutrophil mechanisms, secretory granules, and neutrophil extracellular traps (NETs), along with decreased gene expression in lymphocytes and clonal expansion of the acquired immune response. Therapies such as convalescent serum and dexamethasone reduced many of the blood expression signatures of COVID-19. Severely ill or deceased patients are marked by various secondary infections, unique gene patterns, dysregulated innate response, and peripheral organ damage not otherwise found in the cohort. High-density transcriptomic data offers shared gene expression signatures, providing unique insights into the immune system and individualized signatures of patients that could be used to understand the patient’s clinical condition. Whole blood transcriptomics provides patient-level insights for immune activation, immune repertoire, and secondary infections that can further guide precision treatment.

Identification of Acetylation, Immune Cell Alterations and Gene Signature As Dynamic Biomarkers of Response to Panobinostat Treatment in Multiple Myeloma Patients

Introduction and aims:Panobinostat is a pan-deacetylase inhibitor (DACi) that affects multiple oncogenic and immune-stimulating genes required for tumour cell growth and survival through acetylation of both histone and non-histone proteins. Evaluation of panobinostat as single agent consolidation in patients post-melphalan 200mg/m2 conditioned autologous stem cell transplant (ASCT) was performed in a Phase II, open label, single arm study, wherein newly diagnosed patients (n=35) with less than complete response following ASCT were enrolled. Eighteen patients (12 responders - R, 6 non-responders - NR) were subsequently selected for correlative analysis to identify dynamic biomarkers that could predict for response to treatment. Acetylation is the key downstream effect of treatment with DACi and was therefore assessed to determine its validity as a pharmacodynamic marker of response. The proportions of different immune subsets at specific time-points during treatment were also assessed to identify differences in the immune composition between R and NR. Gene transcription studies were performed to detect differentially regulated genes/pathways.MethodsFor all correlative studies, screening sample (Pre-ASCT), post - ASCT, end of cycle 3 (EOC3) and EOC6 were utilised. Peripheral blood mononuclear cells (PBMC) from post-ASCT, EOC3 and EOC6 were processed to determine acetylation status of lysine residues (K) in histone 2A - H2A (K5), H2B (K5), H3 (K9, K14, K18 and K56), H4 (K5, K8, K12 and K16) and tubulin in CD3+ (T-lymphocytes) utilising flow cytometry. Changes were measured using mean fluorescence intensity (MFI) of the geometric mean. The MFI from a sample was normalized to the respective control sample and expressed as a fold difference. PBMC from all 4 time points were also assessed for proportions of different subsets of T-lymphocytes (T-helper, cytotoxic T-cells and T-regs) and natural killer (NK) cells using flow cytometry. Whole bone marrow (BM) aspirates were collected in PAXgene blood RNA tubes and extracted RNA was subjected to 100 bp paired-end total RNA sequencing.Results and conclusion:Panobinostat-induced acetylation in T-lymphocytes was significantly higher at EOC3 (H2B K5, H3 K9, H3 K14, H3 K56, H4 K8, H4 K12, H4 K16 (p<0.01)) and EOC6 (all histones assessed, p<0.05) in R, with no differences in NR at EOC3 and EOC6 compared to post-ASCT, suggesting that acetylation could be utilised as a pharmacodynamic biomarker of response to panobinostat. Assessment of T-cell proportions in this annotated sample set indicated that the ratio of CD4/CD8 T-cells was significantly higher at EOC3 in R compared to NR (p<0.01), consistent with published literature, which demonstrates that the ratio of CD4/CD8 is significantly lower in patients with poor prognostic tumors. Furthermore, MFI of CD57 in cytotoxic cells was higher in EOC3 and EOC6 compared to post-ASCT in R while it was downregulated in NR (p<0.05), also suggesting an increased cytotoxic activity for T-cells in R. Absolute numbers of NK cells increased significantly in NR at EOC3 and EOC6, while this was significantly reduced in R at EOC3 and EOC6 compared to post-ASCT (p<0.01). This indicates that immunogenic cell death of NK cells appear to occur in the R in response to treatment. Total RNA-sequencing studies indicated that minimal changes were induced between pre-ASCT and EOC3 in NR (6 genes), while comparison of R (Pre-ASCT) to EOC3 and EOC6 demonstrated that 2482 and 2814 genes, respectively, were regulated. A total of 984 genes were differentially regulated in both EOC3 and EOC6 compared to pre-ASCT. Notably, HMGB1, a critical mediator of immunogenic cell death was significantly upregulated in R at EOC3 compared to pre-ASCT sample, consistent with our observations of NK cell proportions. Further analysis revealed that a total of 13 genes were differentially regulated in R at Post-ASCT, EOC3 and EOC6 compared to the pre-ASCT. In conclusion, we present potential pharmacodynamic biomarkers of clinical benefit (acetylation of histones, CD4/CD8 ratio and changes in immune subsets) using PBMC. A subset of genes that can be assessed from whole BM biopsy are also being assessed to determine a genetic signature for response. DisclosuresSpencer:Janssen: Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding.