scholarly journals A Transcriptome Fingerprinting Assay for Clinical Immune Monitoring

2019 ◽  
Author(s):  
Matthew C Altman ◽  
Nicole Baldwin ◽  
Elizabeth Whalen ◽  
Taha Al-Shaikhly ◽  
Scott Presnell ◽  
...  
Keyword(s):  
Lupus Erythematosus ◽  
Large Scale ◽  
Biomarker Discovery ◽  
Transcriptional Profiling ◽  
Transcript Abundance ◽  
Immune Monitoring ◽  
Immune Functions ◽  
Functional Interpretation ◽  
Clinical Tools

ABSTRACTBackgroundWhile our understanding of the role that the immune system plays in health and disease is growing at a rapid pace, available clinical tools to capture this complexity are lagging. We previously described the construction of a third-generation modular transcriptional repertoire derived from genome-wide transcriptional profiling of blood of 985 subjects across 16 diverse immunologic conditions, which comprises 382 distinct modules.ResultsHere we describe the use of this modular repertoire framework for the development of a targeted transcriptome fingerprinting assay (TFA). The first step consisted in down-selection of the number of modules to 32, on the basis of similarities in changes in transcript abundance and functional interpretation. Next down-selection took place at the level of each of the 32 modules, with each one of them being represented by four transcripts in the final 128 gene panel. The assay was implemented on both the Fluidigm high throughput microfluidics PCR platform and the Nanostring platform, with the list of assays target probes being provided for both. Finally, we provide evidence of the versatility of this assay to assess numerous immune functionsin vivoby demonstrating applications in the context of disease activity assessment in systemic lupus erythematosus and longitudinal immune monitoring during pregnancy.ConclusionsThis work demonstrates the utility of data-driven network analysis applied to large-scale transcriptional profiling to identify key markers of immune responses, which can be downscaled to a rapid, inexpensive, and highly versatile assay of global immune function applicable to diverse investigations of immunopathogenesis and biomarker discovery.

Large-scale transcriptional profiling of chemosensory neurons identifies receptor-ligand pairs in vivo

2015 ◽  
Vol 18 (10) ◽  
pp. 1455-1463 ◽  
Author(s):  
Benoît von der Weid ◽  
Daniel Rossier ◽  
Matti Lindup ◽  
Joël Tuberosa ◽  
Alexandre Widmer ◽  
...  
Keyword(s):  
Large Scale ◽  
Transcriptional Profiling ◽  
Receptor Ligand ◽  
Chemosensory Neurons

scholarly journals Protein Deimination Signatures in Plasma and Plasma-EVs and Protein Deimination in the Brain Vasculature in a Rat Model of Pre-Motor Parkinson’s Disease

2020 ◽  
Vol 21 (8) ◽  
pp. 2743 ◽  
Author(s):  
Marco Sancandi ◽  
Pinar Uysal-Onganer ◽  
Igor Kraev ◽  
Audrey Mercer ◽  
Sigrun Lange
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Rat Model ◽  
Lupus Erythematosus ◽  
Biomarker Discovery ◽  
Alcoholic Fatty Liver ◽  
Brain Vasculature ◽  
Micro Rnas ◽  
The Brain

The identification of biomarkers for early diagnosis of Parkinson’s disease (PD) is of pivotal importance for improving approaches for clinical intervention. The use of translatable animal models of pre-motor PD therefore offers optimal opportunities for novel biomarker discovery in vivo. Peptidylarginine deiminases (PADs) are a family of calcium-activated enzymes that contribute to protein misfolding through post-translational deimination of arginine to citrulline. Furthermore, PADs are an active regulator of extracellular vesicle (EV) release. Both protein deimination and extracellular vesicles (EVs) are gaining increased attention in relation to neurodegenerative diseases, including in PD, while roles in pre-motor PD have yet to be investigated. The current study aimed at identifying protein candidates of deimination in plasma and plasma-EVs in a rat model of pre-motor PD, to assess putative contributions of such post-translational changes in the early stages of disease. EV-cargo was further assessed for deiminated proteins as well as three key micro-RNAs known to contribute to inflammation and hypoxia (miR21, miR155, and miR210) and also associated with PD. Overall, there was a significant increase in circulating plasma EVs in the PD model compared with sham animals and inflammatory and hypoxia related microRNAs were significantly increased in plasma-EVs of the pre-motor PD model. A significantly higher number of protein candidates were deiminated in the pre-motor PD model plasma and plasma-EVs, compared with those in the sham animals. KEGG (Kyoto encyclopedia of genes and genomes) pathways identified for deiminated proteins in the pre-motor PD model were linked to “Alzheimer’s disease”, “PD”, “Huntington’s disease”, “prion diseases”, as well as for “oxidative phosphorylation”, “thermogenesis”, “metabolic pathways”, “Staphylococcus aureus infection”, gap junction, “platelet activation”, “apelin signalling”, “retrograde endocannabinoid signalling”, “systemic lupus erythematosus”, and “non-alcoholic fatty liver disease”. Furthermore, PD brains showed significantly increased staining for total deiminated proteins in the brain vasculature in cortex and hippocampus, as well as increased immunodetection of deiminated histone H3 in dentate gyrus and cortex. Our findings identify EVs and post-translational protein deimination as novel biomarkers in early pre-motor stages of PD.

scholarly journals FlowCT for the analysis of large immunophenotypic datasets and biomarker discovery in cancer immunology

2021 ◽  
Author(s):  
Cirino Botta ◽  
Catarina Da Silva Maia ◽  
Juan-José Garcés ◽  
Rosalinda Termini ◽  
Cristina Perez ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Maintenance Therapy ◽  
Large Scale ◽  
Biomarker Discovery ◽  
Immune Monitoring ◽  
T Cell Subsets ◽  
Multidimensional Data ◽  
Modeling Tools ◽  
Immune Biomarkers

Large-scale immune monitoring is becoming routinely used in clinical trials to identify determinants of treatment responsiveness, particularly to immunotherapies. Flow cytometry remains one of the most versatile and high throughput approaches for single-cell analysis; however, manual interpretation of multidimensional data poses a challenge to capture full cellular diversity and provide reproducible results. We present FlowCT, a semi-automated workspace empowered to analyze large datasets that includes pre-processing, normalization, multiple dimensionality reduction techniques, automated clustering and predictive modeling tools. As a proof of concept, we used FlowCT to compare the T cell compartment in bone marrow (BM) vs peripheral blood (PB) of patients with smoldering multiple myeloma (MM); identify minimally-invasive immune biomarkers of progression from smoldering to active MM; define prognostic T cell subsets in the BM of patients with active MM after treatment intensification; and assess the longitudinal effect of maintenance therapy in BM T cells. A total of 354 samples were analyzed and immune signatures predictive of malignant transformation in 150 smoldering MM patients (hazard ratio [HR]: 1.7; P <.001), and of progression-free (HR: 4.09; P <.0001) and overall survival (HR: 3.12; P =.047) in 100 active MM patients, were identified. New data also emerged about stem cell memory T cells, the concordance between immune profiles in BM vs PB and the immunomodulatory effect of maintenance therapy. FlowCT is a new open-source computational approach that can be readily implemented by research laboratories to perform quality-control, analyze high-dimensional data, unveil cellular diversity and objectively identify biomarkers in large immune monitoring studies.

183 EFFECT OF WELL IN WELL CULTURE OF BOVINE EMBRYOS ON GENE EXPRESSION PROFILE

2009 ◽  
Vol 21 (1) ◽  
pp. 190 ◽  
Author(s):  
N. Ghanem ◽  
M. Hoelker ◽  
C. Phatsara ◽  
K. Schellander ◽  
D. Tesfaye
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Relative Abundance ◽  
Large Scale ◽  
Gene Expression Pattern ◽  
Cdna Array ◽  
Transcript Abundance ◽  
Oxidoreductase Activity ◽  
Group Culture

To culture embryos in small groups, the well in well culture system (miniwells harboring 1 single embryo within the well) has been developed previously. In this work, we aimed to examine the effects of the microenvironment provided by well in well culture and embryo density on the relative abundance of transcripts in the resulting embryos. Cumulus–oocyte complexes (COCs) were aspirated from small follicles (2 to 8 mm), and COCs were cultured in 400 μL of modified TCM (TCM-199, Sigma, Taufkirchen, Germany) supplemented with 12% heat-inactivated estrous cow serum and 10 μg mL–1 of FSH (FSH-p, Sheering, Kenilworth, NJ, USA) for 24 h at 39°C in a humidified atmosphere with 5% CO2 in air. Fertilization was performed in Fert-TALP supplemented with 1 μg mL–1 of heparin. Zygotes were allocated randomly in 2 groups, namely: well in well culture (16 miniwells of 0.7 diameter and deepness each containing 1 embryo per well) and group of 16 (group culture of 16 embryos per well). Six pools each containing 20 Day 7 blastocysts derived from the first 2 groups were used to investigate large-scale gene expression analysis using BlueChip cDNA-Array. Three pools each containing 5 blastocysts were used for Array data validation by real-time PCR using primers specific to 5 selected genes (ATP5, PLAC8, KRT8, S100A10, and ZP3). During validation in vivo-derived bovine blastocysts were included to be used as standard. Significance Analysis of Microarray identified 75 transcripts differentially expressed between the 2 groups. Blastocysts derived from well in well culture were found to be enriched with genes regulating different molecular functions including structural constituent of ribosome (RPS29), protein binding (Cul1), calcium ion binding (S100A10, NPTX2), nitric oxide synthase regulator activity (HSPCA), and RNA polymerase II transcription factor activity (UHRF1). However, blastocysts derived from group of 16 culture were found to be enriched with genes involved in oxidoreductase activity (ALOX15, AKR1B), cytochrome-c oxidase activity (COX7A2), hydrogen ion transporting ATP synthase activity (ATP5O), transcription (PTTG1), and cell redox homeostasis (TXN). According to their biological process, genes enriched in blastocysts derived from well in well culture belong to small molecule transport and signal transduction, whereas most downregulated genes have a metabolic function. Comparison of the transcript abundance of the 5 selected genes in the 3 embryo groups showed that the expression of ATP5, PLAC8, and KRT8 in embryos from well in well culture resembles to the relative abundance in blastocyst derived from in vivo culture. However, with respect to the expression of S100A10 and ZP3 genes, blastocysts derived from group culture showed similarity with embryos derived from in vivo. In conclusion, microenvironment affects the gene expression pattern of the resulting embryos.

scholarly journals Development of a Comprehensive Antibody Staining Database using a Standardized Analytics Pipeline

2019 ◽  
Author(s):  
El-ad David Amir ◽  
Brian Lee ◽  
Paul Badoual ◽  
Martin Gordon ◽  
Xinzheng V. Guo ◽  
...  
Keyword(s):  
Large Scale ◽  
Biomarker Discovery ◽  
Staining Intensity ◽  
Immune Monitoring ◽  
Mass Cytometry ◽  
Data Set ◽  
Mait Cells ◽  
Antibody Staining ◽  
Online Resource ◽  
Analysis Platform

AbstractLarge-scale immune monitoring experiments (such as clinical trials) are a promising direction for biomarker discovery and responder stratification in immunotherapy. Mass cytometry is one of the tools in the immune monitoring arsenal. We propose a standardized workflow for the acquisition and analysis of large-scale mass cytometry experiments. The workflow includes two-tiered barcoding, a broad lyophilized panel, and the incorporation of a fully automated, cloud-based analysis platform. We applied the workflow to a large antibody staining screen using the LEGENDScreen kit, resulting in single-cell data for 350 antibodies over 71 profiling subsets. The screen recapitulates many known trends in the immune system and reveals potential markers for delineating MAIT cells. Additionally, we examine the effect of fixation on staining intensity and identify several markers where fixation leads to either gain or loss of signal. The standardized workflow can be seamlessly integrated into existing trials. Finally, the antibody staining data set is available as an online resource for researchers who are designing mass cytometry experiments in suspension and tissue.

Purification of the Antihemophilic Factor by Gel Filtration on Agarose

1969 ◽  
Vol 22 (03) ◽  
pp. 577-583 ◽  
Author(s):  
M.M.P Paulssen ◽  
A.C.M.G.B Wouterlood ◽  
H.L.M.A Scheffers
Keyword(s):  
Factor Viii ◽  
Plasma Proteins ◽  
Large Scale ◽  
Gel Filtration ◽  
Large Scale Preparation ◽  
Scale Preparation ◽  
Antihemophilic Factor

SummaryFactor VIII can be isolated from plasma proteins, including fibrinogen by chromatography on agarose. The best results were obtained with Sepharose 6B. Large scale preparation is also possible when cryoprecipitate is separated by chromatography. In most fractions containing factor VIII a turbidity is observed which may be due to the presence of chylomicrons.The purified factor VIII was active in vivo as well as in vitro.

The Effect of Active Site-inhibited Factor VIIa on Tissue Factor-initiated Coagulation Using Platelets before and after Aspirin Administration

1997 ◽  
Vol 78 (04) ◽  
pp. 1202-1208 ◽  
Author(s):  
Marianne Kjalke ◽  
Julie A Oliver ◽  
Dougald M Monroe ◽  
Maureane Hoffman ◽  
Mirella Ezban ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Active Site ◽  
Large Scale ◽  
Thrombin Generation ◽  
Factor Viia ◽  
Dependent Manner ◽  
Antithrombotic Agent ◽  
Before And After ◽  
Ic50 Values

SummaryActive site-inactivated factor VIIa has potential as an antithrombotic agent. The effects of D-Phe-L-Phe-L-Arg-chloromethyl ketone-treated factor VIla (FFR-FVIIa) were evaluated in a cell-based system mimicking in vivo initiation of coagulation. FFR-FVIIa inhibited platelet activation (as measured by expression of P-selectin) and subsequent large-scale thrombin generation in a dose-dependent manner with IC50 values of 1.4 ± 0.8 nM (n = 8) and 0.9 ± 0.7 nM (n = 7), respectively. Kd for factor VIIa binding to monocytes ki for FFR-FVIIa competing with factor VIIa were similar (11.4 ± 0.8 pM and 10.6 ± 1.1 pM, respectively), showing that FFR-FVIIa binds to tissue factor in the tenase complex with the same affinity as factor VIIa. Using platelets from volunteers before and after ingestion of aspirin (1.3 g), there were no significant differences in the IC50 values of FFR-FVIIa [after aspirin ingestion, the IC50 values were 1.7 ± 0.9 nM (n = 8) for P-selectin expression, p = 0.37, and 1.4 ± 1.3 nM (n = 7) for thrombin generation, p = 0.38]. This shows that aspirin treatment of platelets does not influence the inhibition of tissue factor-initiated coagulation by FFR-FVIIa, probably because thrombin activation of platelets is not entirely dependent upon expression of thromboxane A2.

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