Dendritic cells (DCs) and monocytes play a central role in pathogen sensing, phagocytosis, and antigen presentation and consist of multiple specialized subtypes. However, their identities and ...interrelationships are not fully understood. Using unbiased single-cell RNA sequencing (RNA-seq) of ~2400 cells, we identified six human DCs and four monocyte subtypes in human blood. Our study reveals a new DC subset that shares properties with plasmacytoid DCs (pDCs) but potently activates T cells, thus redefining pDCs; a new subdivision within the CD1C
subset of DCs; the relationship between blastic plasmacytoid DC neoplasia cells and healthy DCs; and circulating progenitor of conventional DCs (cDCs). Our revised taxonomy will enable more accurate functional and developmental analyses as well as immune monitoring in health and disease.
Increasing knowledge about the heterogeneity of mRNA expression within cell populations highlights the need to study transcripts at the level of single cells. We present a method for detection and ...genotyping of individual transcripts based on padlock probes and in situ target-primed rolling-circle amplification. We detect a somatic point mutation, differentiate between members of a gene family and perform multiplex detection of transcripts in human and mouse cells and tissue.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to angiotensin converting enzyme 2 (ACE2) enabling entrance of the virus into cells and causing the infection termed coronavirus ...disease of 2019 (COVID-19). Here, we investigate associations between plasma ACE2 and outcome of COVID-19. This analysis used data from a large longitudinal study of 306 COVID-19 positive patients and 78 COVID-19 negative patients (MGH Emergency Department COVID-19 Cohort). Comprehensive clinical data were collected on this cohort, including 28-day outcomes. The samples were run on the Olink® Explore 1536 platform which includes measurement of the ACE2 protein. High admission plasma ACE2 in COVID-19 patients was associated with increased maximal illness severity within 28 days with OR = 1.8, 95%-CI: 1.4-2.3 (P < 0.0001). Plasma ACE2 was significantly higher in COVID-19 patients with hypertension compared with patients without hypertension (P = 0.0045). Circulating ACE2 was also significantly higher in COVID-19 patients with pre-existing heart conditions and kidney disease compared with patients without these pre-existing conditions (P = 0.0363 and P = 0.0303, respectively). This study suggests that measuring plasma ACE2 is potentially valuable in predicting COVID-19 outcomes. Further, ACE2 could be a link between COVID-19 illness severity and its established risk factors hypertension, pre-existing heart disease and pre-existing kidney disease.
Current assays for somatic mutation analysis are based on extracts from tissue sections that often contain morphologically heterogeneous neoplastic regions with variable contents of normal stromal ...and inflammatory cells, obscuring the results of the assays. We have developed an RNA-based in situ mutation assay that targets oncogenic mutations in a multiplex fashion that resolves the heterogeneity of the tissue sample. Activating oncogenic mutations are targets for a new generation of cancer drugs. For anti-EGFR therapy prediction, we demonstrate reliable in situ detection of KRAS mutations in codon 12 and 13 in colon and lung cancers in three different types of routinely processed tissue materials. High-throughput screening of KRAS mutation status was successfully performed on a tissue microarray. Moreover, we show how the patterns of expressed mutated and wild-type alleles can be studied in situ in tumors with complex combinations of mutated EGFR, KRAS and TP53. This in situ method holds great promise as a tool to investigate the role of somatic mutations during tumor progression and for prediction of response to targeted therapy.
We have developed an approach for simultaneous detection of individual endogenous protein modifications and mRNA molecules in single cells in situ. For this purpose we combined two methods previously ...developed in our lab: in situ proximity ligation assay for the detection of individual protein interactions and -modifications and in situ detection of single mRNA molecules using padlock probes. As proof-of-principle, we demonstrated the utility of the method for simultaneous detection of phosphorylated PDGFRβ and DUSP6/MKP-3 mRNA molecules in individual human fibroblasts upon PDGF-BB stimulation. Further we applied drugs disrupting the PDGFRβ signaling pathway at various sites to show that this combined method can concurrently monitor the molecular effect of the drugs, i.e. inhibition of downstream signaling from the targeted node in the signaling pathway. Due to its ability to detect different types of molecules in single cells in situ the method presented here can contribute to a deeper understanding of cell-to-cell variations and can be applied to e.g. pinpoint effector sites of drugs in a signaling pathway.
Understanding the dynamics of the human proteome is crucial for developing biomarkers to be used as measurable indicators for disease severity and progression, patient stratification, and drug ...development. The Proximity Extension Assay (PEA) is a technology that translates protein information into actionable knowledge by linking protein-specific antibodies to DNA-encoded tags. In this report we demonstrate how we have combined the unique PEA technology with an innovative and automated sample preparation and high-throughput sequencing readout enabling parallel measurement of nearly 1500 proteins in 96 samples generating close to 150,000 data points per run. This advancement will have a major impact on the discovery of new biomarkers for disease prediction and prognosis and contribute to the development of the rapidly evolving fields of wellness monitoring and precision medicine.
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•Novel tool for protein discovery and quantification.•PEA technology in combination with high-throughput sequencing read-out.•Parallel measurement of nearly 1500 proteins.•Innovative, miniaturized, and automated sample preparation protocol.
Wik et al. presents a groundbreaking method based on the PEA technology in combination with NGS readout to measure nearly 1500 validated proteins in parallel. The protocol contains an innovative and automated sample preparation workflow generating close to 150,000 data points per run while consuming minimal amount of sample. Using dual DNA tags as readout enables high multiplex detection with exceptional specificity. This protocol meets all the requirements for a proteomic tool of the 21st century healthcare.
Analysis at the single-cell level is essential for the understanding of cellular responses in heterogeneous cell populations, but it has been difficult to perform because of the strict requirements ...put on detection methods with regard to selectivity and sensitivity (i.e., owing to the cross-reactivity of probes and limited signal amplification). Here we describe a 1.5-d protocol for enumerating and genotyping mRNA molecules in situ while simultaneously obtaining information on protein interactions or post-translational modifications; this is achieved by combining padlock probes with in situ proximity ligation assays (in situ PLA). In addition, we provide an example of how to design padlock probes and how to optimize staining conditions for fixed cells and tissue sections. Both padlock probes and in situ PLA provide the ability to directly visualize single molecules by standard microscopy in fixed cells or tissue sections, and these methods may thus be valuable for both research and diagnostic purposes.
Mechanisms underlying severe coronavirus disease 2019 (COVID-19) disease remain poorly understood. We analyze several thousand plasma proteins longitudinally in 306 COVID-19 patients and 78 ...symptomatic controls, uncovering immune and non-immune proteins linked to COVID-19. Deconvolution of our plasma proteome data using published scRNA-seq datasets reveals contributions from circulating immune and tissue cells. Sixteen percent of patients display reduced inflammation yet comparably poor outcomes. Comparison of patients who died to severely ill survivors identifies dynamic immune-cell-derived and tissue-associated proteins associated with survival, including exocrine pancreatic proteases. Using derived tissue-specific and cell-type-specific intracellular death signatures, cellular angiotensin-converting enzyme 2 (ACE2) expression, and our data, we infer whether organ damage resulted from direct or indirect effects of infection. We propose a model in which interactions among myeloid, epithelial, and T cells drive tissue damage. These datasets provide important insights and a rich resource for analysis of mechanisms of severe COVID-19 disease.
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16% of COVID-19 patients display an atypical low-inflammatory plasma proteomeSevere COVID-19 is associated with heterogeneous plasma proteomic responsesDeath of virus-infected lung epithelial cells is a key feature of severe diseaseLung monocyte/macrophages drive T cell activation, together promoting epithelial damage
Filbin et al. use plasma proteomics in 306 coronavirus disease 2019 (COVID-19) patients and 78 symptomatic controls over time to better understand the role of circulating immune cells and tissue cells in inflammation, disease severity, and survival. They propose a model in which interactions among myeloid, epithelial, and T cells drive tissue damage.
Abstract
Background
People with human immunodeficiency virus (PWH) demonstrate increased atherosclerotic cardiovascular disease (ASCVD). Statins are being studied to prevent ASCVD in human ...immunodeficiency virus (HIV), but little is known regarding the effects of statins on a broad range of inflammatory and cardiovascular proteins in this population.
Methods
We used a highly specific discovery proteomic approach (Protein Extension Assay), to determine statin effects on over 350 plasma proteins in relevant ASCVD pathways among HIV and non-HIV groups. Responses to pitavastatin calcium were assessed in 89 PWH in the INTREPID trial and 46 non-HIV participants with features of central adiposity and insulin resistance. History of cardiovascular disease was exclusionary for both studies.
Results
Among participants with HIV, PCOLCE (enzymatic cleavage of type I procollagen) significantly increased after pitavastatin therapy and PLA2G7 (systemic marker of arterial inflammation) decreased. Among participants without HIV, integrin subunit alpha M (integrin adhesive function) and defensin alpha-1 (neutrophil function) increased after pitavastatin therapy and PLA2G7 decreased. At baseline, comparing participants with and without HIV, differentially expressed proteins included proteins involved in platelet and endothelial function and immune activation.
Conclusions
Pitavastatin affected proteins important to platelet and endothelial function and immune activation, and effects differed to a degree within PWH and participants without HIV.
We assessed the effects of pitavastatin on a broad array of proteins using a highly specific proximity extension assay (PEA) in HIV and metabolic syndrome non-HIV patients. Our results highlight effects on key inflammatory, collagen, neutrophil, and integrin functions.
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