Introduction: Testing for active SARS-CoV-2 infection is a fundamental tool in the public health measures taken to control the COVID-19 pandemic. Because of the overwhelming use of SARS-CoV-2 reverse transcription (RT)-PCR tests worldwide, the availability of test kits has become a major bottleneck and the need to increase testing throughput is rising. We aim to overcome these challenges by pooling samples together, and performing RNA extraction and RT-PCR in pools. Methods: We tested the efficiency and sensitivity of pooling strategies for RNA extraction and RT-PCR detection of SARS-CoV-2. We tested 184 samples both individually and in pools to estimate the effects of pooling. We further implemented Dorfman pooling with a pool size of eight samples in large-scale clinical tests. Results: We demonstrated pooling strategies that increase testing throughput while maintaining high sensitivity. A comparison of 184 samples tested individually and in pools of eight samples showed that test results were not significantly affected. Implementing the eight-sample Dorfman pooling to test 26 576 samples from asymptomatic individuals, we identified 31 (0.12%) SARS-CoV-2 positive samples, achieving a 7.3-fold increase in throughput. Discussion: Pooling approaches for SARS-CoV-2 testing allow a drastic increase in throughput while maintaining clinical sensitivity. We report the successful large-scale pooled screening of asymptomatic
Stimulation of TAM (TYRO3, AXL and MERTK) Receptor Tyrosine Kinases promotes tumor progression through numerous cellular mechanisms. TAM cognate ligands GAS6 and PROS1 (for TYRO3 and MERTK) are secreted by host immune cells, an interaction which may support tumor progression. Here we reveal an unexpected antimetastatic role for myeloid-derived PROS1, directly suppressing the metastatic potential of lung and breast tumor models. Pros1 deletion in myeloid cells led to increased lung metastasis, independent of primary tumor infiltration. PROS1-cKO BMDMs led to elevated TNFα, IL-6, Nos2 and IL-10 via modulation of the Socs3-NFκB pathway. Conditioned medium from cKO BMDMs enhanced EMT, ERK, AKT and STAT3 activation within tumor cells, and promoted IL-10 dependent invasion and survival. Macrophages isolated from metastatic lungs modulated T cell proliferation and function, as well as expression of costimulatory molecules on dendritic cells in a PROS1-dependent manner. Inhibition of MERTK kinase activity blocked PROS1mediated suppression of TNFα and IL-6, but not of IL-10. Overall, using lung and breast cancer models, we identify the PROS1-MERTK axis within BMDMs as a potent regulator of adaptive immune responses with a potential to suppress metastatic seeding, and reveal IL-10 regulation by PROS1 to deviate from that of TNFα and IL-6.
Myeloid‐derived suppressor cells (MDSCs) are heterogenous populations of immature myeloid cells that can be divided into two main subpopulations, polymorphonuclear (PMN) MDSCs and monocytic (M) MDSCs. These cells accumulate during chronic inflammation and induce immunosuppression evident in an array of pathologies such as cancer, inflammatory bowel disease, and infectious and autoimmune diseases. Herein, we describe methods to isolate and characterize MDSCs from various murine tissue, as well as to phenotype blood‐derived MDSCs from patients. The protocols describe methods for isolation of total MDSCs and their subpopulations, for characterization, and for evaluation of their distribution within tissue, as well as for assessing their maturation stage by flow cytometry, immunofluorescence analyses, and Giemsa staining. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Single‐cell suspension generation from different tissue Alternate Protocol 1: Single‐cell suspension generation from subcutaneous melanoma tumors Basic Protocol 2: Characterization of MDSC phenotype Basic Protocol 3: Cell separation using magnetic beads: Separating pan‐MDSCs or PMN‐MDSC and M‐MDSC subpopulations Alternate Protocol 2: Staining and preparing MDSCs for sorting Support Protocol: PMN‐MDSC and M‐MDSC gating strategy in mouse Basic Protocol 4: Immunofluorescence analysis of MDSCs Basic Protocol 5: Handling human blood samples and characterizing human MDSCs Alternate Protocol 3: Flow cytometry staining of thawed human whole blood samples
Elevated osteoclast (OC) activity is a major contributor to inflammatory bone loss (IBL) during chronic inflammatory diseases. However, the specific OC precursors (OCPs) responding to inflammatory cues and the underlying mechanisms leading to IBL are poorly understood. We identified two distinct OCP subsets: Ly6ChiCD11bhi inflammatory OCPs (iOCPs) induced during chronic inflammation, and homeostatic Ly6ChiCD11blo OCPs (hOCPs) which remained unchanged. Functional and proteomic characterization revealed that while iOCPs were rare and displayed low osteoclastogenic potential under normal conditions, they expanded during chronic inflammation and generated OCs with enhanced activity. In contrast, hOCPs were abundant and manifested high osteoclastogenic potential under normal conditions but generated OCs with low activity and were unresponsive to the inflammatory environment. Osteoclasts derived from iOCPs expressed higher levels of resorptive and metabolic proteins than those generated from hOCPs, highlighting that different osteoclast populations are formed by distinct precursors. We further identified the TNF-α and S100A8/A9 proteins as key regulators that control the iOCP response during chronic inflammation. Furthermore, we demonstrated that the response of iOCPs but not that of hOCPs was abrogated in tnf-α−/− mice, in correlation with attenuated IBL. Our findings suggest a central role for iOCPs in IBL induction. iOCPs can serve as potential biomarkers for IBL detection and possibly as new therapeutic targets to combat IBL in a wide range of inflammatory conditions.
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