Rationale: In life-threatening coronavirus disease (COVID-19), corticosteroids reduce mortality, suggesting that immune responses have a causal role in death. Whether this deleterious inflammation is primarily a direct reaction to the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or an independent immunopathologic process is unknown. Objectives: To determine SARS-CoV-2 organotropism and organ-specific inflammatory responses and the relationships among viral presence, inflammation, and organ injury. Methods: Tissue was acquired from 11 detailed postmortem examinations. SARS-CoV-2 organotropism was mapped by using multiplex PCR and sequencing, with cellular resolution achieved by in situ viral S (spike) protein detection. Histologic evidence of inflammation was quantified from 37 anatomic sites, and the pulmonary immune response was characterized by using multiplex immunofluorescence. Measurements and Main Results: Multiple aberrant immune responses in fatal COVID-19 were found, principally involving the lung and reticuloendothelial system, and these were not clearly topologically associated with the virus. Inflammation and organ dysfunction did not map to the tissue and cellular distribution of SARS-CoV-2 RNA and protein between or within tissues. An arteritis was identified in the lung, which was further characterized as a monocyte/myeloid-rich vasculitis, and occurred together with an influx of macrophage/monocyte-lineage cells into the pulmonary parenchyma. In addition, stereotyped abnormal reticuloendothelial responses, including excessive reactive plasmacytosis and iron-laden macrophages, were present and dissociated from viral presence in lymphoid tissues. Conclusions: Tissue-specific immunopathology occurs in COVID-19, implicating a significant component of the immune-mediated, virus-independent immunopathologic process as a primary mechanism in severe disease. Our data highlight novel immunopathologic mechanisms and validate ongoing and future efforts to therapeutically target aberrant macrophage and plasma-cell responses as well as promote pathogen tolerance in COVID-19.

Purpose: Nucleoside analogues form the backbone of many therapeutic regimens in oncology and require the presence of intracellular enzymes for their activation. A ProTide is comprised of a nucleoside fused to a protective phosphoramidate cap. ProTides are easily incorporated into cells whereupon the cap is cleaved and a preactivated nucleoside released. 3′-Deoxyadenosine (3′-dA) is a naturally occurring adenosine analogue with established anticancer activity in vitro but limited bioavailability due to its rapid in vivo deamination by the circulating enzyme adenosine deaminase, poor uptake into cells, and reliance on adenosine kinase for its activation. In order to overcome these limitations, 3′-dA was chemically modified to create the novel ProTide NUC-7738. Experimental Design: We describe the synthesis of NUC-7738. We determine the IC50 of NUC-7738 using pharmacokinetics (PK) and conduct genome-wide analyses to identify its mechanism of action using different cancer model systems. We validate these findings in patients with cancer. Results: We show that NUC-7738 overcomes the cancer resistance mechanisms that limit the activity of 3′-dA and that its activation is dependent on ProTide cleavage by the enzyme histidine triad nucleotide-binding protein 1. PK and tumor samples obtained from the ongoing first-in-human phase I clinical trial of NUC-7738 further validate our in vitro findings and show NUC-7738 is an effective proapoptotic agent in cancer cells with effects on the NF-κB pathway. Conclusions: Our study provides proof that NUC-7738 overcomes cellular resistance mechanisms and supports its further clinical evaluation as a novel cancer treatment within the growing pantheon of anticancer ProTides.

ERISM continuously maps dynamic changes in the forces podocytes exert on their substrate in a model of acute kidney damage.

Background: Tissue inflammation is associated with organ dysfunction and death in Covid-19. The efficacy of dexamethasone in preventing mortality in critical Covid-19 suggests that inflammation has a causal role in death. Whether this deleterious inflammation is a direct response to the presence of SARS-CoV-2, or an independent immuno-pathologic process, is unknown. Methods: Tissue was acquired from detailed post-mortem examinations conducted on 11 well characterised hospitalised patients with fatal Covid-19. SARS-CoV-2 organotropism was mapped at an organ level by multiplex PCR and sequencing, with cellular resolution achieved by in situ viral spike (S) protein detection. Histological evidence of inflammation and organ injury was systematically examined, and the pulmonary immune response characterized with multiplex immunofluorescence. Findings: SARS-CoV-2 was detected across a wide variety of organs, most frequently in the respiratory tract but also in numerous extra-pulmonary sites. Minimal histological evidence of inflammation was identified in non-pulmonary organs despite frequent detection of viral RNA and protein. At a cellular level, viral protein was identified without adjacent inflammation in the intestine, liver and kidney. Severe inflammatory change was restricted to the lung and reticulo-endothelial system. Diffuse alveolar damage, pulmonary thrombi and a monocyte/myeloid-predominant vasculitis were the predominant pulmonary findings, though there was not a consistent association between viral presence and either the presence or nature of the inflammatory response within the lung. Immunophenotyping revealed an influx of macrophages, monocytes and T cells into pulmonary parenchyma. Bone marrow examination revealed plasmacytosis, erythroid dysplasia and iron-laden macrophages. Plasma cell excess was also present in lymph node, spleen and lung. These stereotyped reticulo-endothelial responses occurred largely independently of the presence of virus in lymphoid tissues. Conclusions: Tissue inflammation and organ dysfunction in fatal Covid-19 do not map to the tissue and cellular distribution of SARS-CoV-2, demonstrating tissue-specific tolerance. We conclude that death in Covid-19 is primarily a consequence of immune-mediated, rather than pathogen-mediated, organ inflammation and injury. Funding: The Chief Scientist Office, LifeArc, Medical Research Scotland, UKRI (MRC).

Nuclear factor erythroid 2-related factor 1 (NFE2L1, NRF1) and nuclear factor erythroid 2-related factor 2 (NFE2L2, NRF2) are distinct oxidative stress response transcription factors, both of which have been shown to perform cytoprotective functions, modulating cell stress response and homeostasis. NAD(P)H:quinone oxidoreductase (NQO1) is a mutual downstream antioxidant gene target that catalyzes the two-electron reduction of an array of substrates, protecting against reactive oxygen species (ROS) generation. NQO1 is upregulated in non-small cell lung cancer (NSCLC) and is proposed as a predictive biomarker and therapeutic target. Antioxidant protein expression of immune cells within the NSCLC tumor microenvironment (TME) remains undetermined and may affect immune cell effector functions and survival outcomes. Multiplex immunofluorescence was performed to examine the co-localization of NQO1, NRF1 and NRF2 within the tumor and TME of 162 chemotherapy-naïve, early-stage NSCLC patients treated by primary surgical resection. This study demonstrates that NQO1 protein expression is high in normal, tumor-adjacent tissue and that NQO1 expression varies depending on the cell type. Inter and intra-patient heterogenous NQO1 expression was observed in lung cancer. Co-expression analysis showed NQO1 is independent of NRF1 and NRF2 in tumors. Density-based co-expression analysis demonstrated NRF1 and NRF2 double-positive expression in cancer cells is associated with improved overall survival.

Podocyte cellular injury and detachment from glomerular capillaries constitute a critical factor contributing to kidney disease. Notably, transcription factors are instrumental in maintaining podocyte differentiation and homeostasis. This study explores the hitherto uninvestigated expression of Nuclear Factor Erythroid 2-related Factor 1 (NFE2L1) in podocytes. We evaluated the podocyte expression of NFE2L1, Nuclear Factor Erythroid 2-related Factor 2 (NFE2L2), and NAD(P)H:quinone Oxidoreductase (NQO1) in 127 human glomerular disease biopsies using multiplexed immunofluorescence and image analysis. We found that both NFE2L1 and NQO1 expressions were significantly diminished across all observed renal diseases. Furthermore, we exposed human immortalized podocytes and ex vivo kidney slices to Puromycin Aminonucleoside (PAN) and characterized the NFE2L1 protein isoform expression. PAN treatment led to a reduction in the nuclear expression of NFE2L1 in ex vivo kidney slices and podocytes.