Cutaneous and systemic hyperinflammation drives maculopapular drug exanthema in severely ill COVID-19 patients.

BACKGROUND: Coronavirus disease-2019 (COVID-19) has been associated with cutaneous findings, some being the result of drug hypersensitivity reactions such as maculopapular drug rashes (MDR). The aim of this study was to investigate whether COVID-19 may impact the development of the MDR. METHODS: Blood and skin samples from COVID-19 patients (based on a positive nasopharyngeal PCR) suffering from MDR (COVID-MDR), healthy controls, non-COVID-19-related patients with drug rash with eosinophilia and systemic symptoms (DRESS), and MDR were analyzed. We utilized imaging mass cytometry (IMC) to characterize the cellular infiltrate in skin biopsies. Furthermore, RNA sequencing transcriptome of skin biopsy samples and high-throughput multiplexed proteomic profiling of serum were performed. RESULTS: IMC revealed by clustering analyses a more prominent, phenotypically shifted cytotoxic CD8+ T cell population and highly activated monocyte/macrophage (Mo/Mac) clusters in COVID-MDR. The RNA sequencing transcriptome demonstrated a more robust cytotoxic response in COVID-MDR skin. However, severe acute respiratory syndrome coronavirus 2 was not detected in skin biopsies at the time point of MDR diagnosis. Serum proteomic profiling of COVID-MDR patients revealed upregulation of various inflammatory mediators (IL-4, IL-5, IL-6, TNF, and IFN-γ), eosinophil and Mo/Mac -attracting chemokines (MCP-2, MCP-3, MCP-4 and CCL11). Proteomics analyses demonstrated a massive systemic cytokine storm in COVID-MDR compared with the relatively milder cytokine storm observed in DRESS, while MDR did not exhibit such features. CONCLUSION: A systemic cytokine storm may promote activation of Mo/Mac and cytotoxic CD8+ T cells in severe COVID-19 patients, which in turn may impact the development of MDR.

ROS Induction Targets Persister Cancer Cells with Low Metabolic Activity in NRAS-Mutated Melanoma.

Clinical management of melanomas with NRAS mutations is challenging. Targeting MAPK signaling is only beneficial to a small subset of patients due to resistance that arises through genetic, transcriptional, and metabolic adaptation. Identification of targetable vulnerabilities in NRAS-mutated melanoma could help improve patient treatment. Here, we used multiomics analyses to reveal that NRAS-mutated melanoma cells adopt a mesenchymal phenotype with a quiescent metabolic program to resist cellular stress induced by MEK inhibition. The metabolic alterations elevated baseline reactive oxygen species (ROS) levels, leading these cells to become highly sensitive to ROS induction. In vivo xenograft experiments and single-cell RNA sequencing demonstrated that intratumor heterogeneity necessitates the combination of a ROS inducer and a MEK inhibitor to inhibit both tumor growth and metastasis. Ex vivo pharmacoscopy of 62 human metastatic melanomas confirmed that MEK inhibitor-resistant tumors significantly benefited from the combination therapy. Finally, oxidative stress response and translational suppression corresponded with ROS-inducer sensitivity in 486 cancer cell lines, independent of cancer type. These findings link transcriptional plasticity to a metabolic phenotype that can be inhibited by ROS inducers in melanoma and other cancers. SIGNIFICANCE: Metabolic reprogramming in drug-resistant NRAS-mutated melanoma cells confers sensitivity to ROS induction, which suppresses tumor growth and metastasis in combination with MAPK pathway inhibitors.

Prolonged Unfrozen Storage and Repeated Freeze-Thawing of SARS-CoV-2 Patient Samples Have Minor Effects on SARS-CoV-2 Detectability by RT-PCR.

Reliable transportation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) patient samples from a swabbing station to a diagnostics facility is essential for accurate results. Therefore, cooling or freezing the samples is recommended in case of longer transportation times. In this study, SARS-CoV-2 detectability by RT-PCR was assessed after prolonged unfrozen storage or repetitive freeze-thawing of SARS-CoV-2 samples. SARS-CoV-2-positive patient swabs stored in viral transport medium were exposed to different temperatures (4°C, 25°C, and 35°C) and to repetitive freeze-thawing, to assess the effect of storage conditions on RT-PCR detection. SARS-CoV-2 RNA was still reliably detected by RT-PCR after 21 days of storage in viral transport medium, even when the samples had been stored at 35°C. The maximum observed change in cycle threshold value per day was 0.046 (±0.019) at 35°C, and the maximum observed change in cycle threshold value per freeze-thaw cycle per day was 0.197 (±0.06). Compared with storage at 4°C, viral RNA levels deviated little but significantly when stored at 25°C or 35°C, or after repeated freeze-thawing. The results of this study indicate that viral RNA levels are relatively stable at higher temperatures and repetitive freeze-thawing.

A Comparative Study of Real-Time RT-PCR-Based SARS-CoV-2 Detection Methods and Its Application to Human-Derived and Surface Swabbed Material.

Real-time RT-PCR remains a gold standard in the detection of various viral diseases. In the coronavirus 2019 pandemic, multiple RT-PCR-based tests were developed to screen for viral infection. As an emergency response to increasing testing demand, we established a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PCR diagnostics platform for which we compared different commercial and in-house RT-PCR protocols. Four commercial, one customized, and one in-house RT-PCR protocols were evaluated with 92 SARS-CoV-2-positive and 92 SARS-CoV-2-negative samples. Furthermore, economical and practical characteristics of these protocols were compared. In addition, a highly sensitive digital droplet PCR (ddPCR) method was developed, and application of RT-PCR and ddPCR methods on SARS-CoV-2 environmental samples was examined. Very low limits of detection (1 or 2 viral copies/µL), high sensitivities (93.6% to 97.8%), and high specificities (98.7% to 100%) for the tested RT-PCR protocols were found. Furthermore, the feasibility of downscaling two of the commercial protocols, which could optimize testing capacity, was demonstrated. Tested commercial and customized RT-PCR detection kits show very good and comparable sensitivity and specificity, and the kits could be further optimized for use on SARS-CoV-2 viral samples derived from human and surface swabbed samples.

Specific Activation of the CD271 Intracellular Domain in Combination with Chemotherapy or Targeted Therapy Inhibits Melanoma Progression.

CD271 (NGFR) is a neurotrophin receptor that belongs to the tumor necrosis receptor (TNFR) family. Upon ligand binding, CD271 can mediate either survival or cell death. Although the role of CD271 as a marker of tumor-initiating cells is still a matter of debate, its role in melanoma progression has been well documented. Moreover, CD271 has been shown to be upregulated after exposure to both chemotherapy and targeted therapy. In this study, we demonstrate that activation of CD271 by a short ß-amyloid-derived peptide (Aß(25-35)) in combination with either chemotherapy or MAPK inhibitors induces apoptosis in 2D and 3D cultures of eight melanoma cell lines. This combinatorial treatment significantly reduced metastasis in a zebrafish xenograft model and led to significantly decreased tumor volume in mice. Administration of Aß(25-35) in ex vivo tumors from immunotherapy- and targeted therapy-resistant patients significantly reduced proliferation of melanoma cells, showing that activation of CD271 can overcome drug resistance. Aß(25-35) was specific to CD271-expressing cells and induced CD271 cleavage and phosphorylation of JNK (pJNK). The direct protein-protein interaction of pJNK with CD271 led to PARP1 cleavage, p53 and caspase activation, and pJNK-dependent cell death. Aß(25-35) also mediated mitochondrial reactive oxygen species (mROS) accumulation, which induced CD271 overexpression. Finally, CD271 upregulation inhibited mROS production, revealing the presence of a negative feedback loop in mROS regulation. These results indicate that targeting CD271 can activate cell death pathways to inhibit melanoma progression and potentially overcome resistance to targeted therapy. SIGNIFICANCE: The discovery of a means to specifically activate the CD271 death domain reveals unknown pathways mediated by the receptor and highlights new treatment possibilities for melanoma.