Correspondence to Dr Sarah H Chung, Rheumatology, University of Washington, Seattle, WA 98195, USA; sarahhc@uw.edu Bonelli et al recently reported reduced humoral responses to the BNT162b2 mRNA ...vaccine in five patients on rituximab therapy; in two patients with repopulated B cells, a low-level Spike protein antibody response occurred, but three patients with no detectable B-cells had no measurable antibody response to the vaccine.1 Of great interest, they reported that interferon-γ T-cell responses to SARS-CoV-2 Spike peptides were present in all five patients, irrespective of the antibody response to the vaccine. HCQ 200 mg one time a day mRNA 43 66 8 66/M RA RTX 1000 mg MTX 20 mg weekly mRNA 36 80 9 62/F SSc RTX 1000 mg MMF 1000 mg two times a day mRNA 110 43¶ 10 57/F SSc RTX 1000 mg HCQ 300 mg one time a day mRNA 13 39 11 64/F SSc RTX 1000 mg Prednisone 5 mg one time a day MMF 1500 mg two times a day MTX 17.5 mg weekly mRNA 12 20 12 45/F SLE RTX 1000 mg HCQ 300 mg one time a day mRNA 39 9 13 34/F SLE BEL 200 mg MMF 1500 mg two times a day mRNA 5 15 14 47/F IgG4-RD RTX 1000 mg Prednisone 8 mg one time a day mRNA 54 41 15 27/F IgA vasculitis RTX 1000 mg – mRNA 67 4 No patient had a history of SARS-CoV-2 clinical infection and/or testing by PCR. *BCDT dose refers to the dose last received prior to COVID-19 vaccination. †mRNA refers to Pfizer-BioNTech vaccine (two doses) or Moderna vaccine series (two doses); viral vector refers to Johnson & Johnson Janssen vaccine (one dose). ‡Vaccine refers to first/initial vaccine dose date, if series. §Vaccine refers to second/last vaccine dose, if series; anti-Spike IgG assessment as detected by Euroimmun IgG assay. ¶As detected by DiaSorin Liaison SARS-CoV-2 S1/S2 IgG assay (LabCorp Seattle). ...more information is available, we recommend that patients on targeted B-cell depleting or suppressive therapy follow local guidelines on COVID-19 prevention as if they were not vaccinated.
Pyroptosis is a programmed process of proinflammatory cell death mediated by caspase-1-related proteases that cleave the pore-forming protein, gasdermin D, causing cell lysis and release of ...inflammatory intracellular contents. The amino acid glycine prevents pyroptotic lysis via unknown mechanisms, without affecting caspase-1 activation or pore formation. Pyroptosis plays a critical role in diverse inflammatory diseases, including sepsis. Septic lethality is prevented by glycine treatment, suggesting that glycine-mediated cytoprotection may provide therapeutic benefit. In this study, we systematically examined a panel of small molecules, structurally related to glycine, for their ability to prevent pyroptotic lysis. We found a requirement for the carboxyl group, and limited tolerance for larger amino groups and substitution of the hydrogen R group. Glycine is an agonist for the neuronal glycine receptor, which acts as a ligand-gated chloride channel. The array of cytoprotective small molecules we identified resembles that of known glycine receptor modulators. However, using genetically deficient Glrb mutant macrophages, we found that the glycine receptor is not required for pyroptotic cytoprotection. Furthermore, protection against pyroptotic lysis is independent of extracellular chloride conductance, arguing against an effect mediated by ligand-gated chloride channels. Finally, we conducted a small-scale, hypothesis-driven small-molecule screen and identified unexpected ion channel modulators that prevent pyroptotic lysis with increased potency compared to glycine. Together, these findings demonstrate that pyroptotic lysis can be pharmacologically modulated and pave the way toward identification of therapeutic strategies for pathologic conditions associated with pyroptosis.
The unfolded protein response (UPR) is an ancient cellular pathway that detects and alleviates protein-folding stresses. The UPR components X-box binding protein 1 (XBP1) and inositol-requiring ...enzyme 1α (IRE1α) promote type I interferon (IFN) responses. We found that
-deficient mouse embryonic fibroblasts and macrophages had impaired antiviral resistance. However, this was not because of a defect in type I IFN responses but rather an inability of
-deficient cells to undergo viral-induced apoptosis. The ability to undergo apoptosis limited infection in wild-type cells.
-deficient cells were generally resistant to the intrinsic pathway of apoptosis through an indirect mechanism involving activation of the nuclease IRE1α. We observed an IRE1α-dependent reduction in the abundance of the proapoptotic microRNA miR-125a and a corresponding increase in the amounts of the members of the antiapoptotic Bcl-2 family. The activation of IRE1α by the hepatitis C virus (HCV) protein NS4B in XBP1-proficient cells also conferred apoptosis resistance and promoted viral replication. Furthermore, we found evidence of IRE1α activation and decreased miR-125a abundance in liver biopsies from patients infected with HCV compared to those in the livers of healthy controls. Our results reveal a prosurvival role for IRE1α in virally infected cells and suggest a possible target for IFN-independent antiviral therapy.
Botanical natural products have been widely consumed for their purported usefulness against COVID-19. Here, six botanical species from multiple sources and 173 isolated natural product compounds were ...screened for blockade of wild-type (WT) SARS-CoV-2 infection in human 293T epithelial cells overexpressing ACE-2 and TMPRSS2 protease (293TAT). Antiviral activity was demonstrated by an extract from Stephania tetrandra. Extract fractionation, liquid chromatography–mass spectrometry (LC-MS), antiviral assays, and computational analyses revealed that the alkaloid fraction and purified alkaloids tetrandrine, fangchinoline, and cepharanthine inhibited WT SARS-CoV-2 infection. The alkaloids and alkaloid fraction also inhibited the delta variant of concern but not WT SARS-CoV-2 in VeroAT cells. Membrane permeability assays demonstrate that the alkaloids are biologically available, although fangchinoline showed lower permeability than tetrandrine. At high concentrations, the extract, alkaloid fractions, and pure alkaloids induced phospholipidosis in 293TAT cells and less so in VeroAT cells. Gene expression profiling during virus infection suggested that alkaloid fraction and tetrandrine displayed similar effects on cellular gene expression and pathways, while fangchinoline showed distinct effects on cells. Our study demonstrates a multifaceted approach to systematically investigate the diverse activities conferred by complex botanical mixtures, their cell-context specificity, and their pleiotropic effects on biological systems.
Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. ...Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1β and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis. In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.
Critically ill patients manifest many of the same immune features seen in coronavirus disease 2019 (COVID-19), including both "cytokine storm" and "immune suppression." However, direct comparisons of ...molecular and cellular profiles between contemporaneously enrolled critically ill patients with and without severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are limited. We sought to identify immune signatures specifically enriched in critically ill patients with COVID-19 compared with patients without COVID-19. We enrolled a multisite prospective cohort of patients admitted under suspicion for COVID-19, who were then determined to be SARS-CoV-2-positive (
= 204) or -negative (
= 122). SARS-CoV-2-positive patients had higher plasma levels of CXCL10, sPD-L1, IFN-γ, CCL26, C-reactive protein (CRP), and TNF-α relative to SARS-CoV-2-negative patients adjusting for demographics and severity of illness (Bonferroni
value < 0.05). In contrast, the levels of IL-6, IL-8, IL-10, and IL-17A were not significantly different between the two groups. In SARS-CoV-2-positive patients, higher plasma levels of sPD-L1 and TNF-α were associated with fewer ventilator-free days (VFDs) and higher mortality rates (Bonferroni
value < 0.05). Lymphocyte chemoattractants such as CCL17 were associated with more severe respiratory failure in SARS-CoV-2-positive patients, but less severe respiratory failure in SARS-CoV-2-negative patients (
value for interaction < 0.01). Circulating T cells and monocytes from SARS-CoV-2-positive subjects were hyporesponsive to in vitro stimulation compared with SARS-CoV-2-negative subjects. Critically ill SARS-CoV-2-positive patients exhibit an immune signature of high interferon-induced lymphocyte chemoattractants (e.g., CXCL10 and CCL17) and immune cell hyporesponsiveness when directly compared with SARS-CoV-2-negative patients. This suggests a specific role for T-cell migration coupled with an immune-checkpoint regulatory response in COVID-19-related critical illness.
Pyroptosis is a cell death process that causes inflammation and contributes to numerous diseases. Pyroptosis is mediated by caspase-1 family proteases that cleave the pore-forming protein gasdermin ...D, causing plasma membrane rupture and release of pathogenic cellular contents. We previously identified muscimol as a small molecule that prevents plasma membrane rupture during pyroptosis via an unidentified mechanism. Here, we show that muscimol has reversible activity to prevent cellular lysis without affecting earlier pyroptotic events. Although muscimol is a well-characterized agonist for neuronal GABA
receptors, muscimol protection is not altered by GABA
receptor antagonists or recapitulated by other GABA
agonists, suggesting that muscimol acts via a novel mechanism. We find that muscimol blocks oligomerization of ninjurin-1, which is required for plasma membrane rupture downstream of gasdermin D pore formation. Our structure-activity relationship studies reveal distinct molecular determinants defining inhibition of pyroptotic lysis compared to GABA
binding. In addition, we demonstrate that muscimol reduces lethality during LPS-induced septic shock. Together, these findings demonstrate that ninjurin-1-mediated plasma membrane rupture can be pharmacologically modulated and pave the way toward identification of therapeutic strategies for pathologic conditions associated with pyroptosis.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently emerged, currently pandemic virus, and the etiologic agent of coronavirus disease 2019 (COVID-19). Clinical testing for ...antibodies to SARS-CoV-2 has rapidly become widespread, but data regarding the interlaboratory performance of these serologic assays are limited.
To describe the development and initial results of the College of American Pathologists (CAP) SARS-CoV-2 Serology Survey.
Members from the CAP Microbiology and Diagnostic Immunology and Flow Cytometry Committees formed a working group to support development of a new proficiency testing survey for anti-SARS-CoV-2 antibody assays. Supplemental questions in the survey assessed the state of SARS-CoV-2 serologic testing among participating laboratories as of July 2020. Results were analyzed for agreement by immunoglobulin (Ig) isotype tested, assay manufacturer, and methodology.
A total of 4125 qualitative results were received from 1110 laboratories participating in the first survey. Qualitative agreement for assays measuring anti-SARS-CoV-2 total antibodies or IgG was greater than 90% for all 3 samples in the survey. Qualitative agreement for IgM and IgA for the negative sample was greater than 95%, but lacked consensus for the other 2 samples.
These initial data suggest overall excellent agreement and comparable performance for most qualitative anti-SARS-CoV-2 IgG and total antibody assays across all participating clinical laboratories, regardless of specific target antigen or assay methodology.
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