Modelling human choices: MADeM and decision‑making

Research supported by FAPESP 2015/50122-0 and DFG-GRTK 1740/2. RP and AR are also part of the Research, Innovation and Dissemination Center for Neuromathematics FAPESP grant (2013/07699-0). RP is supported by a FAPESP scholarship (2013/25667-8). ACR is partially supported by a CNPq fellowship (grant 306251/2014-0)

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Utility of kidney organoids for disease modeling and therapeutic development

Thesis (Ph.D.)--University of Washington, 2022Chronic kidney disease (CKD) affects 1 in 7 adults and is the 10th leading cause of death in the United States in 2022. Kidney transplant and dialysis remain the leading treatment strategies for kidney failure despite their expensive and outdated technological innovation. Stem cell derived human kidney organoids aim to provide a vital tool to study complex diseases ranging from infectious to genetic, and a translationally relevant system to discover and probe novel therapeutic pathways to improve our ability to treat kidney disease. CKD is the greatest risk factor for developing severe COVID-19, the 3rd leading cause of death in the US in 2022, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been reported to cause acute kidney injury in 1 in 4 hospitalized COVID-19 patients. Utilizing genome-edited kidney organoids, SARS-CoV-2 variants, and clinical data, we investigated viral tropism, mechanism, and therapeutic approaches in the context of the kidney. SARS-CoV-2 infected proximal tubules in kidney organoids via angiotensin converting enzyme 2 (ACE2). Infected organoids produced replication competent virus and displayed apoptotic responses in the context of polycystic kidney disease (PKD), a genetic cause of CKD. Cross-validation of gene expression patterns in organoids reflects proteomic signatures of COVID-19 in the urine of critically ill patients indicating interferon pathway upregulation. SARS-CoV-2 viral variants alpha, beta, gamma, kappa, and delta exhibit comparable levels of infection in kidney organoids. Replication is reduced by remdesivir treated and infection blocked by treatment with de novo–designed spike binder peptides. This work clarifies the impact SARS-CoV-2 infection has on the kidney and enables the assessment of viral fitness and emerging therapies in the context of infectious disease. Autosomal dominant PKD is a genetic kidney disease affecting 1 in every 400-1000 people worldwide, causing progressive fluid-filled cyst production followed by fibrosis in the kidneys, liver, and other organs, resulting in organ failure. PKD is caused by mutations in the polycystin proteins polycystin-1 (PC1) or polycystin-2 (PC2), but the molecular pathway causing cystogenesis remains elusive. Genome-edited PKD organoids phenocopy cystogenesis and previously identified that the myosin inhibitor blebbistatin resulted in cyst enlargement. I discovered that treatment with the myosin activator, EMD 57033 (EMD), prevented cyst growth and that treatment when cysts were already established was able to slow cyst expansion. Live-imaging of EMD-treated organoids expressing fluorescently-tagged non-muscle myosin II B (NMIIB-GFP) revealed increased apical-basal tubule contractility of PKD organoid tubules compared to controls, indicating that the PKD organoid tubules were poised to contract and may have intrinsic contractile dysfunction. Analysis of the slowly progressing Pkd1RC/RC mouse model reveals a concomitant expansion of phosphorylated myosin light chain 2 (pMLC2) expressing stromal pericytes and cyst growth, suggesting that a therapeutic reducing pMLC2 expression may rescue kidney fibrosis later in disease. Together, this work suggests both a tubular and stromal myosin contribution to PKD pathogenesis that can be therapeutically targeted using myosin activators early in disease and pMLC2 inhibitors in later stage disease progression. In conclusion, our studies of PKD and COVID-19 reveal great utility of kidney organoids for disease modeling and therapeutic development, advancing the translational applications of organoid technology

A virus-packageable CRISPR screen identifies host factors mediating interferon inhibition of HIV.

UNLABELLED: Interferon (IFN) inhibits HIV replication by inducing antiviral effectors. To comprehensively identify IFN-induced HIV restriction factors, we assembled a CRISPR sgRNA library of Interferon Stimulated Genes (ISGs) into a modified lentiviral vector that allows for packaging of sgRNA-encoding genomes in trans into budding HIV-1 particles. We observed that knockout of Zinc Antiviral Protein (ZAP) improved the performance of the screen due to ZAP-mediated inhibition of the vector. A small panel of IFN-induced HIV restriction factors, including MxB, IFITM1, Tetherin/BST2 and TRIM5alpha together explain the inhibitory effects of IFN on the CXCR4-tropic HIV-1 strain, HIV-1LAI, in THP-1 cells. A second screen with a CCR5-tropic primary strain, HIV-1Q23.BG505, described an overlapping, but non-identical, panel of restriction factors. Further, this screen also identifies HIV dependency factors. The ability of IFN-induced restriction factors to inhibit HIV strains to replicate in human cells suggests that these human restriction factors are incompletely antagonized. EDITORIAL NOTE: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editors assessment is that all the issues have been addressed (see decision letter)

Multivalent designed proteins neutralize SARS-CoV-2 variants of concern and confer protection against infection in mice.

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to arise and prolong the coronavirus disease 2019 (COVID-19) pandemic. Here, we used a cell-free expression workflow to rapidly screen and optimize constructs containing multiple computationally designed miniprotein inhibitors of SARS-CoV-2. We found the broadest efficacy was achieved with a homotrimeric version of the 75-residue angiotensin-converting enzyme 2 (ACE2) mimic AHB2 (TRI2-2) designed to geometrically match the trimeric spike architecture. Consistent with the design model, in the cryo-electron microscopy structure TRI2-2 forms a tripod at the apex of the spike protein that engaged all three receptor binding domains simultaneously. TRI2-2 neutralized Omicron (B.1.1.529), Delta (B.1.617.2), and all other variants tested with greater potency than the monoclonal antibodies used clinically for the treatment of COVID-19. TRI2-2 also conferred prophylactic and therapeutic protection against SARS-CoV-2 challenge when administered intranasally in mice. Designed miniprotein receptor mimics geometrically arrayed to match pathogen receptor binding sites could be a widely applicable antiviral therapeutic strategy with advantages over antibodies in greater resistance to viral escape and antigenic drift, and advantages over native receptor traps in lower chances of autoimmune responses

Effect of Antiplatelet Therapy on Survival and Organ Support–Free Days in Critically Ill Patients With COVID-19

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