Mutual antagonism between CD44 and integrins in glioblastoma cell traction and migration.

Cell migration is the major driver of invasion and metastasis during cancer progression. For cells to migrate, they utilize the actin-myosin cytoskeleton and adhesion molecules, such as integrins and CD44, to generate traction forces in their environment. CD44 primarily binds to hyaluronic acid (HA) and integrins primarily bind to extracellular matrix (ECM) proteins such as collagen. However, the role of CD44 under integrin-mediated conditions and vice versa is not well known. Here, we performed traction force microscopy (TFM) on U251 cells seeded on collagen I-coated polyacrylamide gels to assess the functional mechanical relationship between integrins and CD44. Performing TFM on integrin-mediated adhesion conditions, i.e., collagen, we found that CD44KO U251 cells exerted more traction force than wild-type (WT) U251 cells. Furthermore, untreated WT and CD44-blocked WT exhibited comparable results. Conversely, in CD44-mediated adhesive conditions, integrin-blocked WT cells exerted a higher traction force than untreated WT cells. Our data suggest that CD44 and integrins have a mutually antagonistic relationship where one receptor represses the other's ability to generate traction force on its cognate substrate.

Biophysical modeling identifies an optimal hybrid amoeboid-mesenchymal phenotype for maximal T cell migration speeds.

Despite recent experimental progress in characterizing cell migration mechanics, our understanding of the mechanisms governing rapid cell movement remains limited. To effectively limit tumor growth, antitumoral T cells need to rapidly migrate to find and kill cancer cells. To investigate the upper limits of cell speed, we developed a new hybrid stochastic-mean field model of bleb-based cell motility. We first examined the potential for adhesion-free bleb-based migration and show that cells migrate inefficiently in the absence of adhesion-based forces, i.e., cell swimming. While no cortical contractility oscillations are needed for cells to swim in viscoelastic media, high-to-low cortical contractility oscillations are necessary for cell swimming in viscous media. This involves a high cortical contractility phase with multiple bleb nucleation events, followed by an intracellular pressure buildup recovery phase at low cortical tensions, resulting in modest net cell motion. However, our model suggests that cells can employ a hybrid bleb- and adhesion-based migration mechanism for rapid cell motility and identifies conditions for optimality. The model provides a momentum-conserving mechanism underlying rapid single-cell migration and identifies factors as design criteria for engineering T cell therapies to improve movement in mechanically complex environments.

Favorable Antiviral Effect of Metformin on Severe Acute Respiratory Syndrome Coronavirus 2 Viral Load in a Randomized, Placebo-Controlled Clinical Trial of Coronavirus Disease 2019.

BACKGROUND: Metformin has antiviral activity against RNA viruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The mechanism appears to be suppression of protein translation via targeting the host mechanistic target of rapamycin pathway. In the COVID-OUT randomized trial for outpatient coronavirus disease 2019 (COVID-19), metformin reduced the odds of hospitalizations/death through 28 days by 58%, of emergency department visits/hospitalizations/death through 14 days by 42%, and of long COVID through 10 months by 42%. METHODS: COVID-OUT was a 2 × 3 randomized, placebo-controlled, double-blind trial that assessed metformin, fluvoxamine, and ivermectin; 999 participants self-collected anterior nasal swabs on day 1 (n = 945), day 5 (n = 871), and day 10 (n = 775). Viral load was quantified using reverse-transcription quantitative polymerase chain reaction. RESULTS: The mean SARS-CoV-2 viral load was reduced 3.6-fold with metformin relative to placebo (-0.56 log10 copies/mL; 95% confidence interval [CI], -1.05 to -.06; P = .027). Those who received metformin were less likely to have a detectable viral load than placebo at day 5 or day 10 (odds ratio [OR], 0.72; 95% CI, .55 to .94). Viral rebound, defined as a higher viral load at day 10 than day 5, was less frequent with metformin (3.28%) than placebo (5.95%; OR, 0.68; 95% CI, .36 to 1.29). The metformin effect was consistent across subgroups and increased over time. Neither ivermectin nor fluvoxamine showed effect over placebo. CONCLUSIONS: In this randomized, placebo-controlled trial of outpatient treatment of SARS-CoV-2, metformin significantly reduced SARS-CoV-2 viral load, which may explain the clinical benefits in this trial. Metformin is pleiotropic with other actions that are relevant to COVID-19 pathophysiology. CLINICAL TRIALS REGISTRATION: NCT04510194.

Glioblastoma Cells Use an Integrin- and CD44-Mediated Motor-Clutch Mode of Migration in Brain Tissue.

Purpose: Glioblastoma (GBM) is an aggressive malignant brain tumor with 2 year survival rates of 6.7% (Stupp et al. in J Clin Oncol Off J Am Soc Clin Oncol 25:4127-4136, 2007; Mohammed et al. in Rep Pract Oncol Radiother 27:1026-1036, 2002). One key characteristic of the disease is the ability of glioblastoma cells to migrate rapidly and spread throughout healthy brain tissue (Lefranc et al. in J Clin Oncol Off J Am Soc Clin Oncol 23:2411-2422, 2005; Hoelzinger et al. in J Natl Cancer Inst 21:1583-1593, 2007). To develop treatments that effectively target cell migration, it is important to understand the fundamental mechanism driving cell migration in brain tissue. Several models of cell migration have been proposed, including the motor-clutch, bleb-based motility, and osmotic engine models. Methods: Here we utilized confocal imaging to measure traction dynamics and migration speeds of glioblastoma cells in mouse organotypic brain slices to identify the mode of cell migration. Results: We found that nearly all cell-vasculature interactions reflected pulling, rather than pushing, on vasculature at the cell leading edge, a finding consistent with a motor-clutch mode of migration, and inconsistent with an osmotic engine model or confined bleb-based migration. Reducing myosin motor activity, a key component in the motor-clutch model, was found to decrease migration speed at high doses for all cell types including U251 and 6 low-passage patient-derived xenograft lines (3 proneural and 3 mesenchymal subtypes). Variable responses were found at low doses, consistent with a motor-clutch mode of migration which predicts a biphasic relationship between migration speed and motor-to-clutch ratio. Targeting of molecular clutches including integrins and CD44 slowed migration of U251 cells. Conclusions: Overall we find that glioblastoma cell migration is most consistent with a motor-clutch mechanism to migrate through brain tissue ex vivo, and that both integrins and CD44, as well as myosin motors, play an important role in constituting the adhesive clutch. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-024-00799-x.

The OTX2 Gene Induces Tumor Growth and Triggers Leptomeningeal Metastasis by Regulating the mTORC2 Signaling Pathway in Group 3 Medulloblastomas.

Medulloblastoma (MB) encompasses diverse subgroups, and leptomeningeal disease/metastasis (LMD) plays a substantial role in associated fatalities. Despite extensive exploration of canonical genes in MB, the molecular mechanisms underlying LMD and the involvement of the orthodenticle homeobox 2 (OTX2) gene, a key driver in aggressive MB Group 3, remain insufficiently understood. Recognizing OTX2's pivotal role, we investigated its potential as a catalyst for aggressive cellular behaviors, including migration, invasion, and metastasis. OTX2 overexpression heightened cell growth, motility, and polarization in Group 3 MB cells. Orthotopic implantation of OTX2-overexpressing cells in mice led to reduced median survival, accompanied by the development of spinal cord and brain metastases. Mechanistically, OTX2 acted as a transcriptional activator of the Mechanistic Target of Rapamycin (mTOR) gene's promoter and the mTORC2 signaling pathway, correlating with upregulated downstream genes that orchestrate cell motility and migration. Knockdown of mTOR mRNA mitigated OTX2-mediated enhancements in cell motility and polarization. Analysis of human MB tumor samples (N = 952) revealed a positive correlation between OTX2 and mTOR mRNA expression, emphasizing the clinical significance of OTX2's role in the mTORC2 pathway. Our results reveal that OTX2 governs the mTORC2 signaling pathway, instigating LMD in Group 3 MBs and offering insights into potential therapeutic avenues through mTORC2 inhibition.

Radiation Therapy and Myeloid-Derived Suppressor Cells: Breaking Down Their Cancerous Partnership.

Radiation therapy (RT) has been a primary treatment modality in cancer for decades. Increasing evidence suggests that RT can induce an immunosuppressive shift via upregulation of cells such as tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). MDSCs inhibit antitumor immunity through potent immunosuppressive mechanisms and have the potential to be crucial tools for cancer prognosis and treatment. MDSCs interact with many different pathways, desensitizing tumor tissue and interacting with tumor cells to promote therapeutic resistance. Vascular damage induced by RT triggers an inflammatory signaling cascade and potentiates hypoxia in the tumor microenvironment (TME). RT can also drastically modify cytokine and chemokine signaling in the TME to promote the accumulation of MDSCs. RT activation of the cGAS-STING cytosolic DNA sensing pathway recruits MDSCs through a CCR2-mediated mechanism, inhibiting the production of type 1 interferons and hampering antitumor activity and immune surveillance in the TME. The upregulation of hypoxia-inducible factor-1 and vascular endothelial growth factor mobilizes MDSCs to the TME. After recruitment, MDSCs promote immunosuppression by releasing reactive oxygen species and upregulating nitric oxide production through inducible nitric oxide synthase expression to inhibit cytotoxic activity. Overexpression of arginase-1 on subsets of MDSCs degrades L-arginine and downregulates CD3ζ, inhibiting T-cell receptor reactivity. This review explains how radiation promotes tumor resistance through activation of immunosuppressive MDSCs in the TME and discusses current research targeting MDSCs, which could serve as a promising clinical treatment strategy in the future.

Glioblastoma cells use an integrin- and CD44-mediated motor-clutch mode of migration in brain tissue.

Glioblastoma (GBM) is an aggressive malignant brain tumor with 2-year survival rates of 6.7% [1], [2]. One key characteristic of the disease is the ability of glioblastoma cells to migrate rapidly and spread throughout healthy brain tissue[3], [4]. To develop treatments that effectively target cell migration, it is important to understand the fundamental mechanism driving cell migration in brain tissue. Here we utilized confocal imaging to measure traction dynamics and migration speeds of glioblastoma cells in mouse organotypic brain slices to identify the mode of cell migration. Through imaging cell-vasculature interactions and utilizing drugs, antibodies, and genetic modifications to target motors and clutches, we find that glioblastoma cell migration is most consistent with a motor-clutch mechanism to migrate through brain tissue ex vivo, and that both integrins and CD44, as well as myosin motors, play an important role in constituting the adhesive clutch.

The microtubule-severing protein UNC-45A preferentially binds to curved microtubules and counteracts the microtubule-straightening effects of Taxol.

Uncoordinated protein 45A (UNC-45A) is the only known ATP-independent microtubule (MT)-severing protein. Thus, it severs MTs via a novel mechanism. In vitro and in cells, UNC-45A-mediated MT severing is preceded by the appearance of MT bends. While MTs are stiff biological polymers, in cells, they often curve, and the result of this curving can be breaking off. The contribution of MT-severing proteins on MT lattice curvature is largely undefined. Here, we show that UNC-45A curves MTs. Using in vitro biophysical reconstitution and total internal fluorescence microscopy analysis, we show that UNC-45A is enriched in the areas where MTs are curved versus the areas where MTs are straight. In cells, we show that UNC-45A overexpression increases MT curvature and its depletion has the opposite effect. We also show that this effect occurs is independent of actomyosin contractility. Lastly, we show for the first time that in cells, Paclitaxel straightens MTs, and that UNC-45A can counteracts the MT-straightening effects of the drug.

The Microtubule Severing Protein UNC-45A Counteracts the Microtubule Straightening Effects of Taxol.

UNC-45A is the only known ATP-independent microtubule (MT) severing protein. Thus, it severs MTs via a novel mechanism. In vitro and in cells UNC-45A-mediated MT severing is preceded by the appearance of MT bends. While MTs are stiff biological polymers, in cells, they often curve, and the result of this curving can be breaking off. The contribution of MT severing proteins on MT lattice curvature is largely undefined. Here we show that UNC-45A curves MTs. Using in vitro biophysical reconstitution and TIRF microscopy analysis, we show that UNC-45A is enriched in the areas where MTs are curved versus the areas where MTs are straight. In cells, we show that UNC-45A overexpression increases MT curvature and its depletion has the opposite effect. We also show that this effect occurs is independent of actomyosin contractility. Lastly, we show for the first time that in cells, Paclitaxel straightens MTs, and that UNC-45A can counteracts the MT straightening effects of the drug. Significance: Our findings reveal for the first time that UNC-45A increases MT curvature. This hints that UNC-45A-mediated MT severing could be due to the worsening of MT curvature and provide a mechanistic understanding of how this MT-severing protein may act. UNC-45A is the only MT severing protein expressed in human cancers, including paclitaxel-resistant ovarian cancer. Our finding that UNC-45A counteracts the paclitaxel-straightening effects of MTs in cells suggests an additional mechanism through which cancer cells escape drug treatment.

Optimal cell traction forces in a generalized motor-clutch model.

Cells exert forces on mechanically compliant environments to sense stiffness, migrate, and remodel tissue. Cells can sense environmental stiffness via myosin-generated pulling forces acting on F-actin, which is in turn mechanically coupled to the environment via adhesive proteins, akin to a clutch in a drivetrain. In this "motor-clutch" framework, the force transmitted depends on the complex interplay of motor, clutch, and environmental properties. Previous mean-field analysis of the motor-clutch model identified the conditions for optimal stiffness for maximal force transmission via a dimensionless number that combines motor-clutch parameters. However, in this and other previous mean-field analyses, the motor-clutch system is assumed to have balanced motors and clutches and did not consider force-dependent clutch reinforcement and catch bond behavior. Here, we generalize the motor-clutch analytical framework to include imbalanced motor-clutch regimes, with clutch reinforcement and catch bonding, and investigate optimality with respect to all parameters. We found that traction force is strongly influenced by clutch stiffness, and we discovered an optimal clutch stiffness that maximizes traction force, suggesting that cells could tune their clutch mechanical properties to perform a specific function. The results provide guidance for maximizing the accuracy of cell-generated force measurements via molecular tension sensors by designing their mechanosensitive linker peptide to be as stiff as possible. In addition, we found that, on rigid substrates, the mean-field analysis identifies optimal motor properties, suggesting that cells could regulate their myosin repertoire and activity to maximize force transmission. Finally, we found that clutch reinforcement shifts the optimum substrate stiffness to larger values, whereas the optimum substrate stiffness is insensitive to clutch catch bond properties. Overall, our work reveals novel features of the motor-clutch model that can affect the design of molecular tension sensors and provide a generalized analytical framework for predicting and controlling cell adhesion and migration in immunotherapy and cancer.

Metformin reduces SARS-CoV-2 in a Phase 3 Randomized Placebo Controlled Clinical Trial.

Current antiviral treatment options for SARS-CoV-2 infections are not available globally, cannot be used with many medications, and are limited to virus-specific targets.1-3 Biophysical modeling of SARS-CoV-2 replication predicted that protein translation is an especially attractive target for antiviral therapy.4 Literature review identified metformin, widely known as a treatment for diabetes, as a potential suppressor of protein translation via targeting of the host mTor pathway.5 In vitro, metformin has antiviral activity against RNA viruses including SARS-CoV-2.6,7 In the COVID-OUT phase 3, randomized, placebo-controlled trial of outpatient treatment of COVID-19, metformin had a 42% reduction in ER visits/hospitalizations/death through 14 days; a 58% reduction in hospitalizations/death through 28 days, and a 42% reduction in Long COVID through 10 months.8,9 Here we show viral load analysis of specimens collected in the COVID-OUT trial that the mean SARS-CoV-2 viral load was reduced 3.6-fold with metformin relative to placebo (-0.56 log10 copies/mL; 95%CI, -1.05 to -0.06, p=0.027) while there was no virologic effect for ivermectin or fluvoxamine vs placebo. The metformin effect was consistent across subgroups and with emerging data.10,11 Our results demonstrate, consistent with model predictions, that a safe, widely available,12 well-tolerated, and inexpensive oral medication, metformin, can be repurposed to significantly reduce SARS-CoV-2 viral load.

Outpatient treatment of COVID-19 and incidence of post-COVID-19 condition over 10 months (COVID-OUT): a multicentre, randomised, quadruple-blind, parallel-group, phase 3 trial.

BACKGROUND: Post-COVID-19 condition (also known as long COVID) is an emerging chronic illness potentially affecting millions of people. We aimed to evaluate whether outpatient COVID-19 treatment with metformin, ivermectin, or fluvoxamine soon after SARS-CoV-2 infection could reduce the risk of long COVID. METHODS: We conducted a decentralised, randomised, quadruple-blind, parallel-group, phase 3 trial (COVID-OUT) at six sites in the USA. We included adults aged 30-85 years with overweight or obesity who had COVID-19 symptoms for fewer than 7 days and a documented SARS-CoV-2 positive PCR or antigen test within 3 days before enrolment. Participants were randomly assigned via 2 × 3 parallel factorial randomisation (1:1:1:1:1:1) to receive metformin plus ivermectin, metformin plus fluvoxamine, metformin plus placebo, ivermectin plus placebo, fluvoxamine plus placebo, or placebo plus placebo. Participants, investigators, care providers, and outcomes assessors were masked to study group assignment. The primary outcome was severe COVID-19 by day 14, and those data have been published previously. Because the trial was delivered remotely nationwide, the a priori primary sample was a modified intention-to-treat sample, meaning that participants who did not receive any dose of study treatment were excluded. Long COVID diagnosis by a medical provider was a prespecified, long-term secondary outcome. This trial is complete and is registered with ClinicalTrials.gov, NCT04510194. FINDINGS: Between Dec 30, 2020, and Jan 28, 2022, 6602 people were assessed for eligibility and 1431 were enrolled and randomly assigned. Of 1323 participants who received a dose of study treatment and were included in the modified intention-to-treat population, 1126 consented for long-term follow-up and completed at least one survey after the assessment for long COVID at day 180 (564 received metformin and 562 received matched placebo; a subset of participants in the metformin vs placebo trial were also randomly assigned to receive ivermectin or fluvoxamine). 1074 (95%) of 1126 participants completed at least 9 months of follow-up. 632 (56·1%) of 1126 participants were female and 494 (43·9%) were male; 44 (7·0%) of 632 women were pregnant. The median age was 45 years (IQR 37-54) and median BMI was 29·8 kg/m2 (IQR 27·0-34·2). Overall, 93 (8·3%) of 1126 participants reported receipt of a long COVID diagnosis by day 300. The cumulative incidence of long COVID by day 300 was 6·3% (95% CI 4·2-8·2) in participants who received metformin and 10·4% (7·8-12·9) in those who received identical metformin placebo (hazard ratio [HR] 0·59, 95% CI 0·39-0·89; p=0·012). The metformin beneficial effect was consistent across prespecified subgroups. When metformin was started within 3 days of symptom onset, the HR was 0·37 (95% CI 0·15-0·95). There was no effect on cumulative incidence of long COVID with ivermectin (HR 0·99, 95% CI 0·59-1·64) or fluvoxamine (1·36, 0·78-2·34) compared with placebo. INTERPRETATION: Outpatient treatment with metformin reduced long COVID incidence by about 41%, with an absolute reduction of 4·1%, compared with placebo. Metformin has clinical benefits when used as outpatient treatment for COVID-19 and is globally available, low-cost, and safe. FUNDING: Parsemus Foundation; Rainwater Charitable Foundation; Fast Grants; UnitedHealth Group Foundation; National Institute of Diabetes, Digestive and Kidney Diseases; National Institutes of Health; and National Center for Advancing Translational Sciences.

RAD-TGTs: high-throughput measurement of cellular mechanotype via rupture and delivery of DNA tension probes.

Mechanical forces drive critical cellular processes that are reflected in mechanical phenotypes, or mechanotypes, of cells and their microenvironment. We present here "Rupture And Deliver" Tension Gauge Tethers (RAD-TGTs) in which flow cytometry is used to record the mechanical history of thousands of cells exerting forces on their surroundings via their propensity to rupture immobilized DNA duplex tension probes. We demonstrate that RAD-TGTs recapitulate prior DNA tension probe studies while also yielding a gain of fluorescence in the force-generating cell that is detectable by flow cytometry. Furthermore, the rupture propensity is altered following disruption of the cytoskeleton using drugs or CRISPR-knockout of mechanosensing proteins. Importantly, RAD-TGTs can differentiate distinct mechanotypes among mixed populations of cells. We also establish oligo rupture and delivery can be measured via DNA sequencing. RAD-TGTs provide a facile and powerful assay to enable high-throughput mechanotype profiling, which could find various applications, for example, in combination with CRISPR screens and -omics analysis.

Main Manuscript for Cell migration simulator-based biomarkers for glioblastoma.

Glioblastoma is the most aggressive malignant brain tumor with poor survival due to its invasive nature driven by cell migration, with unclear linkage to transcriptomic information. Here, we applied a physics-based motor-clutch model, a cell migration simulator (CMS), to parameterize the migration of glioblastoma cells and define physical biomarkers on a patient-by-patient basis. We reduced the 11-dimensional parameter space of the CMS into 3D to identify three principal physical parameters that govern cell migration: motor number - describing myosin II activity, clutch number - describing adhesion level, and F-actin polymerization rate. Experimentally, we found that glioblastoma patient-derived (xenograft) (PD(X)) cell lines across mesenchymal (MES), proneural (PN), classical (CL) subtypes and two institutions (N=13 patients) had optimal motility and traction force on stiffnesses around 9.3kPa, with otherwise heterogeneous and uncorrelated motility, traction, and F-actin flow. By contrast, with the CMS parameterization, we found glioblastoma cells consistently had balanced motor/clutch ratios to enable effective migration, and that MES cells had higher actin polymerization rates resulting in higher motility. The CMS also predicted differential sensitivity to cytoskeletal drugs between patients. Finally, we identified 11 genes that correlated with the physical parameters, suggesting that transcriptomic data alone could potentially predict the mechanics and speed of glioblastoma cell migration. Overall, we describe a general physics-based framework for parameterizing individual glioblastoma patients and connecting to clinical transcriptomic data, that can potentially be used to develop patient-specific anti-migratory therapeutic strategies generally.

Multiscale models of integrins and cellular adhesions.

Computational models of integrin-based adhesion complexes have revealed important insights into the mechanisms by which cells establish connections with their external environment. However, how changes in conformation and function of individual adhesion proteins regulate the dynamics of whole adhesion complexes remains largely elusive. This is because of the large separation in time and length scales between the dynamics of individual adhesion proteins (nanoseconds and nanometers) and the emergent dynamics of the whole adhesion complex (seconds and micrometers), and the limitations of molecular simulation approaches in extracting accurate free energies, conformational transitions, reaction mechanisms, and kinetic rates, that can inform mechanisms at the larger scales. In this review, we discuss models of integrin-based adhesion complexes and highlight their main findings regarding: (i) the conformational transitions of integrins at the molecular and macromolecular scales and (ii) the molecular clutch mechanism at the mesoscale. Lastly, we present unanswered questions in the field of modeling adhesions and propose new ideas for future exciting modeling opportunities.

Photoresponsive Hydrogels for Studying Mechanotransduction of Cells.

Hydrogels are important platform materials for in vitro cellular studies. Mechanistic studies on durotaxis, the directional movement of a cell affected by a spatial gradient of stiffness of the underlying substrate, requires materials such as polyacrylamide, polyethylene glycol, or PDMS, in which the stiffness can be controlled in a spatiotemporal manner. Here, we describe the synthesis of an o-nitrobenzyl-based photocleavable cross-linker and its incorporation into a polyacrylamide hydrogel to render it photoresponsive. Precise control over the physical properties of the gel allows observation of glioblastoma durotaxis under surface stiffness conditions relevant to the actual brain environment.

Impact of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Vaccination and Booster on Coronavirus Disease 2019 (COVID-19) Symptom Severity Over Time in the COVID-OUT Trial.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination has decreasing protection from acquiring any infection with emergence of new variants; however, vaccination continues to protect against progression to severe coronavirus disease 2019 (COVID-19). The impact of vaccination status on symptoms over time is less clear. METHODS: Within a randomized trial on early outpatient COVID-19 therapy testing metformin, ivermectin, and/or fluvoxamine, participants recorded symptoms daily for 14 days. Participants were given a paper symptom diary allowing them to circle the severity of 14 symptoms as none (0), mild (1), moderate (2), or severe (3). This is a secondary analysis of clinical trial data on symptom severity over time using generalized estimating equations comparing those unvaccinated, SARS-CoV-2 vaccinated with primary vaccine series only, or vaccine-boosted. RESULTS: The parent clinical trial prospectively enrolled 1323 participants, of whom 1062 (80%) prospectively recorded some daily symptom data. Of these, 480 (45%) were unvaccinated, 530 (50%) were vaccinated with primary series only, and 52 (5%) vaccine-boosted. Overall symptom severity was least for the vaccine-boosted group and most severe for unvaccinated at baseline and over the 14 days (P < .001). Individual symptoms were least severe in the vaccine-boosted group including cough, chills, fever, nausea, fatigue, myalgia, headache, and diarrhea, as well as smell and taste abnormalities. Results were consistent over Delta and Omicron variant time periods. CONCLUSIONS: SARS-CoV-2 vaccine-boosted participants had the least severe symptoms during COVID-19, which abated the quickest over time. Clinical Trial Registration. NCT04510194.

Randomized Trial of Metformin, Ivermectin, and Fluvoxamine for Covid-19.

BACKGROUND: Early treatment to prevent severe coronavirus disease 2019 (Covid-19) is an important component of the comprehensive response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. METHODS: In this phase 3, double-blind, randomized, placebo-controlled trial, we used a 2-by-3 factorial design to test the effectiveness of three repurposed drugs - metformin, ivermectin, and fluvoxamine - in preventing serious SARS-CoV-2 infection in nonhospitalized adults who had been enrolled within 3 days after a confirmed diagnosis of infection and less than 7 days after the onset of symptoms. The patients were between the ages of 30 and 85 years, and all had either overweight or obesity. The primary composite end point was hypoxemia (≤93% oxygen saturation on home oximetry), emergency department visit, hospitalization, or death. All analyses used controls who had undergone concurrent randomization and were adjusted for SARS-CoV-2 vaccination and receipt of other trial medications. RESULTS: A total of 1431 patients underwent randomization; of these patients, 1323 were included in the primary analysis. The median age of the patients was 46 years; 56% were female (6% of whom were pregnant), and 52% had been vaccinated. The adjusted odds ratio for a primary event was 0.84 (95% confidence interval [CI], 0.66 to 1.09; P = 0.19) with metformin, 1.05 (95% CI, 0.76 to 1.45; P = 0.78) with ivermectin, and 0.94 (95% CI, 0.66 to 1.36; P = 0.75) with fluvoxamine. In prespecified secondary analyses, the adjusted odds ratio for emergency department visit, hospitalization, or death was 0.58 (95% CI, 0.35 to 0.94) with metformin, 1.39 (95% CI, 0.72 to 2.69) with ivermectin, and 1.17 (95% CI, 0.57 to 2.40) with fluvoxamine. The adjusted odds ratio for hospitalization or death was 0.47 (95% CI, 0.20 to 1.11) with metformin, 0.73 (95% CI, 0.19 to 2.77) with ivermectin, and 1.11 (95% CI, 0.33 to 3.76) with fluvoxamine. CONCLUSIONS: None of the three medications that were evaluated prevented the occurrence of hypoxemia, an emergency department visit, hospitalization, or death associated with Covid-19. (Funded by the Parsemus Foundation and others; COVID-OUT ClinicalTrials.gov number, NCT04510194.).