Baricitinib plus Remdesivir for Hospitalized Adults with Covid-19

BACKGROUND: Severe coronavirus disease 2019 (Covid-19) is associated with dysregulated inflammation. The effects of combination treatment with baricitinib, a Janus kinase inhibitor, plus remdesivir are not known. METHODS: We conducted a double-blind, randomized, placebo-controlled trial evaluating baricitinib plus remdesivir in hospitalized adults with Covid-19. All the patients received remdesivir ( \u3c /=10 days) and either baricitinib ( \u3c /=14 days) or placebo (control). The primary outcome was the time to recovery. The key secondary outcome was clinical status at day 15. RESULTS: A total of 1033 patients underwent randomization (with 515 assigned to combination treatment and 518 to control). Patients receiving baricitinib had a median time to recovery of 7 days (95% confidence interval [CI], 6 to 8), as compared with 8 days (95% CI, 7 to 9) with control (rate ratio for recovery, 1.16; 95% CI, 1.01 to 1.32; P = 0.03), and a 30% higher odds of improvement in clinical status at day 15 (odds ratio, 1.3; 95% CI, 1.0 to 1.6). Patients receiving high-flow oxygen or noninvasive ventilation at enrollment had a time to recovery of 10 days with combination treatment and 18 days with control (rate ratio for recovery, 1.51; 95% CI, 1.10 to 2.08). The 28-day mortality was 5.1% in the combination group and 7.8% in the control group (hazard ratio for death, 0.65; 95% CI, 0.39 to 1.09). Serious adverse events were less frequent in the combination group than in the control group (16.0% vs. 21.0%; difference, -5.0 percentage points; 95% CI, -9.8 to -0.3; P = 0.03), as were new infections (5.9% vs. 11.2%; difference, -5.3 percentage points; 95% CI, -8.7 to -1.9; P = 0.003). CONCLUSIONS: Baricitinib plus remdesivir was superior to remdesivir alone in reducing recovery time and accelerating improvement in clinical status among patients with Covid-19, notably among those receiving high-flow oxygen or noninvasive ventilation. The combination was associated with fewer serious adverse events. (Funded by the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov number, NCT04401579.)

Remdesivir for the Treatment of Covid-19 - Preliminary Report

BACKGROUND: Although several therapeutic agents have been evaluated for the treatment of coronavirus disease 2019 (Covid-19), none have yet been shown to be efficacious. METHODS: We conducted a double-blind, randomized, placebo-controlled trial of intravenous remdesivir in adults hospitalized with Covid-19 with evidence of lower respiratory tract involvement. Patients were randomly assigned to receive either remdesivir (200 mg loading dose on day 1, followed by 100 mg daily for up to 9 additional days) or placebo for up to 10 days. The primary outcome was the time to recovery, defined by either discharge from the hospital or hospitalization for infection-control purposes only. RESULTS: A total of 1063 patients underwent randomization. The data and safety monitoring board recommended early unblinding of the results on the basis of findings from an analysis that showed shortened time to recovery in the remdesivir group. Preliminary results from the 1059 patients (538 assigned to remdesivir and 521 to placebo) with data available after randomization indicated that those who received remdesivir had a median recovery time of 11 days (95% confidence interval [CI], 9 to 12), as compared with 15 days (95% CI, 13 to 19) in those who received placebo (rate ratio for recovery, 1.32; 95% CI, 1.12 to 1.55; P \u3c 0.001). The Kaplan-Meier estimates of mortality by 14 days were 7.1% with remdesivir and 11.9% with placebo (hazard ratio for death, 0.70; 95% CI, 0.47 to 1.04). Serious adverse events were reported for 114 of the 541 patients in the remdesivir group who underwent randomization (21.1%) and 141 of the 522 patients in the placebo group who underwent randomization (27.0%). CONCLUSIONS: Remdesivir was superior to placebo in shortening the time to recovery in adults hospitalized with Covid-19 and evidence of lower respiratory tract infection. (Funded by the National Institute of Allergy and Infectious Diseases and others; ACCT-1 ClinicalTrials.gov number, NCT04280705.)

Increased survival in B-cell-deficient mice during experimental cerebral malaria suggests a role for circulating immune complexes

The pathogenesis of malaria, an insect-borne disease that takes millions of lives every year, is still not fully understood. Complement receptor 1 (CR1) has been described as a receptor for Plasmodium falciparum, which causes cerebral malaria in humans. We investigated the role of CR1 in an experimental model of cerebral malaria. Transgenic mice expressing human CR1 (hCR1(+)) on erythrocytes were infected with Plasmodium berghei ANKA and developed cerebral malaria. No difference in survival was observed in hCR1(+) mice compared to wild-type mice following infection with P. berghei ANKA; however, hCR1 detection was significantly diminished on erythrocytes between days 7 and 10 postinfection. hCR1 levels returned to baseline by day 17 postinfection in surviving animals. Immunoblot assays revealed that total erythrocyte hCR1 levels were diminished, confirming that immune complexes in association with erythrocyte hCR1 were likely removed from erythrocytes in vivo by clearance following immune adherence. Decreases in hCR1 were completely dependent on C3 expression, as mice treated with cobra venom factor (which consumes and depletes C3) retained hCR1 on erythrocytes during C3 depletion through day 7; erythrocyte hCR1 decreases were observed only when C3 levels recovered on day 9. B-cell-deficient mice exhibit a marked increase in survival following infection with P. berghei ANKA, which suggests that immune complexes play a central role in the pathogenesis of experimental cerebral malaria. Together, our findings highlight the importance of complement and immune complexes in experimental cerebral malaria. IMPORTANCE Cerebral malaria is a deadly complication of infection with Plasmodium falciparum. Despite its high prevalence, relatively little is understood about its pathogenesis. We have determined that immune complexes are generated and deposited on erythrocytes specifically expressing human complement receptor 1 in a mouse model of cerebral malaria. We also provide evidence demonstrating the importance of immunoglobulins in the pathogenesis of cerebral malaria in mice. These findings may have important implications in human cerebral malaria

Role of toll-like receptor 2 (TLR2) in neutrophil activation: GM-CSF enhances TLR2 expression and TLR2-mediated interleukin 8 responses in neutrophils

In vitro studies as well as clinical trials indicate that the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) enhance the ability of neutrophils (polymorphonuclear leukocytes) to eliminate microbial organisms. Toll-like receptor (TLR) proteins, homologs of the Drosophila protein Toll, have been found on the surface of mammalian cells and are important in the responses of macrophages to bacterial, viral, and fungal antigens. TLR4 is critical for the response to lipopolysaccharide (LPS) of gram-negative bacteria, while TLR2 is important for response to gram-positive bacteria, bacterial peptides, and yeast zymosan. We demonstrate that TLR2, but very little TLR4, is present on the surface of human neutrophils. In addition we demonstrate that GM-CSF and G-CSF dramatically up-regulate TLR2 and CD14 surface expression. GM-CSF treatment also up-regulates TLR2 and CD14 mRNA levels in neutrophils. In addition to increasing receptor expression, GM-CSF treatment enhanced the interleukin 8 (IL-8) secretion and superoxide priming responses of neutrophils to stimulation with TLR2 ligands, including zymosan, peptidoglycan, and lipoarabinomannan. The human monocyte response to crude bacterial LPS is composed of a TLR4-specific response to the pure LPS component and a TLR2-dependent response to associated lipopeptides. The removal of TLR2 lipopeptide components from LPS by phenol re-extraction substantially reduced both the IL-8 and superoxide response of the stimulated neutrophils, indicating that, unlike monocytes, the neutrophil response is preferentially directed to TLR2 ligands. Thus, our studies demonstrate that GM-CSF dramatically enhances the functional response of neutrophils to TLR2 ligands, including LPS-associated lipopeptides

Human nasal wash RNA-Seq reveals distinct cell-specific innate immune responses in influenza versus SARS-CoV-2

BACKGROUND Influenza A virus (IAV) and SARS-CoV-2 are pandemic viruses causing millions of deaths, yet their clinical manifestations are distinctly different. METHODS With the hypothesis that upper airway immune and epithelial cell responses are also distinct, we performed single-cell RNA sequencing (scRNA-Seq) on nasal wash cells freshly collected from adults with either acute COVID-19 or influenza or from healthy controls. We focused on major cell types and subtypes in a subset of donor samples. Results Nasal wash cells were enriched for macrophages and neutrophils for both individuals with influenza and those with COVID-19 compared with healthy controls. Hillock-like epithelial cells, M2-like macrophages, and age-dependent B cells were enriched in COVID-19 samples. A global decrease in IFN-associated transcripts in neutrophils, macrophages, and epithelial cells was apparent in COVID-19 samples compared with influenza samples. The innate immune response to SARS-CoV-2 appears to be maintained in macrophages, despite evidence for limited epithelial cell immune sensing. Cell-to-cell interaction analyses revealed a decrease in epithelial cell interactions in COVID-19 and highlighted differences in macrophage-macrophage interactions for COVID-19 and influenza. Conclusions Our study demonstrates that scRNA-Seq can define host and viral transcriptional activity at the site of infection and reveal distinct local epithelial and immune cell responses for COVID-19 and influenza that may contribute to their divergent disease courses. Funding Massachusetts Consortium on Pathogen Readiness, the Mathers Foundation, and the Department of Defense (W81XWH2110029) COVID-19 Expansion for AIRe Program

Properdin Levels in Individuals with Chemotherapy-Induced Neutropenia

BACKGROUND: Neutrophils produce and carry key components of the alternative pathway (AP) of complement, including properdin (P). The effect of chemotherapy-induced absolute neutropenia on circulating P levels and AP function has not been previously established. METHODS: We prospectively measured free P levels in serum from 27 individuals expected to develop neutropenia after administration of chemotherapy for hematological malignancies in preparation for hematopoietic stem cell transplantation and here describe the relationship between serum P levels and the neutrophil count over time. RESULTS: When the absolute neutrophil count (ANC) was \u3e 500 cells/mm3 pre-chemotherapy, P levels were significantly higher than P levels associated with an ANC ≤500 cells/mm3 (median values 8392 ng/mL and 6355 ng/mL, respectively; P = .001). Pairwise comparison between pre-chemotherapy P levels and P levels at initial or last documented neutropenia before recovery showed a significant decline (P \u3c .0001). No correlation was observed between P levels during neutropenia and after recovery of neutropenia in 20 subjects for which postneutropenia samples were obtained. A small but significant (P = .02) decrease in AP hemolytic activity was noted between baseline (preneutropenia) and samples obtained at the onset of neutropenia, but only with low (6.25%) and not higher (12.5 or 25%) serum concentrations. CONCLUSIONS: A decline in P levels and AP activity could contribute to the increased risk of infection in neutropenic patients and warrants further study

Importance of extra- and intracellular domains of TLR1 and TLR2 in NFkappa B signaling

Recognition of ligands by toll-like receptor (TLR) 2 requires interactions with other TLRs. TLRs form a combinatorial repertoire to discriminate between the diverse microbial ligands. Diversity results from extracellular and intracellular interactions of different TLRs. This paper demonstrates that TLR1 and TLR2 are required for ara-lipoarabinomannan- and tripalmitoyl cysteinyl lipopeptide-stimulated cytokine secretion from mononuclear cells. Confocal microscopy revealed that TLR1 and TLR2 cotranslationally form heterodimeric complexes on the cell surface and in the cytosol. Simultaneous cross-linking of both receptors resulted in ligand-independent signal transduction. Using chimeric TLRs, we found that expression of the extracellular domains along with simultaneous expression of the intracellular domains of both TLRs was necessary to achieve functional signaling. The domains from each receptor did not need to be contained within a single contiguous protein. Chimeric TLR analysis further defined the toll/IL-1R domains as the area of crucial intracellular TLR1-TLR2 interaction

Phomopsis bougainvilleicola prepatellar bursitis in a renal transplant recipient

Prepatellar bursitis is typically a monomicrobial bacterial infection. A fungal cause is rarely identified. We describe a 61-year-old man who had received a renal transplant 21 months prior to presentation whose synovial fluid and surgical specimens grew Phomopsis bougainvilleicola, a pycnidial coelomycete

US201 Study: A Phase 2, Randomized Proof-of-Concept Trial of Favipiravir for the Treatment of COVID-19

Background: Favipiravir is used to treat influenza, and studies demonstrate that it has antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: We performed a randomized, open-label, multicenter, phase 2 proof-of-concept trial of favipiravir in hospitalized adult patients with polymerase chain reaction (PCR)-positive coronavirus disease 2019 (COVID-19). Patients were randomized to standard of care (SOC) or favipiravir treatment (1800mg per os twice a day [b.i.d.] on day 1, followed by 1000mg b.i.d. for 13 days). The primary end point was time to viral clearance on day 29. Results: Fifty patients were enrolled and stratified by disease severity (critical disease, severe disease, or mild to moderate disease). Nineteen patients were censored from the event of viral clearance based on being SARS-CoV-2 PCR-negative at the study outset, being PCR-positive at day 29, or because of loss to follow-up. Data from the 31 remaining patients who achieved viral clearance show enhanced viral clearance in the favipiravir group compared with the SOC group by day 29, with 72% of the favipiravir group and 52% of the SOC group being evaluable for viral clearance through day 29. The median time to viral clearance was 16.0 days (90% CI, 12.0 to 29.0) in the favipiravir group and 30.0 days (90% CI, 12.0 to 31.0) in the SOC group. A post hoc analysis revealed an effect in the subgroup of patients who were neutralizing antibody-negative at randomization. Treatment-emergent adverse events were equally distributed between the groups. Conclusions: We demonstrate that favipiravir can be safely administered to hospitalized adults with COVID-19 and believe that further studies are warranted. ClinicalTrialsgov registration: NCT04358549

Single-cell analysis of upper airway cells reveals host-viral dynamics in influenza infected adults [preprint]

Influenza virus infections are major causes of morbidity and mortality. Research using cultured cells, bulk tissue, and animal models cannot fully capture human disease dynamics. Many aspects of virus-host interactions in a natural setting remain unclear, including the specific cell types that are infected and how they and neighboring bystander cells contribute to the overall antiviral response. To address these questions, we performed single-cell RNA sequencing (scRNA-Seq) on cells from freshly collected nasal washes from healthy human donors and donors diagnosed with acute influenza during the 2017-18 season. We describe a previously uncharacterized goblet cell population, specific to infected individuals, with high expression of MHC class II genes. Furthermore, leveraging scRNA-Seq reads, we obtained deep viral genome coverage and developed a model to rigorously identify infected cells that detected influenza infection in all epithelial cell types and even some immune cells. Our data revealed that each donor was infected by a unique influenza variant and that each variant was separated by at least one unique non-synonymous difference. Our results demonstrate the power of massively-parallel scRNA-Seq to study viral variation, as well as host and viral transcriptional activity during human infection