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BACKGROUND. Hyaluronan (HA), an extracellular matrix glycosaminoglycan, has been implicated in the pathophysiology of COVID-19 infection, pulmonary hypertension, pulmonary fibrosis, and other diseases, but is not targeted by any approved drugs. We asked whether hymecromone (4-methylumbelliferone [4-MU]), an oral drug approved in Europe for biliary spasm treatment that also inhibits HA in vitro and in animal models, could be repurposed as an inhibitor of HA synthesis in humans. METHODS. We conducted an open-label, single-center, dose-response study of hymecromone in healthy adults. Subjects received hymecromone at 1200 (n = 8), 2400 (n = 9), or 3600 (n = 9) mg/d divided into 3 doses daily, administered orally for 4 days. We assessed safety and tolerability of hymecromone and analyzed HA, 4-MU, and 4-methylumbelliferyl glucuronide (4-MUG; the main metabolite of 4-MU) concentrations in sputum and serum. RESULTS. Hymecromone was well tolerated up to doses of 3600 mg/d. Both sputum and serum drug concentrations increased in a dose-dependent manner, indicating that higher doses lead to greater exposures. Across all dose arms combined, we observed a significant decrease in sputum HA from baseline after 4 days of treatment. We also observed a decrease in serum HA. Additionally, higher baseline sputum HA levels were associated with a greater decrease in sputum HA. CONCLUSION. After 4 days of exposure to oral hymecromone, healthy human subjects experienced a significant reduction in sputum HA levels, indicating this oral therapy may have potential in pulmonary diseases where HA is implicated in pathogenesis. TRIAL REGISTRATION. ClinicalTrials.gov NCT02780752. FUNDING. Stanford Medicine Catalyst, Stanford SPARK, Stanford Innovative Medicines Accelerator program, NIH training grants 5T32AI052073-14 and T32HL129970., Introduction Hyaluronan (HA), an extracellular matrix glycosaminoglycan, plays an important role in inflammation (1, 2). Within injured and infected tissues, HA is produced by stromal cells in response to inflammatory [...]

Bacterial infection can lead to acidosis of the local microenvironment, which is believed to exacerbate disease pathogenesis; however, the mechanisms by which changes in pH alter disease progression are poorly understood. We test the hypothesis that acidosis enhances respiratory epithelial cell death in response to infection with Pseudomonas aeruginosa. Our findings support the idea that acidosis in the context of P. aeruginosa infection results in increased epithelial cell cytotoxicity due to ExoU intoxication. Importantly, enforced maintenance of neutral pH during P. aeruginosa infection demonstrates that cytotoxicity is dependent on the acidosis. Investigation of the underlying mechanisms revealed that host cell cytotoxicity correlated with increased bacterial survival during an acidic infection that was due to reduced bactericidal activity of host-derived antimicrobial peptides. These findings extend previous reports that the activities of antimicrobial peptides are pH-dependent and provide novel insights into the consequences of acidosis on infection-derived pathology. Therefore, this report provides the first evidence that physiological levels of acidosis increase the susceptibility of epithelial cells to acute Pseudomonas infection and demonstrates the benefit of maintaining pH homeostasis during a bacterial infection. [ABSTRACT FROM AUTHOR]

Circulating monocytes are recruited to the tumor microenvironment, where they can differentiate into macrophages that mediate tumor progression. To reach the tumor microenvironment, monocytes must first extravasate and migrate through the type-1 collagen rich stromal matrix. The viscoelastic stromal matrix around tumors not only stiffens relative to normal stromal matrix, but often exhibits enhanced viscous characteristics, as indicated by a higher loss tangent or faster stress relaxation rate. Here, we studied how changes in matrix stiffness and viscoelasticity, impact the three-dimensional migration of monocytes through stromal-like matrices. Interpenetrating networks of type-1 collagen and alginate, which enable independent tunability of stiffness and stress relaxation over physiologically relevant ranges, were used as confining matrices for three-dimensional culture of monocytes. Increased stiffness and faster stress relaxation independently enhanced the 3D migration of monocytes. Migrating monocytes have an ellipsoidal or rounded wedge-like morphology, reminiscent of amoeboid migration, with accumulation of actin at the trailing edge. Matrix adhesions and Rho-mediated contractility were dispensable for monocyte migration in 3D, but migration did require actin polymerization and myosin contractility. Mechanistic studies indicate that actin polymerization at the leading edge generates protrusive forces that open a path for the monocytes to migrate through in the confining viscoelastic matrices. Taken together, our findings implicate matrix stiffness and stress relaxation as key mediators of monocyte migration and reveal how monocytes use pushing forces at the leading edge mediated by actin polymerization to generate migration paths in confining viscoelastic matrices.

Human immunodeficiency virus 1 (HIV-1) infection remains a global health concern, with nearly 30 million people on antiretroviral (ARV) treatment. Integrase strand-transfer inhibitors (INSTIs) that block HIV-1 integrase are crucial components of first-line combination ARV therapies recommended in most international guidelines and have significantly improved HIV-1 treatment due to their efficacy and safety. This study evaluates potential off-target effects of INSTIs on recombinase activating genes (RAG1 and RAG2), which are essential for adaptive immune system function. We performed a comprehensive assessment of the off-target effects of clinically approved INSTIs on RAG activity, utilizing both biochemical and cellular assays. We purified the first catalytically active recombinant human core RAG1-RAG2 complex and tested it in the presence of the co-factor human HMGB1 protein for the gel-based biochemical RAG DNA cleavage assay. Additionally, we optimized an extrachromosomal V(D)J recombination cellular assay using murine mCherry-core RAG1, full-length murine mCherry-RAG2, and a plasmid substrate green fluorescent protein (GFP) reporter system, transfecting them into cells in the absence or presence of inhibitors. This setup enabled high-throughput analysis of V(D)J recombination for multiple compounds in a dose-response manner via flow cytometry. Physiologically relevant concentrations of INSTIs were examined for their potential impact on RAG activity and V(D)J recombination, with approved INSTIs showing minimal to no effects on recombinase activity. Consequently, the findings support the continued use of INSTIs in HIV-1 treatment without substantial concern for adverse effects on V(D)J recombination and immune system function.IMPORTANCEINSTIs are a crucial component of antiretroviral treatments for HIV-1 infection. This study provides a careful and thorough analysis of the impact of approved INSTIs on recombinase activating gene (RAG1 and RAG2) activity, which plays a pivotal role in the adaptive immune system. The concentrations tested were derived from several clinical studies and accounted for the maximum free fraction of the drug available in patients. This approach ensures that our findings are directly applicable to clinical scenarios by providing meaningful insights into the potential drug side effects in patients. We developed biochemical and cellular assays to measure the impact of INSTIs on RAG activity. All tested INSTIs did not inhibit RAG at supratherapeutic concentrations in the RAG1/RAG2 biochemical cleavage and cellular V(D)J recombination assays. Our assessment supports the continued use of INSTIs in HIV-1 treatments without concern for adverse effects.

Thick, viscous respiratory secretions are a major pathogenic feature of COVID-19 disease, but the composition and physical properties of these secretions are poorly understood. We characterized the composition and rheological properties (i.e. resistance to flow) of respiratory secretions collected from intubated COVID-19 patients. We find the percent solids and protein content are greatly elevated in COVID-19 compared to heathy control samples and closely resemble levels seen in cystic fibrosis, a genetic disease known for thick, tenacious respiratory secretions. DNA and hyaluronan (HA) are major components of respiratory secretions in COVID-19 and are likewise abundant in cadaveric lung tissues from these patients. COVID-19 secretions exhibit heterogeneous rheological behaviors with thicker samples showing increased sensitivity to DNase and hyaluronidase treatment. In histologic sections from these same patients, we observe increased accumulation of HA and the hyaladherin versican but reduced tumor necrosis factorâ€"stimulated gene-6 (TSG6) staining, consistent with the inflammatory nature of these secretions. Finally, we observed diminished type I interferon and enhanced inflammatory cytokines in these secretions. Overall, our studies indicate that increases in HA and DNA in COVID-19 respiratory secretion samples correlate with enhanced inflammatory burden and suggest that DNA and HA may be viable therapeutic targets in COVID-19 infection.