Gilbert's syndrome leads to elevated bilirubin after initiation of elexacaftor/tezacaftor/ivacaftor in people with cystic fibrosis.

Nine people with cystic fibrosis (pwCF) were found to have isolated elevations in serum total bilirubin after starting elexacaftor/tezacaftor/ivacaftor (ETI) that were associated with Gilbert's Syndrome. In longitudinal examination, total bilirubin levels increased substantially after initiation of ETI without elevations in liver transaminases in those with this syndrome. Because elevated bilirubin levels in Gilbert's Syndrome are benign, ETI was able to be continued in these individuals. Genetic testing for this relatively common syndrome should be strongly considered for pwCF experiencing isolated hyperbilirubinemia after starting ETI, since appropriate diagnosis may help pwCF avoid unnecessary interruption in this therapy with significant health benefits in CF.

Lack of Type I Interferon Signaling Ameliorates Respiratory Syncytial Virus-Induced Lung Inflammation and Restores Antioxidant Defenses.

Respiratory syncytial virus (RSV) infection in mouse and human lung is associated with pathogenic inflammation and oxidative injury. RSV impairs antioxidant responses by increasing the degradation of transcription factor NF-E2-related factor 2 (NRF2), which controls the expression of several antioxidant enzymes (AOEs). In addition to its protective effects, type I IFNs have been increasingly recognized as important mediators of host pathogenic responses during acute respiratory viral infections. We used a mouse model of RSV infection to investigate the effect of lack of type I interferon (IFN) receptor on viral-mediated clinical disease, airway inflammation, NRF2 expression, and antioxidant defenses. In the absence of type I IFN signaling, RSV-infected mice showed significantly less body weight loss and airway obstruction, as well as a significant reduction in cytokine and chemokine secretion and airway inflammation. Lack of type I IFN receptor was associated with greatly reduced virus-induced promyelocytic leukemia lung protein expression, which we showed to be necessary for virus-induced NRF2 degradation in a cell model of infection, resulting in restoration of NRF2 levels, AOE expression, and airway antioxidant capacity. Our data support the concept that modulation of type I IFN production and/or signaling could represent an important therapeutic strategy to ameliorate severity of RSV-induced lung disease.

Selective Blockade of TNFR1 Improves Clinical Disease and Bronchoconstriction in Experimental RSV Infection.

Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis in infants and young children. Although some clinical studies have speculated that tumor necrosis factor (TNF)-α is a major contributor of RSV-mediated airway disease, experimental evidence remains unclear or conflicting. TNF-α initiates inflammation and cell death through two distinct receptors: TNF-receptor (TNFR)1 and TNFR2. Here we delineate the function of TNF-α by short-lasting blockade of either receptor in an experimental BALB/c mouse model of RSV infection. We demonstrate that antibody-mediated blockade of TNFR1, but not TNFR2, results in significantly improved clinical disease and bronchoconstriction as well as significant reductions of several inflammatory cytokines and chemokines, including IL-1α, IL-1ß, IL-6, Ccl3, Ccl4, and Ccl5. Additionally, TNFR1 blockade was found to significantly reduce neutrophil number and activation status, consistent with the concomitant reduction of pro-neutrophilic chemokines Cxcl1 and Cxcl2. Similar protective activity was also observed when a single-dose of TNFR1 blockade was administered to mice following RSV inoculation, although this treatment resulted in improved alveolar macrophage survival rather than reduced neutrophil activation. Importantly, short-lasting blockade of TNFR1 did not affect RSV peak replication in the lung. This study suggests a potential therapeutic approach for RSV bronchiolitis based on selective blockade of TNFR1.

Increased Lung Catalase Activity Confers Protection Against Experimental RSV Infection.

Respiratory syncytial virus (RSV) infection in mouse and human lung is associated with oxidative injury and pathogenic inflammation. RSV impairs antioxidant responses by increasing the degradation of transcription factor NRF2, which controls the expression of several antioxidant enzyme (AOE) genes, including catalase. Since catalase is a key enzyme for the dismutation of virus-mediated generation of hydrogen peroxide (H2O2) we developed a model of intranasal supplementation of polyethylene glycol-conjugated catalase (PG-CAT) for RSV-infected mice. The results of our study show that PG-CAT supplementation was able to increase specific enzymatic activity along with reduction in H2O2 in the airways and had a significant protective effect against RSV-induced clinical disease and airway pathology. PG-CAT treated mice showed amelioration in airway obstruction, reduction in neutrophil elastase and inflammation. Improved airway hyperresponsiveness was also observed in mice that received PG-CAT as a treatment post-viral inoculation. In addition, PG-CAT greatly reduced the concentration of inflammatory cytokines and chemokines, including IL-1, TNF-α, IL-9, CXCL1, CCL2, and CCL5 in the bronchoalveolar lavage fluid of RSV-infected mice, without increasing viral replication in the lung. In conclusion, catalase supplementation may represent a novel pharmacologic approach to be explored in human for prevention or treatment of respiratory infections caused by RSV.

A Polymorphism in the Catalase Gene Promoter Confers Protection against Severe RSV Bronchiolitis.

Respiratory syncytial virus (RSV) infection is associated with oxidative lung injury, decreased levels of antioxidant enzymes (AOEs), and the degradation of the transcription factor NF-E2-related factor 2 (NRF2), a master regulator of AOE expression. Single nucleotide polymorphisms (SNPs) in AOE and NRF2 genes have been associated with various lung disorders. To test whether specific NRF2 and/or AOE gene SNPs in children with RSV lower respiratory tract infection were associated with disease severity, one hundred and forty one children <24 month of age with bronchiolitis were assessed for seven AOE and two NRF2 SNPs, and data were correlated with disease severity, which was determined by need of oxygen supplementation and intensive care support. One SNP in the promoter region of the catalase gene, rs1001179, which is associated with higher enzyme expression, was significantly underrepresented (p = 0.01, OR 0.38) among patients with moderate to severe RSV bronchiolitis, suggesting a protective effect against disease severity. Our results suggest that increasing catalase expression/activity could exert a protective role in the context of RSV infection and represent a potential novel therapeutic target to ameliorate viral-induced lung disease.

Antiviral and Immunomodulatory Activity of Silver Nanoparticles in Experimental RSV Infection.

Respiratory syncytial virus (RSV) is an important etiological agent of respiratory infection in children for which no specific treatment option is available. The RSV virion contains two surface glycoproteins (F and G) that are vital for the initial phases of infection, making them critical targets for RSV therapeutics. Recent studies have identified the broad-spectrum antiviral properties of silver nanoparticles (AgNPs) against respiratory pathogens, such as adenovirus, parainfluenza, and influenza. AgNPs achieve this by attaching to viral glycoproteins, blocking entry into the host cell. The objective of this study was to evaluate the antiviral and immunomodulatory effects of AgNPs in RSV infection. Herein we demonstrate AgNP-mediated reduction in RSV replication, both in epithelial cell lines and in experimentally infected BALB/c mice. Marked reduction in pro-inflammatory cytokines (i.e., IL-1α, IL-6, TNF-α) and pro-inflammatory chemokines (i.e., CCL2, CCL3, CCL5) was also observed. Conversely, CXCL1, G-CSF, and GM-CSF were increased in RSV-infected mice treated with AgNPs, consistent with an increase of neutrophil recruitment and activation in the lung tissue. Following experimental antibody-dependent depletion of neutrophils, the antiviral effect of AgNPs in mice treated was ablated. To our knowledge, this is the first in vivo report demonstrating antiviral activity of AgNPs during RSV infection.

Student Evaluation of Interprofessional Experiences between Medical and Graduate Biomedical Students.

BACKGROUND: Interprofessional education (IPE) has fostered increased collaboration and appreciation for different disciplines among health professionals but has yet to be established in a translational research setting. Interprofessional experiences (IPEx) implemented early in student training could increase translational research productivity. METHODS AND FINDINGS: Ten students involved in an IPE curriculum wrote autoethnographic accounts that were coded and emergent themes were grouped through constant comparative analysis. IPE led to improvements in communication, trust, appreciation, and an increased desire to seek IPE in future careers. Challenges included administrative barriers and interpersonal conflicts. CONCLUSIONS: Participants found IPE beneficial to their careers and developed a respect for each other's discipline. To implement IPE, institutions should consider possible administrative challenges and inclusion of conflict management training.

Respiratory syncytial virus induces NRF2 degradation through a promyelocytic leukemia protein - ring finger protein 4 dependent pathway.

Respiratory syncytial virus (RSV) is the most important cause of viral acute respiratory tract infections and hospitalizations in children, for which no vaccine or specific treatments are available. RSV causes airway mucosa inflammation and cellular oxidative damage by triggering production of reactive oxygen species and by inhibiting at the same time expression of antioxidant enzymes, via degradation of the transcription factor NF-E2-related factor 2 (NRF2). RSV infection induces NRF2 deacetylation, ubiquitination, and degradation through a proteasome-dependent pathway. Although degradation via KEAP1 is the most common mechanism, silencing KEAP1 expression did not rescue NRF2 levels during RSV infection. We found that RSV-induced NRF2 degradation occurs in an SUMO-specific E3 ubiquitin ligase - RING finger protein 4 (RNF4)-dependent manner. NRF2 is progressively SUMOylated in RSV infection and either blocking SUMOylation or silencing RNF4 expression rescued both NRF2 nuclear levels and transcriptional activity. RNF4 associates with promyelocytic leukemia - nuclear bodies (PML-NBs). RSV infection induces the expression of PML and PML-NBs formation in an interferon (INF)-dependent manner and also induces NRF2 - PMN-NBs association. Inhibition of PML-NB formation by blocking IFN pathway or silencing PML expression resulted in a significant reduction of RSV-associated NRF2 degradation and increased antioxidant enzyme expression, identifying the RNF4-PML pathway as a key regulator of antioxidant defenses in the course of viral infection.

Hydrogen Sulfide: A Novel Player in Airway Development, Pathophysiology of Respiratory Diseases, and Antiviral Defenses.

Hydrogen sulfide (H2S) is a biologically relevant signaling molecule in mammals. Along with the volatile substances nitric oxide (NO) and carbon monoxide (CO), H2S is defined as a gasotransmitter. It plays a physiological role in a variety of functions, including synaptic transmission, vascular tone, angiogenesis, inflammation, and cellular signaling. The generation of H2S is catalyzed by cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST). The expression of CBS and CSE is tissue specific, with CBS being expressed predominantly in the brain, and CSE in peripheral tissues, including lungs. CSE expression and activity are developmentally regulated, and recent studies suggest that CSE plays an important role in lung alveolarization during fetal development. In the respiratory tract, endogenous H2S has been shown to participate in the regulation of important functions such as airway tone, pulmonary circulation, cell proliferation or apoptosis, fibrosis, oxidative stress, and inflammation. In the past few years, changes in the generation of H2S have been linked to the pathogenesis of a variety of acute and chronic inflammatory lung diseases, including asthma and chronic obstructive pulmonary disease. Recently, our laboratory made the critical discovery that cellular H2S exerts broad-spectrum antiviral activity both in vitro and in vivo, in addition to independent antiinflammatory activity. These findings have important implications for the development of novel therapeutic strategies for viral respiratory infections, as well as other inflammatory lung diseases, especially in light of recent significant efforts to generate controlled-release H2S donors for clinical therapeutic applications.

Hydrogen Sulfide Is an Antiviral and Antiinflammatory Endogenous Gasotransmitter in the Airways. Role in Respiratory Syncytial Virus Infection.

Hydrogen sulfide (H2S) is an endogenous gaseous transmitter whose role in the pathophysiology of several lung diseases has been increasingly appreciated. Our recent studies in vitro have shown, we believe for the first time, that H2S has an important antiviral and antiinflammatory activity in respiratory syncytial virus (RSV) infection, the leading cause of bronchiolitis and viral pneumonia in children. Our objective was to evaluate the therapeutic potential of GYY4137, a novel slow-releasing H2S donor, for the prevention and treatment of RSV-induced lung disease, as well as to investigate the role of endogenous H2S in a mouse model of RSV infection. Ten- to 12-week-old BALB/c mice treated with GYY4137, or C57BL/6J mice genetically deficient in the cystathionine γ-lyase enzyme, the major H2S-generating enzyme in the lung, were infected with RSV and assessed for viral replication, clinical disease, airway hyperresponsiveness, and inflammatory responses. Our results show that intranasal delivery of GYY4137 to RSV-infected mice significantly reduced viral replication and markedly improved clinical disease parameters and pulmonary dysfunction compared with the results in vehicle-treated control mice. The protective effect of the H2S donor was associated with a significant reduction of viral-induced proinflammatory mediators and lung cellular infiltrates. Furthermore, cystathionine γ-lyase-deficient mice showed significantly enhanced RSV-induced lung disease and viral replication compared with wild-type animals. Overall, our results indicate that H2S exerts a novel antiviral and antiinflammatory activity in the context of RSV infection and represent a potential novel pharmacological approach for ameliorating virus-induced lung disease.

Biological functionalization of drug delivery carriers to bypass size restrictions of receptor-mediated endocytosis independently from receptor targeting.

Targeting of drug carriers to cell-surface receptors involved in endocytosis is commonly used for intracellular drug delivery. However, most endocytic receptors mediate uptake via clathrin or caveolar pathways associated with ≤200-nm vesicles, restricting carrier design. We recently showed that endocytosis mediated by intercellular adhesion molecule 1 (ICAM-1), which differs from clathrin- and caveolae-mediated pathways, allows uptake of nano- and microcarriers in cell culture and in vivo due to recruitment of cellular sphingomyelinases to the plasmalemma. This leads to ceramide generation at carrier binding sites and formation of actin stress-fibers, enabling engulfment and uptake of a wide size-range of carriers. Here we adapted this paradigm to enhance uptake of drug carriers targeted to receptors associated with size-restricted pathways. We coated sphingomyelinase onto model (polystyrene) submicro- and microcarriers targeted to clathrin-associated mannose-6-phosphate receptor. In endothelial cells, this provided ceramide enrichment at the cell surface and actin stress-fiber formation, modifying the uptake pathway and enhancing carrier endocytosis without affecting targeting, endosomal transport, cell-associated degradation, or cell viability. This improvement depended on the carrier size and enzyme dose, and similar results were observed for other receptors (transferrin receptor) and cell types (epithelial cells). This phenomenon also enhanced tissue accumulation of carriers after intravenous injection in mice. Hence, it is possible to maintain targeting toward a selected receptor while bypassing natural size restrictions of its associated endocytic route by functionalization of drug carriers with biological elements mimicking the ICAM-1 pathway. This strategy holds considerable promise to enhance flexibility of design of targeted drug delivery systems.