Transcriptional Changes in Chronic Rhinosinusitis with Asthma Favor a Type 2 Molecular Endotype Independent of Polyp Status.

BACKGROUND: Data regarding the inflammatory profile of patients with asthma and chronic rhinosinusitis (CRS-A) with (CRSwNP-A) and without (CRSsNP-A) nasal polyposis remain limited. OBJECTIVE: Define and compare systemic transcriptional changes in patients with CRS-A to those with non-asthma-related CRS with (CRSwNP) and without nasal polyposis (CRSsNP). METHODS: Thirty-four patients with CRS-A (n=19) and CRS (n=15) were prospectively enrolled into an observational study. Demographic information and subjective and objective disease severity measures were recorded. Multiplex gene expression analysis of mRNA extracted from peripheral blood was performed. A total of 594 genes associated with innate/adaptive immunity were analyzed using NanoString technology. Gene expression ratios were reported for genes that were differentially expressed among these cohorts. Linear regression analysis was used to compare the mRNA transcript copy numbers for each gene with disease severity. RESULTS: There was no significant difference in age, gender, nasal polyposis, or health-related quality of life measures between the two groups (p>0.05). HLA class II histocompatibility antigen, DRB3-1 beta chain (HLA-DRB3) was significantly upregulated in the peripheral blood of patients with CRSsNP-A compared to CRSsNP, whereas chemokine (C-C motif) ligands 4 (CCL4) and zinc finger protein helios (IKZF2) were significantly upregulated in CRSwNP-A compared to CRSwNP (p<0.05). CONCLUSION: Patients with CRSsNP-A demonstrate a molecular endotype associated with a Th2-dominant inflammatory profile compared to CRSsNP. Patients with CRSwNP-A similarly demonstrate an overrepresentation of genes associated with Th2-driven inflammation compared to patients with CRSwNP.

A longitudinal interprofessional simulation curriculum for critical care teams: Exploring successes and challenges.

Interprofessional care teams are the backbone of intensive care units (ICUs) where severity of illness is high and care requires varied skills and experience. Despite this care model, longitudinal educational programmes for such workplace teams rarely include all professions. In this article, we report findings on the initial assessment and evaluation of an ongoing, longitudinal simulation-based curriculum for interprofessional workplace critical care teams. The study had two independent components, quantitative learner assessment and qualitative curricular evaluation. To assess curriculum effectiveness at meeting learning objectives, participant-reported key learning points identified using a self-assessment tool administered immediately following curricular participation were mapped to session learning objectives. To evaluate the curriculum, we conducted a qualitative study using a phenomenology approach involving purposeful sampling of nine curricular participants undergoing recorded semi-structured interviews. Verbatim transcripts were reviewed by two independent readers to derive themes further subdivided into successes and barriers. Learner self-assessment demonstrated that the majority of learners, across all professions, achieved at least one intended learning objective with senior learners more likely to report team-based objectives and junior learners more likely to report knowledge/practice objectives. Successes identified by curricular evaluation included authentic critical care curricular content, safe learning environment, and team comradery from shared experience. Barriers included unfamiliarity with the simulation environment and clinical coverage for curricular participation. This study suggests that a sustainable interprofessional curriculum for workplace ICU critical care teams can achieve the desired educational impact and effectively deliver authentic simulated work experiences if barriers to educational engagement and participation can be overcome.

Bioengineered lysozyme in combination therapies for Pseudomonas aeruginosa lung infections.

There is increasing urgency in the battle against drug-resistant bacterial pathogens, and this public health crisis has created a desperate need for novel antimicrobial agents. Recombinant human lysozyme represents one interesting candidate for treating pulmonary infections, but the wild type enzyme is subject to electrostatic mediated inhibition by anionic biopolymers that accumulate in the infected lung. We have redesigned lysozyme's electrostatic potential field, creating a genetically engineered variant that is less susceptible to polyanion inhibition, yet retains potent bactericidal activity. A recent publication demonstrated that the engineered enzyme outperforms wild type lysozyme in a murine model of Pseudomonas aeruginosa lung infection. Here, we expand upon our initial studies and consider dual therapies that combine lysozymes with an antimicrobial peptide. Consistent with our earlier results, the charge modified lysozyme combination outperformed its wild type counterpart, yielding more than an order-of-magnitude reduction in bacterial burden following treatment with a single dose.

Glutaredoxin-1 attenuates S-glutathionylation of the death receptor fas and decreases resolution of Pseudomonas aeruginosa pneumonia.

RATIONALE: The death receptor Fas is critical for bacterial clearance and survival of mice after Pseudomonas aeruginosa infection. OBJECTIVES: Fas ligand (FasL)-induced apoptosis is augmented by S-glutathionylation of Fas (Fas-SSG), which can be reversed by glutaredoxin-1 (Grx1). Therefore, the objective of this study was to investigate the interplay between Grx1 and Fas in regulating the clearance of P. aeruginosa infection. METHODS: Lung samples from patients with bronchopneumonia were analyzed by immunofluorescence. Primary tracheal epithelial cells, mice lacking the gene for Grx1 (Glrx1(-/-)), Glrx1(-/-) mice treated with caspase inhibitor, or transgenic mice overexpressing Grx1 in the airway epithelium were analyzed after infection with P. aeruginosa. MEASUREMENTS AND MAIN RESULTS: Patient lung samples positive for P. aeruginosa infection demonstrated increased Fas-SSG compared with normal lung samples. Compared with wild-type primary lung epithelial cells, infection of Glrx1(-/-) cells with P. aeruginosa showed enhanced caspase 8 and 3 activities and cell death in association with increases in Fas-SSG. Infection of Glrx1(-/-) mice with P. aeruginosa resulted in enhanced caspase activity and increased Fas-SSG as compared with wild-type littermates. Absence of Glrx1 significantly enhanced bacterial clearance, and decreased mortality postinfection with P. aeruginosa. Inhibition of caspases significantly decreased bacterial clearance postinfection with P. aeruginosa, in association with decreased Fas-SSG. In contrast, transgenic mice that overexpress Grx1 in lung epithelial cells had significantly higher lung bacterial loads, enhanced mortality, decreased caspase activation, and Fas-SSG in the lung after infection with P. aeruginosa, compared with wild-type control animals. CONCLUSIONS: These results suggest that S-glutathionylation of Fas within the lung epithelium enhances epithelial apoptosis and promotes clearance of P. aeruginosa and that glutaredoxin-1 impairs bacterial clearance and increases the severity of pneumonia in association with deglutathionylation of Fas.

Bioengineered lysozyme reduces bacterial burden and inflammation in a murine model of mucoid Pseudomonas aeruginosa lung infection.

The spread of drug-resistant bacterial pathogens is a growing global concern and has prompted an effort to explore potential adjuvant and alternative therapies derived from nature's repertoire of bactericidal proteins and peptides. In humans, the airway surface liquid layer is a rich source of antibiotics, and lysozyme represents one of the most abundant and effective antimicrobial components of airway secretions. Human lysozyme is active against both Gram-positive and Gram-negative bacteria, acting through several mechanisms, including catalytic degradation of cell wall peptidoglycan and subsequent bacterial lysis. In the infected lung, however, lysozyme's dense cationic character can result in sequestration and inhibition by polyanions associated with airway inflammation. As a result, the efficacy of the native enzyme may be compromised in the infected and inflamed lung. To address this limitation, we previously constructed a charge-engineered variant of human lysozyme that was less prone to electrostatic-mediated inhibition in vitro. Here, we employ a murine model to show that this engineered enzyme is superior to wild-type human lysozyme as a treatment for mucoid Pseudomonas aeruginosa lung infections. The engineered enzyme effectively decreases the bacterial burden and reduces markers of inflammation and lung injury. Importantly, we found no evidence of acute toxicity or allergic hypersensitivity upon repeated administration of the engineered biotherapeutic. Thus, the charge-engineered lysozyme represents an interesting therapeutic candidate for P. aeruginosa lung infections.

Detecting bacterial lung infections: in vivo evaluation of in vitro volatile fingerprints.

The identification of bacteria by their volatilomes is of interest to many scientists and clinicians as it holds the promise of diagnosing infections in situ, particularly lung infections via breath analysis. While there are many studies reporting various bacterial volatile biomarkers or fingerprints using in vitro experiments, it has proven difficult to translate these data to in vivo breath analyses. Therefore, we aimed to create secondary electrospray ionization-mass spectrometry (SESI-MS) pathogen fingerprints directly from the breath of mice with lung infections. In this study we demonstrated that SESI-MS is capable of differentiating infected versus uninfected mice, P. aeruginosa-infected versus S. aureus-infected mice, as well as distinguish between infections caused by P. aeruginosa strains PAO1 versus FRD1, with statistical significance (p < 0.05). In addition, we compared in vitro and in vivo volatiles and observed that only 25-34% of peaks are shared between the in vitro and in vivo SESI-MS fingerprints. To the best of our knowledge, these are the first breath volatiles measured for P. aeruginosa PAO1, FRD1, and S. aureus RN450, and the first comparison of in vivo and in vitro volatile profiles from the same strains using the murine infection model.

Hemolytic phospholipase C inhibition protects lung function during Pseudomonas aeruginosa infection.

RATIONALE: The opportunistic pathogen Pseudomonas aeruginosa causes both acute and chronic lung infections and is particularly problematic in patients with cystic fibrosis and those undergoing mechanical ventilation. Decreased lung function contributes significantly to morbidity and mortality during P. aeruginosa infection, and damage inflicted by P. aeruginosa virulence factors contributes to lung function decline. OBJECTIVES: We sought to describe direct contribution of a bacterial phospholipase C/sphingomyelinase, PlcHR, to alteration of host lung physiology and characterize a potential therapeutic for protection of lung function. METHODS: We infected C57Bl/6 mice with P. aeruginosa wild-type or isogenic plcHR deletion strains and measured lung function using computer-controlled ventilators. For in vivo testing, miltefosine was delivered intraperitoneally 1 hour after infection. Infection and respiratory endpoints were at 24 hours after infection. MEASUREMENTS AND MAIN RESULTS: P. aeruginosa wild-type infection caused significant lung function impairment, whereas the effects of a ΔplcHR strain infection were much less severe. Surfactometry analysis of bronchoalveolar lavage fluid indicated that PlcHR decreased pulmonary surfactant function. Miltefosine has structural similarity to the PC and sphingomyelin substrates of PlcHR, and we found that it inhibits the cleavage of these choline-containing lipids in vitro. Miltefosine administration after P. aeruginosa infection limited the negative effects of PlcHR activity on lung function. CONCLUSIONS: We have directly linked production of a single virulence factor in P. aeruginosa with effects on lung function, and demonstrated that the inhibitor miltefosine protects lung function from PlcHR-dependent surfactant dysfunction.

Enhanced antimicrobial activity of engineered human lysozyme.

Lysozymes contain a disproportionately large fraction of cationic residues, and are thereby attracted toward the negatively charged surface of bacterial targets. Importantly, this conserved biophysical property may inhibit lysozyme antibacterial function during acute and chronic infections. A mouse model of acute pulmonary Pseudomonas aeruginosa infection demonstrated that anionic biopolymers accumulate to high concentrations in the infected lung, and the presence of these species correlates with decreased endogenous lysozyme activity. To develop antibacterial enzymes designed specifically to be used as antimicrobial agents in the infected airway, the electrostatic potential of human lysozyme (hLYS) was remodeled by protein engineering. A novel, high-throughput screen was implemented to functionally interrogate combinatorial libraries of charge-engineered hLYS proteins, and variants with improved bactericidal activity were isolated and characterized in detail. These studies illustrate a general mechanism by which polyanions inhibit lysozyme function, and they are the first direct demonstration that decreasing hLYS's net cationic character improves its antibacterial activity in the presence of disease-associated biopolymers. In addition to avoiding electrostatic sequestration, at least one charge-engineered variant also kills bacteria more rapidly in the absence of inhibitory biopolymers; this observation supports a novel hypothesis that tuning the cellular affinity of peptidoglycan hydrolases may be a general strategy for improving kinetics of bacterial killing.