Aerosol Delivery of a Novel Recombinant Modified Superoxide Dismutase Protein Reduces Oxidant Injury and Attenuates Escherichia coli Induced Lung Injury in Rats.

Background: Acute respiratory distress syndrome (ARDS) is a life-threatening respiratory failure syndrome with diverse etiologies characterized by increased permeability of alveolar-capillary membranes, pulmonary edema, and acute onset hypoxemia. During the ARDS acute phase, neutrophil infiltration into the alveolar space results in uncontrolled release of reactive oxygen species (ROS) and proteases, overwhelming antioxidant defenses and causing alveolar epithelial and lung endothelial injury. Objectives: To investigate the therapeutic potential of a novel recombinant human Cu-Zn-superoxide dismutase (SOD) fusion protein in protecting against ROS injury and for aerosolized SOD delivery to treat Escherichia coli induced ARDS. Methods: Fusion proteins incorporating human Cu-Zn-SOD (hSOD1), with (pep1-hSOD1-his) and without (hSOD1-his) a fused hyaluronic acid-binding peptide, were expressed in E. coli. Purified proteins were evaluated in in vitro assays with human bronchial epithelial cells and through aerosolized delivery to the lung of an E. coli-induced ARDS rat model. Results: SOD proteins exhibited high SOD activity in vitro and protected bronchial epithelial cells from oxidative damage. hSOD1-his and pep1-hSOD1-his retained SOD activity postnebulization and exhibited no adverse effects in the rat. Pep1-hSOD1-his administered through instillation or nebulization to the lung of an E. coli-induced pneumonia rat improved arterial oxygenation and lactate levels compared to vehicle after 48 hours. Static lung compliance was improved when the pep1-hSOD1-his protein was delivered by instillation. White cell infiltration to the lung was significantly reduced by aerosolized delivery of protein, and reduction of cytokine-induced neutrophil chemoattractant-1, interferon-gamma, and interleukin 6 pro-inflammatory cytokine concentrations in bronchoalveolar lavage was observed. Conclusions: Aerosol delivery of a novel recombinant modified SOD protein reduces oxidant injury and attenuates E. coli induced lung injury in rats. The results provide a strong basis for further investigation of the therapeutic potential of hSOD1 in the treatment of ARDS.

Delayed MSC therapy enhances resolution of organized pneumonia induced by antibiotic resistant Klebsiella pneumoniae infection.

Introduction: Mesenchymal stromal cells (MSC) are a promising therapeutic for pneumonia-induced sepsis. Here we sought to determine the efficacy of delayed administration of naïve and activated bone marrow (BM), adipose (AD), and umbilical cord (UC) derived MSCs in organized antibiotic resistant Klebsiella pneumosepsis. Methods: Human BM-, AD-, and UC-MSCs were isolated and expanded and used either in the naïve state or following cytokine pre-activation. The effect of MSC tissue source and activation status was assessed first in vitro. Subsequent experiments assessed therapeutic potential as a delayed therapy at 48 h post infection of rodents with Klebsiella pneumoniae, with efficacy assessed at 120 h. Results: BM-, AD-, and UC-MSCs accelerated epithelial healing, increased phagocytosis, and reduced ROS-induced epithelial injury in vitro, with AD-MSCs less effective, and naïve MSCs more effective than pre-activated MSCs. Delayed MSC administration in pre-clinical organized Klebsiella pneumosepsis had no effect on physiologic indices, but enhanced resolution of structural lung injury. Delayed therapy with pre-activated MSCs reduced mRNA concentrations of fibrotic factors. Naïve MSC treatment reduced key circulating cell proportions and increased bacterial killing capacity in the lungs whereas pre-activated MSCs enhanced the phagocytic index of pulmonary white cells. Discussion: Delayed MSC therapy enhanced resolution of lung injury induced by antibiotic resistant Klebsiella infection and favorably modulated immune cell profile. Overall, AD-MSCs were less effective than either UC- or BM-MSCs, while naïve MSCs had a more favorable effect profile compared to pre-activated MSCs.

Nebulized mesenchymal stem cell derived conditioned medium ameliorates Escherichia coli induced pneumonia in a rat model.

Background: Mesenchymal stem cells (MSC) have shown immense therapeutic promise in a range of inflammatory diseases, including acute respiratory distress syndrome (ARDS), and are rapidly advancing through clinical trials. Among their multimodal mechanisms of action, MSCs exert strong immunomodulatory effects via their secretome, which contains cytokines, small molecules, extracellular vesicles, and a range of other factors. Recent studies have shown that the MSC secretome can recapitulate many of the beneficial effects of the MSC itself. We aimed to determine the therapeutic capacity of the MSC secretome in a rat bacterial pneumonia model, especially when delivered directly to the lung by nebulization which is a technique more appropriate for the ventilated patient. Methods: Conditioned medium (CM) was generated from human bone marrow derived MSCs in the absence of antibiotics and serum supplements. Post-nebulization lung penetration was estimated through nebulization of CM to a cascade impactor and simulated lung and quantification of collected total protein and IL-8 cytokine. Control and nebulized CM was added to a variety of lung cell culture models and injury resolution assessed. In a rat E. coli pneumonia model, CM was instilled or administered by nebulization and lung injury and inflammation assessed at 48 h. Results: MSC-CM was predicted to have good distal lung penetration and delivery when administered by nebulizer. Both control and nebulized CM reduced NF-κB activation and inflammatory cytokine production in lung cell culture, while promoting cell viability and would closure in oxidative stress and scratch wound models. In a rat bacterial pneumonia model, both instilled and nebulizer delivered CM improved lung function, increasing blood oxygenation and reducing carbon dioxide levels compared to unconditioned medium controls. A reduction in bacterial load was also observed in both treatment groups. Inflammatory cytokines were reduced significantly by both liquid and aerosol CM administration, with less IL-1ß, IL-6, and CINC1 in these groups compared to controls. Conclusion: MSC-CM is a potential therapeutic for pneumonia ARDS, and administration is compatible with vibrating mesh nebulization.

Differential Effects of Cytokine Versus Hypoxic Preconditioning of Human Mesenchymal Stromal Cells in Pulmonary Sepsis Induced by Antimicrobial-Resistant Klebsiella pneumoniae.

Background: Pulmonary sepsis is a leading cause of hospital mortality, and sepses arising from antimicrobial-resistant (AMR) bacterial strains are particularly difficult to treat. Here we investigated the potential of mesenchymal stromal cells (MSCs) to combat established Klebsiella pneumoniae pneumosepsis and further evaluated MSC preconditioning and pre-activation methods. Methods: The potential for naïve and preconditioned MSCs to enhance wound healing, reduce inflammation, preserve metabolic activity, and enhance bacterial killing was assessed in vitro. Rats were subjected to intratracheal K. pneumoniae followed by the intravenous administration of MSCs. Physiological indices, blood, bronchoalveolar lavage (BAL), and tissues were obtained 72 h later. Results: In vitro assays confirmed that preconditioning enhances MSC function, accelerating pulmonary epithelial wound closure, reducing inflammation, attenuating cell death, and increasing bacterial killing. Cytomix-pre-activated MSCs are superior to naïve and hypoxia-exposed MSCs in attenuating Klebsiella pneumosepsis, improving lung compliance and oxygenation, reducing bacteria, and attenuating histologic injuries in lungs. BAL inflammatory cytokines were reduced, correlating with decreases in polymorphonuclear (PMN) cells. MSCs increased PMN apoptosis and the CD4:CD8 ratio in BAL. Systemically, granulocytes, classical monocytes, and the CD4:CD8 ratio were reduced, and nonclassical monocytes were increased. Conclusions: Preconditioning with cytokines, but not hypoxia, enhances the therapeutic potential of MSCs in clinically relevant models of K. pneumoniae-induced pneumosepsis.

Multiple Dosing and Preactivation of Mesenchymal Stromal Cells Enhance Efficacy in Established Pneumonia Induced by Antimicrobial-Resistant Klebsiella pneumoniae in Rodents.

Antimicrobial-resistant (AMR) bacteria, such as Klebsiella species, are an increasingly common cause of hospital-acquired pneumonia, resulting in high mortality and morbidity. Harnessing the host immune response to AMR bacterial infection using mesenchymal stem cells (MSCs) is a promising approach to bypass bacterial AMR mechanisms. The administration of single doses of naïve MSCs to ARDS clinical trial patient cohorts has been shown to be safe, although efficacy is unclear. The study tested whether repeated MSC dosing and/or preactivation, would attenuate AMR Klebsiella pneumonia-induced established pneumonia. Rat models of established K. pneumoniae-induced pneumonia were randomised to receive intravenous naïve or cytomix-preactivated umbilical cord MSCs as a single dose at 24 h post pneumonia induction with or without a subsequent dose at 48 h. Physiological indices, bronchoalveolar lavage (BAL), and tissues were obtained at 72 h post pneumonia induction. A single dose of naïve MSCs was largely ineffective, whereas two doses of MSCs were effective in attenuating Klebsiella pneumosepsis, improving lung compliance and oxygenation, while reducing bacteria and injury in the lung. Cytomix-preactivated MSCs were superior to naïve MSCs. BAL neutrophil counts and activation were reduced, and apoptosis increased. MSC therapy reduced cytotoxic BAL T cells, and increased CD4+/CD8+ ratios. Systemically, granulocytes, classical monocytes, and the CD4+/CD8+ ratio were reduced, and nonclassical monocytes were increased. Repeated doses of MSCs-particularly preactivated MSCs-enhance their therapeutic potential in a clinically relevant model of established AMR K. pneumoniae-induced pneumosepsis.

Aerosolized Pulmonary Delivery of mRNA Constructs Attenuates Severity of Escherichia coli Pneumonia in the Rat.

Acute respiratory distress syndrome (ARDS), a rapid onset inflammatory lung disease with no effective specific therapy, typically has pathogenic etiology termed pneumonia. In previous studies nuclear factor-κB (NF-κB) inhibitor α super-repressor (IκBα-SR) and extracellular superoxide dismutase 3 (SOD3) reduced pneumonia severity when prophylactically delivered by viral vector. In this study, mRNA coding for green fluorescent protein, IκBα-SR, or SOD3 was complexed with cationic lipid, passed through a vibrating mesh nebulizer, and delivered to cell culture or directly to rats undergoing Escherichia coli pneumonia. Injury level was then assessed at 48 h. In vitro, expression was observed as early as 4 h in lung epithelial cells. IκBα-SR and wild-type IκBα mRNAs attenuated inflammatory markers, while SOD3 mRNA induced protective and antioxidant effects. In rat E. coli pneumonia, IκBα-SR mRNA reduced arterial carbon dioxide (pCO2) and reduced lung wet/dry ratio. SOD3 mRNA improved static lung compliance and alveolar-arterial oxygen gradient (AaDO2) and decreased bronchoalveolar lavage (BAL) bacteria load. White cell infiltration and inflammatory cytokine concentrations in BAL and serum were reduced by both mRNA treatments compared to scrambled mRNA controls. These findings indicate nebulized mRNA therapeutics are a promising approach to ARDS therapy, with rapid expression of protein and observable amelioration of pneumonia symptoms.

Hypercapnia in the critically ill: insights from the bench to the bedside.

Carbon dioxide (CO2) has long been considered, at best, a waste by-product of metabolism, and at worst, a toxic molecule with serious health consequences if physiological concentration is dysregulated. However, clinical observations have revealed that 'permissive' hypercapnia, the deliberate allowance of respiratory produced CO2 to remain in the patient, can have anti-inflammatory effects that may be beneficial in certain circumstances. In parallel, studies at the cell level have demonstrated the profound effect of CO2 on multiple diverse signalling pathways, be it the effect from CO2 itself specifically or from the associated acidosis it generates. At the whole organism level, it now appears likely that there are many biological sensing systems designed to respond to CO2 concentration and tailor respiratory and other responses to atmospheric or local levels. Animal models have been widely employed to study the changes in CO2 levels in various disease states and also to what extent permissive or even directly delivered CO2 can affect patient outcome. These findings have been advanced to the bedside at the same time that further clinical observations have been elucidated at the cell and animal level. Here we present a synopsis of the current understanding of how CO2 affects mammalian biological systems, with a particular emphasis on inflammatory pathways and diseases such as lung specific or systemic sepsis. We also explore some future directions and possibilities, such as direct control of blood CO2 levels, that could lead to improved clinical care in the future.

The role of cells and their products in respiratory drug delivery: the past, present, and future.

INTRODUCTION: Cell-based delivery systems offer considerable promise as novel and innovative therapeutics to target the respiratory system. These systems consist of cells and/or their extracellular vesicles that deliver their contents, such as anti-microbial peptides, micro RNAs, and even mitochondria to the lung, exerting direct therapeutic effects. AREAS COVERED: The purpose of this article is to critically review the status of cell-based therapies in the delivery of therapeutics to the lung, evaluate current progress, and elucidate key challenges to the further development of these novel approaches. An overview as to how these cells and/or their products may be modified to enhance efficacy is given. More complex delivery cell-based systems, including cells or vesicles that are genetically modified to (over)express specific therapeutic products, such as proteins and therapeutic nucleic acids are also discussed. Focus is given to the use of the aerosol route to deliver these products directly into the lung. EXPERT OPINION: The use of biological carriers to deliver chemical or biological agents demonstrates great potential in modern medicine. The next generation of drug delivery systems may comprise 'cell-inspired' drug carriers that are entirely synthetic, developed using insights from cell-based therapeutics to overcome limitations of current generation synthetic carriers.

Future Trends in Nebulized Therapies for Pulmonary Disease.

Aerosol therapy is a key modality for drug delivery to the lungs of respiratory disease patients. Aerosol therapy improves therapeutic effects by directly targeting diseased lung regions for rapid onset of action, requiring smaller doses than oral or intravenous delivery and minimizing systemic side effects. In order to optimize treatment of critically ill patients, the efficacy of aerosol therapy depends on lung morphology, breathing patterns, aerosol droplet characteristics, disease, mechanical ventilation, pharmacokinetics, and the pharmacodynamics of cell-drug interactions. While aerosol characteristics are influenced by drug formulations and device mechanisms, most other factors are reliant on individual patient variables. This has led to increased efforts towards more personalized therapeutic approaches to optimize pulmonary drug delivery and improve selection of effective drug types for individual patients. Vibrating mesh nebulizers (VMN) are the dominant device in clinical trials involving mechanical ventilation and emerging drugs. In this review, we consider the use of VMN during mechanical ventilation in intensive care units. We aim to link VMN fundamentals to applications in mechanically ventilated patients and look to the future use of VMN in emerging personalized therapeutic drugs.

Nebulized Mesenchymal Stem Cell Derived Conditioned Medium Retains Antibacterial Properties Against Clinical Pathogen Isolates.

Background: Mesenchymal stem/stromal cells (MSCs) have demonstrated promise in pathogenic acute respiratory distress syndrome models and are advancing to clinical efficacy testing. Besides immunomodulatory effects, MSC derived conditioned medium (CM) has direct antibacterial effects, possibly through LL-37 and related secreted peptide activity. We investigated MSC-CM compatibility with vibrating mesh technology, allowing direct delivery to the infected lung. Methods: MSC-CM from bone marrow (BM) and umbilical cord (UC) MSCs were passed through the commercially available Aerogen Solo nebulizer. Known colony forming units of Escherichia coli, Staphylococcus aureus, and multidrug resistant Klebsiella pneumoniae clinical isolates were added to MSC-CM in an orbital shaker and antibacterial capacity assessed through OD600 spectrophotometry. To exclude the possible effects of medium depletion on bacteria proliferation, MSC-CM was concentrated with a 3000 Da cutoff filter, diluted with fresh media, and retested against inoculum. Enzyme-linked immunosorbent assay was used to quantify levels of antimicrobial peptides (AMPs) and IL-8 present at pre- and postnebulization. Results: Both BM and UC MSC-CM inhibited proliferation of all pathogens, and this ability was retained after nebulization. Concentrating and reconstituting CM did not affect antibacterial properties. Interestingly, LL-37 protein did not appear to survive nebulization, although other secreted AMPs and an unrelated protein, IL-8, were largely intact. Conclusion: MSC-CM is a potent antimicrobial agent and is compatible with vibrating mesh nebulization delivery. The mechanism is through a secreted factor that is over 3000 Da in size, although it does not appear to rely solely on previously identified peptides such as LL-37, hepcidin, or lipocalin-2.

'Degraded' RNA profiles in Arthropoda and beyond.

The requirement for high quality/non-degraded RNA is essential for an array of molecular biology analyses. When analysing the integrity of rRNA from the barnacle Lepas anatifera (Phylum Arthropoda, Subphylum Crustacea), atypical or sub-optimal rRNA profiles that were apparently degraded were observed on a bioanalyser electropherogram. It was subsequently discovered that the rRNA was not degraded, but arose due to a 'gap deletion' (also referred to as 'hidden break') in the 28S rRNA. An apparent excision at this site caused the 28S rRNA to fragment under heat-denaturing conditions and migrate along with the 18S rRNA, superficially presenting a 'degraded' appearance. Examination of the literature showed similar observations in a small number of older studies in insects; however, reading across multiple disciplines suggests that this is a wider issue that occurs across the Animalia and beyond. The current study shows that the 28S rRNA anomaly goes far beyond insects within the Arthropoda and is widespread within this phylum. We confirm that the anomaly is associated with thermal conversion because gap-deletion patterns were observed in heat-denatured samples but not in gels with formaldehyde-denaturing.