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.

Nebulised mesenchymal stem cell derived extracellular vesicles ameliorate E. coli induced pneumonia in a rodent model.

BACKGROUND: Mesenchymal stem cell (MSC) derived extracellular vesicles (EVs) have been proposed as an alternative to cell therapy, creating new possible delivery modalities such as nebulisation. We wished to investigate the therapeutic potential of directly nebulised MSC-EVs in the mitigation of Escherichia coli-induced pneumonia. METHODS: EV size, surface markers and miRNA content were assessed pre- and post-nebulisation. BEAS2B and A459 lung cells were exposed to lipopolysaccharide (LPS) and treated with nebulised bone marrow (BM) or umbilical cord (UC) MSC-EVs. Viability assays (MTT) and inflammatory cytokine assays were performed. THP-1 monocytes were stimulated with LPS and nebulised BM- or UC-EVs and phagocytosis activity was measured. For in vivo experiments, mice received LPS intratracheally (IT) followed by BM- or UC-EVs intravenously (IV) and injury markers assessed at 24 h. Rats were instilled with E. coli bacteria IT and BM- or UC-EVs delivered IV or by direct nebulisation. At 48 h, lung damage was assessed by physiological parameters, histology and inflammatory marker presence. RESULTS: MSC-EVs retained their immunomodulatory and wound healing capacity after nebulisation in vitro. EV integrity and content were also preserved. Therapy with IV or nebulised MSC-EVs reduced the severity of LPS-induced lung injury and E. coli-induced pneumonia by reducing bacterial load and oedema, increasing blood oxygenation and improving lung histological scores. MSC-EV treated animals also showed lower levels of inflammatory cytokines and inflammatory-related markers. CONCLUSIONS: MSC-EVs given IV attenuated LPS-induced lung injury, and nebulisation of MSC-EVs did not affect their capacity to attenuate lung injury caused by E. coli pneumonia, as evidenced by reduction in bacterial load and improved lung physiology.

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.

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.

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.

A new multiplex SARS-CoV-2 antigen microarray showed correlation of IgG, IgA, and IgM antibodies from patients with COVID-19 disease severity and maintenance of relative IgA and IgM antigen binding over time.

Zoonotic spillover of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to humans in December 2019 caused the coronavirus disease 2019 (COVID-19) pandemic. Serological monitoring is critical for detailed understanding of individual immune responses to infection and protection to guide clinical therapeutic and vaccine strategies. We developed a high throughput multiplexed SARS-CoV-2 antigen microarray incorporating spike (S) and nucleocapsid protein (NP) and fragments expressed in various hosts which allowed simultaneous assessment of serum IgG, IgA, and IgM responses. Antigen glycosylation influenced antibody binding, with S glycosylation generally increasing and NP glycosylation decreasing binding. Purified antibody isotypes demonstrated a binding pattern and intensity different from the same isotype in whole serum, probably due to competition from the other isotypes present. Using purified antibody isotypes from naïve Irish COVID-19 patients, we correlated antibody isotype binding to different panels of antigens with disease severity, with binding to the S region S1 expressed in insect cells (S1 Sf21) significant for IgG, IgA, and IgM. Assessing longitudinal response for constant concentrations of purified antibody isotypes for a patient subset demonstrated that the relative proportion of antigen-specific IgGs decreased over time for severe disease, but the relative proportion of antigen-specific IgA binding remained at the same magnitude at 5 and 9 months post-first symptom onset. Further, the relative proportion of IgM binding decreased for S antigens but remained the same for NP antigens. This may support antigen-specific serum IgA and IgM playing a role in maintaining longer-term protection, important for developing and assessing vaccine strategies. Overall, these data demonstrate the multiplexed platform as a sensitive and useful platform for expanded humoral immunity studies, allowing detailed elucidation of antibody isotypes response against multiple antigens. This approach will be useful for monoclonal antibody therapeutic studies and screening of donor polyclonal antibodies for patient infusions.

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.

Enhancement strategies for mesenchymal stem cells and related therapies.

Cell therapy, particularly mesenchymal stem/stromal (MSC) therapy, has been investigated for a wide variety of disease indications, particularly those with inflammatory pathologies. However, recently it has become evident that the MSC is far from a panacea. In this review we will look at current and future strategies that might overcome limitations in efficacy. Many of these take their inspiration from stem cell niche and the mechanism of MSC action in response to the injury microenvironment, or from previous gene therapy work which can now benefit from the added longevity and targeting ability of a live cell vector. We will also explore the nascent field of extracellular vesicle therapy and how we are already seeing enhancement protocols for this exciting new drug. These enhanced MSCs will lead the way in more difficult to treat diseases and restore potency where donors or manufacturing practicalities lead to diminished MSC effect.

Improved diagnosis of SARS-CoV-2 by using nucleoprotein and spike protein fragment 2 in quantitative dual ELISA tests.

The novel coronavirus, severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), is the causative agent of the 2020 worldwide coronavirus pandemic. Antibody testing is useful for diagnosing historic infections of a disease in a population. These tests are also a helpful epidemiological tool for predicting how the virus spreads in a community, relating antibody levels to immunity and for assessing herd immunity. In the present study, SARS-CoV-2 viral proteins were recombinantly produced and used to analyse serum from individuals previously exposed, or not, to SARS-CoV-2. The nucleocapsid (Npro) and spike subunit 2 (S2Frag) proteins were identified as highly immunogenic, although responses to the former were generally greater. These two proteins were used to develop two quantitative enzyme-linked immunosorbent assays (ELISAs) that when used in combination resulted in a highly reliable diagnostic test. Npro and S2Frag-ELISAs could detect at least 10% more true positive coronavirus disease-2019 (COVID-19) cases than the commercially available ARCHITECT test (Abbott). Moreover, our quantitative ELISAs also show that specific antibodies to SARS-CoV-2 proteins tend to wane rapidly even in patients who had developed severe disease. As antibody tests complement COVID-19 diagnosis and determine population-level surveillance during this pandemic, the alternative diagnostic we present in this study could play a role in controlling the spread of the virus.

Intra-vital imaging of mesenchymal stromal cell kinetics in the pulmonary vasculature during infection.

Mesenchymal stem/stromal cells (MSCs) have demonstrated efficacy in pre-clinical models of inflammation and tissue injury, including in models of lung injury and infection. Rolling, adhesion and transmigration of MSCs appears to play a role during MSC kinetics in the systemic vasculature. However, a large proportion of MSCs become entrapped within the lungs after intravenous administration, while the initial kinetics and the site of arrest of MSCs in the pulmonary vasculature are unknown. We examined the kinetics of intravascularly administered MSCs in the pulmonary vasculature using a microfluidic system in vitro and intra-vital microscopy of intact mouse lung. In vitro, MSCs bound to endothelium under static conditions but not under laminar flow. VCAM-1 antibodies did not affect MSC binding. Intravital microscopy demonstrated MSC arrest at pulmonary micro-vessel bifurcations due to size obstruction. Retention of MSCs in the pulmonary microvasculature was increased in Escherichia coli-infected animals. Trapped MSCs deformed over time and appeared to release microvesicles. Labelled MSCs retained therapeutic efficacy against pneumonia. Our results suggest that MSCs are physically obstructed in pulmonary vasculature and do not display properties of rolling/adhesion, while retention of MSCs in the infected lung may require receptor interaction.

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.

Mesenchymal Stem/Stromal Cells Therapy for Sepsis and Acute Respiratory Distress Syndrome.

Sepsis and acute respiratory distress syndrome (ARDS) constitute devastating conditions with high morbidity and mortality. Sepsis results from abnormal host immune response, with evidence for both pro- and anti-inflammatory activation present from the earliest phases. The "proinflammatory" response predominates initially causing host injury, with later-phase sepsis characterized by immune cell hypofunction and opportunistic superinfection. ARDS is characterized by inflammation and disruption of the alveolar-capillary membrane leading to injury and lung dysfunction. Sepsis is the most common cause of ARDS. Approximately 20% of deaths worldwide in 2017 were due to sepsis, while ARDS occurs in over 10% of all intensive care unit patients and results in a mortality of 30 to 45%. Given the fact that sepsis and ARDS share some-but not all-underlying pathophysiologic injury mechanisms, the lack of specific therapies, and their frequent coexistence in the critically ill, it makes sense to consider therapies for both conditions together. In this article, we will focus on the therapeutic potential of mesenchymal stem/stromal cells (MSCs). MSCs are available from several tissues, including bone marrow, umbilical cord, and adipose tissue. Allogeneic administration is feasible, an important advantage for acute conditions like sepsis or ARDS. They possess diverse mechanisms of action of relevance to sepsis and ARDS, including direct and indirect antibacterial actions, potent effects on the innate and adaptive response, and pro-reparative effects. MSCs can be preactivated thereby potentiating their effects, while the use of their extracellular vesicles can avoid whole cell administration. While early-phase clinical trials suggest safety, considerable challenges exist in moving forward to phase III efficacy studies, and to implementation as a therapy should they prove effective.

Umbilical Cord-Derived CD362+ Mesenchymal Stromal Cells Attenuate Polymicrobial Sepsis Induced by Caecal Ligation and Puncture.

Mesenchymal stromal cells (MSCs) have a multimodal, immunomodulatory mechanism of action and are now in clinical trials for single organ and systemic sepsis. However, a number of practicalities around source, homogeneity and therapeutic window remain to be determined. Here, we utilised conditioned medium from CD362+-sorted umbilical cord-human MSCs (UC-hMSCs) for a series of in vitro anti-inflammatory assays and the cryopreserved MSCs themselves in a severe (Series 1) or moderate (Series 2+3) caecal ligation and puncture (CLP) rodent model. Surviving animals were assessed at 48 h post injury induction. MSCs improved human lung, colonic and kidney epithelial cell survival following cytokine activation. In severe systemic sepsis, MSCs administered at 30 min enhanced survival (Series 1), and reduced organ bacterial load. In moderate systemic sepsis (Series 2), MSCs were ineffective when delivered immediately or 24 h later. Of importance, MSCs delivered 4 h post induction of moderate sepsis (Series 3) were effective, improving serum lactate, enhancing bacterial clearance from tissues, reducing pro-inflammatory cytokine concentrations and increasing antimicrobial peptides in serum. While demonstrating benefit and immunomodulation in systemic sepsis, therapeutic efficacy may be limited to a specific point of disease onset, and repeat dosing, MSC enhancement or other contingencies may be necessary.

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.

ß-Glucan extracts from the same edible shiitake mushroom Lentinus edodes produce differential in-vitro immunomodulatory and pulmonary cytoprotective effects - Implications for coronavirus disease (COVID-19) immunotherapies.

Coronavirus pneumonia is accompanied by rapid virus replication, where a large number of inflammatory cell infiltration and cytokine storm may lead to acute lung injury, acute respiratory distress syndrome (ARDS) and death. The uncontrolled release of pro-inflammatory cytokines, including interleukin (IL)-1ß and IL-6, is associated with ARDS. This constituted the first study to report on the variability in physicochemical properties of ß-glucans extracts from the same edible mushroom Lentinus edodes on the reduction of these pro-inflammatory cytokines and oxidative stress. Specifically, the impact on the immunomodulatory and cytoprotective properties of our novel in 'house' (IH-Lentinan, IHL) and a commercial (Carbosynth-Lentinan, CL) Lentinan extract were investigated using in vitro models of lung injury and macrophage phagocytosis. CL comprised higher amounts of α-glucans and correspondingly less ß-glucans. The two lentinan extracts demonstrated varying immunomodulatory activities. Both Lentinan extracts reduced cytokine-induced NF-κB activation in human alveolar epithelial A549 cells, with the IHL extract proving more effective at lower doses. In contrast, in activated THP-1 derived macrophages, the CL extract more effectively attenuated pro-inflammatory cytokine production (TNF-α, IL-8, IL-2, IL-6, IL-22) as well as TGF-ß and IL-10. The CL extract attenuated oxidative stress-induced early apoptosis, while the IHL extract attenuated late apoptosis. Our findings demonstrate significant physicochemical differences between Lentinan extracts, which produce differential in vitro immunomodulatory and pulmonary cytoprotective effects that may also have positive relevance to candidate COVID-19 therapeutics targeting cytokine storm.

Umbilical cord-derived CD362+ mesenchymal stromal cells for E. coli pneumonia: impact of dose regimen, passage, cryopreservation, and antibiotic therapy.

BACKGROUND: Mesenchymal stromal cells (MSCs) demonstrate considerable promise for acute respiratory distress syndrome (ARDS) and sepsis. However, standard approaches to MSC isolation generate highly heterogeneous cell populations, while bone marrow (BM) constitutes a limited and difficult to access MSC source. Furthermore, a range of cell manufacturing considerations and clinical setting practicalities remain to be explored. METHODS: Adult male rats were subject to E. coli-induced pneumonia and administered CD362+ umbilical cord (UC)-hMSCs using a variety of cell production and clinical relevance considerations. In series 1, animals were instilled with E. coli and randomized to receive heterogeneous BM or UC-hMSCs or CD362+ UC-hMSCs. Subsequent series examined the impact of concomitant antibiotic therapy, MSC therapeutic cryopreservation (cryopreserved vs fresh CD362+ UC-hMSCs), impact of cell passage on efficacy (passages 3 vs 5 vs 7 vs 10), and delay of administration of cell therapy (0 h vs 6 h post-injury vs 6 h + 12 h) following E. coli installation. RESULTS: CD362+ UC-hMSCs were as effective as heterogonous MSCs in reducing E. coli-induced acute lung injury, improving oxygenation, decreasing bacterial load, reducing histologic injury, and ameliorating inflammatory marker levels. Cryopreserved CD362+ UC-hMSCs recapitulated this efficacy, attenuating E. coli-induced injury, but therapeutic relevance did not extend beyond passage 3 for all indices. CD362+ UC-hMSCs maintained efficacy in the presence of antibiotic therapy and rescued the animal from E. coli injury when delivered at 6 h + 12 h, following E. coli instillation. CONCLUSIONS: These translational studies demonstrated the efficacy of CD362+ UC-hMSCs, where they decreased the severity of E. coli-induced pneumonia, maintained efficacy following cryopreservation, were more effective at early passage, were effective in the presence of antibiotic therapy, and could continue to provide benefit at later time points following E. coli injury.

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.