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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.

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.

Introduction: Patients who survive early stage sepsis may progress to late stage sepsis which is characterised by immunosuppression believed to be due, in part, to immune cell paralysis. We have previously shown that Mesenchymal Stem Cells (MSCs) are effective in preclinical models of early stage sepsis (Devaney et al., 2015) and to restore macrophage function in early clinical sepsis. We sought to develop in vivo and in vitro late stage sepsis models to assess if MSCs could restore immune cell homeostasis in this setting. Methods: We established an in vivo model of pulmonary sepsis by intratracheal administration of K.pneumoniae to Sprague-Dawley rats. MSCs were administered at day two and immune cell and cytokine secretion profiles in the Bronchoalveolar Lavage (BAL) and blood were analysed at day five. An in vitro endotoxin tolerance model was then established using immune cell lines and primary macrophages from blood and BAL exposed to repeated high levels of endotoxin. Results: MSCs improve paralysed immune cell responsiveness to an endotoxin stimulus ex vivo as demonstrated by a restoration of cytokine secretion in cells isolated from treated animal models (Figure 1). Conclusions: MSCs have the potential to reverse late sepsis induced immune cell dysfunction caused by prolonged exposure to bacterial endotoxin ex vivo.

Acute respiratory distress syndrome (ARDS) continues to have a high mortality with no effective specific therapy. Anti-inflammatory and anti-oxidant therapeutic proteins have previously shown promise in in vivo ARDS models. Similarly, targeting cell permeability (‘leakiness’) associated with ARDS has shown potential. A synthetic mRNA vector for rapid production of protein, coupled with direct delivery to the injured lung via nebulisation, could overcome these limitations. mRNA complexed with a novel transfection reagent (Factor Bioscience Ltd.) encoding green fluorescent protein (GFP), anti-inflammatory and anti-oxidant therapeutic proteins was administered to aseptic or septic rat lungs through a vibrating mesh nebuliser (Aerogen), via the flexiVent (SCIREQ). Animals were harvested and physiological and histological parameters were assessed. An in vitro model of cell permeability employing Calu-3 epithelial cells was also examined using TIE2 and ANGPT1 mRNA targets. A system incorporating Ussing chambers facilitating the measurement of specific ionic transport mechanisms, and by extension fluid dynamics across cell monolayers, was enrolled. GFP mRNA expressed rapidly in lung epithelial cells (

Bacterial infection remains the main cause of acute respiratory distress syndrome and is a leading cause of death and disability in critically ill patients Here we report on the use of purified beta-glucan (lentinan) extracts from Lentinus edodes (Shiitake) mushroom that can reduce infection by a multidrug-resistant clinical isolate of Klebsiella pneumoniae in a rodent pneumonia model, likely through immunomodulation Adult male Sprague-Dawley rats were subjected to intra-tracheal administration of K pneumoniae to induce pulmonary sepsis and randomized to three groups;vehicle control (Vehicle, n = 12), commercial lentinan (CL, n = 8) or in-house extracted lentinan (IHL, n = 8) were administered intravenously 1 h postinfection Physiological parameters and blood gas analysis were measured, bacterial counts from bronchoalveolar-lavage (BAL) were determined, along with differential staining of white cells and measurement of protein concentration in BAL 48 h after pneumonia induction Use of IHL extract significantly decreased BAL CFU counts Both CL and IHL extractions reduced protein concentration in BAL Use of IHL resulted in an improvement in physiological parameters compared to controls and CL In conclusion, administration of lentinan to treat sepsis-induced lung injury appears safe and effective and may exert its effects in an immunomodulatory manner

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.

Adhesive proteins of barnacle cement have potential as environmentally friendly adhesives owing to their ability to adhere to various substrates in aqueous environments. By understanding the taxonomic breath of barnacles with different lifestyles, we may uncover commonalities in adhesives produced by these specialized organisms. The 19 kDa cement protein (cp19k) of the stalked barnacle Pollicipes pollicipes was expressed in Escherichia coli BL21 to investigate its adhesive properties. Initial expression of hexahistidine-tagged protein (rPpolcp19k-his) yielded low levels of insoluble protein. Co-overproduction of E. coli molecular chaperones GroEL-GroES and trigger factor (TF) increased soluble protein yields, although TF co-purified with the target protein (TF-rPpolcp19k-his). Surface coat analysis revealed high levels of adsorption of the TF-rPpolcp19k-his complex and of purified E. coli TF on both hydrophobic and hydrophilic surfaces, while low levels of adsorption were observed for rPpolcp19k-his. Tag-free rPpolcp19k protein also exhibited low adsorption compared to fibrinogen and Cell-Tak controls on hydrophobic, neutral hydrophilic and charged self-assembled monolayers under surface plasmon resonance assay conditions designed to mimic the barnacle cement gland or seawater. Because rPpolcp19k protein displays low adhesive capability, this protein is suggested to confer the ability to self-assemble into a plaque within the barnacle cement complex. This article is part of the theme issue ‘Transdisciplinary approaches to the study of adhesion and adhesives in biological systems’.

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 CO2to 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 CO2on multiple diverse signalling pathways, be it the effect from CO2itself 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 CO2concentration and tailor respiratory and other responses to atmospheric or local levels. Animal models have been widely employed to study the changes in CO2levels in various disease states and also to what extent permissive or even directly delivered CO2can 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 CO2affects 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 CO2levels, that could lead to improved clinical care in the future.

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 barnacleLepas 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.

McCarthy SD, Waters SM, Kenny DA, Diskin MG, Fitzpatrick R, Patton J, Wathes DC, Morris DG. Negative energy balance and hepatic gene expression patterns in high-yielding dairy cows during the early postpartum period: a global approach. Physiol Genomics 42A: 188-199, 2010. First published August 17, 2010; doi:10.1152/physiolgenomics.00118.2010.-In high-yielding dairy cows the liver undergoes extensive physiological and biochemical changes during the early postpartum period in an effort to re-establish metabolic homeostasis and to counteract the adverse effects of negative energy balance (NEB). These adaptations are likely to be mediated by significant alterations in hepatic gene expression. To gain new insights into these events an energy balance model was created using differential feeding and milking regimes to produce two groups of cows with either a mild (MNEB) or severe NEB (SNEB) status. Cows were slaughtered and liver tissues collected on days 6-7 of the first follicular wave postpartum. Using an Affymetrix 23k oligonucleotide bovine array to determine global gene expression in hepatic tissue of these cows, we found a total of 416 genes (189 up- and 227 downregulated) to be altered by SNEB. Network analysis using Ingenuity Pathway Analysis revealed that SNEB was associated with widespread changes in gene expression classified into 36 gene networks including those associated with lipid metabolism, connective tissue development and function, cell signaling, cell cycle, and metabolic diseases, the three most significant of which are discussed in detail. SNEB cows displayed reduced expression of transcription activators and signal transducers that regulate the expression of genes and gene networks associated with cell signaling and tissue repair. These alterations are linked with increased expression of abnormal cell cycle and cellular proliferation associated pathways. This study provides new information and insights on the effect of SNEB on gene expression in high-yielding Holstein Friesian dairy cows in the early postpartum period.

Peer-reviewed Differences in genetic selection criteria for dairy cows internationally have led to divergence in the Holstein-Friesian breed. The objective of this study was to compare hepatic expression of genes of the somatotropic axis in the North American Holstein-Friesian and the New Zealand Holstein-Friesian strains of dairy cow at early and mid lactation. Mature cows of both the North American Holstein-Friesian (n = 10) and New Zealand Holstein-Friesian (n = 10) strains were selected. Liver tissue was collected by percutaneous punch biopsy from all cows at 35 and 140 d postpartum, representing early and mid lactation, respectively. Total RNA was extracted and the hepatic expression of genes involved in the control of the somatotropic axis was examined. Abundance of insulin-like growth factor (IGF)-1 mRNA was greater in the New Zealand strain, concomitant with a tendency for increased expression of acid-labile subunit mRNA. Across strains, mRNA abundance of IGF-binding protein-1, IGF-binding protein-2, and growth hormone receptor 1A decreased from d 35 to 140 postpartum, whereas expression of IGF-1 and acid-labile subunit tended to increase. Abundance of suppressor of cytokine signaling-3 mRNA was increased at d 140 postpartum. Both the strain of Holstein-Friesian cow and the stage of lactation influenced expression of genes controlling the somatotropic axis in hepatic tissue.

In the study presented, the hypothesis that ligands of the insulin-like growth factor (IGF) family, as well as their binding proteins (BPs), are temporally regulated and are altered by elevated progesterone (P4) and/or pregnancy was tested. Heifers detected in standing estrus following synchronization ( n = 210, day 0), were artificially inseminated ( n = 140) or left as noninseminated cyclic controls ( n = 70). On day 3, half of each group were randomly assigned to receive a P4-releasing intravaginal device resulting in four treatment groups, pregnant and cyclic heifers with high and normal P4 concentrations on either day 5, 7, 13, or 16 of the estrous cycle/early pregnancy. Quantitative real-time PCR and immunohistochemistry were performed on endometrial homogenate and uterine cross sections to measure transcript abundance and protein localization respectively ( n = 5 per treatment per time point). No effect of day, treatment, or their interactions was observed for IGF2, IGFBP4, and - 5 ( P > 0.05). IGF1 and IGFBP6 expression decreased, while IGF1R and IGFBP2 expression increased ( P < 0.05) as the days of the cycle or pregnancy progressed. The expression of IGFBP2 was increased by elevated P4; in addition, localization of both IGF2 and IGFBP2 protein in the luminal and superficial glandular epithelium displayed a P4 × day interaction. In conclusion it is proposed that decreased IGF1 and IGFBP6 expression with a coordinate increase in IGF1R and IGFBP2 as the estrous cycle/early pregnancy progresses, along with other factors, are required to establish a uterine environment that promotes the growth and development of the conceptus prior to implantation. In addition, the increased protein abundance of both IGF2 and IGFBP2 observed in heifers supplemented with P4 contributes to the enhanced conceptus elongation observed in this model.