Blood gene expression predicts intensive care unit admission in hospitalised patients with COVID-19.

Background: The COVID-19 pandemic has created pressure on healthcare systems worldwide. Tools that can stratify individuals according to prognosis could allow for more efficient allocation of healthcare resources and thus improved patient outcomes. It is currently unclear if blood gene expression signatures derived from patients at the point of admission to hospital could provide useful prognostic information. Methods: Gene expression of whole blood obtained at the point of admission from a cohort of 78 patients hospitalised with COVID-19 during the first wave was measured by high resolution RNA sequencing. Gene signatures predictive of admission to Intensive Care Unit were identified and tested using machine learning and topological data analysis, TopMD. Results: The best gene expression signature predictive of ICU admission was defined using topological data analysis with an accuracy: 0.72 and ROC AUC: 0.76. The gene signature was primarily based on differentially activated pathways controlling epidermal growth factor receptor (EGFR) presentation, Peroxisome proliferator-activated receptor alpha (PPAR-α) signalling and Transforming growth factor beta (TGF-ß) signalling. Conclusions: Gene expression signatures from blood taken at the point of admission to hospital predicted ICU admission of treatment naïve patients with COVID-19.

Delayed induction of type I and III interferons mediates nasal epithelial cell permissiveness to SARS-CoV-2.

The nasal epithelium is a plausible entry point for SARS-CoV-2, a site of pathogenesis and transmission, and may initiate the host response to SARS-CoV-2. Antiviral interferon (IFN) responses are critical to outcome of SARS-CoV-2. Yet little is known about the interaction between SARS-CoV-2 and innate immunity in this tissue. Here we apply single-cell RNA sequencing and proteomics to a primary cell model of human nasal epithelium differentiated at air-liquid interface. SARS-CoV-2 demonstrates widespread tropism for nasal epithelial cell types. The host response is dominated by type I and III IFNs and interferon-stimulated gene products. This response is notably delayed in onset relative to viral gene expression and compared to other respiratory viruses. Nevertheless, once established, the paracrine IFN response begins to impact on SARS-CoV-2 replication. When provided prior to infection, recombinant IFNß or IFNλ1 induces an efficient antiviral state that potently restricts SARS-CoV-2 viral replication, preserving epithelial barrier integrity. These data imply that the IFN-I/III response to SARS-CoV-2 initiates in the nasal airway and suggest nasal delivery of recombinant IFNs to be a potential chemoprophylactic strategy.

Interferon regulatory factor 8 regulates expression of acid ceramidase and infection susceptibility in cystic fibrosis.

Most patients with cystic fibrosis (CF) suffer from acute and chronic pulmonary infections with bacterial pathogens, which often determine their life quality and expectancy. Previous studies have demonstrated a downregulation of the acid ceramidase in CF epithelial cells resulting in an increase of ceramide and a decrease of sphingosine. Sphingosine kills many bacterial pathogens, and the downregulation of sphingosine seems to determine the infection susceptibility of cystic fibrosis mice and patients. It is presently unknown how deficiency of the cystic fibrosis transmembrane conductance regulator (CFTR) connects to a marked downregulation of the acid ceramidase in human and murine CF epithelial cells. Here, we employed quantitative PCR, western blot analysis, and enzyme activity measurements to study the role of IRF8 for acid ceramidase regulation. We report that genetic deficiency or functional inhibition of CFTR/Cftr results in an upregulation of interferon regulatory factor 8 (IRF8) and a concomitant downregulation of acid ceramidase expression with CF and an increase of ceramide and a reduction of sphingosine levels in tracheal and bronchial epithelial cells from both human individuals or mice. CRISPR/Cas9- or siRNA-mediated downregulation of IRF8 prevented changes of acid ceramidase, ceramide, and sphingosine in CF epithelial cells and restored resistance to Pseudomonas aeruginosa infections, which is one of the most important and common pathogens in lung infection of patients with CF. These studies indicate that CFTR deficiency causes a downregulation of acid ceramidase via upregulation of IRF8, which is a central pathway to control infection susceptibility of CF cells.

Trends in nontuberculous mycobacteria infection in children and young people with cystic fibrosis.

Nontuberculous mycobacteria (NTM) infection is of growing concern in cystic fibrosis (CF). UK CF Registry data were analyzed from 2016 to 2018. Prevalence of infection stabilized in the pediatric age-group during this period but remained substantially higher than in 2010. Allergic bronchopulmonary aspergillosis and Pseudomonas aeruginosa infection were associated with NTM infection.

Clinical and molecular characterization of the R751L-CFTR mutation.

Cystic fibrosis (CF) arises from mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in progressive and life-limiting respiratory disease. R751L is a rare CFTR mutation that is poorly characterized. Our aims were to describe the clinical and molecular phenotypes associated with R751L. Relevant clinical data were collected from three heterozygote individuals harboring R751L (2 patients with G551D/R751L and 1 with F508del/R751L). Assessment of R751L-CFTR function was made in primary human bronchial epithelial cultures (HBEs) and Xenopus oocytes. Molecular properties of R751L-CFTR were investigated in the presence of known CFTR modulators. Although sweat chloride was elevated in all three patients, the clinical phenotype associated with R751L was mild. Chloride secretion in F508del/R751L HBEs was reduced compared with non-CF HBEs and associated with a reduction in sodium absorption by the epithelial sodium channel (ENaC). However, R751L-CFTR function in Xenopus oocytes, together with folding and cell surface transport of R751L-CFTR, was not different from wild-type CFTR. Overall, R751L-CFTR was associated with reduced sodium chloride absorption but had functional properties similar to wild-type CFTR. This is the first report of R751L-CFTR that combines clinical phenotype with characterization of functional and biological properties of the mutant channel. Our work will build upon existing knowledge of mutations within this region of CFTR and, importantly, inform approaches for clinical management. Elevated sweat chloride and reduced chloride secretion in HBEs may be due to alternative non-CFTR factors, which require further investigation.

Recombinant Acid Ceramidase Reduces Inflammation and Infection in Cystic Fibrosis.

Rationale: In cystic fibrosis the major cause of morbidity and mortality is lung disease characterized by inflammation and infection. The influence of sphingolipid metabolism is poorly understood with a lack of studies using human airway model systems.Objectives: To investigate sphingolipid metabolism in cystic fibrosis and the effects of treatment with recombinant human acid ceramidase on inflammation and infection.Methods: Sphingolipids were measured using mass spectrometry in fully differentiated cultures of primary human airway epithelial cells and cocultures with Pseudomonas aeruginosa. In situ activity assays, Western blotting, and quantitative PCR were used to investigate function and expression of ceramidase and sphingomyelinase. Effects of treatment with recombinant human acid ceramidase on sphingolipid profile and inflammatory mediator production were assessed in cell cultures and murine models.Measurements and Main Results: Ceramide is increased in cystic fibrosis airway epithelium owing to differential function of enzymes regulating sphingolipid metabolism. Sphingosine, a metabolite of ceramide with antimicrobial properties, is not upregulated in response to P. aeruginosa by cystic fibrosis airway epithelia. Tumor necrosis factor receptor 1 is increased in cystic fibrosis epithelia and activates NF-κB signaling, generating inflammation. Treatment with recombinant human acid ceramidase, to decrease ceramide, reduced both inflammatory mediator production and susceptibility to infection.Conclusions: Sphingolipid metabolism is altered in airway epithelial cells cultured from people with cystic fibrosis. Treatment with recombinant acid ceramidase ameliorates the two pivotal features of cystic fibrosis lung disease, inflammation and infection, and thus represents a therapeutic approach worthy of further exploration.

Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells.

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) hydration, mucus viscosity and activity of antimicrobial peptides, key parameters involved in innate defense of the lungs. This is of primary relevance in the field of chronic respiratory diseases such as cystic fibrosis (CF) where these parameters are dysregulated. While different groups have studied ASL pH both in vivo and in vitro, their methods report a relatively wide range of ASL pH values and even contradictory findings regarding any pH differences between non-CF and CF cells. Furthermore, their protocols do not always provide enough details in order to ensure reproducibility, most are low throughput and require expensive equipment or specialized knowledge to implement, making them difficult to establish in most labs. Here we describe a semi-automated fluorescent plate reader assay that enables the real-time measurement of ASL pH under thin film conditions that more closely resemble the in vivo situation. This technique allows for stable measurements for many hours from multiple airway cultures simultaneously and, importantly, dynamic changes in ASL pH in response to agonists and inhibitors can be monitored. To achieve this, the ASL of fully differentiated primary human airway epithelial cells (hAECs) are stained overnight with a pH-sensitive dye in order to allow for the reabsorption of the excess fluid to ensure thin film conditions. After fluorescence is monitored in the presence or absence of agonists, pH calibration is performed in situ to correct for volume and dye concentration. The method described provides the required controls to make stable and reproducible ASL pH measurements, which ultimately could be used as a drug discovery platform for personalized medicine, as well as adapted to other epithelial tissues and experimental conditions, such as inflammatory and/or host-pathogen models.

Epidemiology of Nontuberculous Mycobacteria Infection in Children and Young People With Cystic Fibrosis: Analysis of UK Cystic Fibrosis Registry.

BACKGROUND: Infection with nontuberculous mycobacteria (NTM) is of growing clinical concern in people with cystic fibrosis (CF). The epidemiology of infection in children and young people remains poorly understood. Our goal was to investigate the epidemiology of NTM infection in the pediatric age group using data from the UK CF Registry. METHODS: Data from 2010-2015 for individuals aged <16 years (23200 observations from 5333 unique individuals) were obtained. Univariate analysis of unique individuals comparing all key clinical factors and health outcomes to NTM status was performed. The significant factors that were identified were used to generate a multivariate logistic regression model that, following step-wise removal, generated a final parsimonious model. RESULTS: The prevalence of individuals with a NTM-positive respiratory culture increased every year from 2010 (45 [1.3%]) to 2015 (156 [3.8%]). Allergic bronchopulmonary aspergillosis (odds ratio [OR], 2.66; P = 5.0 × 10-8), age (OR, 1.08; P = 3.4 × 10-10), and intermittent Pseudomonas aeruginosa infection (OR, 1.51; P = .004) were significantly associated with NTM infection. CONCLUSIONS: NTM infection is of increasing prevalence in the UK pediatric CF population. This study highlights the urgent need for work to establish effective treatment and prevention strategies for NTM infection in young people with CF.

Medical Countermeasures for Children in Radiation and Nuclear Disasters: Current Capabilities and Key Gaps.

OBJECTIVE: Despite children's unique vulnerability, clinical guidance and resources are lacking around the use of radiation medical countermeasures (MCMs) available commercially and in the Strategic National Stockpile to support immediate dispensing to pediatric populations. To better understand the current capabilities and shortfalls, a literature review and gap analysis were performed. METHODS: A comprehensive review of the medical literature, Food and Drug Administration (FDA)-approved labeling, FDA summary reviews, medical references, and educational resources related to pediatric radiation MCMs was performed from May 2016 to February 2017. RESULTS: Fifteen gaps related to the use of radiation MCMs in children were identified. The need to address these gaps was prioritized based upon the potential to decrease morbidity and mortality, improve clinical management, strengthen caregiver education, and increase the relevant evidence base. CONCLUSIONS: Key gaps exist in information to support the safe and successful use of MCMs in children during radiation emergencies; failure to address these gaps could have negative consequences for families and communities. There is a clear need for pediatric-specific guidance to ensure clinicians can appropriately identify, triage, and treat children who have been exposed to radiation, and for resources to ensure accurate communication about the safety and utility of radiation MCMs for children. (Disaster Med Public Health Preparedness. 2019;13:639-646).

Sphingolipids as targets for inhalation treatment of cystic fibrosis.

Studies over the past several years have demonstrated the important role of sphingolipids in cystic fibrosis (CF), chronic obstructive pulmonary disease and acute lung injury. Ceramide is increased in airway epithelial cells and alveolar macrophages of CF mice and humans, while sphingosine is dramatically decreased. This increase in ceramide results in chronic inflammation, increased death of epithelial cells, release of DNA into the bronchial lumen and thereby an impairment of mucociliary clearance; while the lack of sphingosine in airway epithelial cells causes high infection susceptibility in CF mice and possibly patients. The increase in ceramide mediates an ectopic expression of ß1-integrins in the luminal membrane of CF epithelial cells, which results, via an unknown mechanism, in a down-regulation of acid ceramidase. It is predominantly this down-regulation of acid ceramidase that results in the imbalance of ceramide and sphingosine in CF cells. Correction of ceramide and sphingosine levels can be achieved by inhalation of functional acid sphingomyelinase inhibitors, recombinant acid ceramidase or by normalization of ß1-integrin expression and subsequent re-expression of endogenous acid ceramidase. These treatments correct pulmonary inflammation and prevent or treat, respectively, acute and chronic pulmonary infections in CF mice with Staphylococcus aureus and mucoid or non-mucoid Pseudomonas aeruginosa. Inhalation of sphingosine corrects sphingosine levels only and seems to mainly act against the infection. Many antidepressants are functional inhibitors of the acid sphingomyelinase and were designed for systemic treatment of major depression. These drugs could be repurposed to treat CF by inhalation.

Ataluren in cystic fibrosis: development, clinical studies and where are we now?

INTRODUCTION: Cystic fibrosis (CF) is one of the most common genetically-acquired life-limiting conditions worldwide. The underlying defect is dysfunction of the cystic fibrosis transmembrane-conductance regulator (CFTR) which leads to progressive lung disease and other multi-system effects. Around 10% of people with CF have a class I nonsense mutation that leads to production of shortened CFTR due to a premature termination codon (PTC). Areas covered: We discuss the discovery of the small-molecule drug ataluren, which in vitro has been shown to allow read-through of PTCs and facilitate synthesis of full-length protein. We review clinical studies that have been performed involving ataluren in CF. Early-phase short-term cross-over studies showed improvement in nasal potential difference. A follow-up phase III randomised controlled trial did not show a significant difference for the primary outcome of lung function, however a post-hoc analysis suggested possible benefit in patients not receiving tobramycin. A further randomised controlled trial in patients not receiving tobramycin has been reported as showing no benefit but has not yet been published in full peer-reviewed form. Expert opinion: A small-molecule approach to facilitate read-through of PTCs in nonsense mutations makes intuitive sense. However, at present there is no high-quality evidence of clinical efficacy for ataluren in people with CF.

ß1-Integrin Accumulates in Cystic Fibrosis Luminal Airway Epithelial Membranes and Decreases Sphingosine, Promoting Bacterial Infections.

Chronic pulmonary colonization with bacterial pathogens, particularly Pseudomonas aeruginosa, is the primary cause of morbidity and mortality in patients with cystic fibrosis (CF). We observed that ß1-integrins accumulate on the luminal membrane of upper-airway epithelial cells from mice and humans with CF. ß1-integrin accumulation is due to increased ceramide and the formation of ceramide platforms that trap ß1-integrins on the luminal pole of bronchial epithelial cells. ß1-integrins downregulate acid ceramidase expression, resulting in further accumulation of ceramide and consequent reduction of surface sphingosine, a lipid that kills bacteria. Interrupting this vicious cycle by triggering surface ß1-integrin internalization via anti-ß1-integrin antibodies or the RGD peptide ligand-or by genetic or pharmacological correction of ceramide levels-normalizes ß1-integrin distribution and sphingosine levels in CF epithelial cells and prevents P. aeruginosa infection in CF mice. These findings suggest a therapeutic avenue to ameliorate CF-associated bacterial infections.

Risk factors for "late-to-test" HIV diagnosis in Riverside County, California.

Patients diagnosed late in the course of HIV infection are at an increased risk of negative health outcomes and are more likely to transmit HIV to others. Using the CDC's definition for AIDS, we analyzed case report data from persons diagnosed with AIDS within 12 months of an HIV diagnosis ("late-to-test") in Riverside County, CA, between 2009 and 2014. Of 1385 HIV cases, 422 (30.5%) were late-to-test. Factors associated with late-to-test were: having no insurance (P = 0.005), being Hispanic (P = 0.002) and being between 45 and 64 years of age (P < 0.001). Females (P = 0.013) and those in the eastern region of Riverside County (P = 0.002) were less likely to be late-to-test. In the absence of universal HIV testing, interventions to decrease late testing are needed.

Human hair follicle dermal sheath and papilla cells support keratinocyte growth in monolayer coculture.

Traditional skin grafting techniques are effective but limited methods of skin replacement. Autologous transplantation of rapidly cultured keratinocytes is successful for epidermal regeneration, but the current gold-standard technique requires mouse fibroblast feeders and serum-rich media, with serum-free systems and dermal fibroblast (DF) feeders performing relatively poorly. Here, we investigated the capacity of human hair follicle dermal cells to act as alternative supports for keratinocyte growth. Dermal papilla (DP) dermal sheath (DS), DF and 3T3 cells were used as inactivated feeder cells for human keratinocyte coculture. Under conditions favouring dermal cells, proliferation of keratinocytes in the presence of either DS or DP cells was significantly enhanced compared with DF cells, at levels comparable to keratinocytes cultured under gold-standard conditions. Secreted protein acidic and rich in cysteine (SPARC) expression increased DS and DP cells relative to DFs; however, further experiments did not demonstrate a role in keratinocyte support.

The critical role of TAK1 in accentuated epithelial to mesenchymal transition in obliterative bronchiolitis after lung transplantation.

Therapies to limit or reverse fibrosis have proven unsuccessful, highlighting the need for a greater understanding of basic mechanisms that drive fibrosis and, in particular, the link between fibrosis and inflammation. It has been shown that pro-fibrotic transforming growth factor ß1 (TGF-ß1)-driven epithelial-to-mesenchymal transition (EMT) can be accentuated by tumor necrosis factor α (TNF-α). TGF-ß-activated kinase 1 (TAK1) is activated by both TGF-ß1 and TNF-α, activating both nuclear factor kappa-light-chain-enhancer of activated B cells and mitogen-activated protein kinase signaling pathways. In this study, we evaluated the potential for TAK1 to modulate the synergistic effect between TGF-ß1 and TNF-α in driving EMT. Co-stimulation with TGF-ß1 and TNF-α induced an accentuated and extended phosphorylation of TAK1 compared to either alone. TAK1 signaled downstream via nuclear factor kappa-light-chain-enhancer of activated B cells, and Jun N-terminal kinase-2, but independent of Jun N-terminal kinase-1, extracellular signal-regulated kinase-1/2, or p38 mitogen-activated protein kinase signaling to drive EMT in bronchial epithelial cells. Blocking either TAK1 or Jun N-terminal kinase-2 inhibited EMT. TAK1 phosphorylation was increased in the airway epithelium of patients with fibrotic airway disease. These data identify factors leading to and affected by accentuated and extended TAK1 phosphorylations potential novel therapeutic targets in inflammation-driven fibrotic diseases.

Fungal infections and antifungal prophylaxis in pediatric cardiac extracorporeal life support.

OBJECTIVE: Infections acquired by children during extracorporeal membrane oxygenation (ECMO) increase mortality. Our aim was to evaluate the effectiveness of prophylactic fluconazole on the incidence of fungal infections and to assess whether hospital-acquired fungal infection is associated with increased in-hospital mortality in pediatric cardiac patients requiring ECMO. METHODS: We retrospectively reviewed a prospectively maintained database and collected data on all hospital-acquired infections in patients supported for cardiac indications at a tertiary children's hospital from 1989 to 2008. RESULTS: ECMO was deployed 801 times in 767 patients. After exclusion criteria were applied, 261 pediatric patients supported for cardiac indications were studied. Fungal infection (blood, urine, or surgical site) occurred in 12% (31/261) of patients, 9 (7%) of 127 patients receiving fluconazole prophylaxis versus 22 (16.4%) of 134 without antifungal prophylaxis (P = .02). Using a multivariable logistic regression model, the absence of fluconazole prophylaxis was associated with an increased risk of fungal infection (odds ratio [OR] = 2.8; 95% confidence intervals [CI], 1.2, 6.7; P = .016). In a multivariable logistic regression model for in-hospital mortality, the presence of fungal infection was associated with increased odds (OR = 3.8; 95% CI, 1.5, 9.6; P = .005) of in-hospital mortality among cardiac patients requiring ECMO, and the absence of antifungal prophylaxis showed a trend toward the same (OR = 1.6; 95% CI, 0.96, 2.8; P = .072). CONCLUSIONS: Children with cardiac disease supported with ECMO who acquire fungal infections have increased mortality. Routine fluconazole prophylaxis is associated with lower rates of fungal infections in these patients.

Transforming Growth Factor-ß1 (TGF-ß1) Driven Epithelial to Mesenchymal Transition (EMT) is Accentuated by Tumour Necrosis Factor α (TNFα) via Crosstalk Between the SMAD and NF-κB Pathways.

Epithelial to mesenchymal transition (EMT) is a process by which an epithelial cell alters its phenotype to that of a mesenchymal cell and plays a critical role in embryonic development, tumour invasion and metastasis and tissue fibrosis. Transforming growth factor-ß1 (TGF-ß1) continues to be regarded as the key growth factor involved in driving EMT however recently tumour necrosis factor α (TNFα) has been demonstrated to accentuate TGF-ß1 driven EMT. In this study we investigate how various signalling pathways contribute to this accentuated effect. A549 cells were treated with TGF-ß1 (10 ng/ml), TNFα (20 ng/ml) or a combination of both for 72 h and EMT assessed. The effect of selective inhibition of the SMAD, MAPK and NF-κB pathways on EMT was assessed. A549 cells treated with TGF-ß1 downregulate the expression of epithelial markers, increase the expression of mesenchymal markers, secrete matrix-metalloproteinases and become invasive. Significantly, TGF-ß1 driven EMT is accentuated by co-treatment with TNFα. SMAD 3 inhibition attenuated TGF-ß1 driven EMT but has no effect on the accentuation effect of TNFα. However, inhibiting IKKß blocked both TGF-ß1 driven EMT and the accentuating action of TNFα. Inhibiting p38 and ERK signalling had no effect on EMT. TNFα accentuates TGF-ß1 driven EMT in A549 cells via a SMAD 2/3 independent mechanism involving the NF-κB pathway independent of p38 and ERK 1/2 activation.

Is CFTR-delF508 really absent from the apical membrane of the airway epithelium?

BACKGROUND: Understanding where mutant CFTR is localised in airway epithelia is essential in guiding the best therapeutic approach to correct the dysfunction of the CFTR protein. The widely held paradigm is that CF patients harbouring the commonest mutation, CFTR-delF508, trap CFTR within the endoplasmic reticulum and target it for degradation. However there are conflicting reports concerning expression and localisation of CFTR-delF508 in lung tissue. To attempt to resolve this fundamental issue we developed a novel approach to measure CFTR-delF508 in the lower airways of patients who have undergone lung transplantation for advanced CF. By sampling CF and non-CF epithelium simultaneously from the same individual, confounding factors of different airway microenvironments which may have influenced previous observations can be overcome. METHODS: Epithelia sampled by bronchial brushing above (CF) and below (non-CF) the bronchial anastomosis were stained for CFTR and the localisation and level of expression assessed (n = 12). RESULTS: There was no significant difference in the proportion of tall columnar cells showing CFTR immunostaining as a discrete band at the apical membrane in cells harbouring the CFTR-delF508 mutation compared to non-CF cells (p = 0.21, n = 12). However, the amount of CFTR expressed at the apical surface was reduced by ∼50% in CF cells compared to non-CF cells (p = 0.04, n = 5). CONCLUSIONS: Our novel observation challenges the prevailing paradigm that CFTR is essentially absent from the apical membrane of respiratory cells harbouring the CFTR-delF508 mutation. Moreover, it raises the possibility that the new generation of CFTR potentiators may offer a realistic therapeutic option for CF patients.

Lung epithelial wound healing in health and disease.

Physiological lung epithelial wound repair is a complex, highly orchestrated process presenting numerous points where dysregulation may occur, leading to the development of several pulmonary disorders. Current studies are limited by a lack of relevant lung injury models, with much work relying on other organ models such as the skin or in vitro cultures. However, much promising investigative work is being undertaken, some of which is described in this article. This article attempts to describe the processes required to heal a severe wound to the airway epithelium, characteristic of several chronic pulmonary disorders, highlighting areas where dysregulation may occur, which in turn leads to the development or continuation of a disease state.

The role of doxorubicin in non-viral gene transfer in the lung.

Proteasome inhibitors have been shown to increase adeno-associated virus (AAV)-mediated transduction in vitro and in vivo. To assess if proteasome inhibitors also increase lipid-mediated gene transfer with relevance to cystic fibrosis (CF), we first assessed the effects of doxorubicin and N-acetyl-l-leucinyl-l-leucinal-l-norleucinal in non-CF (A549) and CF (CFTE29o-) airway epithelial cell lines. CFTE29o- cells did not show a response to Dox or LLnL; however, gene transfer in A549 cells increased in a dose-related fashion (p < 0.05), up to approximately 20-fold respectively at the optimal dose (no treatment: 9.3 x 10(4) +/- 1.5 x 10(3), Dox: 1.6 x 10(6)+/-2.6 x 10(5), LLnL: 1.9 x 10(6) +/- 3.2 x 10(5)RLU/mg protein). As Dox is used clinically in cancer chemotherapy we next assessed the effect of this drug on non-viral lung gene transfer in vivo. CF knockout mice were injected intraperitoneally (IP) with Dox (25-100 mg/kg) immediately before nebulisation with plasmid DNA carrying a luciferase reporter gene under the control of a CMV promoter/enhancer (pCIKLux) complexed to the cationic lipid GL67A. Dox also significantly (p < 0.05) increased expression of a plasmid regulated by an elongation factor 1alpha promoter (hCEFI) approximately 8-fold. Although administration of Dox before lung gene transfer may not be a clinically viable option, understanding how Dox increases lung gene expression may help to shed light on intracellular bottle-necks to gene transfer, and may help to identify other adjuncts that may be more appropriate for use in man.