Noel G. McElvaney

Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis.

We have administered a recombinant adenovirus vector (AdCFTR) containing the normal human CFTR cDNA to the nasal and bronchial epithelium of four individuals with cystic fibrosis (CF). We show that this vector can express the CFTR cDNA in the CF respiratory epithelium in vivo. With doses up to 2 × 109 pfu, there was no recombination/complementation or shedding of the vector or rise of neutralizing antibody titres. At 2 × 109 pfu, a transient systemic and pulmonary syndrome was observed, possibly mediated by interleukin-6. Follow-up at 6–12 months demonstrated no long term adverse effects. Thus, it is feasible to use an adenovirus vector to transfer and express the CFTR cDNA in the respiratory epithelium of individuals with CF. Correction of the CF phenotype of the airway epithelium might be achieved with this strategy.

Neutrophil elastase in respiratory epithelial lining fluid of individuals with cystic fibrosis induces interleukin-8 gene expression in a human bronchial epithelial cell line.

The respiratory manifestations of cystic fibrosis (CF) are characterized by neutrophil-dominated airway inflammation. Since a variety of inflammatory stimuli are capable of inducing bronchial epithelial cells to express the gene for IL-8, a cytokine that attracts and activates neutrophils, mediators in respiratory epithelial lining fluid (ELF) of CF individuals might induce IL-8 production by epithelial cells, thus recruiting neutrophils to the airways. BET-1A human bronchial epithelial cells at rest or incubated with normal ELF showed little IL-8 gene expression, but after incubation with CF ELF, a marked increase in IL-8 transcript levels was observed. CF ELF contained high levels of neutrophil elastase (NE) and various serine protease inhibitors prevented CF ELF from inducing IL-8 gene expression in BET-1A cells, suggesting that NE was the dominant inducer for IL-8 production in CF ELF. The addition of purified NE caused BET-1A cells to increase IL-8 gene transcription with accumulation of mRNA transcripts and to release IL-8-like neutrophil chemotactic activity. These observations suggest a self-perpetuating inflammatory process on the CF bronchial surface where NE released by neutrophils induced the bronchial epithelium to secrete IL-8, which in turn recruits additional neutrophils to the bronchial surface.

Aerosol α1 -antitrypsin treatment for cystic fibrosis

Abstract In cystic fibrosis neutrophil-dominated inflammation on the respiratory epithelial surface results in a chronic epithelial burden of the destructive enzyme, neutrophil elastase. α1-antitrypsin (α1AT), the main inhibitor of neutrophil elastase in lung, was given in aerosol form to 12 cystic fibrosis patients. It suppressed neutrophil elastase in the respiratory epithelial lining fluid (ELF) and restored the ELF anti-neutrophil elastase capacity when ELF α1AT reached 8 μmol/l. This treatment also reversed the inhibitory effect of cystic fibrosis ELF on pseudomonas killing by neutrophils, which suggests that it may augment host defence in cystic fibrosis.

Modulation of airway inflammation in cystic fibrosis. In vivo suppression of interleukin-8 levels on the respiratory epithelial surface by aerosolization of recombinant secretory leukoprotease inhibitor.

Based on the knowledge that neutrophil elastase (NE) in cystic fibrosis (CF) epithelial lining fluid (ELF) can induce human bronchial epithelial cells to express the gene for interleukin 8 (IL-8), an 8.5-kD neutrophil chemoattractant, we have evaluated CF ELF for the presence of IL-8, and investigated the ability of aerosolized recombinant secretory leukoprotease inhibitor (rSLPI) to suppress NE, and hence IL-8, levels on the respiratory epithelial surface in CF. Enzyme-linked immunoassay revealed 21.9 +/- 4.8 nM IL-8 in CF ELF compared with none in normals. Active NE was detectable in ELF of all individuals with CF and was significantly decreased (P < 0.03) after aerosolization of rSLPI. Human bronchial epithelial cells exposed to CF ELF recovered before rSLPI therapy expressed IL-8 mRNA transcripts, but ELF recovered after rSLPI therapy induced far less bronchial epithelial cell IL-8 gene expression. Consistent with this, rSLPI aerosol therapy caused a marked reduction in CF ELF IL-8 levels (P < 0.05) and neutrophil number (P < 0.02). There was also a clear association between CF ELF active NE and IL-8 levels (r = 0.94). These data suggest that rSLPI therapy not only suppresses respiratory epithelial NE levels, but also breaks a cycle of inflammation on the CF epithelial surface.

Neutrophil elastase up-regulates interleukin-8 via toll-like receptor 4.

Cystic fibrosis is characterised in the lungs by high levels of neutrophil elastase (NE). NE induces interleukin‐8 (IL‐8) expression via an IL‐1 receptor‐associated kinase signalling pathway. Here, we show that these events involve the cell surface membrane bound toll‐like receptor 4 (TLR4). We demonstrate that human embryonic kidney (HEK)293 cells transfected with a TLR4 cDNA (HEK‐TLR4) express TLR4 mRNA and protein and induce IL‐8 promoter activity in response to NE. Treatment of both HEK‐TLR4 and human bronchial epithelial cells with NE decreases TLR4 protein expression. Furthermore, a TLR4 neutralising antibody abrogates NE‐induced IL‐8 production, and induces tolerance to a secondary lipopolysaccharide stimulus. These data implicate TLR4 in NE induced IL‐8 expression in bronchial epithelium.

TLR-Induced Inflammation in Cystic Fibrosis and Non-Cystic Fibrosis Airway Epithelial Cells

Cystic fibrosis (CF) is a genetic disease characterized by severe neutrophil-dominated airway inflammation. An important cause of inflammation in CF is Pseudomonas aeruginosa infection. We have evaluated the importance of a number of P. aeruginosa components, namely lipopeptides, LPS, and unmethylated CpG DNA, as proinflammatory stimuli in CF by characterizing the expression and functional activity of their cognate receptors, TLR2/6 or TLR2/1, TLR4, and TLR9, respectively, in a human tracheal epithelial line, CFTE29o−, which is homozygous for the ΔF508 CF transmembrane conductance regulator mutation. We also characterized TLR expression and function in a non-CF airway epithelial cell line 16HBE14o−. Using RT-PCR, we demonstrated TLR mRNA expression. TLR cell surface expression was assessed by fluorescence microscopy. Lipopeptides, LPS, and unmethylated CpG DNA induced IL-8 and IL-6 protein production in a time- and dose-dependent manner. The CF and non-CF cell lines were largely similar in their TLR expression and relative TLR responses. ICAM-1 expression was also up-regulated in CFTE29o− cells following stimulation with each agonist. CF bronchoalveolar lavage fluid, which contains LPS, bacterial DNA, and neutrophil elastase (a neutrophil-derived protease that can activate TLR4), up-regulated an NF-κB-linked reporter gene and increased IL-8 protein production in CFTE29o− cells. This effect was abrogated by expression of dominant-negative versions of MyD88 or Mal, key signal transducers for TLRs, thereby implicating them as potential anti-inflammatory agents for CF.

Phase 2 Clinical Trial of a Recombinant Adeno-Associated Viral Vector Expressing α1-Antitrypsin: Interim Results

Recombinant adeno-associated virus (rAAV) vectors offer promise for the gene therapy of α(1)-antitrypsin (AAT) deficiency. In our prior trial, an rAAV vector expressing human AAT (rAAV1-CB-hAAT) provided sustained, vector-derived AAT expression for >1 year. In the current phase 2 clinical trial, this same vector, produced by a herpes simplex virus complementation method, was administered to nine AAT-deficient individuals by intramuscular injection at doses of 6.0×10(11), 1.9×10(12), and 6.0×10(12) vector genomes/kg (n=3 subjects/dose). Vector-derived expression of normal (M-type) AAT in serum was dose dependent, peaked on day 30, and persisted for at least 90 days. Vector administration was well tolerated, with only mild injection site reactions and no serious adverse events. Serum creatine kinase was transiently elevated on day 30 in five of six subjects in the two higher dose groups and normalized by day 45. As expected, all subjects developed anti-AAV antibodies and interferon-γ enzyme-linked immunospot responses to AAV peptides, and no subjects developed antibodies to AAT. One subject in the mid-dose group developed T cell responses to a single AAT peptide unassociated with any clinical effects. Muscle biopsies obtained on day 90 showed strong immunostaining for AAT and moderate to marked inflammatory cell infiltrates composed primarily of CD3-reactive T lymphocytes that were primarily of the CD8(+) subtype. These results support the feasibility and safety of AAV gene therapy for AAT deficiency, and indicate that serum levels of vector-derived normal human AAT >20 μg/ml can be achieved. However, further improvements in the design or delivery of rAAV-AAT vectors will be required to achieve therapeutic target serum AAT concentrations.

Association of IL-10 polymorphism with severity of illness in community acquired pneumonia.

Background: The influence of genetic polymorphisms of interleukin (IL)-10, tumour necrosis factor (TNF)-α, and IL-6 gene promoters on severity of systemic inflammatory response syndrome (SIRS) associated with community acquired pneumonia (CAP) was studied. Methods: Using PCR-RFLP analysis we analysed a −1082G/A single nucleotide polymorphism (SNP) of the anti-inflammatory IL-10 gene, a −308G/A SNP of the pro-inflammatory TNF-α gene and a −174G/C SNP of the IL-6 gene. Illness severity was stratified according to SIRS score, calculated by presence of up to four physiological indices: temperature, white blood cell count, heart rate and respiratory rate (non-SIRS, SIRS 2, SIRS 3, and SIRS 4). Results: A statistically significant stepwise increase in frequency of the IL-10 G allele, associated with higher expression of the gene, was observed in patients with increasing severity of illness from non-SIRS (n=19) to SIRS 2 (n=17), SIRS 3 (n=33) and SIRS 4 (n=24). This was primarily due to a higher frequency of the GG genotype with increasing severity from non-SIRS through to SIRS 4. IL-10 G allele frequency was also increased in patients who died as a result of CAP (n=11) compared with CAP survivors (n=82) (p=0.01). No association was seen between the TNF-α −308G/A and IL-6 −174G/C SNPs and disease. Additionally, no interaction between all three SNP genotypes and disease severity was observed. Conclusions: A polymorphism affecting IL-10 expression may influence the severity of illness in patients with CAP.

Inactivation of Human β-Defensins 2 and 3 by Elastolytic Cathepsins

β-Defensins are antimicrobial peptides that contribute to the innate immune responses of eukaryotes. At least three defensins, human β-defensins 1, 2, and 3 (HBD-1, -2, and -3), are produced by epithelial cells lining the respiratory tract and are active toward Gram-positive (HBD-3) and Gram-negative (HBD-1, -2, and -3) bacteria. It has been postulated that the antimicrobial activity of defensins is compromised by changes in airway surface liquid composition in lungs of patients with cystic fibrosis (CF), therefore contributing to the bacterial colonization of the lung by Pseudomonas and other bacteria in CF. In this report we demonstrate that HBD-2 and HBD-3 are susceptible to degradation and inactivation by the cysteine proteases cathepsins B, L, and S. In addition, we show that all three cathepsins are present and active in CF bronchoalveolar lavage. Incubation of HBD-2 and -3 with CF bronchoalveolar lavage leads to their degradation, which can be completely (HBD-2) or partially (HBD-3) inhibited by a cathepsin inhibitor. These results suggest that β-defensins are susceptible to degradation and inactivation by host proteases, which may be important in the regulation of β-defensin activity. In chronic lung diseases associated with infection, overexpression of cathepsins may lead to increased degradation of HBD-2 and -3, thereby favoring bacterial infection and colonization.

Activation of Endoplasmic Reticulum-Specific Stress Responses Associated with the Conformational Disease Z α1-Antitrypsin Deficiency

Conformational diseases are a class of disorders associated with aberrant protein accumulation in tissues and cellular compartments. Z α1-antitrypsin (A1AT) deficiency is a genetic disease associated with accumulation of misfolded A1AT in the endoplasmic reticulum (ER) of hepatocytes. We sought to identify intracellular events involved in the molecular pathogenesis of Z A1AT-induced liver disease using an in vitro model system of Z A1AT ER accumulation. We investigated ER stress signals induced by Z A1AT and demonstrated that both the ER overload response and the unfolded protein response were activated by mutant Z A1AT, but not wild-type M A1AT. Interestingly, activation of the unfolded protein response pathway required an additional insult, whereas NF-κB activation, a hallmark of the ER overload response, was constitutive. These findings have important implications for the design of future therapeutics for Z A1AT liver disease and may also impact on drug design for other conformational diseases.