We report gene transfer to the Edinburgh insertional mutant mouse (cf/cf), delivering CFTR cDNA-liposome complexes into the airways by nebulization. We show full restoration of cAMP related chloride responses in some animals and demonstrate, in the same tissues, human CFTR cDNA expression. Overall, a range of correction was seen with restoration of about 50% of the deficit between wild type mice and untreated cf/cf controls. We report modest correction in the intestinal tract following direct instillation and provide initial encouraging safety data for both the respiratory and intestinal tract following the liposome mediated gene delivery. The non-viral nature and potentially lower immunogenicity of DNA-liposomes suggest that this may offer a therapeutic alternative to adenoviral therapies.

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Background-Transforming growth factor 1 is implicated in the pathogenesis of lung fibrosis. It promotes extracellular matrix accumulation by increasing procollagen synthesis and reducing degradation. TGF 1 gene and protein expression increase in experimental lung fibrosis, and TGF 1 antibodies attenuate fibrosis in mice. The role of other TGF isoforms is unclear. This study aimed to localise TGF 1 and TGF 3 gene expression in fibrotic human lung and compare it with that in normal human lung. Methods-Lung tissue from patients with cryptogenic fibrosing alveolitis and fibrosis associated with systemic sclerosis was examined by in situ hybridisation. Macroscopically normal lung from carcinoma resections was used as control tissue. Digoxigenin labelled riboprobes were synthesised from TGF isoform specific cDNA templates. Results-The digoxigenin labelled riboprobes were sensitive and permitted precise cellular localisation of mRNA transcripts. TGF 1 and TGF 3 mRNA transcripts were widespread in normal lung and localised to alveolar macrophages and bronchiolar epithelium. TGF 1 but not TGF 3 mRNA was detected in mesenchymal and endothelial cells. In fibrotic lung tissue mRNA transcripts for both isoforms were also detected in metaplastic type II cells. TGF 1 gene expression was enhanced in some patients. TGF 3 was expressed in fibrotic lung but was not consistently altered compared with controls. Conclusion-TGF 1 mRNA transcripts were localised in normal and fibrotic human lung and TGF 3 gene expression in human lung fibrosis was shown for the first time. The results suggest that TGF 1 may play the predominant role in pathogenesis. It is suggested that TGF 1 should be the primary target of anticytokine treatments for pulmonary fibrosis. (Thorax 2001;56:549-556)

D Di iv ve er rs se e c ce el ll lu ul la ar r T TG GF F--β 1 1 a an nd d T TG GF F--β 3 3 g ge en ne e e ex xp pr re es ss si io on n i in n n no or rm ma al l h hu um ma an n a an nd d m mu ur ri in ne e l lu un ng g This procedure was technically simple, providing excellent resolution. TGF-β 1 and TGF-β 3 messenger ribonucleic acid (mRNA) transcripts were detected in a wide variety of cells. In human lung, mRNA for both isoforms was localized to bronchiolar epithelium and alveolar macrophages. TGF-β 1 , but not TGF-β 3 mRNA was detected in mesenchymal and endothelial cells. In murine tissue, TGF-β 1 , mRNA was localized to bronchiolar epithelium, Clara cells, mesenchymal cells, pulmonary endothelium and alveolar cells, including macrophages. TGF-β 3 mRNA was similarly distributed but not detected in endothelium.In summary, using a nonisotopic technique in lung tissue, we have detailed the cells expressing the transforming growth factor-β 1 and β 3 genes in human and murine lung. There was widespread expression of these cytokines in normal lung consistent with autocrine or paracrine roles in regulating cellular turnover, immune defence and matrix protein metabolism.

Streptococcus pneumoniae infections are common, but how they cause host tissue injury and death is incompletely understood. Immunization with pneumolysin, a thiol-activated toxin produced by the pneumococcus, partially protects animals during subsequent infection. The mechanism by which pneumolysin contributes to disease is not known. The aim of the present investigation was to determine the histologic changes induced by recombinant pneumolysin in the rat lung and to compare them with the changes induced by live organisms. Injection of either toxin (200 or 800 ng) or bacteria into the apical lobe bronchus was associated with the development of a severe lobar pneumonia restricted to the apical lobe. The changes induced by the toxin were greater at the higher concentration, and changes were most severe in those animals in which there was partial ligation of the apical lobe bronchus. The pneumonitis was less severe following injection of a modified toxin with decreased hemolytic activity, generated by site-directed mutagenesis of the cloned pneumolysin gene, indicating that this property of the toxin was important in generating pulmonary inflammation. There was still considerable pneumonitis after injection of a modified toxin with decreased capacity to activate complement.