Stephen L. Hart

Cyclooxygenase-2 Deficiency Results in a Loss of the Anti-Proliferative Response to Transforming Growth Factor-β in Human Fibrotic Lung Fibroblasts and Promotes Bleomycin-Induced Pulmonary Fibrosis in Mice

Prostaglandin E(2) (PGE(2)) inhibits fibroblast proliferation and collagen production. Its synthesis by fibroblasts is induced by profibrotic mediators including transforming growth factor (TGF)-beta(1). However, in patients with pulmonary fibrosis, PGE(2) levels are decreased. In this study we examined the effect of TGF-beta(1) on PGE(2) synthesis, proliferation, collagen production, and cyclooxygenase (COX) mRNA levels in fibroblasts derived from fibrotic and nonfibrotic human lung. In addition, we examined the effect of bleomycin-induced pulmonary fibrosis in COX-2-deficient mice. We demonstrate that basal and TGF-beta(1)-induced PGE(2) synthesis is limited in fibroblasts from fibrotic lung. Functionally, this correlates with a loss of the anti-proliferative response to TGF-beta(1). This failure to induce PGE(2) synthesis is because of an inability to up-regulate COX-2 mRNA levels in these fibroblasts. Furthermore, mice deficient in COX-2 exhibit an enhanced response to bleomycin. We conclude that a decreased capacity to up-regulate COX-2 expression and COX-2-derived PGE(2) synthesis in the presence of increasing levels of profibrotic mediators such as TGF-beta(1) may lead to unopposed fibroblast proliferation and collagen synthesis and contribute to the pathogenesis of pulmonary fibrosis.

In-vitro activity of S. lavandulaefolia (Spanish sage) relevant to treatment of Alzheimer's disease

Salvia lavandulaefolia Vahl. (Spanish sage) essential oil and individual monoterpenoid constituents have been shown to inhibit the enzyme acetylcholinesterase in‐vitro and in‐vivo. This activity is relevant to the treatment of Alzheimers disease, since anticholinesterase drugs are currently the only drugs available to treat Alzheimers disease. Other activities relevant to Alzheimers disease include antioxidant, anti‐inflammatory and estrogenic effects. Results of in‐vitro tests for these activities are reported here for S. lavandulaefolia extracts, the essential oil and its major constituents. Antioxidant activity (inhibition of bovine brain liposome peroxidation) was found in the EtOH extract of the dried herb (5 mg mL−1) and the monoterpenoids (0.1 M) α‐ and β‐pinene and 1,8‐cineole. Thujone and geraniol had lower antioxidant effects, while camphor had no antioxidant effects. Possible anti‐inflammatory activity (eicosanoid inhibition in rat leucocytes) was found in the EtOH extract (50 μg mL−1) and was shown by the monoterpenoids α‐pinene and geraniol (0.2 mM), but not 1,8‐cineole, thujone or camphor. Possible estrogenic activity (via induction of β‐galactosidase activity in yeast cells) was found in the essential oil (0.01 mg mL−1) and the monoterpenoid geraniol (0.1–2 mM). 1,8‐Cineole, α‐ and β‐pinene and thujone did not exhibit estrogenic activity in this analysis. These results demonstrate that S. lavandulaefolia, its essential oil and some chemical constituents have properties relevant to the treatment of Alzheimers disease and provide further data supporting the value of carrying out clinical studies in patients with Alzheimers disease using this plant species.

Severity of Lung Injury in Cyclooxygenase-2- Deficient Mice Is Dependent on Reduced Prostaglandin E 2 Production

Levels of prostaglandin E(2) (PGE(2)), a potent inhibitor of fibroblast function, are decreased in the lungs of patients with pulmonary fibrosis, which has been shown to be because of limited expression of cyclooxygenase-2 (COX-2). To further investigate the relative importance of COX-2 and PGE(2) in the development of fibrosis we have used a selective COX-2 inhibitor and COX-2-deficient ((-/-) and (+/-)) mice in studies of bleomycin-induced lung fibrosis. We demonstrate in wild-type mice that bleomycin-induced lung PGE(2) production is predominantly COX-2 mediated. Furthermore, COX-2(+/-) mice show limited induction of PGE(2) and an enhanced fibrotic response with increased lung collagen content compared with wild-type mice after bleomycin injury (P < 0.001). In contrast, COX-2(-/-) mice show increased levels of lung PGE(2), compared with wild-type mice after injury (P < 0.05), because of compensatory up-regulation of COX-1, which appears to be associated with macrophage/monocytes but not fibroblasts derived from these mice. COX-2(-/-) mice show an enhanced and persistent inflammatory response to bleomycin, however the fibrotic response to injury was unaltered compared with wild-type animals. These data provide further direct evidence for the importance of up-regulating COX-2 and PGE(2) expression in protecting against the development of fibrosis after lung injury.

Integrin-mediated transfection with peptides containing arginine-glycine-aspartic acid domains.

Two synthetic peptides comprising an RGD moiety for integrin binding and a polylysine moiety for DNA binding were tested for transfection efficiency under a variety of different conditions. Binding of target cells to the peptide was shown to be strongly dependent on cyclisation of the peptides via cysteine residues. Low (10 μ M) concentrations of chloroquine, added to assist endocytic exit, unexpectedly reduced transfection efficiency in two of the cell lines tested, COS-7 and ECV304. However, transfection efficiency increased at higher chloroquine concentrations and exceeded that in the absence of chloroquine in the case of the COS-7 and A375M cell lines. With the ECV304 cell line, optimum transfection occurred in the absence of chloroquine. Transfection efficiency of the peptides was greatest at peptide:DNA ratios of 4:1 (w/w), which were calculated to generate complexes containing approximately 5000 peptide molecules per plasmid. This represented approximately a 6:1 ratio of positive to negative charges. Peptide 5 was shown to have a higher transfection efficiency under most conditions, possibly because of more efficient stabilisation of cyclisation by two cysteine–cysteine bonds.

An integrin-targeted non-viral vector for pulmonary gene therapy.

Gene therapy offers potential for the treatment of severe respiratory diseases. However, the vectors which are currently available have drawbacks limiting their therapeutic application. Here we report on an integrin-targeted, non-viral gene delivery system for pulmonary gene transfer. We demonstrate that this vector can deliver the lacZ reporter gene to the lung, transfecting bronchial epithelium and parenchymal cells with similar efficiency to an adenoviral vector and with greater efficiency than a cationic liposome. In addition, vector administration can be repeated leading to further gene expression without inducing inflammation. The advantages of this novel gene delivery system provide considerable potential for targeted gene therapy in vivo.

Pharmacological and antimicrobial studies on different tea‐tree oils (Melaleuca alternifolia, Leptospermum scoparium or Manuka and Kunzea ericoides or Kanuka), originating in Australia and New Zealand

Three different species of Myrtaceae growing in Australia and New Zealand are known as ‘Tea‐tree’: the Australian Tea tree (Melaleuca alternifolia), the New Zealand Manuka (Leptospermum scoparium) and Kanuka (Kunzea ericoides). All three essential oils are used by aromatherapists, although only Melaleuca has been tested for toxicity, and its antimicrobial effects studied. The pharmacology and antimicrobial activity of the three ‘tea‐tree’ oils was determined using guinea‐pig ileum, skeletal muscle (chick biventer muscle and the rat phrenic nerve diaphragm) and also rat uterus in vitro. Differences were shown between the three essential oils in their action on smooth muscle: Manuka had a spasmolytic action, while Kanuka and Melaleuca had an initial spasmogenic action. Using the diaphragm, Manuka and Melaleuca decreased the tension and caused a delayed contracture; Kanuka had no activity at the same concentration. The action on chick biventer muscle was, however, similar for all three oils, as was the action on the uterus, where they caused a decrease in the force of the spontaneous contractions. The latter action suggests caution in the use of these essential oils during childbirth, as cessation of contractions could put the baby, and mother, at risk. The comparative antimicrobial activity showed greater differences between different samples of Manuka and Kanuka than Melaleuca samples. The antifungal activity of Kanuka was inversely proportional to its strong antibacterial activity, whilst Manuka displayed a stronger antifungal effect, though not as potent as Melaleuca. The antioxidant activity of Manuka samples was more consistent than that of Kanuka, while Melaleuca showed no activity. The variability in the Manuka and Kanuka essential oils suggests caution in their usage, as does the fact that the oils have not been tested for toxicity.

High Yield Incorporation of Plasmid DNA within Liposomes: Effect on DNA Integrity and Transfection Efficiency

Effective use of liposomes in gene therapy requires high yield incorporation of nucleic acids within vesicles which protect their content from nuclease attack and facilitate transfection: To that end, pGL2 plasmid DNA (3.99 x 10(6) Daltons) expressing the luciferase reporter gene was incorporated quantitatively (40-92% of the DNA used) by a mild procedure into neutral and negatively or positively charged multilamellar liposomes which offered considerable protection from deoxyribonuclease attack. Smaller vesicles (210-383 nm diameter) produced from such liposomes, retained much of the original content of DNA which was still significantly inaccessible to the enzyme. Liposomal plasmid DNA was found to retain its structural integrity and to transfect cells in vitro in relation to the size and surface charge of the vesicles. Such DNA-incorporating liposome constructs could prove effective for plasmid DNA expression in vivo.

A nonviral vector system for efficient gene transfer to corneal endothelial cells via membrane integrins.

BACKGROUND Genetic manipulation of allografts to suppress their ability to induce rejection is a promising approach for controlling rejection responses. A key to this approach is the development of appropriate DNA vectors. We are developing nonviral DNA vector systems based on synthetic peptides containing an integrin-binding segment for cellular targeting and a polylysine segment for DNA binding. METHODS Two such peptides have been tested for their ability to deliver the beta-galactosidase reporter gene to the corneal endothelial cells of the rabbit, pig, and man. One peptide was derived from a phage display library, the other from the integrin-binding moiety of the toxin from the American pit viper, Crotalus molossus molossus. Corneas were cultured overnight and then exposed to the DNA/peptide vector under a variety of conditions involving different DNA concentrations, chloroquine concentrations, times of exposure, presence of serum, and presence of polyanion buffers. Expression of the beta-galactosidase gene was determined after 3 additional days in culture. Effects of the treatment on the viability of the endothelium were examined by confocal microscopy. RESULTS We report that approximately 30% of corneal endothelial cells can be transfected with our optimal protocol using the molossin-based vector. Transfection is dependent on the presence of chloroquine and is inhibited by polyanion buffers such as HEPES. Viability of the corneal endothelium was excellent, except if corneas were incubated at high concentrations of chloroquine (0.5 mM) for prolonged periods (24 hr). CONCLUSIONS Synthetic peptides containing both an integrin targeting and a DNA-binding moiety are promising as simple and highly versatile DNA vectors for use in corneal transplantation.

A preliminary study of the effect of essential oils on skeletal and smooth muscle in vitro

The pharmacological activity of nine commercial essential oils was studied on the rat isolated phrenic nerve diaphragm preparation and compared with activity on field-stimulated guinea-pig ileum preparations. The essential oils at final bath concentrations of 2 x 10(-5) and 2 x 10(-4) g/ml produced four different effects on skeletal muscle, whilst only a contracture with or without a decrease in response to field stimulation in smooth muscle. The first type of effect on skeletal muscle involved a contracture and inhibition of the twitch response to nerve stimulation shown by a sample of clary sage, dill, fennel, frankincense and nutmeg; a second, shown by thyme produced a contracture without a change in the twitch response; a third, shown by lavender reduced the twitch response alone and the fourth, shown by camphor, increased the size of the twitch response. Angelica root oil at the highest concentration studied showed no response on skeletal muscle.

Transbronchial biopsies provide longitudinal evidence for epithelial chimerism in children following sex mismatched lung transplantation

Background: Recent reports have shown evidence of host derived parenchymal engraftment in several human allografts including the lung, leading to speculation that stem cell therapy may be useful for lung repair in diseases such as cystic fibrosis (CF). To date, previous studies have looked at single surgical or autopsy specimens and no longitudinal studies have been reported. The aim of this study was to assess whether transbronchial biopsies could be used to study the time course of chimerism following lung transplantation. Methods: Specimens of archived transbronchial lung biopsies from five time points taken for clinical purposes from two boys who had received a sex mismatched heart-lung transplant for end stage CF were examined. Sections were dual stained for cytokeratin (epithelium) and a mixture of leucocyte common antigen and CD68 for inflammatory cells. Co-localisation of cells containing a Y chromosome was confirmed by fluorescent in situ hybridisation. Results: Evidence of chimerism was found in up to 6.6% of epithelial cells in bronchial (median 1.4% (range 0–6.6)) and alveolar (median 3.6% (range 2.3–5.5) tissue without apparent evidence of fusion. This engraftment was seen as early as 3 weeks and remained relatively constant up to 37 months. Conclusions: This study has demonstrated proof of principle for long term chimerism in lung epithelium. Transbronchial biopsies may provide a new method for studying the kinetics of stem cell engraftment in the lung.