We describe the use of cationic, pH-sensitive liposomes to mediate the efficient transfer of DNA into a variety of cells in culture. Cationic lipids, containing an amine with a pK within the physiologic range of 4.5 to 8, were synthesized and incorporated with dioleoylphosphatidylethanolamine into liposomes. Acid conditions promoted DNA-binding, DNA-incorporation, and DNA-induced fusion by these cationic, pH-sensitive liposomes. Transfection efficiency in cultured cells was dependent on endosomal acidification in a manner akin to acidic-induced endosomal release of viruses. These liposomes constitute a promising new class of reagents for gene therapy.

Mycoplasma synoviae is a major pathogen of poultry, causing synovitis and respiratory infection. A cluster of 45-to 50-kDa membrane proteins is immunodominant in strain WVU-1853. Four distinct proteins were identified in this cluster by high-pressure liquid chromatography. Monoclonal antibodies and monospecific antisera against each established that they fell into two groups, MSPA and MSPB, each containing two members distinguishable by a difference in hydrophobicity. A 25-to 30-kDa membrane protein (MSPC) was shown to be antigenically related to the MSPB proteins. Considerable variation in the size and expression of MSPA and MSPB was observed among different strains of M. synoviae. Examination of expression in colonies of strain WVU-1853 established that both MSPA and MSPB (and MSPC) were phase variable. Immunostaining of MSPB (and MSPC) with monoclonal antibodies exhibited quantal variation, with three distinct levels observed between and within colonies. Hemadsorption by M. synoviae colonies was also found to be phase variable, with some colonies exhibiting sectorial expression of hemadsorption. Monospecific antisera against MSPA inhibited hemagglutination, but neither monoclonal antibodies nor monospecific antisera against MSPB could inhibit hemagglutination. However, loss of the capacity to hemadsorb by individual clones was associated with loss of expression of both MSPA and MSPB. These findings have elucidated the complexity of structure, function, and expression of the 45-to 50-kDa membrane protein cluster of M. synoviae, and they suggest that all members of the cluster may be involved in adhesion.

The implantation of cells genetically modified to express tyrosine hydroxylase has been proposed for the treatment of Parkinson's disease. Tyrosine hydroxylase converts tyrosine to L-DOPA and endogenous decarboxylase activity then converts L-DOPA to the neurotransmitter dopamine, which alleviates the symptoms of Parkinson's disease. Immortalized cells have been successfully used as intracerebral vehicles for transgene expression of tyrosine hydroxylase, but the tumorigenic potential of these cells prevents their application in humans. Intracerebral expression of this enzyme has also been achieved using primary cells like skin fibroblasts, but the ameliorating effect on a rat model for Parkinson's disease lasted for only a few weeks. We have found that co-transplantation of cultured myoblasts and myotubes enabled reporter genes to be expressed intracerebrally at high and stable levels. Here we show that the intracerebral transplantation of plasmid-transfected primary muscle cells can substantially reduce for the long-term the asymmetric rotational behaviour in the rat model.

1. Nitric oxide (NO) has important roles in physiological vasodilatation, cytotoxicity and vascular disease. Nitric oxide and prostacyclin (PGIz), both released from the endothelium, act synergistically to inhibit platelet aggregation and adhesion. These autacoids also inhibit the adhesion and migration of leucocytes and, in some arteries, they synergize in terms of vasodilatation.2. The development of atherosclerosis and hyperlipaemiaper se is accompanied by impairment of endothelium-dependent vasodilatation.3. Atherosclerosis is associated with marked changes in the activity of isoforms of NO synthase (NOS) in the artery wall, including increased expression of the NOS2 (inducible) isoform in complex human lesions as well as in the neointima of experimental animal models.4. Failure of NO release from the endothelium with normal physiological stimuli, which has been attributed to a defect in the operation of the endothelial NOS (NOS3), provides conditions propitious for leucocyte adhesion, vasospasm, thrombosis and, in addition, may promote increased proliferation of intimal cells.5. Nitric oxide and superoxide anions generated by inflammatory cells in atherosclerosis react to form cytodestructive peroxynitrite radicals, potentially causing injury to the endothelium and myocytes, and this may be a factor in apoptosis of cells leading to plaque rupture. 6. We have been able to reverse these NO defects with therapeutic agents, including angiotensin-converting enzyme inhibitors, antagonists of platelet-activating factor and NO donor compounds, all offering promise in protecting against some manifestations of vascular disease.