No abstract

A major hurdle to adenovirus (Ad)-mediated gene transfer is that the target tissue lacks sufficient levels of receptors to mediate vector attachment via its fiber coat protein. Endothelial and smooth muscle cells are primary targets in gene therapy approaches to prevent restenosis following angioplasty or to promote or inhibit angiogenesis. However, Ad poorly binds and transduces these cells because of their low or undetectable levels of functional Ad fiber receptor. The Ad-binding deficiency of these cells was overcome by targeting Ad binding to ␣ v integrin receptors that are sufficiently expressed by these cells. In order to target ␣ v integrins, a bispecific antibody (bsAb) that comprised a monoclonal Ab to the FLAG peptide epitope, DYKDDDDK, and a monoclonal Ab to ␣ v integrins was constructed. In conjunction with the bsAb, a new vector, AdFLAG, which incorporated the FLAG peptide epitope into its penton base protein was constructed. Complexing AdFLAG with the bsAb increased the ␤-glucuronidase transduction of human venule endothelial cells and human intestinal smooth muscle cells by seven-to ninefold compared with transduction by AdFLAG alone. The increased transduction efficiency was shown to occur through the specific interaction of the complex with ␣ v integrins. These results demonstrate that bsAbs can be successfully used to target Ad to a specific cellular receptor and thereby increase the efficiency of gene transfer.

Vascular endothelial growth factor (VEGF) is a critical stimulus for both retinal and choroidal neovascularization, and for diabetic macular edema. We used mouse models for these diseases to explore the potential of gene transfer of soluble VEGF receptor-1 (sFlt-1) as a treatment. Intravitreous or periocular injection of an adenoviral vector encoding sFlt-1 (AdsFlt-1.10) markedly suppressed choroidal neovascularization at rupture sites in Bruch's membrane. Periocular injection of AdsFlt-1.10 also caused significant reduction in VEGF-induced breakdown of the blood-retinal barrier, but failed to significantly inhibit ischemia-induced retinal neovascularization. Periocular delivery of an adenoviral vector encoding pigment epithelium-derived factor (PEDF), another secreted protein, resulted in high levels of PEDF in the retinal pigmented epithelium and choroid, but not in the retina. This may explain why periocular injection of AdsFlt-1.10 inhibited choroidal, but not retinal neovascularization. Periocular delivery offers potential advantages over other routes of delivery and the demonstration that sFlt-1 enters the eye from the periocular space in sufficient levels to achieve efficacy in treating choroidal neovascularization and retinal vascular permeability is a novel finding that has important clinical implications. These data suggest that periocular gene transfer of sFlt-1 should be considered for treatment of choroidal neovascularization and diabetic macular edema.

The lipopolysaccharide (LPS) of Bradyrhizobiumjaponicum 61A123 was isolated and partially characterized.Phenol-water extraction of strain 61A123 yielded LPS exclusively in the phenol phase. The water phase contained low-molecular-weight glucans and extracellular or capsular polysaccharides. The LPSs from B. japonicum 61A76, 61A135, and 61A1O1C were also extracted exclusively into the phenol phase. The LPSs from strain USDA 110 and its Nod-mutant HS123 were found in both the phenol and water phases. The LPS from strain 61A123 was further characterized by polyacrylamide gel electrophoresis, composition analysis, and 'H and 13C nuclear magnetic resonance spectroscopy. Analysis of the LPS by polyacrylamide gel electrophoresis showed that it was present in both high-and low-molecular-weight forms (LPS I and LPS II, respectively). Composition analysis was also performed on the isolated lipid A and polysaccharide portions of the LPS, which were purified by mild acid hydrolysis and gel filtration chromatography. The major components of the polysaccharide portion were fucose, fucosamine, glucose, and mannose. The intact LPS had small amounts of 2-keto-3-deoxyoctulosonic acid. Other minor components were quinovosamine, glucosamine, 4-0-methylmannose, heptose, and 2,3-diamino-2,3-dideoxyhexose. The lipid A portion of the LPS contained 2,3-diamino-2,3-dideoxyhexose as the only sugar component. The major fatty acids were I-hydroxymyristic, lauric, and oleic acids. A long-chain fatty acid, 27-hydroxyoctacosanoic acid, was also present in this lipid A. Separation and analysis of LPS I and LPS II indicated that glucose, mannose, 4-0-methylmannose, and small amounts of 2,3-diamino-2,3-dideoxyhexose and heptose were components of the core region of the LPS, whereas fucose, fucosmine, mannose, and small amounts of quinovosamine and glucosamine were components of the LPS 0-chain region.

Despite intensive studies of the Trichophyton mentagrophytes species complex, its taxonomy still causes confusion. In this study, more than 70 dermatophytes were analyzed based on nuc rDNA ITS1-5.8S-ITS2 (ITS), D1–D2 domains of nuc 28S rDNA (D1D2), and β-tubulin gene (TUBB) sequences to clarify phylogenetic relationships in the complex. This demonstrated that strains of the complex were divided into three major lineages with high statistical support: (i) T. benhamiae and related species; (ii) T. simii and two related species, T. quinckeanum and T. schoenleinii; and (iii) T. mentagrophytes, T. interdigitale, and related species. The major lineages could be further divided into 18 phylogroups, representing either individual species or phylogenetically distinct groups within species. Among strains of T. benhamiae, African isolates American Type Culture Collection (ATCC) 28064 and 28065 formed a phylogenetically distinct phylogroup from their type strain and were considered a distinct species. Strains of T. mentagrophytes were divided into at least four phylogroups based on combined sequence analysis, but some phylogroups showed closer relationships to T. interdigitale, T. equinum, and T. tonsurans when compared by individual genes. This indicates that identifying those species with one gene could lead to incorrect results. For rapid identification of those dermatophytes, each phylogroup was tested by matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) mass spectrometry using a database with customized reference spectra of each phylogroup. This system was able to identify all the tested strains to species level with higher than 91% accuracy, except for strains of T. interdigitale. The three phylogroups of T. benhamiae were well distinguished from one another with high identification accuracy, whereas phylogroups of T. mentagrophytes were often cross-identified to one another or to T. interdigitale. Further research should improve identification accuracy for some species, but the results suggested that MALDI-TOF MS could be a rapid and efficient identification tool for closely related dermatophytes in the T. mentagrophytes species complex.