Topical treatment with liposomes containing T4 endonuclease V protects human skin in vivo from ultraviolet-induced upregulation of interleukin-10 and tumor necrosis factor-alpha.

Exposing human skin to ultraviolet radiation causes DNA damage, sunburn, immune alterations, and eventually, skin cancer. We wished to determine whether liposomes containing a DNA repair enzyme could prevent any of the acute effects of irradiation when applied after ultraviolet exposure. Fifteen human patients with a prior history of skin cancer were exposed to two minimal erythema doses of ultraviolet radiation on their buttock skin. Liposomes containing T4 endonuclease V or heat-inactivated enzyme were applied immediately and at 2, 4, and 5 h after ultraviolet irradiation. Transmission electron microscopy after anti-T4 endonuclease V-staining and immunogold labeling on biopsies taken at 6 h after ultraviolet exposure revealed that the enzyme was present within cells in the skin. Immunohistochemical DNA damage studies suggested a trend toward improved DNA repair at the active T4 endonuclease V liposome-treated test sites. Although the active T4 endonuclease V liposomes did not significantly affect the ultraviolet-induced erythema response and microscopic sunburn cell formation, they nearly completely prevented ultraviolet-induced upregulation of interleukin-10 and tumor necrosis factor-alpha RNA message and of interleukin-10 protein. These studies demonstrate that liposomes can be used for topical intracellular delivery of small proteins to human skin and suggest that liposomes containing DNA repair enzymes may provide a new avenue for photoprotection against some forms of ultraviolet-induced skin damage.

Reproducible and efficient murine CNS gene delivery using a microprocessor-controlled injector.

To develop a reproducible gene transfer method for the murine CNS we evaluated delivery of various gene vehicles using mechanical or manual stereotaxic intracranial inoculation. A microprocessor controlled microsyringe pump (The World Precision Instruments/UltraMicroPump) programmable for volume, rate and syringe size and designed to dispense nanoliter and picoliter volumes was compared to a standard manual deliver method. Gene transfer efficiency of two viral vectors, two synthetic cationic lipid molecules, and naked DNA were evaluated in mice injected unilaterally in two brain regions. Animals received 1 microl over 10 min. of either HSVlac (1 x 10(5) b.f.u), AdLac (1 x 10(5) p.f.u), Tfx-10 or Tfx-20 (2.6 microg DNA in 2.0 microl Tfx; 1:1 charge ratio of DNA to liposome), or naked DNA (HSVlac plasmid, 10 microg/microl). After 4 days, animals from each group were perfused and tissue prepared for X-gal histochemical detection of beta-galactosidase expression. Blue cells were observed in the HSV, Adenovirus, and Tfx-20 groups only at the injection site in animals injected using the UMP. Animals injected manually exhibited fewer blue cells and positive cells were not restricted to the injection site. To quantify expression, tissue punches harvested from the injection sites as well as other brain regions were analyzed using a chemiluminescent reporter assay to detect beta-galactosidase (Galacto-Light). These data indicated increased activity in all animals injected with a lacZ containing vector via the UMP as compared to manual delivery: A 41% increase in the expression levels of beta-gal in HSVlac infected animals (p = 0.0029); a 29% increase in Adlac infected animals (p = 0.01); a 56% increase in Tfx-10 transduced animals (p = 0.04); a 24% increase in Tfx-20 transduced animals (p = 0.01); and a 69% increase in naked DNA gene transfer (p = 0.05). Total beta-galactosidase activity was greatest in HSVlac infected mice followed by Adlac > Tfx-20 > Tfx-10 = naked DNA.

Radiation dose to the lens of eye and thyroid gland in paranasal sinus multislice CT.

CT has become an established examination in the evaluation of the paranasal sinuses. Until recently this was achieved by the direct coronal technique on conventional and single slice helical scanners. With the advent of multislice technology, thin slice axial CT with excellent coronal and sagittal reconstructions is now the norm. We describe a study designed to evaluate the radiation dose to the lens of the eye and thyroid gland in the axial and coronal planes on a Siemens Volume Zoom quad slice scanner at 140 kV and effective mAs of 100 using 1 mm collimation. Thermoluminescent dosimeters were placed on the eyelid and thyroid gland of 29 patients scanned axially in the supine position and a further 28 patients scanned coronally in the prone position with gantry tilt. The results show mean doses of 35.1 mGy (lens) and 2.9 mGy (thyroid gland) in the coronal plane compared with 24.5 mGy (lens) and 1.4 mGy (thyroid gland) in the axial plane. Results obtained from a head phantom and from using the ImPACT CT dose calculator were comparable. The kV and mAs were then reduced to 120 and 40, respectively, and the axial study repeated using the head phantom and predicted doses using the ImPACT CT dose calculator. The low dose scanning technique revealed a lens dose of 9.2 mGy and thyroid dose of 0.4 mGy. The eye dose on a multislice scanner is still substantially less than the threshold dose of 0.5-2 Gy for detectable lens opacities. These results indicate that, in addition to the established perceived advantages of multislice axial sinus CT, i.e. patient comfort, no artefact from dental amalgam and reproducible true coronal images, should be included a decreased radiation dose to both the eye lens and thyroid gland compared with direct coronal scanning.

Identification of the cohesion molecule, contact sites B, of Dictyostelium discoideum.

Polyspecific antibodies were raised against vegetative cells of Dictyostelium discoideum, strain Ax2. Monovalent (Fab') fragments of antibodies CMC 1, 5, 7 and 12 blocked completely the cohesion of vegetative cells. Antibody CMC 1 was studied in detail. The Fab' of this blocked the cohesion of aggregation-competent cells by 40%. It also caused some loss of cell contact in aggregation streams. In so doing the contacts that remained were mostly at the ends of the cells. Immunofluorescence showed that CMC 1 Fab' bound to both the cytoplasm and the surface of fixed cells. It also bound to the surface of live cells. A control (N Fab') also bound to the cell surface but did not block vegetative cell cohesion. An extract of vegetative cells was obtained using the detergent Triton X-100. D. discoideum proteins were immunoprecipitated from this extract using protein A-Sepharose and CMC 1 immunoglobulin G (IgG). These immobilized proteins absorbed the cohesion-blocking activity of CMC 1 Fab'. About 30 proteins were obtained when the Triton-soluble fraction was immunoprecipitated with IgG of CMC 1, 5, 7 and 12. Five of these were found to be cell surface proteins by the technique of lactoperoxidase-catalysed radio-iodination. These proteins had molecular weights of 178 000, 166 000, 126 000 and 64 000. CMC 12 IgG immunoprecipitated an additional cell surface protein of 46 000 molecular weight. Slices of polyacrylamide gel containing each of the five proteins identified as possible contact sites were fixed, washed and incubated with CMC 1 Fab'. Gel that contained protein of 178 000, 166 000 and 64 000 molecular weight had no effect on the activity of CMC 1 Fab'. However, Fab' that had been incubated with gel containing protein of 126 000 molecular weight no longer blocked cell cohesion.

Monomers of the Neurospora plasma membrane H+-ATPase catalyze efficient proton translocation.

Liposomes prepared by sonication of asolectin were fractionated by glycerol density gradient centrifugation, and the small liposomes contained in the upper region of the gradients were used for reconstitution of purified, radiolabeled Neurospora plasma membrane H+-ATPase molecules by our previously published procedures. The reconstituted liposomes were then subjected to two additional rounds of glycerol density gradient centrifugation, which separate the H+-ATPase-bearing proteoliposomes from ATPase-free liposomes by virtue of their greater density. The isolated H+-ATPase-bearing proteoliposomes in two such preparations exhibited a specific H+-ATPase activity of about 11 mumol of Pi liberated/mg of protein/min, which was approximately doubled in the presence of nigericin plus K+, indicating that a large percentage of the H+-ATPase molecules in both preparations were capable of generating a transmembrane protonic potential difference sufficient to impede further proton translocation. Importantly, quantitation of the number of 105,000-dalton ATPase monomers and liposomes in the same preparations by radioactivity determination and counting of negatively stained images in the electron microscope indicated ATPase monomer to liposome ratios of 0.97 and 1.06. Because every liposome in the preparations must have had at least one ATPase monomer, these ratios indicate that very few of the liposomes had more than one, and simple calculations show that the great majority of active ATPase molecules in the preparations must have been present as proton-translocating monomers. The results thus clearly demonstrate that 105,000-dalton monomers of the Neurospora plasma membrane H+-ATPase can catalyze efficient ATP hydrolysis-driven proton translocation.

Enzyme therapy of xeroderma pigmentosum: safety and efficacy testing of T4N5 liposome lotion containing a prokaryotic DNA repair enzyme.

Xeroderma pigmentosum (XP) is a rare genetic disease in which patients are defective in DNA repair and are extremely sensitive to solar UV radiation exposure. A new treatment approach was tested in these patients, in which a prokaryotic DNA repair enzyme specific for UV-induced DNA damage was delivered into the skin by means of topically applied liposomes to supplement the deficient activity. Acute and chronic safety testing in both mice and humans showed neither adverse reactions nor significant changes in serum chemistry or in skin histology. The skin of XP patients treated with the DNA repair liposomes had fewer cyclobutylpyrimidine dimers in DNA and showed less erythema than did control sites. The results encourage further clinical testing of this new enzyme therapy approach.