In vivo gene therapy in young and adult RPE65-/- dogs produces long-term visual improvement

Defects in the RPE65 gene, which is selectively expressed in the retinal pigment epithelium (RPE), result in blindness and gradual photoreceptor cell degeneration. Experiments were conducted to assess the efficacy of gene replacement therapy in restoring retinal function in RPE65-/- dogs. Long-term effects of RPE65 gene therapy were assessed using visual behavioral testing and electroretinographic (ERG) recordings at 10-12 weeks and 6-9 months after surgery in five affected dogs. Subretinal injections of similar dosages of two constructs were performed in affected dogs at the ages of 4-30 months: rAAV.RPE65 into one eye and, in four of five dogs, rAAV.GFP contralaterally. Before surgery all RPE65-/- dogs were behaviorally blind with either no or very low-amplitude ERG responses to light stimuli. Marked improvements in visual behavior and ERG responses were observed as early as 4 weeks after surgery in affected animals. Except for light-adapted 50 Hz ERG flicker responses, all ERG parameters tested increased significantly in the eyes treated with the rAAV.RPE65 construct at the early follow-up. Gradual progressive improvements in ERG responses were observed in the RPE65-treated eyes over time. An unexpected finding was that on long-term follow-up, marked improvement of photopic ERG responses were also observed in the contralateral control eye in both young and older dogs. These results are promising for future clinical trials of human patients with retinal degenerative diseases, such as Leber congenital amaurosis, that result from RPE65 gene defects.     

Detection of human immunodeficiency virus in infected CEM cells using fluorescent DNA probes and a laser-based computerized image cytofluorometry system

Quantitative nonradioactive methods to measure the human immunodeficiency virus (HIV) in individually infected cells are needed for the direct assessment of HIV infection, for the evaluation of antiviral chemotherapy and for testing the efficacy of vaccines. As a first step in accomplishing this goal, we built an argon ion laser-based computerized image cytofluorometry (ALCIC) system and determined this instrument's ability to quantitatively measure HIV nucleotides in infected lymphocytes. ALCIC consisted of a 43-mW argon ion laser connected to a Zeiss Universal microscope via a fiberoptic cable, a charged coupled-device video camera, a video frame grabber and array processor and a Micro Vax II computer using computer programs written in FORTRAN. HIV RNA and DNA were detected in infected CEM lymphocytes in culture by in situ hybridization using acetylaminofluorene (AAF)-labeled HIV-DNA probes, a rabbit anti-AAF antibody and a fluorescein-labeled goat anti-rabbit antibody. ALCIC measurements showed that 61% of the CEM cells were infected and that quantitative differences were distinguishable within this group. The levels of fluorescein isothiocyanate (FITC) fluorescence were sixfold or more greater than that observed with the same system using a 100-W mercury lamp for illumination; the improved intensity of the laser-based system is due to greater excitation intensity of the laser and the matching of the excitation spectrum to the peak wavelength of FITC. ALCIC has potential clinical value for determining the effect of antiviral agents on HIV infection and for assessing the susceptibility of different cell types to infection.     

Vasopressin conformational fluctuations: a molecular dynamics study

Molecular dynamics simulations have been used to study the conformational fluctuations of the oligopeptide hormone vasopressin. Starting coordinates for these simulations were built upon the crystal structure of pressinoic acid, the cyclic ring moiety of vasopressin, recently determined by x-ray diffraction. Coordinates for the additional tripeptide “tail” of vasopressin were selected by arbitrary positioning of this segment using interactive computer graphics. Two such starting configurations were minimized to relax strains, and long dynamics simulations (20 and 40 ps) in vacuo were then conducted following extensive heating and equilibration sequences (36 ps). In these studies, vasopressin was found to undergo few substantial conformational changes at 300 K on the time scale simulated, in contrast to the results of a shorter previous simulation, but comparable structural transitions were observed during the equilibration periods. The pressinoic acid structure was found to be a reasonably stable possible conformation for vasopressin in vacuum on this time scale.     

Two Thousand and What? Anthropological Moments and Methods for the Next Century

Ethnography and Human Development: Context and Meaning in Social Inquiry. Richard Jessor. Anne Colby, and Richard A. Shweder, eds. Chicago: University of Chicago Press, 1996. 516 pp
Interpretive Anthropology: Ethnographic Practices for the 21st Century. Norman K. Denzin. Thousand Oaks, CA: Sage, 1997. 325 pp.