A novel endothelial cell-based gene therapy platform for the in vivo delivery of apolipoprotein E.

A major focus in gene therapy has been the use of recombinant viruses to deliver genes in vivo. Although this approach shows much promise, there are many safety concerns associated with the use of viral materials in the treatment of human diseases. Our alternative cell-based gene therapy approach utilizes endothelial cells (Pro 175) isolated from the murine embryonic yolk sac. These endothelial cells were evaluated for their potential use in gene therapy as a gene delivery platform. As a test model, we used these cells to deliver apolipoprotein E (apoE) in the murine apoE knockout atherosclerosis model. The lack of apoE protein in these animals results in high levels of serum cholesterol and formation of severe aortic plaques and lesions at a young age. After transplantation of the apoE secreting Pro 175 endothelial cells into apoE-deficient mice, serum cholesterol levels were measured at 2 week intervals. During the 3 months after the initiation of these experiments, levels of cholesterol in the animals having received the apoE secreting endothelial cells were statistically lower compared with the levels of age-matched controls having received non-secreting endothelial cells. Concomitant with cholesterol reduction, atherosclerotic aortic plaques were noticeably reduced in the experimental apoE+ animals. These results highlight the potential of these unique endothelial cells as an efficient delivery platform for somatic gene therapy.     

Kinetics of CD4+ T cell repopulation of lymphoid tissues after treatment of HIV-1 infection

Potent combinations of antiretroviral drugs diminish the turnover of CD4+ T lymphocytes productively infected with HIV-1 and reduce the large pool of virions deposited in lymphoid tissue (LT). To determine to what extent suppression of viral replication and reduction in viral antigens in LT might lead correspondingly to repopulation of the immune system, we characterized CD4+ T lymphocyte populations in LT in which we previously had quantitated viral load and turnover of infected cells before and after treatment. We directly measured by quantitative image analysis changes in total CD4+ T cell counts, the CD45RA+ subset, and fractions of proliferating or apoptotic CD4+ T cells. Compared with normal controls, we documented decreased numbers of CD4+ T cells and increased proliferation and apoptosis. After treatment, proliferation returned to normal levels, and total CD4+ T and CD45RA+ cells increased. We discuss the effects of HIV-1 on this subset based on the concept that renewal mechanisms in the adult are operating at full capacity before infection and cannot meet the additional demand imposed by the loss of productively infected cells. The slow increases in the CD45RA+ CD4+ T cells are consistent with the optimistic conclusions that (i) renewal mechanisms have not been damaged irreparably even at relatively advanced stages of infection and (ii) CD4+ T cell populations can be partially restored by control of active replication without eradication of HIV-1.     

In situ amplification of visna virus DNA in tissue sections reveals a reservoir of latently infected cells.

Maedi and visna are, respectively, the pulmonary and neurological manifestations of slowly progressive infections of sheep caused by retroviruses of the lentivirus subfamily. Lentivirus infections are also persistent infections in which host defenses are generally not successful in eliminating the infectious agent because of restricted viral gene expression in many infected cells. In this report, we describe a method for amplifying and detecting viral DNA in tissue sections which has made it possible to verify experimentally the postulated existence of this reservoir of latently infected cells, as well as to estimate the actual number of cells which harbor viral genomes in infected tissues. In the discussion, we present a simple mathematical model that relates this number to the rate at which inflammatory lesions develop. This model can account for both the slow progression of natural infections and for the rapid accumulation of inflammatory foci in the high dosage experimental system analysed in our studies.     

Characterization of neonatal personnel time inputs and prediction from clinical variables--a time and motion study.

OBJECTIVE: To characterize and predict personnel time inputs to neonatal intensive care using infant characteristics from chart review. STUDY DESIGN: For 12 hours each day, observers timed all direct care, charting, discussions, and procedures for 154 infants. Time inputs were correlated with 40 infant characteristics and resource markers, as well as the Score for Neonatal Acute Physiology (SNAP) for that day of care. RESULTS: Nurses accounted for 76%, respiratory therapists 8%, fellows 5%, nurse practitioners 7% and attendings 5% of total time invested in patient care. Nurses and respiratory therapists spent proportionately more time in direct patient care. In regression models, a limited number of variables explained 36% of the variance in time input per patient for respiratory therapists (p<0.0001), 42% for nurses (p<0.0001), and 23% for physicians and nurse practitioners (p<0.0001). CONCLUSIONS: Total labor inputs can be accurately predicted through the use of a limited number of clinical characteristics. This technique should be routinely employed to improve the accuracy of economic evaluations. Nursing accounts for the majority of time invested in neonatal care. Improved efficiency in neonatology is thus most likely to be generated by interventions that reduce direct nursing time.     

Influence of fever on cefazolin pharmacokinetics

The influence of fever on the pharmacokinetics of cefazolin was investigated in patients with acute febrile diseases. Nine patients were included in the study. Antibiotic serum concentrations were determined using high performance liquid chromatograpy (HPLC). An analog computer and the SIMULINK software package were used to identify the pharmacokinetic model and PCNONLIN software package to obtain the secondary parameters. In 6 patients a two-compartment pharmacokinetic model of cefazolin was observed during fever and after defervescence. In 2 patients a two-compartment model changed to a one-compartment after defervescence, and a one-compartment model was observed in one patient during both periods. Cefazolin-treated patients with a two-compartment model (6/9) had higher Cmax, mean steady state serum concentrations (Css), and area under the plasma concentration-time curve (AUC(0-->infinity)), smaller central compartment volume (V1), and lower clearance (Cl) during fever. The varying distribution of antibiotics during fever probably reflects different hemodynamic responses to fever.