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One of the main obstacles for effective human gene therapy for hematopoietic disorders remains the achievement of an adequate number of genetically corrected blood cells. One approach to this goal is to incorporate drug resistance genes into vectors to enable in vivo selection of hematopoietic stem cells (HSCs). Although a number of drug resistance vectors enable HSC selection in murine systems, little is known about these systems in large animal models. To address this issue, we transplanted cells transduced with dihydrofolate resistance vectors into 6 rhesus macaques and studied whether selection of vector-expressing cells occurred following drug treatment with trimetrexate and nitrobenzylmercaptopurineriboside-phosphate. In some of the 10 administered drug treatment courses, substantial increases in the levels of transduced peripheral blood cells were noted; however, numbers returned to baseline levels within 17 days. Attempts to induce stem cell cycling with stem cell factor and granulocyte-colony stimulating factor prior to drug treatment did not lead to sustained enrichment for transduced cells. These data highlight an important species-specific difference between murine and nonhuman primate models for assessing in vivo HSC selection strategies and emphasize the importance of using drugs capable of inducing selective pressure at the level of HSCs.

A comparison was made of the effectiveness of popular disinfectants (Cavicide, Cidexplus, Clorox, Exspor, Lysol, Renalin, and Wavicide) under conditions prescribed for disinfection in the respective product labels on Pseudomonas aeruginosa either in suspension or deposited onto surfaces of metallic or polymeric plastic devices. The testing also included 7 nonformulated germicidal agents (glutaraldehyde, formaldehyde, peracetic acid, hydrogen peroxide, sodium hypochlorite, phenol, and cupric ascorbate) commonly used in disinfection and decontamination. Results showed that P. aeruginosa is on average 300-fold more resistant when present on contaminated surfaces than in suspension. This increase in resistance agrees with results reported in studies of biofilms, but unexpectedly, it precedes biofilm formation. The surface to which bacteria are attached can influence the effectiveness of disinfectants. Viable bacteria attached to devices may require dislodging through more than a one-step method for detection. The data, obtained with a sensitive and quantitative test, suggest that disinfectants are less effective on contaminated surfaces than generally acknowledged.

This study was prompted by toxic responses to disinfecting agents reported in patients after surgical procedures and in sensitized health care personnel. We evaluated the cytotoxicity of seven substances used in the formulation of common liquid chemical disinfectants and sterilants. We found that a standard method based on direct microscopic examination of cell cultures was insensitive and may result in an underestimation of the risk that disinfectants pose to health care personnel or patients who are exposed to these substances. Using independent quantitative tests measuring the integrity of the cellular membrane, metabolic activity, or cell growth, we found that there is a several-hundredfold difference in the relative toxicity of various disinfecting substances. The concentration toxic in 50% of the cell population (TC(50)) that was found for each disinfectant was similar in a variety of cell lines from human, monkey, or mouse origin. Statistical analysis of TC(50)s suggests that liquid disinfecting agents could be classified in three main groups according to their relative toxicity, with: (1) mild (TC(50) > 1 mM, including phenol, hydrogen peroxide, and formaldehyde); (2) moderate (1mM > TC(50) > 0.1 mM, sodium hypochlorite); and (3) severe (TC(50) < 0.1 mM, glutaraldehyde, cupric ascorbate, and peracetic acid) toxicity. These data suggest a vast difference in the potential risk of various disinfectants and sterilants. The data presented in this study should help to define the relative toxic risk of different disinfecting substances to patients and health care personnel and assist in the selection of safer microbicidal formulations.

This study compares the effects of various concentrations of salt or serum in the killing of Bacillus subtilis spores by either glutaraldehyde, sodium hypochlorite, cupric ascorbate, hydrogen peroxide, peracetic acid, formaldehyde, or phenol. Salt affected only glutaraldehyde, its sporicidal activity increasing with an increase in concentration of sodium bicarbonate or sodium chloride. The sporicidal activity of glutaraldehyde was minimal when the concentrations of aldehyde groups and lysine residues from protein were similar. We present an equation describing the effect of serum on spore survival as a function of glutaraldehyde concentration that fits the data with a regression coefficient of 0.9. Cupric ascorbate and peracetic acid were inhibited by serum, but this effect was linked to a rise in pH. Sodium hypochlorite was the agent most sensitive to protein, with its sporicidal activity nearly disappearing in the presence of 2% serum or an equivalent amount of purified protein.

Noninvasive imaging of a reporter gene is a new and promising technique to quantify transgene expression after gene therapy. This study was performed to demonstrate visualization of lentiviral-marked cells by PET.We transduced nonhuman primate CD34+ hematopoietic cells with a lentiviral vector expressing a PET reporter gene, the mutant viral herpes simplex virus type 1-thymidine kinase (HSV1-sr39tk) gene. 1-(2-Fluoro-2-deoxy-beta-D-arabinofuranosyl)-76Br-5-bromouracil (76Br-FBAU) was used as the substrate for the viral tk enzyme. Upon phosphorylation, 76Br-FBAU was retained by cells and imaged by PET. The long half-life of 76Br, 16.2 h, permitted us to perform extended pharmacokinetic and imaging studies.76Br-FBAU was retained in vascular tissues of the animals with transplanted tk lentiviral vector-transduced CD34+ cells. Elimination of 76Br-FBAU was through renal and hepatic excretion.Noninvasive molecular imaging using PET will help us, in the future, to define the contribution and distribution of cells and their progeny to tissue repair and development.

This study compared the activity of commercial liquid sterilants and disinfectants on Bacillus subtilis spores deposited on three types of devices made of noncorrodible, corrodible, or polymeric material. Products like Renalin, Exspor, Wavicide-01, Cidexplus, and cupric ascorbate were tested under conditions specified for liquid sterilization. These products, at the shorter times indicated for disinfection, and popular disinfectants, like Clorox, Cavicide, and Lysol were also studied. Data obtained with a sensitive and quantitative test suggest that commercial liquid sterilants and disinfectants are less effective on contaminated surfaces than generally acknowledged.

Background: The relatively limited variety of surfaces and geometries challenged in current sporicidal testing reduces the predictive value of these analyses when extrapolated to the wide variety of medical devices. The unknown spore load being challenged and the qualitative nature (growth/no growth) of those tests further prevent precise comparison among liquid chemical disinfectants. Hence, the relative activity of different chemical substances has not been clearly established, hindering selection of the best agent for each clinical situation. Methods: A micromethod was developed to assess sporicidal activity against Bacillus subtilis spores deposited on three different medical devices: carbon-steel dental burs silicone-rubber medical catheters, and titanium-alloy dental abutment screws. The spore load on each device and the recovery after three analytical steps were quantitatively assessed with spores radiolabeled with carbon 14 menthionine. Results: The killing of 2 to 7 × 10 6 spores loaded on three different devices and exposed to glutaraldehyde, fomaldehyde, copper ascorbate, hydrogen peroxide, peracetic acid, sodium hypochlorite, or phenol for 30 minutes at 20° C ranged from a 10 3 -fold decrease for 10% hydrogen peroxide to zero decrease for 5% phenol. Our results suggest that the nature of the surface being challenged may affect the sporicidal activity of some chemical agents. Conclusion: The quantitative data presented allow comparison of the sporicidal effect of different liquid chemical agents. These findings may help prevent an overestimation of sporicidal activity and possible transmission of pathogens from the surface of improperly decontaminated medical devices.