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A retrovirus vector containing an enhanced green fluorescent protein complimentary DNA (EGFP cDNA) was used to mark and dynamically follow vector-expressing cells in the peripheral blood of bone marrow transplanted X-linked severe combined immunodeficient dogs. CD34(+) cells isolated from young normal dogs were transduced, using a 2 day protocol, with an amphotropic retroviral vector that expressed enhanced green fluorescent protein (EGFP) and the canine common gamma chain (gammac) cDNAs. Following transplantation of the transduced cells, normal donor peripheral blood lymphocytes (PBL) appeared by 1 month post-bone marrow transplant (BMT) and rescued three of five treated dogs from their lethal immunodeficiency. PCR and flow cytometric analysis of post-BMT PBL documented the peripheral EGFP expressing cells as CD3(+) T cells, which varied from 0% to 28%. Sorting of EGFP(+) and EGFP(-) peripheral blood T cells from two dogs, followed by vector PCR analysis, showed no evidence of vector shutdown. EGFP expression in B cells or monocytes was not detected. These marking experiments demonstrate that the transduction protocol did not abolish the lymphoid engraftment capability of ex vivo transduced canine CD34(+) cells and supports the potential utility of the MSCV retroviral vector for gene transfer to XSCID affected canine hematopoietic progenitor cells (HPC).

We have used a murine MSCV-based bicistronic retroviral vector, containing the common gamma chain (gammac) and enhanced green fluorescent protein (EGFP) cDNAs, to optimize retroviral transduction of canine cells, including an adherent canine thymus fibroblast cell line, Cf2Th, as well as normal canine CD34(+) bone marrow (BM) cells. Both canine cell types were shown to express Ram-1 (the amphotropic retroviral receptor) mRNA. Supernatants containing infectious viruses were produced using both stable (PA317) and transient (Phoenix cells) amphotropic virus producer cell lines. Centrifugation (spinfection) combined with the addition of polybrene produced the highest transduction efficiencies, infecting approximately 75% of Cf2Th cells. An average of 11% of highly enriched canine CD34(+) cells could be transduced in a protocol that utilized spinfection and plates coated with the fibronectin fragment CH-296 (Retronectin). Indirect assays showed the vector-encoded canine gammac cDNA produced a gammac protein that was expressed on the cell surface of transduced cells. This strategy may result in the transduction of sufficient numbers of CD34(+) BM cells to make the treatment of canine X-linked severe combined immunodeficiency and other canine genetic diseases feasible.

Historically, the dog has been a valuable model for bone marrow transplantation studies, with many of the advances achieved in the dog being directly transferable to human clinical bone marrow transplantation protocols. In addition, dogs are also a source of many well-characterized homologues of human genetic diseases, making them an ideal large animal model in which to evaluate gene therapy protocols. It is generally accepted that progenitor cells for many human hematopoietic cell lineages reside in the CD34+ fraction of cells from bone marrow, cord blood, or peripheral blood. In addition, CD34+ cells are the current targets for human gene therapy of diseases involving the hematopoietic system. In this study, we have isolated and characterized highly enriched populations of canine CD34+ cells isolated from dogs 1 week to 3 months of age. Bone marrow isolated from 2- to 3-week-old dogs contained up to 18% CD34+ cells and this high percentage dropped sharply with age. In in vitro 6-day liquid suspension cultures, CD34+ cells harvested from 3-week-old dogs expanded almost two times more than those from 3-month-old dogs and the cells from younger dogs were also more responsive to human Flt-3 ligand (Flt3L). In culture, the percent and number of CD34+ cells from both ages of dogs dropped sharply between 2 and 4 days, although the number of CD34+ cells at day 6 of culture was higher for cells harvested from the younger dogs. CD34+ cells harvested from both ages of dogs had similar enrichment and depletion values in CFU-GM methylcellulose assays. Canine CD34+/Rho123lo cells expressed c-kit mRNA while the CD34+/Rhohi cells did not. When transplanted to a sub-lethally irradiated recipient, CD34+ cells from 1- to 3-week-old dogs gave rise to both myeloid and lymphoid lineages in the periphery. This study demonstrates that canine CD34+ bone marrow cells have similar in vitro and in vivo characteristics as human CD34+ cells. In addition, ontogeny-related functional differences reported for human CD34+ cells appear to exist in the dog as well, suggesting pediatric CD34+ cells may be better targets for gene transfer than adult bone marrow. The demonstration of similarities between canine and human CD34+ cells enhances the dog as a large, preclinical model to evaluate strategies for improving bone marrow transplantation protocols, for gene therapy protocols that target CD34+ cells, and to study the engraftment potential of various cell populations that may contain hematopoietic progenitor cell activity.

Our laboratory has identified an X-linked severe combined immunodeficiency (XSCID) in dogs that is the result of mutations in the common gamma chain (gammac) subunit of the interleukin-2 (IL-2), IL-4, IL-7, IL-9, IL-15, and IL-21 receptors. Canine XSCID, unlike genetically engineered gammac-deficient mice, has a clinical and immunologic phenotype virtually identical to human XSCID, suggesting species-specific differences exist in the role of the gammac and its associated cytokines in mice in comparison to their role in humans and dogs. This review compares and contrasts thymopoiesis and postnatal T cell development in gammac-deficient (XSCID) dogs raised in a conventional environment, with gammac-deficient dogs raised in a gnotobiotic environment. Therapy to accelerate T cell regeneration following hematopoietic stem cell transplantation or gene therapy is also discussed.

Background and Objectives: Benzodiazepine agonists and azaperone derivatives are used clinically as anxiolytics but have different neuroreceptor mechanisms of action. This study evaluated clinical pharmacodynamic approaches to distinguishing these two classes of compounds., Methods: Healthy volunteers received single oral doses of placebo, the benzodiazepine agonist triazolam (0.25 mg) or the azaperone anxiolytic buspirone (20 mg), in a double-blind, three-way crossover study. Ratings of mood and sedation, performance on the digit symbol substitution test (DSST), and quantitative measures of electroencephalographic (EEG) beta activity (13 to 31.75 cycles/sec) determined by fast-Fourier transform were obtained at multiple times after dosage., Results: Triazolam significantly increased self- and observer-rated sedation, impaired DSST performance, impaired recall, and increased EEG beta activity. Pharmacodynamic changes were significantly intercorrelated; all effects were maximal 1 to 2 hours after dosage but were indistinguishable from placebo by 8 hours. Buspirone did not alter the EEG or DSST performance but did increase self-ratings of sedation and feeling "spacey" and impaired memory function; these effects generally were quantitatively less than with triazolam. Peak plasma triazolam concentrations preceded maximum pharmacodynamic effects; the mean plasma effect site equilibration half-life was 9.4 minutes. Kinetic-dynamic modeling procedures yielded significant relationships between hypothetical effect site triazolam concentrations and pharmacodynamic changes., Conclusions: Quantitative analysis of the EEG clearly distinguishes a typical benzodiazepine agonist from a nonagonist anxiolytic, in clinically relevant dosage, whose pharmacodynamic actions do not involve benzodiazepine receptor occupancy. EEG effects associated with triazolam are intercorrelated with other pharmacodynamic measures.

Background: Elderly persons frequently appear to be sensitive to the effects of many drugs that depress the central nervous system. We studied the effect of age on the pharmacokinetics and pharmacodynamics of the benzodiazepine hypnotic agent triazolam, now the most frequently prescribed hypnotic drug in the United States., Methods: Twenty-six healthy young subjects (mean age, 30 years) and 21 healthy elderly subjects (mean age, 69 years) participated in a four-way crossover study. After a single-blind adaptation trial with placebo, each subject received, in random order and in double-blind fashion, single doses of placebo, 0.125 mg of triazolam, and 0.25 mg of triazolam. For 24 hours after the administration of each of the three study medications, plasma triazolam levels were determined and psychomotor performance, memory, and degree of sedation were assessed., Results: Plasma triazolam concentrations increased in proportion to the dose, but the elderly subjects had higher plasma concentrations due to reduced clearance of the drug. The degree of sedation as rated by an observer and the reduction in the subjects' performance on the digit-symbol substitution test were both greater in the elderly than in the young subjects after they were given the same doses. The relation of the plasma triazolam concentration to the degree of impairment was similar for the two groups. As part of the study, information was presented 1 1/2 hours after the administration of the drugs; the subjects' ability to recall the information 24 hours later was impaired by both doses of triazolam, and the percent decrease was similar in the young and elderly groups., Conclusions: Triazolam caused a greater degree of sedation and greater impairment of psychomotor performance in healthy elderly persons than in young persons who received the same dose. These effects resulted from reduced clearance and higher plasma concentrations of triazolam rather than from an increased intrinsic sensitivity to the drug. On the basis of these results, the dosage of triazolam for elderly persons should be reduced on average by 50 percent.