Artificial capillary culture: expansion and retroviral transduction of CD4+ T-lymphocytes for clinical application.

An artificial capillary culture/transduction technique has been developed for application in a phase I gene therapy clinical trial for HIV. The trial protocol involves isolation of CD4+ T-lymphocytes from a genetically matched HIV negative twin, retroviral transduction of equal numbers of cells with the ribozyme therapeutic and control genes, and expansion in Cellmax artificial capillary modules. Preclinical studies showed transduction efficiencies in the range of 3-30%, with preferential expansion of CD4+ lymphocytes over a culture period of 10-14 days. Over this time period, an average yield of 1.7 x 10(9) lymphocytes was readily attainable from 5 x 10(7) CD8-depleted lymphocytes. In addition, a sensitive and reliable quantitative competitive PCR method was developed to assess the levels of transduction before infusion into the recipient. The transduction data suggest that the efficiency of retroviral transduction was affected by the presence of inhibitory factors present in the virus preparations or generated as a result of the transduction process itself. It is hypothesised that the method of transduction could significantly affect the extent of this inhibition, and thus impact on clinical efficacy of retrovirus mediated gene therapy.

Production of an early release, M(r) 36,000 monokine by stimulated rat macrophages.

Supernatants from rat peritoneal macrophage cultures stimulated with bacterial products contain a M(r) 36,000 factor that protects immature cortical thymocytes from loss of viability over a 4-hr incubation period in vitro. This effect could not be produced with purified transforming growth factor-beta or recombinant interleukin-6 (IL-6). Further, the partially purified M(r) 36,000 fraction was inactive in bioassays for IL-1 and tumour necrosis factor. Maximal production of the factor occurred 2 hr after the addition of 20 micrograms/ml of lipopolysaccharide, as assessed by the titre resulting in 100% protection of thymocytes in a viability assay. The detection of protective activity within 5 min after addition of the stimulant could be attributed to the release of intracellular stores but protein synthesis was required to account for the increasing titre up to peak levels. The titre fell rapidly after 2 hr so that activity could not be detected at 4 hr. This profile of release was refractory to repeated stimulation with lipopolysaccharide. Conjoint addition of lipopolysaccharide and indomethacin, did, however, allow release in response to subsequent challenge. Related to this finding, prostaglandin E2 completely inhibited the release of protective activity.