BACKGROUND: Genetically modified T-lymphocytes are potential therapeutic agents for the treatment of various disorders. Successful retroviral infection of primary murine T-lymphocytes is a prerequisite to the study of adoptive cell therapies in a small animal model. The definition of factors controlling retroviral infection of T-lymphocytes would also be useful to better understand retroviral diseases. However, retroviral-mediated gene transfer into murine primary T-cells has remained elusive. METHODS: In order to define the requirements for stable and efficient gene transfer in primary murine T-lymphocytes, we investigated factors capable of affecting retroviral infection. These include activation conditions (using mitogens or monoclonal antibodies), culture conditions (including media composition and cytokine addition), timing of viral exposure, retroviral receptor selection (ecotropic, amphotropic, or vesicular stomatitis virus G (VSV-G) glycoprotein receptor), and viral titer. RESULTS: We show that efficient gene transfer can be achieved in murine T-lymphocytes, provided that a number of favorable conditions are met, in particular optimized T-cell activation conditions, optimal timing of infection, adequate interleukin-2 concentration and T-cell density, and a high viral titer. On a particulate basis, we find that ecotropic particles are more effective than amphotropic or VSV-G-pseudotyped particles, and recommend the use of a specifically selected retroviral packaging cell line. CD4+ T-cells are equally or more infectible than CD8+ lymphocytes, depending on the activation conditions. The Th1 and Th2 subsets are comparably susceptible to retroviral infection, in contrast to what has been reported in some instances of human T-cell infection by human immunodeficiency virus (HIV). CONCLUSIONS: We establish conditions that enable efficient retroviral-mediated gene transfer in murine primary T-lymphocytes. T-lymphocytes are not uniformly susceptible to retroviral infection depending on the mode of T-cell activation. These findings have implications for devising approaches to the study of T-cell biology, adoptive cell therapies, and the pathophysiology of retroviral diseases in mouse models.

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Phagocytes from both vertebrate and invertebrate species use "coiling" phagocytosis.

Coiling phagocytosis has been observed previously only by chance, and there has been no systematic investigation of this uptake mechanism. Therefore, a comparative electron microscopical study was performed. Different human and murine cell populations, phagocytes from various vertebrate and invertebrate species, and predatory amoebae were incubated with Borrelia burgdorferi, one of the microbes known to induce coiling phagocytosis, to study the uptake mechanisms used. In this model, coiling phagocytosis was observed with both vertebrate and invertebrate species but not with amoebae. With cells from humans and mice, this uptake mechanism was restricted to phagocytic cells of myeloid origin. The coiled membrane gaps did not give rise to phagosomes; instead, membrane fusion was followed by membrane dissipation. Thus, coiling of B. burgdorferi apparently is an alternative uptake mechanism used by metazoan phagocytes, involving special membrane processing. However, coiling phagocytosis may show different features with different microbes.

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PubMedDate not supplied

Common polymorphism in a highly variable region upstream of the human lactase gene affects DNA-protein interactions.

In most mammals lactase activity declines after weaning when lactose is no longer part of the diet, but in many humans lactase activity persists into adult life. The difference responsible for this phenotypic polymorphism has been shown to be cis-acting to the lactase gene. The causal sequence difference has not been found so far, but a number of polymorphic sites have been found within and near to the lactase gene. We have shown previously that in Europeans there are two polymorphic sites in a small region between 974 bp and 852 bp upstream from the start of transcription, which are detectable by denaturing gradient gel electrophoresis (DGGE). In this study, analysis of individuals from five other population groups by the same DGGE method reveals four new alleles resulting from three additional nucleotide changes within this very small region. Analysis of sequence in four primate species and comparison with the published pig sequence shows that the overall sequence of this highly variable human region is conserved in pigs as well as primates, and that it lies within a 1kb region which has been shown to control lactase downregulation in pigs. Electrophoretic mobility shift assay (EMSA) studies were carried out to determine whether common variation affected protein-DNA binding and several binding activities were found using this technique. A novel two base-pair deletion that is common in most populations tested, but is not present in Europeans, caused no change in binding activity. However, a previously published C to T transition at -958bp dramatically reduced binding activity, although the functional significance of this is not clear.

Adult↗