The immune-enhancing effects of dietary fibres and prebiotics.

The gastrointestinal tract is subjected to enormous and continual foreign antigenic stimuli from food and microbes. This organ must integrate complex interactions among diet, external pathogens, and local immunological and non-immunological processes. It is critical that protective immune responses are made to potential pathogens, while hypersensitivity reactions to dietary antigens are minimised. There is increasing evidence that fermentable dietary fibres and the newly described prebiotics can modulate various properties of the immune system, including those of the gut-associated lymphoid tissues (GALT). This paper reviews evidence for the immune-enhancing effects of dietary fibres. Changes in the intestinal microflora that occur with the consumption of prebiotic fibres may potentially mediate immune changes via: the direct contact of lactic acid bacteria or bacterial products (cell wall or cytoplasmic components) with immune cells in the intestine; the production of short-chain fatty acids from fibre fermentation; or by changes in mucin production. Although further work is needed to better define the changes, mechanisms for immunomodulation, and the ultimate impact on immune health, there is convincing preliminary data to suggest that the consumption of prebiotics can modulate immune parameters in GALT, secondary lymphoid tissues and peripheral circulation. Future protocols on the physiological impact of consuming prebiotics should be designed to include assessments of the gut microflora, gut physiology and the function and composition of the various regions of GALT.

Efficient retrovirus-mediated gene transfer to transplantable human bone marrow cells in the absence of fibronectin.

The low frequency of transplantable hematopoietic stem cells in adult human bone marrow (BM) and other differences from cord blood stem cells have impeded studies to optimize the retroviral transduction of stem cells from adult sources. To address this problem, first a cytokine combination was defined that would both maximize the kinetics of adult BM CD34(+)CD38(-) cell mitogenesis and minimize the period of prestimulation required for the transduction of these cells by a MSCV-GFP/neo(r) virus in tissue culture dishes in the absence of fibronectin. Three days of stimulation with flt3-ligand, Steel factor, interleukin (IL)-3, and hyper-IL-6 proved both necessary and sufficient to obtain 83% +/- 2% GFP(+) CD34(+)CD38(-) cells, 75% +/- 10% G418-resistant clonogenic progenitors, and 50% +/- 20% transduced long-term culture-initiating cells as recovered 48 hours after a single exposure to virus. Moreover, this was accompanied by a several-fold increase in viral receptor (pit-1) messenger RNA transcripts in the target cells. Using this prestimulation protocol, repeated daily exposure to new virus (3x) did not alter the proportion of transduced cells over that obtained with a single exposure. Adult human BM cells able to engraft immunodeficient (NOD/SCID-beta(2)M(-/-)) mice were also efficiently transduced (10%-20% GFP(+) human lymphoid and myeloid cells present 6-8 weeks after transplant) using a 6-day prestimulation and infection protocol. A clinically useful efficiency of retrovirus-mediated gene transfer to transplantable adult human BM stem cells can thus be obtained with a protocol that allows their semisynchronous activation into cycle and concomitant increased expression of virus receptor transcripts before virus exposure.

High-efficiency retroviral transduction of mammalian cells on positively charged surfaces.

The efficiency of retroviruses as transducing agents has been appreciated for many years, particularly for hematopoietic cell targets for which alternative strategies applicable to adherent cells are not effective. Advances in vector design, pseudotyping, and infection conditions have eliminated the need to cocultivate the target cells with virus-producing cells. Nevertheless, improvements are still needed for many applications, including those with a therapeutic or clinical cell-tracking objective. In this study we show that more positively charged surfaces, including those designed for the culture of anchorage-dependent cells, allow measurable levels of adhesion by different pseudotypes of retroviruses, which can result in increased gene transfer efficiencies to a variety of target cells including normal primary human hematopoietic cells as well as human leukemic cell lines and rat and murine fibroblasts. In the experiments with primary human cells, equal aliquots of enriched CD34+ cord blood cells were first stimulated for 2 days with cytokines (Flt3 ligand, Steel factor, IL-3, IL-6, and G-CSF) and then exposed for 4 days to a green fluorescent protein (GFP)- and Neo(r)-encoding retrovirus produced in PG13 cells. Both the final yield (approximately 300% relative to initial numbers), and the proportion (approximately 60%) of transduced CD34+ cells, colony-forming cells, and long-term culture-initiating cells were the same for cells infected either in tissue culture dishes or in fibronectin-coated petri dishes. Similar proportions (approximately 10%) and absolute yields of GFP+ human cells were also found in multilineage engrafted NOD/SCID mice assessed 6 to 8 weeks after being transplanted with these two types of transduced, but unselected, cells. These findings suggest a new and simpler approach for achieving high gene transfer efficiencies to hematopoietic cells.