Eukaryotic expression plasmid transfer from the intracellular bacterium Listeria monocytogenes to host cells.

The facultative intracellular, Gram-positive bacterium Listeria monocytogenes invades phagocytic and non-phagocytic cells from the tissues and organs of a wide variety of animals and humans. Here, we report the use of these bacteria as vehicles for gene transfer. Eukaryotic expression plasmids were introduced into the nucleus of host cells following lysis of the intracytosolic, plasmid-carrying bacteria with antibiotics. Cell lines of different tissues and species could be transfected in this way. We examined bacterial properties required for delivery of the expression plasmids and found that this was strictly dependent on the ability of these bacteria to both invade eukaryotic cells and egress from the vacuole into the cytosol of the infected host cells. Macrophage-like cell lines or primary, peritoneal macrophages proved to be almost refractory to Listeria-mediated gene transfer. Thus, attenuated L. monocytogenes represents a serious candidate for consideration as a DNA-transfer vehicle for in vivo somatic gene therapy. The potential for oral administration of L. monocytogenes and the ease in producing and cultivating recombinant strains are further attributes that make its use as a gene transfer vehicle attractive.

T cell phenotypes of the normal nasal mucosa: induction of Th2 cytokines and CCR3 expression by IL-4.

Mucosal environments such as that of the nose are points of first contact between the human organism and its environment. At these sites the immune system must be regulated to differentiate between and respond appropriately to pathogens and harmless contaminants. T cell-driven immune responses broadly fall into Th1- or Th2-type phenotypes, with increasing evidence that the recruitment of these T lymphocyte subsets is mediated by selective expression of specific chemokine receptors. We have investigated the immunology of the normal nasal mucosa. We show that nasal T cell lines from normal individuals, expanded by culture in IL-2, show reduced expression of the Th2-type cytokines IL-4 and IL-5 compared with lines derived from the blood of the same subjects. These T cells also show reduced expression of the Th2-selective chemokine receptor, CCR3, but similar levels of CCR4 compared with the blood-derived lines. This apparent suppression of Th2 cytokine and CCR3 expression by nasal T cells was reversed by addition of IL-4 to the culture medium. These data are consistent with the presence of a nasal mucosal microenvironment that suppresses Th2 responses and may represent a protective measure against atopic allergic disease in humans and a favoring of Th1 responses to infectious agents. In contrast, T cell expression of CCR1 was higher in the nose than in the blood regardless of the culture medium cytokine environment in keeping with a role for this receptor in tissue homing or lymphocyte activation.

T cells from human allergen-induced late asthmatic responses express IL-12 receptor beta 2 subunit mRNA and respond to IL-12 in vitro.

IL-12 suppresses proallergic Th2-type cytokine production and induces Th1-type cytokine production by peripheral blood T cells from subjects with allergic disease. The objective of the present study was to examine the relevance of these findings to target organ T cell responses in human asthma. Bronchoalveolar lavage (BAL) and PBMC were collected from atopic asthmatics 24 h after fiberoptic allergen challenge of a segmental bronchus. BAL T cells and PBMC were cultured with allergen in the presence of recombinant IL-12 or IFN-gamma, and cytokines were measured in culture supernatants after 6 days. IL-5 production by BAL T cells and PBMC was inhibited by IL-12 and, to a lesser extent, by IFN-gamma. IL-12 also induced IFN-gamma production by BAL T cells and PBMC. The effects of IL-12 nor IFN-gamma on IL-5 production could not be reversed by neutralizing anti-IFN-gamma or anti-IL-12 mAbs, respectively. Thus, the effect of neither IL-12 nor IFN-gamma appeared to be mediated through induction of the other cytokine. In situ hybridization revealed that approximately one-third of BAL T cells expressed mRNA transcripts encoding the IL-12R beta 2 subunit following allergen challenge. Thus, human T cells obtained from BAL during asthmatic late responses, like T cells in the peripheral circulation, remain susceptible to immunomodulation by IL-12. These findings raise the possibility that IL-12 may hold therapeutic potential in allergic diseases such as asthma.

Oral somatic transgene vaccination using attenuated S. typhimurium.

An attenuated strain of S. typhimurium has been used as a vehicle for oral genetic immunization. Eukaryotic expression vectors containing truncated genes of ActA and listeriolysin--two virulence factors of Listeria monocytogenes--have been used to transform S. typhimurium aroA. Multiple or even single oral immunizations with such transformants induced excellent cellular and humoral responses. In addition, protective immunity was induced with listeriolysin transformants. The quality of the responses suggested a transfer of plasmid DNA from the bacterial carrier to the host. Such transfer was unequivocally shown in vitro with primary peritoneal macrophages. We describe a highly versatile system for antigen delivery, identification of protective antigens for vaccination, and efficient generation of antibodies against the product of open reading frames present on virtually any DNA segment.