Bactofection of mammalian cells by Listeria monocytogenes: improvement and mechanism of DNA delivery.

Bacteria-mediated transfer of plasmid DNA into mammalian cells (bactofection) is a potent approach to express plasmid-encoded heterologous proteins (protein antigens, toxins or enzymes) in a large set of different cell types including phagocytic and nonphagocytic mammalian cells. Previously, we have described a Listeria monocytogenes-mediated DNA delivery system, which releases plasmid DNA directly into the cytosol of mammalian cells by partial self-destruction of the carrier bacteria. Here we report on a second generation of this phage lysin supported bactofection system, which is greatly improved with respect to plasmid stability, transfer efficacy and biosafety. In this case, DNA release is initiated by spontaneous bacterial lysis in the infected cells cytosol which is subsequently enhanced by the simultaneously released phage lysin produced by the intracellular carrier bacteria. Bacteria that are capable of cell-to-cell spread are found to be much more efficient in bactofection than their non spreading counterparts.

[Treatment of lichen planus pemphigoides with acitretin and pulsed corticosteroids]. / Therapie des Lichen planus pemphigoides durch Acitretin und pulsweise verabreichtes Kortikosteroid.

A patient with lichen planus pemphigoides first developed multiple pruritic papules and subsequently, tense blisters on trunk and extremities. Histopathologic examination of a skin biopsy demonstrated both the typical changes of lichen planus and subepidermal blisters as in bullous pemphigoid. Direct immunofluorescence microscopy revealed both cytoid bodies and linear C3 deposits at the dermal-epidermal junction. By indirect immunofluorescence microscopy on 1 M NaCl-split-skin, circulating autoantibodies labeled the epidermal side of the split. Immunoblot analysis showed binding of the antibodies to the cell-derived soluble 120 kD domain of the 180 kD bullous pemphigoid antigen and to a recombinant form of the immunodominant NC16A region of this protein. When treated with pulsed intravenous corticosteroids, the patient continued to develop new papules and blisters, but when oral acitretin was added, the skin lesions cleared. The immunoblot reactivity of the patient's autoantibodies well reflected disease activity, while the indirect immunofluorescence microscopy titers did not.

Interaction of Neisseria meningitidis with human dendritic cells.

Infection with Neisseria meningitidis serogroup B is responsible for fatal septicemia and meningococcal meningitis. The severity of disease directly correlates with the production of the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), IL-6, and IL-8. However, the source of these cytokines has not been clearly defined yet. Since bacterial infection involves the activation of dendritic cells (DCs), we analyzed the interaction of N. meningitidis with monocyte-derived DCs. Using N. meningitidis serogroup B wild-type and unencapsulated bacteria, we found that capsule expression significantly impaired neisserial adherence to DCs. In addition, phagocytic killing of the bacteria in the phagosome is reduced by at least 10- to 100-fold. However, all strains induced strong secretion of proinflammatory cytokines TNF-alpha, IL-6, and IL-8 by DCs (at least 1,000-fold at 20 h postinfection [p.i.]), with significantly increased cytokine levels being measurable by as early as 6 h p.i. Levels of IL-1beta, in contrast, were increased only 200- to 400-fold at 20 h p.i. with barely measurable induction at 6 h p.i. Moreover, comparable amounts of cytokines were induced by bacterium-free supernatants of Neisseria cultures containing neisserial lipooligosaccharide as the main factor. Our data suggest that activated DCs may be a significant source of high levels of proinflammatory cytokines in neisserial infection and thereby may contribute to the pathology of meningococcal disease.

Differential expression of Rel/NF-kappaB and octamer factors is a hallmark of the generation and maturation of dendritic cells.

A key feature of maturation of dendritic cells is the down-regulation of antigen-processing and up-regulation of immunostimulatory capacities. To study the differential expression of transcription factors in this process, we investigated the nuclear translocation and DNA binding of Rel/NF-kappaB and octamer factors during in vitro generation and maturation of dendritic cells compared with macrophage development. RelB was the only factor strongly up-regulated during the generation of both immature dendritic cells and macrophages. Cytokine-induced maturation of dendritic cells resulted in an increase in nuclear RelB, p50, p52, and especially c-Rel, whereas cytokine-treated macrophages responded poorly. This up-regulation of NF-kappaB factors did not correlate with lower levels of cytosolic NF-kappaB inhibitors, the IkappaBs. One IkappaB, Bcl-3, was strongly expressed only in mature dendritic cells. Furthermore, generation and maturation of dendritic cells led to a continuous down-regulation of the octamer factor Oct-2, whereas monocytes and macrophages displayed high Oct-2 levels. A similar pattern of maturation-induced changes in transcription factor levels was found in cultured murine epidermal Langerhans cells, suggesting a general physiological significance of these findings. Finally, this pattern of differential activation of Rel and octamer factors appears to be suitable in determining the maturation stage of dendritic cells generated by treatment with different cytokine combinations in vitro. (Blood. 2000;95:277-285)