Escherichia coli ghosts promote innate immune responses in human keratinocytes.

Bacterial ghosts (BGs) as non-living bacterial envelopes devoid of cytoplasmic content with preserved and intact inner and outer membrane structures of their living counterparts have been used to study the ability of their surface components for the induction of antimicrobial peptides and pro-inflammatory cytokines in human primary keratinocytes (KCs). Quantitative real-time PCR analysis revealed that incubation of KCs with BGs generated from wild-type Escherichia coli induced the mRNA expression of antimicrobial psoriasin (S100A7c) in a BGs particle concentration-dependent manner. Using immunoblot analysis we showed that BGs generated from the flagellin-deficient (ΔFliC) E. coli strain NK9375 were as effective as its isogenic wild-type (wt) E. coli strain NK9373 to induce psoriasin expression when normalized to BG particles being taken up by KCs. However, results obtained from endocytic activity of KCs reflect that internalization of BGs is greatly dependent on the presence of flagellin on the surface of BGs. Moreover, BGs derived from wt E. coli NK9373 strongly induced the release of the pro-inflammatory cytokines IL-6 and IL-8, compared to ΔFliC E. coli NK9375 BGs. Taken together, obtained data demonstrate that non-living BGs possessing all bacterial bio-adhesive surface properties in their original state while not posing any infectious threat have the capacity to induce the expression of innate immune modulators and that these responses are partially dependent on the presence of flagellin.

Impact of lactic acid bacteria on oxidative DNA damage in human derived colon cells.

It is assumed that reactive oxygen species (ROS) play a key role in inflammatory bowel diseases and colon cancer and a number of studies indicate that lactic acid bacteria (LAB) possess antioxidant properties and may prevent these diseases. In the present study, we developed a model which allowed us to investigate the prevention of oxidative DNA damage in human derived colon (HT29) cells by LAB. Furthermore, we investigated if these effects correlate with superoxide (O2(-)) resistance of the strains. The protective properties of 55 strains were monitored in single cell gel electrophoresis (SCGE) assays. After preincubation of the cells with LAB (60 min), oxidative damage was induced by exposure to plumbagin (5.0 microM, 120 min) which releases O2(-) or by hydrogen peroxide (50 microM, 10 min); O2(-) resistance was monitored in plate growth inhibition assays. 25 strains (45%) reduced plumbagin induced DNA migration while only few of them (20%) were protective towards hydrogen peroxide induced damage. The strongest effects (up to 60% reduction of O2(-) induced DNA migration) were observed with representatives of the species Streptococcus thermophilus. The prevention of DNA damage in the colon cells by the bacteria did not correlate with their O2(-) resistance. Additional experiments indicate that the reduction of oxidative damage is only seen with viable bacteria but not with heat inactivated cells and that it takes also place when the colon cells are separated from the LAB by permeable filter membranes indicating that the bacteria release ROS protective factors into the medium. Dose response experiments showed that the protection depends on the concentration of the bacteria; significant effects were observed with titers 3 x 10(6-7)cells/ml. Unexpectedly, we found that a substantial fraction of the strains (13%) induced DNA damage in untreated cells, some of them increased also the effects of the ROS generating chemicals. Preliminary experiments with tetramethylbenzidine (TMB) agar indicate that this phenomenon may be due to release of hydrogen peroxide by the bacteria. Overall, our study shows that the impact of LAB on DNA damage in human derived colon cells is ambivalent; while the majority of strains was protective against oxidative damage some of them induced per se pronounced DNA migration. Since the effects were seen with bacterial concentrations which may be reached in the intestinal tract after consumption of fermented milk products, it is likely that the effects we observed in the present study are relevant for humans.

Bacterial Ghosts as antigen and drug delivery system for ocular surface diseases: Effective internalization of Bacterial Ghosts by human conjunctival epithelial cells.

The purpose of the presented investigation was to examine the efficiency of the novel carrier system Bacterial Ghosts (BGs), which are empty bacterial cell envelopes of Gram-negative bacteria to target human conjunctival epithelial cells, as well as to test the endocytic capacity of conjunctival cells after co-incubation with BGs generated from different bacterial species, and to foreclose potential cytotoxic effects caused by BGs. The efficiency of conjunctival cells to internalize BGs was investigated using the Chang conjunctival epithelial cell line and primary human conjunctiva-derived epithelial cells (HCDECs) as in vitro model. A high capacity of HCDECs to functionally internalize BGs was detected with the level of internalization depending on the type of species used for BGs generation. Detailed analysis showed no cytotoxic effect of BGs on HCDECs independently of the used bacterial species. Moreover, co-incubation with BGs did not enhance expression of both MHC class I and class II molecules by HCDECs, but increased expression of ICAM-1. The high rates of BG's internalization by HCDECs with no BG-mediated cytotoxic impact designate this carrier system to be a promising candidate for an ocular surface drug delivery system. BGs could be useful for future therapeutic ocular surface applications and eye-specific disease vaccine development including DNA transfer.

Bacterial ghosts (BGs)--advanced antigen and drug delivery system.

Bacterial ghosts (BGs) are empty bacterial envelopes of Gram-negative bacteria produced by controlled expression of cloned gene E, forming a lysis tunnel structure within the envelope of the living bacteria. BGs are devoid of cytoplasmic content and possess all bacterial bio-adhesive surface properties in their original state while not posing any infectious threat. BGs are ideally suited as an advanced drug delivery system (ADDS) for toxic substances in tumor therapy. The inner space of BGs can be loaded with either single components or combinations of peptides, drugs or DNA which provides an opportunity to design new types of (polyvalent) drug delivery vehicles. Uptake of BGs loaded with Doxorubicin (Dox) by CaCo2 cells led to effective Dox release from endo-lysosomal compartments and accumulation in the nucleus. Viability and proliferative capacity of the cells were significantly decreased (2-3 orders of magnitude) after internalization of Dox loaded BGs as compared to cells incubated with free Dox. The same effect was observed with leukemia cells. Melanoma cells also revealed a high capability to internalize BGs. These results indicate that BGs are able to target a range of types of cancer. BGs have also been investigated as DNA delivery vectors. Studies show DNA loaded BGs are efficiently phagocytosed and internalized by both professional APCs and tumor cells with up to 82% of cells expressing the plasmid-encoded reporter gene. Our studies with BGs as an ADDS system contribute (i) to optimize drug delivery for the treatment of cancer; (ii) define specific conditions for selection and preparation of BG formulations; (iii) and provide a background for the clinical application of BGs in cancer therapy.

The Bacterial Ghost platform system: production and applications.

The Bacterial Ghost (BG) platform technology is an innovative system for vaccine, drug or active substance delivery and for technical applications in white biotechnology. BGs are cell envelopes derived from Gram-negative bacteria. BGs are devoid of all cytoplasmic content but have a preserved cellular morphology including all cell surface structures. Using BGs as delivery vehicles for subunit or DNA-vaccines the particle structure and surface properties of BGs are targeting the carrier itself to primary antigen-presenting cells. Furthermore, BGs exhibit intrinsic adjuvant properties and trigger an enhanced humoral and cellular immune response to the target antigen. Multiple antigens of the native BG envelope and recombinant protein or DNA antigens can be combined in a single type of BG. Antigens can be presented on the inner or outer membrane of the BG as well as in the periplasm that is sealed during BG formation. Drugs or supplements can also be loaded to the internal lumen or periplasmic space of the carrier. BGs are produced by batch fermentation with subsequent product recovery and purification via tangential flow filtration. For safety reasons all residual bacterial DNA is inactivated during the BG production process by the use of staphylococcal nuclease A and/or the treatment with ß-propiolactone. After purification BGs can be stored long-term at ambient room temperature as lyophilized product. The production cycle from the inoculation of the pre-culture to the purified BG concentrate ready for lyophilization does not take longer than a day and thus meets modern criteria of rapid vaccine production rather than keeping large stocks of vaccines. The broad spectrum of possible applications in combination with the comparably low production costs make the BG platform technology a safe and sophisticated product for the targeted delivery of vaccines and active agents as well as carrier of immobilized enzymes for applications in white biotechnology.