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Biomedical subjects

Werner Lubitz

Publications and source records attributed to Werner Lubitz.

At least 19 recordsLinked to original sources
PubMed2006

Bacterial ghosts as a novel advanced targeting system for drug and DNA delivery.

Although there are powerful drugs against infectious diseases and cancer on the market, delivery systems are needed to decrease serious toxic and noncurative side effects. In order to enhance compliance, several delivery systems such as polymeric micro- and nanoparticles, liposomal systems and erythrocyte ghosts have been developed. Bacterial ghosts representing novel advanced delivery and targeting vehicles suitable for the delivery of hydrophobic or water-soluble drugs, are the main focus of this review. They are useful nonliving carriers, as they can carry different active substances in more than one cellular location separately and simultaneously. Bacterial ghosts combine excellent natural or engineered adhesion properties with versatile carrier functions for drugs, proteins and DNA plasmids or DNA minicircles. The simplicity of both bacterial ghost production and packaging of drugs and/or DNA makes them particularly suitable for the use as a delivery system. Further advantages of bacterial ghost delivery vehicles include high bioavailability and a long shelf life without the need of cold-chain storage due to the possibility to freeze-dry the material.

Animals↗
PubMed2005

Comparative immunogenicity of the hepatitis B virus core 149 antigen displayed on the inner and outer membrane of bacterial ghosts.

Two membrane compartments of Escherichia coli ghosts, representing empty bacterial cell envelopes, were investigated as carriers of foreign antigens. By subcutaneous immunisation of mice the immunogenicity of bacterial ghosts carrying the Hepatitis B virus core 149 protein (HBcAg-149) as model antigen anchored either in the inner or the outer membrane of E. coli was compared. Both systems induced significant immune responses against the foreign target antigen, the HBcAg-149, in mice. Results indicate that bacterial ghosts provide an excellent carrier system for antigen delivery.

Animals↗
PubMed2005

Bacterial ghosts as antigen delivery vehicles.

The bacterial ghost system is a novel vaccine delivery system unusual in that it combines excellent natural intrinsic adjuvant properties with versatile carrier functions for foreign antigens. The efficient tropism of bacterial ghosts (BG) for antigen presenting cells promotes the generation of both cellular and humoral responses to heterologous antigens and carrier envelope structures. The simplicity of both BG production and packaging of (multiple) target antigens makes them particularly suitable for use as combination vaccines. Further advantages of BG vaccines include a long shelf-life without the need of cold-chain storage due to their freeze-dried status, they are safe as they do not involve host DNA or live organisms, they exhibit improved potency with regard to target antigens compared to conventional approaches, they are versatile with regards to DNA or protein antigen choice and size, and as a delivery system they offer high bioavailability.

Adjuvants, Immunologic↗
PubMed2005

Immobilization of plasmid DNA in bacterial ghosts.

The development of novel delivery vehicles is crucial for the improvement of DNA vaccine efficiency. In this report, we describe a new platform technology, which is based on the immobilization of plasmid DNA in the cytoplasmic membrane of a bacterial carrier. This technology retains plasmid DNA (Self-Immobilizing Plasmid, pSIP) in the host envelope complex due to a specific protein/DNA interaction during and after protein E-mediated lysis. The resulting bacterial ghosts (empty bacterial envelopes) loaded with pDNA were analyzed in detail by real time PCR assays. We could verify that pSIP plasmids were retained in the pellets of lysed Escherichia coli cultures indicating that they are efficiently anchored in the inner membrane of bacterial ghosts. In contrast, a high percentage of control plasmids that lack essential features of the self-immobilization system were expelled in the culture broth during the lysis process. We believe that the combination of this plasmid immobilization procedure and the protein E-mediated lysis technology represents an efficient in vivo technique for the production of non-living DNA carrier vehicles. In conclusion, we present a "self-loading", non-living bacterial DNA delivery vector for vaccination endowed with intrinsic adjuvant properties of the Gram-negative bacterial cell envelope.

DNA↗
PubMed2005

Detection of single nucleotide polymorphisms in coagulation factor XI deficient patients by multitemperature single-strand conformation polymorphism analysis.

Factor XI (FXI) deficiency is a rare inherited disorder which can cause bleeding complications especially in case of hemostatic challenge and/or in tissues with high fibrinolytic activity. A number of causative mutations have been described in FXI deficient individuals which have been detected by various screening methods. In this study, we present the application of the multitemperature single-strand conformation polymorphism analysis (MSSCP) on the FXI gene, a recently developed methodology for the detection of single nucleotide exchanges. We analyzed a total of 217 polymerase chain reaction (PCR) fragments from the promoter region as well as from exons 1-7 and 11-15 and compared the results to automatic fluorescent sequencing. A total of 29 PCR fragments showed single nucleotide exchanges in conventional fluorescent sequencing, representing 10 different mutations (nine missense mutations, one small deletion) and four frequent polymorphisms. With MSSCP electrophoresis at a standard temperature profile (gel temperature 35-20-10 degrees C) we were able to detect 13 of 14 (93%) different nucleotide exchanges in 25 of 29 PCR fragments (86%). Hence, the detection rate for genetic variations in the FXI gene was 86%. To evaluate the reproducibility, MSSCP was performed twice for 174 PCR fragments and the consistency between two electrophoretic runs was 99%. We conclude that the MSSCP is a sensitive, fast, and cost effective screening method for the detection of FXI gene mutations.

DNA Mutational Analysis↗
PubMed2005

DNA-loaded bacterial ghosts efficiently mediate reporter gene transfer and expression in macrophages.

There is a demand for efficient and safe DNA delivery vehicles mediating gene transfer and expression. We present bacterial ghosts as a novel platform technology for DNA delivery and targeting of macrophages. Bacterial ghosts are cell envelopes of gram-negative bacteria that are devoid of the cytoplasmic content. Escherichia coli ghosts were loaded with plasmid DNA and linear double-stranded DNA. Confocal laser scanning microscopy and flow cytometry confirmed that the DNA localized to the inner lumen of bacterial ghosts and was not associated with the outer surface of the bacteria. Up to approximately 6000 plasmids could be loaded per single ghost and the amount of loaded DNA correlated with the DNA concentration used for loading. E. coli ghosts loaded with plasmids encoding the enhanced green fluorescent protein (EGFP) targeted efficiently murine macrophages (RAW264.7) and mediated effective gene transfer. The EGFP was expressed by more than 60% of the macrophages as measured by flow cytometry detecting the green fluorescence and immunocytochemical staining with antibodies specific for EGFP.

Animals↗
PubMed2005

Bacterial ghosts as an oral vaccine: a single dose of Escherichia coli O157:H7 bacterial ghosts protects mice against lethal challenge.

Enterohemorrhagic Escherichia coli (EHEC) is a bacterial pathogen that is associated with several life-threatening diseases for humans. The combination of protein E-mediated cell lysis to produce EHEC ghosts and staphylococcal nuclease A to degrade DNA was used for the development of an oral EHEC vaccine. The lack of genetic material in the oral EHEC bacterial-ghost vaccine abolished any hazard of horizontal gene transfer of resistance genes or pathogenic islands to resident gut flora. Intragastric immunization of mice with EHEC ghosts without the addition of any adjuvant induced cellular and humoral immunity. Immunized mice challenged at day 55 showed 86% protection against lethal challenge with a heterologous EHEC strain after single-dose oral immunization and 93.3% protection after one booster at day 28, whereas the controls showed 26.7% and 30% survival, respectively. These results indicate that it is possible to develop an efficacious single-dose oral EHEC bacterial-ghost vaccine.

Animals↗
PubMed2005

Delivery of Chlamydia vaccines.

The plethora of ocular, genital and respiratory diseases of Chlamydia, including nongonococcal urethritis, cervicitis pelvic inflammatory disease, ectopic pregnancy, tubal factor infertility, conjunctivitis, blinding trachoma and interstitial pneumonia, and chronic diseases that may include atherosclerosis, multiple sclerosis, adult onset asthma and Alzheimer's disease, still pose a considerable public health challenge to many nations. Although antibiotics are effective against Chlamydia when effectively diagnosed, asymptomatic infections are rampart, making clinical presentation of complications often the first evidence of an infection. Consequently, the current medical opinion is that an effective prophylactic vaccine would constitute the best approach to protect the human population from the most severe consequences of these infections. Clinical and experimental studies have demonstration that Chlamydia immunity in animals and humans is mediated by T cells and a complementary antibody response, and the completion of the genome sequencing of several isolates of Chlamydia is broadening our knowledge of the immunogenic antigens with potential vaccine value. Thus, major advances have been made in defining the essential elements of a potentially effective subunit vaccine design and parameters for evaluation. However, the challenge to develop effective delivery systems and human compatible adjuvants that would boost the immune response to achieve long-lasting protective immunity remains an elusive objective in chlamydial vaccine research. In response to evolving molecular and cellular technologies and novel vaccinology approaches, considerable progress is being made in the construction of novel delivery systems, such as DNA and plasmid expression systems, viral vectors, living and nonliving bacterial delivery systems, the use of chemical adjuvants, lipoprotein constructs and the codelivery of vaccines and specific immuno-modulatory biological agonists targeting receptors for chemokines, Toll-like receptors, and costimulatory molecules. The application of these novel delivery strategies to Chlamydia vaccine design could culminate in timely achievement of an efficacious vaccine.

Animals↗
PubMed2004

Modulation of gene expression by promoter mutants of the lambdacI857/pRM/pR system.

Gene expression driven by the p(R) promoter of the lambdacI857/p(RM)/p(R) system results from inactivation of the temperature-sensitive CI857 repressor. The CI857 repressor, whose gene is transcribed by the divergently orientated p(RM) promoter, is destabilised at temperatures above 30 degrees C. In this study, the lambdacI857/p(RM)/p(R) system was modified by the introduction of a single (A-32G) and a double mutation (A-32G and T-41C). The mutated lambdap(R) expression modules, 32G and 32G/41C, tightly repressed the highly lethal phage PhiX174 lysis gene E at temperatures up to 37 and 39 degrees C, respectively. Expression of protein E and subsequent lysis of Escherichia coli was still induced by a temperature up-shift to 42 degrees C. The impact of the mutations on gene expression levels driven by the lambdap(R) and p(RM) promoters was evaluated at various temperatures using the lacZ reporter gene. Results indicate that the A-32G mutation confers a lambdap(R) promoter-down phenotype. The additional increase in the temperature stability of the 32G/41C expression system is due to the T-41C mutation leading to a higher p(RM) activity. The described lambdap(R) expression modules can be used to obtain a defined expression level at a given temperature and to tightly repress in particular highly lethal genes at different bacterial growth temperatures.

Bacteriophage lambda↗
PubMed2004

T cell-specific immune response induced by bacterial ghosts.

BACKGROUND: Bacterial ghosts, genetically inactivated Gram-negative bacterial pathogens, possess significant advantages over commonly used vaccination technologies. The autolysis of the bacteria, by the expression of a cloned viral gene, results in empty bacterial envelopes through the expulsion of cytoplasmic content. Immunostimulatory properties are generally presented through the targeting of professional antigen-presenting cells (APCs), such as macrophages and dendritic cells (DCs). MATERIAL/METHODS: This study investigated the interactions between porcine antigen-presenting cells and bacterial ghosts derived from the bacterial pathogen Actinobacillus pleuropneumoniae. The maturation process of DCs and their generation of immune responses to bacterial ghosts was shown by the expression of activation markers on their surface, as well as in the functional tests. RESULTS: A population of porcine APCs was generated from PBS by incubation with rpo-GMCSF and rh-IL-4. The cells expressed SWC3, MIL-2, CD80/86 molecules, as well as a high level of MSA3 molecules. The internalization of bacterial ghosts by the cells resulted in increased expression of MSA3 molecules. The capacity of T cells to proliferate when induced by bacterial ghosts was 4 times higher in the cultures including APCs than in cultures stimulated with bacterial ghosts only. CONCLUSIONS: We found that antigen-presenting cells have the capacity to stimulate specific T cells after the internalization and processing of Actinobacillus ghosts, as demonstrated by a strong specific T-cell response generated against the ghost antigens.

Actinobacillus pleuropneumoniae↗
PubMed2004

Bacterial ghost technology for pesticide delivery.

Bacterial ghosts are nondenaturated empty cell envelopes of Gram-negative bacteria produced by E-mediated lysis. Such envelopes from the plant-adhering bacterium Pectobacterium cypripedii were tested for their ability to adhere to plant material and to be used as carriers for pesticide delivery. We show, using fluorescence-labeled P. cypripedii ghosts, that depending on the target plants 55 or 10% (rice or soya, respectively) of the applied bacterial ghosts was retained on the leaves after heavy simulated rain (84 mm). Furthermore, the bacterial ghosts could be loaded with the lipophilic triazole fungicide tebuconazole. In subsequent plant experiments in the glass house, the efficacy of the loaded bacterial ghost for resistance to rainfall and the protective and curative effects against the pathogens Erysiphe graminis, Leptosphaeria nodorum, and Pyrenophora teres on barley and wheat and against Sphaerotheca fuliginea on cucumber were tested. The bacterial ghosts were compared primarily with a commercial tebuconazole formulation, a wettable powder, as it has similar physical characteristics. The comparison revealed similar effects and showed consistently higher or comparable efficacy against the pathogens. The standard operational comparison with the most protective, cereal specific emulsion of oil in water displayed that the bacterial ghosts had equal to or lower efficacy than the emulsion. This study confirmed the potential of bacterial ghost platform technology as a new alternative carrier system for pesticides.

Bacteria↗
PubMed2004

A novel recombinant multisubunit vaccine against Chlamydia.

The administration of an efficacious vaccine is the most effective long-term measure to control the oculogenital infections caused by Chlamydia trachomatis in humans. Chlamydia genome sequencing has identified a number of potential vaccine candidates, and the current challenge is to develop an effective delivery vehicle for induction of a high level of mucosal T and complementary B cell responses. Vibrio cholerae ghosts (VCG) are nontoxic, effective delivery vehicles with potent adjuvant properties, and are capable of inducing both T cell and Ab responses in mucosal tissues. We investigated the hypothesis that rVCG could serve as effective delivery vehicles for single or multiple subunit chlamydial vaccines to induce a high level of protective immunity. rVCG-expressing chlamydial outer membrane proteins were produced by a two-step genetic process, involving cloning of Omp genes in V. cholerae, followed by gene E-mediated lysis of the cells. The immunogenicity and vaccine efficacy of rVCG-expressing single and multiple subunits were compared. Immunologic analysis indicated that i.m. immunization of mice with either vaccine construct induced a strong mucosal and systemic specific Th1 response against the whole chlamydial organism. However, there was an immunogenic advantage associated with the multiple subunit vaccine that induced a higher frequency of Th1 cells and a relatively greater ability to confer protective immunity, compared with the single subunit construct. These results support the operational theory that the ability of a vaccine to confer protective immunity against Chlamydia is a function of the level of Th1 response elicited.

Animals↗
PubMed2004

Bacterial ghosts are an efficient delivery system for DNA vaccines.

Mass implementation of DNA vaccines is hindered by the requirement of high plasmid dosages and poor immunogenicity. We evaluated the capacity of Mannheimia haemolytica ghosts as delivery system for DNA vaccines. In vitro studies showed that bacterial ghosts loaded with a plasmid carrying the green fluorescent protein-encoding gene (pEGFP-N1) are efficiently taken up by APC, thereby leading to high transfection rates (52-60%). Vaccination studies demonstrated that ghost-mediated delivery by intradermal or i.m. route of a eukaryotic expression plasmid containing the gene coding for beta-galactosidase under the control of the CMV immediate early gene promoter (pCMVbeta) stimulates more efficient Ag-specific humoral and cellular (CD4(+) and CD8(+)) immune responses than naked DNA in BALB/c mice. The use of ghosts also allows modulating the major Th response from a mixed Th1/Th2 to a more dominant Th2 pattern. Intravenous immunization with dendritic cells loaded ex vivo with pCMVbeta-containing ghosts also resulted in the elicitation of beta-galactosidase-specific responses. This suggests that dendritic cells play an important role in the stimulation of immune responses when bacterial ghosts are used as a DNA delivery system. Bacterial ghosts not only target the DNA vaccine construct to APC, but also provide a strong danger signal, acting as natural adjuvants, thereby promoting efficient maturation and activation of dendritic cells. Thus, bacterial ghosts constitute a promising technology platform for the development of more efficient DNA vaccines.

Animals↗
PubMed2004

Bacterial ghosts as novel advanced drug delivery systems: antiproliferative activity of loaded doxorubicin in human Caco-2 cells.

Systemic application of anticancer drugs often causes severe toxic side effects. To reduce the undesired effects, advanced drug delivery systems are needed which are based on specific cell targeting vehicles. In this study, bacterial ghosts from Mannheimia haemolytica were used for site-specific delivery of doxorubicin (DOX) to human colorectal adenocarcinoma cells (Caco-2). Bacterial ghosts are non-denatured envelopes of Gram-negative bacteria with fully intact surface structures for specific attachment to mammalian cells. The in vitro release profile of DOX-ghosts demonstrated that the loaded drug was non-covalently associated with the bacterial ghosts and that the drug delivery vehicles themselves represent a slow release system. Adherence studies showed that the M. haemolytica ghosts more efficiently than E. coli ghosts targeted the Caco-2 cells and released the loaded DOX within the cells. Cytotoxicity assays revealed that the DOX-ghosts exhibited potent antiproliferative activities on Caco-2 cells as the DOX associated with ghosts was two magnitude of orders more cytotoxic than free DOX provided in the medium at the same concentrations. Notably, a significant reduction in the cell viability was measured with DOX-ghosts at low DOX concentrations, which had no inhibitory effect when applied as free DOX after incubation for 16 h or when applied at higher concentrations for only 10 min to the cells. As the higher antiproliferative effects of DOX on Caco-2 cells were mediated by the specific drug targeting properties of the bacterial ghosts, the bacterial ghost system represents a novel platform for advanced drug delivery.

Caco-2 Cells↗
PubMed2004

Bacterial ghosts--biological particles as delivery systems for antigens, nucleic acids and drugs.

Despite the exponential rate of discovery of new antigens and DNA vaccines resulting from modern molecular biology and proteomics, the lack of effective delivery technology is a major limiting factor in their application. The bacterial ghost system represents a platform technology for antigen, nucleic acid and drug delivery. Bacterial ghosts have significant advantages over other engineered biological delivery particles, owing to their intrinsic cellular and tissue tropic abilities, ease of production and the fact that they can be stored and processed without the need for refrigeration. These particles have found both veterinary and medical applications for the vaccination and treatment of tumors and various infectious diseases.

Animals↗
PubMed2004

Microarray-based identification of bacteria in clinical samples by solid-phase PCR amplification of 23S ribosomal DNA sequences.

The rapid identification of the bacteria in clinical samples is important for patient management and antimicrobial therapy. We describe a DNA microarray-based PCR approach for the quick detection and identification of bacteria from cervical swab specimens from mares. This on-chip PCR method combines the amplification of a variable region of bacterial 23S ribosomal DNA and the simultaneous sequence-specific detection on a solid phase. The solid phase contains bacterial species-specific primers covalently bound to a glass support. During the solid-phase amplification reaction the polymerase elongates perfectly matched primers and incorporates biotin-labeled nucleotides. The reaction products are visualized by streptavidin-cyanine 5 staining, followed by fluorescence scanning. This procedure successfully identified from pure cultures 22 bacteria that are common causes of abortion and sterility in mares. Using the on-chip PCR method, we also tested 21 cervical swab specimens from mares for the presence of pathogenic bacteria and compared the results with those of conventional bacteriological culture methods. Our method correctly identified the bacteria in 12 cervical swab samples, 8 of which contained more than one bacterial species. Due to the higher sensitivity of the on-chip PCR, this method identified bacteria in five cervical swab samples which were not detected by the conventional identification procedure. Our results show that this method will have great potential to be incorporated into the routine microbiology laboratory.

Abortion, Veterinary↗
PubMed2004

Minicircle DNA immobilized in bacterial ghosts: in vivo production of safe non-viral DNA delivery vehicles.

DNA as an active agent is among the most promising technologies for vaccination and therapy. However, plasmid backbone sequences needed for the production of pDNA in bacteria are dispensable, reduce the efficiency of the DNA agent and, most importantly, represent a biological safety risk. In this report we describe a novel technique where a site-specific recombination system based on the ParA resolvase was applied to a self-immobilizing plasmid system (SIP). In addition, this system was combined with the protein E-specific lysis technology to produce non-living bacterial carrier vehicles loaded with minicircle DNA. The in vivo recombination process completely divided an origin plasmid into a minicircle and a miniplasmid. The replicative miniplasmid containing the origin of replication and the antibiotic resistance gene was lost during the subsequently induced PhiX174 gene E-mediated lysis process, which results in bacterial ghosts. The minicircle DNA was retained in these empty bacterial cell envelopes during the lysis process via the specific interaction of a membrane anchored protein with the minicircle DNA. Using this novel platform technology, a DNA delivery vehicle--consisting of a safe bacterial carrier with known adjuvant properties and minicircle DNA with an optimized safety profile--can be produced in vivo in a continuous process. Furthermore, this study provides the basis for the development of an efficient in vitro minicircle purification process.

Bacteria↗