DE10042853A1 - Detection and influencing of the expression or function of CD95 / CD95L in infections - Google Patents

Abstract

The invention relates to the use of a substance for the detection of CD95 and / or CD95L or of members of the signal transduction cascade of CD95 and / or CD95L for the detection of susceptibilities to diseases which are associated with an infection. Furthermore, the invention encompasses the use of an active ingredient for the prevention and treatment of infections, in particular bacterial infections, the active ingredient influencing the expression and / or the function of CD95 and / or CD95L or members of the signal transduction cascade of CD95 and / or CD95L and thereby induces apoptosis in infected cells.

Description

The present invention relates to the use of a Substance for the detection of CD95 and / or CD95L (Fas and / or FasL) or by members of the signal transduction cascade of CD95 and / or CD95L and the use of an active ingredient for Influencing CD95 and / or CD95L for prevention and prevention act of diseases with a degraded Resistance to infections, especially bacterial infections tion related.

A variety of pathogens (parasites, bacteria, viruses) penetrate cells in the infected organism and withdraw thus gaining access through the immune system. In the cells then multiply to after destruction of the host cell to infect other cells. In this way, an explo guaranteed multiplication of the pathogens, the last endangered the health and survival of the host organism.  

Various pathogenic germs, such as some bacteria, viruses and Parasites drive the infected host cells into the programmed cell death (apoptosis) (1, 2). A paradigm for bacteria-induced apoptosis is Shigella flexneri, which according to Invasion induces apoptosis by activating caspases (3). Further investigations covered the triggering of apoptosis by other pathogens, such as Salmonella typhimurium, enteropathogenic Escherichia coli, Listeria monocytogenes and Staphylococcus aureus (4-7). The molecular mechanisms which mediate apoptosis and its meaning for the The course of the disease has so far been largely unclear.

Accordingly, the invention has the task of Clarification of molecular mechanisms new diagnostic and Finding treatment approaches and providing active ingredients len for therapeutic and diagnostic use can be used for infections.

One of the clinically most important bacteria is the bacterium Pseudomonas aeruginosa, as there are various infections, especially pneumonia and sepsis in cystic Fibrosis and immune deficient patients. there it is often a serious, life-threatening infection lungs in particular.

Surprisingly, the example of this bacterium could Mechanism through which the infected are shown Host cells driven into programmed cell death and thus the pathogens themselves are destroyed.

Accordingly, the object of the invention Subject matter of independent claims 1-4, 13, 17 and 21 solved. Preferred designs are in the dependent Claims 5-12, 14-16, 18-20 and 22 called. The content  all of these claims are hereby incorporated by reference Content of the description made.

According to the invention, at least one active ingredient can be used for prevention and treatment of infections, especially bacterial Infections, used in eukaryotic cells. This active substance influences expression after application and / or the function of CD95 and / or CD95L or members the signal transduction cascade of CD95 and / or CD95L, in particular to stimulate the the signal transduction cascade comes. This results in induction of apoptosis in the infected cells. It can with this active ingredient such. B. by genetically modified mutants of CD95 and / or CD95L or of in particular downstream members of the signal transduction casas kade of CD95 and / or CD95L. It is Z. B. possible with the help of genetic engineering to produce active ingredients that either a) a higher activity than their physiological Equivalents, or b) no interaction with theirs physiological inhibitors (e.g. I-CAD) can enter. Another option is to provide the course occurring signal elements. Inhibitors of the Signal transduction cascade are inhibited, so activation of the corresponding signal transduction cascade de to enable.

According to the invention, at least one substance can be used for detection the expression and / or function of CD95 and / or CD95L or from members of the CD95 signal transduction cascade and / or CD95L can be used in eukaryotic cells. It is particularly concerned with the detection of vulnerabilities against diseases associated with an infection, in particular a bacterial infection, ge reconsidered. This can be, for. B. the detection of genetic  Dispositions for certain diseases as a result of Mutations e.g. B. on the CD95 receptor or on the CD95 ligand, act. It may be vulnerable to this diagnosed over certain pathogens, so that at corresponding symptoms quickly concluded from an infection can be sen. So z. B. started with the therapy before bronchoalveolar or trachial lavage is made. This method is done by rinsing with physiological saline cell material from cavities or hollow organs (e.g. bronchi, digestive tract, Douglas Space and bladder) won, processed, and at Infek microbiologically examined. In the case of the substance that Proof of expression and / or function is used, can it also z. B. is an antibody against CD95 and / or CD95L or against members of the signal transducer tion cascade of CD95 and / or CD95L, and in a detection method known to the expert, such as. B. ELISA (enzyme-linked immunosorbent assay) can be used can. Other methods, such as a known target protein can be demonstrated quantitatively, are the expert common.

A method for the prevention and treatment of Infections, especially bacterial infections, bean sprucht. In this method, an active ingredient influences the Expression and / or function of CD95 and / or CD95L or by members of the CD95 signal transduction cascade and / or CD95L in eukaryotic cells. The Stimulates induction of apoptosis in the infected cells, so that the pathogens are destroyed and consequently too the infection subsides.  

According to the invention, a method for the detection of Susceptibility to diseases with an infectious disease on, especially related to a bacterial infection stand, claimed. In this procedure, a substance exhibits the expression and / or function of CD95 and / or CD95L or by members of the CD95 signal transduction cascade and / or CD95L, so that based on this proof a statement can be made early as to whether due to e.g. B. a genetic defect an increased susceptibility to about infectious diseases. So it could e.g. B. due to a defect in the CD95 receptor and / or in the CD95 Ligands or on stimulators of the CD95 / CD95L system no apoptosis occur, so infected cells don't can be fought and those suffering from sepsis die. Furthermore, e.g. B. a genetic defect in the acid Sphingomyelinase, a known activator of the CD95 / CD95L- System, the activation of the CD95 / CD95L system and consequently prevent the apoptosis of infected cells with the known consequences for the infected organism.

In a preferred embodiment of the invention, the members of the signal transduction cascade of CD95 and / or CD95L are in particular JNK, NFκB, PLA ₂ , FAD, Caspase 8, Caspase 3, CAD and / or I-CAD. The CD95L (FasL) recognized by CD95 (Fas) is known to be a homotrimer. When CD95L binds to CD95, it induces the trimerization of the receptor, which then brings its so-called death domains together in the cytoplasmic part. The above-mentioned downstream members of the CD95 / CD95L signal transduction cascade either bind directly to these death domains or are activated by proteins bound to them. CAD is activated as one of the last members of this chain, which then migrates into the cell nucleus and splits the DNA present there into the 200 base pair fragments that are characteristic of apoptosis. The invention allows the conclusion that by activating this cascade it is possible to drive infected cells into apoptosis and consequently to fight the infection.

In a preferred embodiment of the invention, the Active ingredient or the substance against CD95 and / or CD95L or against members of the CD95 signal transduction cascade and / or CD95L directed. It can be under other about antisense sequences, genetically modified Mutants of the members of the signal cascade, so-called "small molecular compounds ", or polynucleotides, which for a Encode peptide which expresses CD95 and / or CD95L or the expression of members of CD95 and / or CD95L influenced, act. In particular, you could known sequences from CD95 and / or CD95L or from members the signal transduction cascade of CD95 and / or CD95L in vectors with strong promoters to create a new one Get expression of the desired molecules. Also one Use of peptides or polypeptides is fundamental possible.

In another preferred embodiment of the invention is the active ingredient or the substance against activators, Inhibitors, regulators and / or biological precursors of CD95 and / or CD95L or by members of the signal transducer tion cascade directed by CD95 and / or CD95L. These assets gates, inhibitors, regulators and / or biological precursors fer can z. B. Transcription factors for the express responsible levels, ceramides and other known ones Activators of the CD95 / CD95L system, but also until now unknown molecules caused by the active ingredient or the sub be influenced and on the expression and / or  Function of CD95 and / or CD95L or members of the signal transduction cascade are involved.

In the present invention, it is possible to use known ones as well to use unknown drugs. With one particularly preferred embodiment, the active ingredient is a so-called "Small molecular compound", especially one with a molecular weight (MG) <1000. In this small molecule Lar compound can be z. B. aminoglycosides such. B. Gentamicin (MG 477.60) act, a group of water-soluble Chen basic aminoglycoside antibiotics from micromonospora purpurea and Mikromonospora echinospora. The gentamicine are, like many other antibiotics in this class, as "pseudo Trisaccharides ". One of their mechanisms of action, Inhibition of protein biosynthesis by binding to the 30S Subunit of bacterial ribosomes, is the same as in the other aminoglycoside antibiotics. Also activated Gentamicin, like Geneticin, another aminoglycoside, the acidic sphingomyelinase, which after this cellular Activation via the CD95 receptor to the outside of the cell membrane transported and there over the released ceramide the so-called receptor capping, d. H. a local concentra tion or aggregation of a specific receptor molecule, taught. This so-called capping leads to a high Receptor density, which in turn in a high concentration of Signaling molecules associated with the receptor intracellularly ren, results. This will be an effective signal transducer tion and a strong activation of the cell via the respective gene receptor possible, which ultimately mediates with CD95 / CD95L ter signal transmission can lead to apoptosis. Also Peptides, polypeptides, polynucleotides or others to the person skilled in the art known active ingredients can be used.  

The invention can be used to make all forms of cranes kungen with an infection, especially a bacteri related infection, detect or related to treat. In particular, these are bacterial Infections with pseudomonas pathogens. It's through this Invention but also all other diseases caused by Induction of apoptosis are treatable.

According to the invention, the active ingredient or the substance can both administered orally, intravenously, topically and / or by inhalation become. The form of administration as well as the dose can be free to get voted. As is known, play in particular Age, gender, as well as other patient factors Role. Also the clinical picture, as well as type and degree of Infection must be taken into account.

The invention further comprises a pharmaceutical combination setting that contains at least one active ingredient that Expression and / or function of CD95 and / or CD95L or of Members of the CD95 and / or signal transduction cascade CD95L influenced, in particular stimulated and possibly a pharmaceutical carrier. It can be the Active ingredient is a polynucleotide, which is a peptide, preferably encodes a polypeptide, which peptide is the Expression of CD95 and / or CD95L or of members of the Signal transduction cascade influenced by CD95 and / or CD95L flows, preferably stimulated. The active ingredient can also be a Be a peptide or a polypeptide, or a small molecular compound, such as the aminoglycosides already mentioned above. Finally, the active ingredient can also be a act so-called antisense sequence, d. H. a sequence that is able to make a double strand duplex with the mRNA form, thereby translating into a polypeptide prevent. The sequence of CD95 and / or CD95L could also be used  themselves can be used to overexpress these proteins to achieve z. B. by incorporation into vectors with strong Promoters. For further features of such a set is referred to the corresponding previous text of the Be spelling referenced.

The invention further comprises a pharmaceutical combination setting, the effective amount of at least one active ingredient has the expression and / or function of Aktivato ren, inhibitors, regulators and / or biological precursors far from CD95 and / or CD95L or from members of the signal transduction cascade influenced by CD95 and / or CD95L, preferably stimulated. This pharmaceutical composition if necessary, a pharmaceutical carrier contain. The activators, inhibitors, regulators and / or biological precursors of CD95 and / or CD95L can e.g. B. the Ceramides, transcription factors already mentioned above, Kinases, as well as other known or as yet unknown Members of the CD95 and / or signal transduction cascade Be CD95L. Also polynucleotides that encode a peptide which expresses activators, inhibitors, regulators gates and / or biological precursors of CD95 and / or CD95L or from members of the signal transduction cascade of CD95 and / or CD95L influenced, preferably stimulated, may be included in such compositions. Also so-called small molecular compounds, which are preferably a Molecular weight (MW) of <1000, and those against assets gates, inhibitors, regulators and / or biological precursors fer from CD95 and / or CD95L or from members of Signal CD95 and / or CD95L transduction cascade, and thereby the expression or function of these proteins stimulate, can be used.  

A number of substances are used today to combat Infections used. This is often used before Substances were tested for resistance. Doing so the sensitivity or resistance of pathogenic germs against the substance in question. So with one Test method in solid or liquid culture media Chemothe rapeutics or antibiotics dissolved, and on these nutrient media the pathogenic germs are coated. After the evaluation this test z. B. regarding specificity and the we efficiency can be decided whether the examined Chemotherapy drug or antibiotic is used. There are In recent years, however, resistant pathogens have increased Germs have occurred and this is a growing problem with treating infections. With the invention Approach can now appear to be unsuitable Chemotherapeutics or antibiotics for a therapeutic to make the application accessible and against such, so far germs classified as resistant.

Finally, the invention also includes a diagnostic kit. This diagnostic kit includes at least one substance that is used for Evidence of expression and / or function of CD95 and / or CD95L or from members of the signal transduction cascade of CD95 and / or CD95L is suitable for detecting vulnerabilities against diseases associated with an infection, especially a bacterial infection stand. Such diagnostics can be targeted in a diagnostic kit are used to identify vulnerabilities to Pseudomonadenerregern.

The features mentioned and other features of the invention result from the following description of preferred th embodiments in connection with the subclaims and figures. Here, the individual characteristics can  realized individually or in combination with each other be light.

The pictures show:

Fig. 1: Pseudomonas aeruginosa induces apoptosis of epithelial cells via CD95 / CD95L

Fig. 2: Pseudomonas aeruginosa infection induces apoptosis via upregulation and activation of CD95 and CD95L on the cell surface

Fig. 3: Bacterially induced apoptosis of epithelial cells protects animals from Pseudomonas aeruginosa-induced sepsis and death

Fig. 4: Apoptosis of lung epithelial cells, but not of immune cells, is crucial for the host response against Pseudo monas aeruginosa

Fig. 5: Different Pseudomonas aeruginosa strains invad ren human epithelial cells

Fig. 6: Pseudomonas aeruginosa invasion into human epithelial cells correlates with an activation of the acid sphingomyelinase

Fig. 7: Genetic deficiency of acid sphingomyelinase prevents invasion of Pseudomonas aeruginosa into human cells

Fig. 8: Activation of the acid sphingomyelinase enables invasion of Pseudomonas aeruginosa in cells with a mutated CFTR molecule. Untreated cystic fibrosis cells almost do not take up Pseudomonas aeruginosa.

material and methods

All cells were cultured in DMEM medium, which with 10% FCS, 2 mM L-glutamine, 100 U / ml penicillin and 100 µg / ml Streptomycin was added. Freshly isolated Fibrobla Most were cultivated for 8 days.

Caspase 8 or -3 activity was determined after lysing the cells in 25 mM HEPES (pH 7.4), 0.2% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 125 mM NaCl, 10 mM sodium fluoride, 10 mM EDTA, 10 mM Na ₃ VO ₄ , 10 mM sodium pyrophosphate and 100 ug / ml aprotinin and leupeptin measured. For the cytochrome C measurement, the cells were removed after 30 min. Incubation on ice in a 200 ul solution consisting of 220 mM mannitol, 68 mM sucrose, 50 mM PIPES-KOH (pH 7.4), 50 mM KCl, 5 mM EGTA, 2 mM MgCl ₂ , 1 mM DTT, 10 µM Homogenized cytochalasin B, 10 µg / ml aprotinin and leupeptin. After centrifugation, the supernatants were separated on a 15% SDS-PAGE gel, blotted and in the presence of 0.5 µg / ml Anti-Caspase-8 (Pharmingen) or Anti-Caspase-3 (Transduction Lab.), Or 1 µg / ml of an anti-cytochrome C (clone 7H8.2C12, Pharmingen) antibody developed, followed by HRP (horse radish peroxidase) coupled protein G and electrochemiluminescence.

In order to detect bacteria in the spleen or lungs, the organs homogenized in 300 µl sterile RPMI, 100 µl this homogenate is applied to an agar plate Cultivated at night and the next day the Pseudomonas aerugi nosa colonies counted.  

Mice were irradiated with 12 Gy and after 3-4 days BMCs transplanted. After a recovery period of 10 weeks the experiments were carried out. Before and after loading the mice were kept sterile at all times.

Results Experiment 1

To gain an insight into the molecular mechanisms, via the Pseudomonas aeruginosa with the host cells intera giert, it was examined whether the triggering of apoptosis for cytotoxicity of Pseudomonas aeruginosa described so far (8) contributes. Human Chang conjunctiva and WI- 38 lung epithelial cells with Pseudomonas aeruginosa strains 762, 769, ATCC 27853, PAO-1 and 696 (cell-bacteria ratio nis 1: 1000) infected. The duration of infection was 30 min. For 762, 90 min. each for 769 or ATCC 27853 and 6 hours for 696 and PAO-1. Apoptosis was diagnosed using DNA fragments determined and via FACS analysis after staining the Cells with FITC-Annexin V or ethidium bromide / propidium iodide or analysis of typical morphological changes in the Fluorescence microscope of ethidium bromide / propidium iodide stained cells confirmed. The mean values ± SD are shown of three independent experiments (* p <0.05, t-test).

The results of Fig. 1A show that actually pronounced apoptosis of human Chang conjunctiva or WI-38 lung epithelial cells occurs within 20 to 360 min. after infection with two well-defined strains of Pseudomonas aeruginosa ATCC 27853 and PAO-1, as well as with clinical isolates 696, 762 and 769 from sputum or urine.

In search of possible mechanisms by which pseudo monas aeruginosa infection triggers apoptosis, the involvement of CD95 receptors / CD95 ligands, one of the most important systems for triggering apoptosis in mammalian cells, was examined (9). For this purpose, pulmonary fibroblasts from normal, GLD or LPR C3H mice were infected as described above and the apoptosis was determined. Since the LPR and GLD mice do not have a functional CD95 / CD95L system (10), the influence of this system on Pseudomonas aeruginosa-induced apoptosis can be examined. The infection shows ( Fig. 1B) that LPR or GLD fibroblasts show almost complete resistance to Pseudomonas aeruginosa-induced apoptosis, while normal fibroblasts are highly susceptible to apoptosis. This shows that Pseudo monas aeruginosa induces apoptosis of mammalian cells preferably via the CD95 / CD95L system.

Since fibroblasts are not the primary in vivo target for Pseudo monas aeruginosa, human chang epithelial cells were examined. In order to switch off the CD95 / CD95L system, recombinant CD95-Fc protein was added, which binds to CD95 and thereby prevents interaction with endogenous ligands (11). Fig. 1B shows that the neutralization of the CD95 / CD95L system by adding 5 ug / ml CD95-Fc protein to human epithelial cells or genetic deficiency on CD95 or CD95L in ex vivo fibroblasts of LPR or GLD mice almost Pseudomonas aeruginosa 762, 769 or ATCC 27853 completely prevented apoptosis. In contrast, otherwise isogenic normal mice were extremely sensitive to induction of apoptosis ( Fig. 1B). This finding confirms the crucial role of the CD95 / CD95L system in triggering cellular apoptosis by Pseumonas aeruginosa.

Experiment 2

Because previous research has shown that the CD95 / CD95L- System can be up-regulated by a series of stimuli (12-14) was examined using flow cytometry, whether Pseudomonas aeruginosa infections the expression of Receptor-ligand pair on the surface of Chang, WI- 38 cells or ex vivo fibroblasts increases. For this purpose Chang cells with Pseudomonas aeruginosa 762 for 15 min. infected and the surface expression of CD95 or CD95 Ligand using flow cytometry after incubation with FITC-labeled monoclonal anti-human CD95 antibodies (CH11, UBI) and anti-human CD95 ligand antibodies (NOK1, Pharmingen) measured. Controls were labeled with FITC Antibodies of the same isotype were carried out. Are shown representative results of four independent Experiments. Similar results were seen after infection with Pseudomonas aeruginosa 769 or ATCC 27853 or according to Infek tion with WI-38 cells or ex vivo mouse fibroblasts Pseudomonas aeruginosa 762 obtained.

Uninfected cells do not express any CD95 or CD95L on the surface. In contrast, Chang showed epithelial cells WI-38 ( Fig. 2A) and ex vivo fibroblasts (2B) infected with Pseudomonas aeruginosa 762, 769 or ATCC 27853 within 15 min. massive expression of the CD95 and the CD95 ligand on the surface. The intracellular localization of the CD95 or CD95L before stimulation could be demonstrated by staining with 0.1% permeabilized cells and semi-quantitative PCR.

In a next step, the typical signal elements the CD95 activation (15) examined. Chang- Epithelial cells with the respective Pseudomonas aeruginosa  Strains infected, and activation of caspase 8 or 3 was based on the occurrence of 18 and 17 kD active frag ment determined. Stable transfection of Chang cells with CrmA (SRSα-crmA) almost completely inhibited that caused by Pseudomo nas aeruginosa-induced death of human Chang cells as well as the cytochrome C release. Transfection of a vector Control (SRSα) had no corresponding influence. Ähnli Results were obtained for WI-38 cells (not ge shows). The mean values ± SD, n = 3 (* p <0.05, t- test). All immunoblots were repeated twice.

The results show a very rapid activation of caspase 8 and caspase 3 by Pseudomonas aeruginosa 762, 769 or ATCC 27853 ( FIG. 2C). Strains 696 or PAO-1 activated caspase 8 and 3 within 3-4 hours of infection. Transfection of Chang and WI-38 cells with an expression vector for CrmA, a viral serpin, which predominantly inhibits caspase 8-like enzymes and thus CD95-mediated apoptosis, almost completely prevented Pseudomonas aeruginosa-induced apoptosis ( FIG. 2C ). In the same way, the release of cytochrome C triggered by infection with Pseudomonas aeruginosa was suppressed by CrmA expression ( FIG. 2C).

Experiment 3

To test the importance of Pseudomonas aeruginosa-induced apoptosis in vivo, LPR, GLD or isogenic wild-type mice were infected intranasally with Pseudomonas aeruginosa 762 or ATCC 27853 (not shown) (16) and the triggering of apoptosis in lung epithelial cells by TUNEL staining certainly. For this purpose, isogenic normal, GLD or LPR C3H mice (12 each) were infected by nasal application of 7 × 10 ⁸ or 13 × 10 ⁹ Colony Forming Units (CFU) Pseudomonas aeruginosa strains 762 or ATCC 27853 (volume 15 μl). Typical results are shown with TUNEL staining from formalin-fixed and parrafin-embedded sections of mice infected with Pseudomonas aeruginosa 762 for 6 hours. Normal mice, but not LPR or GLD mice, showed apoptosis of lung epithelial cells, especially in small bronchi, as early as 3 hours after infection and with a maximum of 6-12 hours after the onset of infection. ATCC 27853 infection resulted in an almost identical image (not shown).

The results show that normal mouse infections resulted in rapid (within 3-4 hours) induction of apoptosis in bronchial epithelial cells ( Fig. 3A). In contrast, no evidence of apoptosis could be found in infected LPR or GLD mice. A small portion of the lungs were excised, weighed, carefully dissected, and then cultured on agar plates after all experiments. Very similar amounts of pathogens were found in the lungs of all infected mice (4 × 10 ⁵ ± 0.5 × 10 ⁵ , 5 × 10 ⁶ ± 1 × 10 ⁵ , 4.7 × 10 ⁵ ± 0.8 × 10 ⁵ Bacteria / g lung tissue in normal, LPR or GLD mice).

To investigate whether apoptosis of lung epithelial cells in Pseudomonas aeruginosa infection is part of the immune system or for bacterial infiltration, normal, LPR or GLD mice infected with Pseudomonas aeruginosa and the development of sepsis and the survival of the animals underneath examined. For this purpose, 12 mice were used as described above infected and continuous over 7 days after infection observed or killed after 36 hours for bacterial sepsis to check. The relationship of Pseudomonas is shown aeruginosa CFUs (colony forming units) produced by the spleen  were taken from CFUs derived from lung tissue have been shown to influence variations in the infection process to rule out.

The results show ( Fig. 3B) that only a few of the wild-type C57 / BL6 mice developed sepsis at this infection dose and only 10% of the animals died within the first 6 days after infection. In contrast, all LPR and GLD mice developed sepsis and 100% of the animals died within 96 hours of infection. The course correlated with Pseudomonas aeruginosa spleen colonization in LPR and GLD mice, but not in control animals ( FIG. 3B), as could be shown by transferring homogenized spleen tissue to agar plates and measuring CFU the next morning. Control experiments with 4 mice, each killed after 6 hours, showed that the lungs of all mice had similar amounts of pathogens (4-6 × 10 ⁵ bacteria / g lung). These results suggest that the high sensitivity of LPR or GLD mice to Pseudomonas aeruginosa infection is related to the inability to apoptose lung epithelial cells.

Experiment 4

Since the high sensitivity of the mice to Pseudomonas aerugi nosa infection could also have been caused by a defective immune system that allows generalized infection with the pathogens, the immune system of normal, GLD or LPR mice was destroyed by radiation and the immune system the wild-type mice were replaced by bone marrow cells (BMC) from the syngeneic LPR or GLD mice. The immune system of LPR and GLD mice was replaced with BMC from the syngeneic normal mice. For this purpose, 21 LPR, GLD or normal mice were irradiated with 12 Gy and then transplanted with 2 × 10 ⁵ BMC from normal mice or LPR or GLD mice. Irradiated animals were marked with an asterisk *, and the transplanted BMCs are identified after the dash (/). After a recovery period of 10 weeks, the animals were infected with Pseudomonas aeruginosa 762, as described under Experiment 3, and the survival of the animals and the development of sepsis were determined (12 animals each) ( FIG. 4A). The animals behaved independently of their immune system: LPR or GLD mice that had been transplanted with normal BMC died or developed sepsis like the non-transplanted control animals. In contrast, normal mice transplanted with BMC from LPR or GLD mice were still resistant to Pseudomonas aeruginosa infection. Representative TUNEL assays from 12 animals killed 6 hours after infection each showed apoptosis of lung epithelial cells in normal mice transplanted with LPR or GLD BMC ( Figure 4B). LPR or GLD mice transplanted with normal BMC behaved like non-transplanted controls and showed no apoptotic lung epithelial cells after infection ( Fig. 4C).

Stimulation of ex vivo splenic lymphocytes for 2d with 10 µg / ml anti-CD3 antibody 2C11 triggers apoptosis of all lymphocytes Animals that have been transplanted with BMC normal mice were. BMC transplantation from LPR or GLD mice prevents activation-induced cell death, a hint on the successful transplant, the means is shown worth ± SD of 24 mice. Each individual animal had a radio tioning graft.  

As the results show, cross transplantation of BMC had no effect on the development of Pseudomonas aeruginosa infection in normal mice transplanted with BMC from LPR or GLD mice. The normal cross-transplanted mice showed the same resistance as non-transplanted normal mice ( Fig. 4A). Likewise, transplanted LPR or GLD mice with normal BMCs were just as sensitive to Pseudomonas aeruginosa as untransplanted LPR or GLD mice and died of sepsis. In accordance with this observation, apoptosis of lung epithelial cells was only observed in normal mice, regardless of whether or not they had been transplanted with BMC from LPR or GLD mice ( Fig. 4B). LPR or GLD mice remained resistant to apoptosis in lung pithelial cells despite transplantation with normal BMC ( Fig. 4C). Successful transplantation was verified by the detection of activation-induced cell death (AICD) in ex vivo splenic lymphocytes ( FIG. 4D). Since the AICD requires the functional expression of the CD95 and CD95L (17), the normal positive response in the LPR or GLD mice with transplanted normal BMC and the lack of response in normal mice with transplanted LPR or GLD BMC served as evidence for the successful transplant.

The present experimental data show that there is direct genetic evidence for the necessary role of the CD95 / CD95L system in the in vivo and in vitro induction of apoptosis in mammalian lung epithelial cells after infection with Pseudomonas aeruginosa. Several studies showed the cytotoxicity of Pseudomonas aeruginosa, which was prevented by deletion of the regulatory exsA gene (18). The mechanisms that trigger cell death have so far been unclear. The present findings show that the upregulation of the CD95 / CD95L is one of the most important mechanisms that trigger cytotoxicity. It is also shown that induction of apoptosis is crucial for the host's defense against Pseudomonas aeruginosa. Apoptosis could be beneficial because apoptotic death of infected cells enables phagocytosis of the bacteria-infected cells by other cells. Phagocytosis of Pseudomonas aeruginosa could enable phagocytic cells to fuse the bacterial phagosome with lysosomes, whose enzymes break down the pathogens. Since apoptosis does not lead to local inflammation, this mechanism of the epithelial cells, together with the phagocytes and other immune mechanisms, allows the pathogens to be removed efficiently from the lungs. In contrast, internalization of Pseudomonas aeruginosa without apoptosis of the host cell (as with LGR or GLD) allows the bacterium to prevent the maturation of the phagosome and the formation of a phagolyso soms. This enables the pathogen to survive and even multiply before it becomes transcytotic. Such intracellular localization of Pseudomonas aeruginosa in cells which are unable to die apoptotically protects the pathogen from the host's immune defense. In addition to this mechanism, stimulation of the CD95 receptor leads to activation of NFκB (19), JNK (20), GADD 153 (21), PLA ₂ (22) or interferes with functions of growth factor receptors (23), which secrete the secretion of Could stimulate cytokines and / or defensins of epithelial cells. Together with the breakdown of Pseudomonas aeruginosa in apoptotic bodies, this mechanism could mediate the defense against Pseudo monas aeruginosa.

Experiment 5

In a next approach, factors that the CD95 / CD95L system, and thus the apoptosis, can activate. It was found that the so-called capping, a local concentration or aggregation of a certain Receptor molecule, the CD95 receptor by the acidic sphingo myelinase or the released ceramide can be mediated can. The capping is done by stimulating cells induced via the corresponding receptor. genetic Deficiency of the acid sphingomyelinase prevents capping of the CD95 receptor.

First, human lung and conjunctival epithelial cells were infected with various Pseudomonas aeruginosa strains and the internalization of the bacteria was measured after 30 minutes of infection by staining with crystal violet. It was found that different human cells (lung and conjunctival epithelial cells, fibroblasts) can be internalized with different infectious Pseudomonas aeruginosa strains ( FIG. 5).

Experiment 6

In a next step, the activity of the acid sphingomyelinase in cell lysates of infected or non-infected cells was determined on the basis of the breakdown of [ ¹⁴ C] sphingomyelin. Tests with immunoprecipitates showed an almost identical result. It was found that the invasion of Pseudomonas aeruginosa into human conjunctival epithelial cells correlated with an activation of the acidic sphingomyelinase ( FIG. 6). The activity of the acid sphingomyelinase was measured in cell lysates and immunoprecipitates of the enzyme. These data from three independent experiments each indicate an extremely important role of acid sphingomyelinase in the internalization of Pseudomonas aeruginosa.

Experiment 7

The crucial role of acidic sphingomyelinase for the invasion of Pseudomonas aeruginosa was demonstrated in experiments with fibroblasts obtained from patients with Niemann Pick Type A disease. These cells have a genetic deficiency of the acid sphingomyelinase and have less than 1% acid sphingomyelinase residual activity compared to normal cells. The invasion of the Pseudomo naden was determined in a so-called polymyxin assay. After a 30-minute infection of normal fibroblasts (+) and Niemann Pick Type A fibroblasts that do not express acidic sphingomyelinase (-), the cells were treated with polymyxin. Since polymyxin is not absorbed into mammalian cells, only extracellular bacteria are killed. After a two-hour incubation period, the polymyxin is washed away, the mammalian cells are broken up with a very mild lysis buffer, the lysates are spread on an agar plate and the number of Pseudomonas aeruginosa colonies that have grown after 24 hours of incubation is determined. It was found that acidic sphingomyelinase deficient cells are hardly able to internalize Pseudomonas aeruginosa, while normal fibroblasts internalize Pseudomonas aerugi nosa very well ( FIG. 7).

Experiment 8

In a next approach, lymphocyte cells that express a mutated CFTR molecule and therefore only very poorly internalize Pseudomonas aeruginosa were examined. For this purpose, lymphocyte cells with a mutated CFTR molecule and normal fibroblasts with an invasive Pseudo monas aeruginosa strain for 30 min. infected. During this time the cells were treated with a pharmaceutical, such as. B. Genta micin, which activates the acid sphingomyelinase, incubated (+) or left untreated (-). It was shown ( FIG. 8) that untreated L cells only take up very few bacteria, while the incubation with the drug strongly stimulates the invasion of Pseudomonas aeruginosa. The invasion efficiency in pharmacon-treated cells is comparable to that of normal fibroblasts. Thus, the invasion of Pseudomonas aeruginosa in CFTR cells was normalized by the drug. This shows that the activation of the acid sphingomyelinase alone is sufficient for the invasion of the bacteria. A mutated and thus inactive CFTR molecule can be avoided by direct stimulation of the acid sphingomyelinase.

From these experimental data it follows that the acidic sphin gomyelinase both in vivo and in vitro by a Pharmakon can be activated. The activation leads to a translocation of the acid sphingomyelinase to the Outside of the cell membrane. The acidic sphingomyeli localized nose like the CD95 receptor in the cap structure. There it mediates receptor capping via ceramide. The Capping then leads to high receptor density, which again to in a high concentration of signaling molecules that with intracellularly associate with the receptor (CD95) results. This makes effective signal transduction and  strong activation of the cell possible, which when activating the CD95 / CD95L systems in the internalization of Pseudomonas aeruginosa and consequently in the apoptosis of the infected cell empties.

LITERATURE

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Claims (22)

1. Use of an active ingredient for prevention or treatment infection, especially bacterial infections tions, wherein the active ingredient expression and / or Function of CD95 and / or CD95L or of members the signal transduction cascade of CD95 and / or CD95L in eukaryotic cells, particularly stimulates, and thereby apoptosis in infected Cells induced. 2. Use of a substance for the detection of expression and / or function of CD95 and / or CD95L or of Members of the CD95 signal transduction cascade and / or CD95L in eukaryotic cells, for recognition of susceptibility to diseases with a Infection, especially a bacterial infection Related.   3. Procedures for preventing or treating infectious diseases NEN, especially bacterial infections characterized in that eukaryotic cells with a Treated drug, the expression and / or Function of CD95 and / or CD95L or of members the signal transduction cascade of CD95 and / or CD95L influenced, in particular stimulated, and thereby the Apoptosis induced in infected cells. 4. Procedures to identify vulnerabilities to Diseases associated with infection, in particular a bacterial infection, characterized in that eukaryotic cells with be brought into contact with a substance that Expression and / or function of CD95 and / or CD95L or of members of the signal transduction cascade of CD95 and / or CD95L. 5. Use or method according to any one of claims 1-4, characterized in that it is the members of the signal transduction cascade of CD95 and / or CD95L to JNK, GADD 153, NFκB, PLA ₂ , FADD, Caspase 8, Caspase 3, CAD and / or I-CAD. 6. Use or method according to one of the preceding Claims, characterized in that the active ingredient or the substance against CD95 and / or CD95L or against Members of the CD95 signal transduction cascade and / or CD95L.   7. Use or method according to one of the preceding Claims, characterized in that the active ingredient or the substance against activators, inhibitors, Regulators and / or biological precursors to CD95 and / or CD95L or by members of the signal transducer tion cascade of CD95 and / or CD95L is directed. 8. Use or method according to one of the preceding Claims, characterized in that the active ingredient or the substance is a polynucleotide which is a Peptide, in particular a polypeptide, encoded, wherein preferably this peptide expresses CD95 and / or CD95L or by members of the signal transducer tion cascade influenced by CD95 and / or CD95L, esp special stimulates. 9. Use or method according to one of claims 1-7, characterized in that the active ingredient or Substance is a peptide, preferably a polypeptide, preferably this peptide expresses CD95 and / or CD95L or by members of the signal transducer tion cascade influenced by CD95 and / or CD95L, esp special stimulates. 10. Use or method according to one of the preceding Claims, characterized in that the active ingredient or the substance a "small molecular compound" preferably a "small molecular compound" with a Molecular weight (MW) <1000. 11. Use or method according to one of the preceding Claims, characterized in that it is in the bacterial infection caused by pseudomonas like bacterial infection.   12. Use or method according to one of the preceding Claims, characterized in that the active ingredient or the substance orally, intravenously, topically and / or by Inhalation can be administered. 13. Pharmaceutical composition comprising an effective Amount of at least one active ingredient, the expression and / or function of CD95 and / or CD95L or member the signal transduction cascade of CD95 and / or CD95L influences, especially stimulates, and given if necessary, a pharmaceutical carrier. 14. Pharmaceutical composition according to claim 13, characterized in that the active ingredient is a poly is nucleotide, which is a peptide, especially a Encoded polypeptide, preferably this peptide Expression of CD95 and / or CD95L or of members the signal transduction cascade of CD95 and / or CD95L influenced, especially stimulated. 15. The pharmaceutical composition according to claim 13, characterized in that the active ingredient is a peptide, preferably a polypeptide, preferably this peptide expresses CD95 and / or CD95L or of members of the signal transduction cascade of CD95 and / or CD95L influenced, in particular stimulated. 16. Pharmaceutical composition according to one of the claims 13-15, characterized in that the active ingredient is a "small molecular compound", preferably a "small molecular compound "with a molecular weight (MG) < Is 1000.   17. Pharmaceutical composition comprising an effective Amount of at least one active ingredient, the expression and / or function of activators, inhibitors, regulators gates and / or biological precursors of CD95 and / or CD95L or by members of the signal transduction cascade de influenced by CD95 and / or CD95L, in particular stimulated, and optionally a pharmaceutical Carrier. 18. The pharmaceutical composition according to claim 17, characterized in that the active ingredient is a polynu is kleotide, which is a peptide, especially a Encoded polypeptide, preferably this peptide Expression of activators, inhibitors, regulators and / or biological precursors to CD95 and / or CD95L or of members of the signal transduction cascade of CD95 and / or CD95L influenced, in particular stimulated. 19. Pharmaceutical composition according to claim 17, characterized in that the active ingredient is a peptide, preferably a polypeptide, preferably this peptide expression of activators, inhibito regulators and / or biological precursors of CD95 and / or CD95L or by members of the Signal transduction cascade influenced by CD95 and / or CD95L, especially stimulated. 20. Pharmaceutical composition according to one of the claims 17-19, characterized in that the active ingredient is a "small molecular compound", preferably a "small molecular compound "with a molecular weight (MG) < Is 1000.   21. Diagnostic kit comprising at least one substance for Evidence of expression and / or function of CD95 and / or CD95L or by members of the signal transducer cascade of CD95 and / or CD95L, for the detection of Susceptibility to diseases with a Infection, especially a bacterial infection Related. 22. Diagnostic kit according to claim 21 for the detection of susceptibility against pseudomonas pathogens.