WO2006032330A1 - Modulation d'annexine et resistance aux infections - Google Patents

Modulation d'annexine et resistance aux infections Download PDF

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WO2006032330A1
WO2006032330A1 PCT/EP2005/008832 EP2005008832W WO2006032330A1 WO 2006032330 A1 WO2006032330 A1 WO 2006032330A1 EP 2005008832 W EP2005008832 W EP 2005008832W WO 2006032330 A1 WO2006032330 A1 WO 2006032330A1
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substance
annexin
expression
function
erythrocytes
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PCT/EP2005/008832
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German (de)
English (en)
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Florian Lang
Phillipp Lang
Stephan Huber
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Eberhard-Karls-Universität Tübingen
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Publication of WO2006032330A1 publication Critical patent/WO2006032330A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4721—Lipocortins
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4718—Lipocortins

Definitions

  • the present invention relates to a substance for therapeu ⁇ tables or prophylactic treatment of infectious diseases, the use of this substance for the manufacture of a medicament for the treatment or prophylaxis of infections, a method for identifying a substance, the tion tion or expression of Annexin modified, a substance identified or identifiable by this process, a process for the preparation of a pharmaceutical composition for the treatment or prophylaxis of infectious diseases, a composition comprising one of the aforementioned substances, a process for detecting a modified, preferably a reduced susceptibility of a living being to infections, and a method for the therapeutic or prophylactic treatment of a living being which is affected by an infectious disease or in which the risk of infection exists.
  • Infectious diseases are infectious and non-contagious diseases in animals and humans with an acute or chronic course, caused by infection with certain pathogens, such as Vi ⁇ ren, bacteria, fungi, protozoa, worms, arthropods, Prio ⁇ nen, etc.
  • pathogens such as Vi ⁇ ren, bacteria, fungi, protozoa, worms, arthropods, Prio ⁇ nen, etc.
  • pathogens such as Vi ⁇ ren, bacteria, fungi, protozoa, worms, arthropods, Prio ⁇ nen, etc.
  • pathogens such as Vi ⁇ ren, bacteria, fungi, protozoa, worms, arthropods, Prio ⁇ nen, etc.
  • the interaction of infection, damaging effect of the pathogens and specific defense of the body leads to a characteristic picture of a certain infectious disease and determines its course.
  • Infectious diseases are the leading causes of death worldwide. In 1995, 17.3 million deaths were caused by the most common infectious diseases: acute respiratory infections, diarrhea, tuberculosis, malaria, hepatitis, AIDS and measles. The phenomenon of more and more widespread infectious diseases worldwide is being watched with concern, and others. called alarming by the World Health Organization.
  • infectious diseases The emergence and manifestation of infectious diseases is to be prevented by means of vaccinations, ie by active immunization. Although with these measures various infectious diseases are eliminated regionally or even worldwide could be eradicated, such as smallpox, there are still a significant number of pathogens. This is partly due to the fact that a corresponding immunotherapy is often ineffective because the pathogens evade access by the immune system through a variety of mechanisms. Another problem associated with active immunization is the often heavy burden on the organism to be immunized.
  • Interferons interfere in a variety of ways with the metabolism, growth and differentiation of cells, causing a variety of side effects.
  • Annexins form a heterogeneous group of calcium-binding proteins.
  • Annexins are also referred to as calelectrines, calpactins, chromanobindins, endonexins, lipocortins or syneasines.
  • Annexins have a molecular weight of about 35 kDa and about 80 amino acid-long conserved fragment that can be repeated up to eight times within a protein.
  • Annexins occur in all eukaryotic organisms, except in yeasts. In the meantime, the annexins are subdivided into subgroups which have hitherto been designated by numbers from I to XIII or 1 to 13.
  • ⁇ nnexins occur in vivo as cytoplasmic soluble proteins:.
  • Annexin II is held responsible for the ⁇ alcium-dependent exo- and endoacytosis; it can bind calcium-dependent to membranes.
  • Annexin XIII carries a myristoyl residue at the N-terminus and appears to be involved in polar vesicle transport in intestinal epithelial cells.
  • Annexin VI seems to control the calcium release in muscle cells after electrical stimulation.
  • the exact function of the annexins is still far from clear.
  • An overview of annexins can be found in Gerke V. and Moss S.E. (2002), "Annexins: from structure to function", Physiol. Rev. 82: pp. 331 to 371.
  • the substance can be defined both chemically and biochemically or biologically. It can therefore be a classically chemically synthesized compound, but also a peptide, or a nucleic acid.
  • the term expression is understood in the broadest sense and covers the entire process of the formation of a functional protein.
  • the expression or function of annexin can be modulated according to the invention by the substance at all levels of biosynthesis, such as transcription, processing of the RNA, translation, folding of the protein, localization of the protein to its physiological location. Annexin can also be modulated by the substance in its activity.
  • modulating is understood as meaning activation, inhibition, amplification, inhibition or complete termination of the expression or physiological function of annexin, for example by a so-called knockout.
  • the substance is designed such that the expression and / or function of annexin is inhibited.
  • inhibition is understood as meaning the complete or partial inhibition of the physiological function or activity or the natural expression of annexin.
  • mice in which by genetic engineering the annexin gene (Annexin hereafter), a drastically higher survival rate after infection with the malaria pathogen compared to their. Have wild-type littermates. Such so-called anziexin knockout mice survive the otherwise deadly murine infection with the parasite Plasmodi ⁇ berghei. In comparison, all corresponding wild type mice died from the same infection.
  • the substance according to the invention is used on the body's own structures and not, as in most antibiotics, in the metabolism of a Influenza attacked, whereas this can easily form resistances aus ⁇ .
  • the risk of the formation of such resistance to the substance according to the invention is therefore extremely low. Since it is the knowledge of the inventors of annexin to a central protein that in. Therefore, the substance according to the invention has a particularly broad spectrum of action, whereas many of the antibiotics known in the prior art are effective only against certain pathogens.
  • the Exfinder have also developed a method for identifying a substance that moci turn the function and / or expression of annexin and comprising the following steps: (al) providing a peptide derived from annexin, or (a2) providing a biological A system expressing an annexin-derived peptide; (bl) contacting the peptide with a test substance under conditions which allow the binding of the test substance to the peptide, or (b2) introducing a test substance into the biological system, and (c) determining whether the test substance is from Step (bl) the function, or from step (b2) modulates the expression of the peptide, preferably inhibited.
  • steps (a1), (b1) and (c) or steps (a2), (b2) and (C) are carried out successively in the process.
  • a substance according to the invention can be identified without necessarily providing the entire annexin protein in steps (a1) and (a2) must, but only a derived peptide.
  • Hierun ⁇ ter is a portion of the totalannexin protein to understand, which is at least responsible for the physiological functions, ie enzymatic or catalytic or other activity of annexin.
  • a substance which modulates the activity or expression of such a peptide also shows activity against the totalannexin protein. It is understood that in the context of this method, the total Tannexinprotein can be used.
  • a substance can be identified by means of a biological system which modifies the expression of annexin.
  • a biological system can be provided by an entire eukaryotic or prokaryotic organism or else by an in vitro expression system comprising bacteria, baculoviruses, cultured cells or Xe ⁇ opus oocytes or from corresponding extracts.
  • step (bl) in order to function as a prodrug, the test substance must bind to the annexin-derived peptide, ie, a condition must be established in which the substance to be tested is at least in close proximity to the peptide and therefore capable of is to influence the activity orress tion of the peptide.
  • Conditions which make it possible to bring the peptide into contact with the test substance or which must prevail in the biological system in order to detect an expression-modulating activity are well known in the field of protein or enzyme biochemistry or molecular biology.
  • conventional physiological or biological buffer systems are used, such as Tris, HEPES or PBS-based buffers, optionally mixed with various salts in suitable concentrations or with further customary additives.
  • step (c) is carried out using the functionality, activity or expression tests known to the person skilled in the art.
  • the method described above has the advantage that the person skilled in the art is given a targeted route with which a virtually unlimited number of substances can be found which can be used for the treatment or prophylaxis of infectious diseases and which are largely free of the disadvantages which have the substances used in the prior art against infectious diseases.
  • the substance is selected from the group consisting of low-molecular-weight substances, antibodies and genetic constructs.
  • Low-molecular-weight active substances or "small molecules" are a collective term for chemical substances having activities in biological systems, the molecular weights of which are generally below about 1,000 to 1,200 D.
  • Such an active substance has the advantage that it can be used by means of
  • such low molecular weight active compounds can be prepared starting from chemically defined or biological lead structures, such as, for example, peptides, using the methods of so-called “rational drug design” or the so-called “rational drug design”.
  • the preparation of low-molecular-weight active substances is familiar to the person skilled in the art, see Böhm et al. (2002), Wirk ⁇ stoffdesign, Spektrum Akademischer Verlag, Heidelberg.
  • Genetic constructs are understood as meaning nucleic acid molecules, such as vectors, plasmids, probes or comparable molecules, which belong to the tools of the molecular biologist and with which the expression or / and function of annexin can be specifically modulated. Such genetic constructs have the advantage that they are much more stable and robust than peptides and can be stored almost indefinitely.
  • the production of a genetic Kostruktes is described, for example, in the paper by Joseph Sambrook and David W. Jossel (a.a.O.).
  • the substance codes for an antisense annexin probe or / and an Armexin RNAi or / and for transdominant inhibitory Annexin.
  • This measure has the advantage that the expression and / or function of annexin is specifically and already modulated or inhibited at a genetic level by means of the so-called antisense technology.
  • An antisense annexin probe is a genetic construct, i. a DNA or RNA sequence complementary to a messenger or functional RNA or DNA encoding annexin. This probe is able to attach itself to the complementary structure which codes for annexin, thereby blocking the translation and / or transcription of the coding region.
  • Annexin RNAi stands for annexin RNA "interference", also called siRNA (for "silencing” RNA).
  • RNAi is a double-stranded RNA (dsRNA) which is largely homologous to the natural messenger RNA.
  • dsRNA double-stranded RNA
  • Such an RNAi which is introduced into an organism or a cell, recognizes and binds the correspondingly homologous mRNA and, via a hitherto still largely unexplained mechanism, leads to a degradation of the latter and to a knockout phenotype with respect to the corresponding gene or gene product; see. Fire et al. (1998), “Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans", Nature 391: pp. 806-811.
  • Transdominant inhibitory annexin protein is characterized by a mutation, for example, a point mutation that causes such a modified annexin to be inoperable.
  • a mutation for example, a point mutation that causes such a modified annexin to be inoperable.
  • the wild-type annexin becomes one not measurable background activity suppressed and thus switched off.
  • the subject of the present invention is also an above-described substance for medical use.
  • the inventors have for the first time provided a substance which modulates or inhibits the expression and / or function of annexin, which is used in the field of medicine.
  • the substance according to the invention is suitable both as a therapeutic and as a prophylactic agent.
  • the substance can, for example, also be used in livestock husbandry in order to create infection resistance in animals.
  • the annexin gene can be eliminated in a targeted manner and the problem of infections by various veterinary pathogens can thus be avoided in a simple manner.
  • the substance according to the invention is suitable for the treatment or / and prophylaxis of infectious diseases, in particular of those which are caused by intracellular pathogens, preferably of parasitemia and / or malaria.
  • the inventors have recognized a universal principle and designed a substance which is effective against a large number of further pathogens, such as against legionella, mycobacteria, viruses, etc.
  • This measure has the advantage that the mechanisms which have been recognized for the first time by the inventors are therapeutically resp be used diagnostically. Namely, cells, for example. Erythrocytes, which have been infected by such agents, from Immune system then accelerates "disposed of" when the annexin protein is inhibited in its expression and / or function.
  • This embodiment of the substance quite specifically creates an active substance which is specifically directed against intracellular pathogens.
  • the substance according to the invention is preferably designed such that it selectively modulates the expression and / or function of annexin in erythrocytes.
  • This measure has the advantage that the substance can be used specifically as a therapeutic agent against such an infection, which takes place via the infestation of erythrocytes, such as malaria. Annexin, which is present in other cells and exerts its physiological function there, is not affected. Side effects are further reduced by this measure.
  • the subject of the present invention is also the use of the substance described above for the production of a medicament for the treatment and / or prophylaxis of infectious diseases, in particular of such infectious diseases caused by intracellular pathogens as malaria.
  • a further subject matter relates to a process for the preparation of a pharmaceutical composition for the treatment or / and prophylaxis of infectious diseases comprising the steps of: (a) providing a substance which modulates the expression and / or function of annexin, and (b) formulation of this substance into a pharmaceutically acceptable carrier, wherein the substance according to the invention or inventively identified substance is used as the substance.
  • the infectious disease is preferably one caused by intracellular pathogens, preferably parasitemia or malaria.
  • a further subject matter of the present invention relates to a composition, preferably a pharmaceutical composition, comprising the substance according to the invention and optionally a pharmaceutically acceptable carrier.
  • the inventors have recognized that the level of annexin expression or annexin function can be used to make a diagnosis or statement about the extent to which the affected organism is susceptible to infections, preferably caused by intracellular pathogens like malaria.
  • Another object of the present invention therefore relates to a method for detecting a modified, preferably reduced susceptibility of a subject for infections, comprising the steps of: (a) providing a biological sample of the animal; (b) analyzing the sample for the presence of a modified, preferably reduced attached annexin function or Annexin expression, and (c) Kor ⁇ relation of a positive finding in step (b) with the diagnosis of a modified, preferably reduced susceptibility of the living organism.
  • Such a method can be used both in humans and in animals, e.g. Breeding animals.
  • a targeted treatment of the living being may follow, in which the substance according to the invention can be used as a therapeutic or else other substances described in the prior art against infectious diseases.
  • the method according to the invention now provides the basis for the decision as to whether, for example, an active immunization appears expedient or whether due to a reduced annexin function or annexin expression there is a reduced susceptibility to an infection and therefore none, if necessary the appropriate organism onerous measures must be taken.
  • the animal has a reduced susceptibility to infections
  • it can also be determined whether an increased susceptibility exists. Indeed, such a determination will be made if the annexin function or annexin expression is increased beyond the normal level.
  • the annexin function is strengthened, for example, even when the protein shows an above-average activity.
  • the modified annexin function or annexin expression of the living organism can have many causes. As such, genetic variations or changes come into question, such as. Polymorphisms or mutations of the annexin gene, in particular the annexin7 gene. In. Further questions arise in the stability of the annexin protein or the coding mRNA.
  • Another subject matter of the present invention relates to a process for the therapeutic and / or prophylactic treatment of a living being which is affected by an infectious disease or / and in which the risk of an infection exists, which comprises the following steps: (a) Providing a substance that reduces the expression and / or function of annexin, and (b) introducing the substance into the animal.
  • such a procedure can be used to treat persons who have increased contact with infectious agents, such as medical personnel or development aid workers. But even breeding animals can be specifically treated with this method and immunized if necessary.
  • the substance described above be used as the substance according to the invention.
  • the introduction of the substance into the living organism can take place either orally or via an injection or in another manner. Short-term treatment of the animal to achieve a reduced susceptibility to infection or possibly immunization is generally sufficient.
  • the infectious disease is preferably one caused by intracellular pathogens, preferably parasitemia or malaria.
  • a further subject matter also relates to methods for therapeutic or / and prophylactic treatment of a living being affected by an infectious disease and / or in which there is a risk of infection, comprising the following steps: providing a substance which has the expression and / or Reduced function of annexin, and (b) introduction of the substance into the animal, in which the expression and / or function of annexin is reduced.
  • FIG. 3 accelerates phosphatidylserine exposure of erythrocytes from carriers of sickle cell anemia and in annexin 7 knockout mice;
  • FIG. 4 Parasitemia and survival of Plasmodium berghei-infected Annexin7 + / ⁇ - and annexini- / mice.
  • the number of erythrocytes and the hemoglobin concentration in Annexin7 ⁇ / mice was slightly but significantly lower than in Annexin7 + / + mice.
  • the other erythrocyte parameters were not significantly different between annexin / '- / and annexin7 + / + mice.
  • DLe number of platelets and the number of leukocytes were equally increased in JLn Annexin7 - / - mice compared to their Annexin7 + / + littermates.
  • Intact erythrocytes show phosphatidylserine symmetry and do not expose phosphatidylserine to their cell surface.
  • activation of Ca 2+ -permeable channels occurs.
  • the subsequent entry of calcium into the cells leads to activation of a Ca 2+ -sensitive scramblase with subsequent exposure to zellobea phosphatidylserine: - area; see. Duraton et al. (2002), "Oxidation induces a Cl (-) - dependent cationic conductance in human red blood cells", J. Physiol., 539: pp.
  • Partial panel (A) shows the original histograms of annexin-binding erythrocytes (cell counts) from Annexin7 + / + (top row) and Annexin7 - / - mice before (left) and after (right) exposure to ionomycin.
  • * indicates a significant difference between the control and Ca 2+ -Ionophorionomycin approaches
  • # indicates a significant difference between erythrocytes from Annexin7 / and Annexin7 + / + -
  • the cells were contained in annexin binding buffer containing 125 mM NaCl, 10 mM HEPES , pH 7.4 and 5 mM CaCl 2 washed the erythrocytes were stained with annexin-flous (Boehringer Mannheim, Germany) at a 1:50 dilution After 10 minutes the samples were diluted 1:.. 5 and diluted metrieanalysis means fürmanncyto- (FACS Calibur from Beckton Dickinson, Heidelberg, Germany) The cells were analyzed by forward scattering and the annexin fluorescence intensity was measured in the fluorescence channel FL-I at an excitation wavelength of 488 nm and an emission wavelength of 530 nm.
  • annexin binding buffer containing 125 mM NaCl, 10 mM HEPES , pH 7.4 and 5 mM CaCl 2 washed the erythrocytes were stained with annexin-flous (Boehringer Mannheim, Germany) at a 1
  • the intracellular Ca 2+ measurements were carried out as described; Andrews et al. (2002), "Phorbol ester stimulates a protein kinase C-mediated agatoxin TK-sensitive calcium permeability pathway in human red blood cells", Blood, 100: 3392-3399.
  • the erythrocytes were supplemented with fluo- 3 / AM (Calbiochem, Bad Soden, Germany) by adding 2 .mu.l of a Fluo-3 / AM stock solution (2.0 mM in dimethylsulfoxide [DMSO]) to 1 ml of erythrocyte solution (0.16% hematocrit in Ringer) bela ⁇ .
  • the cells were incubated at 37 0 C for 15 minutes with vigorous agitation and protected from light. a further 2 .mu.l of fluorescence 3 / AM were added and it was followed by a further incubation for 25 minutes. Fluo-3 / AM-loaded erythrocytes were at 1000 centrifuged for 3 minutes at 22 ° C.
  • Example 4 "Disposal" of ionomycin-treated erythrocytes from Armexin knockout mice from the blood
  • Macrophages are endowed with receptors specific for phosphatidylserine. It has been suggested that erythrocytes which expose phosphatidylserine on their surface are recognized, bound, phagocytized and degraded by macrophages.
  • FIG. 2 shows in partial illustration (A) the result of an experiment in which the annexin binding of erythrocytes from wild-type mice before (left) and after exposure to Ca 2+ ionophore ionomycin (10 ⁇ M, 1 hour) by means of FACS analysis was determined.
  • Partial figure (B) shows the result of an experiment The cells from (A) were labeled with the surface marker CFSE (5-carboxyfluorescinediacetatsuccinyl ester, Molecular Probes, Eugene, Oregon, USA).
  • CFSE surface marker
  • Plasmodium infection involves the induction of oxidative stress in the host cell; see. Duranton et al. (2003), "Electrophysiological properties of the Plasmodium falciparum-induced cationic conductance of human erythrocytes", Cell Physiol. Biochem., 13: pp. 189 to 198. This activates erythrocyte ion channels and calcium influx, Duranton et (2004), "Organic osmolyte permeabilities of the malaria-induced anionic conductances in human erythrocytes", J. Gen. Chem. Physiol. 123: pp. 417-426. It was therefore examined whether the infection triggers phosphatidylserine exposure.
  • FIG. 3 shows in the partial illustrations (A) and (B) the result of an experiment showing the annexin binding of human erythrocytes before and 48 hours after infection with Plasmodium falciparum.
  • Plasmodium falciparum activates endogenous Cl (-) channels of human erythrocytes by membrane oxidation ", EMBO 21: pp. 22-30) Human RBCs (blood group 0+) drew from blood banks.
  • the parasites were treated as previously described with a hematocrit of 5% and a parasitemia of 2 to 10% in RPMI 1640 medium mixed with Albumax II (0.5%, Gibco, Düsseldorf, Germany) in an atmosphere of 90% N 2 , 5 % CO 2 , 5% O 2 cultured.
  • Partial image (A) shows the original histograms of parasitemia in Annexin7 + / + (upper row) and Annexin7 - / - mice (lower row) 9, 20 and 26 days after infection with Plasmodium berghei.
  • Partial figure (C) shows the result of an experiment with which the survival of Annexin7 + / + - (unfilled
  • mice All wild-type mice died from malaria infection, demonstrating that Plasmodium berghei persisted in annexin7 - / - erythrocytes but was kept in check by the immune system of the infected Ai3rzexin7 knockout animal.

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Abstract

L'invention concerne une substance pour le traitement thérapeutique et/ou prophylactique de maladies infectieuses, l'utilisation de cette substance pour produire un médicament servant au traitement et/ou à la prévention d'infections, un procédé servant à identifier une substance qui modifie la fonction ou l'expression de l'annexine, une substance identifiée ou identifiable par ce procédé, un procédé servant à produire une composition pharmaceutique pour le traitement ou la prévention de maladies infectieuses, une composition présentant l'une des substances susmentionnées, un procédé servant à établir une sensibilité modifiée et, de préférence, réduite d'un être vivant à des infections, ainsi qu'un procédé pour le traitement thérapeutique et/ou prophylactique d'un être vivant touché par une maladie infectieuses ou risquant d'être infecté.
PCT/EP2005/008832 2004-09-21 2005-08-13 Modulation d'annexine et resistance aux infections WO2006032330A1 (fr)

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EP0741144A1 (fr) * 1994-11-11 1996-11-06 International Reagents Corporation Anticorps monoclonal antiannexine-v, procede pour produire cet anticorps, et utilisation de cet anticorps
WO2002017857A2 (fr) * 2000-09-01 2002-03-07 Philadelphia, Health And Education Corporation Methodes et compositions d'inhibition de l'angiogenese

Patent Citations (4)

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EP0741144A1 (fr) * 1994-11-11 1996-11-06 International Reagents Corporation Anticorps monoclonal antiannexine-v, procede pour produire cet anticorps, et utilisation de cet anticorps
DE19541284A1 (de) * 1995-11-06 1996-05-30 Kalden Joachim Robert Prof Dr Verfahren zur Immunmodulation
WO1997017084A1 (fr) * 1995-11-06 1997-05-15 Joachim Robert Kalden Medicament, notamment pour moduler la reponse immunitaire dans la lutte contre des virus, des tumeurs, des bacteries et des parasites
WO2002017857A2 (fr) * 2000-09-01 2002-03-07 Philadelphia, Health And Education Corporation Methodes et compositions d'inhibition de l'angiogenese

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OKAFUJI T ET AL: "Antisense-mediated regulation of Annexin VII gene expression during the transition from growth to differentiation in Dictyostelium discoideum", GENE: AN INTERNATIONAL JOURNAL ON GENES AND GENOMES, ELSEVIER, AMSTERDAM, NL, vol. 189, no. 1, 11 April 1997 (1997-04-11), pages 49 - 56, XP004059558, ISSN: 0378-1119 *
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