EP0809690A1 - Mise en culture in vitro de souches de trypanosomes pleomorphes - Google Patents

Mise en culture in vitro de souches de trypanosomes pleomorphes

Info

Publication number
EP0809690A1
EP0809690A1 EP96904791A EP96904791A EP0809690A1 EP 0809690 A1 EP0809690 A1 EP 0809690A1 EP 96904791 A EP96904791 A EP 96904791A EP 96904791 A EP96904791 A EP 96904791A EP 0809690 A1 EP0809690 A1 EP 0809690A1
Authority
EP
European Patent Office
Prior art keywords
trypanosomes
cultivation
culture medium
pleomorphic
trypanosome
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96904791A
Other languages
German (de)
English (en)
Inventor
Michael Boshart
Erik Vassella
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Original Assignee
Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Max Planck Gesellschaft zur Foerderung der Wissenschaften eV filed Critical Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Publication of EP0809690A1 publication Critical patent/EP0809690A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/10—Protozoa; Culture media therefor
    • C12N1/105—Protozoal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/90—Protozoa ; Processes using protozoa
    • 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
    • G01N2333/44—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from protozoa

Definitions

  • the present invention relates to a method for the in vitro cultivation of trypanosome strains without a previous adaptation phase, the use of the culture medium or fractions or components thereof obtainable by cultivation for the production of a therapeutic agent and a biological in vitro test system for the determination of effector substances which are responsible for the transition induce or / and inhibit a rapidly dividing trypanosome form to an incapable trypanosome form.
  • Trypanosomes are flagellated unicellular organisms that live as parasites in the Elutbahn of mammals and in tropical Africa and South America from insects, e.g. Tsetse flies being transferred from one host to another. They are the causative agent of African sleeping sickness in humans and various livestock diseases such as, in particular, the Nagana and are therefore of great medical, veterinary and economic importance.
  • the parasites occur in two morphologically and biochemically distinguishable forms. Because of their morphology, the rapidly dividing parasites are referred to as "long slenders".
  • the "short stumpy” forms that result from differentiation have irreversibly lost the ability to divide cells. Only if the "short stumpy" trypanosomes get through the blood meal of a Tsetse fly in the fly stomach can they differentiate further and continue the life cycle as procyclical forms. In the mammalian bloodstream, the "short stumpy" trypanosomes die after a few days and therefore cannot maintain the infection in the host.
  • the "long slender” forms are differentiated into “short stumpy” forms by an Known signal that correlates with the number of parasites in the blood. This seems to be a mechanism to limit parasitemia (number of parasites per ml of blood) in a way that ensures the survival of the host and thus the spread of the parasite.
  • Naturally occurring trypanosomes are always pleomorphic (diverse), which means that they can differentiate from "long slender” into “short stumpy” forms. To date, these strains cannot be efficiently propagated as bloodstream forms in any in vitro cell culture system. Only variants, certain monomorphic forms, which have been adapted to the cultivation conditions in the laboratory through a multi-week adaptation or selection phase, have so far been able to be cultivated and propagated in vitro. Such culture forms have lost the ability to differentiate into "short stumpy" forms or are severely defective therein.
  • the method according to the invention enables the cultivation of pleomorphic trypanosomes or monomorphic trypanosomes which have never been cultivated in vitro.
  • Such strains only grow in conventional liquid media after an adaptation period of several weeks during which massive cell death and selection take place. Cultivation in a liquid or semi-solid culture medium which contains a polymeric matrix makes these adaptation and selection steps unnecessary.
  • This high molecular or polymer matrix is preferably selected from polypeptides and / or polysaccharides, in particular from polysaccharides based on agar or / and dextran.
  • the method according to the invention allows pleomorphic trypanosomes to be cultivated in vitro.
  • the "long slender” trypanosomes in this culture system differentiate into “short stumpy” forms when a certain cell density is exceeded.
  • Extensive controls show that "stumpy" forms produced in vitro correspond to "stumpy” forms from the blood of infected animals in all parameters tested so far.
  • the invention thus furthermore relates to a process for the in vitro cultivation of pleomorphic trypanosomes, which is characterized in that pleomorphic trypanosomes capable of proliferation are spread onto the surface of a semi-solid culture medium and incubated under conditions in which the trypanosomes multiply .
  • the cultivation is preferably carried out by light microscopy until it occurs. Trypanosome colonies.
  • the process according to the invention enables the cultivation of the proliferation-capable "long slender” form until it is differentiated into the proliferation-incapable "short stumpy” form.
  • the method according to the invention is based on the fact that the cultivation of pleomorphic wild-type trypanosome strains is surprisingly possible as colonies on the surface of semi-solid culture media.
  • an agar-based culture medium e.g. Agar or agarose plates.
  • An example of a suitable medium is the HMI-9 medium, but other known media suitable for cultivating trypanosomes can also be used in principle.
  • Another object of the present invention is the use of the method described above as an assay system for determining effector substances which influence the growth or / and the differentiation of trypanosome strains, i.e. induce and / or inhibit. Of particular importance are those effector substances which have a growth-inhibiting or / and differentiation-inducing effect.
  • a particularly preferred application of the assay system is the determination and characterization of the signal substance SIF, which can be obtained from a conditioned culture medium which has already been used for the cultivation of trypanosomes.
  • a method for enriching a signaling substance (SIF) which stimulates the differentiation of pleomorphic trypanosome strains which is characterized in that trypanosomes are grown in vitro in a culture medium, the culture medium is separated from the cells and the signaling substance is extracted from the Culture medium enriched by conventional biochemical methods.
  • suitable biochemical enrichment methods are chromatographic methods such as gel chromatography, affinity chromatography, ion exchange chromatography, HPLC, reverse phase HPLC or combinations thereof.
  • the enrichment is preferably carried out in an inert Atmosphere.
  • SIF is contained, for example, in a heat-resistant, low-molecular fraction of the conditioned culture medium.
  • SIF or analogues of this substance are excellently suited for blocking the multiplication of trypanosomes in the mammalian bloodstream by inducing the differentiation into "short stumpy” forms. Since "short stumpy" forms themselves only have a limited lifespan of a few days, the administration of pharmacological doses of SIF or analogues leads to the elimination of the parasites and is therefore of great therapeutic benefit.
  • the above-mentioned bio-assay can also be used to search for other effector substances, e.g. for substances that stimulate the signal transduction cascade that mediates the SIF signal.
  • the present invention therefore furthermore relates to a therapeutic composition which contains, as active substance, a conditioned culture medium which was used for the cultivation of trypanosomes, or fractions or constituents of this medium, if appropriate together with customary pharmaceutical auxiliaries.
  • the composition preferably additionally contains one or more further active substances which are suitable for controlling trypanosome infections, for example germanin, pentamidine or tryparsamide.
  • the composition is preferably administered over a period of at least one week in order to ensure that the "long slender" forms are converted as completely as possible to the "short stumpy" forms which are no longer capable of reproduction.
  • Yet another object of the present invention is the use of the conditioned culture medium or of fractions or constituents of this medium for producing an agent for combating trypanosome infection, in particular an agent for combating sleeping sickness, caused by the trypanosome species T.gambiense or T.rhodes-iense, or an agent for combating the Nagana, which is caused by the trypanosome species T.brucei brucei.
  • Fig. 1 the growth of a pleomorphic trypanosome strain
  • Fig. 2 shows the plating efficiency of pleomorphic trypanosomes after in vitro cultivation.
  • 1.6 x HMI-9 medium is modified from the published method by Hirumi and Hirumi (1989) Journal of Parasitology 75; 985-989.
  • the agarose plates are produced based on the published method by Vern B. Curruthers and George A.M. Cross (1992) Proc. Natl. Acad. Be. USA 89, 8818-8821.
  • a 1.78% agarose solution (1.3 g low melting point SeaPlaque GTG agarose, biozyme, catalog no. 50112 in 73 ml bidistilled water) is autoclaved at 120 ° C for 20 minutes and then cooled to 37 ° C .
  • 127 ml of 1.6x HMI-9 medium preheated to 37 ° C. are added to this solution.
  • 20 ml of this mixture are transferred to 100 x 15 mm petri dishes without bubbles.
  • the agarose plates solidify for 60 minutes at room temperature with the lid closed. This is followed by drying the surface of the agar plate e.g. for approx. 20 minutes with the cover removed in a laminar flow of the Heraeus Lamin Air HB 2948 type.
  • Monomorphic trypanosome strains can be cultured in simply concentrated HMI-9 medium (Vern B. Curruthers and George AM Cross (1992) Proc. Natl. Acad. Sei. USA 89, 8818-8821).
  • the conditioned medium of such cultures (containing the SIF) is obtained by centrifuging the cells and then sterilizing them.
  • This medium or the fractions obtained therefrom with the usual biochemical processes are enriched ad 5% with Serum Plus "and ad 15% with fetal calf serum (see Section A).
  • Pleomorphic trypanosomes are either isolated directly from the blood of infected mice or rats or obtained from one of the cultures described here (by washing the plates with simply concentrated HMI-9 medium) and diluted to the appropriate cell density. 100 .mu.l of the cell suspension are distributed on the surface of the plate by means of a rotary motion using a sterile Drigalski spatula. The plates are then transferred to an incubator (eg of the Heraeus 6000 type) and incubated at 37 ° C. in 5% CO 2 and 100% atmospheric humidity. After 2 to 3 hours (optional) and after overnight incubation, the liquid collected at the edge of the petri dish is aspirated using a sterile pipette. After 2 days, clearly visible colonies are formed under light microscopy (40 x magnification).
  • an incubator eg of the Heraeus 6000 type
  • Figure 1 shows a comparison of the growth of a pleomorphic trypanosome strain (curves 1 and 2) and a monomorphic trypanosome strain (curve 3) when cultivated on a semi-solid agar medium in vitro. It can be seen that the number of cells per plate in monomorphic trypanosomes is approximately at least a factor 10 2 higher than in pleomorphic trypanosomes is. This result is to be interpreted in such a way that pleomorphic trypanosomes differentiate from a form capable of proliferation into a form incapable of proliferation from a certain cell density.
  • pleomorphic trypanosomes are also possible in liquid medium which contains a polymeric matrix such as agarose. This finding was obtained for two types of agarose with different gelling temperatures (semi-liquid agarose and liquid (ultra-low-geling) agarose).
  • the assay system described in Example 1 for determining effector substances which influence the growth or differentiation of pleomorphic trypanosome strains can be considerably simplified and improved, in particular miniaturized. For this purpose, faster testing e.g. possible in the course of the purification of the signal substance s SIF.
  • Monomorphic trypanosome strains which had never been cultivated in vitro, only grew in conventional HMI-9 liquid medium after an adaptation period of several weeks, in which massive cell death and a selection for bloodstream forms take place. This adaptation and selection can be dispensed with by culture on agarose plates according to Example 1.
  • Heat-resistant low molecular weight fraction of the conditioned medium described in Example 1 was prepared. This heat-resistant kidney molecular fraction was sufficient to inhibit the growth of pleomorphic trypanosomes and to induce differentiation.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Toxicology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La présente invention concerne un procédé pour la mise en culture in vitro de souches de trypanosomes, l'utilisation du milieu de culture obtenu ou de fractions ou de constituants de celui-ci pour préparer un agent thérapeutique, et un système de tests biologiques in vitro pour déterminer les effecteurs qui induisent et/ou inhibent la modification d'une forme à division rapide de trypanosome en une forme de trypanosome incapable de division.
EP96904791A 1995-02-15 1996-02-15 Mise en culture in vitro de souches de trypanosomes pleomorphes Withdrawn EP0809690A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19505056A DE19505056C1 (de) 1995-02-15 1995-02-15 In vitro Kultivierung von pleomorphen Trypanosomenstämmen
DE19505056 1995-02-15
PCT/EP1996/000651 WO1996025485A1 (fr) 1995-02-15 1996-02-15 Mise en culture in vitro de souches de trypanosomes pleomorphes

Publications (1)

Publication Number Publication Date
EP0809690A1 true EP0809690A1 (fr) 1997-12-03

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EP96904791A Withdrawn EP0809690A1 (fr) 1995-02-15 1996-02-15 Mise en culture in vitro de souches de trypanosomes pleomorphes

Country Status (3)

Country Link
EP (1) EP0809690A1 (fr)
DE (1) DE19505056C1 (fr)
WO (1) WO1996025485A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19505056C1 (de) * 1995-02-15 1996-03-14 Max Planck Gesellschaft In vitro Kultivierung von pleomorphen Trypanosomenstämmen

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19505056C1 (de) * 1995-02-15 1996-03-14 Max Planck Gesellschaft In vitro Kultivierung von pleomorphen Trypanosomenstämmen

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9625485A1 *

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Publication number Publication date
WO1996025485A1 (fr) 1996-08-22
DE19505056C1 (de) 1996-03-14

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