Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Protozoa – media therefor
Reexamination Certificate
1995-11-09
2002-10-01
Graser, Jennifer E. (Department: 1645)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Protozoa, media therefor
C435S243000, C435S244000, C435S258300, C435S260000, C435S252400, C435S113000, C435S007220
Reexamination Certificate
active
06458581
ABSTRACT:
A subject of the invention relates to a process for the in vitro culture of different stages of the developmental cycle of a parasite. It also relates to the parasitic forms obtained and their biological uses.
By culture is meant, in the description which follows and the claims, both the adaptation of the parasitic form by successive passages in a given medium, and complete differentiation when it occurs for the adaptation and the culture itself of the parasitic form.
It is known that parasites constitute a real plague causing, by the intermediary of vectors, the infection of millions of people and animals.
Thus, leishmaniases, which are widespread throughout the world, are caused by protozoan of the Leishmania genus which are usually transmitted by a sand fly, Phlebotomus. Leishmaniases of the Old World and those of the New World are usually distinguished according to their geographical localization. They have very diverse clinical forms which differ significantly by their seriousness and their effect on health. Cutaneous, mucocutaneous (attacking nasal, buccal mucous and those of the ears) and visceral leishmaniases are distinguished.
As another parasite having a devastating effect, there can also be mentioned
Trypanosoma cruzi,
responsible for Chagas' disease. In South America, it causes the infection of millions of people. Over 150 species of wild and domestic animals can be counted which can serve as hosts to the parasite which is transmitted to man by a bug, namely a Triatoma, which feeds on blood.
The infection can pass unnoticed for several years until the trypanosomas attack the nervous system, the heart or the digestive system.
The development cycle of many parasites includes various parasitic stages. That of Leishmania, for example, includes two stages having important differences at the structural, morphological, biochemical, immunological and physiological level, namely
in a vector insect, a flagellated form, called promastigote, which multiplies by scissiparity before acquiring its infectious form, for the mammalian host, also called metacyclic,
in a mammalian host, a non-flagellated stage, called amastigote, which exclusively parasitizes mononuclear phagocyte cells.
The differentiation into amastigotes occurs after attachment and penetration of the promastigotes into the monocytes. But only the amastigote forms persist and multiply inside the phagolysosome of the macrophages of the infected host.
In
T. cruzi,
this cycle includes three different parasitic stages, an epimastigote form, multiplication form of the vector insect, and the amastigote and trypomastigote forms, which are present in the infected host.
At present, most of the research carried out on the diagnosis of these parasitoses and the development of vaccines is conducted on the promastigote form whose production in culture is easy and quick.
Now, the only form present in the infected host is the amastigote form which, by persisting throughout the infection, triggers the immune response and participates in the development of the pathology. The danger of systematically extrapolating the experimental results obtained with the cultured promastigote forms within the scope of immuno-prophylactic, diagnostic or therapeutic studies aimed at the amastigote forms will be well understood.
In order to respond to this problem, various authors have been interested in obtaining amastigote forms. Thus the obtaining of amastigotes from tissues of experimentally infected animals or from cultures of infected macrophages have been reported. But this involves long and tedious isolation methods, which moreover lead to a limited number of parasites being obtained, which are often degenrated and which are incapable of multiplying and of surviving for longer than 2 to 3 days.
Furthermore, such amastigote forms are contaminated with cells, fragments and molecules derived from the macrophages, tissues or plasma of the host, designated hereafter by “cellular contaminants”, limiting or making impossible the realization of many studies.
Culturing processes under axenic conditions, that is to say in the absence of any cellular contaminant, have been proposed for some species of leishmanias and for
T. cruzi,
but they do not allow an abundant and continual source of amastigote forms to be made available, and do not appear to be generally applicable to a large number of species and to different germs.
An aim of the invention is to resolve the above disadvantages and to provide more satisfactory experimental models by producing the desired parasitic stages in specific culture media, of totally defined simplified composition.
The invention relates in particular to providing a process generally applicable to a large number of species of a given parasite, allowing homogeneous populations of a given parasitic stage to be produced in a continuous manner and in unlimited quantities.
It relates more particularly to a process allowing these stages to be obtained in a form which is free from any cellular and seric contaminant, having the characteristic of not containing any macromolecules. By macromolecules is meant, in the description and the claims which follow, non-dialyzable molecules with a cut-off threshold of 3 kDa, that is to say having an apparent molecular weight of greater than 3 kDa (for example seric protein such as albumin).
It also relates to the in vitro production of the complete development cycle of parasites under axenic and aseric conditions.
According to another aspect, the invention relates to the new parasitic forms obtained, corresponding to the different stages of the parasitic cycle and, for each stage, to the different phases of their growth.
According to another aspect, the invention relates to the applications of the parasitic stages obtained and of the products produced or isolated from these stages, in particular in the domain of the diagnosis of parasitoses, immunoprophylaxis and screening of drug activities.
The process according to the invention, for the in vitro culture of different stages of tissular parasites, such as leishmania,
T. cruzi
, or also hematoprotozoa is characterized in that it is carried out in an axenic and aseric, liquid single-phase culture medium, free from macromolecules (non-dialyzable at a cut-off threshold of 3 kDa) and that for obtaining amastigote forms, this medium is buffered at a pH of 5.5 to 6.5 and has an osmolarity of at least 400 milliosmoles/kg of liquid, and in particular from 400 to 550 milliosmoles/kg of liquid, or that, for obtaining promastigote forms, this medium is buffered at a pH of 7 to 7.5 and has an osmolarity of at least 300 milliosmoles/kg of liquid, and in particular from 300 to 380 milliosmoles/kg of liquid.
The pH value of these media, within the range indicated above, ensures that the culture conditions are strictly controlled.
In the case of the culture of amastigotes, when the pH is greater than 6.5, a tendency towards the retransformation of the amastigotes into promastigotes is in fact noted and when it is less than 5.5, a poor growth is observed.
According to a preferred method of the invention, for obtaining amastigote forms, a culture medium is used containing a base medium, produced essentially from:
at least one culture medium for insect cells which has added to it inorganic salts, of Hanks' salts type,
products which are sources of amino acids, such as L-glutamine and soja bean extracts,
sugars, such as D-glucose.
As soja bean extact, there can advantageously be used that marketed under the Trade mark trypto casein soja®.
The culture medium for insect cells is advantageously the medium 199 H marketed by GIBCO.
Different compositions of this 199 H
R
medium are given in the GIBCO BRL catalogue page 48, 1992 edition.
A specially preferred composition for the production of base media carries the reference 042-01181 on page 48 of this catalogue, 1992 edition. The 199H M medium is more especially used, to which NaHCO
3
and L-glutamine are added.
This medium, to which the above compounds are added, is advantageously buffered, for exam
Graser Jennifer E.
Institut francais de recherche scientifique pour le developpemen
Nixon & Vanderhye P.C.
LandOfFree
Process for the in vitro culture of different stages of... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Process for the in vitro culture of different stages of..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Process for the in vitro culture of different stages of... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2990962