FR2745974A1 - MODIFIED ANIMAL, USES AND METHOD FOR MEASURING THE EFFECTIVENESS OF A THERAPEUTIC PROCESS - Google Patents

Abstract

A non-human mammal genetically modified without affecting its genome and having any two of the following pathological signs: (a) a blood-brain barrier that is open or permeable to water-soluble blood molecules non-specific for the cerebral parenchyma, (b) an astrocyte disorder revealed particularly by physical network disorganisation, the disappearance of perivascular feet around the capillaries, and gliosis, (c) microglial cell activation, (d) demyelinisation plates, particularly in the brain stem and/or the cerebral white matter, and (e) central nervous system glial and endothelial cell lesions, as well as the uses of said animal, are disclosed.

Description

 The present invention relates to the production of a mammalian, non-human, modified animal, having a pathophysiology similar to that of patients suffering from multiple sclerosis (MS), as well as to the uses of the animal thus obtained.

 Alteration of the blood brain barrier (BBB) appears to be a crucial element in the development of multiple sclerosis (MS) lesions (Gay & Esiri 1991, Hawkins & col 1991, Thompson & col 1992, Poser 1993). It seems that a genetic predisposition, combined with a pathogenic agent, possibly of viral origin, can cause these lesions then that an inflammatory and auto-immune demyelinating process develops (Wasksman 1985).

The observations of Perron & col (1989, 1991) of reverse transcriptase (RT) activity, and of the presence of viral particles in leptomeningeal cell lines and monocytes in culture of MS patients, provide arguments compatible with a viral, more particularly retroviral, etiology of MS. A hypothesis recently verified by the present inventors is that, in MS, monocytes probably infected with a retrovirus are capable of releasing gliotoxic molecules in vitro. Indeed, the supernatant of monocytes taken from MS patients in the pushing phase and placed in culture in vitro, added to the culture medium of explants of embryonic rat cortex has shown a toxicity targeted preferentially on astrocytes (see application for PCT / FR95 / 00178 patent filed in the name of the Applicant and the content of which is incorporated in the present description by reference). The purification of the supernatant of monocytes from MS patients, active in vitro, revealed a toxic factor represented mainly by a fraction of 17 Kd and an associated minority band of 21 Kd. This factor induces apoptotic death of astrocyte lines, and also of oligodendrocytes, in vitro (see patent application PCT / FR95 / 00178). These two fractions are also found in the cerebrospinal fluid (CSF) and sera of patients suffering from MS (Dobransky & col submitted).

 The present invention relates to a mammalian, non-human, modified animal, capable of being obtained by inoculation with the toxic factor defined above, and in which pathophysiological lesions, in particular of the brain, are observed, of the same nature as those observed in patients with multiple sclerosis.

 By modified animal according to the invention means an animal which, compared to a control animal of the same family, of the same genus and of the same species, has undergone a voluntary modification. This voluntary modification does not affect the animal's genome and in particular the animal is not transgenic. It is essentially characterized by a pathophysiological condition linked to an infection of the animal with the gliotoxic factor as obtained from biological fluid taken from patients suffering from multiple sclerosis.

The various objects of the invention are as follows:
- a non-human mammalian animal, modified without affecting its genome, having at least any of the following signs or abnormalities: a blood-brain barrier open or permeable to water-soluble molecules of the blood not specific for the cerebral parenchyma; an astrocytic attack notably revealed by the disorganization of physiological networks, by a disappearance of the astrocytic feet around the capillary vessels and by gliosis; activation of microglial cells; demyelination plaques, especially present in the brainstem and / or cerebellar white matter; damage to glial cells and endothelial cells of the central nervous system, in particular a modification of the morphology and / or at least partial fragmentation of the DNA of glial cells of the astrocytic type, at a distance from the injection site;
- a non-human mammalian animal, modified without affecting its genome, capable of being obtained by inoculation of the gliotoxic factor as obtained in a culture of monocytes or in a biological fluid of patients suffering from multiple sclerosis
a process for obtaining an animal as defined above, comprising the steps consisting in
* have an infectious amount of gliotoxic factor, as obtained from a culture of monocytes or from a biological fluid from a patient suffering from multiple sclerosis,
* inoculate said infectious amount to the animal
- the use of an animal of the invention to measure the effectiveness of a therapeutic process, in particular a drug, anti-inflammatory or antitoxic agent
- the use of an animal of the invention, to measure the effectiveness of a therapeutic process in particular of a medicinal agent, intended for the treatment of multiple sclerosis
- the use of an animal of the invention to determine the harm or harmlessness of a therapeutic process, in particular a drug agent, on the pathological brain of the animal described
a method for measuring the effectiveness of a therapeutic method, in particular of a drug, anti-inflammatory agent, comprising the steps consisting in:
* have an animal of the invention,
* administer said therapeutic process to the animal,
* measure the effectiveness of the therapeutic process by observing the reduction or absence of the pathological sign (s) described above, compared with a sick control animal having received the gliotoxic factor, but not having received the therapeutic process, and with reference to a normal control treated by said therapeutic process but not having received a gliotoxic factor;
a method for measuring the effectiveness of a therapeutic method, in particular a medicinal agent, intended for the treatment of multiple sclerosis, comprising the steps consisting in:
* have an animal of the invention,
* administer said therapeutic process to the animal,
* measure the effectiveness of the therapeutic process by observing the reduction or absence of the pathological sign (s) described above, compared with a sick control animal having received the gliotoxic factor, but not having received said therapeutic process, and with reference to a normal control treated by said therapeutic process but not having received a gliotoxic factor
a method for determining the harmlessness or harmlessness of a therapeutic method, in particular of a medicinal agent, on the pathological brain of the animal of the invention, comprising the steps consisting in:
* have an animal of the invention,
* administer said therapeutic process to the animal,
* measure the response to the therapeutic process by histopathological observation studying the criteria previously described.

 Another use of the modified animal according to the invention lies in obtaining hybridomas capable of producing monoclonal antibodies directed against the gliotoxic factor and originating from B lymphocytes taken from said animal and fused with tumor cells adapted to the obtaining hybridomas (rat / rat or rat / mouse hybridomas according to Kohler and Milstein 1975).

Thus, the invention relates to a process for the production of monoclonal antibodies directed against gliotoxic factor and / or against molecules induced or modified by the effects of gliotoxic factor, comprising the steps consisting in fusing B lymphocytes taken from an animal of the invention, with appropriate tumor cells, and to have produced by the hybridoma derived after selection, said antibodies.

 The subject of the invention is also a monoclonal antibody directed against the gliotoxic factor and / or against molecules induced or modified by the effects of the gliotoxic factor, capable of being obtained by the above method.

 Preferably the animal modified according to the present invention belongs to the family of muridae, in particular it is a mouse or a rat. But the animal of the invention could be a monkey or a guinea pig.

 The experimental part below grouping examples 1 to 3 in relation to FIGS. 1 to 11 illustrates the pathological effect of the gliotoxic factor in vivo in rats, and the determination of this effect makes it possible to establish the link with the processes. lesions observed in MS. In Examples 1 to 2, the evolution of the lesions affecting the astrocytes is examined, after intraventricular injection of this factor in the rat.

Figure 1 is a photograph showing the organization and orientation of periventricular astrocytes after injection of the toxic factor
Fig. 1A: controls: the GFAP + cells close to the left lateral ventricle are of the fibrous type; the cell bodies are densely marked; the extensions are relatively thick; GFAP + cells are oriented parallel to the cutting plane (vertical-frontal section);
Fig. 1B: rats treated with the toxic factor the GFAP + cells observed near the left lateral ventricle have an enlarged and / or polymorphic soma with a disorganization of the cytoplasmic extensions; the orientation of these cells is perpendicular to the section plane and the organization of the cells is palisade;
Fig. 1C: at higher magnification of FIG. 1B, we observe a normal astrocyte
Fig. 1D: at higher magnification of FIG. 1B, an astrocyte is observed whose cytoplasmic extensions are oriented towards the left lateral ventricle;
Bar: A, B (20 ssm) C, D (60 ssm)
Figure 2 illustrates nodules in the CNS of rats treated with the toxic factor
Fig. 2A: in the striatum of the control rats, the GFAP + cells are small, star-shaped, the extensions are short and thin
Fig. 2B: in rats treated with factor, the astrocytic bodies are enlarged, and have abnormal cytoplasmic extensions (very fine and numerous);
Fig. 2C: in rats treated with factor, nodules are observed in the striatum
Bar: 20 ssm
Fissure 3 represents the organization of periventricular astrocytes following the injection of the gliotoxic factor
Fig. 3A: in control rats, the astrocytic extensions form GFAP + immunoreactive patches around the vessels
Fig. 3B: in the treated rats, the astrocytic extensions around the vessels regressed or disappeared; we also observe a "depletion" of the vascular environment in astrocytes
Fig. 3C: in treated rats, near certain vessels, foci of astrocytic gliosis are formed; astrocytes are densely labeled with
GFAP; the cytoplasmic extensions are thicker;
Bar: 40 ssm
Figure 4 shows the distribution of astrocytes in the cortex, 10 days after injury
Fig. 4A: in the control animals, in the marginal zone of the piriform cortex, the astrocytes have the appearance of "candles" and their cytoplasmic extensions are oriented towards the pia perpendicular to its surface
Fig. 4B: in layer I of the piriform cortex in the treated rats, there is a structural modification of the GFAP + cells which have lost their physiological orientation; we observe a disorganization of the glia limitans established by the astrocytic prolongations
Fig. 4C: in control animals, in layers II and III of the cortex, the GFAP + cells have a star shape; numerous cytoplasmic extensions occupy the intercellular space
Fig. 4D: in the treated rats, there is a regression of the cytoplasmic prolongations, an increase in the intercellular space and a loss of the "star" morphology of the astrocytes
Bar: 40 ssm
Fissure 5 highlights the fragmentation of cellular DNA by the TUNEL technique after treatment with the toxic factor
Fig. 5A: in the treated rats, the TUNEL + cells are observed in the wall of the lateral ventricles
Fig. 5B: in treated rats, observation in the white matter of the cerebellum
Fig. 5C: in treated rats, observation in the layer of grains of the cerebellum
Fig. 5D: in treated rats, observation in arachnoid vessels
Fig. 5E: in the treated rats, it is observed that in the cerebellum, the fragmented DNA is found in the cytoplasm;
Fig. 5F: in the treated rats, the TUNEL + cells are observed in the choroid plexus, with hematoxylin counter staining
Bar: 40 ssm
Fissure 6: in rats treated with the toxic factor, GFAP positive cells are TUNEL positive; endothelial cells are sometimes also
TUNEL positive
Fig. 6A: GFAP + cells (blue), also TUNEL + (brown) in the cortex
Fig. 6B: in the striatum; the absence of cytoplasmic extensions and the attenuation of the GFAP labeling are observed
Fig. 6C: doubly labeled GFAP / TUNEL cell in the cerebellum
Fig. 6D: doubly labeled cells (FVIII / TUNEL) in the vascular wall; the marking of
FVIII is very weak
Bar: 40 ssm
Fiqure 7 is a photograph showing the opening of the blood-brain barrier in rats treated with the toxic factor
Fig. 7A: in a large periventricular zone, a diffusion of immunoglobulins from the blood is observed; immunostaining is diffuse
Fig. 7B: at higher magnification, an IgG halo is observed around a vessel
Bar: A 20 ssm, B 40 Um
Fissure 8 represents microglial-macrophagic reactivity
Fig. 8A: in the controls, OX42 + cells are observed in the striatum; the microglia is in quiescent form, the marking is weak; very few cells are observed
Fig. 8B: in the treated animals, there is a morphological modification of the OX42 + cells and an increase in their number
Fig. 8C: at higher magnification of FIG. 8B, quiescent microglial cell
Fig. 8D: at higher magnification of FIG. 8B, branched reactive microglial cell
bar: A, B, 20 ssm C, D, 40 Mm
Figure 9 illustrates foci of reactive microglia in the brainstem
Fig. 9A: focus of OX42 + cells in the brainstem
Fig. 9B: at higher magnification, the OX42 + cells are reactive of the branched and pseudopodic type
Bars: A 10 ssm B 20 ssm
Figure 10 illustrates myelin degradation
Fig. 10A: white substance of the cerebellum where there is an attenuation of the immunostaining in a large well defined area
Fig. OB: bundle of myelin in the cerebellum where there is a strong myelin degradation
Fig. 10C: enlargement of Fig. OB: very clear myelin degradation
Bar: A, B, 10 ssm C 20 ssm
Figure 11 represents a co-detection of astrocytes and TUNEL + cells compared to astrocytes in patient biopsies (MS)
Fig. 11A: reactive astrocyte (blue) surrounded by TUNEL + cells (brown)
Fig. llB: small GFAP + cells, weakly labeled by anti-GFAP, lacking cytoplasmic extensions and located at the periphery of the plate
Fig. 11C: TUNEL + cells in the vascular wall, endothelial phenotype
Bar: 40 ssm
EXAMPLE 1: DESCRIPTION OF THE MATERIAL AND
TECHNIQUES USED
a) The gliotoxic factor
CSF or urine samples, or monocytes, are taken from MS patients.

The toxic factor is partially purified after treatment on an ion exchange column, then on an exclusion separation column, in accordance with Example 11 of patent application PCT / FR95 / 00178. The gliotoxic factor consists mainly of a light fraction of 17 Kd and a heavy fraction of 21 Kd, both of which have a strong affinity for concanavalin.

 The separation of the gliotoxic factor is detailed below, from crude culture supernatants of monocytes / macrophages from patients suffering from MS.

* Preparation of monocyte / macrophage cultures of MS patients
Culture medium includes RPMI1640 (Boehringer), penicillin-streptomycin (bioMérieux), L-glutamine (bioMérieux), sodium pyruvate (Boehringer), non-essential amino acids 100x (Boehringer), serum human AB taken from healthy donors and seronegative for all viruses transmissible by known blood derivatives (see
PERRON et al., The Lancet, vol 337, pages 862-863, April 6, 1991).

 The lymphoid cells are cultured in 75 cm 3 Primaria culture flasks (Falcon) after being separated from the plasma and the other blood figured elements by centrifugation on a Ficoll gradient (Lymphoprep, Flow). To obtain these lymphoid cells, 50 ml of blood are taken by venipuncture on a sterile heparinized tube (lithium heparin). The blood and heparin are well mixed as soon as the blood is drawn.

Alternatively, blood can be drawn into tubes containing EDTA. It is then important to immediately transport the tubes maintained at +4 OC to the laboratory, where they will be handled under a "biohazard" laminar flow culture hood under sterile conditions. For a culture of monocytes, an "RPMI" medium is prepared which comprises 100-150 ml of RPMI 1640 medium, a mixture of penicillin and streptomycin, 4% of Lglutamine, 1% of sodium pyruvate, 1% of non-amino acids -Essential Boehringer (100X). Also prepared are 3 sterile 50 ml conical bottom tubes (Falcon) containing 10 ml of the "RPMI" medium described above, and 4 sterile 50 ml tubes with 20 ml of Ficoll at the bottom. The heparinized tubes are opened to pipette the blood, place it in the tubes containing medium and mix it gently with the medium described above. 5 ml of "RPMI" medium are taken and the wall of the heparinized tubes is rinsed. This rinsing must be accompanied by a light scraping using the end of the plastic pipette in order to detach cells which may have adhered to the walls of the tube, and place it in the tubes containing the blood diluted in the "RPMI" medium, by gently mixing the contents by successive aspirations / repressions. These operations should be repeated until the heparinized tubes are clean. It is then necessary to deposit very gently (without swirling) the blood diluted in the medium "RPMI", on the surface of the Ficoll in the 50 ml tubes then use medium "RPMI" to rinse the rest of the diluted blood and recover it as previously to gently place it on the surface of the tubes with the Ficoll. Then, and without shaking the Ficoll, the tubes must be placed in centrifuge cups, equilibrated using tubes filled with water, and centrifuged at + 150C for 20 minutes at 1800 rpm, with a deceleration mode slow. After centrifugation, the tubes are recovered in which a pipette is gently inserted up to the top of the "Ficoll / plasma" interface and the whitish layer located above the Ficoll is gently sucked in by concentric circles from the walls , then by describing "zig-zags" across the surface of the
Ficoll. The aspirated medium is placed in 50 ml tubes, diluted in at least 3 times the volume of RPMI medium and mixed gently by inverting the sterile stoppered tubes. The tubes are then centrifuged at +15 ° C. for 10 minutes at 1800 rpm, with a slow deceleration mode. After centrifugation, the supernatant from these tubes is discarded by pouring slowly but regularly, while ensuring that the whitish pellet of cells does not come off. The pellet is resuspended in 10 ml of "RPMI" medium by successive aspirations / discharges and the suspension is centrifuged at +15 "C for 10 minutes at 1800 rpm, with a slow deceleration mode. By sampling or by 50 ml of blood collected, two small electropositive plastic culture flasks (Falcon "PRIMARIA") of 75 cm3 and 10 ml of "RPMI" medium are prepared, to which 15% of human serum "AB" (SH) described above will have been added. centrifugation, the supernatant is removed as above, the pellet is gently resuspended in 5 ml of "RPMI" medium with 15% SH and the resuspended cells are distributed in the vials placed flat and barely lifted. The suspension is immediately distributed by stirring each flask flat. The centrifuge tubes are rinsed with 5 ml of "RPMI" medium at 15% SH, and the suspension is added and distributed in the two flasks, as before. Advantageously, all the media used for these steps are 37 "C (warmed in a water bath). Once the bottles are closed, they are kept flat in a humid oven at 370C with 5% C02 until the next morning. The following morning, it is advisable to aspirate well all the supernatant with the cells in suspension, to rinse the flasks twice with 4 ml of RPMI alone, leaving to "soak" for 5 minutes each time and slowly raising the flask before aspirate all the remaining medium, in order to eliminate the non-adherent cells. The bottles are then filled with 5 ml of RPMI medium containing 15% of SH, they are replaced in the oven and care is taken not to move them for 4 h. From this stage, it is always advisable to fill the bottles placed upright by directing the jet on the upper wall so as not to detach the cells in the course of adhesion, then, subsequently, affected by a possible cytopathogenic effect. The cell suspensions thus collected 24 h after the cultivation, are centrifuged at + 150C for 10 minutes at 1800 rpm, with a slow deceleration mode. Optionally, the cell pellet can be taken up in fetal calf serum with 10%
DMSO (Dimethyl sulfoxide) to be frozen at -800C or in liquid nitrogen according to a procedure for maintaining viable cells. The corresponding supernatant is then centrifuged at 3000 rpm for 30 min in order to remove the cell debris, and the clarified supernatant is aliquoted, listed as a 24 h culture sample, ie D1, then stored in the freezer at -800C. After 48 h in the oven, the bottles are taken out, the supernatant is gently aspirated, and, as before, centrifuged at 3000 rpm for 30 minutes in order to remove the cellular debris. The clarified supernatant is aliquoted, listed as a sample after 3 days of culture, ie
D3, then stored in the freezer at -800C. The flasks are immediately filled with 5 ml of RPMI medium at 5% SH and replaced in the oven. From this moment, the culture medium contains only 5% of SH and this proportion will be used for all medium renewals. The media from the flasks are then removed, stored under aliquots of clarified medium of cellular debris, at -800C as before, and replaced by "RPMI" medium at 5% SH, every three or four days, until no adherent and refractive cells remaining under microscopic observation in the bottle no longer persist.

* Separation of the gliotoxic factor
The culture supernatant samples are pooled (D1 to D final) after thawing and are previously heated 30 min at 560C and centrifuged 10 min at 1500 rpm, then the supernatant is recovered and, optionally, dialyzed at 40C in 2 times 20 volumes of D-PBS buffer, a first time for 2 h, and a second overnight. The supernatant thus collected constitutes the sample from which the gliotoxic factor will be separated, according to the technique described in patent application PCT / FR95 / 00178.

b) Intraventricular brain injections
The experiments are carried out on spleens
Lewis, adults (2 months), weighing between 200-250 g at the start of the experiment. The animals are anesthetized with chloral hydrate (400 mg / kg) injected intraperitoneally.

 The injections of the toxic solution, 5 to 10 Mg of active proteins in 5 41 of a sterile PBS (phosphate buffered saline) solution are carried out by stereotaxic methods, in the left lateral ventricle, (AP: 0.8, L : 1.5. P: 4). The injections are made using a micropipette (22 ssm in diameter) attached to a 25 41 Hamilton syringe.

 The animals are sacrificed 10 days (n = 9) after the operation. Six control animals are injected under the same conditions with 5 41 of a sterile PBS solution, and are sacrificed at the same time.

All animals receive a lethal dose of chloral hydrate (0.5 g / kg), then perfused transcardially through the ascending aorta with a solution of 4% paraformaldehyde in 0.12 M phosphate buffer. brains with cervical cord are postfixed in the same fixative for one hour at 40C, then cryoprotected in a 10% sucrose solution in 0.12 M phosphate buffer for 2 days, then frozen in isopentane cooled in liquid nitrogen at -40 C and stored at -80 C. Slides with vertical-frontal cryostat, 10 µm thick, serialized every 100 ssm are collected on 1% gelatin slides.

Some cups are colored purple
Cresyl, or hematoxylin eosin.

c) Antibodies
To mark the astrocytes, an anti-glial acid fibrillar glial protein (anti-GFAP) monoclonal antibody (BOEHRINGER) diluted to 1/200 is used. Myelin is visualized by an anti-basic myelin protein antibody (anti-MBP) (BOEHRINGER) diluted to 1/500. Monoclonal antibodies OX42 diluted 1/500 (SEROTEC) are used to identify microglial macrophagic cells.

 When the BBB is opened, specific elements of the blood are detected in the brain parenchyma such as immunoglobulins (Dusart & col 1993). A rat anti-immunoglobulin antibody (AMERSHAM) diluted to 1/200 is used to visualize a possible rupture of the BBB.

 An anti-FVIII antibody is used to visualize endothelial cells.

 In all cases, the sections are incubated with the primary antibodies at the concentrations indicated overnight at 40C, they are diluted in PBS 0.1 M 2% BSA (bovine serum albumin), 0.3% Triton-X. The sections intended to be marked with the anti-MBP are degreased beforehand in absolute alcohol containing 5% acetic acid, for 25 minutes at 40C, then immersed in alcohol baths at 95 and then at 70, for 5 minutes for each, and rinsed several times in PBS.

 The secondary antibody (anti-biotinylated mouse IgG, AMERSHAM) diluted to 1/200 is placed in the presence of the sections, for approximately 2 hours at room temperature.

 The sections are then incubated with the streptavidin-biotin / peroxidase complex (1/200). The peroxidase activity is revealed in the presence of its specific substrate H2O2 and of DAB. To saturate endogenous peroxidases, the sections are incubated for 10 minutes in 0.1 M phosphate buffer (pH = 7.4) containing 0.4% hydrogen peroxide, then are immersed in a PBS solution containing 2% of BSA for 30 minutes before being incubated with primary antibodies.

Certain sections treated with anti antibodies
GFAP, anti-FVIII are revealed according to the ABC-AP technique.

These cuts are then treated according to the TUNEL method described below.

d) TUNEL method (Gavrieli & col 1992)
The sections are treated with proteinase K (BOEHRINGER) (20 Ug / ml) for 15 minutes, then incubated with TdT and biotinylated UTp, for 1 hour glial cells and endothelial cells. For this purpose we describe the progression of the macrophagic microglial response, the state of the BBB, myelin and astrocytic morphology.

a) Impact of the toxic factor on astrocytes
The lesion is bilateral, and affects the distribution, orientation and organization of all of the rat's CNS astrocytes. Astrocytes show profound morphological changes which vary according to their location. The lesions will be described, starting from the periventricular spaces towards the subpial spaces.

In accordance with Figures 1B, 2B, 2C, 3B and 3C, there are several cases
- GFAP-positive cells (GFAP +) of abnormal morphology, with long cytoplasmic extensions, are organized in palisades and are oriented towards the lateral ventricles (Fig. 1B);
- by moving away from the ventricular spaces, in particular in the striatum, we observe GFAP cells of fibrous appearance densely nested one inside the other, forming nodules (Fig. 2B, 2C). These nodules are observed in all of the telencephalic regions
- the immunoreactive zones, normally formed by astrocytic extensions around the vessels, are not visible around most of the vessels in rats treated with the toxic factor (Fig. 3B)
- one observes astrocytes presenting reactive forms, characterized by an enlargement of their somas and a thickening of their prolongations, they are densely marked with GFAP and often arrange themselves in cellular foci near certain vessels (Fig. 3C)
- in the cortex, the GFAP + cells located in the deep layers lose their extensions and become scarce; in the surface layers, the astrocytes whose cytoplasmic prolongations orient themselves towards the pia normally have an abnormal morphology and an anarchic orientation (Fig. 4B).

The distribution of astrocytes in control rats is normal and comparable to that described by
Kalman & Hajos 1989. Astrogliosis, in controls without gliotoxin (that is to say to whom a placebo was injected), is limited at the injection site.

b) Fragmentation of cellular DNA induced by the toxic factor in the CNS of the rat (demonstrated by the TUNEL method)
Cells giving a positive response in the method of TUNEL (TUNEL +) are observed throughout the CNS of the rat. They are more frequent in the wall of the ventricles where they correspond to ependymal type cells (Fig. 5A). They are observed in the cerebellum (Fig. 5B), some are localized in the layer of grains (Fig. 5C), others in the walls of the blood vessels located in the subarachnoid spaces (Fig. 5D) and intraparenchymatous, and also in the choroid plexus (Fig. 5E). Some TUNEL + cells have fragmented DNA in their cytoplasm. Other TUNEL + cells are observed in the choroid plexus (Fig. 5F).

 GFAP + cells are TUNEL +. They are observed throughout the CNS (Fig. 6A, 6B, 6C). TUNEL + cells labeled with FVIII are also observed (FIG. 6D). It should be noted that the labeling of FVIII in this case is very weak.

 No case of DNA fragmentation was observed in the control animals. One or two TUNEL + cells are sometimes visible at the injection site of the placebo solution. However, the presence of TUNEL + cells disseminated in the cerebral parenchyma is specific to gliotoxin + animals.

c) Opening of the blood-brain barrier
A diffusion of immunoglobulins from the blood is observed in the cerebral parenchyma, in all the animals treated with the toxic factor. It is more marked in the periventricular spaces, around certain vessels (Fig. 7A, 7B). This phenomenon was not observed in the controls.

d) Microglial-macrophagic reactivity
The OX42 + cells have characteristic reactive forms, a distinction is made between branched and pseudopodic forms. They are more numerous compared to what is observed in the control rats (Fig. 8B). In certain regions of the brainstem and cerebellum, small foci of OX42 + cells are observed (Fig. 9A, 9B).

e) Toxic factor-induced demyelination in rats
Demyelination areas are visualized by attenuation of the labeling of MBP in well-defined foci. These foci are very clearly observable in the brainstem as well as the cerebellar white matter (Fig. 10A). Cases of myelin degradation are also observed scattered in practically the whole
CNS of the rat (Fig. 10B, 10C).

EXAMPLE 3: INTERPRETATION OF ACQUIRED RESULTS
The toxic factor isolated and purified from
CSF, urine or monocyte culture, from MS patients, injected into the rat CSF induces lesions affecting glial and also endothelial CNS cells. The toxic factor is probably distributed by the CSF which circulates in the ventricles, then in the subarachnoid spaces. It is absorbed by the arachnoid vessels, then the venous sinuses. TUNEL + cells are located in the ventricular walls (ependymal type cells) and in arachnoid vessels. Involvement of the ependymal cells would modify the interface between the CSF and the brain, which is likely to promote the formation of lesions in the periventricular spaces. In MS, lesion plaques are located near the ventricular system and are common around the venules.

Some authors have mentioned the existence of an agent of an enzymatic, or immune or other nature, diffusing
CSF or blood, in the brain (see Marburg 1936,
Lumsden 1970). The injected toxic factor seems to circulate in the CSF, and has a targeted action on certain cell types. It would therefore be diffusible from the CSF to the cerebral parenchyma where it affects certain cell populations.

 The lesion affecting astrocytes is complex.

A fragmentation of astrocyte DNA is observed suggesting death by apoptosis of these cells. Astrocytes induce the formation and maintenance of the BBB, through their perivascular cytoplasmic extensions (astrocytic feet), even if they do not constitute it (Stewart & Wiley 1981). The alteration of the perivascular astrocytes suggests a retraction of the astrocytic feet which can consequently modify the tightness of the BBB. Also the fragmentation of the DNA of cerebral endothelial cells suggests modifications of the intrinsic properties of these cells which could lead to an opening of the BBB. The detection of blood immunoglobulins in the rat brain is proof of this. It is possible that it is the morphological modifications of the perivascular astrocytes, which occur very early, which lead to the modification of the phenotype of the endothelial cells and therefore to the alteration of the BBB.

 Several authors insist on anomalies at the level of the blood vessels in the chronic plaques of MS (Jellinger 1969, Weller 1985, Gay & Esiri 1991).

The endothelial cells of the capillaries contain immunoglobulins and α1-antiglobulin indicating the increase in transendothelial vesicular transport at the periphery of the plates. We also noted the passage of humoral and cellular immune mediators, also testifying to an opening of the BBB (Compston & col 1989).

Astrogliosis has also been observed near certain vessels and a very marked proliferation of fibrous astrocytes throughout the rat's CNS. Astrocytes, after stimulation, produce a variety of immunoregulatory molecules. They include
IL-1 (Fontana & col 1982), 1'-IL-6 (Frei & col 1989), IFN- (Tedeschi & col 1986) and also TNF-a (Robbins & col 1987, Sawada & col 1989, Chung & col 1990). TNF-a is toxic to endothelial cells (Deguchi & col 1989, Ishii & col 1992). It has an important role in the progression of lesions in MS (Sharief & col 1991).

There is a correlation between the opening of the BBB and the production rate of TNF-a in patients with MS (Sharief & Tompson 1992). TNF-a has a direct cytotoxic effect on oligodendrocytes and causes myelin destruction (Selmaj & Raine 1988). It induces proliferation of astrocytes in vitro (Barna & col 1990).

 We also know that astrocytes are found at the crossroads of inflammatory reactions. They represent the resident immunocompetent cells in the CNS (Fontana & col 1987). A primary alteration of astrocytes in the lesional processes of MS could both destabilize the homeostatic balance of the CNS, and trigger a cascade of inflammatory events which would lead to demyelination.

 Soluble factors of monocytic or lymphoid origin could alter the biological activity of astrocytes (Chung & col 1990) and induce cytokines, some potentially demyelinating.

 Macrophagic microglial proliferation was observed in rats treated with the toxic factor. This reactivity is observed in the form of OX42 + foci located in the white matter, and more in the brainstem. The activated microglia also produces cytokines, such as TNF-a (Frei & Fontana 1989, Hetier & col 1990), IL-6 (Frei & col 1989) and others. Macrophagic microglial reactivity has been described in MS and could participate in the demyelination processes (Woodroofe & col 1986, Esiri & col 1987).

Demyelination is also observed. The demyelination areas are very clearly delimited in the brainstem and the cerebellar white matter. These aspects are very close to those observed in MS. The results obtained suggest that the toxic factor isolated from the CSF of patients
MS induces large lesions in the CNS of the rat.

These lesions are similar in nature and distribution to some of the lesions observed in the
MS, in particular demyelination, fibrillar astrogliosis, macrophagic microglial reactivity and the opening of the BBB with the formation of edema objectified by the diffusion of intraparenchymal plasma immunoglobulins.

 These results show the interest of using this new experimental animal model to study the neuropathology of MS and to develop therapeutic strategies to inhibit the synthesis or block the action of the gliotoxic factor and to counteract its effects in a model. in vivo.

EXAMPLE 4: EVALUATION OF THE EFFICIENCY
THERAPEUTICS OF A TREATMENT OF MULTIPLE SCLEROSIS A
HELP FROM THE GLIOTOXIN INDUCED ANIMAL MODEL
First, a purified gliotoxic factor sample is prepared as described in Example 1 and in the patent application
PCT / FR95 / 00178.

 Second, a series of female Lewis rats is selected according to the criteria described in Example 1.

 Thirty or more of these animals receive on day 0 a dose of gliotoxic factor by stereotaxic intraventricular injection, according to the protocol described in Example 1. On days 0, 1, 3, 8, and 10 (for example) after injection gliotoxic factor, they receive an adequate dose of a therapeutic agent such as Rolipram, interferon p, azathioprine, cyclophosphamide, an anti-gliotoxin antiserum, or any other therapeutic means to be evaluated. This series "A" corresponds to sick animals (gliotoxin +) treated with an active product or process, and can be reproduced in several variants to evaluate an optimal quantitative and qualitative administration protocol among all the variants.

 Thirty (or more) of these animals receive on day 0 a dose of gliotoxic factor by stereotaxic intravencular injection, according to the protocol described in Example 1, but receive an inactive placebo of the treatment defined in series A, under strictly conditions equivalent. This series "B" corresponds to sick animals (gliotoxin +) constituting an untreated positive control.

 Thirty (or more) of these animals receive a placebo injection of gliotoxic factor on day 0 (injection buffer with serum albumin), but receive the active therapeutic protocol defined in series A. This series "C" corresponds to healthy animals (gliotoxin -) constituting a treated negative control which makes it possible to evaluate the effects possibly linked to the active treatment, apart from the effect of the gliotoxic factor.

 Therapeutic efficacy is evaluated by sacrificing animals of series A, B and C, on day 10, one month, three months, six months, or even a year if necessary, after the injection of the gliotoxic factor or its placebo, according to the protocol described in Example 1.

 The brains of the animals are studied according to the technical descriptions of Example 1.

The following criteria are used in particular for the comparison between animals of series A, B and C:
General aspects
- condition
- weightloss
- behavioral changes.

Cerebral histopathology (away from the injection site):
- morphological and histological anomalies of the astrocytes (as described in the previous examples)
- DNA fragmentation (TUNEL positive technique) mainly in astrocytic cells, but possibly and in particular ependymal or endothelial
- opening of the BBB visualized by at least one intra-parenchymal diffusion zone of serum immunoglobulins
- macrophagic activation of microglial cells;
- morphological and histological anomalies of myelin structures and / or oligodendrocytes
- presence or absence of lymphocyte infiltrates, in particular perivascular.

 Therapeutic efficacy has been demonstrated if a significant difference (reproducible in a number of statistically analyzable animals) exists between the frequency, intensity, surface area, summation, of the above-described anomalies observed in animals of series A ( gliotoxin + / active treatment) and those of series B (gliotoxin + / placebo treatment).

 The therapeutic efficacy is all the better as the difference between the observations reported in series A and those reported in series C, becomes non-significant according to the same criteria as above.

 This efficiency can thus be evaluated according to the different treatment application protocols (series "A" with variants) and the efficacy in the long term, as a function of the time elapsed after injection of the gliotoxic factor, is evaluated by sacrificing animals of each series. at increasing times as mentioned above.

EXAMPLE 5: OBTAINING SPECIFIC ANTIBODIES
GLIOTOXIC ANTI-FACTOR AND / OR DIRECTED AGAINST
NOLECULES INDUCED OR MODIFIED BY FACTOR EFFECTS
GLIOTOXIC
Injection of gliotoxic factor (intracerebral, intraperitoneal, or IP, intravenous, or IV, intramuscular, or IM, or intradermal, or ID) in a series of animals according to Example 1.

 Recalls by IP, IV, ID or IM injections after one month, two months, three months or more.

 Removal of the animal's spleen (organ) after one month, two months, three months, four months or more.

 Verification of neuropathological damage according to Example 1. Conservation of fixed brain sections for the study of the antibodies produced.

 Extraction of lymphocytes from the spleen and recovery in culture medium with a view to fusion with transformed cells suitable for obtaining hybridomas secreting immunoglobulins.

 Selection of viable merged clones.

Screening of specificities of antibodies produced by hybridomas
Gliotoxin
1- activity inhibition test:
The antibody suspensions produced by the different clones are added to culture wells containing a solution of gliotoxic factor as defined for the bioassay described in the patent application.
PCT / FR95 / 00178. Suspensions of control antibodies from animals injected with a gliotoxin placebo are added to control wells. The gliotoxic activity is measured in each well in accordance with the description of the aforementioned patent application. Any significant difference obtained with an antibody suspension showing a gliotoxic activity lower than the “floor” control value (average of the wells with control antibodies minus three standard deviations), shows an inhibiting anti-gliotoxin activity.

 These inhibiting antibodies are of interest for the detection of the gliotoxic factor (ELISA, etc.), but also for anti-gliotoxin therapy (possibly, after humanization of the antibodies by substituting the constant part of the antibody with a chain of Human Ig and retaining the variable part of the animal antibody).

2- Serological test
A fraction of gliotoxin is exposed to the various antibody suspensions according to an appropriate serological technique and the specific antibodies are revealed by a positive reaction.

 Alternatively, a source of recombinant gliotoxin or synthetic peptides derived from its sequence can be used for this screening.

 The hybridoma clones of interest are subsequently cultured to produce monoclonal antibodies.

Tissue antigens
Serological tests can be applied to antigens extracted from the tissues of sick animals and compared with equivalent tissues from control animals to identify molecules induced or modified by the effect of the gliotoxic factor.

 These antibodies may have a diagnostic or even therapeutic interest.

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Claims (15)

 1. Mammalian animal, non-human, modified without affecting its genome, characterized in that it has a blood-brain barrier open or permeable to water-soluble molecules of the blood not specific for the cerebral parenchyma.  2. A mammalian, non-human animal, modified without affecting its genome, characterized in that it has demyelination plaques, in particular present in the brain stem and / or the cerebellar white matter.  3. A mammalian, non-human animal, modified without affecting its genome, characterized in that the glial cells and endothelial cells of the central nervous system of the animal are damaged, in particular the DNA of glial cells of the astrocytic type is at least partially fragmented.  4. A mammalian, non-human animal, modified without affecting its genome, characterized in that it has at least any two of the pathological signs following a blood-brain barrier open or permeable to water-soluble molecules of the blood not specific for the cerebral parenchyma; demyelination plaques, especially present in the brainstem and / or cerebellar white matter; lesions of glial cells and endothelial cells of the central nervous system, in particular a modification of the morphology and / or at least partial fragmentation of the DNA of glial cells of the astrocytic type.  5. A mammalian, non-human animal, modified without affecting its genome, characterized in that it is capable of being obtained by inoculation of the gliotoxic factor as obtained in a biological fluid from patients suffering from multiple sclerosis.  6. Animal according to any one of the preceding claims, characterized in that it belongs to the family of muridae, in particular the animal is a rat.  7. Method for obtaining an animal according to any one of the preceding claims, comprising the steps consisting in  - have an infectious quantity of gliotoxic factor, as obtained from a biological fluid from a patient suffering from multiple sclerosis,  - inoculate said infectious amount to one animal.  8. Use of an animal according to any one of claims 1 to 6, for measuring the therapeutic effectiveness of a therapeutic process, in particular of a medicinal, anti-inflammatory agent.  9. Use of an animal according to any one of claims 1 to 6, for measuring the therapeutic effectiveness of a therapeutic process, in particular of a medicinal agent, intended for the treatment of multiple sclerosis.  10. Use of an animal according to any one of claims 1 to 6, for determining the harmlessness or harmlessness of a therapeutic process, in particular of a medicinal agent, on the pathological brain of the animal.  11. Method for measuring the effectiveness of a therapeutic process, in particular of a medicinal, anti-inflammatory agent, characterized in that it comprises the steps consisting in:  - have an animal as defined according to any one of claims 1 to 6,  - administer said therapeutic process, in particular a medicinal agent to the animal,  - measure the effectiveness of the therapeutic process by observing the reduction or absence of the pathological sign (s) defined in claims 1 to 4, compared with a sick control animal having received gliotoxic factor, but not having received the process therapeutic, and with reference to a normal control treated by said therapeutic process but not having received a gliotoxic factor.  12. Method for measuring the effectiveness of a therapeutic process, in particular of a medicinal agent, intended for the treatment of multiple sclerosis, characterized in that it comprises the steps consisting in:  - have an animal as defined according to any one of claims 1 to 6,  - administer said therapeutic process to the animal,  - measure the effectiveness of the therapeutic process by observing the reduction or absence of the pathological sign (s) defined in claims 1 to 4, compared with a sick control animal having received gliotoxic factor, but not having received the process therapeutic, and with reference to a normal control treated by said therapeutic process but not having received a gliotoxic factor.  13. Method for determining the harmlessness or harmlessness of a therapeutic process, in particular of a medicinal agent, on the pathological brain of an animal as defined in any one of claims 1 to 6, characterized in that '' it includes the steps of  - dispose of said animal  - administer said therapeutic process to the animal,  - measuring the response of the therapeutic process by histopathological observation of the pathological sign or signs defined in claims 1 to 4.  14. Process for the production of monoclonal antibodies directed against gliotoxic factor and / or against molecules induced or modified by the effects of gliotoxic factor, comprising the steps consisting in fusing B lymphocytes taken from an animal as defined in one any one of claims 1 to 6, with tumor cells suitable for obtaining hybridomas, and for producing by the hybridoma derived selected said antibodies.  15. Monoclonal antibody directed against the gliotoxic factor and / or against molecules induced or modified by the effects of the gliotoxic factor, capable of being obtained by the method according to claim 14.