Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1999-03-29
2001-08-07
Chan, Christina Y. (Department: 1644)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S007210, C435S040500
Reexamination Certificate
active
06270953
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the determination, detection and quantification, in a biological sample, of a gliotoxic factor such as associated with multiple sclerosis.
2. State of the Art
A “biological sample” is understood, in particular, as being a specimen of the biological fluid type, live tissue or tissue fragment, mucus sample, organ or organ fragment type, or any culture supernatant obtained with the aid of an aforementioned specimen.
In accordance with document WO-A-95/21859, which is an application filed in the name of the applicant, a factor which is cytotoxic for glial cells (astrocytes, oligodendrocytes and microgliocytes), and which is termed gliotoxic factor below, has been isolated and/or characterized. While this factor is, in particular, associated with multiple sclerosis, it could also be associated with other neurodegenerative or autoimmune diseases.
In the absence of a complete sequence for this gliotoxic factor, the latter can be characterized either using a process (among others) which enables the factor to be isolated or purified or using different biological, biochemical or chemical properties or characteristics.
According to document WO-A-95/21859, this gliotoxic factor is characterized by all the following properties, considered separately or in combination:
it possesses a toxic activity for human or animal astrocytic cells, with its effect being that of eliciting cytomorphological disruption of their network of intermediate filaments and/or degradation of the proteins of these intermediate filaments and/or cell death, in particular by apoptosis,
its activity is associated with at least one glycoprotein,
this gliotoxic factor consists in the main, if not entirely, of a light-weight fraction which is centred on an apparent molecular weight of approximately 17 kD; this light-weight fraction is resistant, under standard non-denaturing conditions, to the hydrolytic action of pronase or trypsin or proteinase K; and this same light-weight fraction exhibits strong affinity for lectins, in particular concanavalin A.
Consequently, the detection and/or quantification of this gliotoxic factor in any biological sample is a worthwhile and effective analytical tool, in particular for the diagnosis of different pathologies, including multiple sclerosis, and the prediction, monitoring and therapy of this disease. Still in accordance with document WO-A-95/21859, the content and description of which are hereby incorporated into the present patent application by reference, a process for detecting and/or quantifying this gliotoxic factor in a biological sample, for example a sample of cerebrospinal fluid, has been described and proposed. Such a process comprises at least the following treatments:
a starting fraction of this sample, which is, where appropriate, enriched in the said gliotoxic factor by any appropriate prior treatment, is to hand,
this starting fraction is incubated with a reference culture medium which, for example, comprises glial cells, in particular immortalized cells, for example astrocytic cells, and
the dead or living glial cells are detected and/or quantified using any appropriate technique, for example using a calorimetric assay employing calcein AM and homodimeric ethidium, respectively.
Such a process, which is implemented for detecting and monitoring the therapy of multiple sclerosis, presupposes the existence of a so-called “invasive” specimen, for example of cerebrospinal fluid, that is to say which requires a prior medical or surgical act.
SUMMARY OF THE INVENTION
It has now been discovered that urine proves to be a particularly favourable biological fluid for detecting gliotoxic activity. This discovery was altogether surprising on account of the complex composition of urine and of its acidic nature and the presence of urea, which features were not, a priori, according to the general knowledge of the skilled person, compatible with detecting such a cellular activity.
It is therefore totally unexpected that, on the basis of this discovery, the inventors have developed a process for determining the gliotoxic factor in urine, which process additionally exhibits the advantage of using a non-invasive specimen, in the same way as any other sample obtained “invasively”, for example a sample of cerebrospinal fluid, for detecting and/or quantifying the said factor in a patient.
Thus, the invention initially relates to a process for detecting and/or quantifying a gliotoxic factor in a biological sample, according to which process a starting fraction of the said sample, where appropriate enriched in the said gliotoxic factor by a prior treatment, is to hand, the said starting fraction is incubated with a reference culture medium comprising macroglial cells, for example immortalized macroglial cells, such as astrocytic cells, and the dead and/or living macroglial cells are detected and/or quantified, with the said biological sample being a urine sample.
A detection and/or quantification process which comprises a step of specifically detecting and/or quantifying the cells which have died by the apoptosis induced by the gliotoxic factor has then been defined.
According to document EP-A-0 731 179, a process is known for detecting preapoptotic lymphocytes by flow cytometry after the intracellular DNA of the said lymphocytes has been labeled. This document illustrates the use of flow cytometry for detecting cells which are in a preapoptotic state and which are non-adherent, that is to say in suspension.
The techniques for detecting apoptosis in adherent cells are not based on the same principle. As practised nowadays, and because of the fragility of apoptotic adherent cells, these techniques are based on visually observing apoptosis in situ by means of microscopy, with the apoptotic cells being counted. By way of example, mention may be made of the article by R. W. Keane, A. Srinivasan, L. M. Foster et al., J. Neurosci. Res., 1997, 48, 168-180, which deals with detecting adherent apoptotic cells of the nervous system.
It therefore proved necessary to have available a process which overcomes the abovementioned drawbacks of the techniques which are currently used for detecting apoptotic cells, that is a process which is, in particular, simple and relatively inexpensive and which, in addition, makes it possible to take into account the constraints associated with the fragility of apoptotic cells.
In addition, according to the invention, a process is supplied for detecting and/or quantifying, in a biological sample, a factor which is cytotoxic for target adherent cells and whose cytotoxicity induces the death of the said cells by apoptosis, according to which process a starting fraction of the said sample, where appropriate enriched in the said toxic factor by a prior treatment, is to hand, the said starting fraction is incubated with a reference culture medium comprising target adherent cells and at least one direct or indirect feature which is associated with the apoptotic adherent cells of all or part of the incubated medium, which feature, if it exists and/or is quantified, categorizes the said biological sample as being positive, that is to say containing the said toxic factor, is detected and/or quantified, by flow cytometry, the adherent cells which have died by apoptosis.
The above-defined process is advantageously applied to detecting the gliotoxic factor, and the target adherent cells are magroglial cells, in particular astrocytic cells.
Thus, according to at least one of the abovementioned features, the following is provided:
a process which is able to comprise a “non-invasive” specimen,
a process which is simple to implement, which is sensitive and which is specific for the gliotoxic factor.
It is known that apoptosis of macroglial cells is characterized, in particular, by:
fragmentation of the cellular DNA of the apoptotic cells;
preservation of the integrity of the cytoplasmic membrane of the apoptotic cells;
alterations to the structure and size of the apoptotic cells.
Using the process of
Malcus-Vocanson Carine
Mandrand Bernard
Perron Herve
Bio Merieux
Chan Christina Y.
Clemens Karen
Oliff & Berridg,e PLC
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