Multiple Sclerosis (2001) 7, 383±388

ã 2001 Arnold All rights reserved 1352 ± 4585/01

www.arnoldpublishers.com/journals

Glial toxicity in urine and multiple sclerosis

C Malcus-Vocanson1, P Giraud2, F Micoud1, V Janin1, MH Charles1, E Broussolle2, G Chazot2,
B Mandrand1 and H Perron*,1
1
bioMeÂrieux-Pierre Fabre, 69280 Marcy L'Etoile, France; 2Department of Neurology D, HoÃpital Neurologique Pierre
Wertheimer, BP Lyon-Montchat, 69394 Lyon Cedex 03, France

The biochemical and biological characterization of a cytotoxic activity targeting macroglial cells (oligodendrocytes and astrocytes), in moncyte
cultures and in CSF of a patient with multiple sclerosis, has previously been described. In further studies, cell-based tests have shown a good
correlation between this glial cytotoxic (gliotoxic) activity, in CSF or in urine, and MS. We now present results obtained with urine samples from
102 MS patients, 51 patients with other neurological disease and 35 healthy subjects using a bioassay set up for the detection of an apoptosis-like
effect induced in a glial cell-line. Signi®cant gliotoxicity was detected in urine from 74/102 MS patients while only 4/51 neurological controls
(P40.001) and never in healthy subjects (P40.001). Given the statistical tendency provided by this bioassay and its technical limitations for
routine testing, it is now used for monitoring the molecular characterization of this `gliotoxic factor'. Its replacement by a speci®c immunoassay
could provide more accurate routine techniques for the detection of this biological marker in MS.
Multiple Sclerosis (2001) 7, 383±388
Keywords: multiple sclerosis; glial cell; cytotoxicity; urine; disease marker

Introduction
The diagnosis of multiple sclerosis relies upon a series
of clinical signs, MRI, biological (CSF) and electro-
physical examinations which must eliminate any
alternative cause (tumoral, metabolic, vascular ... )
and bring converging `positive' data. According to
international criteria revised with the introduction of
MRI,1 these data should be suf®cient for establishing a
diagnosis of MS.
It is generally accepted that MRI and oligoclonal
bands (OBs) are the most sensitive complementary
examinations for MS diagnosis.2 ± 5 MRI is also fre-
quently used for the follow-up of MS patients e.g. in
therapeutic trials.5 ± 7 Nevertheless MRI hypersignals,
OBs or abnormal evoked potentials are not speci®c for
MS and no test or examination taken alone allows a
diagnostic con®rmation. Despite established criteria,
MS diagnosis can remain undetermined in a small
proportion of cases and several months or years of
evoluation are usually necessary before a de®nite
con®rmation becomes possible.
The natural history of MS has been studied
extensively for clinical features or laboratory measure-
ments that might predict, anticipate, or parallel the
course of the disease all along its evolution or
treatment.8,9 For example, several clinical or biological
characteristics seem to predict a future progressive
course: a later age at onset, increased number of
relapses in the last 5 years,8,10 a rise in the level of
urinary myelin basic protein-like material.11 Other
*Correspondence: H Perron, bioMeÂrieux Pierre-Fabre, R&D
department, 69280 Marcy L'etoile, France
Received 26 September 2000; revised 9 July 2001; accepted
12 July 2001
urinary markers can also correlate disease activity,
among which biological markers of macrophage
activation or production of free radicals have been
explored.12,13
The recent identi®cation of a cytotoxic factor
targeting glial cells, called gliotoxin, in monocyte
cultures and body ¯uids (CSF, urine) from patients
with MS may provide alternative marker(s).14
Such cytotoxic activity was originally characterized
in MS monocyte cultures,15 and further detected in
CSF16 and urine.17 It was found to induce early
intermediate ®laments network disorganization and
delayed apoptotic-like death of macroglial cells
(oligodendrocytes and astrocytes) in primary fetal
brain explant cultures. It did not directly affect
neurons and leptomeningeal cells in the explanted
cultures as well as other cell types tested separately,
apart from schwann cells on which a mild effect was
observed.15,18 Interestingly, such apoptotic events
involving astroglial cells rather exclusively were
observed in biopsied MS brain.19
Physico-chemical analysis showed that gliotoxic
activity was (i) associated with a protein fraction
migrating at an apparent molecular weight of
21+17 kDa on 12% SDS ± PAGE gels, (ii) resistant to
proteases in physiological conditions, (iii) resistant to
heating at 708C but inactivated at 1008C and (iv) was
probably associated with a glycosylated protein.15,18 A
cell-based test for the detection of this activity was ®rst
set up with a practicable macroglial cell-line in
preliminary attempts, consisting in an astrocyte cell-
line from SV40 large T-transgenic mice.20 Assays for
the detection of the corresponding gliotoxic activity in
MS CSF were set up and showed a good correlation
with MS diagnosis and disease activity.16 In control
 Gliotoxicity in MS urine
C Malcus-Vocanson et al
384
analyses, common `cytotoxic' cytokins have been ruled progressive group was not taken into account for
out as candidates responsible for this activity.18 evaluation of disease activity, as well as one case for
Gliotoxicity (identical physico-chemical characteristics which no objective report could ascertain the number
and biological effects) was found in urine from MS of relapses.
patients, suggesting glomerular passage in kidneys, In the MS population tested, 53% of patients
and preliminary data on MS and control urines also received a treatment at the time of sampling:
indicated good correlation with MS diagnosis.17 immunosuppressive agents (26%), corticosteroids
We now present results obtained on a larger series (48%), both corticosteroids and immunosuppressive
of urines from patients with MS or other neurological agents (26%).
diseases and healthy individuals, with the same The OND population consisted of 51 patients with
protocol which quanti®es an apoptosis-like effect non-MS neurological diseases including three cases of
induced in a reference macroglial cell line and discuss Guillain-Barre syndrome, ®ve cases of myopathy or
alternative strategies developed to such bioassay for myasthenia gravis, one case of pontine myelinolysis,
future applications. six cases of epileptic seizures, three cases of meningi-
tis, seven cases of brain tumors, ®ve cases of
Parkinson's disease, two cases of multiple system
Patients, materials and methods atrophy, seven cases of stroke, two cases of benign
Patients cranial injury, ®ve cases of radiculopathy, one case of
One hundred and ninety-one urine samples have been polyneuropathy, two cases of Chiari malformation, and
collected from patients with MS, other neurological two cases of de®nite psychiatric diseases, and were
diseases (OND), and healthy controls. collected either in the Neurological Hospital of Lyon,
The MS population consisted of 102 patients with France (Profs Chazot, Broussolle) or in Croix Rousse
de®nite MS according to Poser's revised criteria.1 Sixty Hospital (Lyon, France, Infectious Diseases Depart-
patients were hospitalized in a neurological depart- ment, Prof Peyramond). Patients age ranged from 22 to
ment (Neurological Hospital of Lyon, France: Profs 81 years (mean, 52 years; details in Table 1).
Chazot, Boussolle, Vighetto, Mauguiere; or Hospital of Thirty-®ve normal control subjects were recruited as
Bourg en Bresse, France: Drs Boulliat, LemaõÃtre). Forty- healthy volunteers from laboratory staff and the
two MS patients were admitted to the Centre MeÂdical general population with the aim of obtaining subjects
Germaine Revel (Saint Maurice sur Dargoire, France), a of similar age to the patients with multiple sclerosis
rehabilitation center specializing in MS. The age of MS (range: 23 ± 57 years; mean: 40 years).
patients ranged from 22 to 74 years (mean, 45 years;
details in Table 1). Each collected urine sample Cells
corresponded to the ®rst morning miction and patients Mouse astrocytic CLTT-1.1 cells20 (SV40 virus-immor-
with urinary tract infections were excluded. Patients talized) were given by Dr P Rouget (College de France,
were classi®ed according to the course of the disease: Paris). Cells were maintained in Dulbecco's Modi®ed
relapsing ± remitting (RR), chronic ± progressive (P) and Eagle's Medium (DMEM) ± Ham's F12 medium (half/
progressive with episodes of relapses (RP). In RR and half) containing non decomplemented FCS (10%) and
RP groups (77 cases), 48 MS patients were examined routinely supplemented with penicillin (100 U/ml) and
during exacerbation, 26 during remission and three streptomycin (100 mg/ml) for cell production. Cultures
could not be classi®ed. In the P group, all cases were were maintained in an atmosphere of 5% CO2 in air and
secondary progressive MS. For the purpose of this saturated humidity and at con¯uency, cells were
study, disease activity was de®ned on the basis of the dissociated by Trypsin 0.1%/EDTA 1/250e, counted in
number of relapses during the 2 previous years: a Tomas' cell and resuspended in DMEM/F12/FCS for
patients were considered as being in an `active phase' either further cell production or bioassay usage.
of the disease when they had experienced two or more
relapses during this period. The (secondary) chronic Detection of apoptosis-like gliotoxic effect
This assay was based on ¯ow cytometry analysis with
propidium iodide (PI) labeling of DNA, using an
Table 1 Patient features adapted technique21 from the method described by
Hotz.22 The test was performed in 6-well tissue culture
Neurological Statistical
MS controls difference plates using 20 000 cells/well resuspended in medium
(n = 102) (n = 51) (Chi-square) as described above (®nal volume/well=2 ml). After
24 h, when astrocytes were adherent to the plastic,
Age (years) urine samples (40 ml/well) were added in triplicate.
530 14 (14%) 4 (8%) NS The wells with or without (plate controls) urine were
30 ± 40 18 (18%) 6 (12%) NS further cultured for 72 h following the addition of
40 ± 50 30 (29%) 13 (25%) NS samples. The cell layer was then subjected to a non-
450 40 (39%) 28 (55%) NS enzymatic dissociation by using Phosphate Buffer
Sex
Saline (PBS) ± EDTA for 20 min at 378C and
male 38 (37%) 26 (51%) NS
female 64 (63%) 25 (49%) NS mechanical dissociation by repeated pipetting. All
centrifugation steps were performed at +108C and
Multiple Sclerosis
 Gliotoxicity in MS urine
C Malcus-Vocanson et al
385
1500 r.p.m. Cells were washed twice with PBS, and inclusion in gliotoxicity studies, was carried out in a
®xed 1 h in 70% ethanol at 7208C. After two washes, previous in vitro evaluation with the same CLTT-1.1
potential fragmented DNA were extracted using 90% astrocyte cell line. As shown in Table 2, it revealed
Na2HPO4 50 mM, 10% citric acid 25 mM solution, that most common cortocosteroid, immunosuppres-
with 0.1% Triton X-100, during 1 h (+48C). After two sive, antaglic or antispatic drugs used in our MS
additional washes with PBS (cells can stay up to a population were inactive. Few active medications
night in PBS), 50 mg/ml Propidium Iodide (PI) and were nonetheless characterized (Table 2) and patients
50 Ul/ml RNase were added on the cell pellet; after taking these drugs were not included in our studies.
15 min incubation period ¯uorescence was quanti®ed The bioassay assessing gliotoxic activity was
using a ¯ow cytometer. Moreover, in order to ®t the performed with urine samples from 102 MS patients
subdiploid peak with apoptotic cells detection and to and 86 controls (other neurological diseases and
eliminate most necrotic cells and debris, the astrocytic normal individuals). Results were compared with
cell line was analyzed in experimental conditions clinical data as summarized in Table 3 (MS and
known to induce cell death after addition of sodium neurological controls). No toxic activity was detected
azide, a substance that induces cell death through a in 35/35 healthy subjects.
non-apoptotic mechanism.23 Incubation of astrocytes Signi®cant gliotoxicity was detected in urine from
with 0.05% sodium azide markedly enhanced the 73% patients with MS taken altogether (77% of
dead cell number after 36 h, and allowed adjusting patients admitted in hospital and 67% of those
gate and markers. admitted in rehabilitation center). Regarding MS
features, the most important proportion of positive
Flow cytometry analysis tests was found in the remitting ± relapsing (RR) form
The protocol used here has been evaluated and of the disease (81%), but was signi®cantly decreased
described elsewhere.21 Brie¯y, the ¯uorescence of cells in remittent-progressive (RP) forms (63%). Interest-
stained with PI was determined using a XL Coulter ingly the two cases having a ®rst attack at the time of
¯uorimeter using a 488 nm wavelength of an ion laser sampling have retrospectively been con®rmed to have
as the excitation source. Three thousand cells in each MS and had a positive test.
sample were analyzed. The red luminescence emission Among 63 urine samples from RR and RP MS
(due to PI) was separated using a long-pass 630 nm patients in an active phase of the disease at sampling,
®lter. Applying this method, non apoptotic (or living) 79% were positive, when only 15% of urine from
cell nuclei show three peaks: one with high ¯uores- patients which did not match these criteria (in non
cence intensity representing cells with a normal active phase) were positive, this difference was highly
chromatin content (G0 or G1 phase of the cell cycle),
and two smaller peaks with hyperpdiploid DNA
content (S phase and G2 or M phase of the cell cycle). Table 2 In vitro cytotoxicity of drugs on CLTT-1.1 astrocyte
In contrast, PI stained at lower intensity apoptotic cell cells
nuclei that can be detected in a broad peak with lower
¯uorescence intensity than the diploid cells. Most Molecule Minimum dose % Cytotoxicity
found cytotoxic (MTT test on
typical necrotic cell fragments were excluded by
on CLTT-1.1 CLTT-1.1
appropriate gate and markers positioning. astrocytes astrocytes)
The result obtained after blind testing with each
urine sample was compared with cellular controls Phenobarbital none ±
from wells without urine in the same plate and was Methotrexate 681 mg/ml 8
considered positive if the percentage of apoptotic cells Labetalol 3 mg/ml 11
was at least 20% higher than the control. Dextropropoxyphen 552 mg/ml 56
Each sample was tested twice in separate experiments Baclofen none ±
and the results thus expressed qualitatively as `positive Amitriptylin 0.80 mg/ml 6
Levodopa 75 mg/ml 21
or negative'. When results were concordant in the two
Clomopramine 10 mg/ml 68
separate assays, they were validated. When they Terbutalin none ±
happened to be discordant they were classi®ed as `not Tocophenol none ±
interpretable' and rejected (three samples in the series). Clonazepam 0.14 mg/ml 11
Aziathioprin none ±
Data analysis and statistics Oxybutynin none ±
Group comparisons were carried out using Chi-square Allopurinol 8 mg/ml 11
test, and non-parametric data were compared using Acetylsalicylat none ±
Mann-Whitney's test. A P value of 50.05 was Carbamazepin none ±
considered statistically signi®cant. Prednisolone none ±
Glicazide 9.6 mg/ml 21
Propericiazine 6 mg/ml 13
Results Buspirone none ±
Bromazepam 0.6 mg/ml 12
Possible interference of common drugs used in Primidone none ±
treatments of patients which were candidate for an

Multiple Sclerosis
 Gliotoxicity in MS urine
C Malcus-Vocanson et al
386
Table 3 Detection of cytotoxic activity in urine

MS Neurological controls
n negative (%) positive (%) n negative (%) positive (%) Stat.

Total 102 27 73 51 92 8 50.001

Rehabilitation center 42 33 67 51 92 8 50.001
Neurological hospital 60 23 77 51 92 8 50.001
Age (years)
530 14 7 93 4 100 0 50.01a
30 ± 40 18 17 83 6 83 17 50.02a
40 ± 50 30 43 57 13 85 15 50.05a
450 40 27 73 28 97 3 50.001
Sex
male 38 39 61 26 92 8 50.001
female 64 20 80 25 88 12 50.001
Clinical form
1st attack 2 0 100
remittent 42 19 81 (1) NSb
remittent/progressive 35 37 63 (2) NSb
chronic ± progressive 23 30 70 (3) NSb
Active disease
(RR+RP) + 63c 21 79 50.001b
(RR+RP) 7 13c 85 15
Relapses
(RR+RP) + 48c 21 79 NSb
(RR+RP) 7 26c 38 62

NS: not signi®cant, RR: remittent form, RP remittent±progressive form. Results are expressed in percentage of total population
in studied group (N). Negative results with 35/35 healthy volunteers are not included in this table. Statistical analysis (Chi-
square P values indicated in columns correspond to the statistical signi®cance of the difference of positive/negative results
observed in MS/control groups on the same line, except: aChi-Square with Yate's correction/bComparison within MS sub-
groups: remittent vs chronic±progressive; remittent vs remittent/progressive; remittent/progressive vs chronic±progressive;
active vs non-active disease at sampling; relapse vs no relapse at sampling/cAs detailed in `Materials and methods', few cases
for which objective examinations were insuf®cient for sub-classi®cation were not taken into account for statistical comparisons:
One case was not classi®ed as active or non-active and three cases were not classi®ed as `with or without relapse'

signi®cant (P50.001). Besides, among MS patients, as
well as in the total studied population, no correlation
at all was found between age and positivity or
negativity.
Gliotoxicity was detected with 4/51 urine from
neurological controls with the following diagnoses:
benign fasciculation syndrome, infectious radiculitis,
Arnold Chiari malformation, neuromyotony. When
urine was collected, none of the corresponding
observations was clinically compatible with MS. The
proportion of positive tests within OND (8%) was
signi®cantly inferior to the 73% of positive MS urines
(P50.01).
Discussion
The present report extends and con®rms our pre-
liminary series17 which had shown an association
between the detection of this gliotoxic activity on
reference passages from CLTT-1 cells20 and multiple
sclerosis.
Interestingly, among ONDs, other diseases affecting
central or peripheral myelin (centropontine myelino-
lysis, Guillain-Barre syndrome) were negative. Notably,
our control series did not include chronic peripheral
neuropathy, which may not be super-imposable to
Multiple Sclerosis
Guillain-Barre cases. Sensitivity and speci®city were
lower than those previously described: 73% and 92%
respectively, instead of 91% and 97%.17 The absence of
positive tests in healthy individuals and the low
proportion of positive patients with OND cases
included in this series, nonetheless con®rmed a good
speci®city. In our previous study,17 MS cases were
exclusively recruited among patients hospitalized in a
neurological department and sampled on admission;
these patients (35 cases) had often been hospitalized in
a context of relapse (all RR forms). In the present study,
the number of positive samples is smaller among
patients recruited in a rehabilitation center than in a
hospital neurological department. The age of the
patients and the duration of the disease are signi®cantly
different in the two groups, particularly because the
majority of patients in this rehabilitation center were
classi®ed as chronic±progressive (secondary form
exclusively) MS at the time of urine collection.
Being more often detected in urine from RR forms
than from progressive forms of MS, and more often in
active than in non-active phase during the present
study (Table 3), this gliotoxicity is likely to re¯ect a
biological activity correlating disease activity.
Nonetheless, since CLTT 1.1 cell phenotype has
already been found to shift towards more or less

responsive sub-cultures in few instances, such a cell-
based `bioassay' is not easily practicable for routine
testing. For this reason, we did not future explore the
concordance `between multiple assays' on replicate
samples for a standardized diagnostic application.
However, given the interesting statistical reseults
obtained with our bioassay in our experimental
conditions, we considered that this study and the
previous ones indicated at least that the molecular
characterization of this gliotoxic activity was worth
being pursued. Quality-controlled CLTT 1.1 subcul-
tures and passages matching our bioassay criteria were
therefore selected and further used to monitor the
puri®cation and molecular characterization of the
`gliotoxic factor' from large volumes of MS urine,
versus control non-MS material.
We have thus identi®ed low-molecular weight
proteins in a ®nal puri®cation step of a `gliotoxic'
fraction, which co-migrated in the previously de-
scribed `21 and 17 kDa' bands on regular SDS ± PAGE
(12%) gels,15,18 but were separated on Tricine gels and
microsequenced. Recombinant proteins have not been
identi®ed which can induce cytotoxicity in the
reference bioassay on CLTT 1.1 cells. Thus, comple-
tion of this `molecular' study now appears a pre-
requisite for further studies and evaluation of
applications relating to this `gliotoxic activity'. Indeed,
a direct `immunological' detection of the factor
corresponding to this gliotoxic activity should reveal
more practicable for diagnostic evaluations in MS in
conditions expected for a marker detected in body
¯uids.24,25 than the `cell-based' bioassay.
Nonetheless, this `cell-based' technology, developed
for the detection of this gliotoxic activity, and the
interesting statistical associations with MS, obtained in
successive studies ± with various bioassay proce-
dures, on different body ¯uids, performed in different
laboratories, by different collaborators, on different
patient series,15 ± 17 ± were also prerequisite for a mole-
cular approach. Indeed, the choice of an adequate
source and quantity of biological material as well as
the set-up of a test procedure adapted to the detection
of the gliotoxic activity in fractions from successive
puri®cation steps, was dependent upon the de®nition
and the optimization of this bioassay. This was
achieved throughout collaborative studies, as pre-
sented here.
Acknowledgements
The authors would like to thank Prof Vighetto, Prof
Mauguiere (neurological departments, Neurological
Hospital of Lyon), Dr Boulliat, De Lemaitre (Neuro-
logical Department, Hospital of Bourg en Bresse),
and their teams, centre me dical Germaine Revel
(Saint Maurice sur Dargoire) nurses and physicians
(Dr Beneton, Dr Chipier, Dr de Parisot, Dr Bouguerra,
Dr Jarrias), Dr Boibieux (Infectious Diseases Depart-
ment, Croix-Rousse Hospital, Lyon, France) for
sample collection. We also would like to thank:
FrancËoise Touraine-Moulin's team (Department of
Gliotoxicity in MS urine
C Malcus-Vocanson et al
387
Imunology, Neurological Hospital of Lyon) for the
access to flow cytometer, and Dr P Rouget and Dr F
Rieger for providing cells. We acknowledge Serge
Comby's team (bioMe rieux SA) for help in statistical
analysis.
References
1 Poser C et al. (1983) New diagnosis criteria for multiple
sclerosis: Guidelines for research protocols. Ann Neurol
13: 227 ± 231.
2 McLean B, Luxton R, Thompson E. (1990) A study of
immunoglobulin G in the cerebrospinal ¯uid of 1007
patients with suspected neurological disease using iso-
electric focusing and the log IgG-index. Brain 113: 1269 ±
1289.
3 Cowdrey G et al. (1993) Isoelectric focusing in an
immobilized pH gradient for the detection of intrathecal
IgG in cerebrospinal ¯uid: sensitivity and speci®city for
the diagnosis of multiple sclerosis. Ann Clin Biochem 30:
463 ± 468.
4 Pirttila T, Nurmikko T. (1995) CSF oligoclonal bands,
MRI and the diagnosis of multiple sclerosis. Acta Neurol
Scand 92: 468 ± 471.
5 Truyen L et al. (1991) Long term follow-up of multiple
sclerosis by standardized, non-contrast-enhanced mag-
netic resonance imaging. J Neurol Sci 106: 35 ± 40.
6 Zhao G et al. (1997) Clinical and magnetic resonance
imaging changes correlate in a clinical trial monitoring
cyclosporine therapy for multiple sclerosis. J Neuroima-
ging 7: 1 ± 7.
7 Truyen L et al. (1997) Speci®c power calculations for
magnetic resonance imaging (MRI) in monitoring active
relapsing remitting multiple sclerosis (MS): implications
for phase II therapeutic trials. Mult Scler 2: 283 ± 290.
8 Weinshenker B, Bass B, Rice G. (1989) The natural
history of multiple sclerosis: a geographically based
study. 2. Predictive value of the early clinical course.
Brain 112: 1419 ± 1428.
9 Weinshenker B. (1995) The natural history of multiple
sclerosis. Neurol Clin 13: 119 ± 146.
10 Runmarker B, Andersen O. (1993) Prognostic factors in a
multiple sclerosis incidence cohort with twenty-®ve
years of follow-up. Brain 116: 117 ± 134.
11 Whitaker J et al. (1995) Urinary myelin basic protein-like
material as a correlate of the progression of multiple
sclerosis. Ann Neurol 38: 625 ± 632.
12 Giovannoni G et al. (1997) Daily urinary neopterin
excretion as a immunological marker of disease activity
in multiple sclerosis. Brain 120: 1 ± 13.
13 Giovannoni G et al. (1999) Increased urinary nitric oxide
metabolites in patients with multiple sclerosis correlates
with early and relapsing disease. Mult Scler 5: 335 ± 341.
14 Perron H. (1998) Retrovirus MSRV et proteine gliotox-
ique: Marqueurs biologiques potentiels dans la scle rose
en plaques? Ann Biol Clin (Paris) 56: 427 ± 438.
15 Menard A et al. (1997) Gliotoxicity, reverse transcriptase
activity and retroviral RNA in monocyte/macrophage
culture supernatants from patients with multiple sclero-
sis. FEBS Lett 413: 477 ± 485.
16 Menard A et al. (1998) Detection of a gliotoxic activity in
the cerebrospinal ¯uid from multiple sclerosis patients.
Neurosci Lett 245: 49 ± 52.
17 Malcus-Vocanson C et al. (1998) A urinary marker for
multiple sclerosis. Lancet 351: 1330.
Multiple Sclerosis
 Gliotoxicity in MS urine
C Malcus-Vocanson et al
388
18 Menard A et al. (1998) A gliotoxic factor in multiple 23 Wyllie A, Morris R, Smith A, Dunlop D. (1984)
sclerosis. J Neurol Sci 154: 209 ± 221. Chromatin cleavage in apoptosis: association with
19 Benjelloun N et al. (1998) Case report: DNA fragmenta- condensed chromatin morphology and dependence on
tion in glial cells in a cerebral biopsy from a multiple macromolecular synthesis. J Pathol 142: 67 ± 77.
sclerosis patient. Cell Mol Biol 44: 579 ± 583. 24 Laman J, Thompson E, Kappos L. (1998) Body ¯uid
20 Galiana E et al. (1990) Establishment of permanent markers to monitor multiple sclerosis: the assays and the
astroglial cell lines, able to differentiate in vitro, from challenges. Mult Scler 4: 266 ± 269.
transgenic mice carrying the polyoma virus large T gene: 25 Sorensen P (1999) Biological markers in body ¯uids for
an alternative approach to brain cell immortalization. activity and progression in multiple sclerosis. Mult Scler
J Neurosci 26: 269 ± 277. 5: 287 ± 290.
21 Micoud F, Mandrand B, Malcus-Vocanson C. (2001)
Comparison of several techniques for the detection of
apoptotic astrocytes in vitro. Cell Prolif 34: 99 ± 113.
22 Hotz M, Gong J, Traganos F, Darzynkiewicz Z. (1994)
Flow cytometric detection of apoptosis: comparison of
the assays of in situ DNA degradation and chromatin
changes. Cytometry 15: 237 ± 244.

Multiple Sclerosis