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Down-Regulation of Survivin Expression in T Lymphocytes After Interferon Beta-1a Treatment in Patients With Multiple Sclerosis
Moad K. Sharief, MD, PhD;
Yemane K. Semra, PhD
Arch Neurol. 2002;59:1115-1121.
ABSTRACT
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Background Treatment with interferon beta reduces clinical exacerbations in multiple
sclerosis (MS) through several immunomodulatory mechanisms that involve the
augmentation of programmed cell death (apoptosis) of peripheral T lymphocytes.
The expression of survivin, a cell cycle–regulated antiapoptosis protein,
is up-regulated in mitogen-stimulated T lymphocytes from patients with MS,
and this expression correlates with MS disease activity.
Objective To evaluate the effect of interferon beta on the expression of survivin
and other apoptosis regulatory molecules in peripheral T lymphocytes from
patients with MS.
Patients and Methods In a prospective, combined clinical and immunologic study, we evaluated
the expression of survivin, Bcl-2 protein, and the death receptor Fas in mitogen-stimulated
T lymphocytes from 26 patients with MS, before and serially after treatment
with interferon beta-1a. We also investigated the long-term effects of interferon
beta-1a on cellular expression of these proteins and T-lymphocyte apoptosis
in a cross-sectional study of 19 patients with MS receiving long-term interferon
beta-1a therapy.
Results Treatment with interferon beta-1a reduced the expression of survivin
in in vitro stimulated T lymphocytes. This reduced expression correlated with
augmented T-cell susceptibility to apoptosis and with clinical response to
treatment. In contrast, interferon beta-1a therapy did not significantly alter
cellular expression of Bcl-2 protein or Fas. This down-regulatory effect of
interferon beta-1a on cellular expression of survivin was maintained after
long-term therapy.
Conclusions Our observations suggest that interferon beta exerts a regulatory effect
on peripheral T lymphocytes through an antiapoptosis mechanism that involves
the down-regulation of cellular survivin expression.
INTRODUCTION
PROGRAMMED CELL death (apoptosis) of lymphocytes is essential for the
proper functioning and homeostasis of the immune system.1
Among other functions, lymphocyte apoptosis is an important antiautoimmune
mechanism that deletes potentially pathogenic autoreactive lymphocytes2 and limits tissue damage in autoimmune diseases, including
multiple sclerosis (MS).3-4 There
is emerging evidence that autoreactive T lymphocytes in MS are more resistant
to apoptosis than cells from healthy individuals or patients with other neurologic
disorders.3, 5-8
Such reduced susceptibility to apoptosis in MS is related to derangements
of the apoptosis machinery at multiple cellular levels that include the death
receptor Fas and Bcl-2 family of oncogenic proteins.9-13
In patients with MS, treatment with interferon beta reduces clinical
exacerbations and lessens the accumulation of disease burden as assessed by
magnetic resonance (MR) imaging.14 The precise
mechanism of action of the 2 recombinant preparations of interferon beta (1a
and 1b) is not clear. These preparations exert multiple regulatory effects
on peripheral lymphocytes that include augmentation of apoptosis15-16
and up-regulation of the death receptor Fas on antigen-specific T lymphocytes.17 Ligation of Fas leads to activation of cellular caspases,
a series of destructive intracellular cysteine proteases that play an essential
role in mediating cell death through cleavage of structural elements of the
cytoplasm and nucleus.18-19 We
recently reported that interferon beta therapy regulates the activity of apical
caspases, such as caspase 8.16 However, its
effect on downstream caspase is currently unknown.
The activity of downstream caspases is partly modulated by survivin,
a recently described cell cycle–regulated antiapoptosis protein. Overexpression
of survivin delays apoptosis induced by various stimuli, whereas antisense-mediated
reductions in survivin sensitize cells to apoptotic stimuli.20
While fetal tissues and normal thymus contain abundant survivin messenger
RNA and protein, no such expression is detected in normal adult tissues.21-22 In contrast, the vast majority of
tumors express survivin at high levels, suggesting that reactivation of survivin
gene expression commonly occurs during oncogenesis.23
However, survivin is not cancer specific, as it also plays an important role
in normal hematopoietic cell survival and proliferation.24-25
Indeed, evidence is emerging that survivin expression contributes to cell
survival early in T-cell activation and also in memory immune responses.26
Overexpression of survivin has been reported in activated T lymphocytes
from patients with relapsing-remitting MS.27
Moreover, this heightened expression in patients with MS seems to correlate
with T-cell resistance to apoptosis,27 and
with clinical features of disease activity.28
On the basis of these observations, we sought to correlate the expression
of survivin in peripheral T lymphocytes from patients with MS after interferon
beta therapy with clinical response to treatment, and with cellular susceptibility
to apoptosis. Addressing these issues would help define the role of survivin
in mediating clinical responses to interferon beta therapy.
PATIENTS AND METHODS
PATIENTS
In a prospective longitudinal study, we investigated cellular apoptosis
and the expression of survivin, Bcl-2, and Fas protein in activated T lymphocytes
from 26 patients with clinically active, relapsing-remitting MS before treatment
with interferon beta-1a (baseline samples) and consecutively after 1, 3, 6,
9, and 12 months of this drug therapy. Their clinical features were presented
earlier.16 In brief, their mean age ±
SD was 32.6 ± 6.2 years, and the mean ± SD disease duration
was 6.3 ± 3.6 years. The criteria for active disease were as follows:
(1) history of at least 2 clearly identified clinical relapses during the
2 years preceding blood collection, and (2) the presence of 1 or more enhancing
lesions on cranial MR imaging at the time when baseline blood samples were
obtained. Twelve patients received Rebif (Serono International SA, Geneva,
Switzerland), 22 µg 3 times weekly; 6 patients received Rebif, 44 µg
3 times weekly; and the remaining 8 patients were treated with Avonex (Biogen,
Inc, Cambridge, Mass), 30 µg once weekly.
In addition to this prospective study, we investigated the long-term
effect of interferon beta-1a on the expression of survivin and other apoptosis
regulatory proteins in a cross-sectional evaluation of another 19 patients
with relapsing-remitting MS. These patients had been receiving interferon
beta-1a (Rebif, 22 µg 3 times weekly) for a mean duration of 4.8 years
(range, 2.5-6.5 years). Their mean age ± SD was 35.7 ± 7.8 years,
and mean disease duration was 7.3 ± 2.6 years. To control for this
treatment group, we collected blood samples from 14 patients with relapsing-remitting
MS who had never received treatment with interferon beta. Their mean age ±
SD was 34.1 ± 8.2 years, and mean disease duration was 4.8 ±
2.9 years. These control patients had had at least 2 clinical relapses during
the 2 years preceding blood collection, and in 8 patients who underwent cranial
MR imaging at the time of sample collection, disease activity was confirmed
by the presence of 1 or more enhancing lesions. No patient from the treatment
or the control groups had received corticosteroid or immunosuppressive therapy
during the 6 months before blood collection. We did not include neurologic
control subjects, as we have already reported a significant difference in
cellular survivin expression between patients with MS and those with other
neurologic disorders.27, 29 However,
we obtained blood samples from 12 healthy individuals to determine the normal
range of cellular survivin expression.
METHODS
Cell Culture and Induction of Apoptosis
Peripheral mononuclear cells were isolated from heparinized blood by
centrifugation on a Ficoll-Hypaque density gradient washed twice and resuspended
in culture medium. In vitro activation of T cells was established by means
of nonspecific stimulation with phytohemagglutinin (1 µg per 106 cells) followed by culture in continuous presence of phytohemagglutinin
and interleukin 2, 100 U/mL, as previously described.30
Flow cytometry analysis of cells after 5 days of culture showed that the proportion
of T cells was more than 94%. Cell viability was determined by trypan blue
dye exclusion assay. Apoptosis was induced on day 5 of culture by incubation
with etoposide, 10 µg/mL (Sigma-Aldrich Corp, St Louis, Mo), or culture
medium as described elsewhere.31 This method
was chosen to account for the fact that survivin, like other inhibitors of
apoptosis proteins, may be involved in the proteolytic processing mediated
by etoposide.32 Apoptosis was quantified by
means of commercial cellular DNA fragmentation immunoassays (Boehringer Mannheim
GmbH, Ingelheim, Germany), as presented in detail elsewhere.12, 29
Quantification of Survivin and Other Proteins
All laboratory analyses were performed blind to clinical data. As we
have already reported that survivin was not usually expressed in resting peripheral
lymphocytes,27 survivin expression in this
study was quantified in T cells that were stimulated in vitro for 5 days.
Cellular contents of survivin were quantified in cell lysates by means of
dot-blot immunoassay, as discussed earlier.27
In brief, cellular lysate equivalents of 5 µg of protein were probed
with affinity-purified antibody raised against a peptide mapping at the carboxy
terminus of human survivin (C-19; Santa Cruz Biotechnology, Inc, Santa Cruz,
Calif), followed by computerized densitometry. To validate cellular expression
of survivin in individual patients, we analyzed signals obtained from Western
blot analysis as described earlier.33 In earlier
experiments, we detected a strong correlation between cellular survivin levels
as determined by the dot-blot assay and corresponding levels measured by Western
blots.27 However, we used dot-blot data for
statistical analyses in this study to avoid gel-to-gel variation and differences
in blotting and exposure time that may occur with Western blotting. Quantification
of Bcl-2 protein content in cell lysates was performed with a commercial immunoassay
(Oncogene Research Products, Calbiochem, CN Biosciences, Inc, Nottingham,
England). We also used commercial assay (Calbiochem, CN Biosciences, Inc),
rather than flow cytometry, to measure cellular Fas protein to maintain consistency
of the assays used in this study.
DATA ANALYSIS
All statistical analyses were performed with the SPSS/PC+ software program
(SPSS Inc, Chicago, Ill). Values were compared, as appropriate, by paired
Student t test, Wilcoxon rank test, analysis of variance,
Kruskal-Wallis test followed by multiple comparisons, and Pearson correlation
test.
RESULTS
PROSPECTIVE CLINICAL EVALUATION
After 12-month follow-up of the prospective group, 18 of the 26 patients
experienced complete cessation or more than 60% reduction in clinical relapse
rate compared with pretreatment levels. Thus, these 18 patients were considered
interferon beta responders, according to published criteria.34-35
This favorable clinical response was verified by the lack of MR imaging activity
in 8 patients who underwent follow-up MR imaging assessment. The remaining
8 of the 26 patients who were included in the prospective study experienced
no or less than 20% reduction in exacerbation rate, and were therefore considered
nonresponders.34
BASELINE CELLULAR SURVIVIN EXPRESSION
The pretreatment (baseline) expression of survivin in mitogen-stimulated
T cells from patients with MS was significantly higher than corresponding
expression in healthy individuals (Figure
1). More specifically, baseline survivin expression was abnormally
high (ie, higher than the cutoff limit in healthy control subjects) in 16
of the 26 patients with MS who were involved in the prospective study (Figure 1). There was no correlation between
survivin expression and the extent of lymphocyte activation, as measured by
proliferation rate. In contrast to high survivin contents, cellular expression
of Bcl-2 and Fas in mitogen-stimulated T cells was relatively similar between
patients with MS and healthy control subjects (Figure 1).
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Figure 1. Cellular expression of survivin
(densitometric units) and Fas and Bcl-2 (units per milligram of protein) in
mitogen-stimulated T lymphocytes from patients with clinically active multiple
sclerosis compared with healthy control subjects. Closed symbols represent
nonresponders to interferon beta-1a therapy. Horizontal bars depict mean values,
and shaded area represents the cutoff limit (mean + 2 SDs) of cellular survivin
expression in healthy control subjects. Asterisk indicates P<.01 compared with corresponding levels in healthy control subjects.
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DYNAMICS OF SURVIVIN EXPRESSION AFTER INTERFERON BETA-1A THERAPY
Baseline survivin expression in the 18 patients with MS who later responded
to interferon beta-1a was comparable to baseline expression in the 8 nonresponders.
However, prospective evaluation showed a progressive decline of survivin expression
after interferon beta-1a therapy in 10 of the 18 treatment responders. Serial
monitoring of treatment responders showed that cellular survivin expression
declined after 6 months of interferon beta-1a therapy and was significantly
lower than baseline levels after 12 months of treatment (Figure 2A). In contrast, cellular survivin in the 8 nonresponders
remained relatively unchanged throughout the study (Figure 2B). Similarly, cellular Bcl-2 expression in patients with
MS was not significantly influenced by interferon beta-1a therapy and was
comparable between interferon beta-1a responders and nonresponders. When a
more stringent definition of interferon beta-1a responders was applied (ie,
complete cessation of clinical and radiographic activity), all responders
showed suppression of survivin expression when compared with nonresponders,
and their mean ± SD survivin expression decreased from a baseline of
90.6 ± 68.7 to 26.3 ± 20.5 densitometric units after 12 months
of therapy (P<.001). There were no differences
in the dynamics of survivin expression between patients receiving the 2 doses
of Rebif, or between the Rebif- and Avonex-treated groups. Cellular survivin
remained undetectable after treatment in the 5 patients whose data are given
in Figure 1 who showed no baseline
expression. There was a tendency for increased Fas expression after interferon
beta-1a therapy (Figure 2), but
this was not statistically significant.
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Figure 2. Cellular expression of survivin
(densitometric units) and Fas and Bcl-2 (units per milligram of protein) in
mitogen-stimulated T cells before treatment with interferon beta-1a (month
0) and serially during 12 months of therapy. Results are mean ± SE.
A, Data from all 18 patients who responded to treatment; B, data from the
8 nonresponders. Blood samples at month 12 were obtained from 8 responders
and 4 nonresponders. Asterisk indicates P<.05
compared with pretreatment levels.
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SURVIVIN EXPRESSION AND LONG-TERM INTERFERON BETA-1A THERAPY
The down-regulatory effect of interferon beta-1a on cellular survivin
expression in treatment responders prompted us to investigate whether such
effect was maintained during long-term treatment. Sixteen patients from the
long-term treatment group were classified as interferon beta-1a responders
(complete cessation or more than 60% reduction in clinical relapse rate),
whereas the remaining 3 patients were considered nonresponders. The duration
of MS was comparable between the 2 groups. Cellular survivin expression in
the 16 treatment responders was significantly lower than corresponding expression
in the 3 nonresponders or in the 14 untreated patients with MS (Figure 3). In contrast, cellular expression of Bcl-2 and Fas proteins
in the long-term treatment group was similar to corresponding expression in
untreated patients or healthy individuals (Figure 3).
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Figure 3. Cellular expression of apoptosis
regulatory proteins in mitogen-stimulated T cells from patients with multiple
sclerosis receiving long-term interferon beta-1a therapy compared with untreated
patients with multiple sclerosis. Closed symbols represent treatment nonresponders.
Horizontal bars depict mean values, and shaded area represents the cutoff
limit (mean + 2 SDs) of cellular survivin expression in healthy control subjects.
Survivin expression is given in densitometric units, and Fas and Bcl-2 in
units per milligram of protein. Asterisk indicates P<.01
compared with corresponding levels in healthy control subjects.
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IN VITRO EFFECT OF INTERFERON BETA-1A ON CELLULAR SURVIVIN
Having established that interferon beta therapy reduces cellular survivin
expression, we sought to validate this down-regulatory effect in vitro. For
this purpose, we analyzed cellular expression of survivin and other apoptosis
regulatory proteins in T cells after mitogen stimulation in the presence or
absence of interferon beta-1a. Results confirmed that adding interferon beta-1a
to the culture medium significantly reduced survivin expression but did not
alter the expression of Bcl-2 protein and up-regulated cellular expression
of Fas protein (Table 1).
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Expression of Apoptosis Regulatory Proteins in Mitogen-Stimulated T
Cells After 24 Hours of Incubation With Interferon Beta-1a (5 µg/mL)
or Culture Medium Alone*
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EFFECT OF INTERFERON BETA-1A ON LYMPHOCYTE APOPTOSIS
We then investigated the relationship between survivin expression and
T-lymphocyte apoptosis to confirm the biological relevance of survivin down-regulation
after treatment with interferon beta. After prospective treatment with this
drug, T-cell susceptibility to etoposide-induced cell death was enhanced in
patients who showed progressive decline in cellular survivin expression (Figure 4). This susceptibility to apoptosis
was independent of cellular Fas expression, and the correlation between survivin
down-regulation and augmented apoptosis was confirmed by T-cell cultures in
the presence of interferon beta-1a (data not shown). We observed a similar
relationship between cellular survivin expression and enhanced apoptosis in
the 16 long-term treatment responders, where etoposide-induced cell death
(mean ± SD DNA fragmentation, 0.92 ± 0.31 A405nm)
was significantly greater than cellular apoptosis in the three nonresponders
with MS (DNA fragmentation, 0.56 ± 0.3 A405nm; P = .03) or from the 14 untreated patients with MS (0.66 ± 0.34
A405nm; P = .04).
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Figure 4. Effect of cellular survivin expression
on etoposide-induced apoptosis of mitogen-stimulated T cells from 14 interferon
beta-1a responders who were studied prospectively. The declining survivin
group represents the 10 patients in whom survivin expression declined during
treatment, whereas the persistent survivin group refers to the 4 patients
in whom cellular survivin was detectable but did not decline after treatment.
Blood samples from the declining survivin group at month 12 were obtained
from 5 patients only. Values represent mean ± SE. Asterisk indicates P<.01 compared with baseline DNA fragmentation.
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COMMENT
The efficacy of interferon beta in the treatment of MS has been established
by several international muticenter trials and involves multiple immunomodulatory
actions. In this study, we investigated the effects of interferon beta-1a
therapy on survivin expression and subsequent apoptosis of mitogen-stimulated
T lymphocytes from both interferon beta-1a responders and nonresponders. Our
results demonstrate that interferon beta therapy preferentially down-regulates
the expression of survivin in mitogen-stimulated T lymphocytes from treatment
responders. Since interferon beta is known to exert antiproliferative actions
that include potent proapoptotic activities,15-16,36-39
our findings are of therapeutic relevance in view of the apoptosis-regulatory
effects of survivin40-41 and the
suggestion that clinical remissions in MS could be induced by apoptotic elimination
of activated T lymphocytes.3
Impairments of activation-induced apoptosis of lymphocytes may play
a role in the perpetuation of the immune response in MS.42-43
Indeed, there is emerging evidence that antigen-specific T lymphocytes from
patients with MS are less susceptible to apoptosis than are healthy cells,
and may therefore maintain a continuous cycle of inflammation and tissue destruction
within the central nervous system.7, 9, 12
Interferon beta is known to inhibit the proliferation of antigen-specific
T lymphocytes,44 probably through apoptosis-regulatory
mechanisms.8 It also augments apoptosis of
activated polyclonal T lymphocytes from patients with MS,15, 45
although this has not yet been confirmed in myelin-specific T-cell lines.17 This interferon beta–mediated increase in T-cell
apoptosis is due to several mechanisms that include up-regulation of Fas or
its ligand,39 induction of genes that down-regulate
protein synthesis,46-47 overexpression
of cellular apoptosis-inducing ligands,37, 48
and down-regulation of apical caspase inhibitors.16
Here, we expand on these mechanisms by reporting that interferon beta down-regulates
T-cell expression of survivin, a potent bifunctional protein that suppresses
apoptosis and regulates cell division.22
Human survivin, a member of the inhibitor-of-apoptosis family, plays
a pivotal role in the regulation of cell death.22, 49
Overexpression of survivin inhibits apoptosis induced by various stimuli and
has the potential to suppress terminal caspases that mediate cell death.18 Survivin is expressed in a cell cycle–regulated
manner, with high levels in the G2/M phase but rapid down-regulation
after cell cycle arrest. Although high expression is seen in most cancers,
survivin is also overexpressed in nonmalignant cells, such as the proliferative
basal layer of normal skin,50 endothelial cells
during angiogenesis,33 and activated and memory
T cells.26 Accordingly, the antiapoptotic properties
of survivin may be necessary to counteract increased apoptosis sensitivity
of proliferating cells during development or homeostasis,51
or to escape their normal proliferative restraints.52
Moreover, evidence is accumulating that derangements of survivin expression
are involved in inflammatory diseases,53 including
MS.27 Indeed, survivin overexpression in T
lymphocytes seems to correlate with MS disease activity.28
These observations help explain our findings that down-regulation of cellular
survivin corresponds with a favorable clinical response to interferon beta
therapy. In this study, we adopted strict criteria for the definition of interferon
beta responsiveness that did not require MR imaging confirmation.34-35 Nonetheless, we used MR imaging monitoring
in a subgroup of patients to confirm favorable therapeutic response. We also
studied unfractionated T lymphocytes, rather than isolated T-cell subpopulations,
to evaluate cell death that reflected interactions among populations. Although
it would be of interest to determine whether interferon beta therapy differentially
modulates survivin expression in T-cell subgroups or antigen-specific lymphocytes,
recent evidence indicates that the drug does not alter survival of myelin-specific
T-cell lines.17
Our findings indicate that reduced cellular survivin expression and
the corresponding increased susceptibility to apoptosis are maintained during
long-term treatment with interferon beta. Since the down-regulatory effect
of interferon beta on cellular survivin expression is confirmed by in vitro
experiments, it is likely that the cellular changes reported herein are functionally
related to interferon beta therapy. These changes also appear to correlate
with clinical response to treatment. However, it is not clear from our data
whether pretreatment levels of cellular survivin would predict clinical response
to interferon beta therapy. Further evaluation is necessary to examine the
clinical relevance of our findings and the molecular mechanisms through which
interferon beta regulates cellular expression of survivin. It is of note,
however, that interferon beta therapy in this study did not modify the expression
of the antiapoptosis protein Bcl-2. This is in agreement with previous reports
that interferon beta activates apoptosis without modulating the expression
of Bcl-2 family proteins.54 We specifically
sought to evaluate Bcl-2 expression because of its unique role among oncogenic
proteins, as it enhances lymphoid cell survival by interfering with apoptosis
rather than promoting cell proliferation.55
Thus, our results implicate survivin, rather than Bcl-2, as the modulator
of T-cell susceptibility to apoptosis during interferon beta therapy. Yet,
our findings of augmented T-cell apoptosis after interferon beta therapy may
be related to the observed increase in cellular Fas expression, a finding
consistent with earlier studies,39, 45, 56
or to down-regulation of the apical caspase inhibitor FLIP (Fas-associated
death domain–like interleukin-1beta–converting enzyme inhibitory
protein).16 Taken together, these observations
and the current results indicate that interferon beta regulates T-cell death
at multiple cellular levels that involve modulation of both apical and downstream
caspase inhibitors.
In conclusion, apoptotic elimination of potentially pathogenic T cells
in MS might accelerate termination of the inflammatory response. Our results
indicate that interferon beta therapy augments apoptosis of T lymphocytes
by down-regulating cellular expression of survivin. Since interferon beta
is known to induce multiple immunomodulatory pathways and apoptosis-regulatory
genes,57 the observed changes in cellular survivin
expression might not represent a primary therapeutic mechanism. Nonetheless,
our findings improve understanding of the therapeutic effects of interferon
beta and suggest that survivin expression is an additional immunologic variable
in MS. The identification of cellular pathways that modulate T-lymphocyte
apoptosis may open avenues to more targeted therapies for MS.
AUTHOR INFORMATION
Accepted for publication March 13, 2002.
Author contributions: Study concept and design (Dr Sharief); acquisition of data (Drs
Sharief and Semra); analysis and interpretation of data (Dr Sharief); drafting of the manuscript (Dr Sharief); critical revision of the manuscript for important intellectual content (Dr Semra); obtaining funding (Dr Sharief); administrative, technical, or material support (Dr Semra); study supervision (Dr Sharief).
This study was supported by a project grant from the Special Trustees
of Guy's Hospital, London, England. Some of the cranial MR images and interferon
beta-1a supplies were sponsored by Serono International SA and Schering-Plough
Ltd, Kenilworth, NJ.
We thank Sara Soudain, Liisa Carey, Osheik Seidi, MD, and June Smalley
for technical assistance and sample collection.
Corresponding author and reprints: Moad K. Sharief, MD, PhD, Department
of Neuroimmunology, Hodgkin Bldg, Guy's Hospital, London SE1 1UL, England
(e-mail: m.k.sharief{at}kcl.ac.uk).
From the Department of Neuroimmunology, Guy's, King's, and St Thomas'
School of Medicine, Guy's Hospital, London, England.
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