More MS news articles for April 2002

American Academy of Neurology
54th Annual Meeting, April 2002

Speakers' Abstracts that contain the words "Multiple Sclerosis"

http://www.aan.com


Multiple Sclerosis: Immunological Effects of Statins In Vitro - A New Therapeutical Approach?

Tuesday, April 16, 2002
Oliver Neuhaus, Siegrid Strasser-Fuchs, Franz Fazekas, Hans-Peter Hartung, Juan J. Archelos Graz, Austria; Düsseldorf, Germany

Objective:

In order to evaluate the potential role of statins as immunomodulators in multiple sclerosis (MS), we studied their immunological effects in vitro in comparison to the established effects of interferon (IFN)-b1b.

Background:

Statins are widely used lipid-lowering agents. Recent in vitro data suggest that statins might also be potent immunomodulatory agents. In addition, lovastatin attenuates disease severity in a rat model of experimental allergic encephalomyelitis.

Design/Methods:

Peripheral blood lymphocytes (PBL) obtained from untreated or IFN-b-treated patients with relapsing-remitting MS or from healthy donors (HD) were stimulated in the presence or absence of lovastatin, simvastatin, mevastatin, IFN-b1b or statins plus IFN-b1b. We analyzed proliferative activity, cytokine production by ELISA and intracellular immunofluorescence, and surface expression of activation markers, adhesion molecules and chemokine receptors.

Results:

Irrespective of the source of the PBL (untreated vs. treated MS vs. HD), all three statins inhibited proliferation of unspecifically stimulated PBL in a dose-dependent manner, simvastatin being most potent, followed by lovastatin and mevastatin. IFN-b1b showed a similar effect, statins and IFN-b1b together added their inhibitory potentials. Statins reduced activation surface markers, increased production of IL-4, and downregulated chemokine receptors both on B and T cells.

Conclusions:

We conclude that statins are effective immunomodulators in vitro equipotent to IFN-b1b that should undergo clinical studies in MS as a putative future treatment option.


Longitudinal Cytokine Responses to Myelin Peptides in Multiple Sclerosis (MS): Persistence and Spreading of Immune Responses

Tuesday, April 16, 2002
Clara M. Pelfrey, Ioana R. Moldovan, Sarah E. Born, Anne C. Cotleur, Matthew Karafa, Jar-Chi Lee, Elizabeth Fisher, Richard A. Rudick Cleveland, OH; Cleveland, OH

Objective:

To examine longitudinal cytokine responses to myelin antigens in MS patients and healthy controls in order to understand the role of persistence and spreading of immune responses in disease progression.

Background:

Myelin-specific immune responses can be found in both MS patients and healthy controls. Consequently, the relevance of these immune responses to MS disease progression is not clear. Unresolved issues include: Which features of the immune response to myelin relate specifically to MS disease maintenance/progression over time; Which myelin responses persist from one timepoint to another; Which myelin responses spread to new epitopes; Whether HLA type plays a role in maintaining responses over time.

Design/Methods:

We performed a 1-year longitudinal study measuring cytokine responses every 3 months in 20 relapsing-remitting MS patients and 27 age/gender matched controls. Using the ELISPOT assay, we determined ex-vivo Interferon-g (IFN-g) and IL-10 production by peripheral blood mononuclear cells in response to 9-mer overlapping peptides. By using peptides that span the entire PLP and MBP molecules, we obtained at each time-point the number and location of epitopes that induced memory T cell responses, the magnitude of the response, whether there was preferential induction of responses to PLP or to MBP, and whether these were associated with the HLA type of the responder.

Results:

At 3 months, 88% of MS patients responded to myelin peptides compared to 53% of controls, which was similar to the baseline proportions. After 3 months, cytokine responses to PLP persisted in 63% of MS patients and new PLP epitopes appeared in 13% of patients. In contrast, MBP responses persisted in only 25% of patients and new MBP responses appeared in 50% of MS patients. In controls, very few responses persisted between timepoints and new responses were equally distributed between MBP and PLP. Among DRB1*1501+ and *0401+ subjects, 4/8 MS patients showed persistence of responses between timepoints, whereas HLA-matched controls showed no persistent responses.

Conclusions:

These data suggest that persistent cytokine responses to the same myelin epitopes over time distinguish MS patients' responses from controls. The data also support the hypothesis that PLP responses occur early, followed by spreading of the immune response to MBP. This finding is relevant to strategies for antigen-specific therapies in MS.

Supported By:

Supported by National Multiple Sclerosis Society grant #RG3005-A-2 and NIH grant #NS 38667-02.


Cytokine Responses to Myelin Peptides Correlate with Clinical Parameters in Multiple Sclerosis

Tuesday, April 16, 2002
Ioana R. Moldovan, Sarah E. Born, Anne C. Cotleur, Matthew T. Karafa, Jar-Chi Lee, Elizabeth Fisher, Richard A. Rudick, Clara M. Pelfrey Cleveland, OH

Objective:

To perform a cross-sectional study involving determinant mapping of interferon (IFN)-g and interleukin (IL)-10 cytokine responses to proteolipid protein (PLP) and myelin basic protein (MBP) in relapsing-remitting multiple sclerosis (MS) patients and matched controls. To identify correlations between cytokine responses and clinical or MRI measures of disease progression.

Background:

There is increasing evidence that T cell responses to myelin-derived antigens play a significant role in the etiology of MS. Putative myelin antigens, like PLP and MBP,can trigger cytokine secretion in autoimmune T lymphocytes from MS patients, but also from healthy blood donors. There is evidence for in vivo activation and clonal expansion of myelin-reactive T cells in MS patients. In the activated state, T cells produce Th1/proinflammatory cytokines, like IFNg, which have been linked to exacerbations of the disease. Th2/antiinflammatory cytokines have a less clear-cut effect in the disease. The relationship between immune responses to myelin antigens and clinical measures of disease progression is poorly understood.

Design/Methods:

By using the ELISPOT assay, we determined ex-vivo IFN-g and IL-10 production by peripheral blood mononuclear cells in response to 9-mer overlapping peptides derived from PLP and MBP in 20 relapsing-remitting MS patients with early disease (disease duration 2.88±1.91 years) and 27 age- and gender-matched controls. Both MS patients and controls were HLA-typed. Our analysis was focused on identifying the number and location of epitopes that induced memory T cell responses, on the magnitude of the response, and on possible skewing of the response to Th1 vs. Th2 cytokines. We examined correlations between cytokine expression and brain parenchymal fraction (BPF), a highly reproducible MRI measure of whole brain brain atrophy.

Results:

80% of MS patients responded to myelin peptides compared to 56% of controls. High IFNg and IL-10 responders were more frequent in the MS patient group. Isolated PLP responses with no MBP response occured in over 1/3 of study subjects. Isolated MBP responses in the absence of PLP responses were very rare. A large majority of responses were directed towards PLP. Within an individual MS patient, PLP responses showed pronounced skewing towards either IFNg or IL-10, whereas MBP responses were mixed. Higher IFNg responses to PLP correlated with more brain atrophy in MS patients (p=0.02). Higher IL-10 responses to PLP correlated with less brain atrophy in the patients (p=0.36).

Conclusions:

The data suggest that PLP responses may play a significant role in initiation/maintenance of early disease. Relationship of the nature of the PLP response to brain atrophy suggests that IFNg responses promote brain tissue injury, while IL-10 responses appear protective. This suggests that inflammatory cytokine responses to myelin antigens have clinical relevance in MS patients.

Supported By:

The National Multiple Sclerosis Society Grant # RG3005-A-2,and NIH grant # NS38667-02.


The Association between Oligoclonal IgG Bands and Corpus Callosal Lesions in Japanese Multiple Sclerosis Patients

Tuesday, April 16, 2002
Ichiro Nakashima, Kazuo Fujihara, Tatsuro Misu, Hidemitsu Miyazawa, Juichi Fujimori, Shigeru Sato, Sadao Takase, Yasuto Itoyama Sendai, Miyagi, Japan

Objective:

To determine whether the presence of cerebrospinal fluid (CSF) oligoclonal IgG bands (OB) is related to corpus callosal lesions in Japanese multiple sclerosis (MS) patients.

Background:

OB is currently a diagnostic laboratory marker supporting the diagnosis of MS, and over 90% of MS patients are positive for OB in Western countries. In contrast, the frequencies of OB in Japanese MS patients have been much lower (35 - 46%) compared with those in Caucasian patients. It is known that the so-called optic-spinal form of MS, which is characterized by selective involvement of the optic nerves and spinal cord and is relatively common in Orientals including Japanese, is rarely positive for OB. These findings suggest that OB may be associated with the presence of brain lesions in MS patients. Since the corpus callosum is frequently involved in Caucasian MS patients, we investigated the association of OB and the callosal lesions in Japanese MS patients.

Design/Methods:

OB was tested in 33 Japanese MS patients (clinically definite MS (30), laboratory-supported definite MS (2), probable MS (1)) by isoelectric focusing methodology using Phast System™. Magnetic resonance imaging (MRI) was performed on a commercially available 1.5-T system. Sagittal 2-mm fast fluid-attenuated inversion-recovery (FLAIR) imaging was added to the routine MR studies of the brain. The images were reviewed for the presence of callosal lesions and subcallosal striations, which appear as notches on the undersurface of the corpus callosum on sagittal views and are considered as a relatively specific indicator for MS.

Results:

Among the 33 patients, 19 (57.6%) patients were OB-positive. Sex ratio, onset age, disease duration, and annual relapsing rate did not differ between OB-positive MS and OB-negative MS. Callosal lesions were seen in 17 (51.5%) out of 33 patients. All of the patients with callosal lesions had other discrete lesions adjacent to the body or temporal horn of the lateral ventricles and subcallosal striations were observed in all except one OB-negative patient. Among 19 OB-positive MS patients, 15 (79.0%) had callosal lesions on MRI. The other 4 OB-positive patients, however, had subcallosal striations. Among 14 OB-negative MS, only 2 (14.3%) had callosal lesions, 4 patients had subcallosal striations without callosal lesions, and 8 had neither callosal lesions nor subcallosal striations. Using Fisher's exact test, the relation between OB and the presence of callosal lesions was highly significant (p<0.001). The IgG index of the patients with callosal lesions (1.025±0.652) was significantly higher than that of the patients without callosal lesions (0.562±0.165) (p<0.001).

Conclusions:

The present study showed a strong association between OB and callosal lesions on sagittal brain fast FLAIR MRI in Japanese MS. The lower frequency of cerebral or callosal lesions in Japanese MS may be related to the low frequency of OB.


Study of the APOE and SCA2 Loci in Multiple Sclerosis Patients of Portuguese Origin

Tuesday, April 16, 2002
Mónica Santos, Maria do Carmo Costa, Maria Edite Rio, Maria José Sá, Marta C. Monteiro, Angela Valença, Alfredo Sá, José Dinis, José Figueiredo, Luís Bigotte de Almeida, António Valongueiro, Isabel Coelho, Anabela Ferro, Jorge Pinto-Basto, Maria Teresa Matamá, Jorge Sequeiros, Patrícia Maciel Porto, Portugal; Lisbon, Portugal; Leiria, Portugal; Viana, Portugal; Braga, Portugal; Almada, Portugal

Objective:

To study the influence of APOE and SCA2 loci in the susceptibility to multiple sclerosis in Portuguese patients.

Background:

Multiple sclerosis (MS) is a disorder of the central nervous system with a highly variable clinical presentation, which affects around 1:500 European young adults. It is a major demyelinating disease that follows a progressive or relapsing-remitting course, having a predilection for the spinal cord, cerebellum and optic nerves. Genetic factors are thought to play a role in susceptibility to MS.
The APOE gene is involved in myelin homeostasis and in several studies decreased CSF apoE levels in patients with MS have been reported, as well as the association of the e4 allele with other neurodegenerative disorders
The SCA2locus, containing a (CAG)n tract, is known to influence the survival of spinocerebellar neurons. Previous studies proposed that the 22 CAG allele of this gene segregated preferentially with MS.

Design/Methods:

Subjects: a sample of 194 patients of Portuguese origin with definite MS diagnosis according to the criteria of Poser et al and 194 healthy controls (HC), matched by sex, age and region of origin. The parents of 92 of these patients were also studied.
Methods: Genomic DNA from all subjects was isolated from peripheral blood using standard protocols. APOE genotyping was performed by PCR-RFLP, in a 8% non-denaturing polyacrilamide gel, using the restriction enzyme HhaI. Typing of the CAG repeat in the SCA2 locus was carried out by hybridization of PCR products, separated in a 6% denaturing polyacrylamide gel, with a (CAG)15 32P-labelled oligonucleotide probe.
Statistical analysis: The genotype frequencies for the APOE gene and the allele frequencies for the SCA2 gene were compared between cases and controls using the Chi-square test. This test was also used to compare genotype frequencies, at these loci, of cases with primary progressive versus other forms of the disease, and of each of these patient subgroups with matched controls. Disequilibrium of transmission was tested for both loci using the TDT-STDT program 1.1 in 92 trios.

Results:

There were no significant differences between cases and controls for both APOE genotype and SCA2 allele frequencies. The comparison of frequencies for APOE and SCA2 between cases with primary progressive versus other forms of the disease, and of each of these patient subgroups with matched controls, did not show significant differences. We also did not found any transmission disequilibrium for any allele of both loci.

Conclusions:

Our study on Portuguese MS patients does not confirm the previously reported influence of the e4 allele of APOE gene on the susceptibility to this disease. Additionally, in our population, the 22 CAG SCA2 allele does not seem to segregate preferentially with MS.

Supported By:

SERONO Portugal


Activated T Lymphocytes Exert Potent Cytotoxic Effect on Human Neurons

Tuesday, April 16, 2002
Fabrizio Giuliani, V. Wee Yong Calgary, AB, Canada

Objective:

To determine whether activated human T cells can kill human neurons and to elucidate the mechanisms of this toxicity.

Background:

MS is considered a T cell-mediated autoimmune disease of the CNS. MS lesions are characterized by the infiltration of inflammatory cells, demyelination, axonal loss and neuronal degeneration. It has been reported by others that CD8+ T cells can induce the apoptosis of rodent neurons via an MHC-I mediated mechanism; MHC-I expression on neurons was induced by pre-treating them with IFNg and tetrodotoxin. Given that neurons have not been found to express MHC-I in vivo, the current study seeks to investigate whether human neurons are also susceptible to T cell cytotoxicity and whether this can occur through a bystander mechanism without the involvement of MHC.

Design/Methods:

We used a co-culture system of human fetal neurons and T cells from allogeneic PBMC or syngeneic splenocytes. T cells were activated with an anti-CD3 antibody (OKT3) for 72h and then incubated with neurons. Following a pre-determined period of co-culture, neurons were stained with mouse anti-MAP-2 antibody conjugated to Cy3. The number of surviving MAP-2 positive neurons was counted using an automatic counting software (ImagePro). In some experiments, neutralizing antibodies to defined antigens were introduced to the co-culture system to modify the killing. Finally, enriched neuronal cultures, in the absence of T cells, were treated with a variety of inflammatory cytokines.

Results:

When activated T lymphocytes were added to neuronal cultures, they aggregated around neuronal elements and death to neurons occurred promptly. By 3h of co-culture, the number of MAP-2 positive neurons was reduced by over 60% when compared to controls (p<0.001). Neuronal toxicity required the activation of T cells since unactivated T lymphocytes did not produce any death to neurons over a 72h period analysed. Allogenic or syngeneic activated T cells were equally deleterious to neurons. The mechanism of T cell mediated neuronal toxicity required cell-cell contact, and was not reproduced by T cell conditioned media. In correspondence, purified TNF-a, IFN-g and IL-1b, alone or in combination, did not affect neuronal survival. Finally, neutralising antibodies to FasL attenuated the toxicity of activated T cells (% of surviving cells in OKT3-treated control alone=17±9; % of surviving cells in 10mg/mL FasL antibody-treated group=62±11.8;p<0.001) , suggesting the involvment of these molecules in T cell-mediated neuronal death.

Conclusions:

These data demonstrate that activated human T cells can kill human neurons in vitro without the apparent involvment of MHC-I. Toxicity was not a result of a graft-versus-host response as demonstrated using the syngeneic co-culture. We suggest that when T cells are appropriately activated, they can traffic into the CNS to induce neuronal death.

Supported By:

Canadian Institute for Health Research.


Regulation and Functional Effects of Monocyte Migration across Human Brain-Derived Endothelial Cells

Tuesday, April 16, 2002
Rosanne Seguin, Katarzyna Biernacki, Alexandre Prat, Jack P. Antel Montreal, QC, Canada

Objective:

We evaluated the molecular mechanisms that regulate passage of ex vivo peripheral blood derived monocytes across an in vitro human brain-derived endothelial cell (HBEC) barrier and the functional consequences of HBEC-monocyte interactions on the properties of both the HBECs and the monocytes.

Background:

Early lesions in the multiple sclerosis (MS) disease process are characterized by focal accumulations of perivascular lymphocytes and macrophages and enhanced permeability of the blood-brain barrier (BBB).

Design/Methods:

We developed an in vitro model of the initial immune cell migration process across the BBB using Boyden chambers coated with a monolayer of HBECs derived from surgically resected adult temporal lobe tissue. Monocytes and T cells were isolated from peripheral blood of healthy donors.

Results:

Monocytes migrate across HBECs in the absence of inflammatory conditions, at rates exceeding those of T cells. Monocyte migration could be inhibited by the addition of blocking antibodies to intercellular adhesion molecule-1 (40% inhibition) and monocyte chemoattractant protein-1 (30 % inhibition). Treatment with tissue inhibitor of metalloproteinase also reduced monocyte migration by over 25%. Following monocyte migration, there was a significant increase (30%) in permeability to soluble molecules (bovine serum albumin) across the HBECs as compared to resting HBECs or HBECs examined following T cell migration. Initial monocyte migration resulted in a subsequent enhanced rate of T cell migration across HBECs. These effects were observed in absence of detectable TNF production by the monocytes. The migration process did not induce the up-regulation of either co-stimulatory molecules or chemokine receptors on the monocytes.

Conclusions:

Monocyte migration across an initially intact human brain endothelium is an active process which renders the endothelium more permeable to soluble molecules and more susceptible to subsequent transmigration by inflammatory cells.

Supported By:

Multiple Sclerosis Society of Canada


Binding Antibodies to Interferon Beta during Treatment of MS Are Biologically and Clinically Relevant

Tuesday, April 16, 2002
Ebrima Gibbs, Gavin MacDonnell, Florian Deisenheimer, Joel Oger Vancouver, BC, Canada; Innsbruck , Austria

Objective:

To examine the incidence, the effect on bioavailability and the effect on clinical effectiveness of Binding Antibodies in MS patients treated with Interferon Beta 1b for 2 years.

Background:

There has been suggestive evidence that neutralizing antibodies reduce the effectiveness of injected Beta interferon 1b in relapsing MS.

Design/Methods:

One hundred relapsing MS patients started on medication in a post-marketing study were followed immunologically by binding antibody assay (sandwich ELISA) and clinically by repeated examinations. Monthly BAB titers were obtained and some samples were also assayed for Neutralizing antibodies (NABs) and for MxA content of lymphocytes.

Results:

Sixty seven out of 100 treated patients developed BABs. Most BABs appeared during the first 6 months of treatment. Maximum was reached at 5 months (78.7% +/- 4.3) (percentage of maximal normalized level +/- SEM). Therafter the titers fell to an average of 32.5% +/- 4.8 at 24 months. Out of 16 randomly chosen patients, 9 were positive for BAB. and 5 of these 9 devlopped NABs. None of the 7 BAB- developped NABs. BAB positives destined to become NAB+ had higher BAB peak titers than those BAB+ who did not become NAB+. Lymphocyte MxA protein was measured; it increased following IFNbeta-1b administration but fell following the appearance of BABs. Finally patients were adjudicated to 2 groups according to their clinical course. Success was considered when no relapse and no increase in EDSS was observed over 2 years; all the others were considered as failures. Clinical information was available for 92 patients. There was 43 "successes" and 49 "failures". Interestingly BABs segregated with failure: 20/33 successes occured in BAB negative and 36/59 failures were BAB positive (X2=4.19, p<.05).

Conclusions:

We conclude that BABs are not only a predictor of future NAB positivity but are also, by themselves, associated with reduced bioavailibility and reduced clinical effectiveness of Interferon beta-1b. As they are clearly much simpler to measure, they could be used clinically on a large scale.

Supported By:

This study was supported by a grant from Berlex Canada and Shering AG.


Clinical Evaluation of Oral Fampridine-SR (Sustained-Release 4-Aminopyridine) in Patients with Chronic Motor-Incomplete Spinal Cord Injury

Tuesday, April 16, 2002
Daniel Lammertse, Virgina Graziani, Mitchell A. Katz, Andrew R. Blight, SCI-F201 Study Group Englewood, CO; Philadelphia, PA; Hawthorne, NY

Objective:

Assess the safety and efficacy of multiple oral doses of fampridine-SR, a sustained-release formulation of fampridine (4-aminopyridine), in patients with chronic motor-incomplete spinal cord injury (SCI).

Background:

Fampridine-SR is a potassium channel blocking agent being investigated for its efficacy to improve neurologic function in patients with chronic SCI and multiple sclerosis.

Design/Methods:

In this double-blind, parallel-group study, a total of 91 patients in 11 centers were randomized to receive fampridine-SR 25 mg bid, fampridine-SR 40 mg bid, or placebo for 8 weeks (2-week dose escalation, 4-week fixed dose, 2-week down-titration). Safety was evaluated from adverse event reports and standard clinical assessments. Efficacy assessments included measures of spasticity (Ashworth scale), bowel and bladder function, male sexual function, Clinical Global Impression, and Subject Global Impression (SGI). Statistical significance was established at P<.025 to adjust for multiple comparisons.

Results:

In total, 78% of patients completed the study. Proportionally, more patients dropped out from the 40 mg bid group. The most frequently reported adverse events for all treatment groups (30%) were hypertonia, generalized spasm, insomnia, dizziness, asthenia, pain, constipation, and headache. One patient in the 40-mg group with a history of traumatic brain injury experienced a seizure; study medication was stopped and no further seizures occurred. Two outcome measures were statistically significant in favor of fampridine-SR 25 mg bid: SGI (P = .02) and number of days with bowel movements (P = .02). Several other outcome measures favored fampridine-SR 25 mg bid (spasticity, number of bladder accidents per day, and male sexual function), but did not reach statistical significance.

Conclusions:

Fampridine-SR 25 mg bid was well tolerated and showed statistically significant improvement on SGI and in bowel function. Further testing of fampridine-SR (25 mg bid) in larger clinical trials is ongoing.


Results of the Extension of a Trial to Assess the Longer Term Safety of Combining Interferon Beta-1a and Glatiramer Acetate

Tuesday, April 16, 2002
Fred Lublin, Monika Baier, Gary Cutter, Chris Bever, Robert Elfont, Omar Khan, Robert Lisak, Henry McFarland, Pan Narayana, John Noseworthy, Hillel Panitch, Stephen Reingold, Michele Weber, John Whitaker, Jerry Wolinsky, Khurram Bashir, Kenneth Johnson New York, NY; Denver, CO; Birmingham, Al; Baltimore, MD; Jenkintown, PA; Detroit, MI; Bethesda, MD; Houston, TX; Rochester, MN; Burlington, VT

Objective:

To determine whether weekly intramuscular treatment with interferon B-1a when used in combination with daily subcutaneous injections of glatiramer acetate is safe in relapsing-remitting (RR) multiple sclerosis (MS) as determined by the frequency of gadolinium-enhancing lesions on MRI scans.

Background:

The commonest employed agents for relapsing MS are interferon beta and glatiramer acetate. We have reported the initial six month safety data of combining these agents and now report the results of extending the study to 12 months.

Design/Methods:

Thirty-three subjects aged 18 to 50 with RR MS, with an MRI consistent with MS, and EDSS between 0 and 5.5, inclusive who have been taking interferon B-1a (IFN) injections for 6 or more months were entered into the study. After a run-in period of 3 months on IFN monotherapy, 31 subjects received additional treatment of glatiramer acetate. Of those, 26 completed 6 months of combination therapy and 16 completed 12 months of combination therapy. At each monthly follow-up visit an MRI scan was done through month 6, then again at months 9 & 12. The EDSS and the Multiple Sclerosis Functional Composite (MSFC) were assessed at the screening visit, month -2, at the start of combination therapy, and every 3 months thereafter.

Results:

The results reject the null hypothesis, that the addition of glatiramer acetate would block the effect of IFN, demonstrated by increasing the average number of gadolinium enhanced MRI lesions by 1 (p<0.001). There is suggestion of increased effectiveness of combination therapy since 8 of the 11 patients (73%) with any gadolinium lesions at baseline showed a decline in lesion number and volume at 6 months. Of the 16 patients completing 12 month follow-up only 2 had lesions present during the final 6 months. In both cases there was a decrease from the previous 6 months in lesion number and volume. A downward trend in Gad lesion numbers and volumes over time was found. Also, a statistically significant improvement in the MSFC over time on combination therapy was evident during the first 6 months. Over 12 months the timed walk showed a statistically significant improvement. There were no exacerbations seen during the final 6 months. No clinical or laboratory safety issues occurred.

Conclusions:

The above results indicate that there is no evidence for increased risk in terms of new lesion load from this combination therapy. Number and volume of gadolinium enhanced lesions showed a decline from baseline to 6 months, as well as from 6 months to 12 months, suggestive of increased effectiveness. Combination of IFN and glatiramer acetate is safe and deserves study in a larger, pivotal trial.

Supported By:

Biogen, Inc, and TevaNeuroscience


The Independent Comparison of Interferon (INCOMIN) Trial: Final Long-Term Results

Tuesday, April 16, 2002
Luca Durelli, Elisabetta Verdun, Pierangelo Barbero, Elisabetta Versino, Angelo Ghezzi, Enrico Montanari, Mauro Zaffaroni, the INCOMIN Trial Study Group Torino, Italy; Gallarate, Italy; Fidenza, Italy; Italy

Objective:

To compare clinical and MRI efficacy of two interferon (IFN) beta formulations in MS.

Background:

Controlled trials have demonstrated efficacy of once-a-week IFN beta-1a or of on-alternate-day IFN beta-1b compared to placebo. Patient's compliance and efficacy may be affected by different formulations or doses, and a direct clinical-MRI comparison of the two IFN betas is needed.

Design/Methods:

Prospective 2-year follow-up of 188 consecutive relapsing-remitting MS patients recruited by 15 MS centers. Patients randomised by independent statisticians blind of patients clinical characteristics to receive IFN beta-1a, 30 mcg intramuscularly once a week, or IFN-beta-1b, 8 million international units subcutaneoulsy on alternate days. Clinical evaluation conducted on an open-label basis, MRI scans analysed blind of the treatment used. Study conducted independently of the pharmaceutical industry. Primary outcome measures: proportion of patients free of relapses, or free of new T2 lesion. Secondary outcomes: relapse rate, corticosteroid-treated relapse rate, proportions of patients with sustained EDSS score progression of 1.0 point or more and of patients free of gadolinium-enhancing lesions, percent changes of T2 disease burden. Data were evaluated according to the intention-to-treat analysis.

Results:

The proportion of patients free of relapses was 51% in the IFN beta-1b group versus 36% in the beta-1a group (p=0.036), a 42% increased probability that patients on IFN beta-1b remained free of relapses compared to those on IFNbeta-1a. Mean annualised relapse rate was lower in IFN beta-1b (0.47) than in IFN beta-1a group (0.73) (p<0.03). Sustained EDSS progression was significantly lesser in patients on IFN beta-1b, with 14% of patients worsening compared with 30% of those on IFN beta-1a: a relative risk reduction of 56% in favour of IFN beta-1b (p=0.005). Of the patients receiving IFN beta-1b, over 24 months 55% were free of new T2 lesions at MRI compared with only 26% of those on IFN beta-1a(P=0.0003). T2 disease burden decreased by 2.8% in IFN beta-1b group, and increased by 11.7% in IFN beta-1a group (p<0.0001). The relative difference between the two treatments increased with time and was particularly pronounced during the second year of follow-up. Side effects occurrence was similar in the two treatment arms, except for a greater proportion of injection site reactions in the IFN beta-1b arm (p<0.001). Treatment was discontinued due to lack of a clinical response by 10 patients on IFN beta-1a and by only 3 of those on IFN beta-1b.

Conclusions:

Blind MRI evaluation confirmed the unblind clinical results indicating that IFN beta-1b reduced clinical and MRI signs of disease activity and progression more effectively than IFN beta-1a. The increasing relative difference between the two treatments during the second year of follow-up underlines the importance of conducting long-term studies, the only ones which, in the context of a chronic disease, may provide clinically relevant results.

Supported By:

Italian Ministry of Health and Italian MS Society.


Beta Interferon Use among Multiple Sclerosis Patients in a Managed Care Setting: Demographics, Cost of Care, Utilization, Health Status, and Quality of Life

Tuesday, April 16, 2002
Stanton B. Elias, Jennifer Elston-Lafata, Lonni R. Schultz, Jackie Reuther, Christina H. Moon Detroit, MI

Objective:

To describe differences in demographics, cost, utilization, health status (HS), and quality of life (QoL) by treatment with beta Interferon (IFN beta) among patients with multiple sclerosis (MS).

Background:

IFN beta was introduced for use in MS patients in 1993. Since that time, there has been controversy concerning the high cost of treatment and whether it is justified by a favorable impact upon overall medical care cost, utilization of health-related resources, HS, and QoL among MS patients. Several studies have followed QoL during early (6-12 months) treatment, or modeled or estimated costs that are incurred or avoided after long term use. The association of IFN beta on cost, HS, and QoL as typically used in an MS population has not been measured.

Design/Methods:

HMO patients with MS receiving care from a multispecialty practice in 1997 were identified (n=229). Automated data sources, medical records and telephone surveys were used to compile information on patient sociodemographic, clinical characteristics, HS (SF-36), QoL (Farmer), satisfaction with care, medical care use, and costs. Generalized Estimating Equations (GEE) approaches were used to test for differences in sociodemographic and comorbidity characteristics by treatment status, and assess differences in cost, HS, QoL, and satisfaction by IFN beta treatment, controlling for patient sociodemographic and comorbidity characteristics.

Results:

During the year, 27.1% of patients were treated with IFN beta. Treated patients were significantly younger (40 vs 47 years), had shorter disease duration (6.6 vs 9.7 years), and more likely to have psychiatric disorders requiring treatment (32 vs 16%). No significant differences were found for male gender (18 vs 29%), Caucasian race (69 vs 72%), full time employment (56 vs 45%) or Charlson Comorbidity scores (.16 vs .22). Median total ($10,251 vs $1,791), pharmacy ($8,606 vs $99) and non-pharmacy ($1,969 vs $1,272) medical care costs were higher for IFN beta treated patients. Although no significant differences were found in office visit frequency (12.1 vs 9.4), MS visit frequency (3.4 vs. 2.3), or ED (26 vs 19%) or hospital (15 vs 8%) use by IFN beta treatment, the trend was always towards higher utilization among IFN beta treated patients.
Only the health perception domain of the SF-36 varied significantly between treated and untreated patients (45.2 vs 51.0), and only the affect domain of the Farmer instrument indicated treated patients exhibited reduced QoL (34.9 vs 37.7). No differences in satisfaction with medical care were found.

Conclusions:

Treatment with IFN beta was an added cost for medical care for MS patients and did not reduce utilization of other medical services. Treatment with IFN beta was not associated with improvement in self-reported HS, QoL or satisfaction scores. Results reported here reflect concurrent associations with IFN beta use. Whether there are potential long term benefits that might accrue from delaying disability are yet unknown.

Supported By:

National Multiple Sclerosis Society


Evidence of Dose Response: Effects of Two Interferon Beta-1a Products on Relapse-Related Parameters in Multiple Sclerosis

Tuesday, April 16, 2002
Mohammad K. Sharief, EVIDENCE Study Group London, United Kingdom

Objective:

To compare the effects of two interferon (IFN) beta-1a products on relapse-related parameters in patients with relapsing-remitting multiple sclerosis (RRMS).

Background:

Two IFN beta-1a products are available for the treatment of RRMS: Rebif® (Serono) and Avonex® (Biogen). The EVIDENCE study was designed to permit a direct comparison of these products. Key data from this prospective, randomised, assessor-blinded study have been presented elsewhere. This presentation describes in more detail results relating to relapses.

Design/Methods:

677 patients with RRMS were randomised to receive Rebif®, 44 mcg three times weekly (tiw), or Avonex®, 30 mcg once weekly (qw), for 24 weeks. To ensure that all relapses were detected, patients were contacted every 2 weeks throughout the study and were required to inform their physicians within 48 hours of the onset of each suspected relapse. The severity of each relapse was assessed and the functional systems affected were noted. In addition, the numbers of hospitalisations and steroid courses needed because of relapses were recorded. All assessing neurologists were blinded to treatment.

Results:

The proportion of patients remaining relapse-free throughout the 24 weeks of the study was 74.9% with Rebif® and 63.3% with Avonex® (p=0.001), and the mean number of relapses per patient was 0.29 with Rebif® and 0.40 with Avonex® (p=0.022). The mean time that elapsed before the first 20% of patients had their first relapse was 118 days with Rebif® and 89 days with Avonex® (p=0.001), and the hazard ratio for relapsing with Rebif® as opposed to Avonex® was 0.63 (p<0.001). The impact of Rebif® on relapses was greater than that of Avonex® for all relapse severity categories. Patients receiving Rebif® and Avonex® were similar with respect to the proportion of relapses affecting each functional system. Only one relapse (Avonex® patient) led to hospitalisation for a reason other than facilitation of steroid administration. The mean number of steroid courses needed per patient was 0.094 with Rebif® and 0.177 with Avonex® (p=0.004).

Conclusions:

In this large, multicentre, randomised, assessor-blinded trial, Rebif®, 44 mcg tiw, had a significantly greater impact than Avonex®, 30 mcg qw, on several relapse-related outcomes, and was not associated with any new or dose-limiting safety concerns. The superiority of Rebif® was not due simply to suppression of mild relapses, but applied across all severity categories. These findings support the hypothesis that dosing schedule has a major impact on the efficacy of IFN beta-1a, and indicate that higher and more frequent doses of IFN beta-1a are beneficial in RRMS.

Supported By:

Serono International SA.


Gray Matter T2 Hypointensity in Multiple Sclerosis: A 2-year Longitudinal Clinical-MRI Study of 79 Patients

Tuesday, April 16, 2002
Rohit Bakshi, Srinivas R. Puli, Christopher W. Tjoa, Robert A. Bermel, Elizabeth Fisher, Richard A. Rudick, Andrew J. Fabiano, Bianca Weinstock-Guttman, Frederick E. Munschauer, Jack H. Simon Buffalo, NY; Cleveland, OH; Denver, CO

Objective:

Study the longitudinal progression of T2 hypointensity and its prognostic value in predicting neurologic disability and brain atrophy over 2 years in multiple sclerosis (MS).

Background:

Gray matter MRI T2 hypointensity (purported iron deposition) has been described in MS and has been related to physical disability, disease course, MRI lesion load, and atrophy in cross-sectional studies. But, no longitudinal studies have tested the progression of T2 hypointensity and its ability to predict the development of disability and brain atrophy.

Design/Methods:

We analyzed 79 placebo patients who completed a 2-year phase III treatment trial of relapsing-remitting MS. Patients had a mean+/-SD age 35+/-6 years and physical disability (EDSS) score 2.4 +/- 0.9 (range 1.0-3.5) at baseline. Brain MRI was obtained at baseline, year 1, and year 2 using conventional spin echo axial T1-/T2-weighted images. T2 hypointensity was determined quantitatively in the thalamus, caudate, globus pallidus, putamen, red nucleus, and dentate nucleus at the Buffalo Neuroimaging Analysis Center. Reliability (coefficients of variation) of the T2 hypointensity data was intraobserver mean 1%, range 0.7% to 1.4% and interobserver mean 0.8%, range 0.6% to 1.7%. Whole brain atrophy (brain parenchymal fraction-BPF) and total T2 hyperintense, gadolinium enhancing, and T1 black hole lesion volumes were determined quantitatively in Cleveland and Colorado. Regression modeling compared the strength of MRI variables to predict dependent variables. Paired Difference tests assessed the 2-year change of T2 hypointensity.

Results:

Baseline T2 hypointensity in several gray matter regions was associated with higher baseline T1 and T2 lesion loads and lower BPF (p=.0001 to .04). Stepwise regression selected low BPF, and T2 hypointensity in the red nucleus and globus pallidus as predictive of baseline EDSS (R-square=.17, p=.007). A regression model containing dentate, caudate, putamen, and thalamus T2 hypointensity predicted prestudy exacerbation rate (R-square=.17, p=.009). Stepwise regression selected baseline thalamus, red nucleus, dentate, and globus pallidus T2 hypointensity as predictive of the 2-year reduction of BPF (R-square=.29, p<.001). Total T1, T2, and enhancing lesions were not selected by any of the regression models as predictive of dependent variables. None of the baseline MRI variables predicted 2-year progression of disability. T2 hypointensity worsened during the 2-year period in the caudate (p=.06) and putamen (p=.45), but the changes were not significant.

Conclusions:

This study provides additional evidence that gray matter T2 hypointensity in MS patients is related to atrophy, lesion load and clinical disease severity. This T2 hypointensity may have prognostic value in predicting the 2-year progression of brain atrophy, suggesting a relationship between iron deposition and subsequent tissue loss.

Supported By:

National Institutes of Health (NIH-NINDS) 1 K23 NS02210-01 (R. Bakshi), American Academy of Neurology Student Interest in Neurology Summer Scholarship (R. Bermel), and Alpha Omega Alpha Student Research Fellowship (R. Bermel).


A Follow-Up Study with Diffusion, Magnetization Transfer and Spectroscopic Imaging of Corpus Callosum in Clinically Isolated Syndromes suggestive of Multiple Sclerosis

Tuesday, April 16, 2002
Jean-Philippe Ranjeva, Jean Pelletier, Sylviane Confort-Gouny, Yann Le Fur, Danielle Ibarrola, Patrick Viout, André Ali Cherif, Patrick J. Cozzone Marseille, France

Objective:

to characterize on follow-up MRI and MRS procedures, the corpus callosum(CC) of patients who underwent clinically isolated syndromes suggestive of multiple sclerosis (CISSMS) from structural and metabolic point of view.

Background:

Morphological callosal impairment is a frequent finding that can be detected in patients with relapsing-remitting MS (RRMS) early in the disease. Neuronal involvement and axonal loss have been demonstrated in early RRMS by Diffusion, Magnetization Transfer and Spectroscopic Imaging .

Design/Methods:

Twenty patients with CISSMS were compared to 22 age and sex matched controls. All subjects were explored on a 1.5T Vision Plus MR imager (Siemens, Erlangen, Germany). The MRI protocol included a sagittal T1-w acquisition, a T2-w sequence in the axial plane followed by two sagittal MT-w flash sequence to calculate the MTR map, and single shot diffusion weighted EPI sequence (b=0, 250 500, 1000 s/mm2) sensitized sequentially in the three directions to compute the mean diffusivity map. Metabolic exploration was performed in the medial sagittal plane with a 2D-SE acquisition weighted CSI sequence. Four ROI in the corpus callosum were drawn on the medialsagittal slice of each modality and referred as the genum, the central anterior part of CC, the central posterior part of CC and the splenium.

Results:

Baseline data have shown a significant decrease in MTR and a significant increase in MD in the CIS group (p<0.1). In the splenium and the central anterior part, a significant decrease of NAA/Cho ratio was also observed (p<.01). A significant increase in MD and a significant decrease in MTR was observed in the CIS group between baseline and month 6. At this stage (M6) NAA/Cho and Cho/Cr ratios came back to values similar to control group while a tendancy of decrease in NAA/Cr was observed (p=0.07).

Conclusions:

These data suggest that the early myelin breakdown observed at the very early stage of MS tend to evolved within the next 6 months to an axonal injury. The results will be confirmed at the 1 year time point.

Supported By:

Association de la Recherche sur la Sclerose en Plaques (ARSEP) et la Fondation Jean et Odette Duranton de Magny (Fondation de France)


Diagnostic MRI Criteria: Prediction of Conversion to CDMS

Tuesday, April 16, 2002
Frederik Barkhof, Massimo Filippi, Giancarlo Comi, the ETOMS Study Group Amsterdam, Netherlands; Milan, Italy

Objective:

To prospectively test the predictive value of MRI criteria as developed by Barkhof et al. [Brain 1997;120:2057] in patients with a first episode consistent with demyelinating disease for progression to clinically definite MS (CDMS) in the ETOMS (Early Treatment of MS) study.

Background:

Several diagnostic MRI criteria have been proposed for the diagnosis of MS. In the recently adopted diagnostic criteria for MS by McDonald et al. [Ann Neurol 2001;50:127], the modified Barkhof criteria have been incorporated. Neither the modification of the criteria, or the proposed cut-off of 3 positive criteria, have yet been tested in a large sample prospectively.

Design/Methods:

ETOMS was a randomized, double-blind, placebo-controlled study of 308 patients equally distributed to 22mcg interferon beta-1a (IFN, Rebif) once weekly and placebo. Baseline MRI scans were assessed for the presence of gadolinium-enhancement (with or without substitution by 9 or more T2 lesions when no gadolinium lesions present), a juxtacortical lesion, an infratentorial lesion, or at least 3 periventricular lesions. The predictive value of the individual criteria and the cumulative number of positive criteria for conversion to CDMS was assessed by Chi-square analysis, and possible treatment interaction by calculation of odds ratios using logistic regression.

Results:

Conversion to CDMS occurred in 43% of patients with gadolinium-enhancement vs. 34% without (p=0.085); similar comparisons were 44% vs. 31% for infratentorial (p=0.031), 40% vs. 35% for juxtacortical (p=0.497), and 41% vs. 17% for more than 3 peri-ventricular lesions (p=0.025). When the lack of a gadolinium lesion was substituted by the presence of 9 or more T2 lesions, the predictive value increased (41% vs. 11%, p=0.008). No significant treatment interaction was noted for any of the parameters. For the cumulative number of modified Barkhof criteria, a nearly linear increase was seen from 25% for 1 abnormal criterion to 47% with 4 abnormal criteria; the most effective cut-off was for 3 or less vs. 4 criteria (p=0.003); for the cumulative number of Barkhof criteria, again no significant treatment interaction was noted.

Conclusions:

This study confirms the validity of the modified Barkhof criteria for conversion to CDMS in a prospective study, and indicates that the efficacy of treatment with Rebif occurs independent of baseline MRI findings.

Supported By:

Serono


Diffusion-Weighted MRI Evidence of Gray Matter Damage in Multiple Sclerosis

Tuesday, April 16, 2002
Andrew J. Fabiano, Robert A. Bermel, Christopher W. Tjoa, Bianca Weinstock-Guttman, Frederick E. Munschauer, Joan M. Feichter, Colleen E. Miller, Eileen Gallagher, Rohit Bakshi Buffalo, NY

Objective:

To assess whether subcortical gray matter damage can be detected by diffusion-weighted MRI in patients with multiple sclerosis (MS) and to compare gray matter diffusion coefficients with clinical findings.

Background:

Injury to the deep gray matter in MS has been suggested from recent studies using PET scanning and by the detection of T2 shortening (proposed iron deposition). This gray matter damage supports a global disease process and may contribute to neurologic dysfunction in patients with MS. Diffusion-weighted MRI (DWI) can noninvasively detect changes in water Brownian motion, offering a novel method to assess tissue damage in a variety of neurologic diseases with a higher sensitivity than conventional MRI. DWI has revealed areas of microscopic disease in the normal appearing white matter (increased diffusion) in patients with MS, but has not been applied to the study of gray matter damage in MS.

Design/Methods:

DWI was performed in 64 patients with clinically definite MS on a General Electric Signa 4x/Lx 1.5-T magnet (Milwaukee,WI) using a single-shot spin-echo echoplanar technique with a TR/TE/NSA 9999/106/1, 5-mm nongapped (contiguous) slices, matrix size 96x128, FOV of 36x27 cm and scan time of 40 seconds, generating 28 axial slices through the brain. Four images per slice location were produced, with a diffusion-weighing b-factor of 0, and 1000 s/mm2 applied in three orthogonal directions. Mean apparent diffusion coeffecient (ADC) maps were reconstructed at the Buffalo Neuroimaging Analysis Center using customized software (Java Image, Xinapse Systems, Leicester, England). The diffusivity was computed separately in the x, y, and z directions, and the results averaged to form the mean ADC. ADC values were obtained from the globus pallidus, putamen, caudate, and thalamus using a standardized region of interest template. Fifty-two patients had relapsing-remitting (RR) and 11 had secondary progressive (SP) MS. Physical disability was assessed using the Expanded Disability Status Scale (EDSS) and ranged from 0 to 8.0 (mean ± SD: 2.8 ± 2.2); patient age ranged from 22 to 59 years (41.4 ± 8.0). Group differences were assessed using t-tests. Correlations were tested using Pearson's or Spearman Rank coefficients.

Results:

ADC was higher in the caudate in SP (0.870x10-3mm2/s ± 0.135x10-3) vs. RR patients (0.764x10-3mm2/s ± 0.083x10-3) (p=.028). ADC in the caudate was positively associated with greater physical disability (higher EDSS score) (rho=.404, p=.001). Age or gender was not related to gray matter ADC in any of the regions.

Conclusions:

Increased water diffusion in the deep gray matter may be associated with clinical markers of disease severity in patients with MS. DWI is potentially useful as a surrogate marker of gray matter tissue damage in MS.

Supported By:

Supported by National Institutes of Health (NIH-NINDS) 1 K23 NS02210-01 (R. Bakshi), American Academy of Neurology Student Interest in Neurology Summer Scholarship (R. Bermel), and Alpha Omega Alpha Student Research Fellowship (R. Bermel).


Evolution of Diffuse and Focal Magnetization Transfer Abnormalities in Multiple Sclerosis

Tuesday, April 16, 2002
Cornelia Laule, Irene M. Vavasour, Kenneth P. Whittall, Joel Oger, Donald W. Paty, David K. B. Li, Alex L. MacKay, Douglas L. Arnold Vancouver, BC, Canada; Montreal, QC, Canada

Objective:

To determine the evolution of magnetization transfer in NAWM of patients with MS

Background:

A range of pathologies is seen in multiple sclerosis (MS), including inflammation, demyelination and axonal loss, which cannot be differentiated on conventional magnetic resonance (MR) images. Magnetization transfer (MT) is a MR technique that investigates the exchange between motionally restricted and mobile protons, allowing for the indirect measurement of tissue structure. In normal healthy white matter, strong interactions between mobile water protons and the restricted protons associated with non-aqueous tissue result in a high magnetization transfer ratio (MTR). Tissue destructive processes, such as demyelination, cause a decrease in MTR.

Design/Methods:

Nine patients with clinically definite MS were enrolled in the study. Patients were scanned 5 times over one year at months 0, 2, 4, 6 and 12. Age and gender matched controls were scanned once. MR examinations for each visit included a conventional dual echo scan, post-contrast gadolinium T1 weighted images and 3D gradient echo experiment with and without a 2000Hz off-resonance MT pulse. Proton density images were registered with MT images with and without the MT pulse, IMT and InoMT, respectively. MTR was defined as MTR=((InoMT-IMT)/InoMT)*100%. Regions of interest were drawn around 10 bilateral normal appearing white matter (NAWM) and normal white matter (NWM) areas in all subjects and in lesions in MS patients. The MTR of NAWM was investigated as a function of disease duration. For new lesions, MTR was examined before and after lesion appearance on conventional MRI.

Results:

The mean NAWM MTR was found to correlate strongly (R=0.93) with the length of time since the patient's first clinical presentation and was well characterized by a linear decrease of -0.16%/year (p<0.0001). The time zero intercept of the NAWM MTR regression was 30.6±0.2%, which was not different from the average MTR of NWM from age and gender matched controls (30.4±0.2%). An additional gradual decrease in NAWM MTR was observed 6 to 12 months before the appearance of a new lesion on conventional MRI, while a more precipitous decrease in MTR was seen 2 to 6 months before the lesion appeared. Those lesions that exhibited pre-lesion MTR decreases showed less MTR recovery than lesions which had no pre-lesion MTR decrease.

Conclusions:

NAWM in MS undergoes a slow progressive decrease in MTR that starts at disease onset and accelerates rapidly in focal areas just prior to lesion appearance on conventional MR images.

Supported By:

MS Society of Canada


A Functional MRI Study of Patients with Clinically Definite Multiple Sclerosis and Atypical Conventional MRI

Tuesday, April 16, 2002
Massimo Filippi, Maria A. Rocca, Maria Codella, Andrea Falini, Mauro Zaffaroni, Angelo Ghezzi, Lucia Moiola, Giuseppe Scotti, Giancarlo Comi Milan, Italy, Italy; Milan, Italy; Gallarate, Italy

Objective:

We used functional magnetic resonance imaging (fMRI) to evaluate the brain pattern of cortical activations during the performance of a simple motor task in patients with clinically definite multiple sclerosis (CDMS) and atypical conventional MRI of the brain.

Background:

FMRI changes have been demonstrated in MS patients with different clinical phenotypes. To which degree these changes are correlated with the extent of microstructural tissue damage has not been clarified yet.

Design/Methods:

We studied 12 right-handed patients with CDMS (10 women and two men; mean age=38.0 years, mean disease duration=7.2 years, median expanded disability status scale score=1.5, range=0.0-6.0). Patients were included if they had normal or near-normal brain MRI scans (defined as three or fewer T2 lesions). None of them had clinical evidence of right hand function impairment at the time of fMRI acquisition or lesions located along the motor pathways on T2-weighted images. Twelve sex- and age-matched right-handed healthy volunteers served as controls. In each subject, we acquired: 1) fMRI during the performance of repetitive flexion-extension of the last four fingers of the right hand; 2) dual-echo turbo spin echo and 3) pulsed-gradient spin-echo (PGSE) echo-planar imaging. Mean diffusivity (MD) histograms of the normal appearing brain tissue (NABT) were produced. FMRI data were analyzed using a random effect analysis (SPM99).

Results:

Although CDMS patients had a very low mean T2-weighted lesion load (0.4 ml), they had a lower NABT MD histogram peak height when compared to healthy subjects (p=0.004). Compared to healthy volunteers, CDMS patients also showed a more significant activation of the contralateral secondary somato-motor cortex (SII). On the contrary, they had significantly reduced activation of the ipsilateral primary somatomotor cortex (SMC), and contralateral inferior frontal gyrus.

Conclusions:

This study suggests that microstructural white matter damage in MS is associated with cortical functional reorganization.


Brain Atrophy in Patients with Clinically Isolated Syndrome

Tuesday, April 16, 2002
Andrea Paolillo, Patrizia Pantano, Francesca Caramia, Maria Cristina Piattella, Silvia Di Legge, Walter Nucciarelli, Isabella Pestalozza, Carlo Pozzilli Rome, Italy

Objective:

To seek MRI parameters predictors of brain atrophy in patients with clinically isolated syndrome (CIS).

Background:

The assessment of brain volume changes provides an objective marker of the progression of Multiple Sclerosis (MS) and appears intriguing in CIS patients. In this population, the relationship between brain atrophy, inflammation and the irreversible axonal damage is debated.

Design/Methods:

Monthly Gd-enhanced MRI scans over a period of 6 months were obtained in 39 consecutive CIS patients with an abnormal MRI scan at baseline. Further MRI scans were performed at 12 and 18 months. Patients who developed a 2nd clinical episode were withdrawn from the study to be treated with disease modifying therapies. For each scan, we obtained measurements of lesion load for Gd-enhancing lesions (Gd-LL), T2 hyperintense lesions (T2-LL) and T1-black holes (T1-LL). Brain volume changes were assessed using the SIENA method. This program estimates percentage brain volume change (PBCV) between two input images, taken of the same subject, at different points in time.

Results:

Twelve of 39 CIS patients showed a 2nd relapse and therefore did not complete the MRI follow-up; 27 patients were followed from baseline to 18 months. They had a median increase of 13% in the T2-LL and of 15% in the T1-LL. There was a decrease of 1.15% in PBCV. The mean monthly Gd-LL (6 months) was significantly associated with PBCV over the study period (r = -0.59, p = 0.001). Exploratory subgroup analyses showed that patients with at least one Gd-enhancing lesion during the first 6 months, (i.e. active patients) (n=16), developed greater brain atrophy (-1,44% vs - 0,67%, p = 0.01) than patients without Gd-enhancing lesions (inactive patients) (n=11).

Conclusions:

The data suggest a robust relationship between the development of brain atrophy and inflammation in patients with CIS. Monthly Gd-enhanced MRI scans over a period of 6 months after the first clinical episode suggestive of MS may help to characterize patients who will develop brain atrophy in short term.


Whole Brain N-Acetylaspartate Concentrations Are Reduced in Patients at Presentation with Clinically Isolated Syndromes Suggestive of MS

Tuesday, April 16, 2002
Massimo Filippi, Marco Bozzali, Anna Gambini, Marco Rovaris, Andrea Falini, Angelo Ghezzi, Vittorio Martinelli, Giuseppe Scotti, Oded Gonen, Robert I. Grossman, Giancarlo Comi Milan, Italy; Gallarate, Italy; New York, NY

Objective:

To quantify the presence and extent of axonal loss/dysfunction in patients at presentation with clinically isolated syndromes (CIS) at risk for subsequent conversion to definite multiple sclerosis (MS), by measuring whole brain N-acetylaspartate (WBNAA) concentration.

Background:

It has been suggested that permanent tissue damage can occur early in the course of MS and have a predictive value for the subsequent disease evolution. WBNAA, which can be considered a global measure of irreversible neuronal loss, was found to be lower in relapsing-remitting MS patients than in normal controls.

Design/Methods:

Eighteen patients with CIS and 10 sex- and age-matched healthy controls were studied. All patients had had a CIS suggestive of demyelination within the three months preceding magnetic resonance (MR) acquisition. During a single session, the following brain MR sequences were acquired in all study subjects: a) 1H-MR spectroscopy (MRS) pulse sequence, based on a four-step cycle of non-selective 180° inversion pulses, to obtain WBNAA spectra; b) dual-echo turbo spin-echo; c) pre- and post-contrast T1-weighted conventional spin-echo. Whole brain volume, T2 and T1 lesion volume (LV) were calculated using a semi-automated segmentation technique. Absolute WBNAA amounts (in mmoles - mM) were calculated using a phantom replacement method and were then corrected for individual subjects' brain volumes.

Results:

In CIS patients, median T2 and T1 LV were 1.7 ml and 0.3 ml, respectively. The average brain volume was significantly lower in CIS patients than in healthy controls (mean values: 1165.4 ml vs 1276.5 ml, p=0.01). The average WBNAA concentration (corrected for brain volume) was also significantly lower in patients than in controls (mean values: 11.1 mM vs 14.3 mM, p=0.002).

Conclusions:

These findings suggest that axonal loss/dysfunction occurs at a very early stage in patients at presentation with CIS suggestive of MS. The magnitude of the reduction of WBNAA concentrations in these patients might be a strong predicting factor of subsequent clinical evolution.


The Evidence of Interferon Dose-Response: European-North American Comparative Efficacy (EVIDENCE) Study: 48 Week Data

Tuesday, April 16, 2002
Hillel Panitch, Patricia Coyle, Gordon Francis, Douglas Goodin, Paul O'Connor, Brian Weinshenker, the EVIDENCE Study Group Burlington, VT; Stony Brook, NY; Norwell, MA; San Francisco, CA; Toronto, ON, Canada; Rochester, MN

Objective:

To assess whether differences in efficacy based on dose of interferon (IFN) and treatment regimen persist to 48 weeks in patients with relapsing-remitting multiple sclerosis (RRMS).

Background:

Although IFN-b therapy is effective in RRMS, debate persists regarding the importance of dose and frequency of administration. At the 24-week primary endpoint, the EVIDENCE study demonstrated significant improvement on relapse and MRI outcome measures for high dose IFNb-1a (44 mcg subcutaneously three times weekly, sc tiw) compared to low dose (30 mcg intramuscularly weekly, im qw). We now present data up to 48 weeks of treatment.

Design/Methods:

This was a prospective, assessor-blinded, comparative study of 677 RRMS patients randomized to treatment with either IFNb-1a 44 mcg sc tiw (Rebif) or IFNb-1a 30 mcg im qw (Avonex). After a screening MRI study, patients had MRI scans every 4 weeks for 24 weeks with and without Gadolinium, plus an additional non-enhanced scan at week 48. Clinical assessments were performed every 12 weeks, and as needed for documentation of relapses. The primary endpoint was the proportion of relapse-free patients. The principal secondary outcome measure was MRI active lesion count (combined unique lesions for 24 weeks based on monthly scans, and T2 active lesion count for 48 weeks based on 6-monthly scans). All analyses were performed according to intention to treat.

Results:

Of the 677 patients enrolled, 339 were randomized to Rebif and 338 to Avonex. All but a single patient received at least one dose of drug. Adherence to study at 48 weeks was 96% for both groups, while adherence to therapy was 91% for both groups. The most common causes for stopping therapy were adverse events (15 Rebif, 8 Avonex) and patient decision (9 Rebif, 15 Avonex). Three patients (all on Avonex) were lost to follow-up. Data on proportion of relapse-free patients, time to first relapse, relapse rate, relapse severity, T2 lesion activity (active T2 lesions, proportion of active scans and proportion of patients free of T2 lesion activity), as well as safety, will be presented.

Conclusions:

This is the first randomized, examiner-blinded comparative study examining the efficacy of two disease modifying drugs in MS. Patient adherence to the study and investigator adherence to the protocol were excellent. IFNb-1a 44mcg administered sc tiw was shown at 24 weeks to provide significant clinical and MRI benefit compared to 30mcg weekly therapy. The 48-week data provide information about the longer-term durability of these beneficial effects.

Supported By:

Serono International SA


Clinical Application of Autologous Stem Cell Transplantation in Severe Multiple Sclerosis Treated with High-Dose Immunosuppressive Thyerapy

Tuesday, April 16, 2002
George H. Kraft, James D. Bowen, Jimmy Y. Cui, Richard A. Nash Seattle, WA

Objective:

To evaluate the possible therapeutic roles of high dose immunosuppressive therapy (HDIT) and rescued with autologous stem cell transplantation (SCT)in the management of severe, non-responsive multiple sclerosis (MS)patients.

Background:

At the present time, there are four FDA approved disease-modifying agents available in the United States. However, none of these completely stops disease progression. All of them produce varying degrees of slowing of the disease process. Our hypothesis was that by ablating the immunoactive cells in an MS patient, and replacing them with non-conditioned naive cells, the disease process could be stopped.

Design/Methods:

we enrolled 26 patients with severe MS including primary progressive-MS (n=7), secondary progressive-MS (n=18), and relapsing remitting-MS (n=1). Their median age was 41 (range 27-60) years. The median EDSS at HDIT was 7.0 (5.0-8.0). Eligibility requirements included an EDSS from 5.0-8.0 and deterioration of one or more points over the previous year. 21 patients had failed previous therapy with interferon-beta, and 15 had failed multiple therapies including copaxone, prednisone and methotrexate. The median follow-up was 12 (3-36) months.

Results:

With a minimum follow-up of 6 months (n=22;1912 months), the majority of patients remain neurologically stable with unchanged EDSS. 6 patients had improvement on EDSS, 5 have had an increase in EDSS. No change was greater than 1.0 EDSS point. Only 2 patients had enhancing lesions on brain MRI at 1 year after SCT. One of these two had a flare of MS associated with the administration of granulocyte-colony stimulating factor (G-CSF). No patient has received further treatment with interferon-beta or copaxone after SCT. Complication included 12 patients of the first 18 patients had an engraftment syndrome involving T38, rash and fatigue. The subsequent 8 patients who received prednisone did not develop this syndrome. One patient had a flare during G-CSF for mobilization, but no more patients experienced MS flare after adding prednisone during the mobilization. One patient developed Guillain-Barre syndrome and pneumonia 17 months after SCT. Another patient had a sustained fever of unknown origin early after transplant and had an associated increase in baseline EDSS of 1.0. The only patient who received rabbit anti-thymocytic globulin (ATG) due to a positive skin test to horse ATG developed CMV disease and EBV- associated post-transplant lymphoproliferative disorder and expired at day 53.

Conclusions:

In conclusion, we believe that HDIT/SCT may become an effective treatment to control severe, progressive and refractory MS. The procedure appears to be effective to date. This and additional studies should be continued to fully assess long term efficacy in these heterogeneous high-risk patients.


Validation of the McDonald Criteria in Patients with Clinically Isolated Syndromes Suggestive of Multiple Sclerosis

Tuesday, April 16, 2002
Catherine M. Dalton, Peter A. Brex, Katherine A. Miszkiel, Gordon T. Plant, Alan J. Thompson, David H. Miller London, United Kingdom

Objective:

The objective of this study was to validate the new McDonald criteria at three months in patients with Clinically Isolated Syndromes (CIS) suggestive of MS who were followed for three years.

Background:

Traditionally MS has been diagnosed on the basis of clinical evidence of dissemination in time and space. New diagnostic criteria of the international panel of McDonald and colleagues incorporate MRI evidence of dissemination in time and space and allow a diagnosis of MS in patients with CIS.

Design/Methods:

Adult patients aged 16-50 years with CIS were recruited and imaged within three months of the onset of symptoms (mean 5 weeks) and again after three months, one year (79 patients) and three years (46 patients). A diagnosis of clinically definite MS was based on the traditional Poser criteria and a diagnosis of MS incorporating MRI findings was made using the McDonald criteria. Using the Poser criteria as the gold standard, the McDonald criteria were evaluated in terms of sensitivity, specificity, positive and negative predictive values.

Results:

38/79 (48%) of patients had a diagnosis of MS at one year using the McDonald criteria compared with 16/79 (20%) who had clinically definite MS. Using baseline and three month scans the McDonald MRI criteria alone have a sensitivity of 59%, specificity of 92%, positive predictive value of 83% and negative predictive value of 76% for the development of clinically definite MS. Specificity is higher than an abnormal baseline MRI brain (30%) or the traditional Barkhof criteria (78%).

Conclusions:

The McDonald criteria more than double the number of patients with a diagnosis of MS at one year. In making a diagnosis of a lifelong condition specificity is more important than sensitivity, therefore the McDonald criteria are valid.

Supported By:

The NMR Unit is supported by Multiple Sclerosis Society of Great Britain and Northern Ireland. CM Dalton was sponsored by elan. Schering AG supported PA Brex.


The Impact of Revised McDonald's Criteria in Predicting Multiple Sclerosis

Tuesday, April 16, 2002
Silvia Di Legge, Maria C. Piattella, Patrizia Pantano, Isabella F. Pestalozza, Valter Nucciarelli, Luigi Bozzao, Gian L. Lenzi, Carlo Pozzilli Rome, Italy

Objective:

To test reliability of the revised McDonald's criteria of Multiple Sclerosis (MS) in a cohort of patients with Clinically Isolated Syndrome (CIS) followed by serial Gd-enhanced MRI.

Background:

To facilitate the diagnosis of MS the revised criteria integrate MRI in the diagnostic scheme. Lesions in the brain detected by MRI can provide evidence of dissemination of lesions in both space (DIS) by Barkhof criteria and dissemination in time (DIT) by the appearance of at least a new Gd-enhanced lesion and/or new T2 lesion in serial MRI.

Design/Methods:

A series of consecutive CIS patients showing at least 3 typical white matter lesions on their brain MRI were followed-up for the first six consecutive months after enrollement with monthly Gd-enhanced brain MRI. Brain MRI were then performed at 12, 18, 24 and 36 months from the inclusion in patients who did not develop a second clinical episode during the study. In patients who developed a second clinical episode the study ended at the time of the relapse. For the present study we analyzed scans performed at baseline, 3, 6 and 12 months of those patients who were observed for at least 18 months.

Results:

We examined 53 patients, 35F and 18M, with a mean age of 30.5 yrs, time since clinical onset of 4.2 months and baseline EDSS of 1.3. During the observation period 19/53 (36%) of patients developed a second clinical episode and 45/53 (85%) developed MS by the McDonald's criteria.
Baseline MRI showed DIS in 89% (47/53) of patients. Out of them, DIT was observed in 28/53 (53%) at 3-month scan, in 40/50 (80%) at 6-month scan and in 31/40 (77.5%) at 12-month scan. At three-month MRI scan, sensitivity of the new McDonald's criteria was 89% (17/19) [95% confidence interval (CI) 75-100%], while specificity was 68% (23/34) [95% CI 52-84%]. Positive predictive value (PPV) and negative predictive value (NPV) were 61% (17/28) and 92% (23/25), respectively.

Conclusions:

These preliminary results obtained in CIS patients show a high sensitivity of the diagnostic McDonald's criteria at three-month MRI scan, while the specificity is low. It is likely that the specificity of the new McDonald's criteria will increase with a longer follow-up. Furthermore, the high NPV at three-month MRI scan suggests that McDonald's criteria can early detect patients who will remain disease-free.


MRI Criteria for Multiple Sclerosis: Evaluation in a Paediatric Cohort

Tuesday, April 16, 2002
Cecil D. Hahn, Manohar M. Shroff, Susan I. Blaser, Brenda L. Banwell Toronto, ON, Canada

Objective:

To investigate whether existing MRI criteria used in the evaluation of multiple sclerosis (MS) in adults are applicable to the paediatric MS population.

Background:

MRI criteria for the evaluation of MS in adults have been proposed and validated by several authors. (Paty et al., Fazekas et al., Barkhof et al.) The validity of these MRI criteria in the paediatric MS population has not been determined.

Design/Methods:

This study was an analysis of children with clinically definite MS (CDMS) by Poser's criteria who are followed in a multidisciplinary paediatric multiple sclerosis clinic at The Hospital for Sick Children. Clinical records were reviewed to identify the dates and clinical features of their first attack and subsequent MS-defining attack. MRI studies performed at the time of each attack were graded according to the criteria of Paty, Fazekas and Barkhof.

Results:

Twenty children with CDMS were identified and 3 were excluded because applicable MRI studies were unavailable for review. Of the remaining 17 children, 13 had MRI studies performed at both attacks, 2 had MRI studies performed only at the first attack, and 2 had MRI studies performed only at the MS-defining attack. 9 children were female. The mean age at diagnosis of CDMS was 11 years (range 6-18 years). The median interval between the first attack and the MS-defining attack was 6 months (range 2-29 months). At the time of the first attack, the criteria of Paty, Fazekas and Barkhof were met by 11 (73%), 10 (67%) and 7 (47%) children, respectively, and 4 children (27%) met none of the criteria. At the time of the MS-defining attack, the criteria of Paty, Fazekas and Barkhof were met by 11 (73%), 12 (80%) and 9 (60%) children, respectively and 3 children (20%) met none of the criteria.

Conclusions:

Recently, McDonald et al. have proposed revised diagnostic criteria for MS which employ the MRI criteria of Barkhof. In the present study of children with CDMS, we have found that less than half met the Barkhof criteria at time of their first attack and only 60% met the Barkhof criteria at the time of their MS-defining attack. The children were more likely to meet the Paty and Fazekas criteria, but still not as likely as has been reported in adults. (Offenbacher et al., Tintore et al.) We conclude that the typical MRI features of adult MS are less common in paediatric MS. The accurate and timely diagnosis of MS in children would be supported by the development of MRI criteria that are validated in the paediatric MS population.


Natalizumab Treatment for Relapsing MS: Further Results of a Randomized, Double-Blind, Placebo-Controlled Trial

Tuesday, April 16, 2002
Catherine Dalton, Paul O'Connor, George Rice, Omar Khan, J. Theodore Phillips, Gareth Barker, David MacManus, Katherine Miszkiel, David Miller, the International Natalizumab (Antegren™) MS Trial Group London, United Kingdom; Toronto, Canada; London, ON, Canada; Detroit, MI; Dallas, TX

Objective:

To assess the effect of monthly IV administrations of natalizumab (3.0 or 6.0 mg/kg) vs placebo over a 6 month treatment period and for an additional 6 months following discontinuation of study treatment on brain lesion activity in patients with relapsing MS.

Background:

The cell adhesion molecule alpha4beta1 integrin, also known as VLA-4, is thought to play an important role in the trafficking of mononuclear cells into sites of inflammation. Through its interaction with vascular cell adhesion molecule-1 (VCAM-1), alpha4beta1 mediates migration of autoreactive lymphocytes into the brain leading to the inflammatory lesions of multiple sclerosis. Recently it has been demonstrated that monthly treatments with natalizumab, an alpha4-integrin antagonist in the class of selective adhesion molecule inhibitors (SAMIs), reduced the occurrence of new gadolinium enhancing MRI lesions and clinical relapses in patients with multiple sclerosis over the six month treatment period (Multiple Sclerosis 2001 (Abstract;7:S16.). The present report describes additional analyses performed during the six months of treatment and follow up.

Design/Methods:

213 patients with relapsing MS were randomized to receive either natalizumab at 3 or 6 mg/kg or placebo IV every 4 weeks for 6 months and were followed for an additional 6 months for safety. Inclusion criteria included relapsing disease with 2 relapses within the last 2 years, an EDSS of 2.0 to 6.5 and no concomitant disease modifying treatment. T1 gadolinium-enhanced and T2-weighted imaging were performed pretreatment, every 4 weeks during the 6 month treatment period and quarterly during the 6 month post-treatment safety follow-up.

Results:

At the six month time point, natalizumab treatment resulted in a statistically significant reduction in the number of new and enlarging T2 lesions, the number of new T1 hypointense lesions and the number of active scans, i.e. containing new enhancing lesions. During the six month follow-up period there were no significant differences in numbers of enhancing brain lesions or relapses between treatment groups. Natalizumab treatment appeared to be well tolerated.

Conclusions:

During the six month treatment period, natalizumab suppressed new T1 gadolinium-enhanced, T2-weighted and T1 hypointense brain lesions as well as clinical relapses. Post treatment MRI and clinical activity resumed to a level similar to placebo. In view of these promising findings, Phase III trials are underway to evaluate the longer term effect on relapses and disability.

Supported By:

Elan Pharmaceuticals Inc. and Biogen Inc.


Safety and Efficacy of Combination Therapy with Interferon Beta-1b and Mitoxantrone in Worsening Multiple Sclerosis Using Monthly Gadolinium Enhanced MRI

Tuesday, April 16, 2002
Douglas R. Jeffery, Neraj B. Chepuri, Jason Rosenburg Winston-Salem, NC

Objective:

To examine the safety and efficacy of combined therapy with Mitoxantrone (MITX)and Interferon Beta-1b (IFNb-1b)in patients with worsening multiple sclerosis (MS) and a subotimal therapeutic response to IFN-b-1b alone.

Background:

Despite the demonstrated efficacy of immunotherapeutic agents in the treatment of MS, many patients show a suboptimal response with continued progression of disability and frequent relapse. Mitoxantrone decreases relapse rates, decreases the progression of disability, decreases the accumulation of new T2 and new gadolinium enhancing lesions in both relapsing-remitting (RR)and secondary progressive (SP)MS. The purpose of this trial was to examine the safety and efficacy of combined therapy with IFNb-1b and MITX in patients with worsening MS not controlled on IFNb-1b alone.

Design/Methods:

Ten patients with RR or SP MS were enrolled in the trial. All patients had to be on IFNbb-1b for at least six months, have at least one new enhancing lesion on a screening MRI, one relapse in the six months prior to study enrty, and be neutralizing antibody negative. All patients were required to have normal cardiac function prior to study entry. Monthly MRI scans using triple dose contrast with 30 min delay between contrast administration and scanning were carried out for three months to obtain baseline numbers of new enhancing lesions (NEL). At the end of the baseline phase, MITX was administered at 12 mg/m2 (month 1), and 5 mg/m2 at month 2 and 3. Dosing was then continued at 5 mg/m2 every third month. Monthly MRI scanning was continued for the duration of the study. The primary outcome measure was the number of NELs. Secondary outcome measures included relapse rates, EDSS, and change in T2 and T1 lesion volume.

Results:

The addition of MITX to therapy with IFN was well tolerated and there no serious adverse events. Mean white blood cell counts decreased to 2,800 at day 14 but returned to normal levels by day 21. Five upper respiratory tract infections occurred in three patients and one patient had a urinary tract infection. No adverse cardiac events were noted. The mean number of NELs per month in the baseline phase was 2.93±2.46. Mean NELs decreased to 1.09±1.5 following the addition of MITX. Median NEL number decreased 81% following the addition of MITX. New enhancing lesion numbers were suppressed to a greater extent with longer duration of exposure to MITX. At the time of the last scan (month 6)only two patients had new enhancing lesions. Relapse rates decreased 74% following the addition of MITX.

Conclusions:

The combination of therapy with IFNb-1b and MITX was well tolerated and there were no serious adverse events. Both clinical and MRI measures of disease activity responded well to the combination. Relapse rates decreased by 74% and by the time of the last scan only two patients had NELs and volumes were very small. While these results are preliminary, they suggest that patients with aggressive forms of MS failing immunotherapy may benefit from the combined use of IFNb-1b and MITX.

Supported By:

This study was supported by grants from Berlex Laboratories and Immunex.


Neuropsychological Features of Pediatric Multiple Sclerosis

Tuesday, April 16, 2002
Brenda L. Banwell, Peter E. Anderson Toronto, ON, Canada

Objective:

To provide a comprehensive assessment of the cognitive profile and academic achievement of a cohort of pediatric MS patients and to compare these results to age-matched normative data.

Background:

Although Multiple Sclerosis (MS) is a disease that primarily affects adults, approximately 2-5% of all MS patients will be diagnosed prior to their 18th birthday. Cognitive deficits are common in adults with MS, and adversely influence patient employment and quality of life. The impact of MS on the developing brain, both on cognition and academic achievement is essentially unknown.

Design/Methods:

Ten children, aged 9-16 years with clinically definite MS as defined by the criteria of Poser et al, were studied. All patients were examined by a pediatric neurologist on the morning of testing to ensure that none were experiencing an MS-related relapse, and all patients were at least 30 days from last corticosteroid exposure. A detailed neuropsychological battery was administered in two 4 hours sessions. Patient, parent and teacher evaluation forms were also completed.

Results:

The mean age at the time of study was 13.2 years (9-16 years), 5 patients were male. Mean time since diagnosis 2.7 years, mean time from first attack was 3.65 years. Relative to normative data, the MS patients performed significantly poorer on some indices from a measure of psychometric intelligence (FSIQ; VIQ, PIQ, VCI) visual motor integration, visual learning and memory, story recall, receptive language, and spelling. Examination of the impact of time since diagnosis (< 2 years, n=4; 2 years, n=6) revealed that participants with a longer time since diagnosis showed even greater deficits on some measures of psychometric intelligence (FSIQ; PIQ; POI), visual perception/construction, visual motor integration, visual memory, and applied mathematical knowledge. These latter findings are consistent with those reported in the adult literature, with a clear deficit in visual-spatial processing in the face of relatively preserved verbal functioning. Overall, however, a wider range of deficits was found in pediatric MS patients than has been documented in studies of MS in adults. Parents and teachers were aware of the child's neurocognitive weaknesses, as well as their socioemotional status and the effect of these variables on scholastic performance.

Conclusions:

Cognitive deficits are common in pediatric MS, are more widespread than in adult MS patients, are more severe in patients with longer duration of disease, and are of sufficient magnitude to influence academic performance.

Supported By:

This study was supported by a pilot project grant from the Canadian Multiple Sclerosis Society.


Safety Profile of Mitoxantrone in a Cohort of 802 Multiple Sclerosis Patients

Tuesday, April 16, 2002
Gilles Edan, Bruno Brochet, David Brassat, Michel Clanet, Pierre Clavelou, Christian Confavreux, Marc Debouverie, Olivier Heinzleff, Christine Lebrun, Catherine Lubetzki, Jean Pelletier, Michel Madigand, Lucien Rumbach, Emmanuelle Leray Rennes, France; Bordeaux, France; Toulouse, France; Clermont-Ferrand, France; Lyon, France; Nancy, France; Paris-Tenon, France; Nice, France; Paris-Salpétrière, France; Marseille, France; St Brieuc, France; Besancon, France

Objective:

To determine the incidence of drug-related adverse events in a cohort of MS patients treated with mitoxantrone.

Background:

MITOX, originally developed as an antineoplastic drug, has been recently approved by the FDA for the treatment of MS. The experience of 12 French MS centers with mitoxantrone from 1992 to 2001 allowed us to evaluate the safety profile of this agent

Design/Methods:

Since 1992, 802 MS patients (302 Relapsing Remitting MS, 351 Secondary Progressive MS, 143 Primary Progressive MS) were treated with MITOX in a consortium of French MS Centers. The median age at MITOX onset for treatment was 39 years; median MS duration before MITOX onset was 8.3 years; median follow-up duration after MITOX onset treatment was 2 years with a follow-up duration more than 2 years for 367 patients. MITOX was administered either monthly for 6 courses in 87% , or every 3 months in 13% of the cohort. The median and the mean cumulative dose of MITOX was 70 mg/m2. 656 consecutive patients completed echocardiograms at 0, 6, 24 or 60 months. Patients underwent clinical and hematological evaluation before every MITOX course and every 6 months after MITOX withdrawal. At baseline, LVEF in all patients was greater than 50%. Treatment was discontinued if LVEF fell below 50%.

Results:

There was no evidence of clinical heart failure in any patient. Twelve patients (1.8%), experienced assymptomatic reduction of LVEF to < 50%. In 3 of these 12 patients, assymptomatic LVEF <50% persisted for follow-up ranging from 4.5-7.0 years. Other adverse events included: 1 case of acute myelogenous leukemia detected 22 months after initiating Mitox, 6 patients with infection associated with ANC < 500, and 10 % of women at risk to lose menses experienced amenorrhea persisting more than 6 months. and up to the last control . There were 11 deaths, none related to therapy. . Most current data for this cohort will be provided at AAN presentation.

Conclusions:

Mitoxantrone was generally well tolerated as administered to this cohort. Continued pharmacovigilence is warranted to ascertain the long term risk of t AL and cardiac toxicity in this population.


Ultra High Field Strength MR Imaging of White Matter at 8 Tesla in Multiple Sclerosis

Tuesday, April 16, 2002
Kottil W. Rammohan, Alayar Kangarlu, Eric Bourekas, Donald Chakeres Columbus, OH

Objective:

To characterize the spectrum of white matter abnormalities that occur in patients with relapsing and progressive multiple sclerosis, and to examine the hypothesis that high-resolution imaging of the white matter in patients with MS at 8 Tesla is superior to conventional imaging at 1.5 Tesla.

Background:

It has been impossible to directly define abnormalities in the normal appearing white matter (NAWM) in patients with progressive MS using conventional imaging at 1.5 Tesla. It is clear that NAWM in patients with progressive MS is not normal. Point-of-interest or whole brain N-acetyl aspartate (NAA), magnetization transfer imaging, magnetization transfer ratios and whole brain apparent diffusion coefficients have all identified abnormalities the NAWM in patients with progressive MS. The potential for high field MR in better defining demyelinating abnormalities remains to be elucidated.

Design/Methods:

Six patients with definite multiple sclerosis (2 relapsing-remitting MS (RRMS),2 primary progressive (PPMS), and 2 secondary progressive (SPMS)) were imaged after informed consent under an IRB. All patients underwent imaging at 1.5 and 8 Tesla using gradient echo and RARE sequences.

Results:

MR images at 8 Tesla were more inhomogeneous compared to the 1.5 Tesla images. The more homogeneous regions on the 8 Tesla images demonstrated better definition, since image matrices of up to 1024 x 1024 were used compared to matrices of 256 X 256 at 1.5 Tesla. This permitted magnifications of lesions 16 times greater than conventional imaging and definition of white matter abnormalities not previously possible. In spite of this greater resolution, preliminary imaging at 8 Tesla did not improve detection of disease burden in patients with PPMS as compared to imaging at 1.5 Tesla. By contrast, imaging of patients with RR and SPMS identified significantly higher disease burden in their white matter at 8 Tesla as compared to 1.5 Tesla. Numerous well defined areas of plaque formation were evident at 8 Tesla in the NAWM in patients with RR and SP MS. Because of the near microscopic (200 micron) definition, a consistent relationship between plaques and vessels was routinely demonstrated, with plaques noted centered around the veins (Dawson's fingers).

Conclusions:

High field MR imaging at 8 Tesla did not improve the detection of lesions in brains of patients with PPMS. On the other hand, patients with RR and SPMS demonstrated improved resolution, permitting better definition of abnormalities in the NAWM. Characterization of such abnormalities will undoubtedly facilitate improved classification of MS patients for evaluation of natural history studies, as well as clinical therapeutic trials. Our preliminary studies at 8 Tesla have established a role for imaging at high field in improving characterization of lesions in patients with MS. Characterization of these white matter abnormalities not previously possible will permit better definition of the NAWM and help to define the progressive nature of the disease in some patients.


MRI Results of the European Interferon Beta-1a Dose-Comparison Study

Wednesday, April 17, 2002
Ernst-Wilhelm Radue, Ludwig Kappos, Nancy Simonian, Mariska Kooijmans, Michel Clanet, Peter Slasor, Richard A. Rudick, the European Interferon Beta-1a Dose-Comparison Study Group Basel, Switzerland; Cambridge, MA; Toulouse, France; Cleveland, OH

Objective:

To report magnetic resonance imaging (MRI) results from the European Interferon Beta-1a (IFNb-1a, AVONEX®) Dose-Comparison Study.

Background:

IFNb-1a 30 mcg IM once weekly slows physical and cognitive disability progression, reduces relapses, reduces conversion to clinically definite MS, and decreases active lesions on MRI. Studies of IFNb at different doses have suggested a ceiling effect. But, until now, studies designed solely to evaluate the efficacy between two doses have not been performed.

Design/Methods:

This was a multinational, randomized, double-blind, parallel-group, dose-comparison study conducted at 38 sites in 10 European countries. Patients with relapsing MS were randomized to receive IFNb-1a 30 mcg (n=402) or 60 mcg (n=400) IM once weekly for 3 years. The first 386 patients enrolled at 28 MRI sites received scans at baseline, months 12, 24, and 36 (annual MRI cohort); 138 of these patients also received scans at Months -4, -3, 4, 5, 6, 10, 11, and 12 (frequent MRI subgroup). MRI endpoints included: number and volume of gadolinium-enhanced (Gd+) lesions for both the annual MRI cohort and the frequent MRI subgroup; and T2-hyperintense lesion accrual, T1 (hypointense) lesion accrual, and brain parenchymal fraction (BPF) for the annual MRI cohort only.

Results:

At baseline, there were no significant differences between groups on any of the MRI parameters. At follow-up, in both doses of IFNb-1a, substantial reductions from baseline in all MRI measures at all time points were found. There were no statistically significant differences nor trends observed between IFNb-1a 30 mcg and 60 mcg with regard to change in T2 lesion volume, change in T1 lesion volume, change in BPF, number and volume of Gd+ lesions, and number of new or enlarging T2 lesions compared with baseline.

Conclusions:

MRI findings corroborate clinical results, which showed no added benefit of IFNb-1a 60 mcg compared with the current commercially available dose of 30 mcg IM once weekly.

Supported By:

Biogen, Inc.


Differences in Brain Atrophy between Early Relapsing Remitting Multiple Sclerosis Patients with Different APOE Genotypes

Wednesday, April 17, 2002
Maria Pia Amato, Maria Letizia Bartolozzi, Benedetta Nacmias, Valentina Zipoli, Marzia Mortilla, Elena Cellini, Silvia Bagnoli, Leonello Guidi, Paolo Lambruschini, Sandro Sorbi, Antonio Federico, Nicola De Stefano Florence, Italy; Empoli, Italy; Siena, Italy

Objective:

To evaluate axonal and tissue damage in relapsing remitting (RR) multiple sclerosis (MS) patients in the early stages of the disease and with different apoliprotein E (APOE) genotypes.

Background:

Recent clinical and imaging studies have raised the still debated hypothesis that MS patients who carry the APOE e4 allele (e3/e4 or e4/e4) may have a more severe disease course with respect to those without the APOE e4 allele (e3/e3). This seems to be related to more extensive tissue destruction and less efficient neuronal maintenance and repair in APOE e4 carriers.

Design/Methods:

We determined the APOE genotype in 27 consecutive RR MS patients with a short disease duration (median = 1 year) and mild disability (median Expanded Disability Status Scale [EDSS] = 1.5. Each MS patient underwent combined proton MR imaging (MRI) and MR spectroscopic imaging (MRSI) examinations in order to obtain measures of cerebral volumes and central brain levels of N-acetylaspartate (NAA, a reliable measure of axonal integrity), respectively. Measures of the MS patient group were compared to those of 18 demographically matched normal controls (NC) using the nonparametric Kruskal-Wallis test of variance. Differences between different patient subgroups and NC were assessed using analysis of variance (ANOVA) followed by pairwise post-hoc comparison using Tukey's HSD procedure to account for multiple comparisons.

Results:

We identified 20 patients with the APOE e3/e3 genotype and 7 patients with the e4 allele. The two patient groups showed no significant differences in age, age at disease onset, EDSS and disease duration (p 0.5) for all measures. Both patient groups showed similar significant (p<0.01) decreases in central brain NAA levels (normalized to creatine [Cr]) with respect to NC (NAA/Cr in e3/e3 MS patients = 2.8 ±0.22, NAA/Cr in e4 MS patients = 2.7 ±0.10, NAA/Cr in NC = 3.1 ±0.20). However, automated measurements of normalized brain volumes (NBV) showed significant decreases (p<0.001) in MS patients carrying the e4 allele with respect to both NC and MS patients without the e4 allele (NBV in e3/e3 MS patients = 1605 ± 39 cc, NBV in e4 MS patients = 1523 ± 038 cc, NBV in NC = 1600 ± 39 cc).

Conclusions:

Preliminary data of this ongoing study could not confirm the hypothesis of more severe disease course in MS patients with APOE e4 genotype. Also, decreases in NAA/Cr, suggesting a relevant axonal damage despite the early disease stage, were similar in the two patient groups. However, significant brain volume loss was found only in early RR MS patients with the APOE e4 genotype. This suggests that the e4 allele can influence more severe tissue destruction in MS patients and that this influence can be seen from early disease stages.


Consensus Guidelines for Standardized MRI Scanning in Multiple Sclerosis

Wednesday, April 17, 2002
P. K. Coyle, CSMC MRI Consensus Committee Stony Brook, NY

Objective:

To develop consensus guidelines for standardized neuroimaging diagnostic protocols, and indications for follow up magnetic resonance imaging (MRI), in multiple sclerosis (MS).

Background:

MRI is usually done as part of the diagnostic workup for MS, and random scans are often obtained to follow patients on treatment. There are no guidelines for such use. Standardized MRI protocols would help maximize the value of individual scans, allow systematic data collection for clinical and comparison studies, and act to guide / standardize MS neurologic practice.

Design/Methods:

An expert consensus meeting on "MRI Protocols for the Diagnosis and Follow Up of MS", sponsored by the Consortium of MS Centers (CMSC) was convened November 3 - 4, 2001, in Vancouver. Participants included MS neurologists and neuroradiologists from the United States, Canada, Europe, and New Zealand, with representation from the American Academy of Neurology, American Society of Neuroradiology, and Radiological Society of North America. A literature review identified important articles for consideration.

Results:

Preliminary results of the consensus meeting propose ten MRI guidelines. The guidelines endorse a standardized MRI protocol to image brain and spinal cord, with specified required and optional features. Images would be obtained on a 1 Tesla or higher machine, using a slice thickness of 3mm ( 1.5mm for 3D sequences), without gaps. Scan orientation would be on the subcallosal line using 3 planes, and using a localizer if available.
Routine follow up MRI is not recommended, although clinical indications for follow up imaging are provided (unexpected worsening, reassessment of disease burden to initiate therapy, suspicion of a second diagnosis). The guidelines provide a prototype radiology report using standardized and consistent common language, describing such features as lesion number, location, size, shape, and character. They recommend that a copy of the MRI be retained permanently.

Conclusions:

These preliminary consensus guidelines are available for review on the CMSC website (MSCARE.ORG). They need to be disseminated widely for discussion and comment, in order to establish standardized practice patterns in MS worldwide.

Supported By:

Sponsored by the Consortium of MS Centers


Functional MRI and Neural Activation Patterns Associated with Recognition Memory Performance in Multiple Sclerosis

Wednesday, April 17, 2002
Lorri J. Lobeck, Julie A. Bobholz, Sally Durgerian, Judy Zaferos, David Miller, Julia Rao, Catherine L. Elsinger, Eric F. Maas, Steve M. Rao Milwaukee, WI

Objective:

This study was done to assess changes in patterns of activation on functional magnetic resonance imaging and their relationship to encoding or retrieval deficits in patients with mutliple sclerosis and abnormal memory function.

Background:

Forty-five to sixty percent of patients with multiple sclerosis will experience cognitive deficits during the course of their illness. The extent of disease burdun or white matter lesions present on conventional T2 weighted MRI scans correlates with the severity of cognitive deficits. Memory functions, including new learning are frequently compromised in cognitively impaired patients. Whether this is due to encoding or retrieval difficulties is not known.

Design/Methods:

15 patients with multiple sclerosis and 8 demographically matched control subjects underwent fMRI and FLAIR imaging. To test encoding memory, subjects made a sematic decision (abstract versus concrete) regarding 60 nouns, presented every 4.5 seconds. Thirty minutes later subjects were tested for recognition memory by testing 60 targets and 60 foils.

Results:

Recognition accuracy was similar for the two groups. However, when investigating the relationship between lesion load and the patterns of neural activation, activation in the bilateral dorsolateral prefrontal cortex and lateral cerebellum was more common in patients with higher lesion load. Lower lesion load was associated with increased activation in the left hipppocampal gyrus.

Conclusions:

Even when recognition memory is normal, the neural systems involved in encoding appear to shift from long-term (left hippocampus) to working memory (bilateral dorsolateral prefrontal cortex and lateral cerebellum) strategies as lesion burden increases in multiple sclerosis.

Supported By:

Biogen
Fourteen Cambridge Center
Cambridge, Massachusetts 02142


T2 Relaxation Measurements of In-Vivo Water Content and Myelin Water Content in Normal Appearing White Matter and Lesions in Multiple Sclerosis

Wednesday, April 17, 2002
Cornelia Laule, Irene M. Vavasour, Joel Oger, Donald W. Paty, David K. B. Li, Alex L. MacKay Vancouver, BC, Canada

Objective:

To measure in-vivo water content and myelin water content of white matter, grey matter and lesions in patients with multiple sclerosis (MS) and controls

Background:

Measurements of the T2 decay curve can provide estimates of total and myelin water content in vivo, which may help in understanding the pathological progression of MS. This study reports total water content and myelin water content in normal white matter (NWM) of controls, normal appearing white and gray matter (NAWM, NAGM) and lesions of MS patients.

Design/Methods:

Thirty-three MS patients (24 RR, 8 SP, 1 PP, mean disease duration = 9yrs) and 15 controls underwent MR examinations. T2 relaxation data were acquired using a 32 echo CPMG experiment. All controls and 18 of the 33 MS patients were scanned in the transverse plane through the genu and splenium of the corpus callosum. Five white matter structures (genu and splenium of the corpus callosum, internal capsule, minor forceps, major forceps) and 6 grey matter structures (cingulate gyrus, insular cortex, cortical grey, thalamus, putamen, caudate) were outlined in these subjects. The remaining 15 MS patients were scanned in various other transverse planes. A total of 185 lesions were outlined in the MS patients. Total water content was estimated from the ratio of signal in the chosen region of interest to that in an internal grey matter standard (putamen or cortical grey). Myelin water content was defined as the fraction of signal with T2 below 50 ms divided by the total signal in the T2 distribution.

Results:

The NAWM total water content was, on average, 1.8% greater than that of NWM, with significant differences occurring in the posterior internal capsules, genu and splenium of the corpus callosum, minor forceps and major forceps (p<0.002). On average, MS lesions had 7.7% higher water content than NAWM (p<0.0001). The average myelin water content was 15% lower in NAWM than NWM, with the largest differences being in the major forceps (p=0.005), minor forceps (p=0.03), genu (p=0.05) and splenium (p=0.02). The myelin water content of MS lesions averaged 50% that of NAWM.

Conclusions:

Normal appearing white matter in MS has a higher water content and lower myelin water content than controls. The cause of the total water content increase in NAWM could be due to either diffuse edema or inflammation. As our understanding of T2 improves, we may be able to distinguish these two pathologies from the shape of the T2 distribution. A simple model of white matter suggests that a 1.8% increase in total water content by edema is not sufficient to account for the observed 15% decrease in myelin water. Therefore, we believe that the white matter of NAWM has diffuse demyelination.

Supported By:

MS Society of Canada


Application of the New McDonald's Diagnostic Criteria in a Cohort of Patients with Clinically Isolated Syndromes

Wednesday, April 17, 2002
Mar Tintore, Alex Rovira, Jordi Rio, Carlos Nos, Elisenda Grivé, Jaume Sastre-Garriga, Imma Pericot, Esther Sánchez, Manuel Comabella, Xavier Montalban Barcelona, Spain

Objective:

To apply the new McDonald's diagnostic criteria in a cohort of patients with clinically isolated syndromes (CIS).

Background:

An international Panel on MS Diagnosis has recently presented revised diagnostic criteria for MS.

Design/Methods:

139 patients with CIS were longitudinally studied (58 optic neuritis, 34 brainstem syndromes, 39 spinal cord syndromes and 8 polyregional syndromes). Brain MRI was performed after the first demyelinating event and repeated after 12 months of follow-up. Criteria proposed by Barkhof et al were applied in the basal MRI. The number of new T2 weighted lesions was also studied in the first year MRI. Poser's and McDonald's diagnostic criteria were applied after a one year follow-up cut point.

Results:

During the first 12 months of follow-up, 15 patients (11%) had developed CDMS. Barkhof's criteria (3 out of 4) showed 71% sensitivity, 69% specificity and 70% accuracy in predicting conversion to CDMS, whereas on adding the alternative condition of at least 2 lesions plus oligoclonal bands, we obtained 87% sensitivity, 56% specificity and 65% accuracy. Seventy-seven patients (55%) fulfilled criteria for dissemination in space according to McDonald's criteria. Sixty-two patients (45%) fulfilled MR criteria for dissemination in time. Fifty-one patients (37%), 10 of whom had previously had one second clinical attack, fulfilled both radiological conditions of dissemination in time and space. Consequently annual MRI anticipated MS diagnosis in 41 patients (29%). At one year follow-up, 40% of the patients (11% because of a second event and 29% according to MR criteria for dissemination in time and space) would be diagnosed as MS according to McDonald's criteria compared 11% with Poser's criteria.

Conclusions:

The alternative condition for dissemination in space (at least 2 lesions plus OB) should be used with caution as the high specificity of Barkhof's criteria may be clearly compromised. McDonald's criteria anticipate the diagnosis of MS in 29% of the patients after one year of follow-up.


Depression in MS: The Relationship to Interferon Therapy

Wednesday, April 17, 2002
Luanne M. Metz, Gordon Francis, Yves Grumser Calgary, AB, Canada; Norwell, MA; Geneva, Switzerland

Objective:

To determine depression and suicide rates in a pooled series of cohorts of MS patients treated with interferon (IFN) beta-1a.

Background:

Depression is common in MS and, with suicide, was linked to IFN therapy based on data in the pivotal IFN beta-1b (Betaseron©) study. Subsequent studies have not confirmed a similar relationship even when objective measures (depression rating scales) were used to evaluate the relationship. Data is presented from a large pooled data set of all MS studies of IFN conducted by Serono.

Design/Methods:

Any event labeled depression by the physician was included. Safety data for 4469 patients (total trial patients [TP]), treated for 6 months to 6 years, was available for analysis. Both randomized controlled trials (RCTP) and uncontrolled studies were included. Only data on serious adverse events (SAE), AE dropouts, and deaths were available from the latter. Both RRMS (n = 3450) and SPMS (n = 1019) patients were included. Patients received either placebo or active drug (3995 Rebif©, 337 Avonex© and 824 placebo patients). Doses ranged from 22mcg weekly (qw) to 44mcg thrice weekly (tiw).

Results:

In RCTP depression was reported within 6 months by 7.6% of placebo patients (n = 824) vs. 4.6% of 22mcg qw (n = 435), 13.4% of 30mcg qw (Avonex) (n = 337), 8.2% of 44mcg qw (n = 98), 9.0% of 22mcg tiw (n = 398), and 11.7% of 44mcg tiw patients (n =727). By 24 months, 26.5% of placebo (n = 392), 24.1% of 22mcg tiw (n=398), and 27.6% of 44 mcg tiw (n = 388) patients had a depressive event (p = 0.75, Fisher's exact test, 44mcg vs. placebo). The proportion of dropouts in RCTP (n =1995) due to depression was 1.35% for IFN and 0.12% for placebo (p = 0.001, Fisher's exact test). For TP the rate was 0.88/100 pt-yr for IFN patients and 0.06/100 pt-yr for placebo patients (p=0.007). Depression was cited as the reason for 32% (27/84) of all IFN RCTP AE dropouts, and 8% (1/12) of placebo AE dropouts (p=0.17), and was twice as common in SPMS as RRMS. Over 2-years suicide attempts were equal in RCTP treatment groups. The rate of suicide in TP was 0.12/100 pt-yr for placebo and 0.08/100 pt-yr for IFN (p=0.62, Poisson regression). There were two suicides in RRMS (one each, placebo and 22 mcg tiw) and six in SPMS (one placebo, one 22mcg qw, two 22mcg tiw and two 44mcg tiw).

Conclusions:

At 6 months, depression varied by IFN dose group. After 2 years of treatment, depression was more frequent overall, but was not significantly different between IFN and placebo. Dropouts due to depression were significantly more common in IFN beta-1a (Rebif©) than placebo treated groups, and were also more common in SPMS than RRMS. The suicide rate was lower on active therapy than placebo. No association between the risk of depression or suicide and interferon therapy was seen but depression is still one of the more common causes to stop therapy. Early identification and treatment of depression may increase adherence to therapy.

Supported By:

Serono.


The Predictive Value of Gadolinum Enhancement for Clinical Status over a 5-10 Year Period

Wednesday, April 17, 2002
Lael A. Stone, Elizabeth Fisher, Gary R. Cutter, Roger Stone, Jennifer McCartin, Joan Ohayon, Nancy D. Richert, Joseph A. Frank, Henry F. McFarland Cleveland, OH; Denver, CO; Bethesda, MD

Objective:

To investigate the predictive value of gadolinium enhancement (GdE) for clinical status in multiple sclerosis (MS) over a 5-10 year period.

Background:

In patients with a clinically isolated demyelinating syndrome, a relationship between amount of T2 weighted MRI abnormality at onset and clinical status over a 5-10 year period has been shown. Although GdE is helpful for diagnostic MRI in MS, little is known about the prognostic value of GdE. This study reports the clinical findings of a cohort of MS patients assessed with 3 consecutive monthly Gd-enhanced MRI scans at baseline and followed up some 6-12 years later.

Design/Methods:

113 MS patients evaluated at the Neuroimmunology Branch (NIB)of the NIH between 1989 and 1994 with three clinical visits and three consecutive monthly Gd-enhanced MRI scans were identified for inclusion in this study: Out of 90 patients contacted, 79 have been evaluated by follow-up clinical examinations and MRI scans. The mean interval between baseline and follow-up was 8.3 years (sd=1.6 range 6-12 years).

Results:

Mean EDSS at entry in this initial group of 79 patients reexamined to date was 3.1 (sd=2.0 range 0.3 to 8.4) and at follow-up was 4.3 (sd= 2.4 range 1 to 9). The majority of the initial group were relapsing remitting MS by clinical course (RRMS-65.3%). Preliminary analyses suggest a relationship between higher levels of GdE at baseline and poorer patient status at follow-up as measured by various measures including the MSFC and its components, in some but not all clinical sub-types of MS. Duration of disease may be an additive factor to GdE as well for prognosis.

Conclusions:

GdE may require additional information on clincial subtype and duration of disease to be useful for prognosis in MS.

Supported By:

National Multiple Sclerosis Society, National Institute of Neurological Disorders and Stroke, and Laboratory for Diagnostic Radiology Research, NIH


Ganglioside Reactive Antibodies in Multifocal Inflammatory Sensory and Motor Neuropathy (MISAM)

Wednesday, April 17, 2002
Armin Alaedini, Howard W. Sander, Norman Latov New York, NY

Objective:

To determine whether anti-ganglioside antibodies are present in patients with acquired multifocal sensory and motor neuropathy.

Background:

Autoimmune mechanisms are suspected in some of the acquired multifocal neuropathies, including multifocal motor neuropathy (MMN), and multifocal acquired demyelinating sensory and motor neuropathy (MADSAM). IgM antibodies to gangliosides have been reported in MMN, but not in multifocal sensory and motor neuropathy.

Design/Methods:

Sera from 218 patients with a variety of chronic neuropathies, and from 76 control subjects (20 with multiple sclerosis, 10 with amyotrophic lateral sclerosis, and 46 healthy individuals) were tested for anti-ganglioside antibodies by a newly developed ganglioside agglutination assay, using polystyrene microparticles coated with a total ganglioside extract from bovine brain. Sera found to be positive by the agglutination assay were also tested by ELISA for IgM and IgG antibodies to GM1, GM2, GD1a, GD1b, GQ1b, and GT1b gangliosides.

Results:

Ten of 20 sera (50%) from patients with multifocal sensory and motor neuropathy were found to be positive for anti-ganglioside antibodies. Electrodiagnostic studies revealed neuropathic dysfunction, affecting legs more than the arms. Focal or multifocal abnormalities were present in 9/10 patients. In 2/10 there were features of demyelination. Sural nerve biopsies in 2 revealed changes consistent with an axonal neuropathy. By ELISA, 5 of the patients had mildly elevated titers of IgG antibodies to one or more gangliosides including GM1, GM2, GD1b, GD1a, and GT1b. None of the control sera were reactive by the agglutination assay or ELISA. Five of 6 patients treated with intravenous gammaglobulins (IVIg), etanercept, or plasmapheresis plus etanercept, showed clinical improvement.

Conclusions:

The presence of anti-ganglioside antibodies in acquired multifocal sensory and motor neuropathy indicates a possible immune mechanism consistent with the observed clinical response to immunomodulating agents. The term multifocal inflammatory sensory and motor neuropathy (MISAM) may be appropriate, given that both axonal and demyelinating features are present. The presence of IgG antibodies suggests a T-cell mediated response. In some of the patients, antibody activity was demonstrated by the agglutination assay, but not by ELISA, possibly due to higher sensitivity of the assay, or because the antibodies are directed at minor or as yet unidentified ganglioside antigens.


Age-Related Disability in Multiple Sclerosis

Wednesday, April 17, 2002
Maria M. Trojano, Maria M. Liguori, Giovanni Bosco G. B. Zimatore, Roberto R. Bugarini, Carlo C. Avolio, Damiano D. Paolicelli, Maria Giovanna M. G. Marrosu, Paolo P. Livrea Bari, Italy; Roma, Italy; Cagliari, Italy

Objective:

The relationship between age and rate of disability progression in a large hospital-based cohort of definite multiple sclerosis (MS) patients was evaluated.

Background:

The clinical course of MS shows evidence of pathological processes and neurological symptoms that are age related.Recent experimental and clinical studies also have demonstrated that pathological and clinical manifestations of autoimmune demyelination are age-dependent.

Design/Methods:

The study population included 1,463 definite MS patients from Apulia (n = 1,039) and Sardinia (n = 424) regions of Italy who were followed for a total period of observation corresponding to 11387.8 person years. At enrollment,clinical and demographic details (age, age at onset, disease duration and EDSS score) were established.Each patient was followed every 6 to 12 months. EDSS was assessed at each visit. Mean EDSS scores for MS patients were calculated according to current age and disease duration within each current age category.The Kruskal-Wallis one-way analysis of variance was used to analyze differences in EDSS scores based on age and disease duration. Association between current age and time to irreversible EDSS scores of 4.0 or 6.0, was assessed using an extended Kaplan-Meier method, with age as a categorical time-varying covariate. For Kaplan-Meier analyses, patients were distributed into three groups based on current age (20 to 35 years, 36 to 50 years, and 51 to 65 years.Polytomus logistic regression analyses were performed to determine the existence of multiplicative effects of covariates.

Results:

EDSS scores significantly increased with current age and disease duration (p = 0.007). A total of 435 patients reached an EDSS score of 4.0 and 180 patients reached an EDSS score of 6.0 by 15 years. Median times to reach EDSS scores of 4.0 and 6.0 were significantly longer among patients 20 to 35 years of age compared with patients 36 to 50 and 51 to 65 years of age (p < 0.0001). Polytomus logistic regression showed significant associations between mean EDSS scores and age at disease onset, current age, and their interaction (p < 0.001). Current age had a larger effect (59% of variability in the model) on disease severity than did age at disease onset. A positive correlation was observed between current age and EDSS score, independent of age at disease onset (bcurrent age = 0.1325), whereas a negative correlation was observed between age at disease onset and EDSS score, independent of current age bage at onset = -0.174). These two estimated effects indicated a positive correlation between disease duration and EDSS score. Furthermore, a multiplicative effect on EDSS score was observed for age at disease onset and current age combined (bage at onset x current age = 0.0013), indicating a significantly faster rate disease progression in older patients.

Conclusions:

Results of the current study demonstrate the impact of age on rate of disability progression in MS, and suggest that an age-adjusted progression index may be a more relevant criterion for defining differences between MS groups.


Induction Treatment with the Monthly Combination Mitoxantrone-Methylprednisolone for 6 Months Followed by a Maintenance Therapy in Worsening Relapsing-Remitting MS: The Clinical Benefits Last at Least 4 Years

Wednesday, April 17, 2002
Emmanuelle Le Page, Marc Coustans, Gregory Taurin, Jacques Chaperon, Gilles Edan Rennes, France

Objective:

To assess the concept of induction therapy by mitoxantrone before initiating other long term disease modifying therapies for worsening relapsing-remitting MS patients

Background:

Mitoxantrone (MITO), administered every 3 months up to the maximum cumulative dose of 140 mg/m2 has recently been approved by the Food and Drug Administration for the treatment of MS.MITO 20 mg is most frequently administered as monthly infusionsx6 months ("induction therapy") to treat MS in France

Design/Methods:

Since 1992, 100 RRMS patients followed in our MS Clinic were treated monthly for 6 months with mitoxantrone IV 20 mg combined with methylprednisolone IV 1g. After mitoxantrone induction , 25 patients received mitoxantrone every 3 months, 53 patients received an other long term modifying therapy (Azathiaprine 19, Interferon beta 21, Methotrexate 9, Glatiramer acetate 4 ) or had no maintenance therapy up to the next relapse ( 22 patients). The median duration of MS before starting mitoxantrone was 3.7 years. Within the 12 months before treatment , the annual relapse rate was 2.8, the mean worsening of EDSS was 2.2. and gado-enhanced lesions were detected in 77 out of 95 patients. The median duration after starting mitoxantrone was 3.8 years (1-8 years). Clinical , MRI and safety data were yearly collected in EDMUS.

Results:

After the induction treatment , the activity of the disease dropped dramatically: The mean number of relapses was 0.3; 0.4 and 0.4 at year 1, 2 and 3 ( 88,3 and 84,5% reduction compared with the annual relapse rate before treatment. p<0,0001). The percentage of relapse free patients were respectively 78%, 63%, 48% and 42.5% at year 1, 2, 3 and 4 . Compared with EDSS at Mitox onset , EDSS was improved of 1 point or more in 58%, 50%, 50% and 40,5% at year 1, 2, 3 and 4 (p<0001). Only 7 out of 80 patients had a Gd+ lesions on the MRI performed after the induction treatment (90% reduction compared with MRI before treatment. P< 0.0001). 15 patients moved to a secondary progressive MS.

Conclusions:

The clinical benefit and the reduction of MRI activity provides support for the notion that MITO as administered in this study may be used effectively as an induction treatment before initiating other long term disease modifying therapies for worsening relapsing-remitting MS patients


Detection of White Matter Pathology in Multiple Sclerosis Using q-Space Analyzed Diffusion Weighted MR Imaging and Spectroscopic Imaging

Wednesday, April 17, 2002
Yaniv Assaf, Joab Chapman, Dafna Ben-Bashat, Yoram Segev, Talma Hendler, Moshe Graif, Amos D. Korczyn, Yoram Cohen Tel Aviv, Israel

Objective:

The objective of this study were to explore the diagnostic capacity of the q-space method in MS and to correlate it to spectroscopic imaging.

Background:

Magnetic resonance imaging (MRI) is the major imaging method for diagnosis and evaluation multiple sclerosis (MS). However, a discrepancy exists between the disease load detected by MRI and the clinical severity of the disease. Indeed, magnetic resonance spectroscopy studies of normal appearing white matter (NAWM) of MS patients have shown that it is abnormal. Previously, we showed that diffusion-weighted imaging (DWI) at high b value provides additional information that seems to be more specific to axonal water and hence increase the ability to detect pathologies in the white matter. One way to analyze the signal decay at high b values is by using q-space analysis. The q-space analysis gives the displacement distribution profile of water molecules in the tissue from which the displacement and probability for zero displacement can be extracted.

Design/Methods:

Here we present q-space MR images (acquired with b values of up to 14,000 s/mm2) on MS (n=6) and control (n=6) human brains. These images were acquired on 1.5T GE Signa MRI scanner using diffusion-EPI pulse-sequence with the following parameters: TR/TE/D/d=1500/167/71/65ms, slice thickness of 4.5mm and Gmax of 2.2 gauss/cm. The q-space images were correlated with NAA/Cr levels derived from 2D spectroscopic imaging (SI) done with the following parameters: TR/TE=1600/135ms,matrix size 16x16, voxel dimensions: 12x11x11mm.

Results:

The q-space images provided a pronounced differentiation between NAWM in controls and MS patients as compared to other MRI techniques (T1, T2 and diffusion tensor images). Mean displacements of 3.4±0.6, 4.9±0.8 and 8.1±2.3 mm were extracted from regions of interests (ROIs) in control patients, NAWM and MS lesions in MS patients, respectively. For the same ROIs, probability for zero displacement of 8.2±0.5, 6.5±0.5 and 5.0±0.8 were extracted. The q-space parameters show overall good correlation with the EDSS of the whole group (r=0.66).
The q-space indeces corrlated well with NAA/Cr values as derived from SI (r=0.61). Using the SI data we performed ROI analysis on two groups of NAWM - one that has normal metabolite distribution (NAA/Cr level higher than 1.8) and one that has abnormal metabolite distribution (NAA/Cr<1.80). The q-space intensities of these ROIs were significantly different (p<0.001) indicating that areas that appear abnormal in the SI are also abnormal in the q-space images.

Conclusions:

q-Space imaging provides a contrast that is based on the probability for displacement of water molecules. These images were shown to be highly sensitive to the integrity of the myelin in white matter. Indeed intensity of the q-space images correlated with NAA/Cr value suggesting that these king of images can provide that same infromation as SI but with much better spatial resolution.


Hospitalization for Neurological Disorders in the United States

Wednesday, April 17, 2002
John K. Lynch, Ann Scher, Karin B. Nelson Bethesda, MD

Objective:

To provide national estimates on the hospitalization of the most common neurological disorders in the United States.

Background:

Neurological disorders are a major cause of morbidity and mortality in the United States. The National Hospital Discharge Survey (NHDS) is a periodic nationwide sample survey of approximately 500 short-stay hospitals in the United States. The NHDS provides information on selected patient hospitalizations including data on patient demographics, diagnostic codes, procedures, length of stay and discharge status. The number of inpatient records sampled between 1990-1999 ranged from 235,000 to 300,000 each year.

Design/Methods:

NHDS data were analyzed for the period 1990-1999 for individuals 17 years of age with a neurological disorder(according to ICD-9 code). The rate of each neurological disorder was calculated as the estimated number of hospital discharges for each disorder among the mid-year resident population in the United States each year and was calculated according to gender and race.

Results:

For the period 1990-99, the most common neurological disorders resulting in hospitalization were (in decreasing order): cerebrovascular diseases, head injury, Alzheimer's disease, epilepsy, migraine, primary brain tumors, multiple sclerosis/demyelinating disorders, Parkinson's disease, CNS infections, polyneuropathy, myoneural disorders, and motor neuron disorders. Over this period, the rates of hospitalization (per 100,000 population) decreased for head injury, epilepsy, migraine, primary brain tumors, and multiple sclerosis and increased for all other neurological disorders. During this period, there was an average 50% decrease in the length of stay for all neurological disorders except head injuries and CNS infections. The male: female rate ratio was greatest for head injury (1.9:1) and primary brain tumors (1.4:1) and least for migraine (1:3), multiple sclerosis (1:2.3), and Alzheimer's disease (1:1.7). The black: white rate ratio was highest for epilepsy (2.1:1), myoneural disorders (1.8:1), CNS infections (1.7), and head injury (1.6:1) and least for primary brain tumors (1:2.3) and Parkinson's disease (1:1.6). The average age at hospitalization for all neurological disorders was lower for blacks compared to whites.

Conclusions:

During the 1990's, the overall rate of hospitalization for the most common neurological disorders was 682.2 per 100,000 persons per year. There was generally an increase in the hospitalization rate for disorders affecting individuals over 50 years of age and a decrease in disorders affecting younger individuals. Lengths of stay for neurological disorders also decreased over the same time period. Blacks were hospitalized at a younger age than whites for most neurological disorders.


Is Neuromyelitis Optica a Separate Disease ? A Comparative Study with Multiple Sclerosis Presenting as Optic Neuritis and Myelitis

Wednesday, April 17, 2002
Jerome de Seze, Christine Lebrun, Tanya Stojkovic, Didier Ferriby, Marc Chatel, Patrick Vermersch Lille, France; Nice, France

Objective:

To compare neuromyelitis optica (NMO) patients with a series of MS patients following the onset of optic neuritis or myelitis, in order to determine the place of NMO in the spectrum of MS.

Background:

NMO associates optic neuritis and myelitis without other neurological signs. Many patients with NMO may be diagnosed as having multiple sclerosis (MS), optic neuritis and myelitis being the inaugural symptom in 20% and 5% of MS cases, respectively. However, there have been no previous studies comparing these two pathologies and it is still unclear if NMO is a separate entity or a syndrome including MS, systemic or infectious diseases.

Design/Methods:

We retrospectively studied 30 patients diagnosed with NMO according to the criteria of Wingerchuk et al. We compared these patients with 50 consecutive MS cases revealed by optic neuritis (n=26) or acute myelitis (n=24). Patients were diagnosed as MS only if a second relapse occurred demonstrating time and space dissemination. We compared the groups in terms of clinical presentation, initial laboratory findings (MRI, visual evoked potentials and CSF) and clinical outcome (evaluated by the expanded disability status scale (EDSS) and the index of progression (IP) =EDSS/duration of the disease).

Results:

NMO patients were older and more frequently female than MS patients but the difference was not significant. CSF and MRI data were different: oligoclonal bands were found in 23% of NMO cases and 88% of MS (p<0.001), abnormal brain MRI data following the criteria of Barkhof et al. were observed in 10% of NMO cases and 66% of MS cases (p<0.001). Clinical outcome was evaluated as more severe in the NMO group (p<0.001 for both EDSS and IP). Only two of the NMO patients met the criteria of MacDonald et al. for MS and one of the MS patients met the criteria for NMO.

Conclusions:

Our study demonstrates that NMO and MS should be considered as two different entities. By strictly applying the criteria for both NMO and MS we were able to distinguish between these two pathologies. This finding could have implications for future therapeutic trials.


Outcomes of Intrathecal Baclofen Therapy in Multiple Sclerosis

Wednesday, April 17, 2002
Francois A. Bethoux, Steven J. Shook, Danuta M. Gogol, Kathleen M. Schwetz, Revere P. Kinkel Cleveland, OH

Objective:

To study the outcome of intrathecal baclofen therapy (ITB) in a population of multiple sclerosis (MS) patients followed in a large MS comprehensive care center.

Background:

Intrathecal baclofen therapy is increasingly used to treat severe spasticity of cerebral or spinal origin in various neurologic diseases, including MS. Since MS patients' symptoms and level of disability are likely to change over time due to progressive central nervous system damage, it is important to evaluate the long-term outcome of ITB in this population. We have not found any report of long-term follow-up of large cohorts of MS patients treated with ITB in the literature.

Design/Methods:

We reviewed medical records of all patients who had an intrathecal baclofen pump implanted from 1990 to present, and were followed in our center. Patients with a diagnosis of MS, and with clinical data available for at least one time point were included in the analysis. Patients treated with a mix of baclofen and pain medications intrathecally were excluded. Clinical outcomes routinely collected at each visit included: ITB rate and dose schedule, lower extremity Ashworth scores, spasms, pain rating, concomitant oral medications, skin status, bowel and bladder management.

Results:

Eighty one patients, who underwent surgery between 10/1990 and 09/2001, were included in the analysis. At the time of surgery, mean age was 49.9 +/- 7.4 years, and mean delay since onset of symptoms was 16.9 +/- 8.4 years. The disease was progressive in all but one patient, and 78% were nonambulatory. Mean follow-up time was 3.7 +/- 2.7 years (range 0.1 - 11.0). Eight patients (10%) were deceased. Mean most current baclofen rate was 266.8 +/- 239.8 mcg/day, and mean lower extremity Ashworth score (averaged on 8 muscle groups) was 0.6 +/- 0.8. Average baclofen rates increased with time, and were significantly correlated with time elapsed since surgery (r=0.31; p=0.004), but not with age or delay since onset of symptoms. Average Ashworth scores remained stable over time, and did not correlate with age, delay since surgery, or delay since onset of symptoms. Comparisons between ambulatory (n=18) and nonambulatory (n=63) patients showed that ambulatory patients had significantly lower baclofen rates and higher Ashworth scores at 1 year. We could not compare long-term outcomes between these two subgroups because most ambulatory patients had 1 year or less of follow-up at the time of the study.

Conclusions:

Our results suggest that intrathecal baclofen therapy remains effective over time in MS patients with severe spasticity, although the dose of baclofen needed to achieve a good control of lower extremity stiffness and spasms may increase. Standardized measures of gait performance, which can be used in routine clinical practice, are needed to fully evaluate the outcome of ambulatory patients treated with ITB, and further long-term outcomes studies are needed in this subgroup of patients.

Supported By:

Not applicable.


Improved MR Sensitivity in Detecting Regional Multiple Sclerosis Brain Injury Measured by Proton Magnetic Resonance Spectroscopy, Diffusion Tensor Imaging and Magnetization Transfer Imaging

Wednesday, April 17, 2002
Joonmi Oh, Daniel Pelletier, Roland G. Henry, Sharmilar Majumdar, Sarah J. Nelson San Francisco, CA; San Francisco, CA

Objective:

To show the potential value of in vivo Proton Magnetic Resonance Spectroscopic Imaging (1H MRSI), Diffusion Tensor Imaging (DTI) and Magnetization Transfer Imaging (MTI) in detecting preferential brain tissue injury in Normal Appearing Corpus Callosum (NACC) compared to central brain in different forms of Multiple Sclerosis (MS).

Background:

Although clinical and MRI differences between Primary Progressive (PP), Relapsing Remitting (RR) and Secondary Progressive (SP) MS are important in monitoring disease activity and treating patients, the etiology underlying differences between those groups remains largely unknown. In MS, 1H MRSI is useful to quantify axonal injury through neuronal marker; N-acetyl aspartate (NAA). Pathologic tissue injury could increase Apparent Diffusion Coefficient (ADC) and decrease MT Ratio (MTR). The CC connects homologous regions of two hemisphere that may be sensitive for detecting fiber track changes.

Design/Methods:

47 MS patients (mean age=46.7 years) with RR/SPMS (n=33) and PPMS (n=14), were studied along 16 controls (mean age=42.3 years). The EDSS of both groups were matched. 2D 1H MRSI was acquired covering a slab of approximately 160 cc centered at the middle of the CC (central brain). Flair DTI and MTI parameters were derived from the same central brain region. A Normal Appearing CC (NACC) Region of Interest (ROI) was manually segmented out. Statistical analyses were performed using multiple comparison Least Square Means (LSM) Tukey HSD tests with age adjustment. The results are reported as LSM±Standard Error (SE) unless otherwise noted.

Results:

A significant reduction of the NAA:Cr ratio in the central brain for the RR/SPMS (1.90±0.02, p=0.008) and PPMS (1.84±0.04, p=0.001) were observed relative to controls (2.01±0.03). A similar pattern was also observed in the NACC for the RR/SPMS (1.96±0.07, p=0.004) relative to controls (2.31±0.09), but not for the PPMS (2.12±0.09, p=0.149). A significant increase of the median ADC in the central brain for the RR/SPMS (788.19±3.61mm^2/s, p=0.0001) and PPMS (780.42±5.21 mm^2/s, p=0.023) were observed relative to controls (763.18±4.85 mm^2/s). A similar pattern was also observed in the NACC for the RR/SPMS (849.81±7.00 mm^2/s, p=0.0002) relative to controls (802.85±9.41 mm^2/s), but not for the PPMS (830.92±10.11 mm^2/s, p=0.055). A significant reduction of the median MTR in the NACC for the RR/SPMS (33.15±0.03%, p = 0.0004) relative to controls (35.38±0.044%), but not in the central brain (34.71±0.02%, p = 0.259) relative to control (35.00±0.02%). The median MTR for the PPMS were similar to control for both NACC (34.63±0.05%, p=0.193) and central brain (35.02±0.02%, p=0.981).

Conclusions:

Based on changes measured in this study between NACC and central brain, we propose that studying the CC in RR/SPMS patients would be more sensitive to detect tissue injury than central brain. Central brain region, however, is more sensitive for the PPMS patients suggesting disease heterogeneity with RR/SPMS.

Supported By:

National MS Society grant RG2655B6/1 and National Institute of Health grant R01 NS39529. Dr. D. Pelletier is a National MS Society Physician Fellowship Awardee.


Clinical Characteristics of Non Responders to Interferon Beta Therapy for Relapsing Remitting Multiple Sclerosis

Wednesday, April 17, 2002
Emmanuelle Waubant, Sandra Vukusic, Christian Confavreux San Francisco, CA; Lyon, France

Objective:

To 1) describe clinical characteristics of patients with relapsing-remitting multiple sclerosis (RRMS) on interferon beta (IFNB) therapy in the Lyon EDMUS database, 2) derive a definition for non-responders to IFNB therapy from this patient population, 3)determine whether there are clinical differences between reponders and non-reponders to IFNB.

Background:

MS is an inflammatory disease for which IFNB has been approved for 7 years. However, some patients may not experience the expected benefit from such therapy. There is currently no consensus to define poor response to IFNB, and consequently it is unknown what percentage of MS patients do not respond to IFNB therapy, nor whether these patients are different from patients doing well on IFNB.

Design/Methods:

The prospectively updated EDMUS database in Lyon was used to obtain information on patients treated with IFNB for MS. Data on all patients who received one of the 3 IFNB approved in Europe for any length of time was extracted from the database at the end of March 2001.

Results:

388 MS patients ever treated by IFNB were identified. Of these, a total of 202 patients had RRMS and had received IFNB for at least 6 consecutive months. 62 of the 202 patients (30%) experienced higher or identical annual relapse rate on IFNB therapy compared to the 2 years and/or the year prior to IFNB therapy. This definition was subsequently adopted to define non-responders to IFNB therapy for further analysis. Non-responders and responders were similar for mean age when MS started (respectively, 27.3 and 27.9 years, p=0.79), disability levels at the time IFNB was initiated (respectively, mean DSS 1.8 and 1.5, p=0.8)), sex ratio (respectively, 20% and 25% of males, p=0.53) and mean time on IFNB (respectively, 27.9 and 28.1 months, p=0.16). At the time IFNB was initiated, responders were older and had a longer mean disease duration than non-responders (respectively, mean age 36 versus 32.7 years, p =0.01; 8.1 versus 5.4 years, p= 0.003). Responders had also a higher mean relapse rate during the year before IFNB therapy was initiated(1.89 versus 1.32, p=0.02). Less often responders converted to secondary progressive MS on IFNB therapy (7.8% versus 16.1%, p=0.07), progressed during IFNB therapy (12.1% versus 27.4%, p=0.007) or discontinued IFNB therapy (24.2% versus 56.4%, p<0.0001) compared to non-responders. Mean annual relapse rate on IFNB was 0.42 for responders and 1.93 for non-responders, representing respectively a 77% decrease and a 46% increase in annual relapse rate compared to the year before IFNB was initiated.

Conclusions:

Clinical profiles of patients with RRMS who respond or not to IFNB therapy may differ. The MRI profile and the biological mechanisms underlying this poor response to IFNB are unknown


Serum Levels of MMP-9 and TIMP-1 Predict MRI Activity in Patients with Secondary Progressive MS and Interferon Therapy Shifts MMP-9/TIMP-1 towards Normal

Wednesday, April 17, 2002
Emmanuelle L. Waubant, Donald E. Goodkin, Alan Bostrom, Peter Bacchetti, Jean Hietpas, Francine Hoffman, Raija Lindberg, David Leppert San Francisco, CA; Seattle, WA; Basel, Switzerland

Objective:

To (1) determine the relationship between serum levels of matrix metalloproteinase-2 (MMP-2) and -9, and tissue inhibitor of MMP-type 1 (TIMP-1) and -2 (TIMP-2) and MRI activity in patients with secondary progressive (SP) multiple sclerosis (MS), and (2) evaluate the effect of interferon beta (IFNB) therapy on these markers.

Background:

High serum levels of MMP-9, an enzyme facilitating migration of T-cells and macrophages across vascular subendothelial basement membranes, and low levels of TIMP-1, its natural inhibitor, predict the appearance of new gadolinium-enhancing (Gd+) lesions in relapsing-remitting (RR) MS. It is not known whether this imbalance persists at later stages of the disease, nor what is the effect of IFNB therapy on this imbalance.

Design/Methods:

Monthly gadolinium-enhanced brain MRIs and three-monthly measures of serum MMP-2 and -9, TIMP-1 and -2 were performed for up to 3 years in 33 patients (21 females) with SPMS participating to the North American multicenter phase III study of IFNB-1b (10 on placebo, 23 on IFNB-1b eod). Within-patient median concentrations were tested for a difference between the two groups using an exact Mann-Whitney test. The relationship between serum levels of MMP-2 and 9, and TIMP 1 and -2, and presence of new Gd+ lesions was evaluated using univariate repeated measures logistic regression models. All time points within 4 weeks of steroid administration were removed from the analysis.

Results:

At baseline, mean age was 47.8 years +/-6.4, mean disease duration 12.8 years +/- 6.9, and median EDSS 6.0 (range 2.5-6.5). During study participation (141.8 weeks +/- 21), mean number of exacerbations was 0.33 +/- 0.6 and mean number of monthly new Gd+ lesions/patient was 1.49 +/- 0.14. 35 treatments with intravenous methylprednisolone 500 mg qd for 3 days were administered. 11 patients failed to show any new Gd+ lesions during the study. Patients who developed new Gd+ lesions during follow-up had higher levels of MMP-9 than patients who did not (respective within-patients medians 356 versus 219 ng/ml, p=0.01). In a univariate analysis, levels of MMP-9/TIMP-1 above within-patient medians predicted new Gd+ lesion on the concurrent scans (OR = 6.49, 95%CI 1.89 to 22.30, p=0.004) and to a lesser extent on the following scan (OR = 3.54, 95%CI 1.36 to 9.16, p=0.011) in the IFNB group. In contrast, levels of MMP-2 and TIMP-2 did not predict occurrence of new Gd+ lesions. Serum levels of TIMP-1 were significantly higher and MMP-9 lower on IFNB compared to placebo (respectively, within-patient median TIMP-1 : 1489 versus1178 ng/ml, p=0.01; within-patient median MMP-9 : 222 versus 361 ng/ml, p=0.03). IFNB did not influence levels of MMP-2 and TIMP-2.

Conclusions:

Serum levels of MMP-9 and TIMP-1, but not MMP-2 and TIMP-2, predict MRI activity in SPMS. IFNB therapy may shift the balance between MMP-9 and TIMP-1 towards normal, thereby decreasing proteolytic activity and at least in part reducing the number of new Gd+ lesions.

Supported By:

This study was supported by the National Multiple Sclerosis Society (USA) (PP0733) and the Swiss Multiple Sclerosis Society


Looking for the Definition of Benign Multiple Sclerosis

Wednesday, April 17, 2002
Marc Coustans, Franck Le Duff, Emmanuelle Leray, Emmanuelle Le Page, Jacques Chaperon, Gilles Edan Rennes, France

Objective:

To determine the best definition of benign multiple sclerosis (BMS) taking either EDSS 2 or 3 at 10, 20, 30 years (y) of disease duration.

Background:

Benign MS has been variably defined in quantitative terms and no consensus had been reached to definite this entity. Benign MS commonly defined as EDSS 2 or 3 after 10 y of disease duration is questionable.

Design/Methods:

In our EDMUS (European database for multiple sclerosis) database, among 1019 clinically definite relapsing remitting MS (RR MS), 695 patients had a disease duration more than 10 y. We analysed evolution of BMS with two definitions: RR MS population with an EDSS 2 and EDSS 3 after 10 y of disease duration and we examined these two groups of patients at 20 y and 30 y disease duration.

Results:

Among the 695 followed more than 10 y, 280 were followed more than 20 y and 100 more than 30 y. Proportion of BMS defined as EDSS 3 was respectively 62.3 %, 36.4 % and 25 %. Proportion of BMS defined as EDSS 2 was respectively 48 %, 26 %, and 16 %.
Moreover among the 433 patients with a definition of BMS as EDSS 3 after 10 y of disease duration, 217 had a disease duration more than 20 y, only 47% (102) of them were still BMS. Among these 102 still BMS patients at 20 y, 49 had 30 y follow-up and only 51% of them (25) were still BMS.
For BMS with EDSS 2 at 10 y (334), 184 had disease duration more than 20 y, 73 were still BMS (39.7%). Among these 73 BMS patients, 41 had 30 y follow-up, only 16 (39%) were still BMS.

Conclusions:

In the population of BMS defined as either EDSS 2 or EDSS 3 at 10 y, a majority of these patients had a worsening of their disability when they are followed at 20 and 30 y and so did not deserve the term of benign MS. Benign MS is a questionable entity.


Short-Term Correlations between Clinical and MRI Findings in Relapsing-Remitting Multiple Sclerosis

Wednesday, April 17, 2002
Marco Rovaris, Giancarlo Comi, David Ladkani, Jerry S. Wolinsky, Massimo Filippi, European/Canadian GA Study Group Milan, Italy; Petah Tiqva, Israel; Houston, TX

Objective:

To investigate the short-term correlations between clinical and magnetic resonance imaging (MRI)-measured disease activity in a large sample of patients with relapsing-remitting (RR) multiple sclerosis (MS) by analysing the dataset from the European/Canadian glatiramer acetate (GA) trial.

Background:

Despite the extensive use of MRI to provide markers of MS activity and accumulated disease burden, the relationship between clinical and MRI findings in patients with established MS has not been fully elucidated yet.

Design/Methods:

The trial was a nine-month, double-blind, placebo-controlled study, where 239 RRMS patients were randomized to receive either 20 mg GA (n=119) or placebo (n=120) by daily subcutaneous injections. Clinical assessment included monthly neurological examinations with Expanded Disability Status Scale (EDSS) score rating and additional visits in case of symptoms suggestive of a relapse. Dual echo, pre- and post-gadolinium (Gd) T1-weighted MRI scans of the brain were obtained at baseline and every month during the follow-up. Gd-enhancing and new T2-hyperintense lesions were counted and total T2-hyperintense and T1-hypointense lesion volumes measured.

Results:

Significant univariate correlations were found between number of relapses during the study period and number of Gd-enhancing lesions at baseline (r=0.25) and during the follow-up (r=0.30) in the study population as a whole. When the two study arms were considered in isolation, the degree of the correlations was higher in the placebo than in the GA group. A multivariable model showed that the independent factors more strongly correlated with the frequency of relapses during the study period were the number of relapses during the two years before study entry and the number of on-trial Gd-enhancing lesions, both in the study population as a whole and in the placebo group. In the whole patient sample, both T2 hyperintense and T1 hypointense lesion volumes at baseline were significantly correlated with baseline EDSS. Changes of MRI lesion volumes and EDSS were significantly correlated in the whole patient sample and in the GA group. Both T2 and T1 lesion volumes at baseline were significant predictors of the amount of MRI activity during the study period. T2 and T1 lesion volumes at baseline were also significantly correlated with the lesion volume changes observed during the subsequent nine months.

Conclusions:

In RRMS, MRI-measured inflammatory activity is only modestly, but significantly correlated with the occurrence of clinical attacks over a short-term period. Clinical and MRI assessment can provide complementary outcome measures for RRMS trials.

Supported By:

TEVA Pharmaceutical Industries, Ltd.


Information Processing Efficiency and Correlative 3T MRI Abnormalities in Multiple Sclerosis

Wednesday, April 17, 2002
Catherine J. Archibald, James N. Scott, Carla J. Wallace, Luanne M. Metz, J. R. Mitchell Calgary, AB, Canada; Toronto, ON, Canada

Objective:

The objective of this pilot study was to examine the relationship between the performance of Multiple Sclerosis (MS) patients on computerized measures of cognitive processing speed and capacity with white matter lesion load and extent of callosal atrophy as measured by 3T MRI.

Background:

Reduced information processing efficiency has been proposed as underlying various cognitive problems in MS. Two measures that systematically tax the speed and efficiency of cognitive processing (Salthouse, Babcock & Shaw, 1991; Sternberg, 1966) were employed to study relapsing-remitting (RRMS) and secondary-progressive (SPMS) patients with moderate disability. These measures detect cognitive changes in mild MS (Archibald & Fisk, 2000) while controlling for perceptual-motor abnormality. The relationship between performance on these measures and MRI has not been previously investigated. Performance on these measures was compared to the 30cm2 total T2 area threshold observed by Rao et al. (1989) above which 80% of MS patients have impairment on standard neuropsychological tests.

Design/Methods:

Twenty patients (7 RRMS; 13 SPMS) with a mean EDSS of 4.2 (sd 1.9) participated in the study. The Sternberg Task required patients to determine whether a test digit was a member of a sequentially presented set of digits ranging from 1-6 at a rate of 1200 ms/digit. The Salthouse task required patients to perform simple mathematical operations (i.e., addition) on a series of digits from 1-9 presented within 1-4 windows displayed at a rate of 2400 ms/digit on a computer screen. Disease characteristics and measures of depression, fatigue and attention were obtained. A 3T MR imaging system was used to generate axial T1, T2 and FLAIR-weighted images. Lesion area was quantified with a computer-assisted program, Segtool (Mitchell, Karlik, Lee & Fenster, 1994).

Results:

Eighty percent had T2 total lesion area above the 30cm2 threshold at which 80% of MS patients are observed to have impairment on neuropsychological tests. Processing speed was significantly slower for the patients than historical normal controls (p<0.05). Lesion area in the posterior fossa was significantly correlated with processing speed (r=0.58; p<0.01). Processing speed was impaired in 7/8 patients (6/7 SPMS) with posterior fossa T2 lesion area 1 cm2 and 2/12 (2/2 RRMS) patients with posterior fossa T2 lesion area < 1 cm2, which suggests a threshold effect for SPMS patients. Callosal length was marginally correlated (p<0.10) with cognitive speed. Patients made more errors on the capacity measure (p<0.05) and their performance was modestly but not significantly correlated with posterior fossa load. There were no significant correlations with the other MRI parameters or EDSS.

Conclusions:

Posterior fossa T2 lesion area was significantly associated with cognitive speed and there was a threshold effect in patients with SPMS which may suggest involvement of cerebellar-frontal circuitry in more advanced disease. Reduced callosal length may also affect cognitive speed. Processing capacity may also be affected by damage to cerebellar-frontal circuitry.

Supported By:

This research was supported by a Pilot Study Award from the Multiple Sclerosis Society of Canada.


Normalized Regional Brain Atrophy Measures in Multiple Sclerosis

Wednesday, April 17, 2002
Laura Locatelli, Robert Zivadinov, Barbara Stival, Alessio Bratina, Attilio Grop, Davide Nasuelli, Ozana Brnabic-Razmilic, Marino Zorzon Trieste, Italy; Heilbronn, Germany

Objective:

The aim of this study was to establish whether normalized measures of regional brain atrophy, correlate better with regional MRI defined brain lesions than an absolute measure of regional brain atrophy in a cross-sectional study.

Background:

Several techniques for measuring regional brain atrophy have been applied to patients with multiple sclerosis (MS). Recently, whole brain atrophy normalized measures have been introduced that correct for head size, and may also minimize the effect of patient positioning in the scanner. The current issue is of great importance, as there is still controversy about the optimal regional brain atrophy measurement for MS studies.

Design/Methods:

We assessed 45 relapsing-remitting (RR) patients with clinically definite MS (median disease duration 12 years) using standard cranial MRI. T1- and T2- lesion loads (LL) of frontal lobes and pons were measured using a reproducible semiautomated technique. The regional brain parenchymal volume (RBPV) of frontal lobes and pons was obtained using computerized interactive program, which incorporates semiautomated and automated segmentation processes. A normalized measure, the regional brain parenchymal fraction (RBPF) was calculated as the ratio of RBPV to the total parenchymal and cerebrospinal fluid (CSF) frontal and pons intraregional volume. Total fractional regional brain volume (TFRBV) was obtained after corrected the total parenchymal and cerebrospinal fluid (CSF) frontal and pons intraregional volume for total intracranial volume.The mean coefficient of variation (CV) for RBPV of the pons was 1% for intra-observer reproducibility and 1.4% for inter-observer reproducibility. The mean CV for RBPV of the frontal lobes was 1.6% for intra-observer reproducibility and 2.1% for inter-observer reproducibility.

Results:

RBPF and TFRBV demonstrated remarkably stronger correlation with quantitative MRI lesion measures compared to the RBPV (r= -0.37, p= 0.011 and r= - 0.40, p= 0.0005 vs. r= - 0.18, p= N.S. for T2-LL of the pons and r= - 0.27, p= 0,046 and r= - 0.31, p= 0.04 vs. r= - 0.09, p= N.S. for T1-LL of the pons. T1-LL of the frontal lobes was related to RBPF (r= -0.32, p= 0.033) and TFRBV (r= -0.29, p= 0.05), whereas RBPV did not show any correlation. There was no significant correlation between T2-LL of the frontal lobes and regional measures.

Conclusions:

The data suggest that normalized regional brain atrophy measures are preferable to absolute measures in cross-sectional studies.


Bright Lesions in the Brain on Non-Contrast T1-Weighted MRI Scans (T1 Shortening) in Multiple Sclerosis

Wednesday, April 17, 2002
Rohit Bakshi, Sonu Suri, Ralph H. B. Benedict, Bianca Weinstock-Guttman, Robert A. Bermel, Christopher W. Tjoa, Andrew J. Fabiano, Michael Santa Maria, Colleen E. Miller, Eileen Gallagher, Joan M. Feichter, Frederick E. Munschauer Buffalo, NY

Objective:

To determined the frequency, topography, and clinical correlation of bright lesions on noncontrast T1-weighted MRI scans (T1 shortening) in the brains of patients with multiple sclerosis (MS). To test whether bright T1 lesions are related to brain atrophy.

Background:

There is anecdotal information on the occurrence of bright lesions on noncontrast T1-weighted MRI scans (T1 shortening) in the brains of patients with multiple sclerosis (MS). The significance of these lesions is not known.

Design/Methods:

We retrospectively studied 145 adult patients with MS (65%relapsing-remitting, 35% secondary progressive). The mean disease duration was 9.6 +/-8.8 yr (range 0-38 yr) and the mean physical disability (EDSS) score was 3.5 +/- 1.9 (range 0-8.5). Bright T1 lesions were defined as areas that were hyperintense vs. the normal white matter on noncontrast T1-weighted axial images and also contained hyperintensity on T2 & FLAIR images. Third ventricle width, whole brain atrophy and total T2 hyperintense and black hole T1 lesion volumes were determined quantitatively using computer-assisted approaches. Regional cortical atrophy was rated on an ordinal rating scale vs. age-matched controls using our previously described method (J Neuroimaging 2001;11:129-136).

Results:

Across the 145 patients, 340 bright T1 lesions were present in the brain (per patient mean 2.3, range 0-9). The most common sites of involvement were the superior/inferior frontal and superior parietal regions. The lesions averaged 0.8 cm in diameter (range 0.2 - 2 cm). In contrast, there were 3075 bright T2 lesions and 885 T1 black holes total across the patients. Two-thirds of the bright T1 lesions showed hyperintensity in the periphery of the plaque. The others were bright in the center of the plaque. The total number of bright T1 lesions correlated with total atrophy (r=.36, p<.001) and third ventricle width (r=.405, p<.001) and within specific regions, superior frontal bright T1 lesions correlated with superior frontal atrophy (r=.28, p<.005). Regression modeling selected occipital, temporal, inferior parietal, and superior parietal bright T1 lesions as related to EDSS score (p<.001). When all MRI variables were placed into regression analysis, brain atrophy was most closely related to physical disability.

Conclusions:

Bright T1 lesions occur in the supratentorial region in the brains of patients with MS, but are less common than bright T2 and dark T1 lesions. Bright T1 lesions are partially related to atrophy, physical disability and disease course and thus may have clinical relevance as a previously under appreciated surrogate marker of tissue damage in MS. Determining the predictive validity of these lesions will require further investigation in prospective studies.

Supported By:

National Institutes of Health (NIH-NINDS) 1 K23 NS02210-01 (R. Bakshi), American Academy of Neurology Student Interest in Neurology Summer Scholarship (R. Bermel), and Alpha Omega Alpha Student Research Fellowship (R. Bermel).


Neuroradiologic Characteristics of Balo's Concentric Sclerosis: A Study of 25 Cases

Wednesday, April 17, 2002
Johnny K. Lokin, Hiroshi Matsuda, Artemio T. Ordinario, Jose C. Navarro, Takeshi Tabira Manila, Philippines; Tokyo, Japan

Objective:

The goal of this study is to describe the neuroimaging characterisitcs of this disease and to establish a neuroradiologic criteria that will enhance pre-mortem diagnosis.

Background:

Although Balo's concentric sclerosis is a rare demyelinating disease, this is the most common demyelinating disease of the central nervous sytem in the Philippines.

Design/Methods:

We studied 25 Filipino patients with acute demyelinating disease of the central nervous system. Neuroimaging materials using cranial computed tomography and conventional spin echo MR images were studied and analyzed using Adobe photoshop versions and Scion image beta version.

Results:

Balo's lesions showed low signal on T1-weighted, high signal on proton density-weighted and very high signal intensity on T2-weighted pulse sequences in all cases. The hallmark concentric and lamellated lesions were identified in 74% of patients. However, large, diffuse and confluent lesions proved to be a more consistent finding. In the absence of concentric lesions, a number of rounded lesions with propensity to coalesce were seen. The frontal and parietal lobe white matter were the most common sites involved, but lesions abutting the lateral ventricular walls were rare. Brain stem, corpus callosum and cerebellum were rarely involved. Lesions did not extend to the cortical gray matter, and occasionally produced a mass effect.

Conclusions:

Our findings indicate that the presence of typical concentric lesions, together with large, rounded, and confluent lesions in the cerebral white matter, and possessing distinct MRI properties are essential elements for the neuroradiologic diagnosis of Balo's disease.

Supported By:

Ministry of Health and Welfare and Ministry of Education,Science and Culture (International Research Program) in Japan.


Proposal of a Diagnostic Algorhytm for Sexual Dysfunctions in MS. A Cross-Sectional Study

Wednesday, April 17, 2002
Francesco Patti, Claudia Giliberto, Alfredo Liberto, Teresa Fiorilla, Mario Reggio, Arturo Reggio, Giancarlo Minaldi Catania, Italy

Objective:

To evaluate the frequency of sexual disturbances in a sample of 239 MS patients consecutivly enrolled.

Background:

Sexual dysfunctions represent a common disorder in MS; however the frequency is poorly reported in literature.

Design/Methods:

We included 239 patients who consecutively entered the MS-center of Catania in the first three month of 2001. All patients were extensively examined in a four-step clinical investigation. Firstly we simply asked about the occurrence of sexual disturbances with the specific item reported by Guy scale. Secondly we investigated sexual hormones abnormalities. As third we studied the vascular functioning of cavernous regions by ecodoppler records in men; finally the urodinamic evaluation corroborated by ano-anal, bulbo-cavernous reflexes and evoked-potentials of pudendal nerves was obtained.

Results:

We excluded from the analyses all patients who had abnormal levels of sexual hormones and / or pathologic features of cavernous ecodoppler records. Fifty-two (25%) MS patients out of 205 complained of sexual disturbances. Men were more frequently involved than women. Sexual disturbances were significantly correlated with EDSS-score ( r = 0.61; p <0.0001) and pyramidal function ( r = 0.8; p = 0.029 ). Urodynamic evaluation, pudendal-evoked potentials and both ano-anal and bulbo-cavernous reflexes resulted impaired in 40 patients (76%) out of 52.

Conclusions:

This study suggests a possible diagnostic algorhytm to detect sexual disturbances in MS. Sexual disfunctions correlate with EDSS worsening and in particular with pyramidal impairment. Clinical examination of MS patients should include investigation of sexual functions in wiew of a possible therapeutical intervention.


Computer-Assisted Volumetric Characterization of Brain Tissue of Multiple Sclerosis Patients Using Multi-Parameter Magnetic Resonance Images

Wednesday, April 17, 2002
Zhaohua Ding, Jana Preiningerova, Christopher J. Cannistraci, Aman Daftary, Gore C. John, Timothy L. Vollmer, Adam W. Anderson New Haven, CT

Objective:

The purpose of this study is to develop an efficient and reliable computer-assisted system for volumetric characterization of brain tissue, and to examine the reproducibility of applying the system to studies of multiple sclerosis(MS) patients involved in clinical trials.

Background:

Quantification of MS lesion load and relevant brain tissue volumetry is critical to clinical studies that monitor the natural progression of MS and assess the efficacy of drug treatment. Manual processing is labor-intensive and prone to considerable intra- and inter-observer variability; it is therefore imperative that automated or semi-automated methods be employed.

Design/Methods:

Axial scans of multi-parameter magnetic resonance images (MRI) were acquired from well characterized MS patients at monthly intervals. Images were filtered and registered for each patient and for each parameter individually, with the MRI exam that showed the best alignment among the parameters as the reference. Brain tissues were classified into white matter, gray matter, cerebrospinal fluid and MS lesion using a fuzzy c-means (FCM) clustering technique. First, training points representative of each tissue type were sampled at certain locations in the reference exam according to our standardized protocol. Image intensities of these training points were then used as initial values for the FCM algorithm, which iteratively determined the tissue type of each pixel. Classification results were reviewed by a well trained operator, who manually deleted false positive lesions if present. As images of all exams from a patient have been registered, the locations of training points in the reference exam could be simply copied to each of the other exams to initialize the FCM algorithm, which proceeded similarly as above. The results of manual editing were also "copied" to all the remaining exams.

Results:

For each exam, total volumes of lesion (LSN), lateral ventricles (VTR) and brain (BRN) were calculated. Brain parenchymal fraction (BPF), defined as the ratio of brain parenchymal volume to brain volume including the ventricles, was also computed. Intra-observer reproducibility of these quantities was tested by a skilled operator who processed all nine exams of two representative cases three times. The coefficient of variation (COV = standard deviation/mean value) was 0.029±0.019, 0.029±0.021, 0.008±0.003, 0.002±0.001 for LSN, VTR, BRN and BPF respectively. Inter-observer reproducibility was tested by three trained operators, with COV shown to be 0.056±0.024, 0.024±0.020, 0.006±0.003, 0.004±0.001 for LSN, VTR, BRN and BPF respectively. Operator processing time from taking training samples to volumetric computation for all nine exams of a patient was approximately four hours on a typical PC computer.

Conclusions:

This study shows that the system we developed is very efficient, and has low intra- and inter-observer variability, which holds great promise as a reliable laboratory and clinical tool for the quantitative study of MS disease burden.

Supported By:

Nancy Davis Foundation, LA, CA


Motor Fatigue in Amyotrophic Lateral Sclerosis (ALS) and Multiple Sclerosis (MS) Are Similar in Terms of Their Decline in Mechanical Force Output but Are Different in Their Myoelectrical Output during Sustained Maximal Voluntary Contraction (MVIC)

Wednesday, April 17, 2002
Mohammed Sanjak, Daryn Belden, Rick Konapacki, Andrew Waclawik, Benjamin R. Brooks Madison, WI; Madsion , WI

Objective:

To compare motor fatigue in ALS and and MS using two neurophysiolgical indicies, the decline in mechanical force output (FFI), and the shift in EMG median frequency (MFS) during 30 sec MVIC.

Background:

Motor fatigue expressed as a reduction in FFI during 30 sec of MVIC has been reported in ALS and MS. Electromyography (EMG) can be also used to study local muscular fatigue and involves measuring changes that occur in the EMG signal in the time and/or frequency domain. During MVIC, the EMG amplitude increases while markers of EMG frequency characteristics [i.e. Median Frequency] shift towards lower values. The increase in EMG amplitude has been suggested to reflect fatigue-induced by increases in motor unit recruitment and/or firing rate, while the decrease in frequency may reflect decreases in muscle fiber conduction velocity (MFCV). As muscular fatigue progresses, changes in the muscle-fiber-membrane permeability interfere with the normal propagation of action potentials, and therefore cause a decrease in the fiber conduction velocity.

Design/Methods:

MVIC was collected from 13 normal control (NC) 13 ALS patients, 13 MS patients, using a load cell attached via a strap to the muscle group being tested. Muscle electrical activity was measured by connecting an EMG preamplifier with integral electrodes to the surface of the skin overlaying the tested muscle group with a reference electrode placed on the back of the hand. The custom made preamplifier is a low-noise, low-bias current, semiconductor-based instrumentation amplifier which allows the stainless-steel electrodes to be placed directly on the skin with minimal surface preparation. The EMG signal is amplified (gain = 1000) and filtered (band pass = 25 to 250 Hz) before being sampled. Given that the task is isometric in nature and by filtering the low frequencies (below 25 Hz), motion artifact is minimized. Both signal voltages are connected to the computer via 12-bit data acquisition board for displayed,digital signal processing (plotting and analyzing)was implemented using custom made software developed with Visual Basic. Decline in force was expressed as FFI = 100 * [1 minus (AUC 25 to30 / AUC 2 to 7)].The compression in the power spectrum of the myoelectric signal in the last 5 second as compared to the first 5 seconds was tracked by Fast Fourier Analysis. The MFS was calculated as MFS = 100 * [1 minus (MF 25 to 30 / MF 2 to 7)].

Results:

FFI was significantly higher in MS and ALS as compared to NC in both muscle groups(p<0.05)indicating excess motor fatigue. MFS, was significantly lower in ALS than MS and NC(p<0.05).

Conclusions:

Motor fatigue in ALS is associated with lower MFS indicating changes in MFCV. This could be due to early loss of fast motor units, or atrophy of the remaining units or both. Peripheral factors in the pathogenesis of fatigue in ALS are implicated. Motor fatigue in MS did not disply these peripheral changes in myoelectrical activities.

Supported By:

Supported in part by Muscular Dystrophy Association [ MDA ] of America, National Institute of General Medical Sciences [ NIH ] University of Wisconsin General Clinical Research Center [ M01 RR03186 ] and Department of Veterans Affairs


Cortical Motor Reorganization after a Single Clinical Attack of Multiple Sclerosis Is Related to Disease Duration and Clinical Presentation

Wednesday, April 17, 2002
Caterina Mainero, Giandomenico D. Iannetti, Francesca Caramia, Silvia Di Legge, Luigi Bozzao, Carlo Pozzilli, Patrizia Pantano Rome, Italy

Objective:

We evaluated patterns of brain activation during a motor task in patients with a single clinical attack of multiple sclerosis (MS) in order to assess whether cortical reorganization occurs in the early phase of the disease. Furthermore the relationship between cerebral activation and both disease duration and clinical presentation was investigated.

Background:

Adaptive functional changes in the cerebral cortex of MS patients during a motor task have been recently described. It is unclear when these changes occur during the course of the disease, and whether a clinically evident motor deficit is necessary for cortical reorganization of motor areas.

Design/Methods:

Out of a consecutive series of patients with clinically isolated syndrome and serial MRI findings indicative of MS, we retrospectively selected 20 right-handed (RH) patients with hemiparesis (group H, n=10) or optic neuritis (group ON, n=10) as onset symptom and no further clinical episode. Ten RH age-matched healthy subjects served as controls (group C). Each subject was submitted to fMRI (1.5 T) during a sequential finger-to-thumb opposition task of the right hand. Image analysis was performed using SPM99 software.

Results:

No significant differences were found in age (31.9±9 vs 31.5±7 years), disease duration (24.3±14 vs 23.9±20 months), T2 lesion load (6.4±3.6 vs 7.3±10 ml) and T1 lesion load (1.0±0.8 vs 0.8±1.1 ml) between group H and ON. Group H showed a significantly higher EDSS score than group ON (1.2±0.8 vs 0.4±0.6).
Three group comparison by ANOVA showed significantly (cluster-level corrected p<.05, uncorrected p<.001) higher activation in H than in ON and C groups. Significant foci were located in the sensory-motor cortex (BA 1-4), parietal cortex (BA 40) and insula of the ipsilateral hemisphere and in the contralateral motor cortex (BA 4/6). No significant foci were found when either the group ON or the group C were tested against the other two. However, the ON group showed, at a lower level of statistical significance (uncorrected p<.01), a higher activation of the contralateral motor-related areas with respect to group C.
Time since clinical onset positively correlated (cluster-level corrected p<.05, uncorrected p<.001) with activation in the ipsilateral sensory-motor cortex (BA 1-4) and supplementary motor area (SMA, BA 6) in group H, and with activation in ipsilateral sensory-motor cortex (BA 1-4) and SMA (BA 6) and contralateral parietal cortex (BA 40) in group ON.

Conclusions:

Our study shows functional adaptive changes during a motor task in MS patients with a single clinical attack. A more severe and specific damage to the motor pathway in patients with a previous hemiparesis could explain the significantly higher involvement of additional motor areas observed in group H than in group ON. Furthermore, the significant correlation between the time since clinical onset and activation in motor areas suggests that cortical reorganization slowly develops along with the subclinical accumulation of tissue damage.


Immunoreactivity to Myelin Epitopes Differs between MS Patients Subgrouped Based on MRI and Clinical Characteristics of the Disease

Wednesday, April 17, 2002
Bibiana Bielekova, Kadom Nadja, Joseph Frank, Henry McFarland, Roland Martin Bethesda, MD

Objective:

To identify MS subgroups based on objective MRI and clinical measurements and determine whether the immune reactivity to different myelin antigens influences the disease phenotype.

Background:

From a clinical standpoint, MS does not appear to represent a single disease entity. Instead, patients differ in terms of their clinical phenotype and prognosis, MRI characteristics of disease as well as response to therapy. In an animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis, two major determinants of disease phenotype are genetic background of the susceptible strain and immunization with different autoantigenic epitopes. In view of this information, we asked the question, whether the T cell reactivity to different autoantigens similarly influences the disease phenotype in patients with MS.

Design/Methods:

We have studied the T cell reactivity to a carefully selected panel of 15 peptides derived from 4 candidate autoantigens in MS in a cohort of 53 untreated MS patients and 15 age-sex and HLA-DR-matched controls. In each studied individual, we have identified the immunodominant autoantigenic epitope based on the results of IL-7-modified primary proliferation, antigen-avidity assays, cytokine phenotype and naive-versus memory- in-vivo origin of the autoreactive T cells. We have also performed quantitative measurements of MRI disease characteristics, and based on these we subgouped MS patients into 4 distinct groups in a blinded fashion.

Results:

The MRI subgrouping of the patients appear to be stable over time and is associated with statistically significant differences in the clinical parameters of the disease.
The immunological assays revealed striking functional differences between MS patients and controls for selected antigenic epitopes, and the same myelin epitopes that proved to be most discriminatory between MS patients and HD, were also most important in discriminating between MS subgroups.

Conclusions:

Our data demonstrate that T cell reactivity to different myelin epitopes appears to be one of the contributing factors determining the phenotype of the disease, and that the reactivity to some epitopes may in fact represent immunomodulatory, rather than encephalitogenic potential. It will be important to establish whether the MS subgrouping based on our proposed criteria remains stable long-term and whether it can serve as a basis for studying the disease heterogeneity in MS.


Immunologic Effects of Mitoxantrone Therapy in Patients with Multiple Sclerosis

Wednesday, April 17, 2002
Samia J. Khoury, Padmanabhan Bharanidharan, Kasia Bourcier, Sandra L. Cook, Lynn Stazzone, Howard L. Weiner Boston, MA; Boston, MA

Objective:

To investigate the change in immunologic markers before and after treatment with Mitoxantrone in patients with chronic progressive MS.

Background:

We have previously reported that treatment with cyclophosphamide pulse therapy was associated with decrease in percentage of IL-12 positive monocytes and increases in IL-4 and IL-5 positive T cells. The mechanisms of action of Mitoxantrone, a recently approved therapy for MS is assumed to be by inhibiting actively proliferating cells. We sought to determine if treatment with Mitoxantrone is associated with changes in cytokine profile of peripheral blood mononuclear cells.

Design/Methods:

29 MS patients with progressive disease (20 females and 9 males) where followed before and after starting Mitoxantrone. Treatment was given at 800 mg/m2 at monthly intervals for 3 months then at 3-monthly intervals. Neurologic examinations were performed prior to start of therapy and at 6-monthly intervals. Pre-treatment evaluations included an echocar-diogram (to be repeated every 6 months) and an MRI of the brain (to be repeated yearly). Blood samples for immunologic studies were obtained just prior to Mitoxantrone infusions. PBMCs were isolated by gradient separation (Ficoll). For monocyte stimulation, the cells were stimulated by rhIFN-g (10ng/mL) for 2 hours and then LPS (100ng/mL) for 22 hours, in the presence of a protein transport inhibitor (monensin, 3mM). For T cell stimulation the cells were incubated with PHA, PMA and ionomycin for 6 hours. The cells were then washed twice with staining and blocking buffers, incubated 20 min. with fluorochrome-labeled surface markers including anti-IL-12Rb1 antibody, fixed with 4% paraformaldehyde solution, washed again, resuspended in permeabilization buffer (with 0.1% saponin), and incubated 30 min with intracytoplasmic anti-IL-12, anti-IL-10, anti-IFN-g, anti-TNF-a, and anti-IL-13 antibodies and corresponding Ig isotype control. The cell acquisition by flow cytometry was gated on the monocyte (CD14+) or the T cell (CD3+) cluster. Statistical analysis was done using Mann-Whitney test.

Results:

Most patients have been followed up to 6 months so far. Unlike our previous observations with cyclophosphamide, we found no change in percentage of IL-12+ monocytes after mitoxantrone therapy. There was a significant decrease in percentage of IL-10+ monocytes at the 6-month time point compared to baseline (p=0.0028) and a significant decrease in percentage of T cells expressing the IL-12Rb1 at the same time point (p=0.0034). There was no change in IFN-g expressing cells and no change in TNF-a+ cells or IL-10+ T cells.

Conclusions:

Longitudinal measurement of immunologic measures in peripheral blood of patients treated with mitoxantrone showed no acute changes in cytokine positive populations after treatment. There was evidence of decrease in IL-10+ monocytes and IL-12Rb1 expressing T cells but only after 3 doses of mitoxantrone, suggesting that these changes may be indirect effects of the therapy rather than directly related to its mechanism of action in MS.

Supported By:

NIH, National Multiple Sclerosis society, and the Nancy Davis Center without Walls


Monitoring Glatiramer Acetate Treatment in Multiple Sclerosis

Wednesday, April 17, 2002
Cinthia Farina, Reinhard Hohlfeld Munich, Germany

Objective:

To verify whether the immunological changes induced by Glatitamer Acetate (GA) treatment reflect its clinical efficacy.

Background:

We described recently that the ex vivo analysis of the blood from GA-treated patients by IFN-g/IL4 Elispot assays allows the definition of a specific immunological response profile for the treated population that was not observed in healthy individuals and untreated multiple sclerosis patients (Farina et al. Brain 2001). Interestingly in this initial study many treated patients were clinical responders and exibited this profile, raising the possibility that it might correlate with the clinical response to GA therapy.
Therefore we decided to perform a small pilot study and we contacted the major German MS centers asking to send us blood samples from all the GA-treated patients that could be identified as clinical nonresponders. These patients either showed an increased exacerbation rate or developed a secondary-progressive course. Over more than one year, we could collect 9 samples from clinical nonresponders.

Design/Methods:

We assessed the ex vivo response to GA of peripheral blood mononuclear cells from 9 clinical nonresponders and 15 clinical responders, using the elispot assays for the detection of IFN-g and IL-4 secreting cells in combination with a standard proliferation assay.

Results:

The immunological response profile defined in Farina et al. Brain 2001 was based on three observations: GA-treated patients displayed a) a reduced proliferative activity to GA, b) a strong activation of IFN-g-producing T cells at high GA in vitro concentration, c) IL-4 production over a wide range of GA concentrations.
Thirteen of 15 (86%) clinical responders met at least 2 of these criteria. On the other side, clinical non-responders showed a trend to mantain proliferation to GA and only two scored positive for IFN-g or IL4 Elispot assay. When considering the whole immunological profile, only 2/9 (22%) clinical nonresponders met two of the criteria.

Conclusions:

The Elipot assay is a promising additional tool for monitoring the treatment response in multiple sclerosis patients treated with GA.

Supported By:

Deutsche Forschungsgemeinschaft (SFB) 571 and Teva Pharma/Hoechst Marion Roussel


Treatment with Glatiramer Acetate (GA) Induces and Restores CD8+ T-Cell Responses in Patients with Multiple Sclerosis

Wednesday, April 17, 2002
Nitin J. Karandikar, Michael P. Crawford, Robert B. Ratts, Jason M. Brenchley, David R. Ambrozak, Amy E. Lovett-Racke, Daniel C. Douek, Richard A. Koup, Michael K. Racke Dallas, TX; Bethesda, MD

Objective:

This study was conducted to characterize glatiramer acetate (GA; Copaxone)-induced immune responses in patients with multiple sclerosis (MS) in order to elucidate the mechanism of action of this drug.

Background:

MS is an inflammatory demyelinating disorder of the central nervous system with features suggestive of T-cell-mediated autoimmunity. Treatment with GA, a random copolymer of alanine, lysine, glutamic acid and tyrosine, decreases the rate of exacerbation in MS. It is believed that GA induces CD4+ Th2-type responses, resulting in suppression of disease-mediating Th1 responses, through cross-reactivity or a bystander effect. However, direct immunophenotyping of responding T-cells has not been undertaken and is important in understanding the mechanism underlying the immunomodulatory effect of the drug.

Design/Methods:

We utilized novel, flow cytometric approaches to characterize the T-cell proliferative response to GA. These assays utilized a cytoplasmic green fluorescent dye, CFSE, which helps to detect proliferating cells. At the same time, cells were stained with fluorescent-tagged antibodies for identification of the proliferating subsets and evaluation of their cytokine profile. Antigen-specific cells were also sorted and analyzed for their functional profile using molecular assays.

Results:

We observed that healthy individuals and untreated MS patients exhibited prominent T-cell proliferative responses to GA. However, these responses were different in distinct subsets of T cells. Whereas GA-induced CD4+ T cell responses were comparable in the two groups, CD8+ responses were significantly lower in untreated patients. GA therapy resulted in the upregulation of these CD8+ responses with restoration to levels seen in healthy individuals. Both CD4+ and CD8+ GA-specific responses were classical HLA-restricted. GA therapy also induced a regulatory cytokine profile in GA-specific CD4+ and CD8+ T cells.

Conclusions:

This study provides the first direct evidence for the presence of GA-specific CD8+ responses. These responses are deficient in untreated MS patients and are upregulated during GA therapy. These findings strongly suggest a role for CD8+ T cells in mediating the effects of immunomodulatory therapy.

Supported By:

This work was supported by grants from the NIH and NMSS.


CCR7-Positive Antigen Presenting Cells and Central Memory T Cells in CNS of Multiple Sclerosis

Wednesday, April 17, 2002
Tatsuro Misu, Kazuo Fujihara, Hiroshi Onodera, Ichiro Nakashima, Feng Juan, Juichi Fujimori, Shigeru Sato, Sadao Takase, Osamu Yoshie, Yasuto Itoyama Sendai, Miyagi, Japan

Objective:

To evaluate distribution of CCR7 positive antigen presenting cells (APC) and central memory T cells (cmT) in the central nervous system (CNS) of multiple sclerosis (MS).

Background:

MS is believed to be a T cell-mediated demyelinating disease of CNS. We previously reported a dominant expression of Th1- over Th2-chemokine receptors in CSF cells during relapses (J Neuroimmunol, 2001;114:207-212). However, how this Th1 dominant immune response is induced in relapse of MS is not fully understood. A newly identified chemokine receptor, CCR7, identifies effector memory T cells (emT, CD4+CCR7-CD45RA-) and central memory T cells (cmT, CD4+CCR7+CD45RA-). EmT can respond to antigen immediately, but cmT are memory reserves which can rapidly differentiate into emT by antigen presentation. Analyses of cmT in CNS and its interaction with APC such as microglia (CD45LCA faint+) and macrophage (CD68+) may reveal a crucial process of initiation of Th1 response related to relapse in MS, but such a study is lacking.

Design/Methods:

1) Mononuclear cells in the peripheral blood (PB) and CSF from 10 patients with MS and 9 healthy controls were analysed with a flow-cytometer for the expression of CD4, CCR7 and CD45RA. 2) We studied mRNA levels of CCR7 and its ligands, EBI1-ligand chemokine (ELC) and secondary lymphoid-tissue chemokine (SLC), in the plaques and normal appearing white matter (NAWM) of five autopsied MS brains and the white matter of two control brains.

Results:

1) The percentage of cmT in CSF of MS was significantly higher that those in PB of MS and controls. In contrast, a significant increase of emT and a significant decrease of naive T cells ( CD4+CCR7+CD45RA) in CSF of MS were noted compared with those in PB of MS and controls. Monocyte/macropharge-gate cells in CSF of MS also expressed CCR7. 2) Much larger amounts of mRNA of CCR7 and ELC were detected in MS plaques than in MS NAWM and control white matters. SLC was not detected in CNS of MS.

Conclusions:

CmT were increased in CSF of MS and mRNA of CCR7 and ELC were detected in MS plaques. Thus, CCR7+APC and cmT may interact in CNS for the presentation of the target myelin antigens of MS. Further studies are needed to clarify the role of cmT and antigen presentation in CNS.

Supported By:

This work was supported by grants-in-aid for General Scientific Research (09470150, 13470131, and 13670626) from the Ministry of Education, Culture, Sports, Science and Technology, a grant from the Neuroimmunological Research Committee, and a research subsidy from the Japan Foundation for Neuroscience and Mental Health.


Chemokine Receptor Expression Differentiates Effector Memory Subsets of MBP Reactive T Cells: Persistence of CCR7 TH2 Cells

Wednesday, April 17, 2002
Peter A. Calabresi, Rameeza Allie, Sung H. Yun, Katharine A. Whartenby Baltimore, MD

Objective:

To characterize the chemokine receptor profile on antigen specific CD4+ T cells during various stages of differentiation from primary proliferation to long-term lines in order to differentiate Th1 and Th2 cytokine effector memory subtypes.

Background:

The chemokine receptors CXCR3, CCR5, and CXCR6 have been associated with Th1 effector memory cells on polyclonally stimulated and cytokine polarized T cells and have been found on T cells in the CSF or demyelinated lesions of MS patients. Furthermore the loss of CCR7 has been linked to a transition stage from central(lymph node homing)memory T cells to cytokine secreting effector T cells, which migrate to tissue sites of inflammation. But there are no studies that have systematically followed expression patterns during differentiation of an antigen reactive T cell.

Design/Methods:

Antigen specific T cell line were generated by the split well method using PBMC derived from healthy donors. We analyzed the chemokine receptors CXCR3, CXCR4, CXCR5, CXCR6, CCR5 and CCR7 by FACS on 135 T cells lines as they differentiated in response to the antigens myelin basic protein, tetanus toxoid, and glatiramer acetate. Chemokine receptors were correlated with secretion of IFNg or IL-5/IL-13 in the supernatants as measured by ELISA.

Results:

At day 21 after identification of antigen specific T cells there was a significant positive correlation between both CXCR3 and CXCR6 and IFNgand negative correlation with IL-5 levels in the supernatant. As expected the loss of CCR7 correlated with conversion to Th1 effector memory status and differentiated recall responses to tetanus as compared to naive responses to myelin basic protein. Surprisingly, Th2 lines were significantly more likely to remain CCR7 positive during short-term culture, and a subset of these expressed the B cell follicle homing receptor CXCR5. All three antigens induced T cells with a heterogeneous array of cytokine and chemokine receptor patterns.

Conclusions:

Antigen specific T cells follow an ordered pattern of chemokine receptor modulation during differentiation into effector memory cells. CXCR6 and CXCR3 are strongly associated with the Th1 phenotype on MBP reactive T cells. The loss of the lymph node homing receptor CCR7 positively correlated with Th1 effector cells, but inversely correlated with short-term Th2 cells. Th2 cells may have different migratory patterns both because of the transitory nature of allergic responses and to provide B cell help within the lymph node follicle. The ability to differentiate antigen specific Th1 and Th2 effector memory cells may be useful to characterize autoreactive T cells in autoimmune diseases such as multiple sclerosis.

Supported By:

NIH RO-1 NS41435-02


IL-18 Is Linked to Raised IFN-g in Multiple Sclerosis and Is Induced by Activated CD4+ T Cells Via CD40-CD40 Ligand Interactions

Wednesday, April 17, 2002
Arnon Karni, Djordje Koldzic, Padmanabhan Bharanidharan, Samia J. Khoury, Howard L. Weiner Boston, MA

Objective:

To investigate the role of IL-18 in IFN-g production in different stages of multiple sclerosis (MS).

Background:

Interferon-g appears to play an important role in the immune pathogenesis of MS, as injection of IFN-g exacerbated MS, increased IFN-g preceeds clinical attacks and there is increased IFN-g in the CNS of MS patients. Immune mechanisms responsible for increased IFN-g are not well understood. IL-18 is produced by non-T cells and with IL-12 is an inducer of IFN-g. IL-18 production by immune cells in MS has not been reported.

Design/Methods:

IL-18 and IFN-g levels were measured following aCD3/CD28 stimulation of peripheral blood mononuclear cells (PBMC) from 39 untreated relapsing remitting (RR) MS patients, 18 untreated secondary progressive (SP) MS patients and 20 healthy controls. The role of endogenous IL-12 and IL-18 on IFN-g production was studied by adding neutralizing aIL-12 and aIL-18 antibodies (Abs) into the cell culture. The concentrations of the neutralizing Abs were chosen to induce maximal suppression of IFN-g production. CD4+ and CD8+ T cells were depleted using Dynabeads. The role of interaction between CD40 ligand and CD40, CD28 and CD80 or CD86 and iCOS and B7RP-1 in the production of IL-18 was studied using appropriate blocking Abs.

Results:

IL-18 secretion was increased in both RR-MS (80.0±13.0 pg/ml, p=0.004) and SP-MS (126.9±25.3 pg/ml, p=0.002) compared to healthy controls (28.5±0.8 pg/ml). IL-18 levels correlated with disease duration in SP-MS (r=0.68, p=0.002), and a similar trend was found in RR-MS patients with disease duration 5 years (p=0.09). In RR-MS, IFN-g levels were reduced (from 3535 pg/ml) by either neutralizing aIL-12 (1496 pg/ml, p=0.003) or aIL-18 (1405 pg/ml, p=0.003). No further reduction was detected with both antibodies. Identical results were observed in healthy controls. However, in SP-MS elevated IFN-g levels (5260 pg/ml with isotype control Ab) were significantly reduced only in the presence of both neutralizing aIL-12 and aIL-18 (646 pg/ml, p=0.007 compared to isotype control, p=0.017 compared to blocking IL-12 alone and p=0.006 compared to blocking IL-18 alone). Elevated IL-18 levels in MS were completely suppressed following depletion of CD4+ T cells but not of CD8+ T cells and by blocking CD40-CD40 ligand interactions by aCD40 ligand Ab.

Conclusions:

Our findings provide a better understanding of the immunologic mechanisms related to the raised IFN-g in SP-MS. The immune cascade which leads to the increased IFN-g in SP-MS begins with activation of CD4+ T cells through the T cell receptor and subsequent up-regulation of CD40 ligand expression which interact with CD40 on antigen presenting cells. This then leads to secretion of IL-12 and IL-18 which increase IFN-g in a linked fashion in RR-MS but act independently to increase IFN-g in SP-MS. Our results have implications for immune therapy of MS, and identify IL-18 as a potential new target of therapy.

Supported By:

National MS Society, NIH and Nancy Davis Foundation. A.K is a National MS Society Fellow.


Chemokine Receptors in Multiple Sclerosis: Increased CXCR6-Bonzo Positive T Cell Expression and Effect of Therapy

Wednesday, April 17, 2002
Padmanabhan Bharanidharan, Arnon Karni, Samia J. Khoury, Howard L. Weiner Boston, MA

Objective:

To investigate the expression of the chemokine receptor CXCR6-Bonzo in patients with multiple sclerosis (MS) and assess the effect of therapy.

Background:

Chemokine receptor CXCR6-Bonzo is established as an differentiation marker of polarized T-helper type-1 cells (Th1) and is not expressed by T- helper type-2 cells (Th2). Bonzo positive T cells are enriched among T cells in tissue sites of inflammation such as rheumatoid joints and inflammed liver. Bonzo may be important in trafficking of effector T cells that mediate type 1 inflammation, making it a potential target for therapeutic modulation of inflammatory diseases such as MS.

Design/Methods:

Peripheral blood was obtained from 44 MS patients and 9 healthy controls. 10 patients were untreated, 17 were treated with interferon-b and 17 with pulse therapy with cyclophosphamide/ methyl prednisolone. After Ficoll-separation isolated peripheral blood mononuclear cells were stained with unlabeled mouse anti-human CXCR6, Fluorescein labelled polyclonal goat anti-mouse IgG1 as seconday staining, Phycoerythrin (PE) labelled mouse anti-human CD4 or mouse anti-human CD8 antibodies. The expression levels were measured by flow cytometry and analyzed by CellQuest software. CD4+CXCR6+ T cells and CD4+ CXCR6- T cells were sorted with a purity 99%. Thereafter the cells were cultured in the presence of plate-bound aCD3/aCD28 and the secretion of interferon-g (IFN-g) and IL-4 were measured by ELISA.

Results:

Surface expression of CXCR6-Bonzo on CD4 cells was significantly higher in untreated MS patients versus healthy controls (10.4 vs. 3.3%, p=0.024). To investigate the functional characteristics of CD4+CXCR6+ T cells, we sorted CD4+ CXCR6+ and CD4+ CXCR6- T cells and found that CD4+ CXCR6+ T- cells secreted higher IFN-g (9721 pg/ml) and lower IL-4 (2.6 pg/ml) as compared with CD4+CXCR6- T cells (IFN-g=4973 pg/ml and IL-4=6.3 pg/ml). Therapy with interferon-b reduced the levels of Bonzo-CXCR6 positive T cells to 6.3% and with pulse cyclophosphamide/ methyl prednisolone to 4.9%. The reduction by cyclophosphamide/ methyl-prednisolone was significant as compared to untreated patients (p=0.039). No significant differences were found in the CD8 population.

Conclusions:

These results demonstrate Th1 polarization using a newly described chemokine receptor that marks Th1 cells. These results provide new avenue to assess immune activation in MS and identify a potential a new target of therapy

Supported By:

National MS Society, NIH and Nancy Davis Foundation.


The Role of DRB5*0101 (DR2a) Versus DRB1*1501 (DR2b) in the Autoimmune Pathogenesis of Multiple Sclerosis

Wednesday, April 17, 2002
Elisabetta Prat, William W. Kwok, Niels Kruse, Maria P. Bettinotti, Henry F. McFarland, Roland Martin Bethesda, MD; Seattle, WA; Wurzburg, Germany

Objective:

The purpose of this study is to investigate the disease relevance of DR2a and DR2b alleles, co-expressed in the multiple sclerosis (MS)-associated HLA-DR15Dw2 haplotype. In particular, the mRNA expression of the two alleles was analyzed by real time RT-PCR on antigen-presenting cells (APC) obtained from HLA-DR15+ healthy donors and MS patients.

Background:

MS is considered a T cell-mediated autoimmune disease triggered by exogenous events in genetically susceptible individuals. Major histocompatibility complex region shows the strongest association to MS and in Caucasians, HLA-DR15Dw2 and -DQw6 are the closest associated.
HLA-DR molecules consist of a polymorphic b-chain encoded by DRB1* or DRB3-5* genes associated with a non-polymorphic a-chain. In the HLA-DR15 haplotype, two b-chains HLA-DRB1*1501 and -DRB5*0101 are co-expressed resulting in two surface heterodimers, DR2b (DRB1*1501) and DR2a (DRB5*0101) that serve as presenting molecules.
While most MS studies examined the role of DR2b, it is clear that both alleles can serve as antigen presenting molecules for myelin basic protein-specific T cells and that the restriction element could influence T cell effector functions. HLA expression levels can directly affect the extent of T cell activation and differential transcriptional activities of HLA-DRB genes have been reported. These findings point out that it is important to dissect which of the alleles in the HLA-DR15 haplotype is functionally relevant for MS.

Design/Methods:

Specific oligonucleotides have been designed to evaluate the expression of the two alleles by real-time RT-PCR. Standard curves were generated for DR2a and DR2b using plasmid DNA. Samples cDNA copy number was calculated based on standard curves. A plasmid DNA standard curve was also generated for b-actin gene, used as internal reference. The study has been conducted on peripheral blood mononuclear cells (PBMC), purified monocytes and B cells obtained from HLA-DR15+ healthy donors and MS patients. Expression has been analyzed under basal conditions and after stimulation of APC with Interferon-g and Interleukin-4 in time-course (T-C) experiments.

Results:

DR2a and DR2b expression was studied on fourteen samples of freshly isolated PBMC and fifteen time-course experiments were performed on PBMC, monocytes or B cells obtained from MS patients or healthy donors. No major differences were found between expression levels of DR2b and DR2a. In T-C experiments the expression of the two alleles was similarly modulated.

Conclusions:

This study shows that DR2a and DR2b alleles are co-expressed at comparable levels in APC obtained from HLA-DR15+ donors, suggesting further evidences for a role of DR2a allele. This contrasts to other haplotypes in which the DRB3*-5*-encoded DR allele is expressed at lower levels. Studies on peripheral APC such as dendritic cells or on thymic APC are in progress to confirm these data and to investigate a possible differential alleles expression in other cell types and tissues that might be important for shaping the T cell receptor repertoire.

Supported By:

U.S. National Multiple Sclerosis Society


Roles of TRAIL in Neuroinflammation

Wednesday, April 17, 2002
Jan D. Lunemann, Sonia Waiczies, Jan Doerr, Orhan Aktas, Klaus P. Wandinger, Ingo Bechmann, Robert Nitsch, Frauke Zipp Berlin, Germany

Objective:

To elucidate the contribution of the TNF-related apoptosis inducing ligand (TRAIL)/TRAIL receptor system in T cell control and CNS damage mechanisms.

Background:

In neuroinflammation, apoptosis takes on two roles by mediating tissue damage on the one hand and determining T cell fate on the other. We could recently demonstrate that TRAIL induces massive apoptosis in human brain cells. Defining the physiologic role of this death receptor/ligand system in both the human brain and on the T cell level could lead to new therapeutic strategies targeting inflammatory diseases of the CNS.

Design/Methods:

Expression of TRAIL and its receptors in the human brain were investigated by RT-PCR, western blot, and double-labeling immunocytochemistry. T cell apoptosis was assessed by specific DNA-fragmentation. T cell proliferation assays were performed by 3H-thymidine uptake and the expression of cell cycle mediators were determined by western blot analysis. Calcium influx upon T cell stimulation was measured by fluorescence spectrophotometry.

Results:

In contrast to other known death ligands, TRAIL was not found to be constitutively expressed in the human brain, whereas its apoptosis mediating receptors could be detected on neurons and oligodendrocytes.
In human T cells, soluble TRAIL did not induce apoptosis, but inhibited activation via blockade of calcium influx. Subsequent inhibition of proliferation could be attributed to downregulation of the cyclin-dependent kinase 4, indicating a G1 arrest of the cell cycle.

Conclusions:

The TRAIL receptor/TRAIL system appears to exert other immunoregulatory than apoptosis induction. However, lack of TRAIL, but presence of its apoptosis mediating receptors in the human brain indicate a potential impact on neuroinflammtory effector mechanisms rather than a role in defense mechanisms of the immune privileged brain. A blockade of TRAIL mediated brain tissue damage might therefore be of therapeutic benefit.

Supported By:

This study was supported by the Gemeinnutzige Hertie-Stiftung and the Deutsche Forschungsgemeinschaft (ZI 448/7-1, BE 2272/1-1, SFB 507/C1).


Short-Term Dynamics of the Peripheral Blood T Cell Receptor V Beta Repertoire in Relapsing-Remitting Multiple Sclerosis (MS)

Wednesday, April 17, 2002
Paolo A. Muraro, Laura Bonanni, Antonello Pantalone, Elisabetta Traggiai, Luca Massacesi, Marco Vergelli, Domenico Gambi Chieti, Italy; Firenze, Italy

Objective:

To identify unbalances and possible short-term changes of T cell receptor (TCR) repertoires of patients with relapsing-remitting multiple sclerosis (MS) and assess their potential relationship with immunopathological events.

Background:

Autoreactive T lymphocytes have been implicated in the pathogenesis of MS. The analysis of TCR usage provides valuable information on the composition of T cell populations that may lead to the identification of pathogenic cells. While the TCR usage of myelin antigen-specific T cells and cerebrospinal fluid cells from MS patients has been extensively investigated, fewer studies focused on the peripheral blood TCR V repertoire. Scarce information in particular is available on the evolution of TCRBV repertoire in MS patients over time.

Design/Methods:

We studied four untreated RR-MS patients who underwent monthly brain magnetic resonance imaging (MRI) for a period of 4 to 6 months. TCR V beta (TCRBV) repertoire was analyzed by semi-quantitative RT-PCR on peripheral blood mononuclear cells (PBMC) obtained monthly at the time of each MRI. T cell responses to MBP were studied in parallel by proliferation assays. As controls, we analyzed the TCRBV repertoires of a population of twenty healthy subjects, five of whom were followed monthly for a period of 4 to 5 months. Identified TCRBV expansions were examined by single-strand conformational polymorphism (SSCP) to assess their clonal composition.

Results:

All MS patients were clinically stable during the observation period. Significant TCRBV expansions were found in 10 out of 24 TCRBV profiles from MS patients, and only in 2 out of a total of 37 TCRBV profiles from healthy controls (P<0.001; Fisher exact test). SSCP analysis showed that 17 out of 21 (80%) TCRBV gene expansions analyzed were oligoclonal, whereas 4 were polyclonal. There was a significant correlation of the presence of TCRBV expansions with the peripheral immune reactivity to MBP (P<0.05), and with inflammatory disease activity detected by MRI (P<0.05), as well as between MBP responses and MRI activity (P<0.05).

Conclusions:

Autoreactive T cell responses against myelin antigens may be implicated in disease-related perturbations of TCR repertoire in MS patients, detectable even in the absence of clinically evident manifestations.

Supported By:

Supported by a grant from the Italian Ministry of University and Research (MURST/1998).


Temporal Variations of Adhesion Molecules and Matrix Metalloproteinases (MMP) during the Course of Multiple Sclerosis (MS)

Wednesday, April 17, 2002
Jorge Correale, María M. Bassani Molinas Buenos Aires, Buenos Aires, Argentina

Objective:

To study temporal variations of adhesion molecules and MMP in patients with MS.

Background:

Infiltration of inflammatory cells into the Central Nervous System requires their migration through the blood-brain barrier (BBB). This process involves the upregulation of cell surface adhesion molecules and the action of MMP, enzymes that digest various collagen components of the extracellular matrix and basement membrane. Although changes in adhesion molecules and MMP levels have been described in MS in cross-sectional studies, little is currently known about their expression in early stages, and the temporal variations occurring during the course of disease.

Design/Methods:

Thirty patients with Clinically Isolated Syndromes (CIS), 20 with optic neuritis, 5 with transverse myelitis and 5 with brainstem lesions, were evaluated at the onset of neurological symptoms and when they developed clinically definite MS (CDMS). Patients were followed up for 6 to 44 months. None had ever received immunomodulatory treatment. Surface expression of LFA-1a, VLA-4 and ICAM-1 on PBMNC and CSF cells was evaluated using flow cytometry. Serum and CSF concentrations of soluble VCAM-1, ICAM-1, and E-selectin were assayed using ELISA. Plasma concentrations of MMP-9 and MMP-2 were also measured by ELISA.

Results:

Nineteen of the patients (14 with optic neuritis, 2 with transverse myelitis and 3 with brainstem syndrome) developed CDMS 18 to 40 months later. Mean LFA-1a and VLA-4 fluorescence on peripheral blood and CSF T cells and monocytes from CIS and CDMS patients was significantly increased compared with control subjects. Moreover, LFA-1a and VLA-4 expression was significantly higher in patients who develop CDMS compared with CIS patients. Interestingly, patients with CIS who failed to developed CDMS and were re-evaluated at the end of the observation period showed similar levels of adhesion molecules in both T cells and monocytes. Besides, in patients with CDMS expression of LFA-1a and VLA-4 molecules on CSF cells correlated significantly with IgG indexes and BBB permeability. CSF soluble VCAM-1 and E-Selectin were raised in patients with CIS and CDMS over control subjects. However, serum levels of these molecules were similar in all three groups. MMP-9 levels in patients with CIS and CDMS were significantly higher than those found in control subjects. Likewise, values of MMP-9 were higher in CDMS than in CIS patients. Patients who failed to develop CDMS and were re-evaluated at the end of the observation period showed similar MMP-9 levels in both determinations. Furthermore, MMP-9 values correlate with IgG indexes, BBB permeability and CSF pleocytosis. In contrast, MMP-2 plasma levels were similar in CIS patients, CDMS patients and control subjects.

Conclusions:

Upregulation of adhesion molecules and MMP-9 is observed early in the course of MS. Moreover, these data suggest that VLA-4, LFA-1a and MMP-9 play a leading role in the evolution of inflammatory demyelinating lesions in patients with CIS who develop CDMS.


Effects of Combination Therapy of Beta-Interferon 1a and Prednisone on Serum Immunologic Markers in Patients with MS

Wednesday, April 17, 2002
Hassan H. Salama, Oldrich J. Kolar, Ying C. Q. Zang, Jingwu Zhang Houston, TX; Indianapolis, IN

Objective:

The objectives are (1) to compare the immunoregulatory effects of beta-IFN alone and combination therapy of beta-IFN with a low daily dose of Prednisone in MS patients and (2) to determine whether the combination therapy provides additional clinical benefits for MS patients.

Background:

The inflammatory/autoimmune processes in MS are associated with T-cell activation and the production of pro-inflammatory cytokines, adhesion molecules and soluble T-cell activation markers,which reflects systemic immune abnormality. Beta-interferon has a proven treatment effect on MS presumably through its regulatory properties on T-cell activation and cytokine production. Prednisone has an immunosuppressive effect by high dose, but no studies about the immunological and clinical effects of combination therapy.

Design/Methods:

A group of 55 clinically definite MS patients with relapsing-remitting or secondary progressive disease and a control group of 27 healthy subjects were included in this study. Patients were randomly assigned to receive either beta-IFN-1a at 30 mcg by weekly i.m.(n=22) or beta-IFN-1a combined with Prednisone at a daily oral dose of 15 mg (n=33). Serum specimens were collected before the initiation of the treatments and 3, 6, 9 and 12 months after the treatments. Control specimens were obtained in parallel from a group of healthy subjects at the same schedule. Specimens were assayed for concentrations of TNF alpha, IL-12, IL-2R, IL-10, CD95, ICAM-1 and binding antibodies to beta-IFN using ELISA. Patients were monitored for clinical exacerbation and expanded disability scale score (EDSS).

Results:

The results revealed that treatment with beta-IFN-1a alone significantly reduced levels of TNF alpha and ICAM-1 but not IL-2R and IL-12. In contrast, the production of IL-10 and CD95 was increased. In group of combination therapy, the inhibition of IL-12, IL-2R, TNF alpha and ICAM-1 was more profound than that monotherapy group. In contrast, the combination therapy appeared to antagonize the up-regulatory effect of beta-IFN-1a on IL-10 and soluble CD95 by suppressing the production of both serum markers. Further results revealed the same occurrence of serum binding antibodies to beta-IFN in both treatment groups (2/22 vs. 3/33) at 6 months and 12 months, suggesting that the addition of Prednisone had no effect on the production of binding antibodies induced by the beta-IFN treatment. Three of 22 patients in the beta-IFN-1a group and 5 of 33 patients in the combination treatment group had a single relapse during the study. Monotherapy group showed a slight reduction in EDSS by 0.1point (p<0.05) after 12 months treatment.

Conclusions:

The combination therapy may have an additional treatment effect on the inflammatory processes in MS by further lowering the production of pro-inflammatory cytokines and T-cell activation state. No difference in the occurrence of binding antibodies to beta-IFN was found between the two treatment groups. The findings are important in designing future clinical trials to evaluate the treatment effect of combination therapy in MS.

Supported By:

Supported by a research grant from Biogen, Inc..


Epitope Analysis of Cerebro-Spinal Fluid IgG in HTLV-1 Associated Myelopathy Patients Using Phage Display Method

Wednesday, April 17, 2002
Juichi Fujimori, Ichiro Nakashima, Kazuo Fujihara, Juan Feng, Masahiro Yamamoto, Naoki Yamamoto, Yusei Shiga, Junichi Onodera, Shigeru Sato, Sadao Takase, Masahiro Asano, Minoru Endo, Yasuto Itoyama Sendai, Miyagi, Japan; Tokyo, Japan

Objective:

The characteristic cerebro-spinal fluid (CSF) findings in patients with HTLV-1-associated myelopathy (HAM) are elevated titers of anti-HTLV-1 antibodies, an elevated IgG index and oligoclonal bands (OB). Previous studies showed that major epitopes of anti-HTLV-1 antibodies in CSF of HAM are the products of HTLV-1 core (gag) and envelope (env) genes. However, it remains to be elucidated whether these anti-HTLV-1 antibodies are dominantly expanding IgG in CSF of the patients. Random peptide libraries displayed on filamentous phages provide an extremely powerful methodology for the selection of ligands binding to monoclonal antibodies. We studied the CSF oligoclonal bands in HAM patients by isoelectric focusing (IEF) and investigated the dominant target antigens of CSF IgG using phage display method.

DESIGN/METHODS: CSF samples from 7 HAM patients (4 women and 3 men, age 40-65 (55.0±8.4) years, disease duration 2-21 (8.5±6.7) years) were analyzed. We tested OB by IEF with PhastSystem™ (Amersham Pharmacia Biotech, Uppsala, Sweden). Only bands present in the CSF but not in the serum were detected as OB.
We investigated the epitopes of CSF IgG using phage display method. We immobilized CSF IgG by protein A-conjugated magnetic beads and detected the presence of consensus amino acid sequences recognized by CSF IgG with a phage display library (Phage Display Peptide Library Kit, New England Biolabs Inc., Bervely, MA) of approximately 40 billion combinations of 12 mer peptides. BLAST was searched for proteins containing homologous sequences, especially with regard to the components of HTLV-1.

Results:

Among 7 patients with HAM, OB was positive in 3 patients. There was no correlation between the existence of OB and the clinical course or the disease duration. CSF from each of 7 patients with HAM selected some common peptide motifs. In four patients, a common peptide motif highly homologous to gp46 237-243 was selected and in two other patients, another common peptide motif highly homologous to gp46 191-199 was selected. Common peptide motifs selected from one patient without OB were not homologous to the component of HTLV-1 and the origin remained unknown.

Conclusions:

Using phage display method, we confirmed that the dominant IgG in CSF of most HAM patients is the one directed against HTLV-1 env gp46 protein regardless of OB status.


Multiple Sclerosis: Immune Alterations during Combined IFN-Beta-1a Plus Compaxone Treatment

Wednesday, April 17, 2002
Hans Link, Yassir Hussien, Alessandra Sanna, Mats Söderström, Yu-Min Huang Stockholm, Sweden; Sassari, Italy

Objective:

To evaluate influence of multiple sclerosis (MS) treatment with IFN-beta1a vs. IFN-beta1a plus Copaxone on surface molecules on HLA-DR+ blood mononuclear cells (MNC) and cytokine production by MNC.

Background:

Cell surface molecules and cytokines of immune cells are believed to play an important role in the proposed immunopathogenesis of MS. Clinical studies have demonstrated beneficial effects of the immunomodulators IFN-b and Copaxone on MS course. But each agent shows only modest clinical efficacy, and not all patients respond to monotherapy. IFN-b and Copaxone act in MS in different ways. We hypothesize that combination therapy with IFN-beta plus Copaxone may be beneficial in patients progressing clinically despite treatment with IFN-b or Copaxone alone.

Design/Methods:

Blood MNC were isolated from 18 untreated patients, 22 treated with IFN-b1a alone, 14 treated with IFN-b1a plus Copaxone, and 23 healthy controls (HC). Two-colour surface staining with flow cytometry was used to analyse surface molecule expression on MNC. ELISA was used to measure cytokine levels in MNC culture supernatants.

Results:

Surface molecules: MS patients compared to HC had higher CD11c (median 35% vs. 23%; p<0.01) and CD80 (11% vs. 5%; p<0.02), and lower CD86 (17% vs. 20%; p<0.04) among HLA-DR+ MNC. Upon subgrouping the MS patients, altered expression of CD11c, CD80 and CD86 was confined to untreated MS patients (41%, 23% and 12%, respectively), and not to the patients treated with IFN-b1a alone or with IFN-b1a plus Copaxone, except for the CD11c molecule in the last group. Untreated MS patients also showed higher CD1a (12%) and lower CD123 (10%) among HLA-DR+ MNC compared to patients treated with IFN-b1a alone (6% and 24%; p<0.05 and p<0.01), and with IFN-b1a plus Copaxone (2.5% and 20%; p<0.001 and n.s.) and HC (6% and 23%; p<0.05 and p<0.01).
Cytokines: MS patients compared to HC had lower IL-10 (p<0.01) and higher IL-12p70 (p<0.01) in MNC culture supernatants. Upon subgrouping, only untreated MS patients had lower IL-10 (p<0.01), higher IL-12p70 (p<0.01) and IFN-g (p<0.05). Patients treated with IFN-b1a alone compared to HC showed no difference for IL-10. Patients treated with IFN-b1a plus Copaxone compared to HC, besides showing no difference for IL-10, also showed no differences for IL-12p70 and IFN-g .

Conclusions:

Treatment with IFN-b1a and Copaxone in combination showed synergistic effects on reducing CD1a+ MNC, augmenting IL-10, and reducing IL-12p70 and IFN-g. Further follow-up studies on combined therapy with IFN-b1a plus Copaxone in relation to clinical outcome in MS are ongoing.


Chronic Inflammatory Demyelinating Polyradiculoneuropathy: Clinical, Electrophysiological and Histopathological Study of 100 Cases

Wednesday, April 17, 2002
Osvaldo J.M. Nascimento, Marcos R.G. Freitas, Tania M. Escada, Maria Teresa Nevares, Myriam D. Hahn Rio de Janeiro, Rio de Janeiro, Brazil

Objective:

To describe the clinical, electrophysiological and histopathological features of idiopathic chronic inflammatory demyelinating polyradiculoneuropathy (CIDP-I) in 100 patients.

Background:

CIDP-I is a demyelinating neuropathy known to be responsive to immune therapy. There are some neuropathies related to CIDP-I, but with quite different clinical and electrophysiological features and immune therapeutic response. Some reports describe a concomitant central nervous system involvement in CIDP-I. There is special interest in the report of large series of CIDP-I being ours the first in a tropical country.

Design/Methods:

In this study we have included patients fulfilling proposed criteria for idiopathic CIDP, referred between 1985 and 1999. Those with distal acquired demyelinating sensory (DADS) polyneuropathy, associated or not with monoclonal gammopathy, and those with multifocal motor neuropathy with conduction block, which we consider separate conditions, were excluded. Neuroconduction (NC) studies were performed according to standard techniques. Superficial nerve biopsies were studied in light and electron microscopy in 62 of the cases.

Results:

There were 56 men and 44 women. Eleven were children. The progressive and relapsing courses were respectively observed in 59 and 41 patients, during a 6 years average period of follow-up. The classical proximal and distal sensorimotor involvement was observed in 86 cases. A multifocal sensorimotor pattern of presentation and progression (multifocal acquired demyelinating sensory and motor [MADSAM] neuropathy) was seen in 14 cases, most involving mainly the upper limbs. Progressive respiratory insufficiency secondary to intercostal muscles and diaphragm involvement was seen in a 8 y.o. child. No patient had symptomatic involvement of CNS. Brain MRI performed in 26 cases did not disclosed demyelination. CSF protein was increased in 82 cases. Modified demyelinating NC criteria were able to recognize all clinically defined cases but 39 did not fulfill the AAN electrophysiological criteria. Nerve biopsy showed demyelinating lesions in 71%, mixed demyelinating and axonal lesions in 21%, and predominatly axonal lesions in 8% of specimens. Mild inflammation was seen in 21 (34%) cases. Ninety-four patients responded to treatment. Two patients died as a consequence of the disease.

Conclusions:

Classical CIDP-I is a distal and proximal neuropathy usually responsive to corticosteroid treatment. Motor and sensory multifocal presentation has the same good response to steroids. Associated demyelinating CNS involvement was not observed in our series, although described in countries of the North Hemisphere. The lack of this association is probably due to the low incidence of multiple sclerosis in our country. Intense axonal loss and progressive course were factors correlated with higher disability and failure to different treatments. Nerve biopsy can be useful only in clinically defined cases of CIDP in which NC and CSF do not fulfill diagnostic criteria, showing active demyelination.


Correlations between Immunologic Markers and MRI Measures of Disease Activity in Patients with Multiple Sclerosis

Wednesday, April 17, 2002
Karim Makhlouf, Charles C. Guttman, Marika J. Hohol, Sandy Cook, Michael J. Olek, Howard L. Weiner, Samia J. Khoury Boston, MA; Toronto, ON, Canada

Objective:

To analyze the correlations between immunologic markers and MRI measures of disease activity in a longitudinal study of patients with chronic progressive (CP) and relapsing-remitting (RR) MS.

Background:

Several studies have reported on the measurement of changes in cytokines or cytokine message in the peripheral blood mononuclear cells (PBMC) of MS patients. There are no studies with systematic longitudinal measurement of cytokine production, cytokine mRNA and MRI measures of disease activity.

Design/Methods:

30 MS patients (15 with RR and 15 with CP MS) from the placebo arm of a randomized, double-blind, placebo-controlled, phase II study of linomide in the treatment of MS were studied. The patients (age range: 18-50) were examined at baseline,12, 24, 36, 48 weeks. Blood samples taken at the same time included: Cytokine production measured by ELISA, in pg/mL: TNF-a (after PHA stimulation), IL-1b (PHA stimulation), IFN-g(AntiCD3 ± IL-4); measurement of cytokine mRNA by RT-PCR normalized to a standard curve for IL-12, TGF-b1, TNF-a, IL-10, IFN-g, and IL-4. MRI scans were done at baseline, then every 4 weeks for 6 months. The following 6 parameters were measured: total number of gadolinium (GD)-enhancing lesions, number of new enhancing lesions, total number and total volume of T2 lesions, number and volume of new T2 lesions.We used a multiple regression model (generalized estimating equation), comparing either each time point to baseline, or each time point to the next. P-values 0.01 were required for significance.

Results:

We found significant correlations between changes in IFN-g message at consecutive time points and the change in number of T2 lesions (p=0.001), and the number (p=0.008) and volume (p=0.003) of new T2 lesions in RR patients. There were also strong correlations between changes in IFN-g message from to baseline values compared to number of new GD+ lesions (p<0.0001) and total number of T2 lesions (p<0.0001) in RR patients, and with number (p<0.0001) and volume (p=0.0014) of new T2 lesions in CP patients. We found a positive correlation between the change in TNF-a message and the change in volume of new T2 lesions on MRI in consecutive time points (p=0.009) in RR patients. The change in TNF-a production (by ELISA) between consecutive time points correlated with the change in number of new GD+ lesions (p=0.008) in RR patients and with total number of GD+ lesions (p=0.0009) in CP patients. There was an inverse correlation between change in TNF-a production and volume of new T2 lesions in CP patients. Changes in TGF-b message correlated inversely with the changes in number of GD+ lesions in RR patients (p=0.0014).

Conclusions:

Several strong correlations were found between the Th1 type cytokines (IFN-g and TNF-a) and MRI parameters, and an inverse correlation was found with the suppressive cytokine TGF-b. This longitudinal study confirms that immune changes in PBMC of MS patients are measurable and correlate with disease activity on MRI.

Supported By:

Upjohn and Pharmacia Inc., the National Multiple Sclerosis Society, the NIH, the Nancy Davis Center Without Walls


Glatiramer Acetate-Specific T Cell Lines Produce Brain-Derived Neurotrophic Factor (BDNF) after Activation upon Antigen Challenge In Vitro: A Novel Mechanism of Action ?

Wednesday, April 17, 2002
Tjalf Ziemssen, Tania Kuempfel, Wolfgang Klinkert, Oliver Neuhaus, Reinhard Hohlfeld Munich, Germany; Graz, Austria

Objective:

To demonstrate a new possible neuroregenerative mechanism of action of Glatiramer Acetate (GA, Copaxone) in the treatment of Multiple Sclerosis (MS).

Background:

GA, a standardized mixture of synthetic polypeptides, has beneficial effects on the clinical course and magnetic resonance imaging-defined brain lesions of patients with MS. As we and others have recently shown, one important mechanism of action may be the induction of GA-specific T-helper 2 (TH2)-like regulatory cells. Recent detailed immunohistochemical analysis of MS lesions revealed that beside various types of brain-derived neurotrophic factor (BDNF)-immunopositive neurons BDNF is primarily present in immune cells (T-cells, microglia, macrophages). On the other hand, the BDNF receptor gp14trkB is found in neurons in the immediate vicinity of MS plaques as well as in reactive astrocytes within the lesions. It is known from animal experiments that activated GA-specific T-cells are able to enter the brain.

Design/Methods:

Longterm GA-specific CD4+ T-cell lines (TCL) were selected from peripheral blood mononuclear cells of a GA-treated patient and a healthy donor by using the split-well technique. The TCL were characterized regarding their cytokine profile by intracellular double-fluorescence flow cytometry and T-cell receptor usage by different antibodies against Vb (variable region). BDNF production of the GA-specific TCL was analyzed after restimulation with GA on protein level by BDNF ELISA and mRNA level by reverse transcriptase-polymerase chain reaction (RT-PCR). Additionally we used intracellular fluorescence flow cytometry to demonstrate BDNF production.

Results:

We examined 2 TH1-, 1 TH0- and 1 TH2-longterm oligoclonal GA-specific TCL with different T-cell receptor usages of a healthy donor and MS patient. All examined GA-specific TCL showed an increased BDNF production in ELISA and in RT-PCR after incubation with GA-pulsed irradiated antigen-presenting cells. By intracellular fluorescence flow cytometry we demonstrated the intracellular production of BDNF in stimulated, but not unstimulated T-cells. In contrast to BDNF, we could not detect a significant production of nerve growth factor (NGF) by activated GA-specific T cells.

Conclusions:

In this paper, we show for the first time that human GA-specific T-cells are able to produce increased amounts of BDNF upon restimulation with GA in vitro independent of their TH1, TH2 oder TH0 phenotype. BDNF is one of the most potent factors supporting neuronal survival and regulating neurotransmitter release and dendritic growth. After reactivation in brain, GA-specific TH2-like regulatory T cells may not only provide protective anti-inflammatory cytokines such as interleukin-(IL)-4, IL-5, IL-13, and transforming growth factor, but also neurotrophic factors such as BDNF. Thus, GA treatment in MS may confer neuroprotection besides bystander suppression.


Myeloid Dendritic Cells Are Activated in Secondary Progressive Multiple Sclerosis

Wednesday, April 17, 2002
Arnon Karni, Samia J. Khoury, Guifung Cai, Gordon Freeman, David Hafler, Howard L. Weiner Boston, MA

Objective:

To investigate the activation and regulation of dendritic cells in multiple sclerosis (MS).

Background:

Dendritic cells (DC) are the most potent antigen presenting cells (APC) and play a central role in initiating immune responses. Thus far only DC derived in vitro from monocytes have been studied in MS. We investigated DCs taken directly from the blood of MS patients and characterized their state of activation and functional status.

Design/Methods:

The expression of HLA-DR, CD80, CD86, CD40, PDL-1 and CD83 and the intracytoplasmic production of IL-12 and TNF-a in CD11c+DC were studied ex-vivo and after overnight culture with 1, 10 and 100 ng/ml of IFN-g, IL-10 or LPS using flow cytometry. 46 untreated stable relapsing remitting MS (RR-MS), 15 untreated secondary progressive MS (SP-MS) and 22 healthy controls (HC) were studied.

Results:

As compared to RR-MS, in SP-MS we found the following: {1} An Increased percentage of CD11c+DC express CD80 and CD40 (CD80=48.4% vs.14.4%, p=0.030 and CD40= 39.4% vs. 20.2%, p=0.05. {2} Increased density of CD80 and CD40 per DC (mean fluorescence intensity-MFI of CD80=815.8 vs. 214.6, p=0.0001 and MFI-CD40=660.2 vs. 221.2, p=0.02). {3} Increased unstimulated IL-12 and TNF-a producing DC (IL-12= 6.7% vs. 0.8%, p=0.001 and TNF-a=10.9% vs. 0.9%, p=0.001). PDL-1, an inhibitory co-stimulatory molecule, is induced upon activation on APC. In stable RR-MS in which there was low expression of immune activation molecules (CD80 and CD40) on CD11c+DC, the percentage of PDL-1+ DC was increased (33.0%) compared with HC (10.3%, p=0.04). CD83 which is a maturation marker for DC was barely detectible in all the study groups (CD83+ DC <1%). We then tested the activation and regulation of DC by adding IFN-g or IL-10 and found: {1} Exogenous IFN-g up-regulated the percentage and density of CD80 in RR-MS and HC but not in SP-MS as CD80 was already expressed maximally prior to stimulation. {2} Exogenous IL-10 down-regulated the increased density of CD80 and CD40 on DC in SP-MS. {3} Exogenous IFN-g increased the percentage of IL-12 and TNF-a producing DC only in SP-MS to 9.0% and 25.6%, respectively. LPS increased the ratio of DC producing IL-12 and TNF-a in all the groups. {4} HLA-DR and CD86 were constitutively highly expressed on DC in all groups without significant differences and are both down-regulated by IL-10 in all the groups.

Conclusions:

CD11c+DC in SP-MS are activated as compared to RR-MS as measured by CD80 and CD40 expression and by production of IL-12 and TNF-a. CD11c+DC in stable RR-MS express high levels of PDL-1, an inhibitory costimulatory molecule. The differential response to IFN-g and LPS suggest that immune up-regulation of DC in SP-MS is related to T helper type-1 cells (IFN-g producers) rather than bacterial infection. These results demonstrate that the activation status of DC may play a key role in determining the type of immune activity in MS as well as response to immune therapy in different stages of the disease.

Supported By:

A.K is a research fellow of the National Multiple Sclerosis Society, NIH grant, Nancy Davis Foundation.


Intrathecal Antibodies to Neurofilament Light (NF-L) Correlate with MRI Markers of Axonal Loss

Wednesday, April 17, 2002
Eli Silber, Axel Petzold, M. Judith Eikelenboom, Richard M. C. Lazeron, Ed J. Tompson, Frederik Barkhof, Gavin Giovannoni, Mohammad Sharief, Chris H. Polman, Bernard M. J. Uitdehaag London, United Kingdom; Amsterdam, Netherlands

Objective:

To assess the relationship between antibodies to the light and heavy neurofilament components (NF-L & NF-H), as well as other possible markers of tissue damage, with MRI markers of inflammation and parenchymal damage.

Background:

Axonal loss is an important contributor to the progression of disability in multiple sclerosis (MS). MRI indicators of axonal loss include hypointense lesions on T1 weighted images (black holes) and cerebral atrophy, measured as a brain parenchymal or ventricular fraction. We have previously described elevated intrathecal production of antibodies to the NF-L, an important axonal cytoskeletal protein, but less so to NF-H in persons with primary (PP) and secondary progressive (SP) MS compared to those with relapsing-remitting (RR) disease. Other purported CSF markers of parenchymal damage include S100b, glial fibrillary acidic protein (GFAP), ferritin, and NF-L.

Design/Methods:

51 MS patients (21 RR; 20 SP; 10 PP) underwent MRI and LP. CSF levels of S100b, GFAP, ferritin, NF-L and NF-H were measured. CSF and serum antibodies to NF-L and NF-H and albumin were measured and indices of intrathecal antibody production calculated. MRI analysis included measurement of T2 hyperintense, T1 hypointense and gadolinium enhancing lesions, as well as the ventricular (VF) and parenchymal (PF) fractions as markers of parenchymal loss.

Results:

There were no detectable differences between levels of proteins measured or the NF-L and NF-H indices between the diagnostic groups. There were significant correlations between the anti-NF-L index and the PF (r= -0.51), T2 lesion load (r=0.41), VF (r= 0.37) and T1 Lesion Load (r= 0.37). In contrast the anti-NF-H index correlated only with the PF (r= -0.39). There were no correlations between any of the other proteins measured and MRI markers of inflammation or tissue damage.

Conclusions:

The correlation between antibodies to NF-L and MRI markers of inflammation and parencymal damage suggest that the anti-NF-L index may be a useful marker of axonal loss in multiple sclerosis.

Supported By:

Dr Silber was supported by the Special Trustees of Guy's & St Thomas' Hospital. This study was supported by a grant from the Friends of MS Research the Netherlands(MS 97-310) and UK MS Society.


Polymorphisms of Interferon g (IFNG) Contribute to Gender-Based Differential Susceptibility to MS

Wednesday, April 17, 2002
Brian G. Weinshenker, An Goris, Orhun H. Kantarci, Shirley Heggarty, Maria G. Marrosu, Colin A. Graham, Stanely A. Hawkins, David D. Hebrink, Elizabeth J. Atkinson, Mariza de Andrade, Koen Vandenbroeck Rochester, MN; Leuven, Belgium; Belfast, United Kingdom; Cagliari, Italy

Objective:

To assess the association of a microsatellite in the interferon g gene (IFNG) with MS and with gender-based differences in susceptibility to MS; to map this trait by linkage disequilibrium (LD) by evaluating other flanking microsatellites and intragenic single nucleotide polymorpisms (SNPs).

Background:

MS is a complex genetic disease that occurs two to three times as frequently in women than men. Interferon g is a Th1 cytokine the expression of which increases preceding exacerbations of MS. Expression of interferon g is higher in women compared to men with inflammatory diseases including MS. The (CA)12 allele of an intron 1 microsatellite has been associated with expression of interferon g. It is also negatively associated with MS susceptibility in men, but not in women in Sardinia.

Design/Methods:

We evaluated 122 unrelated Olmsted County MN patients and 244 ethnicity-matched controls, 312 Northern Irish MS patients and 192 healthy controls, and 217 Sardinian unrelated MS patients and their parents using eight microsatellites and three intragenic SNPs (established or novel). Association studies used case-control design for Olmsted County and Northern Irish populations; affected family-based controls (AFBAC) estimated the allele frequency in Sardinia. Mantel-Haenszel statistic was used to analyze the combined results from the three populations. A one-sided transmission disequilibrium test was used to test for negative association in male Sardinian patients; LD was calculated using the Arlequin v.2000 software; two marker haplotypes were constructed for adjacent markers.

Results:

In each population, the (CA)12 allele of the intron-1 CA repeat at position 761 of intron-1 was associated with MS susceptibility exclusively in men. The overall odds ratio for men heterozygotes was 0.55 (95% CI: 0.33-0.91) in the three populations and 0.25 (0.12-0.48) for homozygotes, suggesting an allele dose effect. Only in the Sardinian population was the association confined to patients with an HLA-associated susceptibility factor (DR3/4-). A two stage LD screen (final resolution of 1 marker per 36 kb) excluded other candidate genes, and bracketed the MS gender-specific susceptibility locus to a 118 kb interval centered on IFNG. A G/A SNP in the untranslated region, in LD with the CA repeat, was also associated with MS susceptibility exclusively in men in Olmsted County; as with the CA repeat, the association was greatest in homozygotes.

Conclusions:

IFNG polymorphisms are associated in case-control studies in three geographically distinct Caucasian populations with susceptibility to MS, exclusively in men. This may partially explain the predilection of women to develop MS. The locus of this gender-specific susceptibility trait is centered on and localized within 118 kb of IFNG itself in the Sardinian population. Two intragenic polymorphisms are associated with this trait. Functional studies are required to determine the specific allele(s) responsible for this association.

Supported By:

National MS Society (RG2870) and the International Federation of MS Societies (Du Pre Exchange grant)


A Basis for p53-Mediated Delayed Immune Injury of Oligodendrocytes in Multiple Sclerosis

Wednesday, April 17, 2002
Karolina Wosik, Josephine Nalbantoglu, Tanja Kuhlmann, Wolfgang Bruck, Jack P. Antel Montreal, QC, Canada; Berlin, Germany

Objective:

To determine mechanisms that underlie rapid and delayed immune injury of oligodendrocytes (OLs) in Multiple Sclerosis (MS).

Background:

OL apoptosis has been described in situ in certain types of MS lesions. We have previously shown that OL cell death can be rapidly induced in vitro by the overexpression of the transcription factor p53. P53 upregulation can occur in response to an array of cell insults, including exposure to inflammatory mediators, viral infection, trauma and ischemia.

Design/Methods:

Apoptosis in OLs in MS brain derived from autopsies and biopsies and its relation to p53 expression was determined by combining in situ tailing, and immunohistochemisty for p53 and myelin oligodendrocyte protein (MOG). For in vitro experiments, we used primary cultures of human adult oligodendrocytes isolated from patients undergoing temporal lobe resection for intractable epilepsy. To obtain upregulation of the transcription factor p53, we infected oligodendrocytes with an adenoviral vector containing a p53 expression cassette. We assessed death receptor expression by RNAse protection assay and Western blotting and apoptotic cell death by nucleosome ELISA.

Results:

Our in situ data demonstrate that the proportion of OLs showing p53 expression is significantly greater (40%) in lesions displaying OL apoptosis when compared to lesions showing OL preservation (4%). Our in vitro results demonstrate that rapid cell death of OLs, induced by high levels of p53, is accompanied by an upregulation of the death receptors Fas, DR4 and DR5. Blocking signalling through these death receptors could inhibit the p53-induced apoptosis. Protection was achieved using a broad-range caspase inhibitor, a caspase 8-specific inhibitor, a viral Bcl-2 homologue and an adenoviral protein characterized to elicit death receptor internalisation. Induction of lower levels of p53 was sublethal for OLs but rendered them susceptible to killing via exogenous FasL and TRAIL, the respective ligands for Fas and DR4/5.

Conclusions:

These data suggest a model whereby an initial sublethal injury or stress to the OL would result in an enhanced susceptibility to subsequent immune mediated injury. We postulate that an initial upregulation of p53 would result in expression of Fas and TRAIL receptors making the OLs susceptible to killing via FasL and TRAIL expressing immune effectors.

Supported By:

This work was supported by the National Cancer Institute of Canada and the Canadian Institute for Health Research


Tolerizing Myelin DNA Vaccination in the Prevention of Experimental Autoimmune Encephalomyelitis: Full Length Versus Minigene Constructs

Thursday, April 18, 2002
Paulo P. Fontoura, Hideki Garren, Pedro J. Ruiz, Lawrence Steinman Lisbon, Portugal; Palo Alto, CA

Objective:

To study the difference between using DNA vaccines encoding a full-length myelin gene as opposed to a minigene encoding the immunodominant epitope for that protein, in the prevention of experimental autoimmune encephalomyelitis (EAE).

Background:

Tolerizing DNA vaccination has been shown to be protective against the induction of autoimmune diseases in animal models. In EAE, preventive DNA vaccination with myelin genes such as proteolipid protein (PLP), myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) has been shown to suppress clinical disease, and to reduce its severity. It is not clear, however, if a tolerizing vaccine should encode the full-length myelin protein, or if it is enough to encode the immunodominant epitope for that protein in order to obtain the same clinical effect. The possibility that epitope spreading occurs in EAE, and that it contributes to clinical relapses, suggests that minigene vaccination might be less effective than a full-length DNA vaccine.

Design/Methods:

DNA vaccines for full-length murine MOG and MOG epitope 35-55 were cloned into the multiple cloning region of the pTargeT vector, driven by the CMV promoter. Large-scale DNA purification was performed, and all vaccines were administered at 100 g/animal intramuscularly, two days after bupivacaine (0.25%) injection into the same site. C57BL/6 female mice were immunized 1 and 2 weeks before the induction of EAE using MOG35-55 peptide in complete Freunds adjuvant, followed by two doses of Bordetella pertussis toxin. Mice were randomly divided into 4 groups: DNA vector alone, full-length MOG DNA, MOG35-55 DNA and carrier alone. m

Results:

Comparison between MOG and MOG35-55 immunized animals revealed a slight reduction in mean clinical severity in the MOG group, together with a reduction in disease incidence. MOG35-55 immunized animals did not recover as well from the first attack of EAE as MOG immunized animals. Lymph node cell proliferative responses against MOG35-55 peptide did not show any significant difference between the groups. An in vitro splenocyte cytokine production ELISA assay showed no significant difference in IL-2 or IFNg levels, but an increase in IL-4 production in MOG immunized animals, compared to MOG35-55 immunized animals.

Conclusions:

Myelin minigene vaccination does not appear to be as efficacious as full-length gene vaccination in the prevention of EAE. Neither vaccine suppresses T cell proliferation against myelin proteins, but full length MOG DNA induces a greater production of Th2 cytokines.

Supported By:

PF was supported by grants from the Luso-American Development Foundation (814/99) and the Gulbenkian Foundation (E/691/99). HG was supported by a NIH grant (1K08AI01492-01). PJR was supported by a NIH National Research Service Award grant (AI07290-15).


Neuropsychological Support for the Concept of White Matter Dementia

Thursday, April 18, 2002
Jose M. Lafosse, John R. Corboy, Maureen A. Leehey, Lauren Seeberger, Christopher M. Filley Denver, CO

Objective:

This study was conducted to investigate the concept of white matter dementia by identifying specific neuropsychological features that distinguish between subcortical diseases that affect primarily white or gray matter.

Background:

Dementia resulting from cerebral white matter involvement has been recognized by clinicians but is not fully characterized. Among the proposed features of white matter dementia is a distinctive profile of a retrieval deficit in declarative memory, similar to subcortical dementia, with preservation of procedural memory, similar to cortical dementia.

Design/Methods:

Sixteen patients with multiple sclerosis (MS), 16 with Huntington's disease (HD), and 16 normal controls (NC) took part. The MS patients had the relapsing-remitting subtype and had used immunomodulatory medications for less than a year. The HD patients were diagnosed on the basis of genetic testing. Groups were matched for age, Mini-Mental State Exam score, and education. Neuropsychological tests included the California Verbal Learning Test (CVLT) and two different skill learning tasks, one of which (Rotary Pursuit) is thought to depend on the striatum while the other (Mirror Drawing) is not.

Results:

ANOVAs on the CVLT indices demonstrated a significant difference among the 3 groups on initial learning, F(2,45)=15.0, p<.01, long delay free recall, F(2,45)=7.99, p<.01, and discriminability, F(2,45)=9.1, p<.01. Post-hoc tests revealed that the HD group scored significantly lower than the MS and NC groups on all 3 variables. Both patient groups performed better on recognition than on recall, indicating more difficulty with retrieval. On Rotary Pursuit, groups were matched on initial performance. Time-on-target scores were analyzed by ANCOVA with variables of group (MS, HD, NC), block (1, 2, 3), and trial (8 trials per block); trials were nested within blocks. The analyses controlled for the effects of movement disorder. Results revealed significant differences among the 3 groups, F(2,42)=9.8, p<.01. As predicted, follow-up tests revealed that the HD group demonstrated less skill learning than the MS group as evidenced by a significant effect of group, F(1,27)=26.27, p<.01, and a significant interaction between group and trial, F(7,196)=2.0, p<.05. The MS and NC groups demonstrated similar levels of skill learning. For Mirror Drawing, completion time scores and error scores were analyzed in separate repeated measures ANCOVAs as with Rotary Pursuit. Results revealed that the MS and HD groups demonstrated learning by both reducing completion time and making fewer errors across blocks and trials. Importantly, all 3 groups showed similar levels of learning.

Conclusions:

HD patients have a learning deficit relative to MS patients and normal individuals. Compared to NC, both MS and HD patients have a retrieval deficit in declarative memory, but only those with HD exhibit a deficit in procedural memory. These results suggest that involvement of subcortical white and gray matter can be differentiated neuropsychologically. These data also support the concept of white matter dementia as a distinct neurobehavioral syndrome.


Effect of Methylprednisolone on Mammalian Neuronal Networks In Vitro

Thursday, April 18, 2002
Matthias Wittstock, Reiner Benecke, Dieter G. Weiss, Dietmar Schiffmann, Uwe K. Zettl Rostock, Germany

Objective:

To study the effect of methylprednisolone on mammalian neuronal network activity in vitro.

Background:

Steroids like methylprednisolone (MP) are widely used for the treatment of immune mediated neurological diseases like multiple sclerosis (MS) and spinal cord (SC) injury. MP produces numerous effects on neuronal excitability, impulse generation, and conduction. Studies of SC network electrical activity showed that MP may protect damaged neurons by stabilizing altered membranes, enhancing lesion resealing, and limiting the spread of ion-mediated damage. Beside their therapeutical properties, high levels of glucocorticoids are toxic to the central nervous system and can produce adverse effects with possible neurological and psychiatric implications.

Design/Methods:

The effect of MP on mammalian neuronal networks was studied in vitro using the microelectrode array (MEA) technique. SC tissue dissociated from 14-15-days old mouse embryos were seeded onto MEA and maintained for one week in modified Eagles medium. The networks developed spontaneous activity after about 5-7 days, stabilised at 3 weeks. The recording device was connected to a multichannel neuronal data acquisition, amplification and recording system. MP (Urbason©, Adventis Pharma, Germany) was diluted with distilled sterile water to get a stock solution of 50 mg MP/1mL and added to the culture medium to yield final concentrations from 10 to 250 mg/mL corresponding to a molarity of 0.0267-0.667 mM. Cultures were incubated at 37°C. The activity was recorded for 30 min.

Results:

Application of MP increased the spontaneous bursting and spiking activity of SC networks grown on MEA. The data of 6 experiments were analysed. MEA age was 39 ±8 days. The experiments showed clearly a dose-dependent excitatory effect on network function occurring within 5 min after appilcation the MP solution. Spiking rate and burst firing were augmented, when increasing MP concentrations were added to the cultures. These effects were followed by a initially reversible and afterwards irreversible shut-off. The minimum shut-off concentration was 208 mg/mL ±74 MP. The effect was reversible by complete medium exchange within the range of 100 to 200 mg/mL MP. At higher MP concentrations the effect was not reversible.

Conclusions:

We demonstrate for the first time a dose-dependent excitatory effect of MP in cultured neuronal networks, followed by a shut-down of electric activity. MP concentrations reached in our model are quite similar to those used in the experimental autoimmune encephalomyelitis (EAE) model and equal to concentrations reached by high-dose MP pulse therapy in MS. This similarity is of special interest, since the effect of MP occurs within 5 min after addition of MP to the culture. The effects of high-dose MP therapy are suggested to be mediated by non-genomic effects with rapid onset changing the physico-chemical membrane properties. Therefore, the used experimental setting may be a promising tool in the evaluation of further potential neuroactive therapeutical agents without use of animal models.


Peroxisome Proliferator-Activated Receptor-gAgonist 15-Deoxy-PGJ2 Ameliorates Experimental Autoimmune Encephalomyelitis

Thursday, April 18, 2002
Asim Diab, Caishu Deng, Rehana Hussain, Paul D. Drew, Michael K. Racke Dallas, TX; Little Rock, AR

Objective:

To examine the role of PPARg in experimental autoimmune encephalomyelitis, an animal model for the human disease, multiple sclerosis.

Background:

Peroxisone proliferator-activated receptors (PPAR) are members of a nuclear receptor superfamily that include receptors for steroids, retinoids, and thyroid hormone. Previous studies dealing with PPARg expression in the immune system have been limited. Studies in experimental models of inflammatory bowel disease and arthritis have shown beneficial effects of PPARg ligands.

Design/Methods:

We examined whether the presence of PPARg agonist 15-deoxy-prostaglandin J2 (15d-PGJ2)during in vitro activation of MBP specific T cells affected proliferation, cytokine production, and their ability to adoptively transfer EAE. Cytokine production was examined using ELISA and ELISPOT. 15d-PGJ2 was used to treat ongoing EAE induced by active immunization. Inflammation in the CNS of treated animals was examined by immunohistochemistry.

Results:

15d-PGJ2 inhibited the proliferation of MBP-specific T cells in vitro. 15d-PGJ2 suppressed IFNg, IL-10, and IL-4 production by MBP-specific T cells and inhibited their ability to adoptively transfer EAE. Examination of the CNS during clinical disease showed PPAR g expression in the inflammatory infiltrate. Administration of 15d-PGJ2 prior to and at the onset of clinical signs of EAE significantly reduced the severity of disease.

Conclusions:

PPARg ligands may be a novel therapeutic agent for diseases such as MS.

Supported By:

This study was supported by NMSS, Yellow Rose Foundation and NIH.


Allergic Responses to Self-Peptides of Myelin in EAE: Evaluation of the Role of Mast Cells and Fc Receptors in Pathogenesis

Thursday, April 18, 2002
Rosetta Pedotti, Dennis Mitchell, Jochen Wedemeyer, Raymond Sobel, Rami Madanat, Guy Hermans, Christopher Lock, Stephen J. Galli, Lawrence Steinman Palo Alto, CA

Objective:

We have recently shown that, in experimental autoimmune encephalomyelitis (EAE), re-exposure of mice during recovery from paralysis to certain myelin peptides can cause fatal anaphylaxis. Aim of our study was to analyze the pathogenesis of allergic reactions to self myelin peptides and to investigate the role of these unexpected responses in the development of EAE. These studies may help identify ways of circumventing this undesirable reaction to therapy, and may potentially identify new targets for the treatment of EAE, and perhaps multiple sclerosis.

Background:

In EAE, re-exposure of mice during recovery from paralysis to certain myelin peptides can cause fatal anaphylaxis. Anaphylaxis appeared to be mediated by anti-myelin antibodies of the IgG1 isotype. IgG1 responses are promoted by Th2 cytokines that are elevated during the remission phase of EAE. The mechanisms underlying allergic responses to self are not fully understood and the role of these responses has never been investigated.

Design/Methods:

We induced EAE with myelin oligodendrocyte glycoprotein (MOG) 35-55 in mast cell-deficient (W/Wv), IL-4 -/-, FcgRIII -/- (FcgRIII a chain -/-), and FcgRIII/FceRI -/- (FcR gchain -/-) mice. Mice were scored daily for signs of EAE. Anaphylaxis was induced by challenge with intraperitoneal injection of MOG 35-55 six weeks after the induction of the disease.

Results:

In IL-4-/- -mice, we observed a worsening of EAE compared to the controls, while in W/Wv mice the disease was similar or slightly worse than that in the +/+ controls. In contrast, in FcgRIII a chain -/- and FcR g chain -/- mice, EAE was ameliorated. There was a decrease in the incidence of anaphylaxis, in the IL-4-/- and in the FcR gchain -/- mice, but not in the W/Wv or in the FcgRIII -/- mice, following challenge during the recovery phase of EAE.

Conclusions:

These results indicate that the mechanism underlying anaphylaxis to self MOG 35-55 peptide may be both IgG1/FcgRIII-and IgE/FceRI-mediated, but that it can occur independently of mast cells. In addition, deletion of FcgRIII or both FcgRIII and FceRI in mice can result in EAE. Our results, obtained in the EAE model, presented interesting correlation with gene microarray analysis we have performed on brain lesions from MS patients, in which we unexpectedly observed a differential expression of genes classically related to allergy. In conclusion, targeting functions of such Fc receptors and of other molecules involved in allergy may provide an effective strategy for treatment of EAE and, possibly, MS.

Supported By:

Supported by the NMSS grant No FG 1365-A-1


Riluzole Improves Motor Function in Experimental Autoimmune Encephalomyelitis (EAE) Mouse Model of Multiple Sclerosis (MS)

Thursday, April 18, 2002
Yossi Gilgun-Sherki, Danniel Offen, Hana Panet, Ronit Mosberg-Galili, Eldad Melamed Bat-Heffer, Israel, Israel; Kefar-Haroeh, Israel, Israel; Petah-Tikva, Israel, Israel; Tel-Aviv, Israel, Israel

Objective:

To examine whether treatment with riluzole, may have beneficial effect in myelin oligodendrocyte glycoprotein (MOG) induced experimental autoimmune encephalomyelitis (EAE).

Background:

Glutamate neurotoxicity was shown to play a role in the pathogenesis of multiple sclerosis (MS). Treatments with AMPA /kainate antagonists resulted in substantial amelioration of EAE, the animal model of MS in mice.

Design/Methods:

EAE induction was performed by immunization of mice with MOG peptide. Mice were observed daily and clinical manifestations of EAE were scored. Riluzole (10mg/kg*2/d i.p.), was injected during or after the appearance of symptoms, while control mice were injected twice a day with saline for the same period.
Hematoxylin and Eosin (H&E) and Luxol Fast Blue (LFB) staining were used for myelin loss assessment. Bielshowesky silver impregnation and Anti-nonphosphorylated antibodies (SMI-32) were used for evaluation of axonal damage. Proliferation of spleen cells in response to MOG was examined 3 weeks after EAE induction.

Results:

Pretreatment with riluzole significantly reduced the clinical severity of the disease, as compared to control mice (mean score 3.25 vs. 0.9, p[1t]0.0001). Moreover riluzole treatment started a week after the appearance of the symptoms, completely stopped the progression of the disease. Pathological and immunohistochemical results were in correlation with the clinical score. No difference between the control and treated groups were observed in T cell proliferative responses to MOG.

Conclusions:

Our results suggest that riluzole treatment, and probably other glutamate antagonists, might be beneficial in the treatment of MS.


Oral Glatiramer Acetate Treatment Reduces EAE Clinical Signs in Monkeys

Thursday, April 18, 2002
Rivka Kreitman, Arthur Malley, Rona Shofti, Ety Klinger Teva Pharmaceutical Industries, Israel; Oragon Primate Center; Medical School, Technion, Israel

Objective:

To test the effect of treatment with oral formulations of glatiramer acetate (GA) on experimental autoimmune encephalomyelitis (EAE) in monkeys.

Background:

GA for injection (Copaxone®) is an approved drug for RR-MS, involving a daily subcutaneous injection. EAE is an accepted model for testing of potential new therapies for MS. Subcutaneous injection of GA was shown to be effective in suppression of disease symptoms in various species including primates. TEVA is presently developing an oral formulation of GA for the treatment of RR- MS. The effects of oral treatment on disease symptoms in monkeys were tested with the clinical formulations (enteric-coated (EC) capsules and tablets). Monkeys were chosen because of the close phylogenetic relationship to humans, the similar pH in the gastro-intestinal track and the relatively large size of the animals.

Design/Methods:

EAE was induced in two monkey strains, Rhesus and Cynomolgus monkeys, by immunization with myelin basic protein (MBP) emulsified in complete Freunds adjuvant (CFA). The Rhesus monkeys were orally treated with various doses of glatiramer acetate administered in EC capsules, and the cynomolgus monkeys were orally treated with the EC tablets. The monkeys were treated on alternate days, starting 10 days prior to disease induction (5 treatments), and daily following disease induction. Monkeys were examined and scored once daily starting from day 10 following disease induction.

Results:

Rhesus monkeys : The EAE clinical signs appeared in Rhesus monkeys in about 24 days post-EAE induction. EAE in Rhesus monkeys is an acute mono-phasic illness leading to death within a short time period. Treatment of Rhesus monkeys with 1, 10 and 20 mg/day of GA in EC capsules, resulted in either complete or partial suppression of EAE clinical symptoms.
Cynomolgus monkeys: The EAE clinical signs appeared in Cynomolgus monkeys within 14 days post-EAE induction. Females were more susceptible to disease induction than males. In Cynomolgus monkeys the course of disease was variable. Under the same induction conditions the monkeys in the EAE control group displayed either a highly acute mono-phasic or a chronic relapsing disease. A dose-response study with GA EC tablets revealed that oral treatment with 5 mg GA/day had no effect on monkey EAE. Oral treatment with 10 mg/day of GA in EC tablets delayed the onset and reduced the severity and duration of the disease. The effect of treatment with 20 mg/day was not reproducible.
Analysis of serum samples revealed that the monkeys from both strains developed antibodies to MBP (the immunizing antigen) but not to GA.

Conclusions:

This study demonstrates that the oral formulations of GA in either EC capsules or tablets are effective in the treatment of acute and CR-EAE in monkeys. The differences in the dose-response relationship could be probably explained by the differences in monkey strains, disease profile and formulation forms. Both GA formulations were found to be safe in toxicology studies and the EC tablets are currently tested in RR MS patients.


Liposomal Prednisolone Pulse Therapy Leads to In Situ T Cell Apoptosis and Is Beneficial in Treatment of Experimental Autoimmune Encephalomyelitis

Thursday, April 18, 2002
Jens Schmidt, Josbert M. Metselaar, Marca H. M. Wauben, Gert Storm, Klaus V. Toyka, Ralf Gold Würzburg, Germany; Utrecht, Netherlands

Objective:

To investigate whether a long-circulating liposome formulation containing prednisolone is superior to methylprednisolone (MP) pulse therapy in induction of T cell (TC) apoptosis in situ.

Background:

In adoptive transfer (AT)- experimental autoimmune encephalomyelitis (EAE) ultra high doses of MP have been shown to be more efficient in induction of TC apoptosis than the "standard" dose of 10mg/kg used in MS therapy.

Design/Methods:

AT-EAE was induced in female Lewis rats by i.v. injection of 107 MBP-specific TC. We used long-circulating liposomes coated with polyethylene glycol, encapsulating prednisolone phosphate. 10mg/kg prednisolone-liposomes (PL) were applied i.v. at 42 hours and 18 hours prior to sacrifice. Another group received 50mg/kg MP i.v. at 18 hours and 6 hours before perfusion. Control rats received empty liposomes and/or saline at equivalent time points. TC or macrophages (Mf) in spinal cord were detected immunohistochemically in paraffin embedded tissue and apoptosis was assesed by the TUNEL assay and by morphological criteria. Student t test for grouped data was used for statistical analysis.

Results:

The rate of TC apoptosis in spinal cord tissue was significantly augmented by PL (39.4 ±6.8%, p<0.0001 vs. 16.1 ±4.3% in the control group, all data given as mean ±SD). MP as an internal control lead to a rate of 30.8 ±8.0% TC apoptosis (p<0.01 vs. controls). As a result of the increase in apoptosis TC infiltration was clearly reduced by PL (45 ±12 TC/mm2), which was statistically significant compared to controls (115 ±51 TC/mm2, p<0.05) as well as compared to MP (96 ±19 TC/mm2, p<0.05). As another aspect of inflammation the Mf infiltration was significantly reduced by PL (31 ±13 Mf/mm2) compared to controls (78 ±37 Mf/mm2, p<0.05) and compared to MP (66 ±25 Mf/mm2, p<0.05). Even though we chose the AT model to investigate rapid mechanisms, we could observe a therapeutical benefit from PL within 42 hours, achieving a clinical score of 2.8 ±0.2 compared to controls (3.2 ±0.3, p<0.01), which was superior to MP (3.2 ±0.3, p<0.05 vs. PL).

Conclusions:

Long-circulating PL given at 10mg/kg augment TC apoptosis in situ rapidly and the reduced infiltration of TC and Mf leads to an ameliorated disease activity of AT-EAE. As the liposomes can extravasate and accumulate in inflamed tissue with a disrupted blood-brain-barrier, PL could be a therapeutic alternative to MP, which needs a higher dosage and would therefore cause more systemic side-effects. These findings may have implications for the treatment of inflammatory autoimmune diseases of the CNS such as multiple sclerosis.


Chlamydia Pneumoniae Is Neurotropic and Increases the Severity of Experimental Allergic Encephalitis

Thursday, April 18, 2002
Subramaniam Sriram, Caigan Du Nashville , TN

Objective:

To examine the neurotropic properties of Chlamydia pneumoniae in experimental allergic encephalitis.

Background:

A close relationship between infections and the development and progression of autoimmunity is well known. Chlamydial infections following exposure to C. pneumoniae are known to be associated with a number of chronic diseases in humans. We have recently shown that C. pneumoniae infection is present in a number of patients with multiple sclerosis. Since there are many similarities in clinical and pathologic features of between MS and its experimental counterpart EAE, we examined the effect of systemic infection of C. pneumoniae on the course of EAE.

Design/Methods:

SJL mice were either immunized with mouse spinal cord homogenate (MSCH) in complete Freunds adjuvant or received myelin basic protein primed lymphocyte in order to induce EAE. Test mice received 5x105 live C. pneumoniae intraperitoneally. Control mice received either an equal number of heat killed organism or live C. trachomatis . Clinical and pathologic scores of disease severity were performed and the presence of C. pneumoniae in the spinal cord was determined by RT-PCR and immunohistochemical techniques.

Results:

Systemic infection with live C. pneumoniae but not heat killed C. pneumoniae or live C. trachomatis worsened acute EAE. Following adoptive transfer of MBP primed cells, all 6 mice infected with C.pneumoniae died of acute EAE, while only 2 of 6 mice died in the uninfected group. Mice infected with C. pneumoniae also showed an increase in the proliferative and pro-inflammatory cytokine response to myelin basic protein in mice immunized with the antigen. However there was no cross reactivity seen between MBP and C. pneumoniae antigens in mice infected with C. pneumoniae alone. Following infection, immunohistochemical studies showed the presence of C. pneumoniae but not C. trachomatis in the CNS of mice with EAE. Immunostaining of the organism was seen in perivascular regions and in the parenchyma of the spinal cord. The presence of replicating organism was confirmed by RT-PCR of the 16S RNA gene using primers specific for C. pneumoniae . Treatment with anti chlamydial agents decreased the severity of paralysis to that seen in uninfected mice with EAE.

Conclusions:

C. pneumoniae in the CNS can accentuate ongoing CNS autoimmune disease. The lack of cross reactivity between chlamydial and myelin antigens, the necessity of live infection and the response to antimicrobial therapy suggests bystander activation of immune cell as a mechanism underlying the clinical worsening of EAE following systemic infection with C. pneumoniae. The possibility of a similar phenomena ocurring in MS needs to be considered.


Tolerizing Myelin DNA Vaccination Reverses Active Experimental Autoimmune Encephalomyelitis by Deviation to the Th2 Pathway

Thursday, April 18, 2002
Paulo P. Fontoura, Hideki Garren, Pedro J. Ruiz, Lawrence Steinman Lisbon, Portugal; Palo Alto, CA

Objective:

To describe the use of myelin DNA vaccination in the treatment of active experimental autoimmune encephalomyelitis (EAE), and the possible role of cytokine co-vaccination in modulating the immune response towards a beneficial Th2 phenotype.

Background:

EAE is a model of T-cell mediated autoimmunity, with many features in common with multiple sclerosis (MS). Myelin-reactive Th1 T cells are central to the pathogenesis of EAE, and immunomodulatory treatments that shift the T cell phenotype to the Th2 pathway are capable of preventing and suppressing active disease. DNA vaccination is a very powerful method of eliciting tolerance against self-antigens, and is capable of preventing EAE in rodent models. Treatment of established EAE, however, would be much more relevant to MS patients, since prevention is not a feasible strategy at present. We hypothesized that using a tolerizing DNA vaccine encoding the immunodominant epitope for EAE, together with the delivery of a functional Th2 cytokine gene as a co-vaccine, would be able to suppress active disease through antigen-specific immunomodulation.

Design/Methods:

DNA vaccines for full-length murine myelin oligodendrocyte glycoprotein MOG) and murine interleukin 4 gene (IL-4) were cloned into the multiple cloning region of the CMV promoter driven pTargeT vector. All vaccines were administered at 100 g/animal intramuscularly, two days after bupivacaine (0.25%) injection into the same site. EAE was induced in C57BL/6 mice using the encephalitogenic peptide MOG35-55 in complete Freunds adjuvant (CFA), followed by two doses of Bordetella pertussis toxin. Mice with active EAE were randomized into four groups and treated with vector DNA alone, MOG encoding DNA, IL-4 encoding DNA, or a combination of MOG and IL-4 encoding DNA. The vaccines were administered at peak of first attack and one week after.

Results:

The administration of a combined MOG+IL-4 DNA vaccine was capable of dramatically suppressing active EAE, even if administered after peak of disease. The average percent incidence was reduced from 78% (vector DNA) to 38%, with a mean peak disease score of 1.8+-0.2 (p value 0.05) and a statistically significant reduction in the mean EAE score at several timepoints after treatment (p 0.05). Treatment with vector, MOG or IL-4 DNA alone was without effect in the disease course. To explore the role of the IL-4 co-vaccine, we performed an in-vitro translation assay using [35S]methionine, polyacrylamide gel electrophoresis (PAGE-SDS) and autoradiography, which confirmed the production of full length IL-4 protein. A Western blot for STAT6 phosphorylation, in draining lymph node lysates after IL-4 immunization, confirmed the local production of functional IL-4 and activation of its receptor.

Conclusions:

Myelin tolerizing DNA vaccination is an effective treatment for EAE when co-administered with a Th2 polarizing cytokine such as IL-4. The combination of the local delivery of IL-4 together with myelin encoding DNA causes the antigen-specific autoreactive T cells to change their phenotype to a protective Th2 phenotype.

Supported By:

PF was supported by grants from the Luso-American Development Foundation (814/99)and the Gulbenkian Foundation (E/691/99). HG was supported by a NIH grant (1K08AI01492-01). PJR was supported by a NIH National Research Service Award grant (AI07290-15).


Patients with Alzheimers Disease Have Increased Antibody Responses to Ab Peptide That Promotes Ab Aggregation.

Thursday, April 18, 2002
Michael Dobbs, Elizabeth Hall, Marina Tuzova, Melina Jones, Carol Anderson, Jerrold Woodward, William Markesbery, Charles Smith, Richard Kryscio, Frederick Schmitt, David Wekstein, Avi Nath Lexington, KY

Objective:

To characterize immune responses to amyloid beta peptide1-42 (Ab) in blood, CSF and brain tissue of patients with Alzheimer's disease (AD) and determine the effect of antibodies on Ab aggregation.

Background:

Animal models for AD show that immune responses to Ab can clear amyloid plaques and prevent neurotoxicity. However immune responses to Ab are poorly characterized in patients with AD.

Design/Methods:

Antibody levels were determined by ELISA to either preaggregated or unaggregated Ab in serum samples from patients with AD (n=16) and compared to age matched controls (n=31), patients with multiple sclerosis (n=11) or patients with HIV infection (n=11). Antibody levels were also determined in CSF obtained at autopsy of another 17 patients with AD and 31 matched controls. T cell proliferation was monitored in response to Ab in AD (n=11) and controls (n=2). Autopsy brain tissue was also stained for presence of IgG in AD (n=3) and matched controls (n=4). To determine if antisera to Ab can modulate its aggregation, IgG was purified and incubated with freshly dissolved Ab for 24 hrs and the amount of aggregation measured using thioflavin T dye.

Results:

Antibody titers to aggregated Ab in patients with AD were markedly elevated (P<0.003) while no significant difference was found in the two groups for antibodies to unaggregated Ab. 3/17 postmortem CSF samples from AD patients and 0/31 controls had antibodies to Ab. No significant T cell proliferation to Ab was seen in AD or control subjects. IgG was localized in several amyloid plaques either in the center, periphery or throughout the plaque. Purified IgG from patients with AD caused increased aggregation of Ab in the aggregation assay compared to controls (P<0.005).

Conclusions:

Patients with AD have increased humoral immune responses to Ab in the serum and CSF. Antibodies to Ab can be localized to the amyloid plaque where they likely enhance aggregation of the peptide.

Supported By:

NIH grants R01NS39253, P20RR15592, NS38428, P50AGO5144


Population Dynamics of Oligodendrocyte Progenitors Cells during Experimental Autoimmune Encephalomyelitis

Thursday, April 18, 2002
Jaime Imitola, Cristina Gutierrez, Evan Y. Snyder, Samia J. Khoury Boston , MA

Objective:

To investigate the dynamics of proliferation, apoptosis, differentiation of oligodendrocyte progenitor cells (OPC) and the relationship with axonal pathology during the experimental autoimmune encephalomyelitis (EAE) model.

Background:

Although previous reports have shown increased numbers and reactivity of NG2 positive OPCs in MS and EAE, it is unclear what the fate of these dividing cells is during progression of the disease. During chronic disease there is incomplete remyelination which is hypothesized to be due to 1) Apoptotic cell death of precursors cells by inflammatory mediators, 2) Lack of lineage progression of OPCs to myelinating oligodendrocytes, 3) Inhibitory effects of astrogliosis, or 4) The loss of axonal signals critical for OPCs differentiation due to axonal damage. However, to date in vivo confirmation of these hypotheses is lacking.

Design/Methods:

C57B/6 mice were injected with 150 mg of MOG peptide 35-55 and pertussis on day 0 and day 2 after immunization. Bromodeoxyuridine (BrDU) 120mg/kg was injected daily i.p. for 5 days beginning on day 20 post immunization or on day 35 during the chronic phase of the disease. Perfusion with paraformaldehyde was performed and the brain and spinal cord used for immunohistochemistry with staining for the specific oligodendrocyte precursor marker NG2,MBP and CNPase (oligodendrocytes), NeuN (neurons), and SMI32, a marker for axonal damage. Dividing and apoptotic cells were counted by fluorescent microscopy, Laser confocal microscopy with Z-reconstruction and morphometric analysis were used to confirm the phenotype of the cells.

Results:

Using morphometric analysis software, we found an increase in the relative amount of NG2 expression in the processes and cytoplasm of OPCs, and a marked proliferation of OPCs in spinal cord and brainstem lesions. Using BrDU labeling index, we found an increase in cell division of NG2 positive cells from 0.7% in control animals to 23% during the acute phase of EAE that was maintained during the chronic phase. We also found an increase in percentage of apoptotic OPCs in the spinal cord from 1% in controls to 24% during acute phase of EAE. To determine if there is OPC cell cycle arrest, we investigated the expression of the cyclin depend kinase inhibitors p21 and p27 during EAE. We found no OPCs expressing p27 and only occasional cells expressing p21, the percentage of new mature oligodendrocytes was less than 1%. We found evidence of axonal damage during EAE with a peak at day 30 post-immunization. Interestingly, reactive OPCs are associated with areas of axonal damage both in the white matter and in the gray matter, in close contact with neurons.

Conclusions:

Our data show that OPCs undergo extensive proliferation during EAE. However this proliferation is accompanied by increased rate of apoptotic cell death, with minimal generation of new oligodendrocytes. Our hypothesis is that apoptotic cell death and abnormalities in differentiation capacity of oligodendrocytes precursors may be important mechanisms for remyelination failure and disease progression in a chronic model of MS.

Supported By:

NIH and Nancy Davis Center without walls


Endogenous Neural Stem Cells Exhibit Repair Potential during Chronic Experimental Autoimmune Encephalomyelitis

Thursday, April 18, 2002
Jaime Imitola, Cristina Gutierrez, Evan Y. Snyder, Samia J. Khoury Boston , MA

Objective:

To investigate the effects of the inflammatory environment on neural stem cells and progenitors cells of adult mice during (EAE).

Background:

Stem cell niches are anatomical and functional microenviroments composed of stem, progenitor and support cells. Such niches in the adult mouse brain are the subventricular zone and the dendate gyrus. These zones are important for generation of neural cell diversity during development and during adulthood. In models of injury, after lysolecithin-induced demyelination and in a monophasic EAE cells from the subventricular zone (SVZ) were shown to engage in proliferation, migration and differentiation. However the behavior and phenotype of stem cells during chronic EAE is unknown.

Design/Methods:

C57B/6 mice were injected with 150 mg of MOG peptide 35-55 and pertussis on day 0 and day 2 after immunization. Bromodeoxyuridine (BrDU) 120mg/kg was injected daily i.p. for 5 days beginning on day 35 during the chronic phase of the disease. Perfusion with paraformaldehyde was performed and the brain and spinal cord used for immunohistochemistry with staining for the specific oligodendrocyte precursor marker NG2, antibodies against MBP (for myelin), Nestin (neural progenitor/stem cells), CNPase (mature oligodendrocytes), and NeuN (neurons). Dividing and migrating cells were counted by fluorescent microscopy, Laser confocal microscopy with Z-reconstruction and morphometric analysis were used to confirm the phenotype of the cells.

Results:

We found evidence of proliferating (BrdU+) cells in areas of inflammatory infiltrates and throughout the subventricular zones of the lateral ventricle and 4th ventricle, the ependymal region of the spinal cord and the dendate gyrus of the hippocampus. BrdU+ cells in the stem cell niches were identified as neural stem/progenitor cells, type B astrocytic stem cells, oligodendrocytes progenitor cells, and oligodendrocytes. The dorsolateral corner of the subventricular zone showed an increase in the numbers of BRDU+ cells from 10% in the control to 40% in EAE mice and we found evidence of emigration of neural progenitor cells from the dorsolateral corner to the corpus callosum, cells were found 150-200 microns from the SVZ compared to 20-30 microns of control animals. EAE increased the migration of neural progenitors through the rostral migratory stream to the olfactory bulb.

Conclusions:

Our data show that neural stem cell niches in the adult central nervous system respond to the inflammatory environment in EAE with proliferation of neural stem/progenitor cells and differentiation of some SVZ stem cell into oligodendrocytes. These results provide a basis for future strategies aimed at improving neural stem cells/progenitors engagement for repair during demyelinating diseases such as multiple sclerosis

Supported By:

Nancy Davis Center without walls and NIH


Experimental Autoimmune Encephalomyelitis after Adoptive Transfer of a Proteolipid Protein Peptide-Specific T-Helper-2 Cell Clone

Thursday, April 18, 2002
Stefan Nessler, Christine Stadelmann, Wolfgang Brueck, Alwina Bittner, Bernhard Hemmer, Norbert Sommer Marburg, Hessen, Germany; Berlin, Berlin, Germany; Merburg, Hessen, Germany

Objective:

To challenge the Th1-Th2 paradigm of experimental autoimmune encephalomyelitis.

Background:

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS). It is classically induced by transfer of CD4+ T-helper 1 (Th1)-cells specific for myelin proteins. A shift towards a T-helper 2 (Th2) response is usually associated with an amelioration of the autoimmune process in this model.

Design/Methods:

All experiments were performed in the SJL mouse strain. As myelin antigen we used the proteolipid protein peptide 139-151. Among more than 100 T cells clones (TCC) only 10% were able to induce clinical EAE. Most TCC had a Th0, some had Th1 phenotype. "3-3" is an encephalitogenic TCC with Th2 phenotype producing IL-4, IL-5, but no IFN-gamma.

Results:

Mice transfered with the Th2-clone "3-3" developed relapsing neurological deficits, similar to Th1- or Th0-induced EAE in this strain. Histologically there were large demyelinating lesions throughout the CNS containing plasma cells and macrophages with taken up myelin, very closely resembling chronic active lesions in MS. Moreover, serum antibodies against PLP peptide were detectable as early as 10 days after T cell clone transfer.

Conclusions:

The data show that - by contrast to the classical paradigm - Th2 cells are capable of inducing a characteristic form of EAE. The findings support the idea of pathogenetic heterogeneity in EAE and may have an impact on new therapeutic approaches.

Supported By:

Gemeinnuetzige Hertie-Sfiftung, Deutsche Forschungsgemeinschaft


Apoptosis of Spinal Cord Neurons in Experimental Autoimmune Encephalomyelitis

Thursday, April 18, 2002
Tanuja Chitnis, Jaime Imitola, Christina Benou, Samia J. Khoury Boston, MA

Objective:

To determine whether experimental autoimmune encephalomyelitis (EAE) is associated with neuronal death and whether such death is due to cell apoptosis or necrosis.

Background:

EAE is an animal model of multiple sclerosis characterized by inflammatory infiltrates in the spinal cord and brain. It has recently been shown that changes consistent with axonal damage are present in the CNS of diseased mice. However, it is unclear whether this pathology is associated with cell death or damage to the neuron itself, or whether these changes are limited to the axon alone. Neuronal cell death may be caused by apoptotic or necrotic mechanisms. Here we investigated the state of the neuronal cell body during EAE, and asked whether changes consistent with an apoptotic mechanism of cell death are present.

Design/Methods:

We immunized wild-type C57BL/6 mice with peptide 35-55 of myelin oligodendrocyte glycoprotein. Mice were perfused at various time points and spinal cords were harvested and fixed. Frozen thick (20mM) longitudinal floating sections were stained using TUNEL stain, anti-NeuN (neuron), anti-activated caspase-3 (apoptosis pathway) and anti-SNAP-25 (synaptic protein) antibodies. Expression of antibodies was assessed using fluorescence microscopy and the identity of the cells confirmed by confocal microscopy with morphometric analysis.

Results:

Spinal cord sections from naïve mice displayed prominent NeuN staining of neuronal nucleus and cytoplasm. Staining for NeuN was significantly decreased in sections from mice harvested on days 8, 17 (acute disease) and 30 (chronic disease) post-immunization, with a progressive decrease in stain intensity and loss of neuronal architecture. We observed apoptotic neurons (NeuN+ and TUNEL+) with maximal numbers in sections from day 8 and 17. Staining for activated caspase-3 was not observed in NeuN positive cells in sections from any timepoint. In addition, we have observed accumulations of staining for SNAP-25, a synaptic protein and constituent of the vesicle-docking complex, in both acute and chronic EAE spinal cord sections. This may indicate synaptic dysfunction during EAE.

Conclusions:

In addition to the reported axonal damage and loss, the autoimmune response in EAE is associated with neuronal cell body architecture disruption and apoptosis, as well as alterations in synaptic proteins. These findings have significant implications for understanding the pathogenic mechanisms of chronic neurological dysfunction in EAE, and should provide the rationale for development of new therapies targeted at the prevention of neuronal and axonal degeneration in multiple sclerosis.

Supported By:

The National Multiple Sclerosis Society, Nancy Davis Center without Walls, and NIH


Handicap-Independent and Severe Nocturnal and Diurnal Oxygene Desaturation in Amyotrophic Lateral Sclerosis. A New Surrogate Marker ?

Thursday, April 18, 2002
Mahmoud Charif, William Camu Montpellier, France, Metropolitan

Objective:

To determine whether amyotrophic lateral sclerosis (ALS) patient may suffer from early, and handicap-independent, oxygene desaturation and nocturnal or diurnal respiratory disturbances.

Background:

The origin of ALS remains largely unknown, except in familial cases carrying an SOD1 mutation. Recently, it has been shown that chronic hypoxia or hypoxemia in transgenic mice, induced by the deletion of the hypoxia-response element of the VEGF promoter, could lead to motoneuron degeneration (Oosthuyse et al., 2001). In the animal, the timing, clinical and pathological features were very similar to ALS in humans. It was demonstrated that the animals suffered from chronic hypoxia or hypoxemia in neural tissues.

Design/Methods:

We performed nocturnal oxymetry of ALS patients (n=79) and controls (n=44) using a NONIN 8500M. Controls were patients with other neurological disorders (multiple sclerosis, parkinson's disease, Alzheimer's disease etc..) matching for age, sex and handicap. The following parameters were documented : pulse, oxymetry, hypoventilations, apnea. All the patients presenting with an apnea syndrom were excluded. In a subgroup of ALS patients, without apnea, we also performed a diurnal examination (n=31).

Results:

There was a marked hypoxemia in ALS with 69 % of the night below 94% saturation vs 35 % in controls (p<0.00001). This hypoxemia did not correlate to handicap (evaluated by ALSFRS) or to vital capacity. All the patients had normal baseline PaO2 and PaCO2. As a large part of the ALS patients had a very steady profile of desaturation all night long, the measurement was prolonged during the day showing similar results with a mean chronic desaturation at 93%. In a few cases, two measurements could be done after a 6 month interval, in which handicap progressed. In all the cases the desaturation profile was the same. Before statistical analysis some patients were excluded because of apnea syndrom. They were 18 ALS patients and 15 controls, confirming that apnea is not a frequent feature in ALS.

Conclusions:

Compared with other patients suffering from similarly disabling neurological disorders, there is a highly significant nocturnal and diurnal oxygene desaturation in ALS that is not explained by the severity of handicap or by a decrease of vital capacity. As this abnormality is present even at very early stages of the disease, the possibility that such a desaturation could be a surrogate marker for ALS has to be considered and further validated. The observed hypoxemia may interestingly parallel with the recently published transgenic mouse model in which the hypoxia-response element of the VEGF promoter was deleted. This could give important clues for ALS etiopathogenesis and treatment.


Myokymic and Neuromyotonic Discharges - A Continuum

Thursday, April 18, 2002
Ludwig Gutmann, Laurie Gutmann, Gauri Pawar Morgantown, WV

Objective:

Myokymic and neuromyotonic discharges are the basis for the clinical phenomena of myokymia and neuromyotonia. These discharges have been defined as separate entities. This study examines this premise and, in particular, addresses the question that they might be a continuum.

Background:

Myokymic and neuromyotonic discharges occur in several clinical disorders in a focal or generalized fashion. They result in the clinical phenomena of myokymia and neuromyotonia. The discharges are characterized by the spontaneous rapid repetitive firing of single motor unit potentials and bursts recurring at regular or irregular intervals. The motor unit potentials within a myokymic burst are defined as firing at 5 - 150 Hz. In the neuromyotonic discharge they fire at 150 - 300 Hz.

Design/Methods:

Recorded myokymic and neuromyotonic discharges were collected at random from a large series of patients that included those with Guillain-Barre syndrome, multiple sclerosis, pontine glioma, focal neuropathies, autoimmune myokymia/neuromyotonia, timber rattlesnake envenomation, and radiation induced myokymia. The firing frequency within each burst of single motor unit potentials was measured.

Results:

Evidence for myokymic and neuromyotonic discharges as a continuum includes (1) both types of discharges could be seen in the same patient (eg. timber rattlesnake envenomation, autoimmune myokymia/neuromyotonia); (2) the same motor unit potential could fire as either type of discharge at different moments within the same patient (eg. autoimmune neuromyotonia); and (3) myokymic discharges were converted to neuromyotonic discharges by decreasing serum ionized calcium (eg. Guillain-Barre syndrome).

Conclusions:

The evidence presented supports the hypothesis that myokymic and neuromyotonic discharges are a continuum and have a similar underlying pathophysiologic mechanism. Both types of discharges appear in patients with either clinical neuromyotonia and myokymia syndromes; the same motor unit potential may fire in either frequency range; and myokymic discharges can be converted to neuromyotonic ones to by altering serum ionized calcium. The distinction between the 2 types of discharges may not be useful.


a(1,3)Fucosyltransferase-VII Deficiency Blocks Lymphocyte Recruitment into the Brain and Experimental Autoimmune Encephalomyelitis

Thursday, April 18, 2002
Laura Piccio, Elio Scarpini, Barbara Rossi, Donatella Ciabini, Linda Ottoboni, Carlo Laudanna, Jonathon W. Homeister, John B. Lowe, Gabriela Constantin Milano, Verona, Italy; Milano, Italy; Verona, Italy; Ann Arbor, MI

Objective:

The goal of this study was to determine the effect of different fucosyltransferase (FucT) deficiency on lymphocyte recruitment in brain venules and on the induction of experimental autoimmune encephalomyelitis (EAE).

Background:

a(1,3)Fucosyltransferases catalyze glycosylation of glycans that ligate endothelial E- and P-selectin and are responsible for leukocyte recruitment to inflammatory sites. Recent evidence suggests that downregulation of fucosyltransferase activity may represent an attractive target for therapeutic intervention aimed at blockade of chronic inflammatory diseases.

Design/Methods:

FucT-deficient mice (FucT-/-) for FucT-IV, FucT-VII and double deficient mice for FucT-IV & FucT-VII were generated on C57BL/6 genetic background. Intravital microscopy studies in brain microcirculation were performed in LPS- or TNFa-treated mice. EAE was induced in wt or FucTs deficient C57BL/6 mice by using MOG35-55.

Results:

Th1 lymphocytes were produced from wt or FucT deficient mice. Lymphocytes isolated from FucT-IV-/- mice efficiently tethered and rolled in brain postcapillary venules, when compared with cells isolated from wt mice. In contrast, primary adhesion of lymphocytes isolated from FucT-VII-/- or FucT-IV-/- & FucT-VII-/- mice was drastically reduced, suggesting that FucT-VII is critical for the recruitment of Th1 cells in brain microcirculation. Actively-induced EAE was significantly inhibited in FucT-IV-/- & FucT-VII-/- mice when compared with wt mice. The effect of single FucT-VII and FucT-IV deficiency on the induction of EAE will be also discussed.

Conclusions:

Our data unveil a critical role for FucT-VII in the recruitment of Th1 lymphocytes into the brain and suggest that fucosyltransferases may represent novel therapeutical targets for autoimmune diseases of the brain.

Supported By:

Fondazione Italiana Sclerosi Multipla, FISM, Genova, Italy; IRCCS Ospedale Maggiore and Associazione Amici del Centro Dino Ferrari, Milano, Italy; European Neurological Society, ENS, Basel, Switzerland.


Gabapentin in the Treatment of Tremor in Multiple Sclerosis

Thursday, April 18, 2002
Charles T. Bakhos, Mazen G. Jabre, Katia G. Habib, Raymond Chemaly, Boulos-Paul W. Bejjani HDF, Beirut, Lebanon; Byblos, Lebanon

Objective:

To assess the efficacy and tolerability of gabapentin in the treatment of tremor in multiple sclerosis.

Background:

Cerebellar tremor in multiple sclerosis is a disabling symptom, which rarely responds to pharmacological treatment. Gabapentin proved effective in essential tremor and in reducing spasticity in multiple sclerosis. The presence of abnormal utterances of GABAergic pathways in cerebellar tremor favors the consideration of gabapentin in the management of tremor in multiple sclerosis.

Design/Methods:

In this 14-week, prospective, single-blind, crossover, placebo controlled study, five multiple sclerosis patients with cerebellar tremor were randomly assigned to either gabapentin or placebo over six weeks with a two-week washout period. Patients were scored at baseline and at after six weeks of treatment using the Tremor Clinical Rating Scale. Evaluations were respectively performed at 1600 mg, 2400 mg of gabapentin or placebo.

Results:

Two patients withdrew prematurely, mostly because of adverse experiences (worsening hypotonia and somnolence). After 2 and 6 weeks of gabapentin therapy, the improvement of the Tremor Clinical Rating Scale score in the remaining three patients was not clinically significant from baseline score (17% and 21%, respectively) or placebo effect (18.5% after both periods). Nevertheless, two patients reported a marked functional improvement when on gabapentin therapy. Most reported adverse events were at 2400 mg of gabapentin and included: somnolence, increased appetite and improved sexual performances.

Conclusions:

Gabapentin was not clinically effective in the treatment of tremor in multiple sclerosis despite the reported functional gain in some patients. Disease stage, progression rate and extend of cerebellar damage can highly influence gabapentin effect. A larger controlled study is warranted to confirm our findings.


Repeated Treatment with Interferon Beta-1a In Vitro Results in the Attenuation of Cellular Responsiveness

Thursday, April 18, 2002
Susan E. Goelz, Stefan Dupont, Jaya Goyal, Marie Green Cambridge, MA

Objective:

To examine how different dosing regimes can effect biological responses to interferon beta (IFNb).

Background:

Interferons exert their biological effects by binding to a multicomponent cell surface receptor (IFNAR1 and IFNAR2). The activated receptor, in turn, induces signaling molecules [Signal Transducers and Activators of Transcription, (STATs)] as well as genes. It is currently unknown how the cells react to prolonged IFN exposure. We investigated the regulation of the IFN receptor, signaling molecules and IFN-induced gene expression in response to high or repeated doses of IFNb-1a using Jurkat T-cells and fresh human PBMCs in vitro.

Design/Methods:

Jurkat cells and an IFN-inducible reporter gene (ISRE-luciferase) were used as a model system to examine the effect of dose (7-1000 IU/mL) and frequency of IFN-beta treatment on cellular responsiveness. STAT and P-STAT levels were determined by western blot analysis. Mx protein (western blot) and mRNA (northern blot) levels were examined in peripheral blood mononuclear cells.

Results:

We show that IFNb treatment can render T cells and normal human PBMCs unresponsive to further IFNb treatment. In Jurkat cells, there is a clear dose-dependent reduction in IFNAR1 cell surface expression and reduction in IFN-dependent Stat-1 phosphorylation in response to relatively modest concentrations of IFNb (starting at 62.5 IU/mL). At this dose of IFN, there is no IFN-dependent gene expression. More interestingly, after extremely low doses (7 IU/mL), IFNAR-1 levels remained normal, but when the cells were retreated with IFNb, IFN-induced gene expression was markedly reduced. Although these pretreated cells remained refractory to further IFN treatment for at least 4 days, they were fully responsive by day 7. The non-responsive state of these cells correlated with high STAT1 and STAT2 levels. Similar results were seen in PBMCs; cells were only partially responsive to retreatment with IFN 48 hours after the initial dose.

Conclusions:

These data show that pretreatment of cells with IFNb can result in alterations in the signal transduction pathway that reduce biological responses to IFNb. Thus, in the context of using IFNs as therapeutic agents, increasing the amount or frequency of IFN may not yield desired biological effects. Therefore, issues concerning the dosage and the frequency of IFNb administration deserve careful consideration.

Supported By:

Biogen, Inc.


Interferon Beta-1a in Combination with Azathioprine and Low Dose Steroids for Relapsing-Remitting Mltiple Sclerosis - Preliminary Clinical and MRI Data from a 2-year Double Blind, Randomized, Placebo-Controlled Study

Thursday, April 18, 2002
Havrdova Eva, Dana Horakova, Jan Krasensky, Veronika Ticha, Iveta Novakova, Manuela Vaneckova, Ondrej Dolezal, Zdenek Seidl, Eva Meluzinova, Eva Pasekova Praha, Czech Republic

Objective:

To test the hypothesis of improving interferon beta (IFNB) efficacy by addition of classical immunosuppressants in the treatment of remittent multiple sclerosis (MS).

Background:

IFNB, azathioprine and steroids have been used for the treatment of relapsing MS, none of them being highly effective in all patients. Preliminary data from various studies and our experience suggest that combination of first-line drugs and drugs used in immunotherapy of other autoimmune diseases may be beneficial. Despite the use of disease-modifying drugs there is increasing evidence concerning ongoing brain atrophy in MS patients, the process which is not fully understood and maybe not connected with inflammatory activity.

Design/Methods:

Inclusion of 105 patients with remitting MS planned for final analysis. 85 patients already signed informed consent and were randomized into 3 groups to receive either 1. IFNB 1-a 30ug i.m./week + placebo A + placebo S, or 2. IFNB 1-a 30ug i.m./week + azathioprine 50mg/d + placebo S, or 3. IFNB 1-a 30ug i.m./week + azathioprine 50mg/d + prednisone 10mg every other day.
MRI scans are done every 8 weeks with 1-2mm slices, T1, T2 and PD weighted images, MTR, automatic measurement of lesion load and atrophy.

Results:

55 patients finished the first year of the study, 40 the second year (age 32.9 8.3 years, disease duration 5.8 5.9 years, relaps rate prior to the study 1.8, Kurtzke EDSS 2.35 1.25). 8 patients dropped out (ongoing disease activity,sustained clinical worsening), were switched to high-dose IFNB or immunosuppressants, and remained in follow-up. Kurtzke EDSS did not change, the relapse rate dropped down from 1.8 to 0.8 after the first year, and to 0.5 after the second year. Though the data are blinded, the baseline MRI data concerning the initial lesion load (LL) were divided into three groups (A.patients with entry LL 4ccm, B.4-10ccm, C: 10ccm). The increase in atrophy differed in these three groups (2 years data): group A with minimal entry LL progressed in atrophy by 0.4%, group B by 3.25%, group C with the highest LL progressed in atrophy by 2.5%. The increase in LL has not correlated (even in individual cases) with the progression in atrophy, being 60-80% in group A+B, and 20% in group C. It may support high variability of pathogenetic mechanisms underlying the development of hyperintense MRI lesion. MTR had a high predicitive value in patients who worsened. The MTR changes preceeded at least 6 months the clinical worsening and the development of typical T2 or FLAIR lesions.

Conclusions:

No unacceptable side-effects were observed in this study. Highly significant 56% drop in relapse rate after one year and 72% drop in two years seems to support the hypothesis on significant profit of combination therapy versus monotherapy. The MRI data support the likelihood to influence MS with early introduced immunomodulatory therapy.

Supported By:

Research project Plasticity of the brain - theoretical and clinical aspects, MSM CR 111100001.
MRI study partially supported by Schering-Plough CR.


Efficacy of Sildenafil Citrate in Men with Erectile Dysfunction and Multiple Sclerosis: Results of an Open-Label Study

Thursday, April 18, 2002
James R. Miller, Clare J. Fowler New York, NY; London, United Kingdom

Objective:

Sildenafil citrate (VIAGRA®) has previously been shown to improve erectile function in men with multiple sclerosis (MS) and erectile dysfunction (ED) in a placebo-controlled trial; this current study evaluates Viagra efficacy during an open-label extension (OLE).

Background:

The incidence of sexual dysfunction and specifically ED in men with MS is high, with reports ranging from 50% to 75%.

Design/Methods:

This was an OLE of a 12-week, double-blind (DB), placebo-controlled trial evaluating the efficacy and safety of Viagra for the treatment of ED in men with MS. Men were eligible if they completed the DB phase or if they discontinued due to lack of efficacy. Overall, 206 patients (placebo, 106; Viagra, 100) entered and 180 patients completed the OLE phase. Because the initial blind was not broken until completion of both phases of the study, all patients started the OLE with 50-mg Viagra. After 24 to 48 weeks (the OLE phase continued until Viagra became commercially available in the study country, but no longer than 48 weeks), efficacy was evaluated with a 3-item Global Efficacy Assessment; Q1: Has the medication you have been taking improved your erections? Q2: If yes, has the improvement in your erections allowed you to engage in successful sexual activity? Q3: When you took a dose of study drug and had sexual stimulation, how often did you get an erection satisfactory for sexual intercourse? Q3 was rated on a scale from 1 to 5, ranging from almost never/never to almost always/always.

Results:

Overall, 95% of patients had improved erections (Q1); of those patients, 95% also reported improved sexual activity (Q2). Patients who had received placebo in the DB phase showed a nearly 4-fold increase (97% vs. 26%) in improved erections (Q1) at the end of the OLE. An improvement in successful sexual activity (Q2) at the end of the OLE was reported by patients from both the DB placebo (96% vs. 73%) and DB Viagra groups (94% vs. 89%). However, since only patients who responded "yes" to Q1 were included here, these percentages are based on fewer patients from the DB placebo group (n=26) than from the DB Viagra group (n=81) answering Q2. For patients who received DB placebo, the frequency of erections more than doubled (mean score 4.35 vs. 1.98) and was similar to the frequency observed in patients who received DB Viagra (mean score 4.26) at the end of the OLE.

Conclusions:

The efficacy of Viagra in patients with ED and MS was sustained over periods ranging from 24 to 48 weeks.

Supported By:

Pfizer Inc


The Registry To Evaluate Mitoxantrone Effects in Worsening MS (RENEW): Status 4/2002

Thursday, April 18, 2002
Donald E. Goodkin, Kate Flanders, Mike Butine, Richard Stead Seattle, WA; Seatte, WA

Objective:

To describe the most recent data regarding cumulative dose and tolerability of Novantrone in the RENEW study.

Background:

On 10/13/2000, Novantrone was approved in the U.S. for treatment for patients with worsening RR, SP, and PR MS. As a post-approval commitment to the FDA, Immunex agreed to develop a registry to facilitate the objectives of RENEW, a multicenter (N=50), open-label, observational safety study of 500 patients with worsening RR, SP and PR MS who initiate commercially available Novantrone according to guidelines in the package insert.

Design/Methods:

Entry criteria: Patients with CD or LSD RR, SP, or PR MS who initiate Novantrone 12mg/m2 within 3 months of site-IRB approval, platelets 100,000 cells/ml, granulocytes <2000 cells/ml, age 18-65 yrs, negative pregnancy test. Exclusion criteria: history of CHF, left ventricular ejection fraction (LVEF) <50%, previous treatment with Novantrone, other anthracenediones or anthracyclines, mediastinal radiotherapy or TLI; AST, ALT, total bilirubin (LFTs) 2x ULN; current UTI or other severe untreated infection, nursing or pregnant women. Procedures: Treatment is administered every third month up to a cumulative dose of 140mg/m2. Medical evaluation, CBC with platelet count, and LFTs are conducted every third month during treatment and every year thereafter for a total of 5 years. LVEF is determined at baseline, prior to each dose above a cumulative dose 100mg/m2, and annually after therapy is discontinued.

Results:

At abstract preparation results were available for 71 patients enrolled from 2/09/01 - 08/20/01: (72% female), mean age 48 (28-64), mean EDSS 6.0 (1.5-8.5), mean LVEF baseline 62% (51-76), mean infusions 2 (range 1-3), mean cumulative dose 18 mg/m2 (range 10-36), RXd relapses 1; SAEs, 2 in one patient (neither therapy-related); patients with CHF 0, LVEF <50% 0, LVEF reduced 10% from baseline 0, therapy related leukemia 0, death 0. 235 patients were enrolled by 11/21/2001. Demographic, clinical, and tolerabilitly data for all patients enrolled by 02/23/01 will be presented.

Conclusions:

Novantrone has been well tolerated by 235 patients enrolled in the RENEW study through 11/21/01. RENEW will provide the most rigorously collected tolerability data in the United States for 500 patients treated with MITO 12mg/m2 every third month to a cumulative dose of 140mg/m2 and follow for 5 years.

Supported By:

Immunex Corporation


Sustained Efficacy of Interferon Beta-1a in Relapsing Multiple Sclerosis: 4-year Results from the European Dose-Comparison Study

Thursday, April 18, 2002
Ludwig Kappos, Michel Clanet, the European Interferon Beta-1a Dose-Conparison Study Group Basel, Switzerland; Toulouse, France

Objective:

To report the 4-year clinical results of interferon beta-1a (IFNb-1a) from the European IFNb-1a (Avonex®) Dose-Comparison Study.

Background:

The European Interferon Beta-1a Dose-Comparison Study was conducted to determine whether a higher weekly dose could improve upon the efficacy of IFNb-1a 30 mcg intramuscular (IM) in reducing the rate of sustained disability progression in patients with relapsing MS. After three years this trial did not detect any difference between the two doses in delaying progression of disability.

Design/Methods:

Patients who completed Part 1 (3 years of treatment) of the European IFNb-1a Dose-Comparison Study were given the option to continue double-blind treatment with IFNb-1a 30 mcg or IFNb-1a 60 mcg intramuscularly (IM) once weekly (Part 2) until the completion of the study. Analysis of 4-year data was performed on the following endpoints: the cumulative rate of sustained disability progression, extent of change in Expanded Disability Status Scale (EDSS) score, relapse rate, the percentage of relapse-free patients, IV steroid use, and NAB formation.

Results:

Of 608 subjects who completed Part 1, 491 subjects had 4-year follow-up and 446 completed 4 years of treatment and follow-up. IFNb-1a 30 mcg and 60 mcg IM once weekly were equally effective for up to 4 years, with no significant differences between doses on any of the clinical endpoints. The Kaplan Meier estimate of percentage of subjects who were free of disability progression by Year 4 was 52% in the 30-mcg group and 57% in the 60-mcg group (rate ratio, 0.89; 95% confidence interval [CI], 0.72 to 1.11; p = 0.32). After 4 years of treatment, the percentage of relapse-free patients was 18% in the IFNb-1a 30-mcg group and 19% in the IFNb-1a 60-mcg group; the difference between doses was not significant (p = 0.85). The incidence of patients who were positive for neutralizing antibodies at any time during the study was 2.3% in the 30-mcg group and 5.8% in the 60-mcg group (p = 0.011).

Conclusions:

The clinical efficacy of IFNb-1a in the 2 doses tested is similar and sustained over 4 years of treatment in patients with relapsing MS.

Supported By:

Biogen, Inc.


Results of the Long-Term (8-year) Prospective, Open Label Trial of Glatiramer Acetate for Relapsing Multiple Sclerosis

Thursday, April 18, 2002
Kenneth P. Johnson, Benjamin B. Brooks, Corey C. Ford, Andrew D. Goodman, Joseph B. Guarnaccia, Robert P. Lisak, Lawrence W. Myers, Hillel S. Panitch, Amy A. Pruitt, Norman Kachuck, Jerry S. Wolinsky Baltimore, MD; Madison, WI; Albuquerque, NM; Rochester, NY; New Haven, CT; Detroit, MI; Los Angeles, CA; Burlington, VT; Philadelphia, PA; Houston, TX

Objective:

To present results of an 8-year prospective open label trial of glatiramer acetate (GA) treatment of relapsing remitting multiple sclerosis.

Background:

In 1991-92, 251 relapsing-remitting multiple sclerosis (RRMS) patients were randomized in a placebo-controlled, double blind (DB) trial of approximately 30 months duration. This trial led to FDA approval of glatiramer acetate (GA) or Copaxone in 1996. At the end of the DB trial, placebo patients were crossed over to GA and a prospective, organized open label study was begun. Now beyond its 8th year, the study is the longest ever, prospective MS investigation.

Design/Methods:

Patients are evaluated for disability status by EDSS every 6 months. They are seen within 7 days of suspected relapse. The same neurologist and nurse coordinator complete the assessment of each patient.

Results:

The 142 patients continuing in the study have a yearly relapse rate of <0.2 or a relapse every 5 years whereas, prior to randomization, the yearly rate was 1.49. The majority, 65.3% of those always on GA, are improved or unchanged from their initial neurologic status while for those who received placebo for the first +/-30 months, 50.4% are improved or unchanged. The mean EDSS level for the entire cohort is 3.1, an increase of .5 step from randomization. When comparing those always on GA (72) with those crossed over after +/-30 months on placebo (70), there are still discernible differences (i.e. disability and brain atrophy) arguing for the need for early therapy. The daily subcutaneous injections of GA are well tolerated and no safety issues have emerged. Dropouts, which occurred for many reasons, such as relocation, pregnancy and disease worsening, will be discussed. The average MS duration was 7.0 years prior to randomization for the entire cohort, so this carefully studied population has had MS for approximately 15 years. Based on the neurologic disability experience documented in several MS natural history studies, one would expect progression in disability to an mean EDSS level of 4 with 50% reaching a level of EDSS 6 (need for full-time walking aids) in 15 years after diagnosis.

Conclusions:

In this long GA trial, the yearly MS relapse rate has fallen to a low level (approximately 1 in 5 years) while the percent of patients worsening has been low especially for patients always on GA. Discernible reduced clinical effects are seen for those who received placebo for +/-30 months. Natural history studies would have predicted a poorer outcome.

Supported By:

Teva Neuroscience, Inc.


A Population-Based Study of IL4 Polymorphisms in MS

Thursday, April 18, 2002
Orhun H. Kantarci, Janet L. Schaefer-Klein, Sara J. Achenbach, Elizabeth J. Atkinson, Cynthia T. McMurray, Brian G. Weinshenker Rochester, MN

Objective:

To study the association of the interleukin-4 gene (IL-4) with susceptibility, gender, age at onset, course and disease severity in MS.

Background:

T-cells that co-express surface B7-1 and intracellular IL-4 are decreased in patients with MS compared to normal subjects (Kipp B, et al. European J Immunol 2000;30:2092-2100). Endogenous IL-4 production increases resistance to EAE (Constantinescu CS, et al. Clin Immunol 2001;98:23-30). IL-4 overexpression in the CNS ameliorates clinical and pathological signs of EAE (Martino G, et al. J Neuroimmunol 2000;107:184-190). IL-4 (chromosome 5q23-31) is, therefore, a candidate gene for the traits related to susceptibility to or clinical features of MS.

Design/Methods:

We comprehensively scanned the promoter, exons 1-4 and their splice junctions by dideoxyfingerprinting (ddF), a hybrid of sequencing and non-denaturing electrophoresis, to detect single-strand conformational polymorphisms in 122 patients from a population-based cohort in Olmsted County, MN and 244 ethnicity and gender matched controls. We identified and analyzed two common single nucleotide polymorphisms (SNPs) in exon 1 [E1(33)CT] and intron 3 [I3(2580)CA] in addition to a previously known variable number of tandem repeat polymorphism in intron-3 [I3(671)VNTR] (allele frequencies 16.5%, 15.5% and 19.5%, respectively). Another SNP in intron-3 was rare [E3(149)CT, 2.5%], and hence was not studied further.

Results:

Not surprisingly, there was significant linkage disequilibrium between all of the polymorphisms in both cases and controls (p<0.001). We found that I3(671)VNTR was associated with susceptibility to MS (p=0.005). The uncommon allele 2 was associated with lower risk (OR:0.63, CI:0.39-1.01, p=0.052) and the frequent allele 1 was associated with increased risk (OR:5.17, CI:0.99-27.05, p=0.031) of MS. A trend for association with susceptibility was observed for I3(2580)SNP (p=0.097) but not for the E1(33)SNP (p=0.149). I3(671)VNTR allele 1 was also associated with more severe disease (p=0.026) in a multivariate model including disease course, gender and age at onset. Being a homozygote for E1(33)SNP was significantly associated with primary progressive (PP) disease course (OR:8.0, CI:1.54-41.47, p=0.005), a result that is fragile due to low number of individuals with PP MS(N=14). Homozygosity for each of the three polymorphisms was associated with a trend towards older age of onset; however, these findings were not statistically significant possibly due to low numbers of homozygotes . We did not confirm the previously reported association (Vandenbroeck K, et al. J Neuroimmunol 1997;76:189-192) between I3(671)VNTR and older age of onset in our study(p0.05). No association with gender was observed with either of the three polymorphisms (p0.05).

Conclusions:

I3(671)VNTR polymorphism of the IL-4 is associated with susceptibility to and severity of MS. These results should be confirmed in other studies.

Supported By:

Advanced fellowship award from the National Multiple Sclerosis Society (Dr. Kantarci). National Multiple Sclerosis Society, grant RG2870 (Dr. Weinshenker).


A Double-Blind, Placebo-Controlled, Modified Crossover Pilot Study of the Effects of Ginkgo Biloba on Cognitive and Functional Abilities in Multiple Sclerosis

Thursday, April 18, 2002
Christopher Kenney, Marc Norman, Mark Jacobson, Sara Lampinen, Dang P. Nguyen, Jody Corey-Bloom La Jolla, CA

Objective:

To determine if Ginkgo biloba affects cognitive and functional abilities in multiple sclerosis (MS) as measured by sensitive neuropsychological and self-report measures.

Background:

Evidence suggests that Ginkgo biloba may slow cognitive decline in dementing conditions, such as Alzheimer disease (Oken, 1998). Cognitive deficits contribute significantly to the functional impairment of patients with MS. To date, few pharmacologic interventions have been shown to stabilize or improve these deficits.

Design/Methods:

A double-blind, placebo-controlled, modified crossover pilot study of 23 patients with mild MS. Cognitive impairment at screening was defined on the basis of performance on the Digit Symbol and Letter-Number Sequencing tasks. One group was randomized to placebo for three months before crossing over to Ginkgo biloba (240 mg/day) for an additional three months. The second group was randomized to Ginkgo biloba for both three-month intervals. An extensive neuropsychological battery, including the Paced Auditory Serial Addition Test (PASAT), California Verbal Learning Test (CVLT II), and Delis-Kaplan Executive Measures Scale (DKEFS), was administered at baseline, 3 months and 6 months. Patients also completed subjective assessments of well-being, including the Beck Depression Index (BDI), the Multiple Sclerosis Quality of Life Index (MSQLI), and the Modified Fatigue Impact Scale (MFIS), at all three time points.

Results:

No statistically significant differences between the placebo- and Ginkgo-treated groups with regard to age, gender, education, or mean EDSS score were seen at baseline in 21 patients who completed three months of treatment. There have been no adverse events. In the Ginkgo-treated group, there was a statistically significant difference (p=0.04) in performance on the PASAT at 3 months (mean 120.2 = +/- 27.1) vs baseline (mean 105.2 +/- 23.6) that was not seen with placebo. There was also a statistically significant (p=0.02) deterioration in the placebo group on category fluency that was not seen in patients treated with Ginkgo. Ginkgo-treated patients showed statistically significant improvement (p=0.03) on the Perceived Deficits Questionnaire (PDQ) of the MSQLI. Interestingly, while there was no correlation between patients' self-report of cognitive abilities on the PDQ and performance on the PASAT, there was an extremely significant (p<0.0001) correlation (r=0.88) between patients' self-report of cognitive abilities and fatigue.

Conclusions:

Ginkgo biloba, in doses of 240 mg/day, is well-tolerated and may show a beneficial effect on attention, memory, and functioning at three months in patients with mild MS. Larger sample sizes and longer durations of treatment will be necessary to confirm and extend these preliminary findings.

Supported By:

Fellowship to Explore Complementary Therapies in the UCSD School of Medicine


A Randomized Study of Low Fat Diet withw-3 Fatty Acid Supplementation in Patients with Relapsing-Remitting Multiple Sclerosis (RRMS)

Thursday, April 18, 2002
Bianca Weinstock-Guttman, Monika Baier, Peterkin LeeKwen, Joan Feichter, Suzanne Dinehart, Jaya Venkatraman, Kulwara Meksawan, Mary Rensel, Pamela Bochiechio, Carol Brownscheidle, Frederick Munschauer, Richard Rudick Buffalo, NY; Denver, CO; Cleveland, OH

Objective:

To determine whether a low fat diet supplemented with w-3 long chain polyunsaturated fatty acids (PUFA) positively affects quality of life in patients with RRMS

Background:

Dietary manipulation is beneficial in patients with cardiovascular disease, and lipid modification has recently attracted attention in autoimmune diseases. PUFA and their derivatives are considered potent modulators of the immune and inflammatory responses. The benefits of dietary intervention in MS have not been rigorously studied.

Design/Methods:

Patients with RRMS were randomized into a 1-year single-blind controlled study comparing two dietary interventions: Group 1 received a very low total fat diet (15% of daily calories) with supplemental w-3 PUFA (6 fish oil capsules/day). Group 2 received the American Heart Association Step I diet (total fat<30% of daily calories) with placebo supplements (6 olive oil capsules). Patients were on interferon b or glatiramer acetate for at least 2 months before entering the study. The Physical Components Summary Scale (PCS) from the Short Form Health Survey Questionnaire (SF 36) was the primary outcome measure supplemented by the Modified Fatigue Impact Scale (MFIS) and the Mental Health Inventory (MHI). Physician rated secondary outcome measures were the Kurtzke EDSS and relapse frequency. Adhesion molecules (ICAM-1, VCAM), prostaglandin PGE2 and leukotriene LTB4 plasma levels were determined using an enzyme linked immunosorbent assay (ELISA).

Results:

This is a preliminary analysis on the first 23 patients. Baseline characteristics were similar in group 1 vs. group 2, except for MFIS: (gender 81.8% vs. 83.3% female; age-mean 44.8 vs. 42; disease duration-3.9 vs. 3.3; EDSS 1.95 vs. 2.09, SF-36/PCS: 44.7 vs. 40.3, MFIS 57.4 vs. 37 (p=0.02), and MHI 86.3 vs. 75). Mean follow-up was 8 months (range 1-12 months). Eight patients completed the 1yr study, 16 completed 6 months. Patients tolerated the diet well, but no significant difference was seen in the quality of life measures examined over time. However, there was a significant decrease in number of relapses in Group 1 patients compared with their relapse rate 1 yr prior to the study: -.64 (SD= 0.5) (p=0.0019). In contrast, there was no difference in Group 2 patients. Significantly less disease progression was seen in group 1 (p= 0.0306). When comparing the levels of immunological parameters, a significant decrease in ICAM-1 from baseline was seen in the 15% diet group (p=0.0098) as well as the 30% group (p=0.0358). PGE2 levels showed a significant decrease only in the 15% diet group (p=0.04). The change in ICAM-1 levels was persistent in the 15% diet group patients that finished their one year study (p=0.04).

Conclusions:

This preliminary analysis suggests that very low fat diet (15%) with supplemental w-3 PUFA was very well tolerated and may have a beneficial effect on disease parameters in patients with RR-MS.

Supported By:

National Multiple Sclerosis Society and Mellen Center-Cleveland Clinic Foundation


Single Centre, DBPC, Randomised Trial of Interferon b1b in Primary Progressive and Transitional Multiple Sclerosis: An Exploratory Phase II Study

Thursday, April 18, 2002
Xavier Montalban, Luis Brieva, Mar Tintore, Cecilia Borras, Jordi Rio, Carlos Nos, Xavier Aymerich, Juli Alonso, Rosalia Horno, M. Jose Vicente, Alex Rovira Barcelona, Spain

Objective:

To investigate safety and hints of efficacy of interferon b1b (IFN) given to patients with primary progressive (PPMS) and transitional multiple sclerosis (TMS)

Background:

The beneficial effects of interferon b have only been shown for patients in the relapsing phase of MS. The role of interferon b in the treatment of patients with secondary progressive MS still remains a controversial issue. The single phase II randomized controlled trial on PPMS using IFN b1a (IM) shows no significant treatment effect on EDSS though some effect on T2 lesion load.

Design/Methods:

73 patients (49 PPMS and 24 TMS) with EDSS scores of 3.0 to 7.0, were enrolled and randomized to receive either 8 million IU of IFN b1b or placebo administered every other day subcutaneously for 2 years. Safety parameters including the Ashworth spasticity, Krupp's fatigue and Depression Inventory scales and blood tests were performed three monthly. Clinical outcomes (EDSS and MS Functional Composite (MSFC)) were also performed three monthly and the Sickness Impact Profile six monthly. MRI measures (T2 and T1-weighted brain lesion load, brain parenchymal fraction (BPF), active lesions, spinal cord atrophy, MTR and spectroscopy) and neuropsychological assessment (BRNB) were undertaken annually.

Results:

Adverse events significantly associated with IFN-b included injection-site reaction, flu-like symptoms and lymphopenia. One patient on the placebo arm died because of pulmonary infection. In all, 96% of the patients reached study end and 93% completed the treatment period of the study. Treatment groups were comparable on all baseline variables. The proportion of patients with confirmed progression at 3 months was 27.8% in the IFN arm and 37.8% in the placebo arm (p= 0.3135). Statistically significant differences were found for T2 (p=0.006) and T1 (p=0.001) lesion load and number of active lesions (p=0.005) in favor of the IFN-treated group. MSFC and BPF data are presented.

Conclusions:

IFN-b1b is safe in the treatment of patients with PPMS and TMS. Our study seems to point to a beneficial effect of IFN-b1b on MRI parameters in this group of patients.


Inflammation and Oligodendrocyte Vulnerability: Interleukin-1 b Excitotoxicity

Thursday, April 18, 2002
Jennifer L. Takahashi, Leonie A. Boven, Christopher Power, Kenneth Warren, Wee Yong Calgary, AB, Canada; Edmonton, AB, Canada

Objective:

1) To evaluate the expression of IL-1 b, a prototypic pro-inflammatory cytokine, in MS to support the relevance of IL-1b in MS pathology. 2) To investigate whether IL-1b can kill mature oligodendrocytes (OL) in OL-astrocyte co-culture. 3) To examine the mechanisms by which IL-1b mediates OL death.

Background:

Glutamate excitotoxicity contributes to the pathology of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Recent reports have demonstrated OL protection, axonal sparing and reduced EAE severity with AMPA/kainate receptor blockade by NBQX. In MS, glutamate excitotoxicity is inferred by the report that glutamate CSF levels are elevated 2-fold in MS patients compared to normal controls. Furthermore, OLs are highly vunerable to AMPA/kainate receptor-mediated toxicity in vitro. Nonetheless, little is known about how excitotoxicity occurs in the context of inflammation. We hypothesize that inflammatory cytokines, represented by IL-1b, may facilitate OL excitotoxicity. For example, IL-1b has been shown to impair the glutamate buffering capacity of astrocytes.

Design/Methods:

1) IL-1b mRNA expression in post mortem brain tissue from 6 cases of MS and 6 normal controls was determined by real-time PCR. 2) A mouse astrocyte-OL co-culture system was used to evaluate the effects of 24hour stimulation with IL-1b at various concentrations in serum-deficient medium. DNA fragmentation, indicative of apoptosis, was determined by TdT-mediated nick end labeling (TUNEL) and further confirmed by chromatin condensation shown by Hoescht staining. Apoptotic OLs (TUNEL+ GalC+) were recorded as a percentage of 200 GalC+ OLs evaluated per well. A minimum of 4 wells comprised each treatment group.

Results:

1) IL-1b mRNA levels were elevated 3-fold in the MS brain tissue versus normal controls (p<0.05). 2) IL-1b induced OL apoptosis in astrocyte-OL co-culture in a dose-dependent manner. At 100U/ml, IL-1b resulted in a 2.5-fold increase in OL apoptosis compared to the control co-culture (9.1% versus 3.6%, respectively, p<0.05). The toxicity of IL-1b in co-culture was attenuated by pre-treatment with IL-1 receptor antagonist (p<0.05), indicating that this toxic effect was mediated through IL-1 receptors. Furthermore, NBQX (p<0.05), but not MK801, decreased IL-1b-induced death, demonstrating that IL-1b toxicity acts in part through an AMPA/kainate receptor mechanism. Inhibition of iNOS with Nw-L-arginine (LNA) had no effect on IL-1b toxicity, suggesting that NO does not contribute to OL death in this system.

Conclusions:

Elevation of IL-1b in the CNS of MS patients may contribute to OL injury and death through a mechanism that involves glutamate excitotoxicity. Current experiments seek to define the chronic source of IL-1b in the CNS of MS patients and to evaluate how IL-1b affects extracellular glutamate homeostasis.

Supported By:

Canadian Institutes of Health Research


Safety and Tolerability of Mitoxantrone Use in MS: Phase IV Experience in 170 Patients Receiving Single or Combination Treatment

Thursday, April 18, 2002
Saud A. Sadiq, Andrew Sylvester, Geraldine Sim New York, NY

Objective:

To establish safety and tolerability of use of mitoxantrone alone and in combination with other disease-modifying agents. In addition to clinically follow subset of patients discontinuing mitoxantrone after receiving cumulative maximum dose of 140mgs/m2.

Background:

Mitoxantrone is a FDA-approved medication for use in relapsing-remitting and secondray progressive MS. Its safety profile when used in combination with other immunomodulator medications is unknown. Also, because of its cardiac toxic effects the life-time maximum dose is limited to 140mgs/m2 and it is not known how long patients in remisson remain inactive after discontinuation of mitoxantrone.

Design/Methods:

We used mitoxantrone in 170 patients with clinically definite MS who continued to have disease activity despite receiving standard first line MS medications. Mitoxantrone was given as an IV infusion at a dose of 12mgs/m2 every 3 months and all patients who received at least two doses are reported. All patients had a CBC drawn before and 10 days post infusion. Every patient had an echocardiogram at start and after every 4 doses of treatment and only patients with LVEF of 50% were treated. Patient groups included those receiving mitoxantrone alone (N=40), or in combination with pulsed IV methylprednisolone (N=50), or monthly IV immunoglobulin gamma (N=40), or weekly IM binterferon 1A (N=40).

Results:

Common adverse effects(10% of patients) included alopecia, nausea, and asthenia of which asthenia was the most disabling. Lecopenia occurred in about 50% of all patients and in the various combination groups except when mitoxantrone was used concurrently with binterferon 1A when almost all patients had WBC counts of < 2000 at 10 days post-infusion. No serious infections occurred although urinary and respiratory infectiopns responding to oral antibiotics were seen in 30% of patients. Asymptomatic cardiac changes on echocardiograhy were seen in 5% of patients.
The most common reason for discontinuing treatment was a lack of efficacy of mitoxantrone seen mostly in patients with primary progressive disease. About 5% of patients discontinued medications because of an unacceptable degree of asthenia. Twenty patients discontinued mitoxantrone after receiving greater than 140 mgs/m2 cumulative dose and follow up periods ranging from 6-9 months show continued disease stabilization. No deaths or life threatening effects were seen in any of the 170 patients.

Conclusions:

Mitoxantrone is safe when used alone and in combination with other disease modifying treatments. It is generally well tolerated but intolerable asthenia occurs in about 5% of all patients. Disease stabilization induced by mitoxantrone appears to last for at least six months post treatment and continued follow-up will provide more definitive data about long term efficacy.

Supported By:

Not supported


Alpha Lipoic Acid Inhibits Matrix Metalloproteinase-9: Implications for Effects on T Cell Migration and Treatment of Multiple Sclerosis

Thursday, April 18, 2002
Gail Marracci, Gabriel McKeon, Richard Jones, Dennis Bourdette Portland, OR

Objective:

To investigate the mechanism of action of alpha lipoic acid (ALA) in suppressing experimental autoimmune encephalomyelitis (EAE) and explore its potential as a therapy for multiple sclerosis (MS)

Background:

ALA is a natural antioxidant that suppresses and treats EAE and inhibits T cell migration into the spinal cord (SC) in SJL mice following immunization with proteolipid protein (PLP) 139-151 (Neurology 56:A67, 2001; manuscript submitted). ALA has several different antioxidant properties, including the ability to chelate metallic ions involved in redox reactions, and therefore might be capable of inhibiting metalloproteinases (MMP) needed for T cells to enter the SC.

Design/Methods:

To test for direct effects on MMP-9, supernatants from antigen stimulated SJL PLP 139-151 specific T cells were analyzed for MMP-9 activity using gelatinase zymography in the presence or absence of varying concentrations of ALA. In addition, human PBMC were stimulated in vitro for 48 hours with Con A in the presence or absence of ALA at varying concentrations and the supernatants from these cultures were tested for MMP-9 activity. Experimental samples were electrophoresed on a 10% Tris-Glycine gel with 0.1% gelatin incorporated as substrate. A clear zone representative of gelatinase activity was visualized after Coomassie blue staining. Zymograms were scanned and band intensity analyzed using OptiQuant software.

Results:

SJL PLP specific T cell supernatants had reduced MMP-9 activity upon incubation with ALA in a dose dependent manner. Zymography revealed a reduction in MMP-9 activity as follows: 0% inhibition occurring at 10 mg/ml, 42% inhibition at 100 mg/ml and 57% inhibition at 400 mg/ml. In addition, supernatants collected from human PBMC stimulated with Con A in the presence of ALA displayed a reduction in MMP-9 activity as follows: 3% inhibition at 25 mg/ml, 51% at 100 mg/ml, 66% at 200 mg/ml, and 97% at 400 mg/ml.

Conclusions:

The ability of ALA to suppress MMP-9 activity may explain its ability to inhibit T cell migration into the spinal cord and its clinical benefit in EAE. ALA also inhibits human MMP-9 activity and therefore might be capable of altering T cell trafficking into the central nervous system in MS. Based on its beneficial effects in EAE and its ability to alter the activity of human MMP-9, ALA may warrant investigation as a treatment for MS.

Supported By:

Supported by NIH grant P50AT00066, Department of Veterans Affairs and the Nancy Davis Center Without Walls


Reduction in Gadolinium Enhancing Lesions, and Clinical Stability in Multiple Sclerosis Patients Receiving a Combination of Cyclophosphamide and Filgrastim (G-CSF) in Preparation for Stem Cell Mobilization

Thursday, April 18, 2002
Mark S. Freedman, Harold L. Atkins, Douglas Arnold, Marjorie J. Bowman, the Canadian MS BMT Study Group Ottawa, ON, Canada; Montreal, QC, Canada

Objective:

To demonstrate the safety of stem cell mobilization for autologous stem cell transplants in multiple sclerosis using a combination of cyclophosphamide together with filgrastim (G-CSF).

Background:

There have been previous reports of clinical exacerbations in both rheumatoid arthritis and multiple sclerosis patients receiving G-CSF alone, in preparation for stem cell mobilization as part of the lead up to autologous stem cell transplantation (ASCT) as a treatment for the autoimmune disease. This has been attributed to activation of inflammatory immune cells by the G-CSF. We postulated that the co-administration of a potent chemotherapeutic agent such as cyclophosphamide with G-CSF would prevent the activation of the immune cells and guard against clinical exacerbations.

Design/Methods:

A phase II study targeting MS as an autoimmune disease with intensive immunoablative therapy and immunological reconstitution using ASCT has begun in a tri-city Canadian study involving 32 MS patients, 24 of whom will receive the active therapy and 8 who will receive "best medical treatment". This non-randomized study began in 2001 with a staggered entry of patients to assure safety. Following an initial back-up bone-marrow harvest, stem cell mobilization is initiated using 4.5g/m2 cyclophosphamide i.v. followed by 10 days of filgrastim (G-CSF) (10mg/kg/day) s.c. Clinical and neurological assessments and MRI studies using gadolinium are performed prior to entry, at baseline and between 7 and 10 days following the initial cyclophosphamide dosing. Stem cells are then collected by leukopheresis.

Results:

All patients were clinically stable at entry and throughout the first phase of treatment. No clinically apparent relapses or worsening was noted. Gadolinium enhancing lesions were seen on MRI in all patients at entry. In all three patients, a marked reduction in the number of enhancing lesions was noted on MRI studies performed during the administration of G-CSF. Successful highly purified stem cell grafts were obtained as well from these first three patients. Detailed results of these and probably a further 2-3 patients will be presented.

Conclusions:

These results confirm the safety of using G-CSF stem cell mobilization in MS patients after treatment using high dose cyclophosphamide, with no observed exacerbations either clinically or by MRI. The high dose cyclophosphamide regimen is probably responsible for the marked reduction observed in the number of gadolinium enhancing lesions. Mobilization using this procedure yields successful grafts for stem cell transplantation.

Supported By:

A grant from the Multiple Sclerosis Foundation of Canada.


Ability of Frequent MRI Activity Measures to Detect a Treatment Effect of Interferon beta-1a in a 50 Patient Cohort

Thursday, April 18, 2002
Guojun Zhao, David K. B. Li, Donald W. Paty, UBC MS/MRI Research Group Vancouver, BC, Canada

Objective:

To examine the ability of using a small patient cohort in detecting the treatment effect of interferon (IFN) on various MRI activity measures in secondary progressive MS (SPMS).

Background:

In a randomized controlled trial of IFN in SPMS (SPECTRIMS, Neurology 2001; 56:1505-1513), treatment reduced median numbers of active lesions per patient per scan with both semiannual T2 activity in 618 patients and 11 monthly combined unique T1 and T2 activity in 283 patients. In the original IFN beta-1b study (Neurology 1993; 43:662-667) a significant reduction in the appearance of new T2 lesions occurred in 52 patients who had frequent MRI (every 6 weeks).

Design/Methods:

50 patients from 1 center, with clinically definite or laboratory-supported SPMS, randomized to receive low dose (LD) 22 or high dose (HD)44 mcg IFN beta-1a (Rebif®) or placebo (PB) self-administered subcutaneously three times a week (tiw), had MRI 4 weeks before first dose and every 4 weeks for 3 years. Transverse, 5-mm thick with 0.5 mm gap, non-contrast enhanced proton density and T2-weighted (T2) and pre- and post-gadolinium (Gd) enhanced T1-weighted (T1) conventional spin echo images were obtained. MRI lesion activity was determined from the sequential review of a patient's entire scan set by a pair of radiologists working together. In case of disagreement, a third senior radiologist reviewed the films and a final consensus was reached. Observers had no knowledge of the patients' treatment or outcomes. MRI endpoints included counts of combined T2 and T1 unique (CU) lesions, T2 lesions and T1-gadolinium enhanced (T1-Gd) lesions. Additionally, acute hypointense lesions on pre-T1W images, so called "black holes" (BH), were identified by one radiologist with reference to previously determined active T2 and T1-Gd lesions. BH were followed for at least 6 months. Comparison of placebo and treated groups of patients on the number of active lesions were analyzed using an analysis of variance on the ranked data (Kruskal-Wallis test).

Results:

Of the 50 patients, 16 were randomized to the HD arm, 15 to the LD arm and 17 to the PB arm. Baseline values for each lesion type were similar between groups. On-study mean and median T2 active lesions were 3.9/1.0, 4.0/1.0 and 14.1/5.0 for HD, LD and PB (both treatment groups combined vs. placebo p < 0.065); for T1-Gd, mean and median active lesions were 6.0/0.0, 4.8/1.0 and 28.1/9.0 for HD, LD and PB(both treatment groups combined vs. placebo p < 0.002); for CU, mean and median active lesions were 6.8/1.5, 5.7/1.0 and 30.0/9.0 for HD, LD and PB(both treatment groups combined vs. placebo p < 0.009); and for BH, mean and median active lesions were 2.7/1.0, 2.7/0.0 and 11.8/5.0 for HD, LD and PB (both treatment groups combined vs.placebo p < 0.048).

Conclusions:

Significant treatment effect of IFN in SPMS can be detected even in a small cohort of 50 patients with frequent scanning over 3 years, for T1-gadolinium enhanced and combined unique active lesions, marginally so for acute hypointense lesions ("black holes") and marginally insignificant for T2 active lesions.

Supported By:

Serono


Tumor Necrosis Factor Induces Necrotic Cell Death of Murine Neural Stem Cells

Thursday, April 18, 2002
Khadir Rhadassi, Jaime Imitola, Evan Y. Snyder, Samia J. Khoury Boston , MA

Objective:

To investigate the in vitro effects of TNF-a on murine neural stem cells

Background:

Neural stem cells (NSC) are uncommitted, multipotent, self-renewing neural progenitors that can be isolated from several regions of the developing and mature brain. However, little is known about the molecular markers and mechanisms that underlie neural stem cell plasticity and response to injury.TNF-a has been shown to be deleterious for oligodendrocytes, however recent reports have shown that TNF-a has beneficial effects on oligodendrocyte progenitors. TNF-a R1 KO mice were reported to have decreased oligodendrocyte progenitor proliferation and decreased ability to repair after chemical demyelination. Others have reported that TNF-a synergizes with IFN-g to impair oligodendrocyte progenitor cell survival. Here we investigated the effects of TNF-a on neural stem cells.

Design/Methods:

The neural stem cell clone C17.2 was isolated from a newborn mouse cerebellum forming multipotent self-renewing clusters that are expanded and used for experiments. C17.2 cells express nestin (a marker for neural stem cells) and fulfill the operational criteria for NSCs. A nestin+ enriched population was obtained by culturing NSCs in DMEM/F12 supplemented with N2 supplement EGF and bFGF (20 ng/ml each). NSCs undergoing apoptosis were distinguished from live and necrotic cells by the use of Annexin-V and 7-amino actinomycin (7- AAD) staining. Live cells were considered as being Annexin-V- and 7-AAD-negative. Apoptotic cells included early (Annexin-V+ and 7-AAD-) and late (Annexin+ and 7-AAD+) apoptotic cells; necrotic cells are only 7-AAD+. TNF-a was used in doses of 2-2000 ng/ml. IFN-g was used at 100 U/ml. L929 and Jurkat cells were used as positive control for TNF-a signaling experiments. The effects of signaling by TNF-a on the TNF adaptor molecule TRAF2 and on phosporylation of ERK were measured.

Results:

We found that neural stem cells express the TNF receptor p75 but not p55 by western blot. In vitro addition of TNF-a induced a dose dependent cell death in neural stem cells, with changes in morphology that were evident at 36-48 hours. These morphological changes were accompanied by decrease in viability by trypan blue exclusion and were more consistent with necrosis than apoptosis as determined by Annexin-V and 7AAD staining. The addition of 100 ng/ml of TNF-a induced phosphorylation at residue 44 and 42 of ERK kinase at 90 minutes that persisted after 24 hours after stimulation. There was no change in expression of TRAF2 at 30 minutes or 24 hours after stimulation. The addition of IFN-g did not increase cell death of neural stem cells.

Conclusions:

Our results show that TNF-a induces necrotic cell death of neural stem cells in vitro. This data is relevant to our understanding of the potential effects of the inflammatory microenviroment on stem cell function

Supported By:

Nancy Davis Center without walls and NIH


Evidence of an Early Dose Effect of Inteferon-b1a on Relapse-Free Outcomes

Thursday, April 18, 2002
Mark S. Freedman, the OWIMS Study Group Ottawa, ON, Canada

Objective:

To examine the evidence for a dose effect of interferon-b1a (Rebif® on relapse-free outcomes at 6 and 12 months in patients treated once a week vs. three times a week with 22mg or 44mg.

Background:

Controversy exists over what may be the optimal dose of interferon-b1a in relapsing-remitting MS. There is evidence that higher more frequent doses may reduce relapses earlier than lower less frequently given doses. Considerable time must pass in order to accumulate enough relapses to appreciate any differences between dosing regimens on relapse number or rate, and studies examining this outcome usually run 1-2 years. In contrast, as time goes by the number of relapse-free patients diminish. Therefore relapse-free outcomes might be a better measure for demonstrating dosing differences earlier in a study. Early changes on relapse-free outcomes should parallel later changes on relapse number or rate.

Design/Methods:

Patients were enrolled in 2 separate randomized double-blind placebo controlled studies using interferon-b1a (Rebif®: 293 patients in OWIMS received either 22 or 44mg once a week; 560 patients in PRISMS received the same doses three times a week. Though the primary outcome of each study was different (MRI activity at 24 weeks for OWIMS and number of relapses at 2 years in PRISMS), we examined secondary outcomes of proportion relapse-free and any increase in this proportion vs. placebo for the different dose regimens at 6 and 12 months of treatment.

Results:

The proportion of patients relapse free at 6 months taking either placebo, or interferon-b1a 22 or 44mg once weekly was 53, 46 and 57%. The proportion relapse-free among patients taking placebo or interferon-b1a 22 or 44mg three times weekly was 41, 58 and 60%. Although there was no statistically significant effect of either interferon-b1a dose given once weekly vs. placebo at 6 months, both doses given three times weekly were significant (p<.05) compared to placebo. At 12 months the proportion relapse-free for the once weekly regimen was 36, 33 and 40% whereas that for the three times weekly regimen was 23, 39 and 45%. There was once again no significant difference between either interferon-b1a dose vs. placebo when given only once a week, but both doses given three times weekly were significantly (p<.05) better than placebo.

Conclusions:

These results show that relapse-free outcomes can differentiate between interferon-b1a dosing regimens as early as 6 months, with more frequently administered doses showing the greater effect. These same conclusions related to dose and frequency of interferon-b1a administration were noted at 4 years in PRISMS and at 3 years in OWIMS, related to relapse number and rate. It may therefore be useful to use relapse-free outcomes in shorter term (< 1 year) studies.

Supported By:

Serono


Changes in Sex-Hormone Pattern during Interferon Beta Therapy in Women with Relapsing-Remitting Multiple Sclerosis

Thursday, April 18, 2002
Valentina Tomassini, Emanuela Onesti, Elisabetta Giugni, Caterina Mainero, Fabiana Marinelli, Bruno Di Pofi, Andrea Paolillo, Carlo Pozzilli Rome, Italy

Objective:

To define sex-hormone profile before and during Interferon beta (IFNb) therapy in women with Relapsing-Remitting Multiple Sclerosis (RRMS) and its relationship with disease activity as measured by brain Gd-enhanced MRI.

Background:

A pathogenetic influence of sex hormones on immune system activity in MS has been suggested by experimental, clinical and MRI evidence; the mechanisms underlying these neuroendocrine/immune interactions are partially known. Moreover, some data indicate a gender-specific response to IFNb in MS. Nevertheless, whether the sex-hormone pattern is influenced by IFNb treatment and hormonal changes are of clinical relevance in MS remains unknown.

Design/Methods:

Serum sex-hormone levels (FSH, LH, 17-b-estradiol, progesterone, testosterone, DHEAs), measured in the follicular and luteal phases during a menstrual cycle, were evaluated in 22 normally menstruating clinically definite RRMS patients before and during IFNb therapy, at month 3 and 6. Brain Gd-enhanced MRI was performed, on the same day as the blood samples. Eighteen healthy age-matched subjects underwent the same hormonal assessment.

Results:

At baseline, no difference in levels of any single hormone was found except for testosterone, which showed significantly lower levels in MS patients than in controls (p=0.006) and, among patients, in those with active MRI scans (p=0.03). A significant decrease in the mean number of Gd-enhancing lesions was found both at month 3 (-84%, p<0.001) and 6 (-93%, p<0.001) of IFNb therapy. At month 3, a significant increase in FSH (+30%, p=0.05) and LH (+24%, p=0.005) levels was observed in the follicular phase, whereas the other sex hormones were either unchanged or slightly decreased. DHEAs levels showed a significant reduction at month 6 (-21%, p=0.015), when compared with the baseline values. No significant correlation was found between changes in MRI activity and changes in sex-hormone levels during the IFNb therapy.

Conclusions:

Our results suggest that sex hormones may influence disease activity in MS by modulating the immune system, as previously reported in experimental models. IFNb therapy may influence the sex hormone pattern either by acting directly on hypothalamic-pituitary axis, and modifying gonadotropin release, or by modulating the steroid receptor expression, thus influencing the hormone-responsiveness in both the CNS and peripheral tissues. Whether the IFNb effects on sex-hormone pattern are of clinical relevance in MS warrants further investigations.


Assessing the Effects of IFNb-1a on Health Related Quality of Life in Secondary-Progressive MS Patients: Results from the IMPACT Study

Thursday, April 18, 2002
Deborah M. Miller, Jeffrey A. Cohen, Elisa C. Tsao, Mariska F. Kooijmans, Nancy A. Simonian Cleveland , OH; Cambridge, MA

Objective:

To determine the impact of IFNb-1a treatment on self-reported health related quality of life (HRQoL) using the Multiple Sclerosis Quality of Life Inventory (MSQLI) in a group of patients with secondary progressive disease. An additonal objective is to assess if HRQoL data are distinct from clinician obtained measures of impairment and disability.

Background:

As previously reported, the IMPACT study demonstrated the benefit of IFNbeta-1a (Avonex) on disease progression as measured by the Multiple Sclerosis Functional Composite (MSFC) in patients with secondary progressive multiple sclerosis (SP-MS). The effect of treatment on health related quality of life (HRQoL) in this study has not been reported in detail. Our objective is to evaluate differences in HRQoL between treated and placebo subjects and to assess if the MSQLI provides information distinct from clinician-derived data. The MSQLI is a comprehensive MS-specfic HRQoL battery developed by a reseach group sponsored by the Consortium of MS Centers and funded by the National Multiple Sclerosis Society. It was initally validated with a group of 300 patients who represented the spectrum of MS-disability and were drawn from 4 MS centers in the US and Canada. The MSQLI includes the generic SF-36 (2 scales), and 9 condition-specific measures. Those disease and symptom-specific measures assess fatigue, bladder control, bowel control, sexual satisfaction, visual impairment, preceived cognitive deficits, mental health, pains and disturbing sensations and, social support.

Design/Methods:

IMPACT was a randomized, double-blind, placebo-controlled, two-arm trial. Subjects were randomized to IFNb-1a (60 mcg) or placebo by weekly IM injection for 24 months. MSFC and EDSS were collected at baseline and every 3 months. MSQLI was collected at basline and months 12 and 24.

Results:

436 subjects were enrolled, 219 to placebo and 217 to IFNb-1a. Eight of 11 MSQLI scales were statistically better and the other 3 trended to improvement in the treatment group. The placebo group trended to worsening in 10 of 11 scales. Consistent with those findings, correlations between MSFC change and MSQLI change over the 2- year period were significantly correlated for 6 of the 11 MSQLI components. The significiant change corrlations ranged from r=-.22 (p=0.003) for the relation between change in the Modified Fatigue Impact Score and MSFC change to r=-.17 (p=.0066)for the relation between pain effects scale change and MSFC change. There were significant differences in 9 of the MSQLI scales when comparing patients who were in the highest and lowest MSFC change quartiles

Conclusions:

Patients who received IFNb-1a had significantly better HRQoL. The consistent trend for association between MSQLI and MSFC supports their validity as outcome measures. None the less, variability in the strength and significance of those associations indicates that the MSQLI and MSFC provide unique information and are distinct outcomes indicators.

Supported By:

This study was conducted with funding provided by Biogen, Inc. Cambridge, MA.


Infliximab in the Treatment of CNS Vasculitis with Uveitis

Thursday, April 18, 2002
Benjamin Turner, Cris S. Constantinescu, Richard J. Powell Nottingham, United Kingdom

Objective:

To study the effects of TNF a neutralisation in CNS vasculitis associated with uveitis.

Background:

CNS vasculitis represents a significant diagnostic and therapeutic challenge. Infliximab (Remicade®) is a monoclonal antibody to tumour necrosis factor (TNF) a, a proinflammatory cytokine, and is used in the treatment of rheumatoid arthritis, Crohn's disease and inflammatory eye disease.

Design/Methods:

We present three patients with uveitis and CNS vasculitis, who demonstrated a good clinical response to infliximab.

Case1. A 30 year-old male with a history of uveitis, acneiform lesions, oral and genital ulcers, treated with cyclosporine and steroids presented with a progressive decline in cognition and gait. On examination he had pyramidal signs and central ataxia.

Case 2. A 72 year-old male had presented three-years previously with sinusitis, anterior uveitis, scleritis, headache, jaw claudication and scalp tenderness, an ESR of 75 mm/hr, C-reactive protein of 85 mg/l, thrombocytosis and negative anti-neutrophil cytoplasmic antibodies. He initially responded to steroids but then deteriorated and developed a cerebral sinus thrombosis consequent upon CNS inflammation.

Case 3. A 54 year-old female had a five-year history of headaches, scalp tenderness, livedo reticularis, retinal vasculitis and uveitis. On examination she had bilateral pyramidal signs with gait ataxia. Investigations revealed a positive anti-nuclear antibody (400 IU/ml), elevated double-stranded DNA antibodies (300 IU/ml), CRP 31mg/L, positive rheumatoid factor (1280 IU/ml) and negative thrombophilia screen. She initially responded to steroids but after two years on prednisolone her symptoms and uveitis worsened. Azathioprine and methotrexate were of no benefit. She improved on cyclophoshamide but could not tolerate the side effects.
The first patient fulfils the criteria for Behcets Syndrome, the second has an unclassifiable vasculitis suggestive of large vessel disease and the third has features suggestive of a connective tissue disease such as systemic lupus erythematosus. These patients were refractory to conventional immunosuppression, and hence were given 3 infusions of infliximab (5 mg/kg) at 0, 2 and 4 weeks with methotrexate 7.5mg weekly and folic acid daily.

Results:

These are the only cases of CNS vasculitis we have treated with infliximab and all three had a clinical improvement. Case 1 and 3 have maintained their improvement several months after the infliximab whilst the uveitis of case 2 relapsed one month after treatment.

Conclusions:

This is the first report of the therapeutic effect of infliximab in CNS vasculitis. In contrast it has been shown that TNF a neutralisation increases exacerbations in multiple sclerosis (MS), highlighting the differing inflammatory mechanisms and response to treatment between CNS vasculitis and MS.


Oligodendrocytes Express Functional Chemokine Receptors

Thursday, April 18, 2002
Martin Stangel, Michael Höpfner, Franziska Zobel, Uta Henke, Hans Scherübl, Dan Nguyen Berlin, Germany; Berlin, Germany, Germany

Objective:

To demonstrate functionality of chemokine receptors on cells of the oligodendroglial cell lineage.

Background:

Chemokines represent a family of chemotactic proteins and activators in leukocytes during physiological and inflammatory processes. A number of chemokine receptors have been demonstrated to be expressed in the CNS on neurons, astrocytes and microglia, but their function in the CNS is poorly understood. GRO-alpha is considered to influence proliferation of cultured oligodendrocyte progenitor cells (OPC) and we have previously demonstrated the expression of its receptor CXCR2 on oligodendroglial cells.

Design/Methods:

RT-PCR and immunocytochemistry was used to demonstrate expression of chemokine receptors on the oligodendrocyte precursor cell line CG-4 and on primary cultures of rat OPC. Fura-imaging was utilized for intracellular Ca2+-monitoring to test for functionality of chemokine receptors.

Results:

We found evidence for the expression of the chemokine receptors CXCR1, CXCR2, CXCR4, CCR1, CCR3, CCR7, and CX3CR, but not for CXCR3, CCR2, CCR4, CCR5, and CCR9/10. There was an intracellular rise in Ca2+ upon treatment of the cells with the chemokines MIP-1alpha (CCL3), Eotaxin (CCL11), and GRO-alpha (CXCL1), demonstrating the functionality of some of the expressed chemokine receptors.

Conclusions:

We have shown that oligodendroglial cells express functional chemokine receptors. These findings demonstrate that the chemokine/chemokine receptor system is probably involved in the regulation of oligodendroglial cells during developmental processes and may have implications for repair mechanisms in demyelinating diseases like multiple sclerosis.

Supported By:

Hertiestiftung


Thyroid Abnormalities Related to Interferon Beta-1a Therapy

Thursday, April 18, 2002
Judith A. Abdalla Geneva, Switzerland

Objective:

Analyze the thyroid safety profile of interferon (IFN) therapy in MS.

Background:

IFN has been suspected of leading to thyroid abnormalities, possibly by an increase in auto-immunity although this remains uncertain. The precise rate and nature of these abnormalities have not been fully explored and conflicting reports exist on the relationship between IFN and thyroid dysfunction.

Design/Methods:

Pooled safety data for IFN-treated (22, 30, 44, 66, and 132 mcg weekly in single or divided doses) and placebo patients from controlled studies are available for analysis (the controlled population). Data were analyzed after 6 (n = 2819, 1995 IFN-treated and 824 placebo patients) and 24 months (n = 1178, 789 IFN-treated and 389 placebo patients) for thyroid function abnormalities. In addition, the overall safety population of 3996 patients was analyzed for AE drop-outs and serious adverse events related to thyroid disease.

Results:

In the pooled safety population, thyroid disorders (de novo or worsening of pre-existing thyroid dysfunction) were more commonly seen in patients in the 132mcg Rebif® dose group than in other treatment groups: 18 cases (0.3%) in 15 patients (2.1%). Seven cases (0.4%) of thyroid dysfunction in six patients (1.8%) were noted by 6 months of therapy with the next most frequent group (the 30mcg group (i.e. Avonex®)). The other dose groups had only 1 or 2 affected patients (i.e. <1%). The 2-year data showed a mild excess of thyroid dysfunction for treated (6.8% for 22mcg, 6.4% for 44mcg) compared to placebo (4.8%) patients. Thyroid abnormalities were more frequent in the second year of therapy. Various thyroid abnormalities were noted with a slight preponderance of increased T4 but without evidence of a dose effect. Less than 1% of patients stopped therapy due to thyroid abnormalities. Five of 3996 patients in the overall population experienced thyroid dysfunction that was considered serious, including two cases of goitre, two patients with hyperthyroidism and one with a thyroid adenoma, representing a rate of 0.07 per 100 patient-years in the overall safety population.

Conclusions:

Thyroid abnormalities are infrequent but show a slight, but non-significant increase in patients treated with IFN compared to those treated with placebo in controlled clinical trials. Onset is later than most other IFN-associated adverse events that typically occur within the first 6 months. Thyroid abnormalities rarely lead to discontinuation of IFN treatment. Thyroid function should be assessed before starting therapy, and if normal, monitoring on therapy is not needed unless clinical symptoms develop.

Supported By:

Serono International S.A.


The Role of Inducible Nitric Oxide Synthase and Macrophages in the Neuropathology of Multiple Sclerosis

Thursday, April 18, 2002
Lauren V. Zollinger, Kenneth E. Hill, John W. Rose Salt Lake City, UT; Salt Lake City, UT

Objective:

We examined the localization of inducible nitric oxide synthase, (iNOS) in conjunction with specific cell markers for macrophages and astrocytes.

Background:

Previous studies suggest the involvement of activated macrophages and reactive oxygen or nitrogen species, however these efforts are limited the in identification and characterization of cell types in relation to lesions.

Design/Methods:

Tissue from four MS patients were classified for disease activity using hematoxylin-eosin staining. Immunohistochemical techniques were applied utilizing specific iNOS, GFAP, MBP fragement, CD14, and high affinity receptor for IgG FcgR1 (CD64) directed antibodies. Control polyclonal and monclonal antibodies were utilized to assure specificity. After the application of additional fluorescent labeled secondary antibodies, the samples were analyzed with confocal microscopy allowing for three color, three-dimensional images of the diseased tissue.

Results:

Large numbers of iNOS+ cells and inflammatory cells were identified in perivascular, parenchymal, and peri-lesional areas. Many of iNOS+ macrophages are CD64+, with CD14+ macrophages also present. The CD64+/iNOS+ and CD14+/iNOS+ cells are most prominent along the active edge lesions. Similar phagocytic cells in this area may be MBP fragment positive. GFAP+ cells producing iNOS are infrequent.
Further from the lesion edge, CD64+ and CD14+ macrophages are not producing iNOS. Though, lipid-layden is this areas may contain MBP fragments.

Conclusions:

INOS+ macrophages are found in perivascular cuffs, parenchyma, and the active edge of lesions. CD64+/iNOS+ and CD14+/iNOS+ cells are present along the active edge of MS lesions. CD64 expression on macrophages is regulated by interferon gamma and is characteristic of phagocytic potential. Lipid layden iNOS+ cells are also observed at the active edge of the lesion. These results support the major contribution of iNOS+ macrophages in demyelination in multiple sclerosis.

Supported By:

Department of Veterans Affairs and Western Institute for Biomedical Research; AAN Medical Student Summer Research Scholarship (LZ)


Outcome of Pregnancy during Treatment with Interferon-Beta-1A (Rebif®) in Patients with Multiple Sclerosis

Thursday, April 18, 2002
Magnhild Sandberg-Wollheim, for the Rebif Investigators Lund, Sweden

Objective:

To review the outcome of pregnancies occurring during treatment with interferon beta-1a (IFN)in patients with multiple sclerosis.

Background:

IFN therapy is effective in patients with MS but due to the known abortifacient potential of IFN, therapy should be stopped during pregnancy. Data however, indicates that relapse rates diminish spontaneously during pregnancy for as yet undetermined reasons. Although pregnancy was an exclusion criterion in clinical trials with IFN beta-1a (Rebif®), a number of pregnancies occurred during the clinical trial programme. Treatment was to be stopped if pregnancy occurred; however, in some cases treatment was not stopped either because the patient was unaware that she was pregnant or because the patient made an explicit request to continue treatment. This presentation reviews the outcome of pregnancies occurring during IFN treatment.

Design/Methods:

Review of individual patient data obtained during controlled clinical trials (including placebo-controlled and extension phases).

Results:

A total of 37 pregnancies were reported during clinical trials (involving approximately 1400 women) with IFN beta-1a at doses ranging from 22mcg weekly to 44mcg thrice weekly. Thirty pregnancies occurred in patients who were receiving, or had recently stopped receiving, active medication. Of the latter, 13 resulted in the delivery of healthy full-term babies and two premature births; there were seven induced abortions (six at the patient's request, one due to fetal death) and seven spontaneous abortions. All of the spontaneous abortions occurred in the early phase of pregnancy. One patient was lost to follow-up.

Conclusions:

Although these data are limited and thus one cannot exclude risk to fetus, they do not provide specific evidence of fetal harm associated with administration of IFN during pregnancy. The incidence and timing of spontaneous abortions was consistent with that seen in the general population. However, animal experiments have indicated some abortifacient potential with IFN. Hence, in the absence of conclusive data, IFN should continue to be regarded as contra-indicated in pregnancy, and treatment should be discontinued if a patient becomes pregnant while receiving interferon therapy. Therapy may restart after pregnancy if the patient is not breast-feeding.


The Spectrum of Demylinating Disorders in Children

Thursday, April 18, 2002
Evangeline Wassmer, Diedre Peake, Girish Gupte, Mary Stonehouse, William P. Whitehouse Birmingham, United Kingdom

Objective:

We studied the clinical features of children with demyelinating disorders.

Background:

The different demylinating disorders have specific clinical, laboratory, electrophysiological and imaging features, yet there are many similarities and possibly some overlap.

Design/Methods:

The children reported in this retrospective study have been seen at the Birmingham Childrens Hospital NHS Trust from December 1995 to March 2000. Patients were identified from the computerised departmental discharge databases. The clinical records, laboratory results and imaging of the children were reviewed.

Results:

79 children were admitted with demylinating disorders (ages 1 to 15 years): 16 with a diagnosis of Guillan-Barre Syndrome (GBS), 2 with Chronic Inflammatory Demylinating Polyradiculoneuropathy (CIDP), 9 with Multiple Sclerosis (MS), 18 with Acute Disseminated Encephalomyelitis (ADEM), 15 with Transverse Myelitis (TM) and 19 with Optic Neuritis (ON).
We noted many similarities between the different disorders and some overlap. A preceding viral illness and the recurrence of the disorder is common. Symptoms at presentation overlap e.g. ON can be part of the presentation of MS and ADEM, yet four children with a diagnosis of ON had other neurological symptoms: ataxia, seizures, reduced conscious level and myelitis (Devic Disease).
Demylinating diseases are generally regarded as selectively involving either the central nervous system (CNS) or the peripheral nervous system (PNS), yet some had involvement of both. A child with ADEM had a peripheral demylinating neuropathy (confirmed by nerve conduction studies). A child with TM had cranial imaging consistent with ADEM. 3 children with GB had upper motor neuron signs consistent with myelitis (confirmed by imaging).

Conclusions:

Our review and the literature confirm that CNS and PNS demyelination can occur in the same patient and that there maybe a common pathogenesis. The similarities in clinical features and investigations suggest that the different disorders may lie on a spectrum.


Epstein-Barr Virus in Pediatric Multiple Sclerosis

Thursday, April 18, 2002
Suad F. Alotaibi, Helen Heurter, Elizabeth Ford-Jones, Brenda L. Banwell Toronto, ON, Canada

Objective:

To study the seropositivity for Epstein-Barr Virus (EBV) in children with Multiple Sclerosis (MS) compared to age- matched controls.

Background:

Multiple sclerosis (MS) is a chronic, demyelinating disease of the CNS. Pathogenesis is complex, and likely multifactorial. Considerable support exists for the hypothesis that MS is an autoimmune disease triggered by prior infection with a common virus, and propagated by mechanisms of molecular mimicry. Infection of human T lymphocytes by EBV induces T-cell surface expression of a variety of proteins also expressed in the CNS. Thus, peripheral priming of T cells by EBV may initiate or propagate the immune mediated attack against these CNS proteins. Increased seropositivity for EBV has been shown in MS-affected adults, although the ubiquitous nature of EBV infection by adulthood has made interpretation of this data difficult. EBV infection is not ubiquitous in early childhood. Thus, if exposure to EBV is important in the pathogenesis of MS, children with MS should have a higher seropositivity rate than age-matched peers.

Design/Methods:

The presence of antibodies directed against the EBV viral capsid antigen (VCA), which is a marker of past infection with EBV, was determined by enzyme linked immunosorbent assay (ELISA) in serum samples from 19 children with MS and 38 age-matched controls. Two control samples for each case, matched within 4 years of case age, were selected from children enrolled the Encephalitis registry at the Hospital for Sick Children. The medical charts of all control patients were reviewed. Control samples were obtained from previously healthy children admitted to the hospital with acute fever and encephalopathy, but without clinical or neuroradiological evidence of demyelination. All MS-affected children met criteria for clinically definite MS, as per Poser et al, and all are followed at the Pediatric Multiple Sclerosis Clinic in our hospital. The proportion of EBV-VCA seropositive cases and controls were compared using the z-test. The Welch t-test was used to compare the average age for each group, and all tests were two-tailed with alpha = 0.05

Results:

Nineteen pediatric MS and 38 control samples were studied. The mean age of the MS patients was 13.5 years (±3.7 SD), mean age of controls was 11.8 (±3.0 SD). Seventeen of 19 MS patients (89.5%) compared to 18 of 38 controls (47.4%) were seropositive for EBV-VCA (p<0.001).

Conclusions:

EBV seropositivity is significantly higher in MS-affected children than in age-matched controls. These results support the hypothesis that early exposure to a putative viral trigger may play a role in the pathogenesis of MS.


Active Human Herpesvirus Six Infections and Multiple Sclerosis: Clinical and Treatment Relationships

Thursday, April 18, 2002
Konstance K. Knox, Lorri J. Lobeck, Eric F. Maas, Donald R. Carrigan Milwaukee, WI

Objective:

The goal of these studies was to assess multiple sclerosis (MS) patients for evidence of active human herpesvirus six (HHV-6) infections and to correlate those findings with the clinical and therapy status of the patients.

Background:

Several laboratories have presented data linking the pathogenesis of MS to active HHV-6 infections. Different diagnostic technologies have been used in these studies including immunohistochemical staining of tissues, polymerase chain reaction analysis of serum samples and isolation of the virus from blood samples. The majority of the work from our laboratory has involved staining of MS patient derived CNS and lymphoid tissues for HHV-6 antigens and isolation of the virus from peripheral blood leukocytes of MS patients by a rapid culture procedure (Knox et al.; Clin Infect Dis 2000; 31:894-903). In the studies described here we have used an alternate methodology, i.e. serum PCR, to address this issue.

Design/Methods:

Blood samples were obtained at the time of new clinical relapse in patients with relapsing-remitting MS and assessed for active HHV-6 infection by serum PCR. Then, several weeks later (mean interval:68 days), a second blood sample was obtained from the same patients and assessed for active HHV-6 infection. Patients'changes of Expanded Disability Status Scale (EDSS) from the relapse and treatments at the time samples were obtained were noted.

Results:

5 of 39 (13%) patients had at least one sample positive for active HHV-6 infection with viral loads varying from 9000 to 500,000 viral genomes per ml of serum. Variant typing of the positive samples was possible with 3 of the 5 positives, and 2 were HHV-6 variant A. Four of the five (80%) positive samples were obtained at the time of relapse whereas only one (20%) positive was observed in a patient after relapse. The HHV-6 positive patients suffered a larger change in their EDSS (mean EDSS change of 1.4) than the HHV-6 negative patients (mean EDSS change of 0.7). It was also found that the patients receiving either beta interferon or glatirimar acetate (copaxone) were less likely to be HHV-6 positive (2/30; 7%) than the patients receiving no treatment 3/9; 33%). Since the majority (75%) of the patients on therapy were receiving beta interferon, the decreased positivity for active HHV-6 may reflect the known antiviral properties of beta interferon.

Conclusions:

The findings presented here demonstrate that active HHV-6 infections can frequently be detected at the time of clinical relapse in patients with relapsing-remitting MS and confirm the work of other laboratories that have used serum PCR to detect such infections. Also, we observed that patients with active HHV-6 infections at the time of relapse show a greater degree of residual disability than HHV-6 negative patients and that patients who are receiving beta interferon or glatirimar acetate therapies are at reduced risk for the occurrence of detectable active HHV-6 infections.

Supported By:

Grant PP0751 from the National Multiple Sclerosis Society


Randomized Trial of Pulse Cyclophosphamide in IFN-B Resistant Active MS

Thursday, April 18, 2002
Derek R. Smith, Bianca Weinstock-Guttman, Jeffrey A. Cohen, Charles Guttmann, Xingchang Wei, William H. Stuart, Howard L. Weiner Boston, MA; Buffalo, NY; Cleveland, OH; Atlanta, GA

Objective:

To test the safety and efficacy of 6 monthly infusions of cyclophosphamide plus methylprednisolone (CY) versus methylprednisolone alone (MP) in MS patients with active disease while on IFN-B therapy.

Background:

Many MS patients have active or breakthrough disease while on treatment with IFN-B. Reports have suggested that the addition of cyclophosphamide pulse therapy may be effective in reducing disease activity in these patients. (Gobbini et al, 1999; Khan et al, 2001; Patti et al, 2001)

Design/Methods:

Patients classified as having active MS while on therapy with IFN-B were treated with a 3 day course of methylprednisolone and then randomized to 6 monthly infusions of CY (800 mg/m2) or MP (1 gm)alone. The patients were continued on IFN-B1a (30 mcg IM weekly) during the 6 month infusion phase and 18 month follow-up phase. IFN-B nonresponders were defined as having at least one of the following within the previous year: 1)sustained increase in EDSS of 1.5 or greater 2)two confirmed attacks 3) two courses of steroids in the past year, or 4)multiple gadolinium enhancing lesions (Gd+) within the past 8 weeks. The primary outcome measures were change in number of Gd+ lesions and change in T2 lesion burden. Single blind clinical assessments and MRIs were obtained at baseline, 3, 6, 12, 18, and 24 months. Gd+ lesions were quantitated centrally by a radiologist who was blinded. An automated segmentation algorithm was applied to dual-echo sequences to obtain T2 lesion burden and atrophy measures.

Results:

59 patients were enrolled and 58 completed the infusion phase. For the primary outcome measure, change in number of Gd+ lesions, we were able to evaluate data from 58 baseline, 58 month 3, and 56 month 6 MRIs. One month after the initial 3 day steroid course, the baseline mean number of Gd+ lesions was balanced between the arms with 0.79 in MP and 0.93 in CY (p=0.71). 10 of 28 baseline scans were Gd+ in MP, and 14 of 30 in CY. The MP arm showed a mean change of +0.57 Gd+ lesions at month 3 and +0.19 at M6 vs baseline, for a total of 21 of 54 Gd+ scans. The CY arm showed a mean change of -0.70 Gd+ lesions at month 3 (p=0.01 vs MP) and -0.77 Gd+ lesions at month 6 (p=0.02 vs MP) for a total of 8 of 60 Gd+ scans. The mean total number of Gd+ lesions at month 3 & month 6 was 1.18 for MP and 0.20 for CY (p=0.001). For change in T2 lesion burden there was a trend in favor of CY (+0.10% versus +0.35% for MP). No differences in atrophy were seen. Treatments were generally well tolerated and clinical data will be presented.

Conclusions:

The addition of six monthly infusions of cyclophosphamide plus methylprednisolone reduces the number of Gd+ MRI lesions versus methylprednisolone alone in MS patients with active disease while on IFN-B therapy.

Supported By:

supported in part by Biogen Inc, Cambridge, MA, USA; Derek Smith, MD supported by NIH 1 K23 NS02 117-01


Loss of Claudin-3 but Not of Claudin-5 from Cerebral Endothelial Tight Junctions Directly Correlates with Blood-Brain Barrier Leakyness In Vivo and In Vitro

Thursday, April 18, 2002
Joerg Kraus, Sefan Hamm, Benedicte Dehouck, Karen Wolburg-Buchholz, Marie-Pierre Dehouck, Hartwig Wolburg, Romeo Cecchelli, Mikio Furuse, Werner Risau, Britta Engelhardt Bad Nauheim, Germany; Lille, France; Tuebingen, Germany; Kyoto, Japan

Objective:

To investigate the subcellular distribution of blood-brain barrier (BBB) tight junction (TJ) molecules which are involved in the development of BBB leakyness.

Background:

In the brain, cerebral endothelial cells are connected by complex P-face associated tight junctions forming a restrictive paracellular diffusion barrier, the blood-brain barrier. During inflammation in the central nervous system (CNS) BBB properties are frequently lost resulting in a dramatic decrease in vascular permeability and formation of brain edema.

Design/Methods:

We addressed this by applying two different approaches. In an in vitro BBB model, bovine brain capillary endothelial cells (BBCE) in co-culture with rat astrocytes form a tight permeability barrier. We measured changes in the paracellular permeability of BBCE by determining permeability rates of 3H-inulin and 14C-sucrose. Moreover, we investigated ultrastructural changes at the tight junctions of BBCE by electron microscopy. Furthermore, we determined the distribution of the TJ molecules of claudin-1, claudin-3, claudin-5, occludin, ZO-1, ZO-2, b-catenin and p120cas in BBCE by immunohistochemistry. In addition, we assessed protein expression of TJ molecules in BBCE by western blotting. In vivo, we also observed the localization of endothelial specific TJ proteins during CNS inflammation in experimental autoimmune encephalomyelitits (EAE).

Results:

Removal of astrocytes from the co-culture of the BBB model resulted in breakdown of this permeability barrier as determined by increased diffusion of 3H-inulin and 14C-sucrose across the endothelial monolayer and opening of the TJs to horseradish peroxidase detected by electron microscopy. Opening of the endothelial TJs was accompanied by the specific loss of claudin-3 immunostaining from the endothelial TJs. Surprisingly, claudin-1 was not localized in the TJs, neither in co-culture nor after removal of astrocytes. The junctional localization of the other investigated TJ proteins was conserved. In vivo, we observed the specific loss of the TJ protein claudin-3 from cerebral vessels during EAE. Again, claudin-1 could not be obtained at the TJ, neither in healthy nor in inflamed microvascular brain vessels whereas the other TJ molecules were localized in the TJ and remained unchanged during EAE.

Conclusions:

Our data demonstrate a direct correlation of the specific loss of claudin-3 from BBB TJs with increased paracellular permeability and suggest that claudin-3 is the central component determining the tightness of BBB TJs.


Safety, Tolerability and Pharmacodynamics of a Recombinant-Human Interferon-Beta-1A Following Oral Administration in Healthy Male Volunteers

Thursday, April 18, 2002
Isabelle Trinchard-Lugan, Thierry Buclin, K. Lapointe, Francois Spertini, Jerome Biollaz, Gordon Francis, Alain Munafo, Arnaud Ythier Geneva, Switzerland; Lausanne, Switzerland; Norwell, MA

Objective:

To assess the safety of orally administered interferon beta-1a (IFN) and determine whether it can induce pharmacodynamic (PD) markers or alter local immune response.

Background:

IFN is an effective treatment for multiple sclerosis (MS) but is currently given by subcutaneous or intramuscular route. Mucosal surfaces of mammals are important immunological sites that have specific immune capabilities and cytokine responsiveness. Studies of orally administered IFN in animals and man have shown PD responses and suggest that ultra-low doses are most effective than high oral doses while recent studies of oral IFN beta in MS failed to show benefit on clinical or magnetic resonance imaging outcomes.

Design/Methods:

Six doses of a parenteral formulation of IFN beta-1a ranging from 0.00022 to 22 mcg, were given by ingestion or oropharyngeal application to 18 male volunteers according to a double-blind, placebo-controlled, cross-over design. Each subject received four treatments (two dose levels and two routes) separated by 2-week wash-out periods. Clinical safety assessments were performed twice weekly and blood samples were drawn repeatedly during the 24 hours following each treatment for assessment of: the ex vivo release by stimulated peripheral blood mononuclear cells of tumour necrosis factor alpha, tumour growth factor beta and interleukins 10 and 12; the lymphocyte proliferation with or without stimulation; and serum concentrations of the IFN beta-inducible pharmacodynamic markers neopterin, beta-2 microglobulin and 2-5A synthetase. Local alteration of mucosal immunity was explored through measurement of immunoglobulin A levels in saliva. Antibodies to IFN were monitored.

Results:

Oral IFN was well tolerated by either route and no antibodies were raised. There were no significant clinical or laboratory changes, except for decreases in hemoglobin and erythrocyte values related to blood sampling. Regardless of the dose administered or route used, no significant pharmacodynamic response was observed.

Conclusions:

Over the dose range tested, the parenteral formulation of IFN-beta-1a administered orally in this study was not absorbed into the circulation to a level sufficient to induce a pharmacodynamic response. This finding is consistent with recent failed clinical trials of oral IFN in MS. Moreover, it appears to disprove the hypothesis that IFN can cause a systemic response by binding to specific receptors in the oro-pharyngeal or gastro-intestinal mucosa or peripheral lymphoid tissues. Further work is needed to determine whether a response can be elicited using a different formulation or different doses.

Supported By:

Serono International SA


MRI Predictors of Clinically Definite MS Based on Analyses of Placebo Patients from the CHAMPS Trial

Thursday, April 18, 2002
Jack H. Simon, CHAMPS Study Group Denver, CO

Objective:

Evaluation of the effectiveness of individual and combined baseline MRI criteria in predicting clinically definite MS (CDMS) and combined CDMS/MRI outcomes over an 18 month followup based on placebo patients from the CHAMPS trial with a positive MRI at the time of their first neurologic event.

Background:

After an isolated neurologic event resembling demyelination, patients with a positive MRI are at increased risk for developing CDMS. Prior studies have suggested that individual and sets of MRI criteria (Paty,Fazekas,Barkhof et al)can increase the accuracy of prediction of CDMS.

Design/Methods:

Entry into trial based on a first neurologic event (clinically isolated syndrome), and a positive MRI with at least two T2-hyperintense lesions, one periventricular and/or ovoid. Evaluation of 190 subjects with standardized baseline MRI (3mm thick, non-gapped slices)and standardized MRI followup of this placebo group(6,12,18 months)until primary CDMS outcome reached or through 18 month MRI followup. All treated at baseline with a standardized protocol including high dose intravenous methyprednisolone. Outcomes included CDMS, combined CDMS/MRI1 outcome( CDMS or MRI with 1 new or enlarging T2 lesions)and CDMS/MRI2(CDMS or MRI with one new or enlarging T2 lesions).

Results:

Irrespective of additional MRI criteria, 27% developed CDMS over 18 months. The strongest individual baseline predictor of CDMS was the presence of enhancing lesions, with 43% of patients with one or more enhancing lesions (GD+) developing CDMS, compared to 23% in the absence of enhancing lesions. The strongest set of predictive criteria were the Barkhof et al. (Barkhof) criteria , with 32% developing CDMS compared to 16% when these criteria were not met.Irrespective of individual or sets of criteria, the majority of patients were positive by 18 months for the CDMS/MRI1 and CDMS/MRI2 outcomes(73 and 84%,respectively). This was increased in the GD+ groups for both CDMS/MRI outcomes(87 and 93%,respectively)and for the Barkhof criteria positive group(79 and 89%,respectively). The majority of patients not meeting the GD+ or Barkhof criteria were also positive for the combined outcomes.

Conclusions:

For patients with a positive MRI at the time of a their initial neurological event both enhancing lesions and the Barkhof criteria are relatively strong predictors for development of CDMS over 18 months, likely identifying patients with an accelerated pace of disease. However our results suggest that even when the enhancing lesion or Barkhof criteria are not met, the vast majority of patients achieve the combined CDMS/MRI outcomes after only a short followup (18 months), suggesting important subclinical MRI activity, which is likely an expression of ongoing demyelinating activity. Current diagnostic criteria for MS do not address this large population of patients in the earliest stages of their disease who do not meet "criteria".

Supported By:

Biogen.Inc. Cambridge, MA


Detectable Atrophy Occurs within Three Months in Relapsing Remitting MS (RRMS)

Thursday, April 18, 2002
Martin Hardmeier, Peter Freitag, Wagenpfeil Stefan, Fisher Elizabeth, Richard A. Rudick, Mariska Kooijmans, Michel Clanet, Ernst W. Radue, Ludwig Kappos Basel, Switzerland; Munich, Germany; Cleveland, OH; Cambridge, MA; Toulouse, France

Objective:

To determine the time interval necessary to detect whole brain atrophy changes in RRMS and to evaluate the impact of inflammation as depicted by Gadolinium (Gd) enhancement.

Background:

Atrophy is believed to be the result of destructive changes in MS. Measurements of atrophy within intervals of six to twelve months have been used in clinical trials to show treatment effects. Short term changes have been attributed to fluctuations in hydrational state, to steroid treatment and edema caused by acute inflammation.

Design/Methods:

129 RRMS patients of the frequent MRI cohort of the rIFN-b-1a (Avonex ®) dose comparison study with complete clinical and MRI baseline data were evaluated. Three scans were performed within four months prior to randomization. No immunomodulatory drug was administered. Eleven patients were treated with intravenous Methylprednisolone (IVMP) for a relapse.
To assess atrophy the brain parenchymal fraction (BPF) was used. BPF is a normalized measure of whole brain volume and is automatically calculated (autosegMS ®). It has been shown to detect brain volume and volume changes reproducibly in yearly measurements.
Volume of T1-Gd-enhancing (T1Gd) lesions and T2 hyperintense lesions (T2LL) was determined by a semiautomated seedgrowing technique.
For statistical evaluation Wilcoxon rank and Spearman rank correlation test were used.

Results:

At baseline median EDSS was 3.5 (range 2.0-5.5); mean age: 38.5 (± 7.3) years. Mean interval between scan 1 and 2 and scan 1 and 3 was 33 days and 76 days respectivly.
Mean and median BPF decreased from scan 1 to 2 (-0.066%, p=0.075), from scan 1 to 3 this decrease was highly significant (-0.218%, p<0.001). The calculated yearly rate is -1.06% (SD 2.51).
Median T1Gd was not significantly different between timepoints.
Cross-sectionally BPF correlates only weakly with T1Gd at one timepoint (r=-0.199, p<0.05), longitudinally no correlations between changes in these parameters were found.
T2LL showed a strong negative correlation to BPF (r=-0.666, p<0.001).
The results of a subanalysis excluding subjects who had received IVMP (n=11) were not significantly different to the results of the main analysis.

Conclusions:

We showed, that BPF has the capability to detect whole brain volume changes in relapsing MS in intervals of less than three months in groups of patients. Presumably these changes reflect the ongoing atrophy process, since they can neither be attributed to inflammation nor to other known causes of short-term changes in brain volume.
Therefore, BPF may be a valuable tool in future Phase II studies with agents expected to have impact on the evolution of destructive MS pathology.

Supported By:

Biogen Inc., Cambridge, MA, USA


Longitudinal Magnetic Resonance Imaging Study on the Effect of Azathioprine in Relapsing-Remitting Multiple Sclerosis Patients Refractory to the Treatment with Interferon b-1b

Thursday, April 18, 2002
Silva Markovic-Plese, Bibiana Bielekova, Nadia Kadom, Nancy D. Richert, Thomas P. Leist, Roland Martin, Henry F. McFarland Bethesda, MD

Objective:

The objective of this study is to assess the effectiveness of azathioprine (AZA) on augmenting the response to interferon b-1b (IFNb-1b) in a cohort of relapsing-remitting multiple sclerosis (RRMS) patients. The number of Gadolinium (Gd)-enhancing magnetic resonance imaging (MRI) lesions was used as a primary outcome measure.

Background:

A subset of RRMS patients continues to have an active disease while maintained on IFNb-1b monotherapy. The use of combination immunotherapy is a frequently considered approach in the subset of patients with an aggressive form of RRMS. AZA is a purine analog whose favorable profile includes limited toxicity, oral administration, availability, and low cost. We proposed to study the combination of IFNb-1b and AZA in RRMS patients refractory to IFNb-1b therapy, using a change in the number of Gd-enhancing lesions as a sensitive primary outcome measure. The critical assessment of the individual response to therapy and an early introduction of combination immunomodulatory therapy may achieve a net immunosupressive effect required for the adequate RRMS disease control.

Design/Methods:

In a longitudinal study of RRMS patients treated with IFNb-1b, a subset of patients refractory to the therapy was identified, using the number of contrast enhancing lesions as a measure of treatment efficacy. A cohort of 6 RRMS patients who had been treated with IFNb-1b for 3 to 35 months (median 17 months), with a continuously high disease activity (median number of Gd-enhancing lesions 12.69 and median exacerbation rate 1.63), were studied on the IFNb-1b and AZA combination therapy for 6 to 35 months (median 15 months). Patients were evaluated using serial monthly MRI scans and comprehensive neurological evaluations. Clinical outcome measures included expanded disability status score (EDSS), progression index (change in EDSS per year), and exacerbation rate. Newly detected and total number of Gd-enhancing lesions, total T2 weighted lesion load and atrophy measures were determined at monthly intervals during the baseline, IFNb-1b monotherapy, and IFNb-1b with AZA combination therapy.

Results:

This study evaluated the effectiveness of add-on AZA therapy in RRMS patients with a high baseline disease activity (median number of new Gd-enhancing lesions 14.51, total number of lesions 18.95 ), using monthly MRI scans as a sensitive outcome measure. The number of new Gd-enhancing lesions was reduced from 9.83 to 3.37 (p=0.006), and the number of total Gd-enhancing lesions from 12.69 to 3.87 (p=0.002) during the combination IFNb-1b and AZA therapy, in comparison to the IFNb-1b monotherapy. Four patients with recurrent exacerbations during the IFNb-1b monotherapy had a reduction in relapse number during the IFNb-1b and AZA combination therapy.

Conclusions:

IFNb-1b and AZA combination therapy provide a synergistic effect on stabilizing the blood brain barrier, as demonstrated by the decreased number of Gd-enhancing lesions.

Supported By:

National Institute of Neurological Diseases and Stroke.


Evidence of Early Cortical Grey Matter Atrophy in Patients with Relapsing Remitting and Primary Progressive Multiple Sclerosis

Thursday, April 18, 2002
Nicola De Stefano, Paul M. Matthews, Massimo Filippi, Giuseppe Iannucci, Angelo Ghezzi, Enrico Montanari, Maria L. Bartolozzi, Leonello Guidi, Antonio Federico, Stephen Smith Siena; Oxford; Milano; Milano, Italy; Gallarate, Italy; Fidenza, Italy; Empoli, Italy; Siena, Italy; Oxford, United Kingdom

Objective:

To assess cortical atrophy in patients with the relapsing and progressive forms of multiple sclerosis (MS) at different disease stages and evaluate its relationship with clinical disability.

Background:

Recent in situ and in vivo studies have shown that neuro-axonal damage is relevant in MS. Axonal transection and apoptotic loss of neurons due to cortical demyelination have been demonstrated in MS brains. Thus, cortical grey matter (GM) pathology may contribute to neurological impairment in MS.

Design/Methods:

Conventional proton T1-weighted MR images were obtained in 90 patients with definite MS who had either the relapsing remitting (RR, n= 65) or the primary progressive (PP, n= 25) form of the disease. These MR images were used to obtain cortical GM normalized for head size using a method for computing measurement of regional brain volume (SIENA, Smith et al. JCAT 2001). The two groups of MS patients were matched for disease duration (RR mean = 7±7.2 years, PP mean = 8±6.5, p= 0.4), but PP patients were significantly (p < 0.001) older and more disabled than RR patients (PP median age = 50 years, RR median ages = 34 years; median EDSS in PP = 4.5, median EDSS in RR = 1.5). In the analyses of the MR data, age differences in the two MS groups were corrected for by using a z-score transformation relative to an age-matched normal control (NC) group for each patient group. The nonparametric Kruskal-Wallis test of variance was used for group analyses and the nonparametric Spearman rank order correlation was used to correlate MR and clinical measures. Data were considered significant at the 0.05 level.

Results:

Cortical GM values were significantly lower in the whole group of MS patients than in NC (GM in MS patients = 0.58±0.05, GM in NC = 0.63±0.03, p<0.0001), but similar in the two patient groups (p= 0.8). Even MS patients grouped for early disease duration (<5 years) or mild disability (EDSS 2) had significantly lower cortical GM values than NC (GM in MS patients with early disease duration = 0.60±0.03, GM in NC = 0.63±0.02, p<0.01; GM in MS patients with low EDSS = 0.59±0.04, GM in NC = 0.63±0.02, p<0.01). Measures of cortical GM showed a significant negative correlation with EDSS scores in the whole group of MS patients (r = -0.55, p< 0.0001), but this relationship was stronger for PP (r = -0.62 p< 0.0001) than for RR patients (r = -0.33 p< 0.1).

Conclusions:

These data confirm that cortical GM abnormalities are significant in MS and may occur even early in the course of the disease. The significant negative correlation between clinical disability and the cortical GM values suggest that, especially in PP MS patients, GM pathology may contribute significantly to neurological impairment.


Aspirin for Multiple Sclerosis-Related Fatigue: Results of a Double-Blind Placebo-Controlled Study

Thursday, April 18, 2002
Dean M. Wingerchuk, Eduardo E. Benarroch, Peter C. O'Brien, B. Mark Keegan, Claudia F. Lucchinetti, John H. Noseworthy, Brian G. Weinshenker, Moses Rodriguez Scottsdale, AZ; Rochester, MN

Objective:

To determine whether aspirin (ASA) reduces the severity of multiple sclerosis-related fatigue.

Background:

Fatigue is the most common symptom of multiple sclerosis (MS) and often the most disabling early in the disease course. Several lines of clinical evidence suggest it is related to the underlying immunopathogenesis of the disease. Pharmacotherapy is currently limited to amantadine and nonspecific central nervous system stimulants that provide little insight into fatigue pathophysiology and are modestly effective for most patients. More effective and targeted therapies are urgently needed.
Following previous clinical observations, we had anecdotal success empirically treating MS fatigue with 650-1900 mg/d of ASA. We performed a randomized, controlled study to objectively evaluate the effects of ASA on MS-related fatigue.

Design/Methods:

Thirty ambulatory patients with significant MS-related fatigue (MS-specific Fatigue Scale score4) of at least 3 months duration were enrolled in this double-blind, placebo-controlled, crossover trial. Patients with clinically significant depression, medical contraindications, or who were using current MS-fatigue therapies were excluded. During the first 6-week phase of the study, 15 patients were randomized to receive 650 mg ASA twice per day (morning and noon) and 15 were randomized to placebo. Following a 2-week washout period, patients crossed over to the other treatment for 6 weeks. Fatigue was measured using descriptive (patient report of clinical change) and continuous measures (visual analog scale, modified and complete forms of the Fatigue Impact Scale, Fatigue Severity Scale, MS-Specific Fatigue Scale). Statistical analysis was performed using McNemar and paired t-tests as appropriate.

Results:

Twenty-four women and six men were enrolled. The mean Expanded Disability Status Scale (EDSS) score was 2.5 (range 0-5.5) and mean fatigue duration was 33 months. Twenty-six patients completed the study. Forty-two percent of patients reported good or excellent improvement in fatigue levels during ASA treatment compared with 11% during the placebo phase (p=0.008). Modified Fatigue Impact Scale scores were also significantly reduced (p=0.035). There were trends toward benefit on all measurement scales. Patient questionnaires indicated that treatment blinding was successfully maintained and there were no significant adverse events.

Conclusions:

ASA may reduce fatigue severity in MS. The results of this controlled study strengthen the concept of MS-related fatigue as an immunologically mediated construct and theoretically implicate several pathophysiological mechanisms involving prostaglandins, cytokines, and the neuroendocrine axis in fatigue generation. Further study is warranted to confirm these results and to explore the mechanisms by which ASA modulates fatigue.

Supported By:

Mayo Clinic and Foundation


Is it Possible To Reduce the Dose of Interferon (IFN) Beta in MS Patients with a Prolonged MRI-Confirmed Absence of Disease Activity ?

Thursday, April 18, 2002
Pierangelo Barbero, Antonio Pipieri, Elisabetta Verdun, Mauro Bergui, Gianluca Isoardo, Andrea Bosio, Marinella Clerico, Angele Cucci, Alessandra Ricci, Bruno Bergamasco, Luca Durelli Torino, Italy

Objective:

To evaluate the effects of reducing IFN beta dose in patients with relapsing-remitting (RR) MS in chronic treatment with IFN beta-1b and with clinical and MRI stabilization.

Background:

Controlled trials have demonstrated the efficacy of IFN beta-1a at the dose of 30 mcg intramuscular once a week or of IFN beta-1b at the dose of 8 million international units (MIU) on alternate days. The chronic administration of a drug on alternate days may affect patient compliance and encourage the decision to reduce IFN beta dose. A study of clinical and MRI effects associated with the reduction of IFN beta dose is needed.

Design/Methods:

Prospective one year follow-up of 27 RR MS patients randomised to gradually switch from on-alternate-day IFN beta-1b to once-a-week IFN beta-1a (13 patients), or to continue on IFN beta-1b (14 patients). Before IFN beta reduction the patients had to be on chronic IFN beta treatment for at least 3 years, and without clinical signs of disease activity during the last 2 years and no MRI activity in last two yearly scans. Clinical outcome measures were exacerbation rate (ER), proportion of patients with exacerbations and with progression of disability (defined as an increased of a 1.0 point of EDSS score from baseline sustained for 3 months). MRI was performed before randomisation and at the end of the one year follow-up. MRI outcome measures were the proportion of patients without MRI activity (i.e., without either new T2 or gadolinium-enhancing lesions), and percent changes of PD/T2 disease burden.

Results:

Both before starting IFN treatment and before the study, there were no differences between demographic, clinical, and MRI parameters of the two groups. One year after the reduction of IFN beta dose clinical outcome measures were, for IFN beta-1a group, ER, 0.9; proportion of patients with exacerbations, 77%; proportion of patients with sustained EDSS worsening, 23%; for IFN beta-1b group, ER, 0.2; proportion of patients with exacerbations, 21%; proportion of patients with sustained EDSS worsening, 0%. The proportion of patients with MRI activity was, for IFN beta-1a group, 85%; for IFN beta-1b group, 36%. Both ER as well as the proportions of patients with exacerbations, with EDSS worsening, or with MRI activity were significantly lower (p=0.05) in the patients who continued on IFN beta-1b compared to those switched to once-a-week beta-1a treatment. Percent median changes from baseline of PD/T2 disease burden were +6.7% in IFN beta-1a, and - 0.9% in IFN beta-1b group (p=0.05).

Conclusions:

IFN beta treatment is a chronic one. The reduction of IFN beta-1b dose is not advisable even in patients with years of absence of clinical and MRI disease activity.


Matrix Metalloproteinase-9 Expression in Sural Nerve Biopsies from Diabetic Patients Differentiates Chronic Inflammatory Demyelinating Polyneuropathy from Diabetic Polyneuropathy

Thursday, April 18, 2002
Stefano Jann, Manuela A. Bramerio, Elio Scarpini, Klaus V. Toyka, Carlo A. Defanti, Claudia Sommer Milan, Italy; Wuerzburg, Germany

Objective:

The aim of this study was to find markers on sural nerve biopsies that would help to distinguish chronic inflammatory demyelinating polyneuropathy in diabetic patients (CIDP-DM) from diabetic polyneuropathy (DP)

Background:

There is growing evidence that diabetic patients have an increased predisposition to CIDP. In these patients, immunosuppressive therapy is indicated. However, differentiating CIDP in diabetic patients from DP may be difficult. Matrix metalloproteinases (MMPs) are a family of calcium-dependent zinc endopeptidases which are able to degrade the basement membrane and thus contribute to tissue destruction and cell invasion. MMPs may be involved in the pathogenesis of Multiple Sclerosis, Guillain Barre-Syndrome and CIDP as effector molecules of blood-brain/nerve-barrier disruption. We therefore hypothesised that MMPs might be a useful marker to differentiate CIDP-DM from DP..

Design/Methods:

Sural nerve biopsies were taken from 10 patients with CIDP-DM and from 5 patients with DP. CIDP-DM patients (fulfilling electrophysiological criteria for CIDP) comprised 8 males and 2 females, mean age 60.7 years, while DP patients were 3 males and 2 females, mean age 67.9 years. The mean duration of diabetes was 5.6 years in CIDP-DM patients and 11.8 years in DP patients; diabetes was type 1 in 4 cases and type 2 in 6 CIDP-DM cases and type 1 in 1 case and type 2 in 4 DP cases. Fiber size distribution histograms were created from semithin sections using computer assisted morphometry. Immunohistochemistry was performed for T-cells, macrophages, von Willebrand factor and MMP-9. Endoneurial and epineurial immunoreactivity was evaluated and quantified separately.

Results:

Small epineurial T-cell infiltrates were present in 5 out of 10 CIDP-DM patients and in none of DP patients. In CIDP-DM 52.9% of endoneurial vessels were immunoreactive for MMP-9 against only 12.7% in DP. Similarly, MMP-9-immunoreactivity was detected in 34.4% of epineurial vessels in CIDP-DM and only in 14.2% in DP. Statistical analysis confirmed a significant increase of MMP-9 expression in endothelial cells in CIDP-DM. Moreover, 5 out of 10 CIDP-DM patients showed endoneurial cells (mainly T cells) strongly positive for MMP-9 while none of the 5 DP patients showed positive cells.

Conclusions:

In this study we demonstrate that expression of MMP-9 is strongly enhanced in CIDP-DM but not in DP. The pattern of MMP-9 immunoreactivity may help to differentiate CIDP-DM from DP and thus allow to select patients for a trial with immunosuppressant drugs.


Baseline Variables Predictive of Conversion from Possible to Clinically Definite MS: ETOMS Results

Thursday, April 18, 2002
Filippo Martinelli-Boneschi, Hans-Peter Hartung, Per Soelberg Sorensen, Gilles Edan, Oscar Fernandez, Luca Durelli, Marco Roveris, Pierrette Seeldrayers, Frederik Barkhof, Massimo Filippi, Otto Hommes, Giancarlo Comi, ETOMS Study Group Milan, Italy; Dusseldorf, Italy; Copenhagen, Denmark; Rennes, France; Sienna, Spain; Turin, Italy; Charlerio, Belgium; Amsterdam, Netherlands; Nijmegen, Netherlands; Europe

Objective:

Examine baseline characteristics that predict which patients with a first episode consistent with demyelinating disease progress to clinically definite MS (CDMS) and to examine differences between those with clinically isolated syndrome (CIS) onset compared to poly-symptomatic (PS) onset.

Background:

Patients with a first episode of CNS dysfunction consistent with MS present with CIS or have symptoms affecting more than one CNS region. The risk of progression to CDMS may vary. Patients in the ETOMS study provide an opportunity to determine whether a differential risk exists as well as to explore which other baseline variables are associated with progression to CDMS.

Design/Methods:

ETOMS was a randomized, double-blind, placebo-controlled study of 308 patients equally distributed to 22mcg interferon beta-1a (IFN, Rebif®) once weekly and placebo. Primary endpoint was the proportion of patients having a second event defining CDMS. Baseline variables were assessed for predictive value for primary outcome and time to conversion. Odds ratios are from logistic regression adjusted for country. Continuous variables were analyzed by ANOVA on ranks while binary variables were analyzed by Mantel-Haenszel test. The relationship between baseline variables and time to CDMS was assessed using the Cox proportional hazards model.

Results:

IFN treated patients had a 24% relative reduction in primary outcome (p = 0.047) at 24 months.Thirty-two percent (98/309) patients had multi-focal onset of symptoms with similar proportions in both treatment groups. The proportion of patients developing CDMS (second attack) was 34% for CIS and 51% for PS patients (OR = 1.99, p = 0.015). Conversion to CDMS was also higher for time since first attack and randomization [< 60 days vs. 60 days] (OR = 2.1, p = 0.029) and baseline T2 lesion number [< 9 vs. 9 or more] (OR = 3.6, p = 0.014). Multi-focal onset (p 0.004), baseline EDSS (p = 0.003), T2 lesion burden (p < 0.001) and baseline T1-Gd lesion activity (p = 0.007) were all prognostic for time to CDMS. Patients with CIS did not show a significant treatment effect (OR = 0.86, p = 0.59) while PS onset patients did (OR = 0.34, p = 0.01). PS onset had more T2 lesions (p < 0.019), more gadolinium enhancing lesions (p=0.009), more hypo-intense T1 lesions (p=0.001) and a greater proportion of patients meeting multiple Barkhof MRI criteria for diagnosis of MS (p = 0.001).

Conclusions:

Patients with PS onset of possible MS have approximately double the odds of progressing to CDMS within 2 years of first attack. PS patients have increased evidence of disease activity as measured by MRI. IFN treatment effect is greater in PS patients than CIS showing the importance of early treatment based on clinical and MRI parameters.


Effect of Glatiramer Acetate Treatment on Multiple Sclerosis Lesions with Different Degrees of Ongoing Inflammation

Thursday, April 18, 2002
Marco Rovaris, Giancarlo Comi, Maria Codella, Lucia Moiola, Angelo Ghezzi, Mauro Zaffaroni, Gianluigi Mancardi, Elena Capello, Francesco Sardanelli, Massimo Filippi Milan, Italy; Gallarate, Italy; Genoa, Italy

Objective:

To assess whether glatiramer acetate (GA) treatment has a graduated effect on multiple sclerosis (MS) disease activity as measured on single dose (SD) and triple dose (TD) gadolinium (Gd)-enhanced magnetic resonance imaging (MRI) scans of the brain

Background:

MS lesions detected by TD Gd-enhanced MRI have milder degrees of tissue disruption than those enhancing after a SD. GA significantly reduces the frequency of SD enhancing lesions in relapsing-remitting (RR) MS.

Design/Methods:

In 20 patients with RRMS, brain MRI scans were obtained every four weeks for two periods of 5 consecutive months, before and 90 days after GA treatment initiation. At each time point, T1-weighted scans were obtained after injection of SD (0.1 mmol/kg of Gd) in one session and TD (0.3 mmol/kg of Gd) in another one, separated by a 24-hour interval. All patients had to have one or more enhancing lesions on the first SD scan performed at study initiation.

Results:

At baseline, the median T2 lesion volume was 10.3 ml (range: 2.3-33.6 ml). The mean numbers of total and new enhancing lesions/patient/month in the baseline period were 3.4 and 3.0 on SD scans, 5.4 and 4.1 on TD scans (p<0.0001). During GA treatment, the mean numbers of total and new enhancing lesions/patient/month dropped to 1.7 (-50%) and 0.9 (-70%) on SD, 3.3 (-39%) and 1.7 (-59%) on TD scans. The mean numbers of new T2 lesions/patient/month were 2.7 and 0.9 before and during treatment, respectively. During the baseline/GA treatment periods, the frequencies of active scans were 76%/56% (SD - total lesions), 64%/37% (SD - new lesions), 87%/69% (TD - total lesions), 76%/51% (TD - new lesions). The effect of treatment was significant on all the MRI measures of MS activity (p<0.0001), without interactions with the Gd dose.

Conclusions:

GA treatment is effective in reducing MRI-measured MS activity, independently of the severity of the accompanying inflammatory process.

Supported By:

TEVA Pharmaceutical Industries, Ltd.


Low-Contrast Sloan Letter Chart Testing as a Candidate Visual Outcome Measure for the Multiple Sclerosis Functional Composite (MSFC)

Friday, April 19, 2002
Laura J. Balcer, Monika L. Baier, Mariska Kooijmans, Jeffrey A. Cohen, Ligia Nano-Schiavi, Gui-Shang Ying, Maureen G. Maguire, Steven L. Galetta, Gary R. Cutter Philadelphia, PA; Denver, CO; Cambridge, MA; Cleveland, OH; Philadelphia, PA; Denver, CO

Objective:

To examine low-contrast Sloan letter chart testing as a candidate visual outcome measure for the Multiple Sclerosis Functional Composite (MSFC).

Background:

Visual dysfunction is one of the most common and disabling aspects of neurologic impairment in multiple sclerosis (MS). The MSFC, a new clinical trial outcome measure, includes a timed 25-foot walk, 9-hole peg test, and the paced auditory serial addition test, but does not yet include a measure of visual function. Adding a visual dimension to the MSFC will likely increase its applicability in MS populations.

Design/Methods:

The design of these studies was cross sectional, including a sub-study of randomized clinical trial. Binocular low-contrast Sloan letter chart (LCSLC) testing and high-contrast visual acuity testing was administered to a subgroup of participants in the International MS Secondary-Progressive Avonex Controlled Trial (IMPACT). Data from an ongoing study of visual outcome measures at the University of Pennsylvania (Penn MS Study: relapsing-remitting MS) were also analyzed.

Results:

Rank-correlations of LCSLC scores (#letters correct at 5%, 2.5%, 1.25% contrast levels) with scores from the MSFC and Expanded Disability Status Scale (EDSS) were significant to a modest-moderate degree, supporting a potential role for LCSLC testing in MS:
IMPACT - 12-month visit:
(N=65; age 47±8 years)
LCSLC vs. MSFC: rs = 0.49 to 0.57, p < 0.0002
LCSLC vs. EDSS: rs = -0.39 to -0.43, p < 0.003
Penn MS Study:
(N=50; age 45±8 years)
LCSLC vs. MSFC: rs = 0.49 to 0.57, p < 0.0006
LCSLC vs. EDSS: rs = -0.37 to -0.40, p < 0.008
Median binocular Snellen acuities were 20/20 (20/16-20/100) in both studies. Among 5 visual outcome measures in the Penn MS Study (including high-contrast visual acuity), LCSLC scores best distinguished MS patients from age-matched controls on the basis of visual function, even following detailed refractions (p<0.006, logistic regression analysis).

Conclusions:

LCSLC testing captures aspects of neurologic impairment in MS not entirely captured by high-contrast visual acuity, EDSS, or MSFC scores, and demonstrates excellent potential as a candidate MSFC visual component based on cross-sectional data. Large-scale, longitudinal studies are needed to determine if LCSLC testing is sensitive to clinical changes and treatment effects over time.

Supported By:

National MS Society RG3208-A-1, NIH RO1 EY-013273, and Biogen, Inc.