Stanton B. Elias

Glatiramer acetate in primary progressive multiple sclerosis: Results of a multinational, multicenter, double-blind, placebo-controlled trial

To determine whether glatiramer acetate (GA) slows accumulation of disability in primary progressive multiple sclerosis.

Erythropoietin treatment improves neurological functional recovery in EAE mice

Erythropoietin (EPO), originally recognized for its central role in erythropoiesis, has been shown to improve neurological outcome after stroke. Here, we investigated the treatment of experimental autoimmune encephalomyelitis (EAE) in mice with EPO. Mice were treated with recombinant human EPO (rhEPO) upon onset of paresis. Neurological functional tests were scored daily by grading of clinical signs (score 0-5). Hematoxylin and eosin (HE) staining of cerebral tissue was performed to detect inflammatory infiltrates. Double staining for Luxol fast blue and Bielshowsky was used to demonstrate myelin and axons, respectively. Immunohistochemistry was performed to measure the expression of bromodeoxyuridine (BrdU, a marker for cell proliferation), NG2 (a marker for oligodendrocyte progenitor cells) and brain-derived neurotrophic factor (BDNF). Treatment with rhEPO significantly improved neurological functional recovery, reduced inflammatory infiltrates and demyelination, and increased oligodendrocyte progenitor cell proliferation and BDNF+ cells compared to the EAE controls. These data indicate that rhEPO treatment improved functional recovery after EAE in mice, possibly, via stimulating oligodendrogenesis, downregulating proinflammatory infiltrates and by elevating BDNF expression.

NEUROLOGICAL FUNCTIONAL RECOVERY AFTER THYMOSIN BETA4 TREATMENT IN MICE WITH EXPERIMENTAL AUTO ENCEPHALOMYELITIS

In the present study, we hypothesized that thymosin beta 4 (Tbeta4) is a potential therapy of multiple sclerosis (MS). To test this hypothesis, SJL/J mice (n=21) were subjected to experimental autoimmune encephalomyelitis (EAE), an animal model of MS. EAE mice were treated with saline or Tbeta4 (6 mg/kg, n=10) every 3 days starting on the day of myelin proteolipid protein (PLP) immunization for total five doses. Neurological function, inflammatory infiltration, oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes were measured in the brain of EAE mice. Double immunohistochemical staining was used to detect proliferation and differentiation of OPCs. Tbeta4 was used to treat N20.1 cells (premature oligodendrocyte cell line) in vitro, and proliferation of N20.1 cells was measured by bromodeoxyuridine (BrdU) immunostaining. Tbeta4 treatment improved functional recovery after EAE. Inflammatory infiltrates were significantly reduced in the Tbeta4 treatment group compared to the saline groups (3.6+/-0.3/slide vs 5+/-0.5/slide, P<0.05). NG2(+) OPCs (447.7+/-41.9 vs 195.2+/-31/mm(2) in subventricular zone (SVZ), 75.1+/-4.7 vs 41.7+/-3.2/mm(2) in white matter), CNPase(+) mature oligodendrocytes (267.5+/-10.3 vs 141.4+/-22.9/mm(2)), BrdU(+) with NG2(+) OPCs (32.9+/-3.7 vs 17.9+/-3.6/mm(2)), BrdU(+) with CNPase(+) mature oligodendrocytes (18.2+/-1.7 vs 10.7+/-2.2/mm(2)) were significantly increased in the Tbeta4 treated mice compared to those of saline controls (P<0.05). These data indicate that Tbeta4 treatment improved functional recovery after EAE, possibly, via reducing inflammatory infiltrates, and stimulating oligodendrogenesis.

Bone marrow stromal cells increase oligodendrogenesis after stroke

Oligodendrocytes are sensitive to ischemic damage. The Sonic hedgehog (Shh) pathway is critical in oligodendrogenesis; Gli1 is the principal effector of Shh signaling. We investigated oligodendrogenesis and Shh/Gli1 pathway activation after bone marrow stromal cell (BMSC) treatment of stroke in rats. Rats were subjected to the middle cerebral artery occlusion (MCAo). BMSCs have been shown to promote functional recovery post stroke. A therapeutic dose of BMSC (3times106 cells) treatment was initiated 1 day after MCAo. Immunohistochemistry was carried out to measure the oligodendrocyte progenitor cells, oligodendrocytes, myelin, and expressions of Shh and Gli1 at 14 days after MCAo. Gene expression of Shh and Gli1 was tested at 2 days after MCAo. An in vitro study was used to investigate the effects of BMSC on a premature oligodendrocyte cell line (N20.1 cells). BMSC treatment significantly increased 04+ oligodendrocytes, MBP+ area, and bromodeoxyuridine (BrdU)+, NG2+, BrdU+-NG2+ cells, and mRNA and protein expressions of Shh and Gli1 in the ipsilateral brain of the MCAo rats than that in phosphate buffered saline (PBS)-treated rats. BMSCs promoted N20.1 cell proliferation and Gli1 mRNA expression, and these effects were abolished by the Shh pathway inhibitor cyclopamine. These data indicate that the BMSC treatment stimulates oligodendrogenesis by activation of the Shh/Gli1 pathway post stroke.

The PROMiSe trial: baseline data review and progress report

The PRO MiSe trial is a multinational, multicentre, double-blind, placebo -controlled trial evaluating the effects of glatiramer acetate treatment over 3 years in patients with primary progressive multiple sclerosis (PPMS). A total of 943 patients were enrolled, and all those remaining on-study had completed at least 24 months as of O ctober 2002. Baseline clinical and MRI character istics and select correlations are reported here. A total of 3.9% of patients exhibited confirmed relapse over 1904 patient-years of exposure, indicating success of efforts to exclude relapsing MS types. O f the 26.3% of patients who have prematurely withdrawn from the study, only 36% discontinued after meeting the study primary endpoint of disease progression. The progression rate in patients in the low Expanded Disability Status Scale (EDSS) stratum (3.0-5.0) observed thus far is markedly lower than the 50% annual progression rate estimate used for determining size and statistical power of the trial; progression was observed in 16.1% of patients with 12 months of study exposure. These early findings raise some concern about the ability of the trial to demonstrate a significant treatment effect, and suggest that the short-term natural history of PPMS may not be as aggressive as previously assumed.

Polyradiculopathy in sarcoidosis.

We present three new and 14 retrospective cases of polyradiculopathy in sarcoidosis. Of these, 71% had weakness and 59% areflexia of the lower extremities, and 35% had sphincter dysfunction. Cases often were associated with central nervous system sarcoidosis. All cases involved thoracolumbar or lumbosacral roots, except a single case of cervical polyradiculopathy. Of 14 treated patients, nine improved with corticosteroids, laminectomy, or both. Polyradiculopathy complicating sarcoidosis: (1) is uncommon; (2) primarily involves thoracic and lumbar roots; (3) may arise from contiguous, hematogenous, or gravitational nerve root sleeve seeding; (4) may be asymptomatic; and (5) may improve with corticosteroids. Differential diagnosis of weakness in patients with sarcoidosis should include nerve root involvement from the primary process by direct sarcoid involvement.

Bone marrow stromal cell therapy reduces proNGF and p75 expression in mice with experimental autoimmune encephalomyelitis.

Demyelination is prominent in experimental autoimmune encephalomyelitis (EAE). The receptor p75 and its high affinity ligand proNGF are required for oligodendrocyte death after injury. We hypothesize that bone marrow stromal cells (BMSCs) provide therapeutic benefit in EAE mice by reducing proNGF/p75 expression. PBS or BMSCs (2 x 10(circumflex)6) were administered intravenously on the day of EAE onset. Neurological function and demyelination areas were measured. Immunohistochemical staining was used to measure apoptotic oligodendrocytes, expression of proNGF and p75, and the relationship between proNGF and p75 in neural cells. proNGF was used to treat oligodendrocytes in culture with or without BMSCs. EAE mice exhibited neurological function deficit and demyelination, and expression of proNGF and p75 was increased. BMSC treatment improved functional recovery, reduced demyelination area and apoptotic oligodendrocytes, decreased expression of proNGF and p75 compared with PBS treatment. proNGF(+) cells colocalized with neural cell markers, while p75 colocalized with an oligodendrocytic marker, and proNGF colocalized with p75. proNGF induced apoptosis of oligodendrocytes in vitro, and p75 antibody blocked this apoptotic activity. BMSCs reduced p75 expression and apoptotic activity in oligodendrocytes with proNGF treatment. BMSC treatment benefits on EAE mice may be fostered by decreasing the cellular expression of proNGF and p75, thereby reducing oligodendrocyte death.

Fingolimod treatment promotes proliferation and differentiation of oligodendrocyte progenitor cells in mice with experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a major demyelinating disease of the central nervous system (CNS) leading to functional deficits. The remyelination process is mediated by oligodendrocyte progenitor cells (OPCs). In this study, we tested the hypothesis that Fingolimod, a sphingosine 1-phosphate (S1P) receptor modulator, stimulates OPC differentiation into mature oligodendrocytes, in addition to its well-known anti-inflammatory effect. Using an animal model of MS, experimental autoimmune encephalomyelitis (EAE), we performed a dose-response study of Fingolimod (0.15 or 0.3mg/kg bw), which was initiated on the day of EAE onset. The neurological function was tested to determine the optimal dose of Fingolimod. Immunofluorescent staining was performed to measure the profile of OPC proliferation and differentiation. The mechanistic premise underlying the therapeutic effect of Fingolimod, was that Fingolimod stimulates the sonic hedgehog (Shh) pathway, and this pathway promotes OPC differentiation. To test this hypothesis, a loss-of-function study using cyclopamine, an inhibitor of the sonic hedgehog (Shh) pathway, was employed in vivo. Protein levels of the Shh pathway were measured by Western blot analysis. We found that Fingolimod treatment (0.3mg/kg bw) significantly decreased cumulative disease score compared to the EAE control group. Concurrently, OPCs and proliferation of OPCs were significantly increased in the white matter of the brain and spinal cord at day 7 and day 30 after EAE onset, and oligodendrocytes, myelination and differentiation of OPCs were significantly increased at day 30 compared with the EAE control group. EAE mice treated with Fingolimod exhibited substantially elevated levels of Shh, its receptor Smoothened and effector Gli1 in the white matter of the CNS. However, combination treatment of EAE mice with cyclopamine-Fingolimod decreased Fingolimod monotherapy elevated protein levels of Smoothened and Gli1, and abolished the effect of Fingolimod on OPC proliferation and differentiation, as well as on neurological function outcome. Together, these data demonstrate that Fingolimod is effective as a treatment of EAE by promoting OPC proliferation and differentiation, which facilitate remyelination. In addition, the Shh pathway likely contributes to the therapeutic effects of Fingolimod on OPCs.

Bone Marrow Stromal Cells Protect Oligodendrocytes From Oxygen-Glucose Deprivation Injury

Oligodendrocyte (OLG) damage leads to demyelination, which is frequently observed in ischemic cerebrovascular diseases. In this study, we investigated the effect of bone marrow stromal cells (BMSCs) on OLGs subjected to oxygen‐glucose deprivation (OGD). N20.1 cells (mouse OLG cell line) were transferred into an anaerobic chamber for 3 hr in glucose‐free and serum‐free medium. After OGD incubation, OLG cultures were divided into the following groups: 1) OGD alone, 2) OLG cocultured with BMSCs, 3) treatment with the phosphoinostide 3‐kinase (PI3k) inhibitor LY294002, 4) LY294002‐treated OLGs with BMSC cocultured, and 5) anti‐p75 antibody‐treated OLGs. After an additional 3 hr of reoxygenation incubation, OLG viability and apoptosis were measured. The mRNA expression in the BMSCs and OLGs was analyzed using quantitative real‐time PCR (RT‐PCR). Serine/threonine‐specific protein kinase (Akt), phosphorylated Akt (p‐Akt), p75, and caspase 3 protein expressions in OLGs were measured by Western blot. Our results suggest that BMSCs produce growth factors, activate the Akt pathway, and increase the survival of OLGs. BMSCs also reduce p75 and caspase 3 expressions in the OGD‐OLGs, which leads to decreased OLG apoptosis. BMSCs participate in OLG protection that may occur with promoting growth factors/PI3K/Akt and inhibiting the p75/caspase pathways. Our study provides insight into white matter damage and the therapeutic benefits of BMSC‐based remyelinating therapy after stroke and demyelinating diseases.

Niaspan treatment improves neurological functional recovery in experimental autoimmune encephalomyelitis mice.

We investigated the treatment of experimental autoimmune encephalomyelitis (EAE) in mice with Niaspan, an agent used to elevate high-density lipoprotein (HDL). EAE mice were treated with Niaspan starting on the immunization or clinical onset day. Neurological functional recovery was significantly increased in the Niaspan treated mice (100 mg/kgbw) compared to the controls. Inflammatory infiltrates were significantly reduced in the Niaspan treatment group compared to the EAE controls. HDL level, intact myelin area, newly formed oligodendrocytes, regenerating axons, gene and protein levels of sonic hedgehog (Shh)/Gli1 were significantly increased in the Niaspan treated mice compared to EAE controls. These data indicate that Niaspan treatment improved functional recovery after EAE, possibly, via reducing inflammatory infiltrates and demyelination areas, and stimulating oligodendrogenesis and axonal regeneration. Niaspan-mediated activation of Shh/Gli1 pathway may promote functional recovery post-EAE.