Tamara Vyshkina

Clinical and pathological effects of intrathecal injection of mesenchymal stem cell-derived neural progenitors in an experimental model of multiple sclerosis

Multiple sclerosis (MS) is associated with irreversible disability in a significant proportion of patients. At present, there is no treatment to halt or reverse the progression of established disability. In an effort to develop cell therapy-based strategies for progressive MS, we investigated the pre-clinical efficacy of bone marrow mesenchymal stem cell-derived neural progenitors (MSC-NPs) as an autologous source of stem cells. MSC-NPs consist of a subpopulation of bone marrow MSCs with neural progenitor and immunoregulatory properties, and a reduced capacity for mesodermal differentiation, suggesting that this cell population may be appropriate for clinical application in the CNS. We investigated whether MSC-NPs could promote repair and recovery after intrathecal injection into mice with EAE. Multiple injections of MSC-NPs starting at the onset of the chronic phase of disease improved neurological function compared to controls, whereas a single injection had no effect on disease scores. Intrathecal injection of MSC-NPs correlated with reduced immune cell infiltration, reduced area of demyelination, and increased number of endogenous nestin-positive progenitor cells in EAE mice. These observations suggest that MSC-NPs may influence the rate of repair through effects on endogenous progenitors in the spinal cord. This study supports the use of autologous MSC-NPs in MS patients as a means of promoting CNS repair.

Variations in mitochondrial DNA copy numbers in MS brains.

The aim of this study is to determine if there is a pathology-related variation in mitochondrial (mt)DNA copy numbers in brains of patients with multiple sclerosis (MS). Our recent study demonstrated an age-dependent but excluded a MS pathology-related increase in the proportion of cytochrome oxidase (COX)-negative cells and deleted mtDNA molecules in postmortem brain tissue specimens of patients and controls (Blokhin et al., Neuromolecular Medicine, in press, 2008). This corollary study further extends our efforts defining mitochondrial contributions to tissue degeneration associated with inflammatory demyelination. Copy number variations of mtDNA molecules were defined by quantifying the mtDNA ND1 gene copies relative to the invariable nuclear ribosomal 18S gene copies (ND1/r18S) using real-time polymerase chain reaction analyses in laser dissected, COX-positive and COX-negative single neurons and glial cells from frozen postmortem normal-appearing gray (NAGM) and white matter (NAWM) regions and chronic active plaques of MS patients, and gray matter (GM) and white matter (WM) regions of age matched non-neurological disease (NND) controls. ND1/r18S values were correlated with tissue regions, pathology, and age. While the ND1/r18S values were similar in NAWM and plaque-containing specimens of MS patients as well as in NAWM of patients and WM of age-matched NND controls, we found significantly higher mtDNA copy number values in neurons of NAGM than in cells of other MS brain regions. The ND1/r18S values were even higher in NAGM than in GM of age-matched NND controls. An age-related decline in ND1/r18S values was also noted in neurons of both MS patients and NND controls. These observations exclude a change in mtDNA copy numbers in plaques, however, suggest a compensatory replication of mtDNA or mitochondria in the cortex with neuroaxonal loss in MS. The age-related decline in mtDNA copy numbers may explain some features of late-onset MS.

Increased promoter methylation of the immune regulatory gene SHP-1 in leukocytes of multiple sclerosis subjects

The protein tyrosine phosphatase, SHP-1, is a negative regulator of proinflammatory signaling and autoimmune disease. We have previously reported reduced SHP-1 expression in peripheral blood leukocytes of subjects with multiple sclerosis (MS). Recent evidence indicates that virus-induced DNA methylation of the SHP-1 promoter is responsible for aberrant silencing of SHP-1 expression and function in hematopoietic cells that might relate to inflammatory diseases. In the present study, bisulfite sequencing of the SHP-1 promoter demonstrated that over a third of MS subjects had abnormally high promoter methylation. As SHP-1 is deficient in MS leukocytes and SHP-1-regulated proinflammatory genes are correspondingly upregulated, we propose that increased SHP-1 promoter methylation may relate in part to decreased SHP-1 expression and increased leukocyte-mediated inflammation in MS.

Autoantibodies and neurodegeneration in multiple sclerosis

Neurodegeneration develops in association with inflammation and demyelination in multiple sclerosis. Available data suggest that the progressive neuroaxonal loss begins in the earliest stages of the disease and underlies the accumulation of clinical disability. The loss of neurons and their processes is driven by a complex molecular mechanism involving cellular and humoral immune histotoxicity, demyelination, reduced neurotrophic support, metabolic impairment, and altered intracellular processes. Here we survey available data concerning the role of autoreactive immunoglobulins in neurotoxicity. A better understanding of molecular pathways leading to immune-mediated neurodegeneration may have key importance in the successful treatment of the disease.

Genetic variants of Complex I in multiple sclerosis

HYPOTHESIS A mitochondrial mechanism contributes to neurodegeneration in multiple sclerosis (MS). Genetic variants of Complex I genes may influence the nature of tissue response to inflammation in the central nervous system (CNS). BACKGROUND Complex I is encoded by seven mitochondrial and 38 nuclear genes. Many of the nuclear genes colocalize with regions where full genome scans detected linkage in MS. Previous studies revealed an association between variants of mitochondrial (mt)DNA encoded subunits of Complex I and MS. Biochemical studies suggested a functional involvement of Complex I in the degenerative processes downstream to inflammatory injury in the CNS. METHODS Patients with all MS phenotypes were included. DNA specimens of affected sib pair, trio and multiplex families were studied. Single nucleotide polymorphisms (SNP) were determined by using the Taqman assay. The association of MS with nuclear DNA encoded alleles and haplotypes of Complex I was tested by the pedigree disequilibrium test (PDT) and by the transmit program in the families. Haplotypes were further investigated by using ldmax (GOLD). The association of mtDNA encoded variants with MS was tested by the Fishers Exact Test. RESULTS The previously identified MS-associated mtDNA variants and haplotypes were not increased in mothers as compared to fathers in these families. However, an association of all clinical phenotypes with haplotypes within NDUFS5 (1p34.2-p33), NDUFS7 (19p13) and NDUFA7 (19p13) was detected. The inclusion of families with primary progressive (PP)-MS phenotype did not modify the outcome and, as a subgroup alone, did not have a sufficient size to draw conclusion regarding phenotype specific associations. CONCLUSIONS SNP haplotypes within Complex I genes are associated with MS. Further studies are needed to refine the identification of disease relevant variants nearby or within these haplotypes. Molecular and functional properties of Complex I subunits may offer novel explanations to better understand the relationship between inflammation and neurodegeneration.

Association of haplotypes in the β -chemokine locus with multiple sclerosis

Linkage studies in multiple sclerosis (MS) identified several susceptibility loci. One of these regions includes chromosome 17q11 where a meta-analysis of data from three genome scans suggested linkage. This region encodes a cluster of genes for β-chemokines or CC chemokine ligands (CCLs), which may be involved in the development of MS lesions. Here we aimed to test if CCL alleles and haplotypes are associated with MS. Using methods of linkage and association, we observed deviations from the expected 50% transmission of haplotypes from unaffected parents to their affected children at CCL2, CCL11–CCL8–CCL13 and CCL3 within the investigated 1.85 MB chromosomal segment. Analyses of the linkage disequilibrium map support that variants with possible relevance to MS can be located within these subregions. Identification of MS associated CCL variants may have direct clinical significance, as it can lead to the design of small competitive antagonists of these molecules with beneficial effects in the treatment of patients with early and active disease.

CD45 (PTPRC) as a candidate gene in multiple sclerosis.

The 77C→ G polymorphism in exon 4 of CD45 or protein tyrosine phosphatase receptor-type C (PTPRC) has been investigated in families with multiple sclerosis (MS) and in several cohorts of sporadic patients and controls, however, with conflicting results. To better understand the role of this functionally important polymorphism in MS, we investigated the transmission of the ‘G’ allele from unaffected parents to their affected children in 176 families by using linkage and association statistics. The ‘G’ allele was transmitted to the affected offspring in five of the seven pedigrees carrying this allele and the TRANSMIT program detected association with a P -0.0342. We conclude that the 77C→ G PTPRC polymorphism is present and preferentially transmitted in a small subgroup (< 5%) of MS families, which may only be detected with complementary methods of analysis.

Clinical safety of intrathecal administration of mesenchymal stromal cell-derived neural progenitors in multiple sclerosis.

BACKGROUND AIMS There is a critical unmet need to develop regenerative therapies for the demyelinating disease multiple sclerosis (MS). We previously characterized the immunoregulatory and trophic properties of neural progenitors derived from bone marrow mesenchymal stromal cells (MSC-NPs) and established that cells derived from MS and non-MS patients alike were therapeutically viable. In an experimental model of MS, intrathecal MSC-NP injection resulted in disease amelioration with decreased T-cell infiltration, and less severe lesion pathology associated with recruitment of resident progenitors to inflammatory sites. In this pilot feasibility study, we investigated safety and dosing of intrathecal MSC-NP therapy in six patients with MS. METHODS Patients with progressive MS and advanced disability who were refractory to all other conventional MS treatments were enrolled in the study. For each dose, MSC-NP cells were cultured from autologous MSCs and tested for quality control before intrathecal administration. Patients were evaluated for adverse events and neurological status to assess safety of the treatment. RESULTS Six patients with progressive MS were treated with between 2 and 5 intrathecal injections of escalating doses of autologous MSC-NPs and were followed an average of 7.4 years after initial injection. There were no safety concerns noted, no serious adverse events, and the multiple dosing regimen was well tolerated. Four of the six patients showed a measurable clinical improvement following MSC-NP treatment. DISCUSSION This pilot study supports preliminary first-in-human safety and tolerability of autologous MSC-NP treatment for MS.

Analyses of a MS-associated haplotype encompassing the CCL3 gene

Our previous studies showed the association of multiple sclerosis with the same marker haplotype encompassing the CCL3 gene in two independent sets of families. Here we present that sequencing of this haplotype and its flanking regions detected no new mutation, but 16 single nucleotide polymorphisms (SNP) and 1 insertion/deletion variant in both affected and unaffected individuals. Transmission distortion analyses of the newly identified variants in the second set of families revealed no individual marker association. In the absence of a single disease relevant variant within the MS associated haplotype and the surrounding linkage disequilibrium block, the highlighted haplotype may itself indicate a functionally relevant allelic combination or interaction.

Phase I Trial of Intrathecal Mesenchymal Stem Cell-derived Neural Progenitors in Progressive Multiple Sclerosis

Background Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system and is one of the leading causes of disability in young adults. Cell therapy is emerging as a therapeutic strategy to promote repair and regeneration in patients with disability associated with progressive MS. Methods We conducted a phase I open-label clinical trial investigating the safety and tolerability of autologous bone marrow mesenchymal stem cell-derived neural progenitor (MSC-NP) treatment in 20 patients with progressive MS. MSC-NPs were administered intrathecally (IT) in three separate doses of up to 1 × 107 cells per dose, spaced three months apart. The primary endpoint was to assess safety and tolerability of the treatment. Expanded disability status scale (EDSS), timed 25-ft walk (T25FW), muscle strength, and urodynamic testing were used to evaluate treatment response. This trial is registered with ClinicalTrials.gov, number NCT01933802. Findings IT MSC-NP treatment was safe and well tolerated. The 20 enrolled subjects completed all 60 planned treatments without serious adverse effects. Minor adverse events included transient fever and mild headaches usually resolving in <24 h. Post-treatment disability score analysis demonstrated improved median EDSS suggesting possible efficacy. Positive trends were more frequently observed in the subset of SPMS patients and in ambulatory subjects (EDSS ≤ 6.5). In addition, 70% and 50% of the subjects demonstrated improved muscle strength and bladder function, respectively, following IT MSC-NP treatment. Interpretation The possible reversal of disability that was observed in a subset of patients warrants a larger phase II placebo-controlled study to establish efficacy of IT MSC-NP treatment in patients with MS. Funding source The Damial Foundation.