Long-Term Clinical and Immunological Effects of Repeated Mesenchymal Stem Cell Injections in Patients With Progressive Forms of Multiple Sclerosis.

Background: Mesenchymal stem cells (MSC) were shown to possess immunomodulatory and neurotrophic effects. Our previous trials, have shown that intrathecal (IT) and intravenous (IV) administration of MSCs were safe and provided indications of beneficial clinical effects. Methods: This is an open prospective study to evaluate the safety and the long-term clinical and immunological effects of multiple injections of autologous MSCs in 24 patients with active-progressive MS. At inclusion, the mean age of the patients was 47.0 ± 9.22, and the mean EDSS score was 6.75 ± 0.68 (range: 5.5-7.5). Patients were initially treated with 1 ×106 MSCS/kg of body weight (IT + IV) and subsequently with up to additional eight courses of MSCs, at intervals of 6-12 months. The duration of the trial was 4 years. Results: No serious, treatment-related adverse events were observed during the follow-up period. Twenty-two of the 24 patients were either stable or improved at the last follow-up visit. Ten patients had a lower than baseline EDSS at the last follow-up (nine were among those who received >2 treatments and one in the subgroup of ≤ 2 treatments, p = 0.04). The mean EDSS score reduced from 6.75 ± 0.68 at baseline to 6.42 ± 0.84 at the last visit, during a median follow-up period of 27.8 months (p = 0.028). Immunological follow-up showed a transient upregulation of CD4+CD25+FoxP3+ cells and downregulation of the proliferative ability of lymphocytes. Conclusions: Repeated MSC treatments in patients with progressive MS were shown safe at the short/intermediate term and induced clinical benefits (especially in patients treated with >2 injections) that lasted for up to 4 years, paralleled by short-term immunomodulatory effects. Clinical Trial Registration: www.ClinicalTrials.gov, identifier: NCT04823000.

Beneficial effects of autologous mesenchymal stem cell transplantation in active progressive multiple sclerosis.

In this study (trial registration: NCT02166021), we aimed to evaluate the optimal way of administration, the safety and the clinical efficacy of mesenchymal stem cell (MSC) transplantation in patients with active and progressive multiple sclerosis. Forty-eight patients (28 males and 20 females) with progressive multiple sclerosis (Expanded Disability Status Scale: 3.0-6.5, mean : 5.6 ± 0.8, mean age: 47.5 ± 12.3) and evidence of either clinical worsening or activity during the previous year, were enrolled (between 2015 and 2018). Patients were randomized into three groups and treated intrathecally (IT) or intravenously (IV) with autologous MSCs (1 × 106/kg) or sham injections. After 6 months, half of the patients from the MSC-IT and MSC-IV groups were retreated with MSCs, and the other half with sham injections. Patients initially assigned to sham treatment were divided into two subgroups and treated with either MSC-IT or MSC-IV. The study duration was 14 months. No serious treatment-related safety issues were detected. Significantly fewer patients experienced treatment failure in the MSC-IT and MSC-IV groups compared with those in the sham-treated group (6.7%, 9.7%, and 41.9%, respectively, P = 0.0003 and P = 0.0008). During the 1-year follow-up, 58.6% and 40.6% of patients treated with MSC-IT and MSC-IV, respectively, exhibited no evidence of disease activity compared with 9.7% in the sham-treated group (P < 0.0001 and P < 0.0048, respectively). MSC-IT transplantation induced additional benefits on the relapse rate, on the monthly changes of the T2 lesion load on MRI, and on the timed 25-foot walking test, 9-hole peg test, optical coherence tomography, functional MRI and cognitive tests. Treatment with MSCs was well-tolerated in progressive multiple sclerosis and induced short-term beneficial effects regarding the primary end points, especially in the patients with active disease. The intrathecal administration was more efficacious than the intravenous in several parameters of the disease. A phase III trial is warranted to confirm these findings.

Cerebrospinal Fluid (CSF) Exchange with Artificial CSF Enriched with Mesenchymal Stem Cell Secretions Ameliorates Experimental Autoimmune Encephalomyelitis.

The complexity of central nervous system (CNS) degenerative/inflammatory diseases and the lack of substantially effective treatments point to the need for a broader therapeutic approach to target multiple components involved in the disease pathogenesis. We suggest a novel approach directed for the elimination of pathogenic agents from the CNS and, in parallel, its enrichment with an array of neuroprotective substances, using a "cerebrospinal fluid (CSF) exchange" procedure, in which endogenous (pathogenic) CSF is removed and replaced by artificial CSF (aCSF) enriched with secretions of human mesenchymal stem cells (MSCs). MSCs produce a variety of neuroprotective agents and have shown beneficial effects when cells are transplanted in animals and patients with CNS diseases. Our data show that MSCs grown in aCSF secrete neurotrophic factors, anti-inflammatory cytokines, and anti-oxidant agents; moreover, MSC-secretions-enriched-aCSF exerts neuroprotective and immunomodulatory effects in neuronal cell lines and spleen lymphocytes. Treatment of experimental-autoimmune-encephalomyelitis (EAE) mice with this enriched-aCSF using an intracerebroventricular (ICV) CSF exchange procedure ("CSF exchange therapy") caused a significant delay in the onset of EAE and amelioration of the clinical symptoms, paralleled by a reduction in axonal damage and demyelination. These findings point to the therapeutic potential of the CSF exchange therapy using MSC-secretions-enriched-aCSF in inflammatory/degenerative diseases of the CNS.

NMO-IgG and AQP4 Peptide Can Induce Aggravation of EAMG and Immune-Mediated Muscle Weakness.

Neuromyelitis optica (NMO) and myasthenia gravis (MG) are autoimmune diseases mediated by autoantibodies against either aquaporin 4 (AQP4) or acetylcholine receptor (AChR), respectively. Recently, we and others have reported an increased prevalence of NMO in patients with MG. To verify whether coexisting autoimmune disease may exacerbate experimental autoimmune MG, we tested whether active immunization with AQP4 peptides or passive transfer of NMO-Ig can affect the severity of EAMG. Injection of either AQP4 peptide or NMO-Ig to EAMG or to naive mice caused increased fatigability and aggravation of EAMG symptoms as expressed by augmented muscle weakness (but not paralysis), decremental response to repetitive nerve stimulation, increased neuromuscular jitter, and aberration of immune responses. Thus, our study shows increased disease severity in EAMG mice following immunization with the NMO autoantigen AQP4 or by NMO-Ig, mediated by augmented inflammatory response. This can explain exacerbation or increased susceptibility of patients with one autoimmune disease to develop additional autoimmune syndrome.

Phos-tau peptide immunization of amyloid-tg-mice reduced non-mutant phos-tau pathology, improved cognition and reduced amyloid plaques.

Tau-immumotherapy has shown promising results in tangle/tauopathy-tg animal models. Here we immunized amyloid-mice (APPSwe/PSEN1dE9-tg, presenting amyloid-plaques, not neurofibrillary-tangles) with phos-tau peptides, previously shown by us to have high efficacy in mutant-tau tauopathy-mice. These amyloid-mice allowed us to test the effect of the vaccine in a model of familial AD patients with mutant amyloid plaque pathology, where tau pathology - once develops - is of non-mutant tau. Fourteen-month-old amyloid-mice were immunized with phos-tau peptides or vehicle. Eight weeks later, amelioration of cognitive impairment was noticed. Histological analysis revealed that the phos (non-mutant)-tau pathology (detected by us in these aged amyloid-mice while not in non-tg-mice), was lower in the phos-tau immunized amyloid-mice than in the non-immunized mice. Interestingly, we detected a decrease in amyloid plaque pathology, probably associated with the increased microglial burden, which surrounded both tau and amyloid pathology. These results point to the added value of immunizing AD-mice with the phos-tau-vaccine, targeting both tau and amyloid pathology, which may have clinical relevance. It also points to the multifaceted interplay between tau/amyloid pathologies.

Circulating microRNAs as biomarkers for rituximab therapy, in neuromyelitis optica (NMO).

BACKGROUND: Neuromyelitis optica (NMO) is a chronic autoimmune disease of the central nervous system (CNS). The main immunological feature of the disease is the presence of autoantibodies to Aquaporin 4 (AQP4+), identified in about 82 % of cases. Currently, there are no reliable biomarkers for monitoring treatment response in patients with NMO. In an effort to identify biomarkers, we analyzed microRNAs (miRNAs) in the blood of rituximab-treated NMO patients before and after therapy. METHODS: Total RNA extracted from whole blood of nine rituximab-responsive NMO patients before and 6 months following treatment was subjected to small RNAseq analysis. The study included an additional group of seven untreated AQP4+ seropositive NMO patients and 15 healthy controls (HCs). RESULTS: Fourteen miRNAs were up regulated and 32 were downregulated significantly in the blood of NMO patients following effective therapy with rituximab (all p < 0.05). Furthermore, we show that expression of 17 miRNAs was significantly higher and of 25 miRNAs was significantly lower in untreated NMO patients compared with HCs (all p < 0.05). Following rituximab treatment, the expression levels of 10 of the 17 miRNAs that show increased expression in NMO reverted to the levels seen in HCs. Six of these "normalized" miRNAs are known as brain-specific/enriched miRNAs. CONCLUSIONS: Specific miRNA signatures in whole blood of patients with NMO might serve as biomarkers for therapy response. Furthermore, monitoring the levels of brain-specific/enriched miRNAs in the blood might reflect the degree of disease activity in the CNS of inflammatory demyelinating disorders.

T-cell responses to distinct AQP4 peptides in patients with neuromyelitis optica (NMO).

BACKGROUND: Although antibodies to aquaporin-4(AQP4) are strongly associated with Neuromyelitis optica (NMO), the sole transfer of these antibodies is not sufficient to induce an NMO-like disease in experimental animals and T-cells and complement are also needed. Initial data indicating the presence of T-cell responses to AQP4 in patients with NMO, have beeen recently reported. OBJECTIVE: To evaluate the T-cell responses to specific AQP4 peptides/epitopes in patients with NMO and multiple sclerosis (MS). METHODS: Peripheral blood mononuclear cells (PBMCs) were obtained from 14 patients fulfilling the criteria for definite NMO and the proliferation responses to one of 15 distinct pentadecapeptides of AQP4, spanning the whole protein (except of its transmembrane parts) were tested by a standard [H3]-thymidine uptake assay and compared with those of 9 healthy controls and 7 MS patients. A cytometric bead array assay (CBA) and flow cytometry were used to evaluate cytokine (IFNγ, IL17, IL2, IL4, IL5, IL10 and TNFα) and chemokine (CXCL8, CCL5, CXCL10, CXCL9, CCL2) secretion by PHA-stimulated PBMCs and AQP4-specific T-cell lines. RESULTS: Four main immunodominant epitopes of the AQP4 protein (p137-151, p222-236, p217-231 and the p269-283) were identified in the NMO group. The first two epitopes (assigned as peptides 3 and 9) showed the highest sensitivity (~60% positivity), whereas the latter two (assigned as peptides 8 and 11), the higher specificity. Longitudinal follow up of 5 patients revealed changes in the epitope-specificities during the course of NMO. T-cell lines specific for the AQP4 peptides, produced from NMO patients (but not healthy donors) secreted mainly IL-17 and IL-10 and less IFNγ. CONCLUSIONS: Our findings indicate that T-cells bearing characteristics of both Th1 and Th17 T-cells and targeting specific immunodominant epitopes of the AQP4 protein might be involved in the pathogenesis of NMO.

Safety and Clinical Effects of Mesenchymal Stem Cells Secreting Neurotrophic Factor Transplantation in Patients With Amyotrophic Lateral Sclerosis: Results of Phase 1/2 and 2a Clinical Trials.

IMPORTANCE: Preclinical studies have shown that neurotrophic growth factors (NTFs) extend the survival of motor neurons in amyotrophic lateral sclerosis (ALS) and that the combined delivery of these neurotrophic factors has a strong synergistic effect. We have developed a culture-based method for inducing mesenchymal stem cells (MSCs) to secrete neurotrophic factors. These MSC-NTF cells have been shown to be protective in several animal models of neurodegenerative diseases. OBJECTIVE: To determine the safety and possible clinical efficacy of autologous MSC-NTF cells transplantation in patients with ALS. DESIGN, SETTING, AND PARTICIPANTS: In these open-label proof-of-concept studies, patients with ALS were enrolled between June 2011 and October 2014 at the Hadassah Medical Center in Jerusalem, Israel. All patients were followed up for 3 months before transplantation and 6 months after transplantation. In the phase 1/2 part of the trial, 6 patients with early-stage ALS were injected intramuscularly (IM) and 6 patients with more advanced disease were transplanted intrathecally (IT). In the second stage, a phase 2a dose-escalating study, 14 patients with early-stage ALS received a combined IM and IT transplantation of autologous MSC-NTF cells. INTERVENTIONS: Patients were administered a single dose of MSC-NTF cells. MAIN OUTCOMES AND MEASURES: The primary end points of the studies were safety and tolerability of this cell therapy. Secondary end points included the effects of the treatment on various clinical parameters, such as the ALS Functional Rating Scale-Revised score and the respiratory function. RESULTS: Among the 12 patients in the phase 1/2 trial and the 14 patients in the phase 2a trial aged 20 and 75 years, the treatment was found to be safe and well tolerated over the study follow-up period. Most of the adverse effects were mild and transient, not including any treatment-related serious adverse event. The rate of progression of the forced vital capacity and of the ALS Functional Rating Scale-Revised score in the IT (or IT+IM)-treated patients was reduced (from -5.1% to -1.2%/month percentage predicted forced vital capacity, P < .04 and from -1.2 to 0.6 ALS Functional Rating Scale-Revised points/month, P = .052) during the 6 months following MSC-NTF cell transplantation vs the pretreatment period. Of these patients, 13 (87%) were defined as responders to either ALS Functional Rating Scale-Revised or forced vital capacity, having at least 25% improvement at 6 months after treatment in the slope of progression. CONCLUSIONS AND RELEVANCE: The results suggest that IT and IM administration of MSC-NTF cells in patients with ALS is safe and provide indications of possible clinical benefits, to be confirmed in upcoming clinical trials. TRIAL REGISTRATION: clinicaltrials.gov Identifiers: NCT01051882 and NCT01777646.

Treatment of a multiple sclerosis animal model by a novel nanodrop formulation of a natural antioxidant.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system and is associated with demyelination, neurodegeneration, and sensitivity to oxidative stress. In this work, we administered a nanodroplet formulation of pomegranate seed oil (PSO), denominated Nano-PSO, to mice induced for experimental autoimmune encephalomyelitis (EAE), an established model of MS. PSO comprises high levels of punicic acid, a unique polyunsaturated fatty acid considered as one of the strongest natural antioxidants. We show here that while EAE-induced mice treated with natural PSO presented some reduction in disease burden, this beneficial effect increased significantly when EAE mice were treated with Nano-PSO of specific size nanodroplets at much lower concentrations of the oil. Pathological examinations revealed that Nano-PSO administration dramatically reduced demyelination and oxidation of lipids in the brains of the affected animals, which are hallmarks of this severe neurological disease. We propose that novel formulations of natural antioxidants such as Nano-PSO may be considered for the treatment of patients suffering from demyelinating diseases. On the mechanistic side, our results demonstrate that lipid oxidation may be a seminal feature in both demyelination and neurodegeneration.

Frequent misdiagnosis of adult polyglucosan body disease.

Adult polyglucosan body disease (APBD) is a rare glycogenosis manifesting progressive spastic paraparesis, sensorimotor polyneuropathy and neurogenic bladder. Misdiagnosis of APBD may lead to unnecessary investigations and to potentially harmful therapeutic interventions. To examine the frequency of misdiagnosis of APBD, we retrospectively reviewed the clinical data of 30 patients diagnosed between 1991 and 2013. Diagnosis was based on the combination of typical clinical and imaging findings, reduced glycogen branching enzyme activity, and the presence of p.Y326S GBE1 mutation. Initial symptoms started in the 5th-6th decade with bladder dysfunction (47 %), gait problems (33 %) or both. Diagnosis of APBD was delayed by 6.8 (±4.8) years. Consistent signs at diagnosis were spasticity in the legs (93 %), decreased or absent ankle reflexes (100 %), bilateral extensor plantar response (100 %) and distal sensory deficit (80 %). Nerve conduction study showed invariable sensorimotor polyneuropathy, and MRI demonstrated cervical spinal cord atrophy (100 %) and leukoencephalopathy (97 %). All 30 patients were initially misdiagnosed. Common misdiagnoses included cerebral small vessel disease (27 %), multiple sclerosis (17 %), amyotrophic lateral sclerosis (17 %) and peripheral neuropathies (20 %). Consequently, 27 % received inappropriate therapy. In addition, lower urinary tract symptoms in 60 % of men were attributed solely to prostatic disorders but did not respond to medical treatment or prostatectomy. These findings suggest that despite limited clinical variability, APBD is invariably misdiagnosed and patients are often mistreated. Physicians' unfamiliarity with the typical clinical and imaging features of APBD appears as the main reason for misdiagnosis.

Corrigendum to "Presymptomatic treatment with acetylcholinesterase antisense oligonucleotides prolongs survival in ALS (G93A-SOD1) mice".

[This corrects the article DOI: 10.1155/2013/845345.].

Increased anti-KIR4.1 antibodies in multiple sclerosis: could it be a marker of disease relapse?

BACKGROUND: Screening of putative autoimmune targets in multiple sclerosis (MS) revealed a proportion of patients carrying antibodies (Abs) against KIR4.1, a potassium channel that shares functional properties with AQP4. Both are localized at the perivascular astrocytic processes. AIMS: To measure anti-KIR4.1 Abs in the serum of MS and neuromyelitis optica (NMO) patients, and to identify the clinical and laboratory characteristics of patients harboring anti-KIR4.1 Abs. METHODS: We measured anti-KIR4.1 Abs in serum, using the peptide KIR4.1 (83-120) enzyme-linked immunosorbent assay (ELISA). RESULTS: Serum levels of anti-KIR4.1 Abs were significantly higher in MS and NMO patients than in healthy controls (HCs); with Abs detected in 21 of 80, 10 of 45, and 2 of 32 individuals, respectively (MS versus HC, p < 0.05). The level of anti-KIR4.1 Abs was significantly higher during MS relapse, versus remission (p = 0.04). The clinical characteristics of our study patients did not vary based on KIR4.1 positivity. CONCLUSIONS: Anti-KIR4.1 Abs were found in similar proportions of patients with MS and NMO, at a significantly higher level than observed in HCs; consequently, the presence of Abs does not discriminate between these demyelinating diseases. However, anti-KIR4.1 Ab levels differed in MS patients during relapse and remission; as such, they may represent a marker of disease exacerbation.

Tumefactive demyelination following in vitro fertilization (IVF).

Tumefactive demyelination (TD) is a solitary cerebral demyelinating lesion clinically and radiologically mimicking brain tumors. It can occur in isolation or may be rarely associated with other demyelinating diseases. The underlying pathogenic mechanisms are unknown. We present the first report of TD following in-vitro fertilization (IVF) in a 36-year-old healthy woman who developed subacute right hemiparesis shortly after a scheduled IVF cycle. Evaluation revealed left hemispheric space-occupying lesion pathologically diagnosed as TD. Treatment with intravenous methylprednisolone promptly resulted in a clinical and radiological improvement maintained thereafter. This report confirms and expands the spectrum of inflammatory demyelinating conditions associated with IVF and suggests possible hormonal influence in the development of TD.

NMO spectrum of disorders: a paradigm for astrocyte-targeting autoimmunity and its implications for MS and other CNS inflammatory diseases.

When studying a rare or orphan disease, we hope to shed light on more prevalent syndromes. Neuromyelitis optica (NMO), also known as Devic's disease, is a rare disease with a prevalence of about 4 in 100,000. Since 2005 when the anti-Aquaporin 4 (AQP4) NMO autoantibody was discovered by Lennon's group at the Mayo clinic, an enormous amount of data have been acquired on the pathogenesis of the disease. A review of the literature showed 47 relevant publications in 2004, compared with 353 in 2013. The auto-antigen AQP4 is expressed on the astrocytic foot processes suggesting a role for astrocytes in the pathogenesis of the disease. However, the astrocytes might play a more active role than has previously been suggested in the immune cascade of NMO pathology. Here we will review epidemiological, clinical diagnostic and therapeutic aspects of NMO and highlight the possible role of astrocytes as major direct and indirect players in the pathogenesis of NMO and related CNS inflammatory diseases.

Severe methylenetetrahydrofolate reductase deficiency: clinical clues to a potentially treatable cause of adult-onset hereditary spastic paraplegia.

IMPORTANCE: Hereditary spastic paraplegia is a highly heterogeneous group of neurogenetic disorders with pure and complicated clinical phenotypes. No treatment is available for these disorders. We identified 2 unrelated families, each with 2 siblings with severe methylenetetrahydrofolate reductase (MTHFR) deficiency manifesting a complicated form of adult-onset hereditary spastic paraparesis partially responsive to betaine therapy. OBSERVATIONS: Both pairs of siblings presented with a similar combination of progressive spastic paraparesis and polyneuropathy, variably associated with behavioral changes, cognitive impairment, psychosis, seizures, and leukoencephalopathy, beginning between the ages of 29 and 50 years. By the time of diagnosis a decade later, 3 patients were ambulatory and 1 was bedridden. Investigations have revealed severe hyperhomocysteinemia and hypomethioninemia, reduced fibroblast MTHFR enzymatic activity (18%-52% of control participants), and 3 novel pathogenic MTHFR mutations, 2 as compound heterozygotes in one family and 1 as a homozygous mutation in the other family. Treatment with betaine produced a rapid decline of homocysteine by 50% to 70% in all 4 patients and, over 9 to 15 years, improved the conditions of the 3 ambulatory patients. CONCLUSIONS AND RELEVANCE: Although severe MTHFR deficiency is a rare cause of complicated spastic paraparesis in adults, it should be considered in select patients because of the potential therapeutic benefit of betaine supplementation.

A novel SCARB2 mutation in progressive myoclonus epilepsy indicated by reduced ß-glucocerebrosidase activity.

Action myoclonus renal failure (AMRF) syndrome is a rare form of progressive myoclonus epilepsy with renal dysfunction related to mutations in the SCARB2 gene. This gene is involved in lysosomal mannose-6-phosphate-independent trafficking of ß-glucocerebrosidase (GC), an enzyme deficient in Gaucher disease. We report a family with myoclonic epilepsy, ataxia and skeletal muscle atrophy but without cognitive impairment or overt renal disease. A novel SCARB2 mutation was indicated by a striking discrepancy between lymphocyte and fibroblast GC activity in the proband evaluated for possible Gaucher disease. Our findings expand the genetic and phenotypic diversity of AMRF and suggest that low GC activity may present an important biochemical clue to the diagnosis of AMRF.

Repeated immunization of mice with phosphorylated-tau peptides causes neuroinflammation.

The recent studies of others and of us showing robust efficacy of anti-tangle immunotherapy, directed against phosphorylated (phos)-tau protein, may pave the way to clinical trials of phos-tau immunotherapy in Alzheimer's-disease and other tauopathies. At this stage addressing the safety of the phos-tau-immunotherapy is highly needed, particularly since we have previously shown the neurotoxic potential of tau-immunotherapy, specifically of full-length unphosphorylated-tau vaccine under a CNS-proinflammatory milieu [induced by emulsification in complete-Freund's-adjuvant (CFA) and pertussis-toxin (PT)] in young wild-type (WT)-mice. The aim of our current study was to address safety aspects of the phos-tau-immunotherapy in both neurofibrillary-tangle (NFT)-mice as well as in WT-mice, under challenging conditions of repeated immunizations with phos-tau peptides under a CNS-proinflammatory milieu. NFT- and WT-mice were repeatedly immunized (7 injections in adult-, 4 in aged-mice) with phos-tau peptides emulsified in CFA-PT. A paralytic disease was evident in the phos-tau-immunized adult NFT-mice, developing progressively to 26.7% with the number of injections. Interestingly, the WT-mice were even more prone to develop neuroinflammation following phos-tau immunization, affecting 75% of the immunized mice. Aged mice were less prone to neuroinflammatory manifestations. Anti-phos-tau antibodies, detected in the serum of immunized mice, partially correlated with the neuroinflammation in WT-mice. This points that repeated phos-tau immunizations in the frame of a proinflammatory milieu may be encephalitogenic to tangle-mice, and more robustly to WT-mice, indicating that - under certain conditions - the safety of phos-tau immunotherapy is questionable.