Panayiota Petrou

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.

Myasthenia gravis-associated neuromyelitis optica-like disease: an immunological link between the central nervous system and muscle?

BACKGROUND Although overt involvement of the central nervous system (CNS) in myasthenia gravis (MG) is considered rare, hyperreflexia is a common and yet unexplained finding. Aquaporin 4 (AQP4), the target autoantigen in neuromyelitis optica, is expressed both in the CNS and in the neuromuscular junction. OBJECTIVES To evaluate the prevalence of even mild CNS involvement in patients with MG and to identify features indicative of neuromyelitis optica-like disease. DESIGN Cohort study. SETTING Outpatient clinic. PATIENTS A cohort of 164 patients with MG. METHODS In 24 patients with MG, signs of CNS involvement were detected; 15 of these patients had at least 1 additional paraclinical indication of neuromyelitis optica-like disease (presence of antibodies against AQP4, pathological visual evoked potentials, or white matter lesions detected on brain and/or spinal magnetic resonance imaging scans) and fulfilled the inclusion and exclusion criteria for our study. RESULTS Of the 15 patients who had at least 1 additional paraclinical indication of neuromyelitis optica-like disease, 14 had abnormal visual evoked potentials, and in 6 of 9 patients in whom magnetic resonance imaging was performed, there was evidence of lesions in the white matter of the brain and/or spinal cord. Anti-AQP4 antibodies were detected in 7 patients (out of the 14 tested). Thymic enlargement (hyperplasia or thymoma) was more frequent in patients with MG who had signs of CNS involvement than in patients with MG who did not. CONCLUSIONS The incidence of CNS involvement in MG is higher than previously reported and is expressed predominantly as a pyramidal syndrome accompanied by optical tract involvement (frequently subclinical). These features bear some resemblance to neuromyelitis optica spectrum disease, supported also by the presence of anti-AQP4 antibodies in 7 of the 14 patients tested. This association may represent a new nosological entity or may indicate that an autoimmune process targeting AQP4 is an integral part of the immunopathogenetic mechanisms in MG.

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.

T-cell reactivity against AQP4 in neuromyelitis optica

Neuromyelitis optica (NMO) is an idiopathic demyelinating disease of the CNS that can be clearly distinguished from multiple sclerosis (MS) by clinical, neuroradiogic, and pathologic criteria and the presence of the highly specific serum autoantibodies against the water channel aquaporin-4 (AQP4).1 Although studies support a central role of the anti-AQP4 antibodies in the pathogenesis of NMO, their exact involvement in the immunopathogenetic cascade of the disease is still not clear, and T cells seem to be equally crucial for the full development of clinical and histopathologic NMO.

Clinical experience with stem cells and other cell therapies in neurological diseases

To overcome the limited capacity of the CNS for regeneration, the theoretical alternative would be to use stem cells for more effective management of chronic degenerative and inflammatory neurological conditions, and also of acute neuronal damage from injuries or cerebrovascular diseases. Although the adult brain contains small numbers of stem cells in restricted areas, this intrinsic stem cell repertoire is small and does not measurably contribute to functional recovery. Embryonic cells carrying pluripotent and self-renewal properties represent the stem cell prototype, but there are additional somatic stem cells that may be harvested and expanded from various tissues during adult life. Stem cell transplantation is based on the assumption that such cells may have the potential to regenerate or support the survival of the existing, partially damaged cells. This review summarizes the state-of-the-art and the clinical worldwide experience with the use of various types of stem cells in neurological diseases.

Hematopoietic stem cell transplantation in multiple sclerosis

It is widely accepted that the main common pathogenetic pathway in multiple sclerosis (MS) involves an immune-mediated cascade initiated in the peripheral immune system and targeting CNS myelin. Logically, therefore, therapeutic approaches to the disease include modalities aiming at downregulation of the various immune elements that are involved in this immunological cascade. Since the introduction of interferons in 1993, more specific immunoactive drugs have been introduced, but still most of them can, at best, effectively modulate only the early relapsing phases of MS. The more progressed phases of the disease are not efficiently amendable by the existing immunomodulatory drugs. Moreover, localized and compartmentized inflammation in the CNS, which seems to be mostly responsible for the chronic axonal damage and resulting progression of disability, is less affected by the current drugs. A more radical approach to suppress all the inflammation in MS, including that into the CNS, could theoretically be achieved with high-dose immunosuppression using strong cytotoxic medications and resetting of the immune system by hematopoietic stem cell transplantation (HSCT). HSCT, both allogeneic and autologous, has been tried as a novel therapeutic approach in various autoimmune diseases. During the last 15 years several (mostly open) clinical studies evaluated the effect of HSTC on MS patients; the published papers showed that a high proportion of the HSCT-treated MS patients were stabilized, or even improved after the transplantation and have generally indicated a beneficial effect on disease progression. In this review, the rationale of HSCT and the summary of the results of the existing clinical trials are presented. Despite the fact that it is difficult to collectively summarize the results of all the trials, due to lack of uniformity in the conditioning and treatment protocols and of completed controlled studies, these clinical studies have provided a strong ‘proof of concept’ for HSCT in MS and have significantly contributed to our understanding of the advantages and disadvantages of each approach and HSCT protocol.

Rare combination of myasthenia and motor neuronopathy, responsive to Msc-Ntf stem cell therapy

A 75-year-old man was referred to the Hadassah Medical Center with a 6-month history of progressive limb weakness, dysarthria, and cognitive deterioration. His past medical history included prostate hyperplasia, hypothyroidism, diabetes mellitus, cardiac arrhythmias controlled by pacemaker implantation (1997), and hypertension. Autoimmune myasthenia gravis (MG) had been diagnosed 2 years earlier, based on symptoms of fluctuating fatigue, dysarthric speech, eyelid ptosis, and seropositivity for muscle acetylcholine receptor (AChR) binding antibody. MRI did not reveal thymic enlargement, and malignancy markers (and whole-body computed tomography) were negative. Moderate improvement followed treatment, initially with intravenous immune globulin and later with low dose corticosteroids, pyridostigmine, and azathioprine. The patient was evaluated previously at the Mayo Clinic (Rochester, Minnesota) and had been diagnosed with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The diagnosis was based on the cognitive exam and clinical features of progressive diffuse upper and lower motor neuron dysfunction with electromyographic findings (low motor amplitudes, no focal slowing or conduction blocks, no postexercise facilitation, presence of diffuse fibrillation and fasciculation potentials, and large complex motor unit potentials in all limbs) fulfilling the El-Escorial criteria for ALS. MRI and positron emission tomography scans of the brain showed moderate atrophy predominantly affecting the frontal and temporal lobes. Spinal fluid was acellular with slightly elevated total protein (50 mg%). Autoimmune serology revealed antibodies specific for muscle (AChR binding 9.47 nmol/L [normal 0.00–0.02]; AChR modulating 100% loss [normal 0–20%]; striational 15,360 [normal <60]) and thyroglobulin, and antinuclear antibody. On admission, the patient had memory impairment and signs of frontotemporal functional deficits, typical of FTD. Dysarthria rendered speech totally incomprehensible. He had mild eyelid ptosis bilaterally (without extraocular muscle weakness or diplopia) and was markedly quadriparetic (confined to wheelchair) with bilateral foot drop. Distal hand and foot muscles were moderately atrophic, and fasciculations were prominent in all limbs. Deep tendon reflexes were brisk in arms and legs (except hypoactive Achilles reflexes), and an extensor plantar response was evoked bilaterally. Superficial sensation was normal, and vibration sense was reduced slightly distally in the legs. Due to the diagnosis of MG, the patient did not meet inclusion criteria for the Hadassah clinical trial in ALS with autologous enhanced mesenchymal stem cells (MSCNTF, BrainstormVR , Petach Tikva; NCT01051882). The Hadassah Ethics Committee issued a special license for treatment on a compassionate basis. MSC-NTF (prepared from the patient’s bone marrow) were injected intrathecally (1.5 3 10 per kilogram of body weight) and at 24 sites along the biceps and triceps muscles of the right arm (1.5 3 10 per site). The intrathecal and intramuscular administration of the cells were chosen based on previous animal and clinical studies from our groups, which showed good migration of the intrathecally injected cells to the CNS and amelioration of the “dying-back” phenomenon by intramuscularly injected MSC in early stages of ALS in the SOD mouse model (unpublished data and Dadon-Nachum et al.). For the next 2 days the patient had a low-grade fever, headache, and was more confused, but at discharge, these problems had completely subsided. Treatment with azathioprine (125 mg/day) was discontinued 1 month before the injection and readministered 30 days after the treatment. Pyridostigmine (60 mg 3 times daily) and low dose oral prednisolone (10 mg/day) were continued. At 1 month after transplantation, the patient and his family reported significant improvement in cognition, speech, and muscle power. He was able to walk at least 20 meters without any support. The dysarthria improved to the extent he was able to clearly deliver a speech to an audience. ALS Functional Score Scale-Revised (ALSFRS-R, performed at all time points by the same evaluator and confirmed by a second senior examiner) score rose from 36 to 44, and respiratory forced vital capacity (FVC) and cognitive function also improved significantly (Supplementary Table 1, which is available online, and Fig. 1). VC 2013 Wiley Periodicals, Inc.

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.

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.

Pseudo-quadriceps sparing in multiple sclerosis

Background:  Sparing of the quadriceps muscle has been reported in various myopathies. In multiple sclerosis (MS) and pyramidal syndromes, in general, such a differential involvement of distinct muscle groups has not been described.