Adi Vaknin-Dembinsky

Identification of tissue-specific cell death using methylation patterns of circulating DNA

Significance We describe a blood test for detection of cell death in specific tissues based on two principles: (i) dying cells release fragmented DNA to the circulation, and (ii) each cell type has a unique DNA methylation pattern. We have identified tissue-specific DNA methylation markers and developed a method for sensitive detection of these markers in plasma or serum. We demonstrate the utility of the method for identification of pancreatic β-cell death in type 1 diabetes, oligodendrocyte death in relapsing multiple sclerosis, brain cell death in patients after traumatic or ischemic brain damage, and exocrine pancreas cell death in pancreatic cancer or pancreatitis. The approach allows minimally invasive monitoring of tissue dynamics in humans in multiple physiological and pathological conditions. Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed a method of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic β-cell DNA was identified in the circulation of patients with recently diagnosed type-1 diabetes and islet-graft recipients; oligodendrocyte DNA was identified in patients with relapsing multiple sclerosis; neuronal/glial DNA was identified in patients after traumatic brain injury or cardiac arrest; and exocrine pancreas DNA was identified in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be determined in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally invasive window for diagnosing and monitoring a broad spectrum of human pathologies as well as providing a better understanding of normal tissue dynamics.

Bone Marrow Mesenchymal Stem Cells: Agents of Immunomodulation and Neuroprotection

Mesenchymal stromal cells (MSC) are part of the bone marrow stem cells repertoire which also includes the main stem cells population of the bone marrow, the hematopoietic stem cells. The main role of MSCs is to support hematopoiesis but they can also give rise to cells of the mesodermal layers. Recently, significant interactions between MSCs and cells from the immune system have been demonstrated: MSCs were found to downregulate T and B lymphocytes, natural killer cells (NK) and antigen presenting cells through various mechanisms, including cell-to cell interaction and soluble factor production. Besides the immunomodulatory effects, MSCs were shown to possess additional stem cells features, such as the self-renewal potential and multipotency. Their debatable transdifferentiation potential to cells of the endo- and exo-dermal layer, including cells of the CNS, may explain in part their reported neuroprotective effects. Studies in vitro and in vivo (in cells cultures and in animal models) have indicated neuroprotective effects. MSCs are believed to promote functional recovery following CNS injury or inflammation, by producing trophic factors that may facilitate the mobilization of endogenous neural stem cells and promote the regeneration or the survival of the affected neurons. These immunomodulatory and neuroprotective features could make MSCs potential candidates for future therapeutic modalities in immune-mediated and neurodegenerative diseases.

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.

Robot-assisted gait training in multiple sclerosis patients: a randomized trial:

Background: Preservation of locomotor activity in multiple sclerosis (MS) patients is of utmost importance. Robotic-assisted body weight-supported treadmill training is a promising method to improve gait functions in neurologically impaired patients, although its effectiveness in MS patients is still unknown. Objective: To compare the effectiveness of robot-assisted gait training (RAGT) with that of conventional walking treatment (CWT) on gait and generalized functions in a group of stable MS patients. Methods: A prospective randomized controlled trial of 12 sessions of RAGT or CWT in MS patients of EDSS score 5–7. Primary outcome measures were gait parameters and the secondary outcomes were functional and quality of life parameters. All tests were performed at baseline, 3 and 6 months post-treatment by a blinded rater. Results: Fifteen and 17 patients were randomly allocated to RAGT and CWT, respectively. Both groups were comparable at baseline in all parameters. As compared with baseline, although some gait parameters improved significantly following the treatment at each time point there was no difference between the groups. Both FIM and EDSS scores improved significantly post-treatment with no difference between the groups. At 6 months, most gait and functional parameters had returned to baseline. Conclusions: Robot-assisted gait training is feasible and safe and may be an effective additional therapeutic option in MS patients with severe walking disabilities.

Preferential increase of B-cell activating factor in the cerebrospinal fluid of neuromyelitis optica in a white population

Background: Anti-aquaporin-4 antibodies are believed to have a central pathogenetic role in neuromyelitis optica (NMO). B-cell activating factor (BAFF) is one of the crucial factors that determines the fate and survival of B cells and may play a role in induction of antibody-mediated autoimmunity. Objectives: To evaluate the blood and cerebrospinal fluid (CSF) levels of BAFF in NMO and multiple sclerosis (MS) patients. Methods: Peripheral blood samples were collected from 21 definite NMO patients, 22 healthy controls and 45 MS patients and CSF from 8 NMO and 11 MS patients. BAFF levels were measured using an ELISA technique. Results: We found significantly higher levels of BAFF in the CSF of NMO patients compared with that in MS (215.6 ± 41 pg/ml in NMO and 77.4 ± 11 pg/ml in MS, p < 0.001). There were no differences in serum BAFF levels between NMO, MS and healthy donors. MS patients treated with interferon-beta (IFNβ) or glatiramer acetate (GA) had significantly higher serum BAFF levels, as compared with untreated patients (1227 ± 203 pg/ml in untreated MS, 2253 ± 83.4 pg/ml in GA-treated, p < 0.01, and 2106 ± 277.9 pg/ml in interferon-treated, p < 0.05) Conclusion: The presence of increased BAFF, a soluble factor associated with B-cell activation in the proximity of the disease target organ (CSF) in NMO, and its increase in association with immunomodulating treatments, may help our understanding of the immunopathogenetic mechanisms involved in this disease and contribute to more successful and targeted therapeutic intervention.

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.

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.

Hematopoietic stem cell transplantation in multiple sclerosis: a review of the clinical experience and a report of an international meeting

Hematopoietic stem cell transplantation (HSCT), both allogeneic and autologous, has become one of the hottest topics in clinical immunology. One of the main autoimmune diseases in which HSCT has been extensively tried during the last decade is multiple sclerosis (MS). A few original papers and many anecdotal reports have indicated a beneficial effect of this treatment in MS, leading to stabilization or improvement in a large proportion of the treated patients. However, although hundreds of MS patients have been treated with HSCT, different conditioning and treatment protocols have been used in each center, making it difficult to organize and summarize the results from all of these small studies. Moreover, there is currently no completed controlled study with HSCT in MS. In this review, the cumulative experiences from several centers and countries in the world are summarized, based on the data presented at a recent international meeting in Moscow, Russia, entitled ‘Stem Cell Transplantation in Multiple Sclerosis: Sharing the Experience’.