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These updated EBMT guidelines review the clinical evidence, registry activity and mechanisms of action of haematopoietic stem cell transplantation (HSCT) in multiple sclerosis (MS) and other immune-mediated neurological diseases and provide recommendations for patient selection, transplant technique, follow-up and future development. The major focus is on autologous HSCT (aHSCT), used in MS for over two decades and currently the fastest growing indication for this treatment in Europe, with increasing evidence to support its use in highly active relapsing remitting MS failing to respond to disease modifying therapies. aHSCT may have a potential role in the treatment of the progressive forms of MS with a significant inflammatory component and other immune-mediated neurological diseases, including chronic inflammatory demyelinating polyneuropathy, neuromyelitis optica, myasthenia gravis and stiff person syndrome. Allogeneic HSCT should only be considered where potential risks are justified. Compared with other immunomodulatory treatments, HSCT is associated with greater short-term risks and requires close interspeciality collaboration between transplant physicians and neurologists with a special interest in these neurological conditions before, during and after treatment in accredited HSCT centres. Other experimental cell therapies are developmental for these diseases and patients should only be treated on clinical trials.

•Periventricular regions are frequently damaged in MS.•An outside-in gradient of damage has been observed in periventricular NAWM in CIS.•We evaluated the association between CSF-OCB and periventricular NAMW distribution.•Presence of CSF-OCB was associated with a steeper outside-in NAWM damage gradient.•There was no association between CSF-OCB and periventricular WM lesions severity.

Mesenchymal stem cells (MSC) display a remarkable ability to modulate the immune response and protect the central nervous system mainly through the release of soluble factors in a paracrine fashion, affecting the functional behavior of cells in the tissues. Here we investigated the effect of the interaction between MSC and microglia in vitro, and we dissected the molecular and cellular mechanisms of this crosstalk. We demonstrated that MSC impair microglia activation by inflammatory cues through the inhibition of the expression and release of inflammatory molecules and stress‐associated proteins. We showed that MSC significantly increase microglial expression and release of molecules associated with a neuroprotective phenotype such as CX3CR1, nuclear receptor 4 family, CD200 receptor, and insulin growth factor 1. Interestingly, MSC can enhance functional changes on microglia as depicted by the increase of intracellular calcium concentration and phagocytic activity. This last event is associated with an increased expression of triggering receptor expressed on myeloid cells‐2, an innate immune receptor involved in phagocytosis in the absence of inflammation. The observed effects on CX3CR1‐expressing microglia are due to the release of CX3CL1 by MSC, driven by inflammatory signals, as demonstrated by the reversal of the observed results when CX3CL1 expression was silenced in MSC or its release was blocked. Finally, we showed that exogenous CX3CL1 induce phenotypic and functional changes of microglia similar to those induced by MSC. These findings demonstrate that MSC instruct, through the release of CX3CL1, microglia responsiveness to proinflammatory signals by modulating constitutive “calming” receptors, typically expressed by “steady‐state microglia” thus switching microglia from a detrimental phenotype to a neuroprotective one. StemCells2012;30:2044–2053

The lack of therapies fostering remyelination and regeneration of the neural network deranged by the autoimmune attack occurring in multiple sclerosis (MS) is raising great expectations about stem cells therapies for tissue repair. Mesenchymal stem cells (MSCs) have been proposed as a possible treatment for MS due to the reported capacity of transdifferentiation into neural cells and their ability at modulating immune responses. However, recent studies have demonstrated that many other functional properties are likely to play a role in the therapeutic plasticity of MSCs, including antiapoptotic, trophic and anti-oxidant effects. These features are mostly based on the paracrine release of soluble molecules, often dictated by local environmental cues. Based on the modest evidence of long-term engraftment and the striking clinical effects that are observed immediately after MSCs administration in the experimental model of MS, we do not favor a major role for transdifferentiation as an important mechanism involved in the therapeutic effect of MSCs.

The recent advances in our understanding of stem cell biology, the availability of innovative techniques that allow large-scale acquisition of stem cells, and the increasing pressure from the multiple sclerosis (MS) patient community seeking tissue repair strategies have launched stem cell treatments as one of the most exciting and difficult challenges in the MS field. Here, we provide an overview of the current status of stem cell research in MS focusing on secured actuality, reasonable hopes and unrealistic myths.

To verify the existence of a symptomatic form of restless legs syndrome (RLS) secondary to multiple sclerosis (MS) and to identify possible associated risk factors.

In recent years much excitement has been generated over the possibility that adult stem cells may attempt repair of the injured central nervous system (CNS), thus setting the stage for their utilisation in the treatment of neurodegenerative disorders. Recent studies have shown that some subsets of stem cells can also modulate the (auto)immune response, thus providing a rationale for their use as therapy for experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). This article reviews the scientific evidence supporting the possible use of neural stem cells (NSCs) and mesenchymal stem cells (MSCs) for the treatment of MS. In addition, possible mechanisms sustaining the beneficial mode of action of haematopoietic stem cells (HSCs) following transplantation in MS individuals are discussed. Overall, it is proposed that limited subsets of adult stem cells may have a dual function that may be effective for the treatment of MS, an autoimmune disease of the CNS where degeneration of neural cells follows inflammation.

We studied the immunoregulatory features of murine mesenchymal stem cells (MSCs) in vitro and in vivo. MSCs inhibited T-cell receptor (TCR)-dependent and -independent proliferation but did not induce apoptosis on T cells. Such inhibition was paired with a decreased interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha production and was partially reversed by interleukin-2 (IL-2). Thus, we used MSCs to treat myelin oligodendrocyte glycoprotein (MOG)35-55-induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6J mice. We injected intravenously 1 × 106 MSCs before disease onset (preventive protocol) and at different time points after disease occurrence (therapeutic protocol). MSC administration before disease onset strikingly ameliorated EAE. The therapeutic scheme was effective when MSCs were administered at disease onset and at the peak of disease but not after disease stabilization. Central nervous system (CNS) pathology showed decreased inflammatory infiltrates and demyelination in mice that received transplants of MSCs. T-cell response to MOG and mitogens from MSC-treated mice was inhibited and restored by IL-2 administration. Upon MSC transfection with the enhanced green fluorescent protein (eGFP), eGFP+ cells were detected in the lymphoid organs of treated mice. These data suggest that the immunoregulatory properties of MSCs effectively interfere with the autoimmune attack in the course of EAE inducing an in vivo state of T-cell unresponsiveness occurring within secondary lymphoid organs. (Blood. 2005; 106:1755-1761)

We studied the immunoregulatory features of murine mesenchymal stem cells (MSCs) in vitro and in vivo. MSCs inhibited T-cell receptor (TCR)-dependent and -independent proliferation but did not induce apoptosis on T cells. Such inhibition was paired with a decreased interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha production and was partially reversed by interleukin-2 (IL-2). Thus, we used MSCs to treat myelin oligodendrocyte glycoprotein (MOG)35-55-induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6J mice. We injected intravenously 1 × 106MSCs before disease onset (preventive protocol) and at different time points after disease occurrence (therapeutic protocol). MSC administration before disease onset strikingly ameliorated EAE. The therapeutic scheme was effective when MSCs were administered at disease onset and at the peak of disease but not after disease stabilization. Central nervous system (CNS) pathology showed decreased inflammatory infiltrates and demyelination in mice that received transplants of MSCs. T-cell response to MOG and mitogens from MSC-treated mice was inhibited and restored by IL-2 administration. Upon MSC transfection with the enhanced green fluorescent protein (eGFP), eGFP+cells were detected in the lymphoid organs of treated mice. These data suggest that the immunoregulatory properties of MSCs effectively interfere with the autoimmune attack in the course of EAE inducing an in vivo state of T-cell unresponsiveness occurring within secondary lymphoid organs. (Blood. 2005; 106:1755-1761)

In patients with primary progressive (PP) multiple sclerosis, brain MRI lesion activity and burden are low, despite the presence of severe neurological impairment. On the contrary, the degree of cord atrophy and diffuse tissue damage in the brain and cervical cord have been found to be associated with clinical disability. Against this background, this study aimed at providing an in vivoindirect assessment of brain and cervical cord pathology in a large cohort of PP multiple sclerosis patients, using conventional MRI and magnetization transfer imaging (MTI). Ninety-one PP multiple sclerosis patients, 36 secondary progressive (SP) multiple sclerosis patients and 30 healthy controls underwent brain and cervical cord MRI scans, using dual echo (brain) or fast short-tau inversion recovery (cervical cord) MTI and T1-weighted sequences. For the brain, T2 hyperintense and T1 hypointense lesion volumes were calculated and the volume of the whole of the brain tissue measured. For the cervical cord, the number and burden of lesions and the cross-sectional area were assessed. MTI scans were post-processed and analysed to obtain magnetization transfer ratio (MTR) histograms from the whole of the brain and cervical cord tissue and from the normal-appearing brain tissue in isolation. In PP multiple sclerosis patients, brain, normal-appearing brain tissue and cervical cord MTR histogram-derived metrics revealed the presence of diffuse tissue damage whose characteristics did not significantly differ from those of SP multiple sclerosis patients, even though SP multiple sclerosis patients had higher MRI-visible lesion burdens. None of the correlations between MRI or MTI measures obtained from the brain and the cord were significant. PP multiple sclerosis patients' disability was significantly, albeit weakly associated with a composite MR model including measures of loss and intrinsic damage of cervical cord tissue. Our data indicate the presence of a diffuse tissue damage undetectable by conventional MRI in PP multiple sclerosis patients, whose extent seems to match that of SP multiple sclerosis patients with similar levels of disability. They also suggest that the severity of multiple sclerosis pathology in the cervical cord is one of the factors contributing to neurological impairment in PP multiple sclerosis.

Mutations in the early growth response 2 (EGR2) gene are associated with some forms of Charcot–Marie–Tooth disease (CMT) and other demyelinating neuropathies. These mutations modify the EGR2 binding to specific DNA sequences suggesting a role in the transcriptional control of myelination-specific genes. Here we show that the D355V mutation, associated with a CMT case combining axonal and demyelinating abnormalities, reduces three times the affinity of EGR2 to its consensus sequence and ten times its affinity to a sequence in the human Cx32promoter. These findings could indicate that this EGR2mutation leads to the development of CMT1 through the transcriptional deregulation of Cx32gene.

Charcot‐Marie‐Tooth 1A (CMT1A) neuropathy is caused by duplication of the peripheral myelin protein 22(PMP22) gene, leading to protein overexpression. Although this protein has a role in regulating Schwann cell growth and peripheral myelin compaction, how altered concentrations of PMP22 impair myelination is unknown. We established dorsal root ganglia (DRG) cultures from a transgenic rat overexpressing PMP22 (PMP22tg) to study the behavior of PMP22tgSchwann cells in early stages of development and myelination. We used reverse transcriptase–polymerase chain reaction and light and electron microscopy to study PMP22 expression and myelin formation. Myelin ultrastructure was evaluated in sural nerves from CMT1A patients to compare experimental and human findings. PMP22tgDRG cultures contained a greater number of internodes devoid of myelin, in the absence of remyelination, and increased periodicity of myelin lamellae compared with normal cultures. Widening of myelin lamellae was also observed in CMT1A biopsy specimens. Our results suggest that both functions of PMP22, in regulating Schwann cell differentiation and contributing to peripheral myelin compaction, are affected by its overexpression. The presence of similar myelin abnormalities in PMP22tgcultures and human nerves emphasizes the importance of developing in vitro models of hereditary neuropathies to study their underlying pathomechanisms.

The common marmoset Callithrix jacchus (C. jacchus) is an outbred species characterized by a naturally occurring bone marrow chimerism and susceptibility to a form of experimental autoimmune encephalomyelitis (EAE) resembling multiple sclerosis (MS). T cell clones specific for the myelin antigen, myelin basic protein (MBP), can be derived from both naive and immunized marmosets and can adoptively transfer EAE to compatible chimeric siblings. Here, we demonstrate that severel different antigenic determinants of MBP are recognized by these encephalitogenic T cell clones. Furthermore, PCR-based analysis of TCR Vβ families does not show the preferential usage of any gene segment. Characterization of third complementarity determining regions (CDR3) fails to demonstrate a recurring motif characteristic of the T cell immune response to MBP in this species. Nevertheless, brief amino acid motifs are shared among marmoset clones and CDR3 sequences from MS samples. These data suggest that, due to its outbred condition, the C. jacchus marmoset mounts a diverse pathogenic response to MBP. However, the findings that certain CDR3 sequences are identically expressed in different animals, or by different T cell clones, suggest that MBP-specific T cell populations may be clonally expanded following chronic antigenic stimulation in vivo.

Hereditary neuropathy with a liability to pressure palsies (HNPP) is an autosomal dominant disorder characterized by recurrent pressure palsies generally precipitated by minor trauma; weakness and paraesthesia usually improve and recover completely in a few months. By Southern blotting and fluorescent in situ hybridization analysis we confirm the presence of a 17p11.2 deletion in familial and in isolated cases of HNPP, suggesting that molecular analysis of the 17p11.2 region could also be a reliable and non-invasive method of diagnosis in sporadic cases, where a correct diagnosis usually requires a nerve biopsy. Although HNPP is a mild disease and not all patients seek medical attention, a presymptomatic diagnosis is useful for assessing the risk during genetic counselling, due to the inheritance of the mutation.

Two patients with Parkinson disease (one anatomically confirmed) presented a disorder of eyelid opening known as 'apraxia of eyelid opening'. The mechanism of this problem appeared to be related to the global slowing of the motor functions found in extrapyramidal syndromes. There is a persistent simultaneous activation of the orbicularis oculi and frontalis muscles. The syndrome is also present in Parkinson's disease as well as in progressive supranuclear palsy.

Hereditary neuropathy with liability to pressure palsies (HNPP) is associated with a deletion in chromosome 17p 11.2, which includes the gene for the peripheral myelin protein 22 (PMP‐22). A “gene dosage” effect is probably the mechanism underlying HNPP, but the amount of PMP‐22 mRNA in sural nerves of HNPP patients is highly variable and the role of PMP‐22 underexpression in impairing myelination has yet to be clarified. We have studied 6 genetically proven HNPP patients, to evaluate the relationship between PMP‐22 mRNA levels, and clinical, neurophysiological, and neuropathological findings. Underexpression of PMP‐22 mRNA correlates with disease severity and with mean axon diameter and g ratio, but not with myelin thickness, number of “tomacula,” or nerve conduction parameters. Our findings further confirm that underexpression of PMP‐22 is the main pathogenetic mechanism underlying the severity of clinical symptoms and signs in HNPP. Smaller axons in sural nerves of HNPP patients with lower PMP‐22 levels suggests that underexpression of PMP‐22 may also affect axon development.

We present a case of generalized chorea, with complete recovery, due to MRI-documented bilateral lacunar infarcts of the caudate nucleus, putamen, and deep frontal white matter. MRI is probably more sensitive than CT in disclosing small deep cerebral infarcts.

Multiple Sclerosis (MS) is a chronic inflammatory demyelinating disease believed to be mediated by autoreactive lymphocytes that invade the Central Nervous System and cause oligodendrocyte, axonal and neuronal damage as well as glial scarring.to and resulting in demyelination, neuronal death and brain atrophy. Hematopoietic Stem Cell Transplantation (HSCT) has been tried in the last 15 years as a therapeutic option in patients with a poor prognosis autoimmune disease not responding to conventional treatments. Worldwide ≥600 patients with MS have been treated with HSCT, most of them having been recruited in small, single center, phase 1–2 uncontrolled trials. Clinical and MRI outcomes from case series reports or Registry-based analyses suggest that a major response is achieved in most patients; quality and duration of response are better in patients transplanted earlier in the relapsing-remitting phase. The intensity of the immunosuppression from transplant treatment may be determined by several factors, such as the use of chemotherapy in the mobilization regimen, the intensity of the conditioning regimen and ex-vivo T-cell depletion.We report here the Italian multi-center experience on 74 MS patients treated with AHSCT between 1996 and 2008, all mobilized with Cyclophosphamide/G-CSF and conditioned with BEAM and rabbit ATG. Clinical and MRI outcomes were reported to the Italian Registry; the median follow-up is 48.3 (range 30–210) months. All patients clinically deteriorated in the year prior to HSCT, with an increase of Extended Disability Scale (EDSS) of at least one point (average EDSS change=1.5 points, range=1–9),Two patients (3.3%) died for transplant-related causes. At 5 years after the transplant, 66% of patients remained stable or improved. Progression free survival (PFS) was slightly better in relapsing-remitting (RR) (5 years PFS=71%) than in secondary-progressive (SP) forms (5 years PFS=62%, p=0.28). Amongst patients with a follow up longer than 1 year, 8 out of 25 RR subjects (31%) had a 6–12 months confirmed EDSS improvement > 1 point as compared to 1 out of 36 (3%) SP patients (p=0.009), Figure 1.Out of 18 cases with a follow up longer than 7 years, 8 (44%) remained stable or had a sustained improvement whilst 10 (56%), after an initial period of stabilization or improvement with a median duration of 3.5 years, showed a slow progression of disability.This study shows in a large cohort of patients with a long follow-up that AHSCT with BEAM/ATG conditioning regimen has a profound effect in suppressing disease progression in aggressive MS cases, unresponsive to conventional therapies. Indeed it results in a sustained improvement of the disability, free of immunosuppression, in a significant amount of RR patients. Clinical improvement is scarcely reported in MS literature as it is rather infrequent with conventional treatments; nevertheless it results in a stable increase of the quality of the life in this subset of young patients and should be considered in the choice of a therapeutic strategy in the early phase of MS.Cuneo: Roche: Consultancy, Speakers Bureau.

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We investigated the contribution of Schwann cell-derived ciliary neurotrophic factor (CNTF) to the pathogenesis of Charcot-Marie-Tooth disease type 1A (CMT1A) and addressed the question as to whether it plays a role in the development of axonal damage observed in the disease, with aging. Ciliary neurotrophic factor was underexpressed in experimental CMT1A but not in other models of hereditary neuropathies. Sciatic nerve crush experiments and dosage of CNTF at different time points showed that expression of this trophic factor remained significantly lower in CMT1A rats than in normal controls; moreover, in uninjured CMT1A sciatic nerves CNTF levels further decreased with ageing, thus paralleling the molecular signs of axonal impairment, that is increased expression of non-phosphorylated neurofilaments and amyloid precursor protein. Administration of CNTF to dorsal root ganglia cultures reduced dephosphorylation of neurofilaments in CMT1A cultures, without improving demyelination. Taken together, these results provide further evidence that the production of CNTF by Schwann cells is markedly reduced in CMT1A. Moreover, the observations suggest that trophic support to the axon is impaired in CMT1A and that further studies on the therapeutic use of trophic factors or their derivatives in experimental and human CMT1A are warranted.

Background. Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder of the adult life, characterized by the progressive loss of cortical and spinal motor neurons and an outcome usually fatal within 3 to 5 yrs, due to respiratory failure, with no effective therapies presently available. Efforts are needed in ALS to find novel treatments, able to reduce or block neuronal loss and/or to rebuild damaged neuronal circuits. Recent studies have raised the interest for the use of bone marrow-derived cells (BMCs) to repair damaged nervous system; this approach seems particularly attractive in ALS, which is characterized by neuronal degeneration. Aims of the study. To evaluate, at the multicenter setting, feasibility, safety and tolerability, and possible benefit of repeated procedures of G-CSF-induced BMC mobilization in ALS patients. Patients and methods. Seven Neurology Centers, along with seven Hematology Centers, are participating to the multicenter “STEMALS” trial, that started in June 2006 and will enrol a total of 28 patients, with 4 mobilization procedures for each patients; this is the interim analysis, after 42 mobilization procedures performed on a total of 22 patients. Patient median age is 56 yrs (range 40–64), 14 are male. The protocol includes four cycles of G-CSF, scheduled at 3 mos. intervals. In each cycle, G-CSF is administered at the dose of 5 μg/kg s.c., twice a day, for 4 consecutive days. At each cycle, CBCs and circulating CD34+ cells are determined, since day 0 through day 6. Results. Overall, 18 patients completed the first G-CSF cycle and all displayed good response; peak values were for WBCs: 41 x103/μL (range 24– 71) at day 3, and for CD34+ve cells: 52/μL (range 8.4–156), at day 4; 16 patients have already completed the second mobilization course, with values of circulating cells matching those of the first cycle (median WBCs/μL: 42x103; median CD34+ve/μL: 60.7); a few patients have undergone the third and fourth cycle, again they consistently displayed high levels of mobilization. Overall, the mobilization procedures were well tolerated, with the exception of a transient increase of growth hormone level in one patient, and a deep venous thrombosis in one patient, both complications did not preclude to conclude the treatment. Conclusion. The STEMALS trial indicate that: i. the use of G-CSF to induce BMC mobilization is safe, well tolerated and feasible in ALS, even at the multicenter level; ii. so far, few and reversible adverse effects have been recorded; iii. peak values of circulating CD34+ve cells indicate that BMC mobilization capacity in ALS is analogous to that commonly observed in the healthy population; iv. there are no signs of impaired mobilization, after repeated courses of G-CSF administration, performed at few month intervals. A longer follow up is still required to verify possible benefits of the repeated BMC mobilization program in ALS.

Background. Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder of the adult life, characterized by the progressive loss of cortical and spinal motor neurons and an outcome usually fatal within 3 to 5 yrs, due to respiratory failure, with no effective therapies presently available. Efforts are needed in ALS to find novel treatments, able to reduce or block neuronal loss and/or to rebuild damaged neuronal circuits. Recent studies have raised the interest for the use of bone marrow-derived cells (BMCs) to repair damaged nervous system; this approach seems particularly attractive in ALS, which is characterized by neuronal degeneration. Aims of the study. To evaluate, at the multicenter setting, feasibility, safety and tolerability, and possible benefit of repeated procedures of G-CSF-induced BMC mobilization in ALS patients.

Haematopoietic Stem Cells Transplantation (HSCT) has been successfully used in the last decade for the treatment of severe Autoimmune Diseases (ADs), including Multiple Sclerosis (MS). A retrospective analysis of 183 Multiple Sclerosis patients recorded in the database of the EBMT is reported. The patients received an autologous HSCT in the period 1995–2004. The disability at the time of HSCT was severe, with a median EDSS of 6.5 (range 3.5–9). Cumulative survival is 86% at 9 years with median follow-up time of 3.5 years. Overall Transplant-Related Mortality (TRM) is 5.3%; no toxic deaths were reported in the 62 patients transplanted after the year 2000. Conditioning regimens including Busulphan were statistically associated to TRM, both in uni- and in multivariate analysis. No toxic deaths were reported among the 53 patients treated with the BEAM/ATG regimen without graft manipulation, irrespective of their clinical condition at the time of the transplant. Sixty-three percent of the 145 patients evaluable for the neurological outcome showed a sustained improvement or stabilization of their disability. A trend of a better clinical outcome in patients transplanted within 5 years of diagnosis was shown. HSCT was shown as an effective procedure to stop disease progression in patients affected by severe, progressive MS; safety and feasibility of the procedure can be significantly improved by appropriate patient selection and choice of transplant regimen.

Haematopoietic Stem Cells Transplantation (HSCT) has been successfully used in the last decade for the treatment of severe Autoimmune Diseases (ADs), including Multiple Sclerosis (MS). A retrospective analysis of 183 Multiple Sclerosis patients recorded in the database of the EBMT is reported. The patients received an autologous HSCT in the period 1995–2004. The disability at the time of HSCT was severe, with a median EDSS of 6.5 (range 3.5–9). Cumulative survival is 86% at 9 years with median follow-up time of 3.5 years. Overall Transplant-Related Mortality (TRM) is 5.3%; no toxic deaths were reported in the 62 patients transplanted after the year 2000. Conditioning regimens including Busulphan were statistically associated to TRM, both in uni- and in multivariate analysis. No toxic deaths were reported among the 53 patients treated with the BEAM/ATG regimen without graft manipulation, irrespective of their clinical condition at the time of the transplant. Sixty-three percent of the 145 patients evaluable for the neurological outcome showed a sustained improvement or stabilization of their disability. A trend of a better clinical outcome in patients transplanted within 5 years of diagnosis was shown. HSCT was shown as an effective procedure to stop disease progression in patients affected by severe, progressive MS; safety and feasibility of the procedure can be significantly improved by appropriate patient selection and choice of transplant regimen.