Silvia Marconi

ADIPOSE-DERIVED MESENCHYMAL STEM CELLS AMELIORATE CHRONIC EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS

Mesenchymal stem cells (MSCs) represent a promising therapeutic approach for neurological autoimmune diseases; previous studies have shown that treatment with bone marrow‐derived MSCs induces immune modulation and reduces disease severity in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Here we show that intravenous administration of adipose‐derived MSCs (ASCs) before disease onset significantly reduces the severity of EAE by immune modulation and decreases spinal cord inflammation and demyelination. ASCs preferentially home into lymphoid organs but also migrates inside the central nervous system (CNS). Most importantly, administration of ASCs in chronic established EAE significantly ameliorates the disease course and reduces both demyelination and axonal loss, and induces a Th2‐type cytokine shift in T cells. Interestingly, a relevant subset of ASCs expresses activated α4 integrins and adheres to inflamed brain venules in intravital microscopy experiments. Bioluminescence imaging shows that α4 integrins control ASC accumulation in inflamed CNS. Importantly, we found that ASC cultures produce basic fibroblast growth factor, brain‐derived growth factor, and platelet‐derived growth factor‐AB. Moreover, ASC infiltration within demyelinated areas is accompanied by increased number of endogenous oligodendrocyte progenitors. In conclusion, we show that ASCs have clear therapeutic potential by a bimodal mechanism, by suppressing the autoimmune response in early phases of disease as well as by inducing local neuroregeneration by endogenous progenitors in animals with established disease. Overall, our data suggest that ASCs represent a valuable tool for stem cell–based therapy in chronic inflammatory diseases of the CNS. STEM CELLS 2009;27:2624–2635

Neuronal differentiation potential of human adipose-derived mesenchymal stem cells

Adult mesenchymal stem cells derived from adipose tissue (A-MSC) have the capacity to differentiate in vitro into mesenchymal as well as endodermal and ectodermal cell lineages. We investigated the neuronal differentiation potential of human A-MSC with a protocol which included sphere formation and sequential culture in brain-derived neurotrophic factor (BDNF) and retinoic acid (RA). After 30 days, about 57% A-MSC showed morphological, immunocytochemical and electrophysiological evidence of initial neuronal differentiation. In fact, A-MSC displayed elongated shape with protrusion of two or three cellular processes, selectively expressed nestin and neuronal molecules (including GABA receptor and tyroxine hydroxilase) in the absence of glial phenotypic markers. Differentiated cells showed negative membrane potential (-60 mV), delayed rectifier potassium currents and TTX-sensitive sodium currents. Such changes were stable for at least 7 days after removal of differentiation medium. In view of these results and the easy availability of adipose tissue, A-MSC may be a ready source of adult MSC with neuronal differentiation potential, an useful tool to treat neurodegenerative diseases.

Novel autoantigens recognized by CSF IgG from Hashimoto's encephalitis revealed by a proteomic approach.

To identify the target of IgG autoimmune response in Hashimotos encephalopathy (HE), we studied the binding of IgG present in serum and cerebro-spinal fluid (CSF) from six patients with HE and 15 controls to human central nervous system (CNS) white matter antigens by 2D-PAGE and immunoblotting and by immunohistochemistry. We found that CSF IgG from HE patients specifically recognized 3 spots, which were identified as dimethylargininase-I (DDAHI) and aldehyde reductase-I (AKRIAI). DDAHI was present in two isoforms recognized respectively by five and four HE patients; immunohistochemistry with anti-DDAHI antiserum depicted endothelial cells in normal human CNS. AKRIAI was recognized by three HE CSF and this enzyme was widely distributed on neurons and endothelia by immunohistochemistry. IgG from HE CSF immunostained both neuronal and endothelial cells in mouse CNS. The presence of these autoantibodies selectively in the CSF of HE patients may have important diagnostic and pathogenetic implications, since the autoimmune response to these enzymes may lead to vascular and/or neuronal damage, two major mechanisms involved in the pathogenesis of HE.

Transketolase and 2′,3′-Cyclic-nucleotide 3′-Phosphodiesterase Type I Isoforms Are Specifically Recognized by IgG Autoantibodies in Multiple Sclerosis Patients

The presence of autoantibodies in multiple sclerosis (MuS) is well known, but their target antigens have not been clearly identified. In the present study, IgG autoreactivity to neural antigens of normal human white matter separated by bidimensional electrophoresis was assessed in serum and cerebrospinal fluid of 18 MuS and 20 control patients. Broad IgG autoreactivity was detected by two-dimensional immunoblotting in all cases to neural antigens, most of which were identified by mass spectrometry. The comparative analysis of MuS and non-MuS reactive spots showed that a restricted number of neural protein isoforms were specifically recognized by MuS IgG. Almost all MuS patients had cerebrospinal fluid IgG directed to isoforms of one of the oligodendroglial molecules, transketolase, 2′,3′-cyclic-nucleotide 3′-phosphodiesterase type I, collapsin response mediator protein 2, and tubulin β4. Interestingly 50% of MuS IgG recognized transketolase, which was mostly localized on oligodendrocytes in human white matter from normal and MuS samples. IgG autoreactivity to cytoskeletal proteins (radixin, sirtuin 2, and actin-interacting protein 1) was prevalent in secondary progressive MuS patients. Among the proteins recognized by serum IgG, almost all MuS patients specifically recognized a restricted number of neuronal/cytoskeletal proteins, whereas 2′,3′-cyclic-nucleotide 3′-phosphodiesterase type I was the oligodendroglial antigen most frequently recognized (44%) by MuS seric IgG. Our immunomics approach shed new light on the autoimmune repertoire present in MuS patients revealing novel oligodendroglial and/or neuronal putative autoantigens with potential important pathogenic and diagnostic implications.

Expression of gangliosides on glial and neuronal cells in normal and pathological adult human brain

Few studies have assessed the glycolipid phenotype of glial cells in the human central nervous system (CNS) in situ. We investigated by immunohistochemistry the expression and cellular distribution of a panel of gangliosides (GM1, GM2, acetyl-GM3, GD1a, GD1b, GD2, GD3, GT1b, GQ1b and the A2B5 antibody) in adult, human normal and pathological brain, namely multiple sclerosis (MS) and other neurological diseases (OND). In normal conditions, we found diffuse expression in the white matter of most gangliosides tested, with the exception of acetyl-GM3, GT1b and GQ1b. By double immunofluorescence with phenotypic markers, GM1 and GD1b were preferentially expressed on GFAP+ astrocytes, GD1a on NG2+ oligodendrocyte precursors, A2B5 immunostained both populations, while GD2 was selectively present on mature oligodendrocytes. In the gray matter, only GM1, GD2 and A2B5 were present on neuronal cells. Interestingly, those gangliosides present on astrocytes in normal conditions were preferentially expressed on NG2+ cells in chronic MS lesions and in OND. Selective expression of GT1b upon astrocytes and NG2+ cells was instead observed in MS lesions, but not in OND. The definition of the glycolipid phenotype of CNS glial cells may be useful to identify distinct biological glial subsets and provide insights on the potential autoantigenic role of gangliosides in CNS autoimmune diseases.

T-Cell cytotoxicity of human Schwann cells: TNFα promotes fasL-mediated apoptosis and IFNγ perforin-mediated lysis

The ability of resident cells to induce apoptosis of invading immune cells is a major regulatory factor operating in immune‐privileged tissues, including the nervous system. We investigated the cellular and molecular factors participating in modulation of immune response in peripheral nerves, focusing on two cytotoxic pathways: fas ligand (fasL) and perforin. fasL and perforin expression was found by immunochemistry on Schwann cells (Sc) in nerve biopsies from patients with chronic inflammatory demyelinating polyneuritis and on human Sc cultures. Treatment of Sc with tumor necrosis factor (TNF) α and interferon (IFN) γ upregulated the expression of both molecules. In a coculture model, Sc exposed to TNFα or IFNγ were able to induce both apoptotic and lytic injury of T‐lymphocytes. Inactivation of fasL with the neutralizing antibody NOK‐2 abolished T‐cell apoptosis induced by Sc treated with TNFα, but not by Sc treated with IFNγ. Conversely, T‐cell lysis was significantly decreased when IFNγ‐activated Sc were treated with concanamycin A, which inhibited perforin release. At variance with T‐lymphocytes, B‐cells were less sensitive to cytokine‐treated Sc toxicity. Thus, Sc exposed to inflammatory cytokines have the capacity of inducing selective damage of T‐lymphocytes and have the potential of regulating the immune response in the peripheral nervous system.

Different content of chitin-like polysaccharides in multiple sclerosis and Alzheimer's disease brains

Chitin is an insoluble N-acetyl-glucosamine polymer coating fungi cell wall and several human parasites. It is hydrolysed by chitotriosidase (Chit); however, as chitin is absent in humans, the significance of human Chit activity is unknown. The level of plasma Chit activity positively correlates with Alzheimers disease (AD) and multiple sclerosis (MS). A recent study revealed the presence of potentially detrimental chitin-like substances in AD brain by Calcofluor histochemistry, whilst its search in MS brains has never been described to date. Through a comparative immunohistochemical analysis we confirm the presence of abundant chitin-like deposition in AD brains but fail to demonstrate it in MS brains. Interestingly, co-localization of beta-amyloid, Calcofluor and the nuclear marker DAPI was observed. Therefore, Chit production in MS patients is induced by mechanisms other than those operating in AD. Microglia-derived Chit activity in MS may counterbalance the naturally occurring glucosamine aggregation, protecting the brain from the chitin-like substance deposition.

Anti-GD2-like IgM autoreactivity in multiple sclerosis patients

Seric IgM autoreactivity in 100 multiple sclerosis (MS) and 106 control (70 of whom had other neurological diseases) patients was assessed either by immunohistochemistry on normal human CNS tissue or to GD2, GD1a, GD3 by ELISA and thin layer chromatography (TLC) techniques. By double immunohistochemistry, we found that 44% of the total MS population showed seric IgM reactivity to oligodendrocytes and myelin, this finding being particularly frequent in patients with secondary progressive MS. In the non-MS cohort, positive signals were seen only in one patient. In all cases, extraction of lipids from CNS sections abolished the immunoreactivity. Among the gangliosides investigated by ELISA, anti-GD2-like IgM autoantibodies were detected in the serum of 30% of MS patients, a subgroup of whom (below 10%) reacted also with GD1a and/or GD3. More than 85% of MS cases with anti-GD2-like IgM immunoreactivity by ELISA showed also IgM anti-oligodendrocyte/myelin staining by immunohistochemistry. However, no immunostaining in MS sera was observed when gangliosides were resolved by TLC. A positive correlation with neurological disability was observed, as the Expanded Disability Status Scale of MS patients with anti-GD2-like IgM autoreactivity by ELISA was significantly worse than seronegative MS cases. The results of the present study enforce the role of glycolipids as potential autoantigens and of IgM autoantibodies in MS pathogenesis.

Gangliosides act as onconeural antigens in paraneoplastic neuropathies

We describe two patients with progressive neuropathy and lung cancer in whom gangliosides (GS) may represent the oncoantigens. Patient 1 had motor neuropathy, high titers of IgG1 and IgG3 to GD1a and GM1, and expansion of circulating gamma-delta T lymphocytes, a T-cell subset responding to glycolipids. Patient 2 presented with Miller-Fisher-like syndrome and IgG3 activity to disialo-GS. In both cases, decreased autoimmune responses and stabilization of neuropathy were accomplished by tumor treatment. By immunohistochemistry, patient 1s IgG bound to his own tumor and to structures of normal nervous system expressing GD1a or GM1. Infiltration of IgG in the same neural structures was found at his autopsy. Regarding cellular immunity, the proportion of gamma-delta T lymphocytes infiltrating carcinoma from patient 1 was significantly higher than in neoplastic controls. These results indicate that GS may represent onconeural antigens in paraneoplastic neuropathy (PNN); their expression on neoplastic tissue may elicit autoimmune responses, which also target neural structures.

Murine adipose-derived mesenchymal stromal cell vesicles: in vitro clues for neuroprotective and neuroregenerative approaches

BACKGROUND AIMS Adipose-derived mesenchymal stromal cells (ASC) are known to promote neuroprotection and neuroregeneration in vitro and in vivo. These biological effects are probably mediated by paracrine mechanisms. In recent years, nanovesicles (NV) and microvesicles (MV) have been shown to play a major role in cell-to-cell communication. We tested the efficacy of NV and MV obtained from ASC in mediating neuroprotection and neuroregeneration in vitro. METHODS We exposed neuronal cells (both cell line and primary cultures) to oxidative stress in the presence or not of NV or MV. RESULTS In this experimental setting, we found that low doses of NV or MV protected neurons from apoptotic cell death. We then assessed the neuroregenerative effect of NV/MV in cerebellar slice cultures demyelinated with lysophosphatidylcholine. We observed that low but not higher doses of NV and MV increased the process of remyelination and activated nestin-positive oligodendroglial precursors. CONCLUSIONS Taken together, our data in vitro support the relevance of ASC vesicles as a source of protecting and regenerating factors that might modulate the microenvironment in neuro-inflammatory as well as in neurodegenerative disorders. The present findings may suggest that stromal cell-derived vesicles might represent a potential therapeutic tool, enabling the safe administration of stromal cell effector factors, avoiding the cellular counterpart.