Bruno Bonetti

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.

Complement-Mediated Demyelination in Patients with IgM Monoclonal Gammopathy and Polyneuropathy

We investigated the role of complement in the pathogenesis of the demyelinating polyneuropathy that occurs in some patients with IgM monoclonal gammopathy. Seven patients with chronic sensorimotor polyneuropathy and IgM monoclonal gammopathy were examined. In six patients, the monoclonal protein recognized an epitope shared by myelin-associated glycoprotein and two peripheral-nerve glycolipids, whereas in one patient, IgM bound to an unidentified myelin antigen. Direct and indirect immunofluorescence and immunoperoxidase assays showed colocalization along the myelin sheaths of peripheral-nerve fibers of monoclonal protein with complement components C1q, C3d, and C5. In addition, terminal-complement complex that was not associated with S protein was detected in myelin sheaths. It appeared that alterations in myelin geometry caused by the separation of myelin lamellae corresponded to sites at which terminal-complement complex was deposited. We conclude that demyelination in polyneuropathy associated with IgM monoclonal gammopathy may be mediated by complement.

Neutrophils promote Alzheimer's disease-like pathology and cognitive decline via LFA-1 integrin

Inflammation is a pathological hallmark of Alzheimers disease, and innate immune cells have been shown to contribute to disease pathogenesis. In two transgenic models of Alzheimers disease (5xFAD and 3xTg-AD mice), neutrophils extravasated and were present in areas with amyloid-β (Aβ) deposits, where they released neutrophil extracellular traps (NETs) and IL-17. Aβ42 peptide triggered the LFA-1 integrin high-affinity state and rapid neutrophil adhesion to integrin ligands. In vivo, LFA-1 integrin controlled neutrophil extravasation into the CNS and intraparenchymal motility. In transgenic Alzheimers disease models, neutrophil depletion or inhibition of neutrophil trafficking via LFA-1 blockade reduced Alzheimers disease–like neuropathology and improved memory in mice already showing cognitive dysfunction. Temporary depletion of neutrophils for 1 month at early stages of disease led to sustained improvements in memory. Transgenic Alzheimers disease model mice lacking LFA-1 were protected from cognitive decline and had reduced gliosis. In humans with Alzheimers disease, neutrophils adhered to and spread inside brain venules and were present in the parenchyma, along with NETs. Our results demonstrate that neutrophils contribute to Alzheimers disease pathogenesis and cognitive impairment and suggest that the inhibition of neutrophil trafficking may be beneficial in Alzheimers disease.

Epilepsy in cerebral glioma: Timing of appearance and histological correlations

Purpose A prospective study of patients with glioma was carried out. Special attention was paid to the first seizure, to the histology, and to the efficacy and prophylactic role of antiepileptic drugs (AEDs). Prognostic factors were analyzed. Methods Between February 1st 2004 and April 1st 2006 patients who underwent surgical treatment at the Neurosurgery Department of Bolzano for primary or recurrent glioma were prospectively followed until April 1st 2007. Seizures at onset occurring in the absence of interictal epileptiform abnormalities were regarded as remote symptomatic seizures and not treated. Results Sixty-four individuals were registered in the study; epilepsy diagnosis was made in 27 cases and in 24 of these seizures were the onset symptom. The correlation with histological grading showed that seizures were more frequent in low-grade glioma and in secondary glioblastoma. Although epilepsy was less frequent in high-grade glioma, in these patients seizures were more difficult to control. Poor seizure control was associated with motor and sensitive focal seizures and presence of neurological deficit. Patients without epilepsy and not taking AEDs never developed seizures during the follow-up. Discussion Our study clearly shows that epilepsy is more frequent in low-grade gliomas but seizures are more difficult to control in high-grade gliomas. In both cases seizures are a quite exclusive symptom at the onset that never appears during the stable course of the disease. Amongst glioblastoma multiforme (GBM), epilepsy is more frequent in GBM developing through progression from low-grade astrocitoma. Moreover, our study strongly indicates that the prophylactic use of AEDs in glioma is not justified.

Single-Cell Phosphoproteomics Resolves Adaptive Signaling Dynamics and Informs Targeted Combination Therapy in Glioblastoma

Intratumoral heterogeneity of signaling networks may contribute to targeted cancer therapy resistance, including in the highly lethal brain cancer glioblastoma (GBM). We performed single-cell phosphoproteomics on a patient-derived in vivo GBM model of mTOR kinase inhibitor resistance and coupled it to an analytical approach for detecting changes in signaling coordination. Alterations in the protein signaling coordination were resolved as early as 2.5 days after treatment, anticipating drug resistance long before it was clinically manifest. Combination therapies were identified that resulted in complete and sustained tumor suppression in vivo. This approach may identify actionable alterations in signal coordination that underlie adaptive resistance, which can be suppressed through combination drug therapy, including non-obvious drug combinations.

Hepatitis C virus infection of peripheral nerves in type II cryoglobulinaemia

Abstract Peripheral neuropathy is a frequent complication in patients suffering from type II mixed cryoglobulinaemia (mCGII), a sort of vasculitis that is strongly associated with hepatitis C virus (HCV) infection and characterised by high concentrations of anti-HCV antibodies and HCV RNA in the cryoprecipitates. We report the finding of HCV RNA in homogenates of nerve biopsies from five such patients, by reverse transcription-polymerase chain reaction (RT-PCR) amplification of different regions of the viral genome. HCV RNA was localized in epineurial cells by in situ RT-PCR. Our data suggest that HCV infection of nerves plays a major role in mCGII-associated neuropathy.

T-cell-mediated epineurial vasculitis and humoral-mediated microangiopathy in cryoglobulinemic neuropathy

We used immunohistochemistry to assess the role of humoral and cellular factors in endoneurial microangiopathy and epineurial vasculitis in 15 nerve biopsies of patients with axonal neuropathy and monoclonal or mixed cryoglobulinemia (CG). Deposition of immunoglobulins and cytolytic complement was detected in endoneurial capillaries of patients with mixed CG. Epineurial inflammatory infiltrates containing beta2-integrin-positive lymphocytes and monocytes surrounded arterioles expressing cell adhesion molecules, thus suggesting a cell-mediated pathogenesis of the epineurial vasculitis. On the other hand, the absence of immune complex deposition and polymorphonuclear elements suggests a minor role for the humoral mechanisms in the formation of the vasculitic lesions. This study indicates that both cell-mediated mechanisms and immune complexes/cryoglobulins are involved, although at different levels, in the pathogenesis of CG neuropathy.

Role of HIV in the pathogenesis of distal symmetrical peripheral neuropathy

We report the results of a clinical, electrophysiological and pathological study conducted in 18 AIDS patients presenting a distal symmetrical predominantly sensory polyneuropathy (DSPN) characterized by painful dysesthesias as main complaint. Onset of the neuropathy was at CDC (Center for Disease Control) stage II in 2 patients, at CDC stage III in 5 patients and at CDC stage IV in the remainder. Electrophysiological investigation confirmed the presence of an axonal alteration in the sensory nerves, but also revealed motor involvement in all cases. The neuropathological features of sensory nerves were fiber loss and axonal degeneration with macrophagic activation. The expression of monocyte-macrophage markers and of major histocompatibily complex class II antigens appeared up-regulated in endoneurial ramified cells, while expression of CR3, a complement receptor involved in the process of phagocytosis, was down-regulated. In six nerve biopsy samples and in two out of five DSPN dorsal root ganglia we found HIV-related mRNA and protein located in scattered cells of the endoneurium which we presume to be macrophages. These data suggest that: (a) DSPN may occur early in the course of the disease and is not limited to later stages; (b) DSPN is not a ganglionitis but is actually a sensory-motor neuropathy; (c) the virus enters the peripheral nervous system and induces changes in the immunocompetent cell population with activation of macrophages. Storage of the virus inside macrophages may act both as a reservoir for the virus and as a putative cause of nerve damage, probably through release of cytotoxins and/or interaction with trophic factors.

Experimental induction of myelin changes by anti-MAG antibodies and terminal complement complex

We investigated the role of anti-myelin-associated glycoprotein (MAG) IgM and complement (C) in the pathogenesis of myelin alterations occurring in patients with anti-MAG-associated polyneuropathy. For this purpose, we separately studied the effects of anti-MAG antibodies and terminal C complex (TCC) after injection into the rabbit sciatic nerve. The two different local treatments produced identical ultrastructural abnormalities such as intramyclinic edema, myelin vesiculation and, in particular, separation of the major dense lines with the formation of widely spaced myelin, a peculiar feature encountered in human peripheral nerve disorders with circulating anti-myelin monoclonal IgM. In nerves treated with anti-MAG IgM ultrastructural myelin alterations were concurrent with activation of the rabbits own C to the formation of TCC. Contrary to the immunological and ultrastructural findings obtained in C-sufficient animals, in C6-deficient rabbits injected with anti-MAG IgM no myelin alterations nor C completion were observed. This study identifies anti-MAG IgM as the mediator and the C as the effector of myelin changes observed in the present model and, for extension, in human neuropathies associated with anti-MAG IgM.