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LABORATORY OF: Neuroimmunobiology
CONTACT PERSON: Prof.Antonio Uccelli (University of Genova and San Martino Hospital)
Phone +39 0105737437 E-mail: auccelli@neurologia.unige.it

Description of Laboratory and Expertise:

The Neuroimmunobiology Unit, directed by Antonio Uccelli M.D., focuses on understanding fundamental immunology and the pathogenesis of human multiple sclerosis and other degenerative diseases of the central nervous system. Dr Uccelli and colleagues have recently characterized the therapeutic potential of bone marrow derived mesenchymal stem cells in the experimental model of multiple sclerosis and are currently investigating the mechanisms of action of bone marrow stem cells in different experimental models.
The Neuroimmunobiology Unit is a three room-laboratory fully equipped to perform cell biology and cell immunology, histology and molecular biology.
Laboratory Expertises include:
Tissue culture, including immune cells and neurons, cellular immunology, including cell separation techniques, proliferation assay, flow cytometry, nucleic acids and proteins extraction from tissues and cells, gel electrophoresis, conventional and real time-PCR, histology and immunohistochemistry, in vivo experimental procedure on animals (mice).

Abstract of Activities:

Specifically, our research goals are as follows:
Characterization of immunoregulatory activity of mesenchymal stem cells in vitro and in vivo
Characterization of the activity of T cells and B cells and their role in experimental models of autoimmunity and multiple sclerosis.
Identification of the mechanisms leading to neural degeneration in multiple sclerosis and amyotrophic lateral sclerosis with focus on the role of microglia.
Pre-clinical evaluation of novel treatments in experimental models of neurological diseases.
Identification of multiple sclerosis associated genes and correlation with clinical findings.

Detailed Research Activities:

Mesenchymal stem cells (MSC) area:

We have recently demonstrated the efficacy of intravenous administration of murine MSC as treatment of chronic and relapsing remitting experimental autoimmune encephalomyelitis (EAE), a prototypical model of autoimmune disease of the central nervous system (CNS) mimicking human multiple sclerosis (MS). We are studying the in vivo MSCs effect on the generation of regulatory T cells and effector “encephalitogenic” Th17 cells in EAE analyzing T cells isolated from lymph-nodes and infiltrated CNS tissue. We are also evaluating the capacity of MSCs to recruit local neural precursors and trigger endogenous neurogenesis, synaptogenesis and promote remyelination.

Previous studies have shown that MSC can exert an anti-apoptotic effect on different cells including neurons. These data have been suggested to investigate about the possible effects of MSC on Amyotrophic lateral sclerosis (ALS), a neurodegenerative disease of motoneurons causing progressive paralysis. Therefore, we are studying the therapeutic potential of MSCs injected (i.v.) in mice expressing mutant human superoxide dismutase (SOD1) with a Gly93Ala substitution [SOD1(+)-G93A(+)], a transgenic animal model of amyotrophic lateral sclerosis (ALS). Through a molecular biology approach we are analyzing the gene expression profile of motor neurons associated tissues such as the motor cortex and the anterior horns of the thoracic spinal cord of mice injected with MSCs. We are also dissecting the role of MSCs on neurotoxic microglia.

Murine MSCs ameliorate experimental autoimmune encephalomyelitis (EAE), through the induction of peripheral T cell tolerance. Nevertheless, the effect of MSC on the physiological immune response to antigens such as tetanus toxoid (TT), is still largely unknown. Thus, we sought analysing the dynamic changes of the humoral and cellular response to TT upon i.v. MSCs administration in Balb/C mice. Upon sacrifice, the frequency of antigen specific plasma cells, memory B cells and T cells are analyzed from the bone marrow, lymph-nodes and spleen. In the latter, the extent of germinal centres formation has been evaluated. Knowing the effects of MSC on primary and secondary immune responses toward non-self and self antigens could be of pivotal importance before moving to a clinical trial on MS subjects and possible other autoimmune disordes.

Although the antiproliferative and immunosuppressive effects of MSCs on T cells have been largely studied, the effect on dendritic cells (DCs) still remains poorly understood. In order to study the effect of murine MSC on maturation, effector functions and subsequent T cells priming, we have generated DC from mouse BM. mMSC have been added at different time points during the in vitro maturation of DC and their phenotype and cytokine secretion have been evaluated. DCs generated without mMSC were activated with LPS in the presence or absence of mMSC. Naïve and memory T lymphocytes specific for an ovalbumin peptide were isolated from DO11.10 transgenic mice and in vitro co-cultured with DCs conditioned by mMSC loaded with OVA.Our data demonstrate that mMSC inhibit the maturation and differentiation of DCs and, in line with these results, impair the capacity to stimulate T cell proliferation. In order to evaluate expression of antigen processing machinery (APM) components by DCs generated with or without MSCs, we are using a panel of mAbs specific for individual APM components. At present we are investigating about the possible role of mMSC on DCs expression of Toll-Like Receptors (TLRs) and analyzing modification of TLRs associated genes by Real Time PCR.

In order to better dissect the mechanisms involved in the modulation of the immune response and the neuroprotective effect mediated by MSCs we are focusing on the possible role of enzymes of the arginine metabolisms, metallothioneins, heme-oxigenase as well as leptin. In addition, to get insights into novel therapeutic MSC-associated molecules, we are performing genes profile analysis of MSCs isolated through laser capture microdissection in the brain tissues of animals with EAE.

Multiple sclerosis area

In order to elucidate the role of Th17 cells in human MS, we have defined a set of surface markers to detect dynamic changes of IL17 producing T cells in the peripheral blood and cerebrospinal fluid of MS individuals over time.

We previously demonstrated that the inflamed CNS is the site of an ongoing germinal center reaction through the detection of well-characterized B cell subsets in the CSF of MS patients. We are analyzing the CSF and CNS tissues in order to functionally characterize B cells and gain insight into their role as Ag presenting cells and local modulators of the immune response through the release of cytokines.

We perform pharmacogenomic analyses of rapidly progressing MS individuals before starting immunotherapies such as the monoclonal antibody Natalizumab (anti-VLA4) and correlate the gene profiles with clinical and MRI data along a two years follow up.

Applications and Developments:

Immunomodulatory activity of MSC suggests the way for the clinical use of these cells in immune-mediated disorders. Based on their capacity to modulate immune responses and promote tissue repair, MSC have been proposed as treatment for autoimmune diseases. It is possible that some of the beneficial effects of MSCs might reflect, in part, the trophic and protective activities they exert on injured cells and tissues, rather than resulting from a true transdifferentiation. In immune-mediated diseases, the protective effects might function in concert with the immunosuppressive and anti-inflammatory activities. A phase I study (a safety study called MESEMS as in MEsenchymal Stem cElls for Multiple Sclerosis) aimed at utilizing haploidentical human MSCs for the treatment of severe, standard therapy resistant, MS patients is currently planned for early 2008 in collaboration with Dr. F. Frassoni (Centre for stem cells and cellular therapy, San Martino Hospital, Genoa)

Ongoing collaborations:

Vito Pistoia, IRCCS Istituto G.Gaslini, Genova, Italy: Immunology of multiple sclerosis and immunoregulatory properties of stem cells

Francesco Frassoni, Centro Cellule Staminali e Dipartimento di Ematologia, Azienda Ospedaliera Ospedale San Martino , Genova, Italy in vivo and in vitro characterization of mesenchymal stem cells

JR Oksenberg , SE Baranzini, Department of Neurology, School of Medicine, University of California, San Francisco, CA, USA. Genetics of MS

Gianbattista Bonanno and Ernesto Fedele, Dipartimento di Medicina Sperimentale, sezione Farmacologia e Tossicologia, Università degli Studi di Genova, Italy: mechanisms of neurodegeneration and synaptic transmission during EAE and other experimental neurological diseases

Pablo Diaz Villoslada, Multiple Sclerosis Center - Department of Neurology, University of Navarra, Spain: Immunology of multiple sclerosis

Antonio Lanzavecchia, Institute for Research in Biomedicine, Bellinzona, Switzerland.Immunology of multiple sclerosis

Elena Zocchi, ABC (Unit of Stem Cell Biochemistry). Paracrine models of interaction between mesenchymal stem cells and neurons.

Most recent and significant publication:

Uccelli A, Pistoia V, Moretta L. Mesenchymal stem cells: a new strategy for immunosuppression? Trends Immunol. 2007 May;28(5):219-26.

Benvenuto F, Ferrari S, Gerdoni E, Gualandi F, Frassoni F, Pistoia V, Mancardi GL, Uccelli A. Human mesenchymal stem cells promote survival of T cells in a quiescent state.
Stem Cells. 2007 Jul;25(7):1753-60.

Gerdoni E, Gallo B, Casazza S, Musio S, Bonanni I, Pedemonte E, Mantegazza R, Frassoni F, Mancardi GL, Pedotti R, Uccelli A. Mesenchymal stem cells effectively modulate pathogenic immune response in experimental autoimmune encephalomyelitis.
Ann Neurol. 2007 Mar;61(3):219-27.

Pedemonte E, Benvenuto F, Casazza S, Mancardi GL, Oksenberg JR, Uccelli A, Baranzini S. Mesenchymal stem cells effectively modulate pathogenic immune response in experimental autoimmune encephalomyelitis.
Ann Neurol. 2007 Mar;61(3):219-27.

Uccelli A, Moretta L, and Pistoia V. Immunoregulatory function of mesenchymal stem cells.
Eur J Immunol. 2006 Oct;36(10):2566-73.

Barcellos LF, Sawcer S, Ramsay PP, Baranzini SE, Thomson G, Briggs F, Cree BCA, Begovich AB, Villoslada P, Montalban X, Uccelli A, Savettieri G, Lincoln RL, DeLoa C, Haines JL, Pericak-Vance MA, Compston A, Hauser SL, Oksenberg JR. Heterogeneity at the HLA-DRB1 locus and risk for multiple sclerosis. Hum Mol Genet. 2006 Sep 15;15(18):2813-24.

Corcione A, Benvenuto F, Ferretti E, Giunti D, Capello V, Cazzanti F, Risso M, Gualandi F, Mancardi GL, Pistoia V, Uccelli A. Human mesenchymal stem cells modulate B-cell functions. Blood. 2006 Jan 1;107(1):367-72.

Zappia E, Casazza S, Pedemonte E, Benvenuto F, Bonanni I, Gerdoni E, Giunti D, Ceravolo A, Cazzanti F, Frassoni F, Mancardi G, Uccelli A. Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T-cell anergy. Blood. 2005 Sep 1;106(5):1755-61.

Uccelli A, Aloisi F, Pistoia V. Unveiling the enigma of the CNS as a B-cell fostering environment. Trends Immunol. 2005 May;26(5):254-9.

Corcione A, Casazza S, Ferretti E, Giunti D, Zappia E, Pistorio A, Gambini C, Mancardi G, Uccelli A*, Pistoia V*. Recapitulation of B cell differentiation in the central nervous system of patients with multiple sclerosis. Proc Natl Acad Sci U S A. 2004 Jul 27;101(30):11064-9.