Publications by authors named "Alessandra Aldinucci"

23 Publications

  • Page 1 of 1

Analysis of a Preliminary microRNA Expression Signature in a Human Telangiectatic Osteogenic Sarcoma Cancer Cell Line.

Int J Mol Sci 2021 Jan 25;22(3). Epub 2021 Jan 25.

Department of Experimental and Clinical Biomedical Sciences, University of Florence, 50134 Florence, Italy.

Telangiectatic osteosarcoma (TOS) is an aggressive variant of osteosarcoma (OS) with distinctive radiographic, gross, microscopic features, and prognostic implications. Despite several studies on OS, we are still far from understanding the molecular mechanisms of TOS. In recent years, many studies have demonstrated not only that microRNAs (miRNAs) are involved in OS tumorigenesis, development, and metastasis, but also that the presence in high-grade types of OS of cancer stem cells (CSCs) plays an important role in tumor progression. Despite these findings, nothing has been described previously about the expression of miRNAs and the presence of CSCs in human TOS. Therefore, we have isolated/characterized a putative CSC cell line from human TOS (TOS-CSCs) and evaluated the expression levels of several miRNAs in TOS-CSCs using real-time quantitative assays. We show, for the first time, the existence of CSCs in human TOS, highlighting the in vitro establishment of this unique stabilized cell line and an identification of a preliminary expression of the miRNA profile, characteristic of TOS-CSCs. These findings represent an important step in the study of the biology of one of the most aggressive variants of OS and the role of miRNAs in TOS-CSC behavior.
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http://dx.doi.org/10.3390/ijms22031163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7866083PMC
January 2021

The TCR Repertoire Reconstitution in Multiple Sclerosis: Comparing One-Shot and Continuous Immunosuppressive Therapies.

Front Immunol 2020 9;11:559. Epub 2020 Apr 9.

Dipartimento di Medicina Sperimentale e Clinica (DMSC), University of Florence, Florence, Italy.

Natalizumab (NTZ) and autologous hematopoietic stem cell transplantation (AHSCT) are two successful treatments for relapsing-remitting multiple sclerosis (RRMS), an autoimmune T-cell-driven disorder affecting the central nervous system that is characterized by relapses interspersed with periods of complete or partial recovery. Both RRMS treatments have been documented to impact T-cell subpopulations and the T-cell receptor (TCR) repertoire in terms of clone frequency, but, so far, the link between T-cell naive and memory populations, autoimmunity, and treatment outcome has not yet been established hindering insight into the post-treatment TCR landscape of MS patients. To address this important knowledge gap, we tracked peripheral T-cell subpopulations (naïve and memory CD4+ and CD8+) across 15 RRMS patients before and after two years of continuous treatment (NTZ) and a single treatment course (AHSCT) by high-throughput TCRß sequencing. We found that the two MS treatments left treatment-specific multidimensional traces in patient TCRß repertoire dynamics with respect to clonal expansion, clonal diversity and repertoire architecture. Comparing MS TCR sequences with published datasets suggested that the majority of public TCRs belonged to virus-associated sequences. In summary, applying multi-dimensional computational immunology to a TCRß dataset of treated MS patients, we show that qualitative changes of TCRß repertoires encode treatment-specific information that may be relevant for future clinical trials monitoring and personalized MS follow-up, diagnosis and treatment regimes. Natalizumab (NTZ) and autologous hematopoietic stem cell transplantation (AHSCT) are two successful treatments for relapsing-remitting multiple sclerosis (RRMS), an autoimmune T-cell-driven disorder affecting the central nervous system that is characterized by relapses interspersed with periods of complete or partial recovery. Both RRMS treatments have been documented to impact T-cell subpopulations and the T-cell receptor (TCR) repertoire in terms of clone frequency, but, so far, the link between T-cell naive and memory populations, autoimmunity, and treatment outcome has not yet been established hindering insight into the posttreatment TCR landscape of MS patients. To address this important knowledge gap, we tracked peripheral T-cell subpopulations (naive and memory CD4+ and CD8+) across 15 RRMS patients before and after 2 years of continuous treatment (NTZ) and a single treatment course (AHSCT) by high-throughput TCRβ sequencing. We found that the two MS treatments left treatment-specific multidimensional traces in patient TCRβ repertoire dynamics with respect to clonal expansion, clonal diversity, and repertoire architecture. Comparing MS TCR sequences with published datasets suggested that the majority of public TCRs belonged to virus-associated sequences. In summary, applying multidimensional computational immunology to a TCRβ dataset of treated MS patients, we show that qualitative changes of TCRβ repertoires encode treatment-specific information that may be relevant for future clinical trials monitoring and personalized MS follow-up, diagnosis, and treatment regimens.
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http://dx.doi.org/10.3389/fimmu.2020.00559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7160336PMC
March 2021

Cytokine inflammatory threat, but not LPS one, shortens GABAergic synaptic currents in the mouse spinal cord organotypic cultures.

J Neuroinflammation 2019 Jun 25;16(1):127. Epub 2019 Jun 25.

International School for Advanced Studies (SISSA/ISAS), 34136, Trieste, Italy.

Background: Synaptic dysfunction, named synaptopathy, due to inflammatory status of the central nervous system (CNS) is a recognized factor potentially underlying both motor and cognitive dysfunctions in neurodegenerative diseases. To gain knowledge on the mechanistic interplay between local inflammation and synapse changes, we compared two diverse inflammatory paradigms, a cytokine cocktail (CKs; IL-1β, TNF-α, and GM-CSF) and LPS, and their ability to tune GABAergic current duration in spinal cord cultured circuits.

Methods: We exploit spinal organotypic cultures, single-cell electrophysiology, immunocytochemistry, and confocal microscopy to explore synaptic currents and resident neuroglia reactivity upon CK or LPS incubation.

Results: Local inflammation in slice cultures induced by CK or LPS stimulations boosts network activity; however, only CKs specifically reduced GABAergic current duration. We pharmacologically investigated the contribution of GABAR α-subunits and suggested that a switch of GABAR α1-subunit might have induced faster GABAR decay time, weakening the inhibitory transmission.

Conclusions: Lower GABAergic current duration could contribute to providing an aberrant excitatory transmission critical for pre-motor circuit tasks and represent a specific feature of a CK cocktail able to mimic an inflammatory reaction that spreads in the CNS. Our results describe a selective mechanism that could be triggered during specific inflammatory stress.
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http://dx.doi.org/10.1186/s12974-019-1519-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6593520PMC
June 2019

CSF/serum matrix metallopeptidase-9 ratio discriminates neuro Behçet from multiple sclerosis.

Ann Clin Transl Neurol 2018 Apr 10;5(4):493-498. Epub 2018 Mar 10.

Department of Neurosciences, Psychology, Drug and Child Health University of Florence Florence Italy.

In neuro Behçet disease with multiple sclerosis-like features, diagnosis could be challenging. Here, we studied the cerebrospinal fluid and serum inflammatory profile of 11 neuro Behçet and 21 relapsing-remitting multiple sclerosis patients. Between the soluble factors analyzed (MMP9, TNF , IL6, CXCL13, CXCL10, CXCL8, IFN , IL10, IL17, IL23, and others) we found MMP9 increased in neuro Behçet serum compared to multiple sclerosis and decreased in cerebrospinal fluid. Furthermore, neuro Behçet analysis of circulating natural killer CD56 subset suggests their potential involvement in increased MMP9 production. We believe that these findings may have a translational utility in clinical practice.
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http://dx.doi.org/10.1002/acn3.538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5899916PMC
April 2018

The effect of strontium chloride on human periodontal ligament stem cells.

Clin Cases Miner Bone Metab 2017 Sep-Dec;14(3):283-293. Epub 2017 Dec 27.

Department of Surgery and Translational Medicine, University of Florence, Florence, Italy.

The complete repair of periodontal structures remains an exciting challenge that prompts researchers to develop new treatments to restore the periodontium. Recent research has suggested strontium ion to be an attractive candidate to improve osteogenic activity. In this study, we have isolated a clonal finite cell line derived from human periodontal ligament (PDL) in order to assess whether and in which way different doses of SrCl (from 0.5 to 500 μg/ml) can influence both the proliferation and the mineralization process, for future application in oral diseases. PDL cells were cloned by dilution plating technique and characterized by FACS. Cell proliferation analysis and mineralization were performed by [H]-thymidine incorporation and spectrofluorometric assay. Results have evidenced that the higher SrCl concentrations tested, from 25 to 500 μg/ml, have increased the proliferation activity after only 24 h of treatment. Interestingly, the same higher concentrations have decreased the mineralization, which was conversely increased by the lower ones, from 0.5 to 10 μg/ml. Our findings suggest the possible use of SrCl in appropriate delivery systems that release, at different time points, the specific dose, depending on the biological response that we want to induce on periodontal ligament stem cells, providing a more efficient periodontal regeneration.
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http://dx.doi.org/10.11138/ccmbm/2017.14.3.283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5762217PMC
December 2017

The effects of Exendin-4 on bone marrow-derived mesenchymal cells.

Endocrine 2018 06 1;60(3):423-434. Epub 2017 Nov 1.

Endocrine Unit, Department of Experimental and Clinical Biomedical Sciences "Mario Serio", University of Florence, Florence, Italy.

Purpose: GLP-1 receptor agonists are antidiabetic drugs currently used in the therapy of type 2 diabetes. Despite several in vitro and in vivo animal studies suggesting a beneficial effect of GLP-1 analogues on bone, in humans their skeletal effects are not clear and clinical studies report conflicting results.

Methods: We differentiated human mesenchymal stromal cells (hMSC) toward the adipogenic and the osteoblastic lineages, analysing the effect of Exendin-4 (EXE) before, during and after specific differentiations.

Results: We showed EXE ability to act selectively on a sub-population of hMSC characterised by a more stem potential, shifting them from G1 to S/M phase of cell cycle. We observed that EXE pre-treatment promotes both adipogenic and osteoblastic differentiations, possibly determined by an increased number of uncommitted progenitors. In fully differentiated cells, EXE affects mature adipocytes by increasing lipolysis, otherwise not altering osteoblasts metabolic activity. Moreover, the increased expression of osteoprotegerin, a modulator of the RANK/RANKL system, observed during osteogenic induction in presence of EXE, could negatively modulate osteoclastogenesis.

Conclusions: Our data suggest a complex action of EXE on bone, targeting the proliferation of mesenchymal progenitors, the metabolism of mature adipocytes and the modulation of osteoclastogenesis. Thus, an overall positive effect of this molecule on bone quality might be hypothesised.
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http://dx.doi.org/10.1007/s12020-017-1430-2DOI Listing
June 2018

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma.

J Vis Exp 2016 10 14(116). Epub 2016 Oct 14.

Department of Surgery and Translational Medicine (DCMT), University of Florence;

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains poor when metastasis has occurred. The Cancer Stem Cell (CSC) theory holds that there is a subset of tumor cells within the tumor that have stem-like characteristics, including the capacity to maintain the tumor and to resist multidrug chemotherapy. Therefore, a better understanding of OS biology and pathogenesis is needed in order to advance the development of targeted therapies to eradicate this particular subset and to reduce morbidity and mortality among patients. Isolating CSCs, establishing cell cultures of CSCs, and studying their biology are important steps to improving our understanding of OS biology and pathogenesis. The establishment of human-derived OS-CSCs from biopsies of OS has been made possible using several methods, including the capacity to create 3-dimensional stem cell cultures under nonadherent conditions. Under these conditions, CSCs are able to create spherical floating colonies formed by daughter stem cells; these colonies are termed "cellular spheres". Here, we describe a method to establish CSC cultures from primary cell cultures of conventional OS obtained from OS biopsies. We clearly describe the several passages required to isolate and characterize CSCs.
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http://dx.doi.org/10.3791/53884DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5092197PMC
October 2016

Establishment and Characterization of a Human Small Cell Osteosarcoma Cancer Stem Cell Line: A New Possible In Vitro Model for Discovering Small Cell Osteosarcoma Biology.

Stem Cells Int 2016 29;2016:3042198. Epub 2016 Aug 29.

Department of Surgery and Translational Medicine (DCMT), University of Florence, 50134 Florence, Italy.

Osteosarcoma (OSA) is the most common primary malignant bone tumor, usually arising in the long bones of children and young adults. There are different subtypes of OSA, among which we find the conventional OS (also called medullary or central osteosarcoma) which has a high grade of malignancy and an incidence of 80%. There are different subtypes of high grade OS like chondroblastic, fibroblastic, osteoblastic, telangiectatic, and the small cell osteosarcoma (SCO). In this study, for the first time, we have isolated, established, and characterized a cell line of cancer stem cells (CSCs) from a human SCO. First of all, we have established a primary finite cell line of SCO, from which we have isolated the CSCs by the sphere formation assay. We have proved their in vitro mesenchymal and embryonic stem phenotype. Additionally, we have showed their neoplastic phenotype, since the original tumor bulk is a high grade osteosarcoma. This research demonstrates the existence of CSCs also in human primary SCO and highlights the establishment of this particular stabilized cancer stem cell line. This will represent a first step into the study of the biology of these cells to discover new molecular targets molecules for new incisive therapeutic strategies against this highly aggressive OSA.
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http://dx.doi.org/10.1155/2016/3042198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5019944PMC
September 2016

The Adaptor Protein Rai/ShcC Promotes Astrocyte-Dependent Inflammation during Experimental Autoimmune Encephalomyelitis.

J Immunol 2016 07 10;197(2):480-90. Epub 2016 Jun 10.

Department of Life Sciences, University of Siena, 2 53100, Siena, Italy;

Th17 cells have been casually associated to the pathogenesis of autoimmune disease. We have previously demonstrated that Rai/ShcC, a member of the Shc family of adaptor proteins, negatively regulates Th17 cell differentiation and lupus autoimmunity. In this study, we have investigated the pathogenic outcome of the Th17 bias associated with Rai deficiency on multiple sclerosis development, using the experimental autoimmune encephalomyelitis (EAE) mouse model. We found that, unexpectedly, EAE was less severe in Rai(-/-) mice compared with their wild-type counterparts despite an enhanced generation of myelin-specific Th17 cells that infiltrated into the CNS. Nevertheless, when adoptively transferred into immunodeficient Rai(+/+) mice, these cells promoted a more severe disease compared with wild-type encephalitogenic Th17 cells. This paradoxical phenotype was caused by a dampened inflammatory response of astrocytes, which were found to express Rai, to IL-17. The results provide evidence that Rai plays opposite roles in Th17 cell differentiation and astrocyte activation, with the latter dominant over the former in EAE, highlighting this adaptor as a potential novel target for the therapy of multiple sclerosis.
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http://dx.doi.org/10.4049/jimmunol.1502063DOI Listing
July 2016

Histamine Regulates Actin Cytoskeleton in Human Toll-like Receptor 4-activated Monocyte-derived Dendritic Cells Tuning CD4+ T Lymphocyte Response.

J Biol Chem 2016 Jul 13;291(28):14803-14. Epub 2016 May 13.

From the Departments of Neuroscience, Psychology, Drugs and Child Health,

Histamine, a major mediator in allergic diseases, differentially regulates the polarizing ability of dendritic cells after Toll-like receptor (TLR) stimulation, by not completely explained mechanisms. In this study we investigated the effects of histamine on innate immune reaction during the response of human monocyte-derived DCs (mDCs) to different TLR stimuli: LPS, specific for TLR4, and Pam3Cys, specific for heterodimer molecule TLR1/TLR2. We investigated actin remodeling induced by histamine together with mDCs phenotype, cytokine production, and the stimulatory and polarizing ability of Th0. By confocal microscopy and RT-PCR expression of Rac1/CdC42 Rho GTPases, responsible for actin remodeling, we show that histamine selectively modifies actin cytoskeleton organization induced by TLR4, but not TLR2 and this correlates with increased IL4 production and decreased IFNγ by primed T cells. We also demonstrate that histamine-induced cytoskeleton organization is at least in part mediated by down-regulation of small Rho GTPase CdC42 and the protein target PAK1, but not by down-regulation of Rac1. The presence and relative expression of histamine receptors HR1-4 and TLRs were determined as well. Independently of actin remodeling, histamine down-regulates IL12p70 and CXCL10 production in mDCs after TLR2 and TLR4 stimulation. We also observed a trend of IL10 up-regulation that, despite previous reports, did not reach statistical significance.
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http://dx.doi.org/10.1074/jbc.M116.720680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4938197PMC
July 2016

Nanomaterial applications in multiple sclerosis inflamed brain.

J Neuroimmune Pharmacol 2015 Mar 24;10(1):1-13. Epub 2015 Jan 24.

Department of Neurofarba, University of Florence, Viale Pieraccini, 6, 50137, Florence, Italy,

In the last years scientific progress in nanomaterials, where size and shape make the difference, has increased their utilization in medicine with the development of a promising new translational science: nanomedicine. Due to their surface and core biophysical properties, nanomaterials hold the promise for medical applications in central nervous system (CNS) diseases: inflammatory, degenerative and tumors. The present review is focused on nanomaterials at the neuro-immune interface, evaluating two aspects: the possible CNS inflammatory response induced by nanomaterials and the developments of nanomaterials to improve treatment and diagnosis of neuroinflammatory diseases, with a focus on multiple sclerosis (MS). Indeed, nanomedicine allows projecting new ways of drug delivery and novel techniques for CNS imaging. Despite the wide field of application in neurological diseases of nanomaterials, our topic here is to review the more recent development of nanomaterials that cross blood brain barrier (BBB) and reach specific target during CNS inflammatory diseases, a crucial strategy for CNS early diagnosis and drug delivery, indeed the main challenges of nanomedicine.
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http://dx.doi.org/10.1007/s11481-015-9588-yDOI Listing
March 2015

Increased CXCL10 expression in MS MSCs and monocytes is unaffected by AHSCT.

Ann Clin Transl Neurol 2014 Sep 29;1(9):650-8. Epub 2014 Aug 29.

Dept. NEUROFARBA, University of Florence Florence, Italy.

Objective: To confirm CXCL10 over production in bone marrow mesenchymal stem cells (MSCs) and circulating monocytes isolated from multiple sclerosis patients (MS) and identify predate cell molecular signature; to extend this analysis after autologous hematopoietic stem cell transplantation (AHSCT) to test if therapy has modifying effects on MSCs and circulating monocytes.

Methods: MSCs and monocytes were isolated from 19 MS patients who undergone AHSCT before and seven of them at least 3 years after transplant. CXCL10 production was detected after LPS/IFN-γ stimulation. TLR4 signaling pathways were investigated by means of transcription factors phosphorylation/activation level. RT-PCR of activated transcription factors was performed to quantify their expression. All experiments were conducted in parallel with 24 matched healthy donors (HD).

Results: CXCL10 expression was significantly increased in both peripheral circulating monocytes and BM MSCs compared to HD. We showed that CXCL10 production is determined by an altered signaling pathway downstream TLR4, with the involvement of STAT-1, NF-κB, p38, JNK, and CREB. All upregulated transcription factors are more phosphorylated in MS patient sample. These features are not modified after AHSCT.

Interpretation: We demonstrated that in MS two different cell lineages are characterized by significantly increased production of CXCL10, due to altered signaling pathways of innate immune reaction mediated by TLR4, probably associated with disease phenotype. This characteristic is not modified by AHSCT, suggesting that when T and B lymphocytes are reset, other possible components of MS pathology, such as CXCL10 over production, do not determine therapy outcome.
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http://dx.doi.org/10.1002/acn3.92DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4241792PMC
September 2014

Carbon nanotube scaffolds instruct human dendritic cells: modulating immune responses by contacts at the nanoscale.

Nano Lett 2013 15;13(12):6098-105. Epub 2013 Nov 15.

Department of NEUROFARBA, University of Florence , 50134 Firenze, Italy.

Nanomaterials interact with cells and modify their function and biology. Manufacturing this ability can provide tissue-engineering scaffolds with nanostructures able to influence tissue growth and performance. Carbon nanotube compatibility with biomolecules motivated ongoing interest in the development of biosensors and devices including such materials. More recently, carbon nanotubes have been applied in several areas of nerve tissue engineering to study cell behavior or to instruct the growth and organization of neural networks. To gather further knowledge on the true potential of future constructs, in particular to assess their immune-modulatory action, we evaluate carbon nanotubes interactions with human dendritic cells (DCs). DCs are professional antigen-presenting cells and their behavior can predict immune responses triggered by adhesion-dependent signaling. Here, we incorporate DC cultures to carbon nanotubes and we show by phenotype, microscopy, and transcriptional analysis that in vitro differentiated and activated DCs show when interfaced to carbon nanotubes a lower immunogenic profile.
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http://dx.doi.org/10.1021/nl403396eDOI Listing
September 2014

Rosiglitazone promotes the differentiation of Langerhans cells and inhibits that of other dendritic cell types from CD133 positive hematopoietic precursors.

Histol Histopathol 2014 03 24;29(3):323-32. Epub 2013 Jul 24.

Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy.

Dendritic cells and their precursors express PPAR-gamma, whose stimulation has inhibitory effects on the maturation and function of dendritic cells in vivo. Dendritic cells can differentiate in vitro from CD133+ progenitors; the influence of PPAR-gamma stimulation on this process is unknown. We have addressed the effect of PPAR-gamma agonist rosiglitazone, at a concentration as used in clinics, on the differentiation of dendritic cells from human CD133+ progenitors. Cells were harvested from cord blood by density gradient and immunomagnetic separation, and cultured for 18 days with fetal calf serum, cytokines and 1 μmol/L rosiglitazone. Analyses included flow cytometry, electron microscopy and mixed lymphocyte reaction. As expected, control cells generated without rosiglitazone were dendritic, expressed MHC-II, CD80, CD83 and CD86 and stimulated mixed reaction potently. A minority of cells expressed the Langerhans cell marker CD207/langerin, but none contained Birbeck granules. With rosiglitazone much fewer cells were generated; they were all dendritic, expressed differentiation and maturation-related antigens in higher percentage and were better stimulators of lymphocytes than those generated without the drug. The vast majority of cells expressed CD207/langerin and many contained Birbeck granules, i.e. were full-fledged Langerhans cells. We conclude that stimulation of PPAR-gamma, while negatively affecting the number of generated cells, promotes the maturation of human cord blood CD133 positive precursors into efficient, immunostimulating dendritic cells with a Langerhans cell phenotype.
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http://dx.doi.org/10.14670/HH-29.323DOI Listing
March 2014

PARP-1 inhibition prevents CNS migration of dendritic cells during EAE, suppressing the encephalitogenic response and relapse severity.

Mult Scler 2011 Jul 22;17(7):794-807. Epub 2011 Feb 22.

Department of Pharmacology, University of Florence, Florence, Italy.

Background: Pharmacological inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1) are currently evaluated in clinical trials for various malignancies but, interestingly, also proved of remarkable efficacy in preclinical models of autoimmune disorders including experimental autoimmune encephalomyelitis (EAE).

Objectives: The objectives of the study were to determine molecular mechanisms underlying suppression of the encephalitogenic response by these drugs; likewise, whether clinically-relevant post-treatment paradigms with PARP-1 inhibitors could prevent EAE relapses.

Methods: Adopted both in vitro techniques (bone marrow-derived cultured DC) as well as in vivo models of chronic or relapsing-remitting (RR) EAE.

Results: We report that two structurally unrelated PARP-1 inhibitors negatively regulated NFκB activation, as well as maturation, cytokine production and APC function of cultured mouse bone marrow-derived dendritic cells (DCs). PARP-1 inhibitors also reduced the number and APC function of DCs migrating in the draining lymph nodes of ovalbumin-immunized mice. In C57Bl mice with chronic EAE or SJL mice with RR EAE, pharmacological inhibition of PARP-1 reduced CNS DC migration and demyelination as well as neurological impairment to an extent similar to that achieved with the potent immunosuppressant cyclosporine A. Remarkably, PARP-1 inhibitors injected after the first phase of disease reduced relapse incidence and severity, as well as the spinal cord number of autoreactive Th17 cells. Under this clinically-relevant treatment paradigm, PARP inhibitors also suppressed epitope spreading of the encephalitogenic response.

Conclusions: Overall, data underscore the potential relevance of PARP-1 inhibitors to MS therapy and suppression of autoimmunity.
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http://dx.doi.org/10.1177/1352458511399113DOI Listing
July 2011

Dendritic cells with lymphocyte-stimulating activity differentiate from human CD133 positive precursors.

Blood 2011 Apr 8;117(15):3983-95. Epub 2011 Feb 8.

Department of Anatomy, Histology and Forensic Medicine, University of Florence, Florence, Italy.

CD133 is a hallmark of primitive myeloid progenitors. We have addressed whether human cord blood cells selected for CD133 can generate dendritic cells, and Langerhans cells in particular, in conditions that promote that generation from CD34(+) progenitors. Transforming growth factor-β1 (TGF-β1) and anti-TGF-β1 antibody, respectively, were added in some experiments. With TGF-β, monocytoid cells were recognized after 7 days. Immunophenotypically immature dendritic cells were present at day 14. After 4 more days, the cells expressed CD54, CD80, CD83, and CD86 and were potent stimulators in mixed lymphocyte reaction; part of the cells expressed CD1a and langerin, but not Birbeck granules. Without TGF-β, only a small fraction of cells acquired a dendritic shape and expressed the maturation-related antigens, and lymphocytes were poorly stimulated. With anti-TGF-β, the cell growth was greatly hampered, CD54 and langerin were never expressed, and lymphocytes were stimulated weakly. In conclusion, CD133(+) progenitors can give rise in vitro, through definite steps, to mature, immunostimulatory dendritic cells with molecular features of Langerhans cells, although without Birbeck granules. Addition of TGF-β1 helps to stimulate cell growth and promotes the acquisition of mature immunophenotypical and functional features. Neither langerin nor Birbeck granules proved indispensable for lymphocyte stimulation.
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http://dx.doi.org/10.1182/blood-2010-08-299735DOI Listing
April 2011

Inhibition of immune synapse by altered dendritic cell actin distribution: a new pathway of mesenchymal stem cell immune regulation.

J Immunol 2010 Nov 1;185(9):5102-10. Epub 2010 Oct 1.

Department of Neurological Sciences, University of Florence, Florence, Italy.

Immune synapse formation between dendritic cells (DCs) and T cells is one of the key events in immune reaction. In immunogenic synapses, the presence of fully mature DCs is mandatory; consequently, the modulation of DC maturation may promote tolerance and represents a valuable therapeutic approach in autoimmune diseases. In the field of cell therapy, bone marrow mesenchymal stem cells (MSCs) have been extensively studied for their immunoregulatory properties, such as inhibiting DC immunogenicity during in vitro differentiation and ameliorating in vivo models of autoimmune diseases (e.g., experimental allergic encephalomyelitis). MSCs seem to play different roles with regard to DCs, depending on cell concentration, mechanism of stimulation, and accompanying immune cells. The aim of this work was to elucidate the immunogenic effects of MSC/DC interactions during DC activation (LPS stimulation or Ag loading). Human monocyte-derived DCs, bone marrow-derived MSCs, and circulating lymphocytes obtained from healthy donors, as well as the laboratory-generated influenza virus hemagglutinin-derived peptide, aa 306-318 peptide-specific T cell line were used for this study. We demonstrate that MSCs mediate inhibition of DC function only upon cell-cell contact. Despite no modification observed in cell phenotype or cytokine production, MSC-treated DCs were unable to form active immune synapses; they retained endocytic activity and podosome-like structures, typical of immature DCs. The transcriptional program induced by MSC-DC direct interaction supports at the molecular pathway level the phenotypical features observed, indicating the genes involved into contact-induced rearrangement of DC cytoskeleton.
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http://dx.doi.org/10.4049/jimmunol.1001332DOI Listing
November 2010

Modulating dendritic cells (DC) from immunogenic to tolerogenic responses: a novel mechanism of AZA/6-MP.

J Neuroimmunol 2010 Jan 24;218(1-2):28-35. Epub 2009 Nov 24.

Department of Neurological Sciences, University of Florence, Italy.

Azathioprine (Aza), 6-Mercaptopurine (6-MP) and 6-Thioguanine (6-TG) are thiopurine drugs widely used as immunosuppressants/anti-inflammatory agents in organ transplantation and chemotherapy. Aza is well tolerated and effective in modifying the course of MS. Here we investigated the action of 6-MP on human dendritic cells (DCs). We described for the first time that 6-MP impairs in vitro differentiation of DCs, has an inhibitory effect during DC activation processes inducing a functionally less immunogenic phenotype. Moreover, 6-MP significantly reduces DC IL-23 production and CCR7 expression, at the same time induces IL-10 augmentation. All these findings add a novel action mechanism in Aza immune modulation.
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http://dx.doi.org/10.1016/j.jneuroim.2009.11.001DOI Listing
January 2010

Differences in mesenchymal stem cell cytokine profiles between MS patients and healthy donors: implication for assessment of disease activity and treatment.

J Neuroimmunol 2008 Aug 17;199(1-2):142-50. Epub 2008 Jun 17.

Department of Neurological Sciences, University of Florence, Viale Pieraccini, 6, 50134 Florence, Italy.

MSCs have been proposed as possible treatment in MS: In this study MSCs obtained from 10 MS patients and 6 healthy donors (HD) were compared in terms of phenotypical and functional characteristics. We show that MSCs isolated from MS and HD differ significantly for IP10 production. Therefore, although MSCs isolated from MS patients exhibit the same properties of HD MSCs in terms of proliferation, phenotype, in vitro differentiation, TLR expression, immunosuppressive ability, inhibition of DC differentiation and activation, the use of autologous MSCs in cell therapy of autoimmune diseases should be submitted to attentive evaluation and treatment.
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http://dx.doi.org/10.1016/j.jneuroim.2008.05.006DOI Listing
August 2008

HCV patients, psychopathology and tryptophan metabolism: analysis of the effects of pegylated interferon plus ribavirin treatment.

Dig Liver Dis 2007 Sep;39 Suppl 1:S107-11

Department of Internal Medicine, Center for Research and Higher Education MCTDNENT, University of Florence, Florence, Italy.

Aims: Depression and other psychiatric disorders are frequent in HCV-infected patients, especially during interferon treatment. The molecular mechanism(s) underlying this finding is still unknown but it has been suggested that HCV and/or interferon administration may increase indoleamine 2,3-dioxygenase (IDO) activity, and reduce plasma tryptophan (TRP) levels and brain serotonin synthesis thus leading to psychopathological disorders.

Methods: We studied 89 subjects: (a) 39 patients with chronic hepatitis C virus (HCV) infection and mild liver damage; (b) 39 healthy controls; and (c) 10 patients with chronic hepatitis B virus (HBV) infection. 15 of the patients with HCV infection were re-evaluated after antiviral treatment with pegylated interferon alpha-2a plus ribavirin leading to viral eradication. We measured serum TRP and kynurenine levels and IDO activity in macrophages. Furthermore, each patient had an accurate psychopathological evaluation.

Results: HCV-infected patients had lower (-28%) serum TRP and kynurenine levels than healthy volunteers or HBV-infected patients with comparable liver damage. Depression and anxiety symptoms were particularly common in HCV patients. After viral clearance, macrophage IDO activity, plasma TRP and kynurenine levels returned toward normal values and psychopathology improved.

Conclusion: Our study shows that HCV patients have reduced serum TRP levels and confirms that they frequently suffer from anxiety and depression-related symptoms. The reduced IDO activity found in the macrophages of these patients suggests that HCV infection may hamper macrophage functions. After successful antiviral treatment, in spite of the expected increase of IDO activity in macrophages, we noticed that TRP and kynurenine plasma levels returned toward physiological levels and psychopathology decreased significantly.
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http://dx.doi.org/10.1016/s1590-8658(07)80021-1DOI Listing
September 2007

A key role for poly(ADP-ribose) polymerase-1 activity during human dendritic cell maturation.

J Immunol 2007 Jul;179(1):305-12

Department of Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy.

Poly(ADP-ribose) (PAR) polymerase (PARP)-1 is a nuclear enzyme regulating protein that functions by targeting PAR chains. Besides its classic role in DNA repair, PARP-1 is emerging as a key transcriptional regulator in different cell types including the immune ones. In this study, we investigated the role of PARP-1 in human dendritic cell (DC) function. We report that both PARP-1 mRNA and protein levels significantly increased during in vitro DC differentiation from monocytes. Of note, inhibitors of PARP-1 such as phenanthridinone and thieno[2,3-c]isoquinolin-5-one reduced expression of CD86 and CD83 in a concentration-dependent manner, having no effects on expression of CD80 and HLA-DR in mature DCs. In the same cultures, PARP-1 inhibitors also reduced production of IL-12 and IL-10. Addition of exogenous IL-12 to the culture medium partially restored CD86 expression in DCs exposed to PARP-1 inhibitors. In line with the role of PAR formation in NF-kappaB-dependent transactivation, we also report that phenanthridinone and thieno[2,3-c]isoquinolin-5-one impaired NF-kappaB and AP-1 subunit DNA binding activity in cellular extract of activated DCs. Finally, we show that PARP-1 inhibitors reduced the T cell allostimulatory activity of mature DCs, and that this reduction was prevented when DCs matured in the presence of PARP-1 inhibitors plus IL-12. Of note, nonproliferating T cells exposed to PARP-1 inhibitor-challenged DCs could undergo efficient proliferation when exposed to a subsequent activation stimulus such as anti-CD3 plus anti-CD-28. Together, data provide evidence for a key role of PARP-1 and poly ADP-ribosylation in DC immunocompetence and underscore the relevance of PARP-1 inhibitors to treatment of immune disorders.
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http://dx.doi.org/10.4049/jimmunol.179.1.305DOI Listing
July 2007

Effects of pixantrone on immune-cell function in the course of acute rat experimental allergic encephalomyelitis.

J Neuroimmunol 2005 Nov 24;168(1-2):111-7. Epub 2005 Aug 24.

Department of Neurological and Psychiatric Sciences, University of Florence, Florence, Italy.

Pixantrone is an immunesuppressor similar to mitoxantrone but with lower cardiotoxicity. We evaluated the effect of pixantrone on B cells and lymphomononuclear cells in the course of acute EAE. Pixantrone reduced the number of B cells and suppressed myelin basic protein (MBP) specific IgG production. In vitro, pixantrone induced apoptosis of rat B lymphocytes in a way similar to mitoxantrone. In addition, pixantrone inhibited antigen specific and mitogen induced lymphomononuclear cell proliferation, as well as IFN-gamma production, during EAE. These findings suggest a similar mechanism of action for pixantrone and mitoxantrone on the effector function of lymphomonocyte B and T cells.
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http://dx.doi.org/10.1016/j.jneuroim.2005.07.010DOI Listing
November 2005