Publications by authors named "Pietro L Poliani"

25 Publications

  • Page 1 of 1

Human and mouse single-nucleus transcriptomics reveal TREM2-dependent and TREM2-independent cellular responses in Alzheimer's disease.

Nat Med 2020 01 13;26(1):131-142. Epub 2020 Jan 13.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA.

Glia have been implicated in Alzheimer's disease (AD) pathogenesis. Variants of the microglia receptor triggering receptor expressed on myeloid cells 2 (TREM2) increase AD risk, and activation of disease-associated microglia (DAM) is dependent on TREM2 in mouse models of AD. We surveyed gene-expression changes associated with AD pathology and TREM2 in 5XFAD mice and in human AD by single-nucleus RNA sequencing. We confirmed the presence of Trem2-dependent DAM and identified a previously undiscovered Serpina3nC4b reactive oligodendrocyte population in mice. Interestingly, remarkably different glial phenotypes were evident in human AD. Microglia signature was reminiscent of IRF8-driven reactive microglia in peripheral-nerve injury. Oligodendrocyte signatures suggested impaired axonal myelination and metabolic adaptation to neuronal degeneration. Astrocyte profiles indicated weakened metabolic coordination with neurons. Notably, the reactive phenotype of microglia was less evident in TREM2-R47H and TREM2-R62H carriers than in non-carriers, demonstrating a TREM2 requirement in both mouse and human AD, despite the marked species-specific differences.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41591-019-0695-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980793PMC
January 2020

DNA threads released by activated CD4 T lymphocytes provide autocrine costimulation.

Proc Natl Acad Sci U S A 2019 04 15;116(18):8985-8994. Epub 2019 Apr 15.

Department of Research, Molecular Immunology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, 20133 Milan, Italy.

The extrusion of DNA traps contributes to a key mechanism in which innate immune cells clear pathogens or induce sterile inflammation. Here we provide evidence that CD4 T cells, a critical regulator of adaptive immunity, release extracellular threads of DNA on activation. These DNA extrusions convey autocrine costimulatory signals to T lymphocytes and can be detected in lymph nodes isolated during the priming phase of experimental autoimmune encephalomyelitis (EAE), a CD4 T cell-driven mouse model of multiple sclerosis. Pharmacologic inhibition of mitochondrial reactive oxygen species (mtROS) abolishes the extrusion of DNA by CD4 T cells, reducing cytokine production in vitro and T cell priming against myelin in vivo. Moreover, mtROS blockade during established EAE markedly ameliorates disease severity, dampening autoimmune inflammation of the central nervous system. Taken together, these experimental results elucidate a mechanism of intrinsic immune costimulation mediated by DNA threads released by activated T helper cells, and identify a potential therapeutic target for such disorders as multiple sclerosis, neuromyelitis optica, and CD4 T cell-mediated disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1822013116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6500139PMC
April 2019

The proneural gene ASCL1 governs the transcriptional subgroup affiliation in glioblastoma stem cells by directly repressing the mesenchymal gene NDRG1.

Cell Death Differ 2019 09 11;26(9):1813-1831. Epub 2018 Dec 11.

Neural Stem Cell Biology Unit, Division of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Scientific Institute, Via Olgettina 58, 20132, Milan, Italy.

Achaete-scute homolog 1 gene (ASCL1) is a gene classifier for the proneural (PN) transcriptional subgroup of glioblastoma (GBM) that has a relevant role in the neuronal-like differentiation of GBM cancer stem cells (CSCs) through the activation of a PN gene signature. Besides prototypical ASCL1 PN target genes, the molecular effectors mediating ASCL1 function in regulating GBM differentiation and, most relevantly, subgroup specification are currently unknown. Here we report that ASCL1 not only promotes the acquisition of a PN phenotype in CSCs by inducing a glial-to-neuronal lineage switch but also concomitantly represses mesenchymal (MES) features by directly downregulating the expression of N-Myc downstream-regulated gene 1 (NDRG1), which we propose as a novel gene classifier of MES GBMs. Increasing the expression of ASCL1 in PN CSCs results in suppression of self-renewal, promotion of differentiation and, most significantly, decrease in tumorigenesis, which is also reproduced by NDRG1 silencing. Conversely, both abrogation of ASCL1 expression in PN CSCs and enforcement of NDRG1 expression in either PN or MES CSCs induce proneural-to-mesenchymal transition (PMT) and enhanced mesenchymal features. Surprisingly, ASCL1 overexpression in MES CSCs increases malignant features and gives rise to a neuroendocrine-like secretory phenotype. Altogether, our results propose that the fine interplay between ASCL1 and its target NDRG1 might serve as potential subgroup-specific targetable vulnerability in GBM; enhancing ASCL1 expression in PN GBMs might reduce tumorigenesis, whereas repressing NDRG1 expression might be actionable to hamper the malignancy of GBM belonging to the MES subgroup.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41418-018-0248-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748080PMC
September 2019

Tuberous sclerosis complex-associated CNS abnormalities depend on hyperactivation of mTORC1 and Akt.

J Clin Invest 2018 04 12;128(4):1688-1706. Epub 2018 Mar 12.

Neural Stem Cell Biology Unit, Division of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Scientific Institute, Milan, Italy.

Tuberous sclerosis complex (TSC) is a dominantly inherited disease caused by hyperactivation of the mTORC1 pathway and characterized by the development of hamartomas and benign tumors, including in the brain. Among the neurological manifestations associated with TSC, the tumor progression of static subependymal nodules (SENs) into subependymal giant cell astrocytomas (SEGAs) is one of the major causes of morbidity and shortened life expectancy. To date, mouse modeling has failed in reproducing these 2 lesions. Here we report that simultaneous hyperactivation of mTORC1 and Akt pathways by codeletion of Tsc1 and Pten, selectively in postnatal neural stem cells (pNSCs), is required for the formation of bona fide SENs and SEGAs. Notably, both lesions closely recapitulate the pathognomonic morphological and molecular features of the corresponding human abnormalities. The establishment of long-term expanding pNSC lines from mouse SENs and SEGAs made possible the identification of mTORC2 as one of the mediators conferring tumorigenic potential to SEGA pNSCs. Notably, in spite of concurrent Akt hyperactivation in mouse brain lesions, single mTOR inhibition by rapamycin was sufficient to strongly impair mouse SEGA growth. This study provides evidence that, concomitant with mTORC1 hyperactivation, sustained activation of Akt and mTORC2 in pNSCs is a mandatory step for the induction of SENs and SEGAs, and, at the same time, makes available an unprecedented NSC-based in vivo/in vitro model to be exploited for identifying actionable targets in TSC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI96342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5873854PMC
April 2018

Chronic Stimulation Unveils Autoreactive Potential of Wiskott-Aldrich Syndrome Protein-Deficient B Cells.

Front Immunol 2017 2;8:490. Epub 2017 May 2.

San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), Division of Regenerative Medicine, Stem Cells and Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy.

Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency caused by mutations in the gene encoding the hematopoietic-specific WAS protein (WASp). WAS is frequently associated with autoimmunity, indicating a critical role of WASp in maintenance of tolerance. The role of B cells in the induction of autoreactive immune responses in WAS has been investigated in several settings, but the mechanisms leading to the development of autoimmune manifestations have been difficult to evaluate in the mouse models of the disease that do not spontaneously develop autoimmunity. We performed an extensive characterization of mice that provided evidence of the potential alteration in B cell selection, because of the presence of autoantibodies against double-stranded DNA, platelets, and tissue antigens. To uncover the mechanisms leading to the activation of the potentially autoreactive B cells in mice, we performed chronic stimulations with toll-like receptors agonists (LPS and CpG) and apoptotic cells or infection with lymphocytic choriomeningitis virus. All treatments led to increased production of autoantibodies, increased proteinuria, and kidney tissue damage in mice. These findings demonstrate that a lower clearance of pathogens and/or self-antigens and the resulting chronic inflammatory state could cause B cell tolerance breakdown leading to autoimmunity in WAS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2017.00490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5411424PMC
May 2017

Alterations in the brain adenosine metabolism cause behavioral and neurological impairment in ADA-deficient mice and patients.

Sci Rep 2017 01 11;7:40136. Epub 2017 Jan 11.

San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCCS San Raffaele Scientific Institute, Milan, Italy.

Adenosine Deaminase (ADA) deficiency is an autosomal recessive variant of severe combined immunodeficiency (SCID) caused by systemic accumulation of ADA substrates. Neurological and behavioral abnormalities observed in ADA-SCID patients surviving after stem cell transplantation or gene therapy represent an unresolved enigma in the field. We found significant neurological and cognitive alterations in untreated ADA-SCID patients as well as in two groups of patients after short- and long-term enzyme replacement therapy with PEG-ADA. These included motor dysfunction, EEG alterations, sensorineural hypoacusia, white matter and ventricular alterations in MRI as well as a low mental development index or IQ. Ada-deficient mice were significantly less active and showed anxiety-like behavior. Molecular and metabolic analyses showed that this phenotype coincides with metabolic alterations and aberrant adenosine receptor signaling. PEG-ADA treatment corrected metabolic adenosine-based alterations, but not cellular and signaling defects, indicating an intrinsic nature of the neurological and behavioral phenotype in ADA deficiency.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep40136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5225479PMC
January 2017

MET inhibition overcomes radiation resistance of glioblastoma stem-like cells.

EMBO Mol Med 2016 05 2;8(5):550-68. Epub 2016 May 2.

Laboratory of Cancer Stem Cell Research, Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Italy Department of Oncology, University of Torino, Candiolo, Italy

Glioblastoma (GBM) contains stem-like cells (GSCs) known to be resistant to ionizing radiation and thus responsible for therapeutic failure and rapidly lethal tumor recurrence. It is known that GSC radioresistance relies on efficient activation of the DNA damage response, but the mechanisms linking this response with the stem status are still unclear. Here, we show that the MET receptor kinase, a functional marker of GSCs, is specifically expressed in a subset of radioresistant GSCs and overexpressed in human GBM recurring after radiotherapy. We elucidate that MET promotes GSC radioresistance through a novel mechanism, relying on AKT activity and leading to (i) sustained activation of Aurora kinase A, ATM kinase, and the downstream effectors of DNA repair, and (ii) phosphorylation and cytoplasmic retention of p21, which is associated with anti-apoptotic functions. We show that MET pharmacological inhibition causes DNA damage accumulation in irradiated GSCs and their depletion in vitro and in GBMs generated by GSC xenotransplantation. Preclinical evidence is thus provided that MET inhibitors can radiosensitize tumors and convert GSC-positive selection, induced by radiotherapy, into GSC eradication.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/emmm.201505890DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5130292PMC
May 2016

Constitutional de novo deletion of the FBXW7 gene in a patient with focal segmental glomerulosclerosis and multiple primitive tumors.

Sci Rep 2015 Oct 20;5:15454. Epub 2015 Oct 20.

Medical Cytogenetics and Molecular Genetics Lab, IRCCS Istituto Auxologico Italiano, Milan, Italy.

Multiple primary malignant neoplasms are rare entities in the clinical setting, but represent an important issue in the clinical management of patients since they could be expression of a genetic predisposition to malignancy. A high resolution genome wide array CGH led us to identify the first case of a de novo constitutional deletion confined to the FBXW7 gene, a well known tumor suppressor, in a patient with a syndromic phenotype characterized by focal segmental glomerulosclerosis and multiple primary early/atypical onset tumors, including Hodgkin's lymphoma, Wilms tumor and breast cancer. Other genetic defects may be associated with patient's phenotype. In this light, constitutional mutations at BRCA1, BRCA2, TP53, PALB2 and WT1 genes were excluded by performing sequencing and MLPA analysis; similarly, we ruled out constitutional abnormalities at the imprinted 11p15 region by methylation specific -MLPA assay. Our observations sustain the role of FBXW7 as cancer predisposition gene and expand the spectrum of its possible associated diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep15454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4612309PMC
October 2015

Cavin-1 and Caveolin-1 are both required to support cell proliferation, migration and anchorage-independent cell growth in rhabdomyosarcoma.

Lab Invest 2015 Jun 30;95(6):585-602. Epub 2015 Mar 30.

1] Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy [2] Interuniversity Institute of Myology (IIM), Rome, Italy.

Rhabdomyosarcoma (RMS) is a childhood soft tissue tumor with broad expression of markers that are typically found in skeletal muscle. Cavin-1 is a recently discovered protein actively cooperating with Caveolin-1 (Cav-1) in the morphogenesis of caveolae and whose role in cancer is drawing increasing attention. Using a combined in silico and in vitro analysis here we show that Cavin-1 is expressed in myogenic RMS tumors as well as in human and primary mouse RMS cultures, exhibiting a broad subcellular localization, ranging from nuclei and cytosol to plasma membrane. In particular, the coexpression and plasma membrane interaction between Cavin-1 and Cav-1 characterized the proliferation of human and mouse RMS cell cultures, while a downregulation of their expression levels was observed during the myogenic differentiation. Knockdown of Cavin-1 or Cav-1 in the human RD and RH30 cells led to impairment of cell proliferation and migration. Moreover, loss of Cavin-1 in RD cells impaired the anchorage-independent cell growth in soft agar. While the loss of Cavin-1 did not affect the Cav-1 protein levels in RMS cells, Cav-1 overexpression and knockdown triggered a rise or depletion of Cavin-1 protein levels in RD cells, respectively, in turn reflecting on increased or decreased cell proliferation, migration and anchorage-independent cell growth. Collectively, these data indicate that the interaction between Cavin-1 and Cav-1 underlies the cell growth and migration in myogenic tumors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/labinvest.2015.45DOI Listing
June 2015

miR-135a Inhibits Cancer Stem Cell-Driven Medulloblastoma Development by Directly Repressing Arhgef6 Expression.

Stem Cells 2015 May;33(5):1377-89

Neural Stem Cell Biology Unit, Division of Regenerative Medicine, Stem Cells and Gene Therapy, Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy; Vita-Salute San Raffaele University, Milan, Italy.

microRNAs (miRNAs) are short noncoding RNAs, which regulate gene expression post-transcriptionally and play crucial roles in relevant biological and pathological processes. Here, we investigated the putative role of miRNAs in modulating the tumor-initiating potential of mouse medulloblastoma (MB)-derived cancer stem cells (CSCs). We first subjected bona fide highly tumorigenic (HT) CSCs as well as lowly tumorigenic MB CSCs and normal neural stem cells to miRNA profiling, which identified a HT CSC-specific miRNA signature. Next, by cross-checking CSC mRNA/miRNA profiles, we pinpointed miR-135a as a potential tumor suppressor gene, which was strongly downregulated in HT CSCs as well as in the highly malignant experimental tumors derived from them. Remarkably, enforced expression of miR-135a in HT CSCs strongly inhibited tumorigenesis by repressing the miR-135a direct target gene Arhgef6. Considering the upregulation of Arhgef6 in human MBs and its involvement in mediating experimental medulloblastomagenesis, its efficient suppression by miR-135a might make available an effective therapeutic strategy to selectively impair the tumorigenic potential of MB CSCs. Stem Cells 2015;33:1377-1389.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/stem.1958DOI Listing
May 2015

Reply: To PMID 24332219.

J Allergy Clin Immunol 2014 Jul 26;134(1):243-4. Epub 2014 Jun 26.

Hospital Pharmacy, Erasmus University Medical Center, Rotterdam, The Netherlands.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jaci.2014.04.032DOI Listing
July 2014

Development of central nervous system autoimmunity is impaired in the absence of Wiskott-Aldrich syndrome protein.

PLoS One 2014 23;9(1):e86942. Epub 2014 Jan 23.

TIGET, San Raffaele Scientific Institute, Milan, Italy ; Milan Unit, Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale delle Ricerche, Milan, Italy.

Wiskott-Aldrich Syndrome protein (WASP) is a key regulator of the actin cytoskeleton in hematopoietic cells. Defective expression of WASP leads to multiple abnormalities in different hematopoietic cells. Despite severe impairment of T cell function, WAS patients exhibit a high prevalence of autoimmune disorders. We attempted to induce EAE, an animal model of organ-specific autoimmunity affecting the CNS that mimics human MS, in Was(-/-) mice. We describe here that Was(-/-) mice are markedly resistant against EAE, showing lower incidence and milder score, reduced CNS inflammation and demyelination as compared to WT mice. Microglia was only poorly activated in Was(-/-) mice. Antigen-induced T-cell proliferation, Th-1 and -17 cytokine production and integrin-dependent adhesion were increased in Was(-/-) mice. However, adoptive transfer of MOG-activated T cells from Was(-/-) mice in WT mice failed to induce EAE. Was(-/-) mice were resistant against EAE also when induced by adoptive transfer of MOG-activated T cells from WT mice. Was(+/-) heterozygous mice developed an intermediate clinical phenotype between WT and Was(-/-) mice, and they displayed a mixed population of WASP-positive and -negative T cells in the periphery but not in their CNS parenchyma, where the large majority of inflammatory cells expressed WASP. In conclusion, in absence of WASP, T-cell responses against a CNS autoantigen are increased, but the ability of autoreactive T cells to induce CNS autoimmunity is impaired, most probably because of an inefficient T-cell transmigration into the CNS and defective CNS resident microglial function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0086942PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3900702PMC
September 2014

Recombination-activating gene 1 (Rag1)-deficient mice with severe combined immunodeficiency treated with lentiviral gene therapy demonstrate autoimmune Omenn-like syndrome.

J Allergy Clin Immunol 2014 Apr 9;133(4):1116-23. Epub 2013 Dec 9.

Department of Hematology, Erasmus University Medical Center, Rotterdam, The Netherlands. Electronic address:

Background: Recombination-activating gene 1 (RAG1) deficiency results in severe combined immunodeficiency (SCID) caused by a complete lack of T and B lymphocytes. If untreated, patients succumb to recurrent infections.

Objectives: We sought to develop lentiviral gene therapy for RAG1-induced SCID and to test its safety.

Methods: Constructs containing the viral spleen-focus-forming virus (SF), ubiquitous promoters, or cell type-restricted promoters driving sequence-optimized RAG1 were compared for efficacy and safety in sublethally preconditioned Rag1(-/-) mice undergoing transplantation with transduced bone marrow progenitors.

Results: Peripheral blood CD3(+) T-cell reconstitution was achieved with SF, ubiquitous promoters, and cell type-restricted promoters but 3- to 18-fold lower than that seen in wild-type mice, and with a compromised CD4(+)/CD8(+) ratio. Mitogen-mediated T-cell responses and T cell-dependent and T cell-independent B-cell responses were not restored, and T-cell receptor patterns were skewed. Reconstitution of mature peripheral blood B cells was approximately 20-fold less for the SF vector than in wild-type mice and often not detectable with the other promoters, and plasma immunoglobulin levels were abnormal. Two months after transplantation, gene therapy-treated mice had rashes with cellular tissue infiltrates, activated peripheral blood CD44(+)CD69(+) T cells, high plasma IgE levels, antibodies against double-stranded DNA, and increased B cell-activating factor levels. Only rather high SF vector copy numbers could boost T- and B-cell reconstitution, but mRNA expression levels during T- and B-cell progenitor stages consistently remained less than wild-type levels.

Conclusions: These results underline that further development is required for improved expression to successfully treat patients with RAG1-induced SCID while maintaining low vector copy numbers and minimizing potential risks, including autoimmune reactions resembling Omenn syndrome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jaci.2013.10.009DOI Listing
April 2014

Inhibition of angiogenesis by β-galactosylceramidase deficiency in globoid cell leukodystrophy.

Brain 2013 Sep;136(Pt 9):2859-75

Department of Molecular and Translational Medicine, School of Medicine, University of Brescia, Italy.

Globoid cell leukodystrophy (Krabbe disease) is a neurological disorder of infants caused by genetic deficiency of the lysosomal enzyme β-galactosylceramidase leading to accumulation of the neurotoxic metabolite 1-β-d-galactosylsphingosine (psychosine) in the central nervous system. Angiogenesis plays a pivotal role in the physiology and pathology of the brain. Here, we demonstrate that psychosine has anti-angiogenic properties by causing the disassembling of endothelial cell actin structures at micromolar concentrations as found in the brain of patients with globoid cell leukodystrophy. Accordingly, significant alterations of microvascular endothelium were observed in the post-natal brain of twitcher mice, an authentic model of globoid cell leukodystrophy. Also, twitcher endothelium showed a progressively reduced capacity to respond to pro-angiogenic factors, defect that was corrected after transduction with a lentiviral vector harbouring the murine β-galactosylceramidase complementary DNA. Finally, RNA interference-mediated β-galactosylceramidase gene silencing causes psychosine accumulation in human endothelial cells and hampers their mitogenic and motogenic response to vascular endothelial growth factor. Accordingly, significant alterations were observed in human microvasculature from brain biopsy of a globoid cell leukodystrophy case. Together these data demonstrate that β-galactosylceramidase deficiency induces significant alterations in endothelial neovascular responses that may contribute to central nervous system and systemic damages that occur in globoid cell leukodystrophy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/brain/awt215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3754455PMC
September 2013

Osteopetrosis rescue upon RANKL administration to Rankl(-/-) mice: a new therapy for human RANKL-dependent ARO.

J Bone Miner Res 2012 Dec;27(12):2501-10

UOS/IRGB, Milan Unit, CNR, Milan, Italy; Humanitas Clinical and Research Center, Rozzano, Italy.

In the last decades the molecular basis of monogenic diseases has been largely unraveled, although their treatment has often remained unsatisfactory. Autosomal recessive osteopetrosis (ARO) belongs to the small group of genetic diseases that are usually treated with hematopoietic stem cell transplantation (HSCT). However, this approach is not effective in the recently identified form carrying mutations in the receptor activator of NF-κB ligand (RANKL) gene. In this subset, therapy replacement approach based on RANKL delivery has a strong rationale. Here we demonstrate that the systematic administration of RANKL for 1 month to Rankl(-/-) mice, which closely resemble the human disease, significantly improves the bone phenotype and has beneficial effects on bone marrow, spleen and thymus; major adverse effects arise only when mice are clearly overtreated. Overall, we provide evidence that the pharmacological administration of RANKL represents the appropriate treatment option for RANKL-deficient ARO patients, to be validated in a pilot clinical trial.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jbmr.1712DOI Listing
December 2012

Anti-CD3ε mAb improves thymic architecture and prevents autoimmune manifestations in a mouse model of Omenn syndrome: therapeutic implications.

Blood 2012 Aug 21;120(5):1005-14. Epub 2012 Jun 21.

Milan Unit, Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale delle Ricerche, Milan, Italy.

Omenn syndrome (OS) is an atypical primary immunodeficiency characterized by severe autoimmunity because of activated T cells infiltrating target organs. The impaired recombinase activity in OS severely affects expression of the pre-T-cell receptor complex in immature thymocytes, which is crucial for an efficient development of the thymic epithelial component. Anti-CD3ε monoclonal antibody (mAb) treatment in RAG2(-/-) mice was previously shown to mimic pre-TCR signaling promoting thymic expansion. Here we show the effect of anti-CD3ε mAb administration in the RAG2(R229Q) mouse model, which closely recapitulates human OS. These animals, in spite of the inability to induce the autoimmune regulator, displayed a significant amelioration in thymic epithelial compartment and an important reduction of peripheral T-cell activation and tissue infiltration. Furthermore, by injecting a high number of RAG2(R229Q) progenitors into RAG2(-/-) animals previously conditioned with anti-CD3ε mAb, we detected autoimmune regulator expression together with the absence of peripheral immunopathology. These observations indicate that improving epithelial thymic function might ameliorate the detrimental behavior of the cell-autonomous RAG defect. Our data provide important therapeutic proof of concept for future clinical applications of anti-CD3ε mAb treatment in severe combined immunodeficiency forms characterized by poor thymus function and autoimmunity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2012-01-406827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3470012PMC
August 2012

Alterations in the adenosine metabolism and CD39/CD73 adenosinergic machinery cause loss of Treg cell function and autoimmunity in ADA-deficient SCID.

Blood 2012 Feb 19;119(6):1428-39. Epub 2011 Dec 19.

San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Via Olgettina 58, Milan, Italy

Adenosine acts as anti-inflammatory mediator on the immune system and has been described in regulatory T cell (Treg)-mediated suppression. In the absence of adenosine deaminase (ADA), adenosine and other purine metabolites accumulate, leading to severe immunodeficiency with recurrent infections (ADA-SCID). Particularly ADA-deficient patients with late-onset forms and after enzyme replacement therapy (PEG-ADA) are known to manifest immune dysregulation. Herein we provide evidence that alterations in the purine metabolism interfere with Treg function, thereby contributing to autoimmune manifestations in ADA deficiency. Tregs isolated from PEG-ADA-treated patients are reduced in number and show decreased suppressive activity, whereas they are corrected after gene therapy. Untreated murine ADA(-/-) Tregs show alterations in the plasma membrane CD39/CD73 ectonucleotidase machinery and limited suppressive activity via extracellular adenosine. PEG-ADA-treated mice developed multiple autoantibodies and hypothyroidism in contrast to mice treated with bone marrow transplantation or gene therapy. Tregs isolated from PEG-ADA-treated mice lacked suppressive activity, suggesting that this treatment interferes with Treg functionality. The alterations in the CD39/CD73 adenosinergic machinery and loss of function in ADA-deficient Tregs provide new insights into a predisposition to autoimmunity and the underlying mechanisms causing defective peripheral tolerance in ADA-SCID.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2011-07-366781DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3426348PMC
February 2012

The matricellular protein SPARC supports follicular dendritic cell networking toward Th17 responses.

J Autoimmun 2011 Dec 29;37(4):300-10. Epub 2011 Sep 29.

Molecular Immunology Unit, Department of Experimental Oncology and Molecular Medicine, AmadeoLab, Fondazione IRCCS Istituto Nazionale Tumori, via Amadeo 42, 20133 Milan, Italy.

Lymphnode swelling during immune responses is a transient, finely regulated tissue rearrangement, accomplished with the participation of the extracellular matrix. Here we show that murine and human reactive lymph nodes express SPARC in the germinal centres. Defective follicular dendritic cell networking in SPARC-deficient mice is accompanied by a severe delay in the arrangement of germinal centres and development of humoral autoimmunity, events that are linked to Th17 development. SPARC is required for the optimal and rapid differentiation of Th17 cells, accordingly we show delayed development of experimental autoimmune encephalomyelitis whose pathogenesis involves Th17. Not only host radioresistant cells, namely follicular dendritic cells, but also CD4(+) cells are the relevant sources of SPARC, in vivo. Th17 differentiation and germinal centre formation mutually depend on SPARC for a proper functional crosstalk. Indeed, Th17 cells can enter the germinal centres in SPARC-competent, but not SPARC-deficient, mice. In summary, SPARC optimizes the changes occurring in lymphoid extracellular matrix harboring complex interactions between follicular dendritic cells, B cells and Th17 cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jaut.2011.09.002DOI Listing
December 2011

Exacerbated experimental autoimmune encephalomyelitis in mast-cell-deficient Kit W-sh/W-sh mice.

Lab Invest 2011 Apr 14;91(4):627-41. Epub 2011 Feb 14.

Molecular Immunology Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy.

Mast cell (MC)-deficient c-Kit mutant Kit(W/W-v) mice are protected against experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, suggesting a detrimental role for MCs in this disease. To further investigate the role of MCs in EAE, we took advantage of a recently characterized model of MC deficiency, Kit(W-sh/W-sh). Surprisingly, we observed that myelin oligodendrocyte glycoprotein (MOG)(35-55)-induced chronic EAE was exacerbated in Kit(W-sh/W-sh) compared with Kit(+/+) mice. Kit(W-sh/W-sh) mice showed more inflammatory foci in the central nervous system (CNS) and increased T-cell response against myelin. To understand whether the discrepant results obtained in Kit(W-sh/W-sh) and in Kit(W/W-v) mice were because of the different immunization protocols, we induced EAE in these two strains with varying doses of MOG(35-55) and adjuvants. Although Kit(W-sh/W-sh) mice exhibited exacerbated EAE under all immunization protocols, Kit(W/W-v) mice were protected from EAE only when immunized with high, but not low, doses of antigen and adjuvants. Kit(W-sh/W-sh) mice reconstituted systemically, but not in the CNS, with bone marrow-derived MCs still developed exacerbated EAE, indicating that protection from disease could be exerted by MCs mainly in the CNS, and/or by other cells possibly dysregulated in Kit(W-sh/W-sh) mice. In summary, these data suggest to reconsider MC contribution to EAE, taking into account the variables of using different experimental models and immunization protocols.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/labinvest.2011.3DOI Listing
April 2011

Epidermal growth factor receptor expression identifies functionally and molecularly distinct tumor-initiating cells in human glioblastoma multiforme and is required for gliomagenesis.

Cancer Res 2010 Oct 21;70(19):7500-13. Epub 2010 Sep 21.

Neural Stem Cell Biology Unit, Division of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Scientific Institute, Milan, Italy.

Epidermal growth factor receptor (EGFR) is a known diagnostic and, although controversial, prognostic marker of human glioblastoma multiforme (GBM). However, its functional role and biological significance in GBM remain elusive. Here, we show that multiple GBM cell subpopulations could be purified from the specimens of patients with GBM and from cancer stem cell (CSC) lines based on the expression of EGFR and of other putative CSC markers. All these subpopulations are molecularly and functionally distinct, are tumorigenic, and need to express EGFR to promote experimental tumorigenesis. Among them, EGFR-expressing tumor-initiating cells (TIC) display the most malignant functional and molecular phenotype. Accordingly, modulation of EGFR expression by gain-of-function and loss-of-function strategies in GBM CSC lines enhances and reduces their tumorigenic ability, respectively, suggesting that EGFR plays a fundamental role in gliomagenesis. These findings open up the possibility of new therapeutically relevant scenarios, as the presence of functionally heterogeneous EGFR(pos) and EGFR(neg) TIC subpopulations within the same tumor might affect clinical response to treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-10-2353DOI Listing
October 2010

Hypomorphic Rag mutations can cause destructive midline granulomatous disease.

Blood 2010 Aug 20;116(8):1263-71. Epub 2010 May 20.

Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

Destructive midline granulomatous disease characterized by necrotizing granulomas of the head and neck is most commonly caused by Wegener granulomatosis, natural killer/T-cell lymphomas, cocaine abuse, or infections. An adolescent patient with myasthenia gravis treated with thymectomy subsequently developed extensive granulomatous destruction of midface structures, palate, nasal septum, airways, and epiglottis. His lymphocyte numbers, total immunoglobulin G level, and T-cell receptor (TCR) repertoire appeared normal. Sequencing of Recombination activating gene-1 (Rag1) showed compound heterozygous Rag1 mutations; a novel deletion with no recombinase activity and a missense mutation resulting in 50% Rag activity. His thymus was dysplastic and, although not depleted of T cells, showed a notable absence of autoimmune regulator (AIRE) and Foxp3(+) regulatory T cells. This distinct Rag-deficient phenotype characterized by immune dysregulation with granulomatous hyperinflammation and autoimmunity, with relatively normal T and B lymphocyte numbers and a diverse TCR repertoire expands the spectrum of presentation in Rag deficiency. This study was registered at www.clinicaltrials.gov as #NCT00128973.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2010-02-267583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2938237PMC
August 2010

Brain lipid composition in grey-lethal mutant mouse characterized by severe malignant osteopetrosis.

Glycoconj J 2009 Aug 10;26(6):623-33. Epub 2008 Sep 10.

Center of Excellence on Neurodegenerative Diseases, Department of Medical Chemistry, Biochemistry and Biotechnology, University of Milan, Milan, Italy.

The grey-lethal mouse (gl/gl) mutant most closely resembles the severe human malignant autosomal recessive OSTM1-dependent form of osteopetrosis that it has been described to be associated with neurological abnormalities. For this reason, we have analyzed the brain lipid composition (gangliosides, neutral glycosphingolipids, phospholipids and cholesterol), from gl/gl mice at different ages of development and compared with wild type mice. Both cholesterol and glycerophospholipid content and pattern in the gl/gl and control mice were very similar. In contrast, significant differences were observed in the content of several sphingolipids. Higher amount of the monosialogangliosides GM2 and GM3, and lower content of sphingomyelin, sulfatide and galactosylceramide were observed in the gl/gl brain with respect to controls. The low content of sphingomyelin, sulfatide and galactosylceramide is consistent with the immunohistochemical results showing that in the grey-lethal brain significant depletion and disorganization of the myelinated fibres is present, thus supporting the hypothesis that loss of function of the OSTM1 causes neuronal impairment and myelin deficit.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10719-008-9179-8DOI Listing
August 2009

Expression studies in gliomas and glial cells do not support a tumor suppressor role for LGI1.

Neuro Oncol 2006 Apr 2;8(2):96-108. Epub 2006 Mar 2.

Department of Experimental Research and Diagnostics, Istituto Nazionale Neurologico C. Besta, Milan 20133, Italy.

Disruptions of LGI1 in glioblastoma (GBM) cell lines and LGI1 mutations in families with autosomal dominant epilepsy imply a role for LGI1 in glial cells as well as in neurons. Although we and others could not find LGI1 mutations in malignant gliomas, our initial studies appeared to support the idea that LGI1 is poorly expressed or absent in these tumors. Microarray data suggested that LGI1 could be involved in the control of matrix metalloproteinases, and we found that tumors derived from U87 glioblastoma cells overexpressing LGI1 were less aggressive than U87 control tumors. To our surprise, we observed that LGI1 expression after differentiation of murine neural stem cells was robust in neurons but negligible in glial cells, in agreement with immunohistochemistry studies on rodent brain. This observation could suggest that the variable levels of LGI1 expression in gliomas reflect the presence of neurons entrapped within the tumor. To test this hypothesis, we investigated LGI1 expression in parallel with expression of the neuronal marker NEF3 by real-time PCR on 30 malignant gliomas. Results showed a strong, positive correlation between the expression levels of these two genes (P < 0.0001). Thus, our data confirm that LGI1 is involved in cell-matrix interactions but suggest that its expression is not relevant in glial cells, implying that its role as a tumor suppressor in gliomas should be reconsidered.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1215/15228517-2005-006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1871933PMC
April 2006

A key regulatory role for histamine in experimental autoimmune encephalomyelitis: disease exacerbation in histidine decarboxylase-deficient mice.

J Immunol 2006 Jan;176(1):17-26

Immunology and Muscular Pathology Unit, National Neurological Institute "C. Besta," Milan, Italy.

Histamine can modulate the cytokine network and influence Th1 and Th2 balance and Ab-isotype switching. Thus, pharmacological blockade or genetic deletion of specific histamine receptors has been shown to reduce the severity of experimental autoimmune encephalomyelitis (EAE), a prototypic Th1-mediated disease with similarities to human multiple sclerosis. To study the comprehensive contribution of endogenous histamine to the expression of EAE, we attempted to induce EAE in histidine decarboxylase-deficient mice, which are genetically unable to make histamine. In this study, we show that EAE is significantly more severe in HDC-/-, histamine-deficient mice, with diffuse inflammatory infiltrates, including a prevalent granulocytic component, in the brain and cerebellum. Unlike splenocytes from wild-type mice, splenocytes from HDC-/- mice do not produce histamine in response to the myelin Ag, whereas production of IFN-gamma, TNF, and leptin are increased in HDC-/- splenocytes in comparison to those from wild-type mice. Endogenous histamine thus appears to regulate importantly the autoimmune response against myelin and the expression of EAE, in this model, and to limit immune damage to the CNS. Understanding which receptor(s) for histamine is/are involved in regulating autoimmunity against the CNS might help in the development of new strategies of treatment for EAE and multiple sclerosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.176.1.17DOI Listing
January 2006
-->