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1.
J Allergy Clin Immunol ; 145(1): 368-378.e13, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31194989

RESUMO

BACKGROUND: Cryopyrin-associated periodic syndromes (CAPS) are a group of autoinflammatory diseases linked to gain-of-function mutations in the NOD-like receptor family, pyrin domain containing 3 (NLRP3) gene, which cause uncontrolled IL-1ß secretion. Proton pump inhibitors (PPIs), which are commonly used as inhibitors of gastric acid production, also have anti-inflammatory properties, protect mice from sepsis, and prevent IL-1ß secretion by monocytes from patients with CAPS. OBJECTIVE: We sought to develop a novel Nlrp3 knock-in (KI) mouse model of CAPS to study amyloidosis, a severe CAPS complication, and test novel therapeutic approaches. METHODS: We generated KI mice by engineering the N475K mutation, which is associated with the CAPS phenotype, into the mouse Nlrp3 gene. KI and wild-type mice received PPIs or PBS intraperitoneally and were analyzed for survival, inflammation, cytokine secretion, and amyloidosis development. RESULTS: Mutant Nlrp3 KI mice displayed features that recapitulate the immunologic and clinical phenotype of CAPS. They showed systemic inflammation with high levels of serum proinflammatory cytokines, inflammatory infiltrates in various organs, and amyloid deposits in the spleen, liver, and kidneys. Toll-like receptor stimulated macrophages from KI mice secreted high levels of IL-1ß, IL-18, and IL-1α but low amounts of IL-1 receptor antagonist. Treatment of KI mice with PPIs had a clear clinical effect, showing a reduction in inflammatory manifestations, regression of amyloid deposits, and normalization of proinflammatory and anti-inflammatory cytokine production by macrophages. CONCLUSION: Nlrp3 KI mice displayed a CAPS phenotype with many characteristics of autoinflammation, including amyloidosis. The therapeutic effectiveness of PPIs associated with a lack of toxicity indicates that these drugs could represent relevant adjuvants to the anti-IL-1 drugs in patients with CAPS and other IL-1-driven diseases.

3.
Stem Cells Transl Med ; 8(1): 22-34, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30184340

RESUMO

Biomimetic scaffolds are extremely versatile in terms of chemical composition and physical properties, which can be defined to accomplish specific applications. One property that can be added is the production/release of bioactive soluble factors, either directly from the biomaterial, or from cells embedded within the biomaterial. We reasoned that pursuing this strategy would be appropriate to setup a cell-based therapy for RANKL-deficient autosomal recessive osteopetrosis, a very rare skeletal genetic disease in which lack of the essential osteoclastogenic factor RANKL impedes osteoclast formation. The exogenously administered RANKL cytokine is effective in achieving osteoclast formation and function in vitro and in vivo, thus, we produced murine Rankl-/- mesenchymal stromal cells (MSCs) overexpressing human soluble RANKL (hsRL) following lentiviral transduction (LVhsRL). Here, we described a three-dimensional (3D) culture system based on a magnesium-doped hydroxyapatite/collagen I (MgHA/Col) biocompatible scaffold closely reproducing bone physicochemical properties. MgHA/Col-seeded murine MSCs showed improved properties, as compared to two-dimensional (2D) culture, in terms of proliferation and hsRL production, with respect to LVhsRL-transduced cells. When implanted subcutaneously in Rankl-/- mice, these cell constructs were well tolerated, colonized by host cells, and intensely vascularized. Of note, in the bone of Rankl-/- mice that carried scaffolds with either WT or LVhsRL-transduced Rankl-/- MSCs, we specifically observed formation of TRAP+ cells, likely due to sRL released from the scaffolds into circulation. Thus, our strategy proved to have the potential to elicit an effect on the bone; further work is required to maximize these benefits and achieve improvements of the skeletal pathology in the treated Rankl-/- mice. Stem Cells Translational Medicine 2019;8:22-34.

4.
Front Immunol ; 8: 490, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28512459

RESUMO

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 Was-/- 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 Was-/- mice, we performed in vivo 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 Was-/- 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.

5.
Ann Rheum Dis ; 76(10): 1648-1656, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28522451

RESUMO

OBJECTIVES: To analyse the prevalence of CECR1 mutations in patients diagnosed with early onset livedo reticularis and/or haemorrhagic/ischaemic strokes in the context of inflammation or polyarteritis nodosa (PAN). Forty-eight patients from 43 families were included in the study. METHODS: Direct sequencing of CECR1 was performed by Sanger analysis. Adenosine deaminase 2 (ADA2) enzymatic activity was analysed in monocyte isolated from patients and healthy controls incubated with adenosine and with or without an ADA1 inhibitor. RESULTS: Biallelic homozygous or compound heterozygous CECR1 mutations were detected in 15/48 patients. A heterozygous disease-associated mutation (p.G47V) was observed in two affected brothers. The mean age of onset of the genetically positive patients was 24 months (6 months to 7 years). Ten patients displayed one or more cerebral strokes during their disease course. Low immunoglobulin levels were detected in six patients. Thalidomide and anti-TNF (tumour necrosis factor) blockers were the most effective drugs. Patients without CECR1 mutations had a later age at disease onset, a lower prevalence of neurological and skin manifestations; one of these patients displayed all the clinical features of adenosine deaminase 2deficiency (DADA2) and a defective enzymatic activity suggesting the presence of a missed mutation or a synthesis defect. CONCLUSIONS: DADA2 accounts for paediatric patients diagnosed with PAN-like disease and strokes and might explain an unrecognised condition in patients followed by adult rheumatologist. Timely diagnosis and treatment with anti-TNF agents are crucial for the prevention of severe complications of the disease. Functional assay to measure ADA2 activity should complement genetic testing in patients with non-confirming genotypes.


Assuntos
Adenosina Desaminase/deficiência , Adenosina Desaminase/genética , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Peptídeos e Proteínas de Sinalização Intercelular/genética , Livedo Reticular/genética , Poliarterite Nodosa/genética , Acidente Vascular Cerebral/genética , Adolescente , Idade de Início , Estudos de Casos e Controles , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Heterozigoto , Homozigoto , Humanos , Imunoglobulinas/sangue , Imunossupressores/uso terapêutico , Lactente , Itália , Livedo Reticular/tratamento farmacológico , Livedo Reticular/enzimologia , Masculino , Linhagem , Poliarterite Nodosa/tratamento farmacológico , Poliarterite Nodosa/enzimologia , Acidente Vascular Cerebral/enzimologia , Talidomida/uso terapêutico , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adulto Jovem
6.
Stem Cells ; 35(5): 1365-1377, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28100034

RESUMO

Autosomal recessive osteopetrosis (ARO) is a severe bone disease characterized by increased bone density due to impairment in osteoclast resorptive function or differentiation. Hematopoietic stem cell transplantation is the only available treatment; however, this therapy is not effective in RANKL-dependent ARO, since in bone this gene is mainly expressed by cells of mesenchymal origin. Of note, whether lack of RANKL production might cause a defect also in the bone marrow (BM) stromal compartment, possibly contributing to the pathology, is unknown. To verify this possibility, we generated and characterized BM mesenchymal stromal cell (BM-MSC) lines from wild type and Rankl-/- mice, and found that Rankl-/- BM-MSCs displayed reduced clonogenicity and osteogenic capacity. The differentiation defect was significantly improved by lentiviral transduction of Rankl-/- BM-MSCs with a vector stably expressing human soluble RANKL (hsRANKL). Expression of Rankl receptor, Rank, on the cytoplasmic membrane of BM-MSCs pointed to the existence of an autocrine loop possibly activated by the secreted cytokine. Based on the close resemblance of RANKL-defective osteopetrosis in humans and mice, we expect that our results are also relevant for RANKL-dependent ARO patients. Data obtained in vitro after transduction with a lentiviral vector expressing hsRANKL would suggest that restoration of RANKL production might not only rescue the defective osteoclastogenesis of this ARO form, but also improve a less obvious defect in the osteoblast lineage, thus possibly achieving higher benefit for the patients, when the approach is translated to clinics. Stem Cells 2017;35:1365-1377.


Assuntos
Diferenciação Celular , Vetores Genéticos/metabolismo , Lentivirus/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteogênese , Ligante RANK/deficiência , Animais , Biomarcadores/metabolismo , Células Clonais , Imunofenotipagem , Camundongos Endogâmicos C57BL , Ligante RANK/metabolismo , Transdução de Sinais , Transdução Genética
7.
Pediatr Rheumatol Online J ; 14(1): 51, 2016 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-27609179

RESUMO

The deficiency of Adenosine Deaminase 2 (DADA2) is a new autoinflammatory disease characterised by an early onset vasculopathy with livedoid skin rash associated with systemic manifestations, CNS involvement and mild immunodeficiency.This condition is secondary to autosomal recessive mutations of CECR1 (Cat Eye Syndrome Chromosome Region 1) gene, mapped to chromosome 22q11.1, that encodes for the enzymatic protein adenosine deaminase 2 (ADA2). By now 19 different mutations in CECR1 gene have been detected.The pathogenetic mechanism of DADA2 is still unclear. ADA2 in a secreted protein mainly expressed by cells of the myeloid lineage; its enzymatic activity is higher in conditions of hypoxia, inflammation and oncogenesis. Moreover ADA2 is able to induce macrophages proliferation and differentiation; it's deficiency is in fact associated with a reduction of anti-inflammatory macrophages (M2). The deficiency of ADA2 is also associated with an up-regulation of neutrophils-expressed genes and an increased secretion of pro-inflammatory cytokines. The mild immunodeficiency detected in many DADA2 patients suggests a role of this protein in the adaptive immune response; an increased mortality of B cells and a reduction in the number of memory B cells, terminally differentiated B cells and plasmacells has been described in many patients. The lack of the protein is associated with endothelium damage; however the function of this protein in the endothelial homeostasis is still unknown.From the clinical point of view, this disease is characterized by a wide spectrum of severity. Chronic or recurrent systemic inflammation with fever, elevation of acute phase reactants and skin manifestations (mainly represented by livedo reticularis) is the typical clinical picture. While in some patients the disease is mild and skin-limited, others present a severe, even lethal, disease with multi-organ involvement; the CNS involvement is rather common with ischemic or hemorrhagic strokes. In many patients not only the clinical picture but also the histopathologic features are undistinguishable from those of systemic polyarteritis nodosa (PAN). Of note, patients with an unusual phenotype, mainly dominated by clinical manifestations suggestive for an immune-disrective condition, have been described.Due to its rarity, the response to treatment of DADA2 is still anecdotal. While steroids can control the disease's manifestations at high dosage, none of the common immunosuppressive drugs turned out to be effective. Biologic drugs have been used only in few patients, without a clear effectiveness; anti-TNF drugs are those associated to a better clinical response. Hematopoietic stem cells transplantation was effective in patients with a severe phenotype.


Assuntos
Adenosina Desaminase/genética , Síndromes de Imunodeficiência , Peptídeos e Proteínas de Sinalização Intercelular/genética , Adenosina Desaminase/deficiência , Diagnóstico Diferencial , Gerenciamento Clínico , Humanos , Síndromes de Imunodeficiência/etiologia , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/fisiopatologia , Síndromes de Imunodeficiência/terapia , Mutação , Dermatopatias/diagnóstico , Dermatopatias/etiologia , Doenças Vasculares/diagnóstico , Doenças Vasculares/etiologia
8.
J Allergy Clin Immunol ; 136(5): 1337-45, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26025129

RESUMO

BACKGROUND: Hyperzincemia and hypercalprotectinemia (Hz/Hc) is a distinct autoinflammatory entity involving extremely high serum concentrations of the proinflammatory alarmin myeloid-related protein (MRP) 8/14 (S100A8/S100A9 and calprotectin). OBJECTIVE: We sought to characterize the genetic cause and clinical spectrum of Hz/Hc. METHODS: Proline-serine-threonine phosphatase-interacting protein 1 (PSTPIP1) gene sequencing was performed in 14 patients with Hz/Hc, and their clinical phenotype was compared with that of 11 patients with pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome. PSTPIP1-pyrin interactions were analyzed by means of immunoprecipitation and Western blotting. A structural model of the PSTPIP1 dimer was generated. Cytokine profiles were analyzed by using the multiplex immunoassay, and MRP8/14 serum concentrations were analyzed by using an ELISA. RESULTS: Thirteen patients were heterozygous for a missense mutation in the PSTPIP1 gene, resulting in a p.E250K mutation, and 1 carried a mutation resulting in p.E257K. Both mutations substantially alter the electrostatic potential of the PSTPIP1 dimer model in a region critical for protein-protein interaction. Patients with Hz/Hc have extremely high MRP8/14 concentrations (2045 ± 1300 µg/mL) compared with those with PAPA syndrome (116 ± 74 µg/mL) and have a distinct clinical phenotype. A specific cytokine profile is associated with Hz/Hc. Hz/Hc mutations altered protein binding of PSTPIP1, increasing interaction with pyrin through phosphorylation of PSTPIP1. CONCLUSION: Mutations resulting in charge reversal in the y-domain of PSTPIP1 (E→K) and increased interaction with pyrin cause a distinct autoinflammatory disorder defined by clinical and biochemical features not found in patients with PAPA syndrome, indicating a unique genotype-phenotype correlation for mutations in the PSTPIP1 gene. This is the first inborn autoinflammatory syndrome in which inflammation is driven by uncontrolled release of members of the alarmin family.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Complexo Antígeno L1 Leucocitário/metabolismo , Erros Inatos do Metabolismo dos Metais/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Adolescente , Alarminas/genética , Alarminas/metabolismo , Calgranulina A/genética , Calgranulina A/metabolismo , Criança , Citocinas/metabolismo , Proteínas do Citoesqueleto/genética , Feminino , Genótipo , Humanos , Complexo Antígeno L1 Leucocitário/genética , Masculino , Erros Inatos do Metabolismo dos Metais/genética , Mutação de Sentido Incorreto/genética , Fenótipo , Fosforilação , Ligação Proteica/genética , Mapas de Interação de Proteínas/genética , Multimerização Proteica , Pirina , Adulto Jovem
9.
J Immunol ; 194(9): 4144-53, 2015 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-25825446

RESUMO

The immune and the skeletal system are tightly interconnected, and B lymphocytes are uniquely endowed with osteo-interactive properties. In this context, receptor activator of NF-κB (RANK) ligand (RANKL) plays a pivotal role in lymphoid tissue formation and bone homeostasis. Although murine models lacking RANK or RANKL show defects in B cell number, the role of the RANKL-RANK axis on B physiology is still a matter of debate. In this study, we have characterized in detail B cell compartment in Rankl(-/-) mice, finding a relative expansion of marginal zone B cells, B1 cells, and plasma cells associated with increased Ig serum levels, spontaneous germinal center formation, and hyperresponse to CD40 triggering. Such abnormalities were associated with an increased frequency of regulatory B cells and augmented B cell-derived IL-10 production. Remarkably, in vivo IL-10-R blockade reduced T cell-triggered plasma cell differentiation and restrained the expansion of regulatory B cells. These data point to a novel role of the RANKL-RANK axis in the regulation of B cell homeostasis and highlight an unexpected link between IL-10 CD40 signaling and the RANKL pathway.


Assuntos
Linfócitos B/imunologia , Interleucina-10/imunologia , Ligante RANK/deficiência , Ligante RANK/imunologia , Animais , Camundongos , Camundongos Knockout
10.
Cell Rep ; 3(6): 1824-31, 2013 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-23770243

RESUMO

Immunoglobulin (Ig) isotype diversification by class switch recombination (CSR) is an essential process for mounting a protective humoral immune response. Ig CSR deficiencies in humans can result from an intrinsic B cell defect; however, most of these deficiencies are still molecularly undefined and diagnosed as common variable immunodeficiency (CVID). Here, we show that extracellular adenosine critically contributes to CSR in human naive and IgM memory B cells. In these cells, coordinate stimulation of B cell receptor and toll-like receptors results in the release of ATP stored in Ca(2+)-sensitive secretory vesicles. Plasma membrane ectonucleoside triphosphate diphosphohydrolase 1 CD39 and ecto-5'-nucleotidase CD73 hydrolyze ATP to adenosine, which induces CSR in B cells in an autonomous fashion. Notably, CVID patients with impaired class-switched antibody responses are selectively deficient in CD73 expression in B cells, suggesting that CD73-dependent adenosine generation contributes to the pathogenesis of this disease.


Assuntos
5'-Nucleotidase/imunologia , Trifosfato de Adenosina/imunologia , Formação de Anticorpos/imunologia , Linfócitos B/imunologia , Switching de Imunoglobulina/imunologia , 5'-Nucleotidase/genética , 5'-Nucleotidase/metabolismo , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Animais , Formação de Anticorpos/genética , Antígenos CD/imunologia , Antígenos CD/metabolismo , Apirase/imunologia , Apirase/metabolismo , Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/imunologia , Linfócitos B/citologia , Linfócitos B/metabolismo , Imunodeficiência de Variável Comum/genética , Imunodeficiência de Variável Comum/imunologia , Imunodeficiência de Variável Comum/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Recombinação Genética
11.
J Bone Miner Res ; 27(12): 2501-10, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22836362

RESUMO

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.


Assuntos
Osteopetrose/tratamento farmacológico , Osteopetrose/genética , Ligante RANK/uso terapêutico , Animais , Células da Medula Óssea/efeitos dos fármacos , Reabsorção Óssea/induzido quimicamente , Osso e Ossos/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Osteopetrose/patologia , Fenótipo , Ligante RANK/administração & dosagem , Ligante RANK/efeitos adversos , Ligante RANK/genética , Receptor Ativador de Fator Nuclear kappa-B/deficiência , Receptor Ativador de Fator Nuclear kappa-B/genética
12.
Blood ; 120(5): 1005-14, 2012 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-22723555

RESUMO

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.


Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Doenças Autoimunes/prevenção & controle , Complexo CD3/imunologia , Imunodeficiência Combinada Severa/terapia , Timo/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Autoimunidade/efeitos dos fármacos , Autoimunidade/genética , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tamanho do Órgão/efeitos dos fármacos , Imunodeficiência Combinada Severa/imunologia , Imunodeficiência Combinada Severa/patologia , Timo/imunologia , Timo/patologia , Timo/ultraestrutura
13.
J Allergy Clin Immunol ; 127(6): 1376-84.e5, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21531013

RESUMO

BACKGROUND: Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency characterized by thrombocytopenia, eczema, infections, autoimmunity, and lymphomas. Transplantation of hematopoietic stem cells from HLA-identical donors is curative, but it is not available to all patients. We have developed a gene therapy (GT) approach for WAS by using a lentiviral vector encoding for human WAS promoter/cDNA (w1.6W) and demonstrated its preclinical efficacy and safety. OBJECTIVE: To evaluate B-cell reconstitution and correction of B-cell phenotype in GT-treated mice. METHODS: We transplanted Was(-/-) mice sublethally irradiated (700 rads) with lineage marker-depleted bone marrow wild-type cells, Was(-/-) cells untransduced or transduced with the w1.6W lentiviral vector and analyzed B-cell reconstitution in bone marrow, spleen, and peritoneum. RESULTS: Here we show that WAS protein(+) B cells were present in central and peripheral B-cell compartments from GT-treated mice and displayed the strongest selective advantage in the splenic marginal zone and peritoneal B1 cell subsets. After GT, splenic architecture was improved and B-cell functions were restored, as demonstrated by the improved antibody response to pneumococcal antigens and the reduction of serum IgG autoantibodies. CONCLUSION: WAS GT leads to improvement of B-cell functions, even in the presence of a mixed chimerism, further validating the clinical application of the w1.6W lentiviral vector.


Assuntos
Linfócitos B/imunologia , Terapia Genética/métodos , Síndrome de Wiskott-Aldrich/imunologia , Síndrome de Wiskott-Aldrich/terapia , Animais , Antígenos T-Independentes/administração & dosagem , Autoanticorpos/sangue , Linfócitos B/metabolismo , Transplante de Medula Óssea , Modelos Animais de Doenças , Expressão Gênica , Vetores Genéticos , Humanos , Lentivirus/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo
14.
J Exp Med ; 207(7): 1525-40, 2010 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-20547828

RESUMO

Hypomorphic RAG mutations, leading to limited V(D)J rearrangements, cause Omenn syndrome (OS), a peculiar severe combined immunodeficiency associated with autoimmune-like manifestations. Whether B cells play a role in OS pathogenesis is so far unexplored. Here we report the detection of plasma cells in lymphoid organs of OS patients, in which circulating B cells are undetectable. Hypomorphic Rag2(R229Q) knock-in mice, which recapitulate OS, revealed, beyond severe B cell developmental arrest, a normal or even enlarged compartment of immunoglobulin-secreting cells (ISC). The size of this ISC compartment correlated with increased expression of Blimp1 and Xbp1, and these ISC were sustained by elevated levels of T cell derived homeostatic and effector cytokines. The detection of high affinity pathogenic autoantibodies toward target organs indicated defaults in B cell selection and tolerance induction. We hypothesize that impaired B cell receptor (BCR) editing and a serum B cell activating factor (BAFF) abundance might contribute toward the development of a pathogenic B cell repertoire in hypomorphic Rag2(R229Q) knock-in mice. BAFF-R blockade reduced serum levels of nucleic acid-specific autoantibodies and significantly ameliorated inflammatory tissue damage. These findings highlight a role for B cells in OS pathogenesis.


Assuntos
Células Produtoras de Anticorpos/imunologia , Células Produtoras de Anticorpos/patologia , Proteínas de Ligação a DNA/metabolismo , Homeostase/imunologia , Imunodeficiência Combinada Severa/imunologia , Imunodeficiência Combinada Severa/patologia , Substituição de Aminoácidos/genética , Animais , Formação de Anticorpos/imunologia , Antígenos/imunologia , Fator Ativador de Células B/metabolismo , Receptor do Fator Ativador de Células B/metabolismo , Células da Medula Óssea/imunologia , Células da Medula Óssea/patologia , Compartimento Celular , Proliferação de Células , Modelos Animais de Doenças , Epitopos/imunologia , Humanos , Memória Imunológica/imunologia , Sistema Linfático/imunologia , Sistema Linfático/patologia , Ativação Linfocitária/imunologia , Camundongos , Plasmócitos/imunologia , Plasmócitos/patologia , Transdução de Sinais/imunologia , Baço/imunologia , Baço/patologia , Linfócitos T/citologia , Linfócitos T/imunologia , Receptores Toll-Like/agonistas
15.
Arthritis Rheum ; 62(9): 2776-86, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20496367

RESUMO

OBJECTIVE: Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent cells characterized by immunomodulatory properties and are therefore considered a promising tool for the treatment of immune-mediated diseases. This study was undertaken to assess the influence of murine BM-MSCs on the activation of B cells in (NZB × NZW)F(1) mice as an animal model of systemic lupus erythematosus (SLE). METHODS: We evaluated the in vitro effects of BM-MSCs on the proliferation and differentiation to plasma cells of splenic mature B cell subsets, namely follicular and marginal zone B cells isolated from (NZB × NZW)F(1) mice. Lupus mice were also treated with BM-MSCs, and serum autoantibodies, proteinuria, histologic changes in the kidney, and survival rates were monitored. RESULTS: BM-MSCs inhibited antigen-dependent proliferation and differentiation to plasma cells of follicular and marginal zone B cells in vitro. This inhibitory effect was dependent on interferon-γ (IFNγ) and was mediated by cell-to-cell contact, involving the programmed death 1 (PD-1)/PD ligand pathway. In vivo treatment with BM-MSCs did not affect the levels of anti-double-stranded DNA antibodies or proteinuria. However, a reduction in glomerular immune complex deposition, lymphocytic infiltration, and glomerular proliferation was observed. CONCLUSION: Our findings indicate that BM-MSCs affect B cell receptor-dependent activation of both follicular and marginal zone B cells from lupus mice. This inhibitory effect is IFNγ-dependent and cell contact-dependent. MSCs in vivo do not affect the production of autoantibodies, the level of proteinuria, or the mortality rates. Nonetheless, the significant improvement in histologic findings in the kidney supports the potential role of MSCs in the prevention of glomerular damage.


Assuntos
Subpopulações de Linfócitos B/patologia , Interferon gama/imunologia , Lúpus Eritematoso Sistêmico/patologia , Células-Tronco Mesenquimais/patologia , Animais , Subpopulações de Linfócitos B/imunologia , Células da Medula Óssea/citologia , Comunicação Celular , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Terapia Baseada em Transplante de Células e Tecidos , Células Cultivadas , Modelos Animais de Doenças , Feminino , Interferon gama/farmacologia , Rim/efeitos dos fármacos , Rim/patologia , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/terapia , Ativação Linfocitária/efeitos dos fármacos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Baço/citologia , Baço/imunologia
16.
Stem Cells ; 26(2): 562-9, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18024418

RESUMO

Human bone marrow multipotent mesenchymal stromal cells are progenitor cells that can be expanded in vitro and differentiate into various cells of mesodermal origin. They contribute to the bone marrow reticular niche, where mature B cells and long-lived plasma cells are maintained. Multipotent mesenchymal stromal cells were recently shown to modulate T- and B-cell proliferation and differentiation, dendritic cell maturation, and natural killer activity. These immunoregulatory properties encouraged a possible use of these cells to modulate autoimmune responses in humans. We studied the influence of bone marrow mesenchymal stem cells on highly purified B-cell subsets isolated from healthy donors and total B cells from pediatric systemic lupus erythematosus patients. Bone marrow mesenchymal stem cells promoted proliferation and differentiation into immunoglobulin-secreting cells of transitional and naive B cells stimulated with an agonist of Toll-like receptor 9, in the absence of B cell receptor triggering. They strongly enhanced proliferation and differentiation into plasma cells of memory B-cell populations. A similar effect was observed in response to polyclonal stimulation of B cells isolated from pediatric patients with systemic lupus erythematosus. This study casts important questions on bone marrow mesenchymal stem cells as a therapeutic tool in autoimmune diseases in which B-cell activation is crucially implicated in the pathogenesis of the disease.


Assuntos
Linfócitos B/patologia , Células-Tronco Hematopoéticas/patologia , Lúpus Eritematoso Sistêmico/patologia , Células-Tronco Mesenquimais/patologia , Linfócitos B/citologia , Linfócitos B/imunologia , Estudos de Casos e Controles , Diferenciação Celular , Proliferação de Células , Separação Celular , Criança , Células-Tronco Hematopoéticas/imunologia , Humanos , Imunoglobulinas/biossíntese , Memória Imunológica , Técnicas In Vitro , Lúpus Eritematoso Sistêmico/imunologia , Ativação Linfocitária , Células-Tronco Mesenquimais/imunologia , Plasmócitos/citologia , Plasmócitos/imunologia , Plasmócitos/patologia , Receptor Toll-Like 9/agonistas
17.
Cancer Lett ; 256(1): 56-63, 2007 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-17611020

RESUMO

DDX1, a gene mapping to the 2p24 region, has been observed to be co-amplified with MYCN in neuroblastoma. Co-amplification of the DDX1 gene is a consequence of the short physical distance between the two genes. Recently, it has been found that neuroblastoma cells can show a low increase in MYCN gene copy number, defined as MYCN gain. We studied 13 neuroblastomas with MYCN gain to evaluate the status of the DDX1 gene. We investigated DDX1/MYCN gain by double-colour FISH on interphase nuclei. All cases showed concomitant low extra copy number of DDX1 and MYCN. Heterogeneous distribution of nuclei displaying DDX1/MYCN gain was observed in almost all tumours, suggesting a clonal evolution of cells with DDX1/MYCN gain. This is the first report that shows DDX1 co-gained with MYCN in neuroblastoma and indicates that DDX1 over-representation is closely associated with an increase in MYCN copy number in neuroblastoma cells. Since DDX1 has already been found co-amplified with MYCN, DDX1 gain seems to be a further rearrangement due to the physical proximity of the two genes. Moreover, all patients with DDX1/MYCN gain show a good overall survival but a high frequency of adverse events.


Assuntos
RNA Helicases DEAD-box/genética , Amplificação de Genes , Neuroblastoma/genética , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , RNA Neoplásico/genética , Dosagem de Genes , Humanos , Hibridização in Situ Fluorescente , Proteína Proto-Oncogênica N-Myc , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Células Tumorais Cultivadas
18.
Hum Hered ; 63(3-4): 205-11, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17317969

RESUMO

OBJECTIVES: The rarity of familial neuroblastoma (NB) has allowed only a few linkage studies, most of which did not show any evidence of linkage to regions involved in somatic alterations or to genes implicated in other neurocristopathies seldom associated with NB. We screened a highly informative family with recurrent NB by genome-wide linkage analysis aimed at identifying chromosomal regions for NB predisposing genes. METHODS: A genome-wide screen was performed using 382 microsatellite markers. Multipoint model-based linkage analysis was carried out under a dominant mode of inheritance for the disease using the 'affected only' approach. RESULTS: Our analysis identified two haplotypes co-segregating with the disease on chromosomes 2p and 12p, and yielded maximum lod-score values of 3.01 (p < 0.0001) for markers on both intervals. CONCLUSIONS: Evidence of linkage was reported at 16p in North American families, whereas our studies excluded this interval and indicated other loci for disease predisposition, thus confirming the remarkable genetic heterogeneity of NB. These results suggest an oligogenic inheritance in NB involving more loci in genetic determination of the disease.


Assuntos
Cromossomos Humanos Par 12 , Cromossomos Humanos Par 2 , Ligação Genética , Predisposição Genética para Doença , Neuroblastoma/genética , Heterogeneidade Genética , Testes Genéticos , Genoma Humano , Genótipo , Humanos , Escore Lod , Perda de Heterozigosidade , Repetições de Microssatélites , Linhagem
19.
FEBS Lett ; 523(1-3): 123-7, 2002 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-12123817

RESUMO

The RET gene is expressed with high tissue and stage specificity during development. Understanding its transcriptional regulation might provide new clues to clarify developmental mechanisms. Here we show that the histone deacetylase inhibitor sodium butyrate (NaB) increases RET transcription in cells displaying low levels of its mRNA, while it has no effect in cells expressing at high levels. Chromatin immunoprecipitation (ChIP) experiments showed increased histone acetylation within the 5' flanking [corrected] region, in particular the Sox10-Pax3 enhancer site, due to NaB. Accordingly, ChIP showed different acetylation levels at the Sox10-Pax3 site associated with cell-line specific RET transcription rates. Concluding, chromatin acetylation targeted to functional sequences in the RET regulatory region may control its transcription.


Assuntos
Butiratos/farmacologia , Cromatina/metabolismo , Proteínas de Drosophila , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Fatores de Transcrição , Acetilação , Sítios de Ligação , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Inibidores de Histona Desacetilases , Humanos , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ret , Receptores Proteína Tirosina Quinases/metabolismo , Fatores de Transcrição SOXE , Transcrição Genética/efeitos dos fármacos
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