Assuntos
5'-Nucleotidase , Apirase , Tolerância Imunológica , Terapia de Imunossupressão , Síndrome de Sézary , Neoplasias Cutâneas , Linfócitos T , Humanos , 5'-Nucleotidase/metabolismo , Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Apirase/metabolismo , Síndrome de Sézary/metabolismo , Síndrome de Sézary/terapia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/terapiaRESUMO
Spinocerebellar ataxias (SCAs) are a heterogeneous group of autosomal-dominant neurodegenerative disorders involving the cerebellum and 23 different genes. We mapped SCA38 to a 56 Mb region on chromosome 6p in a SCA-affected Italian family by whole-genome linkage analysis. Targeted resequencing identified a single missense mutation (c.689G>T [p.Gly230Val]) in ELOVL5. Mutation screening of 456 independent SCA-affected individuals identified the same mutation in two further unrelated Italian families. Haplotyping showed that at least two of the three families shared a common ancestor. One further missense variant (c.214C>G [p.Leu72Val]) was found in a French family. Both missense changes affect conserved amino acids, are predicted to be damaging by multiple bioinformatics tools, and were not identified in ethnically matched controls or within variant databases. ELOVL5 encodes an elongase involved in the synthesis of polyunsaturated fatty acids of the ω3 and ω6 series. Arachidonic acid and docosahexaenoic acid, two final products of the enzyme, were reduced in the serum of affected individuals. Immunohistochemistry on control mice and human brain demonstrated high levels in Purkinje cells. In transfection experiments, subcellular localization of altered ELOVL5 showed a perinuclear distribution with a signal increase in the Golgi compartment, whereas the wild-type showed a widespread signal in the endoplasmic reticulum. SCA38 and SCA34 are examples of SCAs due to mutations in elongase-encoding genes, emphasizing the importance of fatty-acid metabolism in neurological diseases.
Assuntos
Acetiltransferases/genética , Metabolismo dos Lipídeos/genética , Mutação/genética , Ataxias Espinocerebelares/genética , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Animais , Ácido Araquidônico/sangue , Cerebelo/patologia , Ácidos Docosa-Hexaenoicos/sangue , Retículo Endoplasmático/metabolismo , Elongases de Ácidos Graxos , Feminino , Ligação Genética , Genótipo , Complexo de Golgi/metabolismo , Haplótipos , Humanos , Itália , Masculino , Camundongos , Pessoa de Meia-Idade , Linhagem , Células de Purkinje/citologiaRESUMO
Systemic sclerosis (SSc) is a chronic autoimmune disease of the connective tissue. The variety and clinical relevance of autoantibodies in SSc patients have been extensively studied, eventually identifying agonistic autoantibodies targeting the platelet-derived growth factor receptor alpha (PDGFRα), and representing potential biomarkers for SSc. We used a resonant mirror biosensor to characterize the binding between surface-blocked PDGFRα and PDGFRα-specific recombinant human monoclonal autoantibodies (mAbs) produced by SSc B cells, and detect/quantify serum autoimmune IgG with binding characteristics similar to the mAbs. Kinetic data showed a conformation-specific, high-affinity interaction between PDGFRα and mAbs, with equilibrium dissociation constants in the low-to-high nanomolar range. When applied to total serum IgG, the assay discriminated between SSc patients and healthy controls, and allowed the rapid quantification of autoimmune IgG in the sera of SSc patients, with anti-PDGFRα IgG falling in the range 3.20-4.67 neq/L of SSc autoantibodies. The test was validated by comparison to direct and competitive anti-PDGFRα antibody ELISA. This biosensor assay showed higher sensibility with respect to ELISA, and other major advantages such as the specificity, rapidity, and reusability of the capturing surface, thus representing a feasible approach for the detection and quantification of high affinity, likely agonistic, SSc-specific anti-PDGFRα autoantibodies.
Assuntos
Autoanticorpos/sangue , Autoanticorpos/imunologia , Biomarcadores/sangue , Técnicas Biossensoriais/métodos , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/imunologia , Escleroderma Sistêmico/imunologia , Adulto , Idoso , Linfócitos B/imunologia , Feminino , Humanos , Limite de Detecção , Masculino , Pessoa de Meia-Idade , Escleroderma Sistêmico/diagnóstico , Sensibilidade e EspecificidadeRESUMO
CD157/BST-1 behaves both as an ectoenzyme and signaling receptor and is an important regulator of leukocyte trafficking and ovarian cancer progression. However, the molecular interactions underpinning the role of CD157 in these processes remain obscure. The biological functions of CD157 and its partnership with members of the integrin family prompted us to assume the existence of a direct interaction between CD157 and an unknown component of the extracellular matrix. Using solid-phase binding assays and surface plasmon resonance analysis, we demonstrated that CD157 binds fibronectin with high affinity within its heparin-binding domains 1 and 2. Furthermore, we found that CD157 binds to other extracellular matrix proteins containing heparin-binding domains. Finally, we proved that the CD157-fibronectin interaction occurs with living cells, where it elicits CD157-mediated cell responses. Indeed, knockdown of CD157 in Met-5A mesothelial cells changed their morphology and cytoskeleton organization and attenuated the activation of intracellular signaling pathways triggered by fibronectin. This led to impaired cell spreading and adhesion to selected extracellular matrix proteins. Collectively, these findings indicate a central role of CD157 in cell-extracellular matrix interactions and make CD157 an attractive therapeutic target in inflammation and cancer.
Assuntos
ADP-Ribosil Ciclase/metabolismo , Antígenos CD/metabolismo , Adesão Celular/fisiologia , Células Epiteliais/citologia , Fibronectinas/metabolismo , ADP-Ribosil Ciclase/química , Antígenos CD/química , Diferenciação Celular/fisiologia , Linhagem Celular , Movimento Celular/fisiologia , Células Epiteliais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Feminino , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/metabolismo , Humanos , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , Transdução de Sinais/fisiologia , Ressonância de Plasmônio de SuperfícieRESUMO
OBJECTIVE: The etiopathogenesis of systemic sclerosis (SSc) is unknown. Platelet-derived growth factor receptors (PDGFRs) are overexpressed in patients with SSc. Because PDGFRα is targeted by the adeno-associated virus type 5 (AAV5), we investigated whether AAV5 forms a complex with PDGFRα exposing epitopes that may induce the immune responses to the virus-PDGFRα complex. METHODS: The binding of monomeric human PDGFRα to the AAV5 capsid was analyzed by in silico molecular docking, surface plasmon resonance (SPR), and genome editing of the PDGFRα locus. AAV5 was detected in SSc lungs by in situ hybridization, immunohistochemistry, confocal microscopy, and molecular analysis of bronchoalveolar lavage (BAL) fluid. Immune responses to AAV5 and PDGFRα were evaluated by SPR using SSc monoclonal anti-PDGFRα antibodies and immunoaffinity-purified anti-PDGFRα antibodies from sera of patients with SSc. RESULTS: AAV5 was detected in the BAL fluid of 41 of 66 patients with SSc with interstitial lung disease (62.1%) and in 17 of 66 controls (25.75%) (P < 0.001). In SSc lungs, AAV5 localized in type II pneumocytes and in interstitial cells. A molecular complex formed of spatially contiguous epitopes of the AAV5 capsid and of PDGFRα was identified and characterized. In silico molecular docking analysis and binding to the agonistic anti-PDGFRα antibodies identified spatially contiguous epitopes derived from PDGFRα and AAV5 that interacted with SSc agonistic antibodies to PDGFRα. These peptides were also able to bind total IgG isolated from patients with SSc, not from healthy controls. CONCLUSION: These data link AVV5 with the immune reactivity to endogenous antigens in SSc and provide a novel element in the pathogenesis of SSc.
Assuntos
Doenças Pulmonares Intersticiais , Escleroderma Sistêmico , Humanos , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Epitopos , Dependovirus/metabolismo , Autoanticorpos , Simulação de Acoplamento Molecular , Escleroderma Sistêmico/patologia , Peptídeos , Pulmão/patologiaRESUMO
CD157, a member of the CD38 gene family, is an NAD-metabolizing ectoenzyme and a signaling molecule whose role in polarization, migration, and diapedesis of human granulocytes has been documented; however, the molecular events underpinning this role remain to be elucidated. This study focused on the role exerted by CD157 in monocyte migration across the endothelial lining and adhesion to extracellular matrix proteins. The results demonstrated that anti-CD157 antibodies block monocyte transmigration and adhesion to fibronectin and fibrinogen but that CD157 cross-linking is sufficient to overcome the block, suggesting an active signaling role for the molecule. Consistent with this is the observation that CD157 is prevalently located within the detergent-resistant membrane microdomains to which, upon clustering, it promotes the recruitment of ß(1) and ß(2) integrin, which, in turn, leads to the formation of a multimolecular complex favoring signal transduction. This functional cross-talk with integrins allows CD157 to act as a receptor despite its intrinsic structural inability to do so on its own. Intracellular signals mediated by CD157 rely on the integrin/Src/FAK (focal adhesion kinase) pathway, resulting in increased activity of the MAPK/ERK1/2 and the PI3K/Akt downstream signaling pathways, which are crucial in the control of monocyte transendothelial migration. Collectively, these findings indicate that CD157 acts as a molecular organizer of signaling-competent membrane microdomains and that it forms part of a larger molecular machine ruled by integrins. The CD157-integrin partnership provides optimal adhesion and transmigration of human monocytes.
Assuntos
ADP-Ribosil Ciclase/metabolismo , Antígenos CD/metabolismo , Movimento Celular/fisiologia , Células Endoteliais/metabolismo , Microdomínios da Membrana/metabolismo , Monócitos/metabolismo , Transdução de Sinais/fisiologia , ADP-Ribosil Ciclase/antagonistas & inibidores , ADP-Ribosil Ciclase/genética , Anticorpos Bloqueadores/farmacologia , Antígenos CD/genética , Antígenos CD18/genética , Antígenos CD18/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Células Endoteliais/citologia , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Fibrinogênio/genética , Fibrinogênio/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Humanos , Integrina beta1/genética , Integrina beta1/metabolismo , Microdomínios da Membrana/genética , Monócitos/citologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Sézary syndrome is a rare subtype of cutaneous T-cell lymphoma characterized by erythroderma, peripheral lymphadenopathies, and circulating atypical cerebriform T-cells. To date, no definite staging system has been developed for these patients. In this retrospective analysis of the archive of the Dermatological Clinic of the University of Turin, Italy, erythrodermic SS patients were classified according to clinical records and photographs into three main presentations: erythematous, infiltrated, or melanodermic. The pattern of erythroderma was found to be associated with disease outcome, as better survivals were recorded in patients with erythematous and infiltrative erythroderma. Patients in the melanodermic group, though less represented in our investigation, seemed to show a worse trend in survival. According to this preliminary evidence, a new prognostic classification, with a revised score specific for Sézary syndrome patients, can be proposed to usefully integrate the current staging system. The correlation displayed in our research will be hopefully confirmed by prospective studies with larger cohorts, with the aim of identifying significant prognostic features in this subset of cutaneous T-cell lymphoma patients.
Assuntos
Dermatite Esfoliativa , Linfoma Cutâneo de Células T , Micose Fungoide , Síndrome de Sézary , Neoplasias Cutâneas , Dermatite Esfoliativa/patologia , Humanos , Linfoma Cutâneo de Células T/patologia , Micose Fungoide/patologia , Estudos Prospectivos , Estudos Retrospectivos , Síndrome de Sézary/patologia , Neoplasias Cutâneas/patologiaRESUMO
BACKGROUND: Reports on the expression of CD38 in Sézary syndrome (SS), erythrodermic primary cutaneous T cell lymphoma with leukemic involvement, are limited. The aim of the present study is the analysis of the expression of CD38 by skin-infiltrating mononuclear cells and circulating T lymphocytes in a cohort of SS patients. METHODS: SS patients diagnosed since 1985 in our clinic were retrospectively analyzed for CD38 expression in biopsy and blood samples by immunohistochemistry and flow cytometry, respectively. RESULTS: SS patients show a predominant CD38-negative phenotype on both skin and blood. A subgroup of patients was found expressing CD38 (12 cases) in either the skin (>25% cell infiltrate) or blood (CD4+CD38+ >50%), among whom 4 in the blood, 7 in the skin, and 1 in both blood and skin. CONCLUSION: The implications of these observations may be twofold: the relevance in basic science is related to a potential role in immune defense regulation, whilst in perspective CD38 may become a target for antibody therapy, considering the availability of different anti-CD38 monoclonal antibodies.
Assuntos
ADP-Ribosil Ciclase 1/imunologia , Biomarcadores Tumorais/sangue , Citometria de Fluxo , Imuno-Histoquímica , Glicoproteínas de Membrana/imunologia , Síndrome de Sézary , Neoplasias Cutâneas , ADP-Ribosil Ciclase 1/genética , Biópsia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/ultraestrutura , Feminino , Humanos , Contagem de Linfócitos , Masculino , Glicoproteínas de Membrana/genética , Pessoa de Meia-Idade , Estudos Retrospectivos , Síndrome de Sézary/imunologia , Síndrome de Sézary/patologia , Pele/imunologia , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/ultraestruturaRESUMO
Several lines of evidence suggest an association between oxidative DNA-damage repair capacity and cancer risk. In particular, a DNA-glycosylase assay for removal of 8-oxoguanine (8-oxoG) in peripheral blood mononuclear cells (PBMC) has been successfully applied to identify populations with increased risk for lung cancer and squamous cell carcinomas of head and neck. In order to verify whether EBV-transformed lymphoblastoid cell lines (LCL) are a suitable surrogate for PBMC in specific DNA-repair phenotypic assays, a validation trial was conducted. PBMC from 20 healthy subjects were collected and an aliquot was transformed with EBV to obtain LCL. The ability of cell-free extracts from both cell types to incise a 3'-fluorescently labelled duplex oligonucleotide containing a single 8-oxoG (OGG assay) was evaluated. Since this activity is mediated predominantly by OGG1, the OGG1 gene expression was also measured. 8-oxoG DNA-glycosylase activity and OGG1 expression were significantly higher (p<0.0001) in LCL than in PBMC. However, while this assay was shown to be robust and reproducible when used on PBMC (intra-assay CV=8%), a high intra-culture variability was observed with LCL (intra-culture CV=16.8%). Neither differences on OGG1 gene expression nor the cell-cycle distribution seemed to account for this variability. Inter-individual variability of OGG activity in PBMC and LCL was not associated with OGG1 gene expression. We have therefore established a non-radioactive cleavage assay that can be easily applied to measure OGG activity in human PBMC. The use of LCL for DNA-repair genotype-phenotype correlation studies seems to be inappropriate, at least with cell-free based functional assays.
Assuntos
DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Reparo do DNA , Guanina/análogos & derivados , Linfócitos/metabolismo , Linhagem Celular , Transformação Celular Viral , Criopreservação , Expressão Gênica , Estudos de Associação Genética , Guanina/metabolismo , Herpesvirus Humano 4 , Humanos , Técnicas In VitroRESUMO
CD157/BST-1 (a member of the ADP-ribosyl cyclase family) is expressed at variable levels in 97% of patients with acute myeloid leukemia (AML), and is currently under investigation as a target for antibody-based immunotherapy. We used peripheral blood and bone marrow samples from patients with AML to analyse the impact of CD157-directed antibodies in AML survival and in response to cytarabine (AraC) ex vivo. The study was extended to the U937, THP1 and OCI-AML3 AML cell lines of which we engineered CD157-low versions by shRNA knockdown. CD157-targeting antibodies enhanced survival, decreased apoptosis and reduced AraC toxicity in AML blasts and cell lines. CD157 signaling activated the PI3K/AKT/mTOR and MAPK/ERK pathways and increased expression of Mcl-1 and Bcl-XL anti-apoptotic proteins, while decreasing expression of Bax pro-apoptotic protein, thus preventing Caspase-3 activation. The primary CD157-mediated anti-apoptotic mechanism was Bak sequestration by Mcl-1. Indeed, the Mcl-1-specific inhibitor S63845 restored apoptosis by disrupting the interaction of Mcl-1 with Bim and Bak and significantly increased AraC toxicity in CD157-high but not in CD157-low AML cells. This study provides a new role for CD157 in AML cell survival, and indicates a potential role of CD157 as a predictive marker of response to therapies exploiting Mcl-1 pharmacological inhibition.
Assuntos
ADP-Ribosil Ciclase/metabolismo , Antígenos CD/metabolismo , Resistencia a Medicamentos Antineoplásicos , Leucemia Mieloide Aguda/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , ADP-Ribosil Ciclase/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/genética , Antimetabólitos Antineoplásicos/toxicidade , Apoptose , Células Cultivadas , Citarabina/toxicidade , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Pessoa de Meia-Idade , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/farmacologia , Células THP-1 , Tiofenos/farmacologiaRESUMO
As first task of a comprehensive investigation on DNA repair genotype-phenotype correlations, the suitability of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) as surrogate of cryopreserved peripheral blood mononuclear cells (PBMCs) in DNA repair phenotypic assays was evaluated. To this aim the amount of DNA damage induced by gamma-rays and DNA repair capacity were evaluated in unstimulated (G(0)) and mitogen-simulated (G(2)) PBMC from 20 healthy subjects and in EBV-transformed LCL obtained from the same individuals. Phosphorylation of histone H2AX, micronuclei and chromosomal aberrations were the end-points investigated. The results obtained show higher basal frequencies of binucleated cells bearing micronuclei and nucleoplasmic bridge (NPB) in LCL with respect to PBMC, suggesting that EBV transformation may be associated with chromosomal instability. After irradiation, higher levels of micronuclei were induced in G(0)-treated PBMC compared to cycling LCL; conversely, NPB were more frequent in LCL than in PBMC. Moreover, higher levels of chromosomal aberrations were observed in G(2)-treated PBMC compared to LCL. Concerning gamma-H2AX measurements, phosphorylation levels 1h after treatment and dephosphorylation kinetics were basically similar in LCL and in PBMC. However, while Spearman's test showed a strong correlation between the results obtained in replicated experiments with PBMC, high inter-experimental variability and poor reproducibility was observed in the experiments performed with LCL, possibly due to the intrinsic instability of LCL. In summary, both the analysis of gamma-H2AX and the evaluation of chromosome damage highlighted a larger inter-experimental variability in the results obtained with LCL compared to PBMC. Noteworthy, the two set of results proved to lack any significant correlation at the individual level. These results indicate that LCL may be unsuitable for investigating genotype-phenotype correlations with phenotypic DNA repair assays, especially when low impact functional genetic variants are involved.
Assuntos
Linhagem Celular , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Leucócitos Mononucleares/efeitos da radiação , Linfócitos , Linhagem Celular Transformada/efeitos da radiação , Transformação Celular Viral , Aberrações Cromossômicas , Citometria de Fluxo/métodos , Fase G2 , Estudos de Associação Genética , Histonas/metabolismo , Linfócitos/efeitos da radiação , FosforilaçãoRESUMO
Short term treatment with low doses of glucocorticoid analogues has been shown to ameliorate neurological symptoms in Ataxia-Telangiectasia (A-T), a rare autosomal recessive multisystem disease that mainly affects the cerebellum, immune system, and lungs. Molecular mechanisms underlying this clinical observation are unclear. We aimed at evaluating the effect of dexamethasone on the induction of alternative ATM transcripts (ATMdexa1). We showed that dexamethasone cannot induce an alternative ATM transcript in control and A-T lymphoblasts and primary fibroblasts, or in an ATM-knockout HeLa cell line. We also demonstrated that some of the reported readouts associated with ATMdexa1 are due to cellular artifacts and the direct induction of γH2AX by dexamethasone via DNA-PK. Finally, we suggest caution in interpreting dexamethasone effects in vitro for the results to be translated into a rational use of the drug in A-T patients.
Assuntos
Processamento Alternativo/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/genética , Ataxia Telangiectasia/patologia , Dexametasona/farmacologia , Processamento Alternativo/genética , Ataxia Telangiectasia/tratamento farmacológico , Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Técnicas de Inativação de Genes , Células HeLa , Histonas/metabolismo , Humanos , Limite de Detecção , Fosforilação/efeitos dos fármacosRESUMO
BACKGROUND: Systemic sclerosis (scleroderma) is characterized by immunologic abnormalities, injury of endothelial cells, and tissue fibrosis. Abnormal oxidative stress has been documented in scleroderma and linked to fibroblast activation. Since platelet-derived growth factor (PDGF) stimulates the production of reactive oxygen species (ROS) and since IgG from patients with scleroderma reacts with human fibroblasts, we tested the hypothesis that patients with scleroderma have serum autoantibodies that stimulate the PDGF receptor (PDGFR), activating collagen-gene expression. METHODS: We analyzed serum from 46 patients with scleroderma and 75 controls, including patients with other autoimmune diseases, for stimulatory autoantibodies to PDGFR by measuring the production of ROS produced by the incubation of purified IgG with mouse-embryo fibroblasts carrying inactive copies of PDGFR alpha or beta chains or the same cells expressing PDGFR alpha or beta. Generation of ROS was assayed with and without specific PDGFR inhibitors. Antibodies were characterized by immunoprecipitation, immunoblotting, and absorption experiments. RESULTS: Stimulatory antibodies to the PDGFR were found in all the patients with scleroderma. The antibodies recognized native PDGFR, inducing tyrosine phosphorylation and ROS accumulation. Autoantibody activity was abolished by preincubation with cells expressing the PDGFR alpha chain or with recombinant PDGFR or by PDGFR tyrosine kinase inhibitors. Stimulatory PDGFR antibodies selectively induced the Ha-Ras-ERK1/2 and ROS cascades and stimulated type I collagen-gene expression and myofibroblast phenotype conversion in normal human primary fibroblasts. CONCLUSIONS: Stimulatory autoantibodies against PDGFR appear to be a specific hallmark of scleroderma. Their biologic activity on fibroblasts strongly suggests that they have a causal role in the pathogenesis of the disease.
Assuntos
Autoanticorpos/sangue , Doenças Autoimunes/imunologia , Imunoglobulina G/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/imunologia , Escleroderma Sistêmico/imunologia , Adulto , Idoso , Animais , Bioensaio , Estudos de Casos e Controles , Células Cultivadas , Colágeno/genética , Colágeno/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Genes ras/fisiologia , Humanos , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Transdução de Sinais , Tirfostinas/farmacologiaRESUMO
CD157/BST1 glycosylphosphatidylinositol-anchored glycoprotein is an evolutionary conserved dual-function receptor and ß-NAD+-metabolizing ectoenzyme of the ADP-ribosyl cyclases gene family. Identified as bone marrow stromal cell and myeloid cell differentiation antigen, CD157 turned out to have a wider expression than originally assumed. The functional significance of human CD157 as an enzyme remains unclear, while it was well established in mouse models. Conversely, the receptor role of CD157 has been clearly delineated. In physiological conditions, CD157 is a key player in regulating leukocyte adhesion, migration and diapedesis. Underlying these functional roles is the ability of CD157 to bind with high affinity selected extracellular matrix components within their heparin-binding domains. CD157 binding to extracellular matrix promotes its interaction with ß1 and ß2-integrins and induces the organization of a multimolecular complex that is instrumental to the delivery of synergistic outside-in signals leading to optimal cell adhesion and migration, both in physiological and in pathological situations. CD157 also regulates cell adhesion and migration and is a marker of adverse prognosis in epithelial ovarian cancer and pleural mesothelioma. This review focuses on human CD157 expression and functions and provides an overview on its role in human pathology and its emerging potential as target for antibody-mediated immunotherapy.
Assuntos
ADP-Ribosil Ciclase/imunologia , Antígenos CD/imunologia , Inflamação/terapia , Neoplasias/terapia , ADP-Ribosil Ciclase/genética , ADP-Ribosil Ciclase/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Adesão Celular , Matriz Extracelular/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Humanos , Imunidade Inata , Imunoterapia , Inflamação/imunologia , Inflamação/metabolismo , Leucócitos/fisiologia , NAD/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismoRESUMO
: Human CD157/BST-1 and CD38 are dual receptor-enzymes derived by gene duplication that belong to the ADP ribosyl cyclase gene family. First identified over 30 years ago as Mo5 myeloid differentiation antigen and 10 years later as Bone Marrow Stromal Cell Antigen 1 (BST-1), CD157 proved not to be restricted to the myeloid compartment and to have a diversified functional repertoire ranging from immunity to cancer and metabolism. Despite being a NAD+-metabolizing ectoenzyme anchored to the cell surface through a glycosylphosphatidylinositol moiety, the functional significance of human CD157 as an enzyme remains unclear, while its receptor role emerged from its discovery and has been clearly delineated with the identification of its high affinity binding to fibronectin. The aim of this review is to provide an overview of the immunoregulatory functions of human CD157/BST-1 in physiological and pathological conditions. We then focus on CD157 expression in hematological tumors highlighting its emerging role in the interaction between acute myeloid leukemia and extracellular matrix proteins and its potential utility for monoclonal antibody targeted therapy in this disease.
Assuntos
ADP-Ribosil Ciclase/metabolismo , Antígenos CD/metabolismo , Células Mieloides/citologia , Células Mieloides/metabolismo , ADP-Ribosil Ciclase/antagonistas & inibidores , ADP-Ribosil Ciclase/química , Imunidade Adaptativa , Antígenos CD/química , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Biomarcadores Tumorais , Suscetibilidade a Doenças , Ativação Enzimática , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/metabolismo , Humanos , Imunidade Inata , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/etiologia , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Modelos Moleculares , Terapia de Alvo Molecular , Células Mieloides/efeitos dos fármacos , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato , Distribuição TecidualRESUMO
BACKGROUND: Human monoclonal antibodies (mAbs) generated as a result of the immune response are likely to be the most effective therapeutic antibodies, particularly in the case of infectious diseases against which the immune response is protective.Human cytomegalovirus (HCMV) is an ubiquitous opportunistic virus that is the most serious pathogenic agent in transplant patients. The available therapeutic armamentarium (e.g. HCMV hyperimmune globulins or antivirals) is associated with severe side effects and the emergence of drug-resistant strains; therefore, neutralizing human mAb may be a decisive alternative in the prevention of primary and re-activated HCMV infections in these patients. RESULTS: The purpose of this study was to generate neutralizing mAb against HCMV from the immunological repertoire of immune donors. To this aim, we designed an efficient technology relying on two discrete and sequential steps: first, human B-lymphocytes are stimulated with TLR9-agonists and IL-2; second, after both additives are removed, the cells are infected with EBV. Using this strategy we obtained 29 clones secreting IgG neutralizing the HCMV infectivity; four among these were further characterized. All of the mAbs neutralize the infection in different combinations of HCMV strains and target cells, with a potency approximately 20 fold higher than that of the HCMV hyperimmune globulins, currently used in transplant recipients. Recombinant human monoclonal IgG1 suitable as a prophylactic or therapeutic tool in clinical applications has been generated. CONCLUSION: The technology described has proven to be more reproducible, efficient and rapid than previously reported techniques, and can be adopted at low overall costs by any cell biology laboratory for the development of fully human mAbs for immunotherapeutic uses.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Infecções por Citomegalovirus/terapia , Citomegalovirus/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/uso terapêutico , Linfócitos B/virologia , Sequência de Bases , Linhagem Celular , Células Cultivadas , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/prevenção & controle , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica , Humanos , Interleucina-2/metabolismo , Dados de Sequência Molecular , Testes de Neutralização , Receptores de IgE/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/uso terapêutico , Reprodutibilidade dos Testes , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/metabolismo , Proteínas do Envelope Viral/metabolismoRESUMO
BACKGROUND: CD157/Bst1 glycoprotein is expressed in >85% of malignant pleural mesotheliomas and is a marker of enhanced tumor aggressiveness. RESULTS: In vitro, mesothelial cells (malignant and non-malignant) released CD157 in soluble form or as an exosomal protein. In vivo, sCD157 is released and can be measured in pleural effusions by ELISA. Significantly higher levels of effusion sCD157 were detected in patients with malignant pleural mesothelioma than in patients with non-mesothelioma tumors or with non-malignant conditions. In our patient cohort, the area under the receiver-operating characteristic curve for sCD157 that discriminated malignant pleural mesothelioma from all other causes of pleural effusion was 0.685, cut-off (determined by the Youden Index) = 23.66 ng/ml (62.3% sensitivity; 73.93% specificity). Using a cut-off that yielded 95.58% specificity, measurement of sCD157 in cytology-negative effusions increased sensitivity of malignant pleural mesothelioma diagnosis from 34.42% to 49.18%. CONCLUSIONS: Evaluation of soluble CD157 in pleural effusions provides a diagnostic aid in malignant mesothelioma. METHODS: Soluble CD157 (sCD157) was detected biochemically in culture supernatants of malignant and non-malignant mesothelial cells, and in pleural effusions from various pathological conditions. An ELISA system was established to measure the concentration of sCD157 in fluids, and extended to analyze sCD157 in pleural effusions from a cohort of 295 patients.
RESUMO
One of the earliest events in the pathogenesis of systemic sclerosis (SSc) is microvasculature damage with intimal hyperplasia and accumulation of cells expressing PDGF receptor. Stimulatory autoantibodies targeting PDGF receptor have been detected in SSc patients and demonstrated to induce fibrosis in vivo and convert in vitro normal fibroblasts into SSc-like cells. Since there is no evidence of the role of anti-PDGF receptor autoantibodies in the pathogenesis of SSc vascular lesions, we investigated the biologic effect of agonistic anti-PDGF receptor autoantibodies from SSc patients on human pulmonary artery smooth muscle cells and the signaling pathways involved. The synthetic (proliferation, migration, and type I collagen gene α1 chain expression) and contractile (smooth muscle-myosin heavy chain and smooth muscle-calponin expression) profiles of human pulmonary artery smooth muscle cells were assessed in vitro after incubation with SSc anti-PDGF receptors stimulatory autoantibodies. The role of reactive oxygen species, NOX isoforms, and mammalian target of rapamycin (mTOR) was investigated. Human pulmonary artery smooth muscle cells acquired a synthetic phenotype characterized by higher growth rate, migratory activity, gene expression of type I collagen α1 chain, and less expression of markers characteristic of the contractile phenotype such as smooth muscle-myosin heavy chain and smooth muscle-calponin when stimulated with PDGF and autoantibodies against PDGF receptor, but not with normal IgG. This phenotypic profile is mediated by increased generation of reactive oxygen species and expression of NOX4 and mTORC1. Our data indicate that agonistic anti-PDGF receptor autoantibodies may contribute to the pathogenesis of SSc intimal hyperplasia.
RESUMO
[This corrects the article on p. 75 in vol. 8, PMID: 28228756.].