RESUMO
BACKGROUND: Clonal mast cell disorders are known to occur in a subset of patients with systemic reactions to Hymenoptera stings. This observation has prompted the question of whether clonal mast cell disorders also occur in patients with idiopathic anaphylaxis (IA). OBJECTIVE: We sought to determine the prevalence of clonal mast cell disorders among patients with IA, criteria to identify those patients who require a bone marrow biopsy, and whether the pathogenesis of IA involves a hyperresponsive mast cell compartment. METHODS: We prospectively enrolled patients with IA (≥3 episodes/y) who then underwent a medical evaluation that included a serum tryptase determination, allele-specific quantitative PCR (ASqPCR) for the KIT D816V mutation, and a bone marrow examination. Mast cells were cultured from peripheral blood CD34+ cells and examined for releasability after FcεRI aggregation. RESULTS: Clonal mast cell disease was diagnosed in 14% of patients referred with IA. ASqPCR for the KIT D816V mutation was a useful adjunct in helping identify those with systemic mastocytosis but not monoclonal mast cell activation syndrome. A modified overall clonal prediction model was developed by using clinical findings, a serum tryptase determination, and ASqPCR. There was no evidence of a hyperresponsive mast cell phenotype in patients with IA. CONCLUSION: Patients with clonal mast cell disease can present as having IA. Distinct clinical and laboratory features can be used to select those patients more likely to have an underlying clonal mast cell disorder (monoclonal mast cell activation syndrome or systemic mastocytosis) and thus candidates for a bone marrow biopsy.
Assuntos
Anafilaxia/genética , Anafilaxia/imunologia , Mastócitos/imunologia , Mastocitose Sistêmica/genética , Mastocitose Sistêmica/imunologia , Mutação de Sentido Incorreto , Proteínas Proto-Oncogênicas c-kit , Adolescente , Adulto , Idoso , Substituição de Aminoácidos , Anafilaxia/patologia , Feminino , Humanos , Masculino , Mastócitos/patologia , Mastocitose Sistêmica/patologia , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/imunologiaRESUMO
Pure red cell aplasia (PRCA) is a rare disorder characterized by inhibition of erythroid precursors in the bone marrow and normochromic, normocytic anaemia with reticulocytopenia. Among 51 PRCA patients, we identified 12 (24%) patients having monoclonal gammopathy, monoclonal gammopathy of undetermined significance or smouldering multiple myeloma, with presence of monoclonal protein or abnormal serum free light chains and atypical bone marrow features of clonal plasmacytosis, hypercellularity and fibrosis. Thus far, three patients treated with anti-myeloma based therapeutics have responded with reticulocyte recovery and clinical transfusion independence, suggesting plasma cells play a key role in the pathogenesis of this specific monoclonal gammopathy-associated PRCA.
Assuntos
Mieloma Múltiplo/diagnóstico , Paraproteinemias/diagnóstico , Aplasia Pura de Série Vermelha/diagnóstico , Adulto , Idoso , Medula Óssea/patologia , Dexametasona/uso terapêutico , Diagnóstico Diferencial , Feminino , Humanos , Imunoglobulinas/sangue , Lenalidomida , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/patologia , Paraproteinemias/patologia , Plasmócitos/fisiologia , Aplasia Pura de Série Vermelha/patologia , Contagem de Reticulócitos , Reticulócitos/fisiologia , Talidomida/análogos & derivados , Talidomida/uso terapêutico , Adulto JovemRESUMO
Hallmarks of cancer, including rapid growth and aneuploidy, can result in non-oncogene addiction to the proteostasis network that can be exploited clinically. The defining example is the exquisite sensitivity of multiple myeloma (MM) to 20S proteasome inhibitors, such as carfilzomib. However, MM patients invariably acquire resistance to these drugs. Using a next-generation shRNA platform, we found that proteostasis factors, including chaperones and stress-response regulators, controlled the response to carfilzomib. Paradoxically, 19S proteasome regulator knockdown induced resistance to carfilzomib in MM and non-MM cells. 19S subunit knockdown did not affect the activity of the 20S subunits targeted by carfilzomib nor their inhibition by the drug, suggesting an alternative mechanism, such as the selective accumulation of protective factors. In MM patients, lower 19S levels predicted a diminished response to carfilzomib-based therapies. Together, our findings suggest that an understanding of network rewiring can inform development of new combination therapies to overcome drug resistance.
Assuntos
Antineoplásicos/metabolismo , Resistencia a Medicamentos Antineoplásicos , Mieloma Múltiplo/enzimologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/metabolismo , Linhagem Celular Tumoral , Humanos , Oligopeptídeos/metabolismoRESUMO
Store-operated Ca(2+) entry is the major route of replenishment of intracellular Ca(2+) in animal cells in response to the depletion of Ca(2+) stores in the endoplasmic reticulum. It is primarily mediated by the Ca(2+)-selective release-activated Ca(2+) (CRAC) channel, which consists of the pore-forming subunits ORAI1-3 and the Ca(2+) sensors, STIM1 and STIM2. Recessive loss-of-function mutations in STIM1 or ORAI1 result in immune deficiency and nonprogressive myopathy. Heterozygous gain-of-function mutations in STIM1 cause non-syndromic myopathies as well as syndromic forms of miosis and myopathy with tubular aggregates and Stormorken syndrome; some of these syndromic forms are associated with thrombocytopenia. Increased concentration of Ca(2+) as a result of store-operated Ca(2+) entry is essential for platelet activation. The York Platelet syndrome (YPS) is characterized by thrombocytopenia, striking ultrastructural platelet abnormalities including giant electron-opaque organelles and massive, multilayered target bodies and deficiency of platelet Ca(2+) storage in delta granules. We present clinical and molecular findings in 7 YPS patients from 4 families, demonstrating that YPS patients have a chronic myopathy associated with rimmed vacuoles and heterozygous gain-of-function STIM1 mutations. These findings expand the phenotypic spectrum of STIM1-related human disorders and define the molecular basis of YPS.
Assuntos
Plaquetas/patologia , Canalopatias/genética , Proteínas de Membrana/genética , Doenças Musculares/genética , Proteínas de Neoplasias/genética , Adulto , Transtornos Plaquetários/genética , Transtornos Plaquetários/metabolismo , Plaquetas/fisiologia , Plaquetas/ultraestrutura , Cálcio/metabolismo , Criança , Pré-Escolar , Dislexia/genética , Dislexia/metabolismo , Eritrócitos Anormais/metabolismo , Exoma/genética , Feminino , Heterozigoto , Humanos , Ictiose/genética , Ictiose/metabolismo , Lactente , Masculino , Pessoa de Meia-Idade , Transtornos de Enxaqueca/genética , Transtornos de Enxaqueca/metabolismo , Miose/genética , Miose/metabolismo , Fadiga Muscular/genética , Doenças Musculares/metabolismo , Mutação , Linhagem , Análise de Sequência de DNA , Baço/anormalidades , Baço/metabolismo , Molécula 1 de Interação Estromal , TrombocitopeniaRESUMO
Episodic angioedema with eosinophilia (Gleich syndrome) is a rare disorder characterized by episodes of angioedema and eosinophilia that occur at monthly intervals and resolve spontaneously without therapy. Despite the striking periodicity of this disorder, its similarity to other cyclic hematopoietic disorders with multilineage involvement has not been assessed. To characterize the involvement of cell lineages in the etiology and pathogenesis of episodic angioedema with eosinophilia, four subjects were evaluated by blood counts and other analyses over the course of 1-2 months. Surface marker expression was assessed on T cells by flow cytometry and clonality by polymerase chain reaction. Intracellular cytokine evaluation, bone marrow and skin biopsies were performed during different parts of the cycle. Cycling of multiple cell lineages, including neutrophils, lymphocytes and eosinophils, was observed in the four subjects with the disorder with a periodicity of 25-35 days. An aberrant CD3(-)CD4(+) T-cell population was detected in all four subjects, and T-cell receptor rearrangement studies showed a clonal pattern in three subjects. A peak of type II cytokines was detected in the serum of subjects prior to the onset of symptoms and eosinophil cycling and corresponded to ex-vivo type II cytokines detected intracellularly in CD3(+)CD4(+)CD154(+) T cells. Although the etiology of episodic angioedema with eosinophilia is not yet known, multiple lineages, including lymphocytes, neutrophils and mast cells, are involved and may be related to disease pathogenesis. Whether these cells act directly or promote eosinophilia and eosinophil activation remains to be elucidated. All subjects gave informed consent and were evaluated under an Institutional Review Board-approved protocol (NCT00001406).
Assuntos
Angioedema/diagnóstico , Linhagem da Célula/imunologia , Eosinofilia/diagnóstico , Adulto , Angioedema/complicações , Angioedema/imunologia , Angioedema/patologia , Antígenos CD/imunologia , Medula Óssea/imunologia , Medula Óssea/patologia , Citocinas/imunologia , Eosinofilia/complicações , Eosinofilia/imunologia , Eosinofilia/patologia , Eosinófilos/imunologia , Eosinófilos/patologia , Feminino , Expressão Gênica , Humanos , Imunofenotipagem , Masculino , Mastócitos/imunologia , Mastócitos/patologia , Pessoa de Meia-Idade , Neutrófilos/imunologia , Neutrófilos/patologia , Periodicidade , Pele/imunologia , Pele/patologia , Síndrome , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
BACKGROUND: Mastocytosis associated with germline KIT activating mutations is exceedingly rare. We report the unique clinicopathologic features of a patient with systemic mastocytosis caused by a de novo germline KIT K509I mutation. OBJECTIVES: We sought to investigate the effect of the germline KIT K509I mutation on human mast cell development and function. METHODS: Primary human mast cells derived from CD34(+) peripheral blood progenitors were examined for growth, development, survival, and IgE-mediated activation. In addition, a mast cell transduction system that stably expressed the KIT K509I mutation was established. RESULTS: KIT K509I biopsied mast cells were round, CD25(-), and well differentiated. KIT K509I progenitors cultured in stem cell factor (SCF) demonstrated a 10-fold expansion compared with progenitors from healthy subjects and developed into mature hypergranular mast cells with enhanced antigen-mediated degranulation. KIT K509I progenitors cultured in the absence of SCF survived but lacked expansion and developed into hypogranular mast cells. A KIT K509I mast cell transduction system revealed SCF-independent survival to be reliant on the preferential splicing of KIT at the adjacent exonic junction. CONCLUSION: Germline KIT mutations associated with mastocytosis drive a well-differentiated mast cell phenotype distinct to that of somatic KIT D816V disease, the oncogenic potential of which might be influenced by SCF and selective KIT splicing.
Assuntos
Mutação em Linhagem Germinativa , Mastócitos/patologia , Mastocitose Sistêmica/genética , Fenótipo , Proteínas Proto-Oncogênicas c-kit/genética , Adulto , Processamento Alternativo , Degranulação Celular/efeitos dos fármacos , Diferenciação Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Células Cultivadas , Feminino , Expressão Gênica , Humanos , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Mastocitose Sistêmica/imunologia , Mastocitose Sistêmica/patologia , Fator de Células-Tronco/farmacologia , Transdução GenéticaRESUMO
BACKGROUND: Although several novel agents are currently in clinical trials for eosinophilic disorders, none has demonstrated efficacy in reducing blood and tissue eosinophilia in all subjects. Additional approaches are clearly needed. OBJECTIVE: We sought to explore the potential of the human eosinophil surface receptor epidermal growth factor-like module containing mucin-like hormone receptor 1 (EMR1) as a therapeutic target for eosinophilic disorders. METHODS: EMR1 expression was assessed in blood and bone marrow specimens from eosinophilic and healthy subjects, cell lines, CD34(+) cells differentiated in vitro, and tissue biopsy specimens by using flow cytometry, quantitative PCR, and immunostaining. Eosinophil targeting by a novel, humanized, afucosylated anti-EMR1 IgG1 was evaluated in vitro by using a natural killer cell-mediated killing assay and in vivo in cynomolgus monkeys. RESULTS: Analysis of blood and bone marrow cells from healthy and eosinophilic donors and in vitro-differentiated CD34(+) cells confirmed restriction of human EMR1 surface and mRNA expression to mature eosinophils. Tissue eosinophils also expressed EMR1. Although EMR1 was highly expressed on eosinophils from all subjects, surface expression was negatively correlated with absolute eosinophil counts (r = -0.46, P < .001), and soluble plasma levels correlated positively with absolute eosinophil counts (r = 0.69, P < .001), suggesting modulation of EMR1 in vivo. Nevertheless, afucosylated anti-EMR1 mAb dramatically enhanced natural killer cell-mediated killing of eosinophils from healthy and eosinophilic donors and induced a rapid and sustained depletion of eosinophils in monkeys. CONCLUSION: EMR1 expression is restricted to mature blood and tissue eosinophils. Targeting of eosinophils with afucosylated anti-EMR1 antibody shows promise as a treatment for eosinophilic disorders.
Assuntos
Anticorpos Monoclonais Murinos/farmacologia , Eosinofilia/tratamento farmacológico , Eosinófilos/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Imunoglobulina G/farmacologia , Glicoproteínas de Membrana/imunologia , Mucinas/imunologia , Receptores Acoplados a Proteínas G/imunologia , Anticorpos Monoclonais Murinos/imunologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/patologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Proteínas de Ligação ao Cálcio , Eosinofilia/imunologia , Eosinofilia/patologia , Eosinófilos/patologia , Feminino , Humanos , Imunoglobulina G/imunologia , Células K562 , Masculino , Glicoproteínas de Membrana/antagonistas & inibidores , Mucinas/antagonistas & inibidores , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Células U937RESUMO
Stem cell factor-dependent KIT activation is an essential process for mast cell homeostasis. The two major splice variants of KIT differ by the presence or absence of four amino acids (GNNK) at the juxta-membrane region of the extracellular domain. We hypothesized that the expression pattern of these variants differs in systemic mastocytosis and that transcripts containing the KIT D816V mutation segregate preferentially to one GNNK variant. A quantitative real-time PCR assay to assess GNNK(-) and GNNK(+) transcripts from bone marrow mononuclear cells was developed. The GNNK(-)/GNNK(+) copy number ratio showed a trend toward a positive correlation with the percentage of neoplastic mast cell involvement, and KIT D816V containing transcripts displayed a significantly elevated GNNK(-)/GNNK(+) copy number ratio. Relative expression of only the GNNK(-) variant correlated with increasing percentage of neoplastic mast cell involvement. A mast cell transfection system revealed that the GNNK(-) isoform of wild type KIT was associated with increased granule formation, histamine content, and growth. When accompanying the KIT D816V mutation, the GNNK(-) isoform enhanced cytokine-free metabolism and moderately reduced sensitivity to the tyrosine kinase inhibitor, PKC412. These data suggest that neoplastic mast cells favor a GNNK(-) variant predominance, which in turn enhances the activating potential of the KIT D816V mutation and thus could influence therapeutic sensitivity in systemic mastocytosis.
Assuntos
Regulação Neoplásica da Expressão Gênica , Mastócitos/metabolismo , Mastocitose Sistêmica/genética , Mastocitose Sistêmica/fisiopatologia , Mutação , Proteínas Proto-Oncogênicas c-kit/genética , Adulto , Idoso , Resistencia a Medicamentos Antineoplásicos/genética , Ativação Enzimática/genética , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/enzimologia , Mastocitose Sistêmica/enzimologia , Pessoa de Meia-Idade , Isoformas de Proteínas/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Estaurosporina/análogos & derivados , Estaurosporina/farmacologiaRESUMO
Selective dehydrogenation of the biomass-derived lignan hydroxymatairesinol (HMR) to oxomatairesinol (oxoMAT) was studied over an Au/Al(2)O(3) catalyst. The reaction was carried out in a semi-batch glass reactor at 343 K under two different gas atmospheres, namely produced through synthetic air or nitrogen. The studied reaction is, in fact, an example of secondary-alcohol oxidation over an Au catalyst. Thus, the investigated reaction mechanism of HMR oxidative dehydrogenation is useful for the fundamental understanding of other secondary-alcohol dehydrogenation over Au surfaces. To investigate the elementary catalytic steps ruling both oxygen-free- and oxygen-assisted dehydrogenation of HMR to oxoMAT, the reactions were mimicked in a vacuum over an Au(28) cluster. Adsorption of the involved molecular species--O(2), three different HMR diastereomers (namely, one SRR and two RRR forms), and the oxoMAT derivative--were also studied at the DFT level. In particular, the energetic and structural differences between SRR-HMR and RRR-HMR diastereomers on the Au(28) cluster were analyzed, following different reaction pathways for the HMR dehydrogenation that occur in presence or absence of oxygen. The corresponding mechanisms explain the higher rates of the experimentally observed oxygen-assisted reaction, mostly depending on the involved HMR diastereomer surface conformations. The role of the support was also elucidated, considering a very simple Au(28) charged model that explains the experimentally observed high reactivity of the Au/Al(2)O(3) catalyst.
Assuntos
Álcoois/química , Ouro/química , Lignanas/química , Oxigênio/química , Adsorção , Catálise , Estrutura Molecular , OxirreduçãoRESUMO
BACKGROUND: MicroRNAs can play an important role in tumorigenesis through post-transcriptional regulation of gene expression, and are not well characterized in follicular lymphoma. DESIGN AND METHODS: MicroRNA profiles of enriched follicular lymphoma tumor cells from 16 patients were generated by assaying 851 human microRNAs. Tandem gene expression profiles were obtained for predicting microRNA targets. RESULTS: The expression of 133 microRNAs was significantly different (> 2-fold; P<0.05) between follicular lymphoma and follicular hyperplasia. Forty-four microRNAs in three groups generated a unique follicular lymphoma signature. Of these, ten microRNAs were increased (miR-193a-5p, -193b*, -345, -513b, -574-3p, -584, -663, -1287, -1295, and -1471), 11 microRNAs were decreased (miR-17*, -30a, -33a, -106a*, -141, -202, -205, -222, -301b, -431*, and -570), and 23 microRNAs formed a group that was increased in most cases of follicular lymphoma but showed lower expression in a subset of cases (let-7a, let-7f, miR-7-1*, -9, -9*, -20a, -20b, -30b, -96, -98, -194, -195, -221*, -374a, -374b, -451, -454, -502-3p, -532-3p, -664*, -1274a, -1274b, and -1260). Higher expression of this last group was associated with improved response to chemotherapy. Gene expression analysis revealed increased expression of MAPK1, AKT1, PRKCE, IL4R and DROSHA and decreased expression of CDKN1A/p21, SOCS2, CHEK1, RAD51, KLF4, BLIMP1 and IRF4 in follicular lymphoma. Functional studies indicated that CDKN1A/p21 and SOCS2 expression is directly regulated by miR-20a/-20b and miR-194, respectively. CONCLUSIONS: Follicular lymphoma is characterized by a unique microRNA signature, containing a subset of microRNAs whose expression correlate with response to chemotherapy. miR-20a/b and miR-194 target CDKN1A and SOCS2 in follicular lymphoma, potentially contributing to tumor cell proliferation and survival.
Assuntos
Perfilação da Expressão Gênica , Linfoma Folicular/genética , MicroRNAs/genética , Linhagem Celular Tumoral , Proliferação de Células , Análise por Conglomerados , Inibidor de Quinase Dependente de Ciclina p21/genética , Regulação Neoplásica da Expressão Gênica , Centro Germinativo/patologia , Humanos , Hiperplasia , Fator 4 Semelhante a Kruppel , Linfoma Folicular/tratamento farmacológico , Linfoma Folicular/metabolismo , MicroRNAs/metabolismo , Estadiamento de Neoplasias , Prognóstico , Proteínas Supressoras da Sinalização de Citocina/genética , Resultado do TratamentoRESUMO
Increased mast cell burden is observed in the inflamed tissues and affected organs and tissues of patients with mast cell proliferative disorders. However, normal mast cells participate in host defense, so approaches to preferentially target clonally expanding mast cells are needed. We found that mammalian target of rapamycin complex 1 (mTORC1) and 2 (mTORC2) are up-regulated in neoplastic and developing immature mast cells compared with their terminally differentiated counterparts. Elevated mTOR mRNA was also observed in bone marrow mononuclear cells of patients exhibiting mast-cell hyperplasia. Selective inhibition of mTORC1 and mTORC2 through genetic and pharmacologic manipulation revealed that, whereas mTORC1 may contribute to mast-cell survival, mTORC2 was only critical for homeostasis of neoplastic and dividing immature mast cells. The cytostatic effect of mTORC2 down-regulation in proliferating mast cells was determined to be via inhibition of cell-cycle progression. Because mTORC2 was observed to play little role in the homeostasis of differentiated, nonproliferating, mature mast cells, these data provide a rationale for adopting a targeted approaching selectively inhibiting mTORC2 to effectively reduce the proliferation of mast cells associated with inflammation and disorders of mast cell proliferation while leaving normal differentiated mast cells largely unaffected.
Assuntos
Homeostase , Mastócitos/metabolismo , Mastocitose Sistêmica/metabolismo , Proteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Western Blotting , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Perfilação da Expressão Gênica , Humanos , Mastócitos/efeitos dos fármacos , Mastocitose Sistêmica/genética , Mastocitose Sistêmica/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Transgênicos , Complexos Multiproteicos , Naftiridinas/farmacologia , Proteínas/antagonistas & inibidores , Proteínas/genética , Interferência de RNA , Proteína Companheira de mTOR Insensível à Rapamicina , Proteína Regulatória Associada a mTOR , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Células Tumorais CultivadasRESUMO
We performed a fluorescent analysis of the binding of Aß to the surface membrane of different types of cells lines such as PC12, GT1-7, and ex vivo neurons. Analyses were performed on sorted cells with membrane bound Aß Competitive binding between Aß phosphatidyl serine- (PtdSer-) specific binder annexin V and an anti-PtdSer antibody provided compelling data confirming the involvement of PtdSer as one of the surface membrane signal molecules for Aß. We found that populations of cells that exhibited high surface membrane binding affinity for Aß also show higher membrane cholesterol levels compared to cells that did not bind Aß. This direct relationship was upheld in cholesterol-enriched or cholesterol-depleted cell membranes. We conclude that the initial process for the cell-selective binding by Aß, to later conversion of elemental Aß units into larger structures such as fibrils or to the potentially toxic ion channel aggregates, is highly influenced by the membrane content of PtdSer and cholesterol in the cell surface membrane.
RESUMO
BACKGROUND: IL-5 plays a central role in the development and maintenance of eosinophilia (EO) and eosinophil activation in a wide variety of eosinophilic disorders. Although IL-5, IL-3, and GM-CSF can modulate the expression of IL-5 receptor α (IL-5Rα) on eosinophils in vitro, little is known about soluble and surface IL-5Rα levels in vivo. OBJECTIVE: To assess soluble and surface IL-5Rα levels in patients with EO and/or mastocytosis. METHODS: Surface IL-5Rα expression was assessed by flow cytometry in blood and/or bone marrow from subjects with EO (n = 39) and systemic mastocytosis (n = 8) and from normal volunteers (n = 28). Soluble IL-5Rα (sIL-5Rα) level was measured in a cohort of 177 untreated subjects and correlated with EO, eosinophil activation, and serum tryptase and cytokine levels. RESULTS: IL-5Rα expression on eosinophils inversely correlated with EO (r = -0.48; P < .0001), whereas serum levels of sIL-5Rα increased with the eosinophil count (r = 0.56; P < .0001) and serum IL-5 (r = 0.40; P < .0001) and IL-13 (r = 0.29; P = .004) levels. Of interest, sIL-5Rα level was significantly elevated in patients with systemic mastocytosis without EO. Although sIL-5Rα levels correlated with serum tryptase levels in these patients, eosinophil activation, assessed by CD69 expression on eosinophils and serum eosinophil-derived neurotoxin levels, was increased compared with that in normal subjects. CONCLUSIONS: These data are consistent with an in vivo IL-5Rα regulatory pathway in human eosinophils similar to that described in vitro and involving a balance between soluble and surface receptor levels. This may have implications with respect to the use of novel therapeutic agents targeting IL-5 and its receptor in patients with EO and/or mastocytosis.
Assuntos
Eosinofilia/metabolismo , Subunidade alfa de Receptor de Interleucina-5/biossíntese , Mastocitose Sistêmica/metabolismo , Adulto , Idoso , Separação Celular , Citocinas/análise , Citocinas/biossíntese , Citocinas/imunologia , Ensaio de Imunoadsorção Enzimática , Neurotoxina Derivada de Eosinófilo/análise , Neurotoxina Derivada de Eosinófilo/biossíntese , Neurotoxina Derivada de Eosinófilo/imunologia , Eosinofilia/imunologia , Eosinófilos/imunologia , Eosinófilos/metabolismo , Feminino , Citometria de Fluxo , Humanos , Subunidade alfa de Receptor de Interleucina-5/imunologia , Masculino , Mastocitose Sistêmica/imunologia , Pessoa de Meia-Idade , Triptases/sangue , Adulto JovemRESUMO
Carbon nanotubes (CNTs) are known to potentiate arterial thrombosis in animal models, which raises serious safety issues concerning environmental or occupational exposure to CNTs and their use in various biomedical applications. We have shown previously that different CNTs, but not fullerene (nC60), induce the aggregation of human blood platelets. To date, however, a mechanism of potentially thrombogenic CNT-induced platelet activation has not been elucidated. Here we show that pristine multiwalled CNTs (MWCNTs) penetrate platelet plasma membrane without any discernible damage but interact with the dense tubular system (DTS) causing depletion of platelet intracellular Ca(2+) stores. This process is accompanied by the clustering of stromal interaction molecule 1 (STIM1) colocalized with Orai1, indicating the activation of store-operated Ca(2+) entry (SOCE). Our findings reveal the molecular mechanism of CNT-induced platelet activation which is critical in the evaluation of the biocompatibility of carbon nanomaterials with blood.
Assuntos
Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Cálcio/metabolismo , Nanotubos de Carbono , Animais , Transporte Biológico/efeitos dos fármacos , Plaquetas/citologia , Plaquetas/fisiologia , Canais de Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Homeostase/efeitos dos fármacos , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Ativação Plaquetária/efeitos dos fármacosRESUMO
Cooperating genetic events are likely to contribute to the phenotypic diversity of KIT-D816V systemic mastocytosis. In this study, 44 patients with KIT-D816V systemic mastocytosis were evaluated for coexisting NRAS, KRAS, HRAS or MRAS mutations. Activating NRAS mutations were identified in 2 of 8 patients with advanced disease. NRAS mutations were not found in patients with indolent systemic mastocytosis. To better understand the clonal evolution of mastocytosis, we evaluated the cell compartments impacted by the NRAS and KIT mutations. Clonal mast cells harbored both mutations. KIT-D816V was not detected in bone marrow CD34(+) progenitors, whereas the NRAS mutation was present. These findings suggest that NRAS mutations may have the potential to precede KIT-D816V in clonal development. Unlike other mature lineages, mast cell survival is dependent on KIT and the presence of these two activating mutations may have a greater impact on the expansion of this cell compartment and in resultant disease severity.
Assuntos
Mastocitose Sistêmica/genética , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Idoso , Anticorpos/imunologia , Anticorpos/metabolismo , Antígenos CD34/genética , Antígenos CD34/imunologia , Medula Óssea/metabolismo , Medula Óssea/patologia , Células Clonais/metabolismo , Células Clonais/patologia , Feminino , Citometria de Fluxo , Humanos , Masculino , Mastócitos/imunologia , Mastócitos/metabolismo , Mastócitos/patologia , Mastocitose Sistêmica/metabolismo , Mastocitose Sistêmica/patologia , Pessoa de Meia-Idade , Mutação , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-kit/metabolismoRESUMO
To elucidate a mechanism of prothrombotic effects of carbon nanotubes (CNTs), we report here that multiwalled CNTs activate blood platelets by inducing extracellular Ca(2+) influx that could be inhibited by calcium channel blockers SKF 96365 and 2-APB. We also demonstrate platelet aggregating activity of different single-walled and multiwalled CNTs. In addition, we show that CNT-induced platelet activation is associated with a marked release of platelet membrane microparticles positive for the granular secretion markers CD62P and CD63.
Assuntos
Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Cálcio/antagonistas & inibidores , Nanotubos de Carbono/química , Inibidores da Agregação Plaquetária/farmacologia , Antígenos CD/sangue , Biomarcadores/sangue , Plaquetas/química , Compostos de Boro/farmacologia , Cálcio/metabolismo , Humanos , Imidazóis/farmacologia , Selectina-P/sangue , Glicoproteínas da Membrana de Plaquetas , Tetraspanina 30 , Fatores de TempoRESUMO
The tumor suppressor role of annexin-A7 (ANXA7) was previously demonstrated by cancer susceptibility in Anxa7(+/-)-mice and by ANXA7 loss in human cancers, especially in hormone-resistant prostate tumors. To gain mechanistic insights into ANXA7 tumor suppression, we undertook an in vitro study in which we compared wild-type (WT)-ANXA7 and dominant-negative (DN)-ANXA7 effects to a conventional tumor suppressor p53 in prostate cancer cells with different androgen sensitivity. Unlike p53 (which caused cell growth arrest and apoptosis to a noticeable extent in benign PrEC), WT-ANXA7 demonstrated profound cytotoxicityin androgen-sensitive LNCaP as well as in the androgen-resistant DU145 and PC3 prostate cancer cells, but not in PrEC. In androgen-sensitive LNCaP, WT-ANXA7 decreased low-molecular-weight (LMW) AR protein forms and maintained higher retinoblastoma 1 (RB1)/phospho-RB1 ratio. In contrast, DN-ANXA7 (which lacks phosphatidylserine liposome aggregation properties) increased LMW-AR forms and hyperphosphorylated RB1 that was consistent with the lack of DN-ANXA7 cytotoxicity. According to the microarray-based Ingenuity Pathways Analysis, a major WT-ANXA7 effect in androgen-sensitive LNCaP constituted of upregulation of the RB1-binding transcription factor E2F1 along with its downstream proapoptotic targets such as ASK1 and ASPP2. These results suggested a reversal of the RBdependent repression of the proapoptotic E2F-mediated transcription. However, DN-ANXA7 increased RB1/2 (but not E2F1) expression and induced the proliferation-promoting ERK5, thereby maintaining the RB-dependent repression of E2F-mediated apoptosis in LNcaP. On the other hand, in androgen-resistant cells, WT-ANXA7 tumor suppressor effects involved PTEN and NFkB pathways. Thus, ANXA7 revived the RB-associated cell survival control and overcame androgen resistance and dysfunctional status of major tumor suppressors commonly mutated in prostate cancer. Published 2009 UICC.
Assuntos
Anexina A7/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias Hormônio-Dependentes/patologia , Próstata/efeitos dos fármacos , Neoplasias da Próstata/patologia , Proteína do Retinoblastoma/metabolismo , Proteínas Supressoras de Tumor/farmacologia , Adenoviridae/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , DNA de Neoplasias/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica , Vetores Genéticos , Humanos , Masculino , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Próstata/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
Alzheimer's disease (AD) is a common, chronic neurodegenerative disease that is thought to be caused by the neurotoxic effect of the Amyloid beta peptides (Abeta). We have hypothesized that the intrinsic Abeta calcium channel activity of the oligomeric Abeta polymer may be responsible for the neurotoxic properties of Abeta, and that Abeta channel blockers may be candidate AD therapeutics. As a consequence of a rational search paradigm based on the model structure of the Abeta channel, we have identified two compounds of interest: MRS2481 and an enatiomeric species, MRS2485. These are amphiphilic pyridinium salts that both potently block the Abeta channel and protect neurons from Abeta toxicity. Both block the Abeta channel with similar potency (approximately 500 nM) and efficacy (100%). However, we find that inhibition by MRS2481 is easily reversible, whereas inhibition by MRS2485 is virtually irreversible. We suggest that both species deserve consideration as candidates for Alzheimer's disease drug discovery.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Fármacos Neuroprotetores/farmacologia , Fenilpropionatos/farmacologia , Piridinas/farmacologia , Doença de Alzheimer/patologia , Animais , Bloqueadores dos Canais de Cálcio/química , Canais de Cálcio/química , Estrutura Molecular , Fármacos Neuroprotetores/química , Células PC12 , Fenilpropionatos/química , Ligação Proteica , Piridinas/química , RatosRESUMO
We studied the effects of a C60 water suspension at 4 microg/mL (nC60) and the water soluble fullerenol C60(OH)24 at final concentrations of 1-100 microg/mL on human umbilical vein endothelial cells (HUVECs) in culture. We found that a 24 hr treatment of HUVECs with C60(OH)24 at 100 microg/mL significantly increased cell surface expression of ICAM-1(CD54) (67 +/- 4% CD54+ cells vs. 19 +/- 2 % CD540 cells in control; p < 0.001). In addition, this treatment induced the expression of tissue factor (CD142) on HUVECs (54 +/- 20% CD142+ cells vs 4 +/- 2% CD142+ cells in control; p = 0.008) and increased exposure of phosphatidylserine (PS) (29 +/- 2% PS+ cells vs. 12 +/- 5% PS+ cells in control; p < 0.001). Analysis of cell cycle and DNA fragmentation (TUNEL) showed that both nC60 and C60(OH)24 caused G1 arrest of HUVECs and C60(OH)24 induced significant apoptosis (21 +/- 2% TUNEL+ cells at 100 microg/mL of C60(OH)24 vs. 4 +/- 2% TUNEL+ cells in control; p < 0.001). We also demonstrated that both nC60 and C60(OH)24 induced a rapid concentration dependent elevation of intracellular calcium [Ca2+]i. This could be inhibited by EGTA, suggesting that the source of [Ca2+]i in fullerene stimulated calcium flux is predominantly from the extracellular environment. In conclusion, fullerenol C60(OH)24 had both pro-inflammatory and pro-apoptotic effects on HUVECs, indicating possible adverse effects of fullerenes on the endothelium.
Assuntos
Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Fulerenos/efeitos adversos , Molécula 1 de Adesão Intercelular/metabolismo , Tromboplastina/metabolismo , Apoptose/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Células Endoteliais/citologia , Expressão Gênica/efeitos dos fármacos , HumanosRESUMO
Digitoxin and other cardiac glycosides are important, centuries-old drugs for treating congestive heart failure. However, the mechanism of action of these compounds is still being elucidated. Calcium is known to potentiate the toxicity of these drugs, and we have hypothesized that digitoxin might mediate calcium entry into cells. We report here that digitoxin molecules mediate calcium entry into intact cells. Multimers of digitoxin molecules also are able to form calcium channels in pure planar phospholipid bilayers. These digitoxin channels are blocked by Al(3+) and La(3+) but not by Mg(2+) or the classical l-type calcium channel blocker, nitrendipine. In bilayers, we find that the chemistry of the lipid affects the kinetics of the digitoxin channel activity, but not the cation selectivity. Antibodies against digitoxin promptly neutralize digitoxin channels in both cells and bilayers. We propose that these digitoxin calcium channels may be part of the mechanism by which digitoxin and other active cardiac glycosides, such as digoxin, exert system-wide actions at and above the therapeutic concentration range.