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1.
Blood ; 132(7): 727-734, 2018 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-29914979

RESUMO

Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder initiated by antibodies to platelet factor 4 (PF4)/heparin complexes. PF4 released from platelets binds to surface glycosaminoglycans on hematopoietic and vascular cells that are heterogenous in composition and differ in affinity for PF4. PF4 binds to monocytes with higher affinity than to platelets, and depletion of monocytes exacerbates thrombocytopenia in a murine HIT model. Here we show that the expression of PF4 on platelets and development of thrombocytopenia are modulated by the (re)distribution of PF4 among hematopoietic and endothelial cell surfaces. Binding of PF4 to platelets in whole blood in vitro varies inversely with the white cell count, likely because of the greater affinity of monocytes for PF4. In mice, monocyte depletion increased binding of PF4 to platelets by two- to three-fold. Induction of HIT in mice caused a transient >80-fold increase in binding of HIT antibody to monocytes vs 3.5-fold increase to platelets and rapid transient monocytopenia. Normalization of monocyte counts preceded the return in platelet counts. Exposure of blood to endothelial cells also depletes PF4 from platelet surfaces. These studies demonstrate a dynamic interchange of surface-bound PF4 among hematopoetic and vascular cells that may limit thrombocytopenia at the expense of promoting prothrombotic processes in HIT.


Assuntos
Antígenos/biossíntese , Plaquetas/metabolismo , Heparina/efeitos adversos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Monócitos/metabolismo , Fator Plaquetário 4/biossíntese , Trombocitopenia/metabolismo , Animais , Plaquetas/patologia , Regulação da Expressão Gênica , Heparina/uso terapêutico , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Camundongos , Monócitos/patologia , Trombocitopenia/induzido quimicamente , Trombocitopenia/patologia
2.
Gynecol Oncol ; 122(3): 648-55, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21684584

RESUMO

OBJECTIVES: Carcinogenesis of the ovary is often associated with endometriosis. We previously demonstrated that antitumor chemokine receptor CXCR3 was upregulated both in endometriosis and ovarian cancers. Currently, little is known about the roles of CXCR3 variants in these ovarian diseases. In this study, we investigated the expression of CXCR3 variants and their corresponding ligands in endometriosis and ovarian cancers. METHODS: The expression patterns of CXCR3 variants (CXCR3A, CXCR3B and CXCR3-alt) and their corresponding ligands were investigated by quantitative RT-PCR, Western blot and in situ hybridization in normal ovaries (n=16), endometriosis (n=12), and clear cell ovarian cancers (n=22) including endometriosis-coexisting cases (n=11). RESULTS: Sequence analysis of purified RT-PCR products confirmed the presence of three CXCR3 variants in human ovaries. Quantitative RT-PCR analysis revealed differential expression patterns of these variants depending on conditions. CXCR3A was upregulated both in endometriosis and cancers. On the other hand, CXCR3-alt was upregulated and CXCR3B was downregulated in cancers compared with endometriosis. The corresponding ligand CXCL11 was upregulated only in the cancers with elevated CXCR3-alt. Another ligand CXCL4 was downregulated in the cancers with suppressed CXCR3B. In situ hybridization demonstrated preferential expression of CXCR3A in cancer cells and infiltrating lymphocytes. CXCR3B and CXCR3-alt were detectable mainly in microvessels. CONCLUSIONS: Collective data suggest that differential expression patterns of CXC chemokines and CXCR3 variants are involved in specific inflammatory microenvironment of ovarian cancers. Altered balance of CXCR3 variants may become helpful information for better understanding of the pathogenesis of ovarian cancers arising from endometriosis.


Assuntos
Quimiocina CXCL11/biossíntese , Endometriose/imunologia , Neoplasias Ovarianas/imunologia , Fator Plaquetário 4/biossíntese , Receptores CXCR3/biossíntese , Adulto , Idoso , Quimiocina CXCL11/genética , Regulação para Baixo , Endometriose/genética , Feminino , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/genética , Fator Plaquetário 4/genética , Isoformas de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Receptores CXCR3/genética
3.
PLoS One ; 15(5): e0232661, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32379796

RESUMO

Platelet factor 4 is a cytokine released into the bloodstream by activated platelets where it plays a pivotal role in etiology and diagnosis of heparin-induced thrombocytopenia. Therefore, a sustainable source of recombinant PF4 with structural and functional similarity to its native form is urgently needed to be used in diagnostic procedures. To this end, a three-in-one primary construct was designed from which three secondary constructs can be derived each capable of employing either type I, type II secretory or cytoplasmic pathways. Protein expression and secretion were performed in Escherichia coli BL21 (DE3) and confirmed by SDS-PAGE and Western blotting. To further enhance protein secretion, the effect of several controllable chemical factors including IPTG, Triton X-100, sucrose, and glycine were individually investigated at the outset. In the next step, according to a fractional factorial approach, the synergistic effects of IPTG, Triton X-100, and glycine on secretion were further investigated. To ascertain the structure and function of the secreted recombinant proteins, dynamic light scattering was utilized to confirm the rPF4 tetramerization and heparin-mediated ultra-large complex formation. Moreover, Raman spectroscopy and Western blotting were exploited to evaluate the secondary and quaternary structures, respectively. The type II secretory pathway was proven to be superior to type I in the case of rPF4 secretion. Supplementation with chemical enhancers improved the protein secretion mediated by the Type II system to approximately more than 500 µg/mL. Large quantities of native rPF4 up to 20 mg were purified as the culture medium was scaled up to 40 mL. Western blotting confirmed the formation of dimers and tetramers in the secreted rPF4 proteins. Dynamic light scattering revealed the rPF4 oligomerization into of larger complexes of approximately 100-1200 nm in size following heparin supplementation, implying proper protein folding and tetramerization. Moreover, the rPF4 secondary structure was found to be 43.5% Random coil, 32.5% ß-sheet, 18.6% α-helix and 4.9% Turn, which is in perfect agreement with the native structure. Our results indicate that the gram-negative type II bacterial secretory system holds a great promise as a reliable protein production strategy with industrial applications. However, further efforts are required to realize the full potential of secretory pathways regarding their application to proteins with distinct characteristics.


Assuntos
Fator Plaquetário 4/biossíntese , Fator Plaquetário 4/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Sistemas de Secreção Tipo II , Clonagem Molecular , Escherichia coli/genética
4.
J Cell Biol ; 96(2): 515-20, 1983 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-6833369

RESUMO

The site of synthesis of platelet-specific proteins remains to be established. With the use of short-term megakaryocyte-enriched cultures, direct evidence was obtained to show that megakaryocytes synthesize the platelet-specific protein, platelet factor 4. A megakaryocyte-enriched fraction of rabbit bone marrow for culture was obtained by centrifugal elutriation and cultured with [3H]leucine. Newly synthesized 3H-platelet factor 4 was sought by copurification with added carrier rabbit platelet factor 4, using heparin agarose affinity chromatography and immunoprecipitation with specific goat anti-rabbit platelet factor 4 antisera. SDS PAGE of the washed immunoprecipitates demonstrated a [3H]leucine-containing peak which migrated identically with purified homogeneous rabbit platelet factor 4. A second, slightly larger molecular-weight protein was identified in the gels also, suggesting that rabbit platelet factor 4 may be synthesized as a larger molecular-weight precursor in rabbit megakaryocytes. These results provide direct evidence that the platelet-specific protein, platelet factor 4, is synthesized in rabbit megakaryocytes before it is packaged into alpha-granules for release in circulating platelets.


Assuntos
Fatores de Coagulação Sanguínea/biossíntese , Megacariócitos/metabolismo , Fator Plaquetário 4/biossíntese , Animais , Células da Medula Óssea , Células Cultivadas , Cromatografia de Afinidade , Heparina/metabolismo , Megacariócitos/ultraestrutura , Microscopia Eletrônica , Coelhos
5.
J Neurochem ; 105(5): 1726-36, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18248618

RESUMO

Signaling through chemokine receptor CXCR3 in the brain has been implicated in various brain diseases, as CXCR3 and its ligands are found under these conditions. Recently, a new chemokine ligand for CXCR3 was reported. In humans, an alternatively spliced variant of CXCR3 expressed on microvascular endothelial cells, named CXCR3b, was shown to bind CXCL4. In the periphery, the cellular expression and functions of CXCL4 are well described but in the brain its expression and function are unknown. Here, we show that brain microglia are a cellular source of CXCL4 in vitro and in vivo under neurodegenerating conditions. Microglial migration induced by CXCL4 is absent in CXCR3-deficient microglia, indicating a role of CXCR3. CXCL4 furthermore attenuates lipopolysaccharide-induced microglial phagocytosis and nitric oxide production in microglia and BV-2 cells. Based on these findings, it is proposed that locally released CXCL4 may control microglia responses.


Assuntos
Regulação da Expressão Gênica/fisiologia , Microglia/metabolismo , Fator Plaquetário 4/biossíntese , Receptores CXCR3/fisiologia , Transdução de Sinais/fisiologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Microglia/fisiologia , Técnicas de Cultura de Órgãos , Fator Plaquetário 4/genética , Receptores CXCR3/genética
6.
J Neuropathol Exp Neurol ; 77(7): 567-576, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29757405

RESUMO

Despite growing evidence for platelets as active players in infection and immunity, it remains unresolved whether platelets contribute to, or are key elements in the development of neuroinflammation. Using the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis, we identified platelet accumulation in the circulation by 7-day postinduction (dpi), ahead of clinical onset which occurs at 13-14 dpi. By inducing platelet depletion between 7 and 16 dpi, we demonstrate an association between platelet accumulation in the spinal cord and disease development. Additionally, we provide evidence for platelet infiltration in the white and gray matter parenchyma, but with different outcomes. Thus, while in white matter platelets are clearly associated with lesions, in gray matter large-scale platelet infiltration and expression of the platelet-specific molecule PF4 are detectable prior to T cell entry. In the retina, platelet accumulation also precedes clinical onset and is associated with significant increase in retinal thickness in experimental relative to control animals. Platelet accumulation increases over the disease course in this tissue, but without subsequent T cell infiltration. These findings provide definitive confirmation that platelet accumulation is key to EAE pathophysiology. Furthermore, they suggest an undescribed and, most importantly, therapeutically targetable mechanism of neuronal damage.


Assuntos
Encefalomielite Autoimune Experimental/patologia , Substância Cinzenta/patologia , Inflamação/sangue , Retina/patologia , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Fator Plaquetário 4/biossíntese , Medula Espinal/patologia , Substância Branca/patologia
7.
Mol Cell Biol ; 24(1): 428-41, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14673175

RESUMO

Ets-1 is important for transcriptional regulation in several hematopoietic lineages, including megakaryocytes. Some transcription factors bind to naked DNA and chromatin with different affinities, while others do not. In the present study we used the megakaryocyte-specific promoters platelet factor 4 (PF4), and glycoprotein IIb (GPIIb) as model systems to explore the properties of Ets-1 binding to chromatin. Chromatin immunoprecipitation assays indicated that Ets-1 binds to proximal regions in the PF4 and GPIIb promoters in vivo. In vitro and in vivo experiments showed that Ets-1 binding to chromatin on lineage-specific promoters does not require lineage-specific factors. Moreover, this binding shows the same order of affinity as the binding to naked DNA and does not require ATP-dependent or Sarkosyl-sensitive factors. The effect of Ets-1 binding on promoter activity was examined using the PF4 promoter as a model. We identified a novel Ets-1 site (at -50), and a novel Sarkosyl-sensitive DNase I-hypersensitive site generated by Ets-1 binding to chromatin, which significantly affect PF4 promoter activity. Taken together, our results suggest a model by which Ets-1 binds to chromatin without the need for lineage-specific accessory factors, and Ets-1 binding induces changes in chromatin and affects transactivation, which are essential for PF4 promoter activation.


Assuntos
Cromatina/metabolismo , Megacariócitos/metabolismo , Fator Plaquetário 4/genética , Glicoproteínas da Membrana de Plaquetas , Proteínas Proto-Oncogênicas/metabolismo , Sarcosina/análogos & derivados , Fatores de Transcrição/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cromatina/efeitos dos fármacos , Detergentes/farmacologia , Regulação da Expressão Gênica , Humanos , Camundongos , Nucleossomos/metabolismo , Fator Plaquetário 4/biossíntese , Complexo Glicoproteico GPIb-IX de Plaquetas/biossíntese , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Regiões Promotoras Genéticas , Proteína Proto-Oncogênica c-ets-1 , Proteínas Proto-Oncogênicas c-ets , Ratos , Sarcosina/farmacologia
8.
Microbes Infect ; 8(4): 974-83, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16488173

RESUMO

In this study, we investigated cytokine expression during experimental pneumococcal meningitis. Mice were intracisternally infected with Streptococcus pneumoniae and treated with ceftriaxone starting at 24 h after infection. At different time points before and after antibiotic therapy, the cytokine expression pattern was determined in mouse brains using protein arrays. Underlining the power of this method, the meningitis-relevant cytokines interleukin-1beta (IL-1beta), IL-6, KC, macrophage inflammatory protein-2 (MIP-2), and monocyte chemoattractant protein-1 (MCP-1/CCL2) were markedly elevated in infected animals. Newly identified proteins during the acute stage of the disease (until 30 h after infection) included lymphotactin (XCL-1), MIP-1gamma (CCL9) and MCP-5 (CCL12), cytokine responsive gene- 2 (CRG-2/CXCL10) and CXCL16, and insulin-like growth factor binding protein 3 (IGFBP3). During later stages, an induction of T-cell activation-3 (TCA-3/CCL1), platelet factor-4 (PF-4/CXCL4) and stromal derived factor-1alpha (SDF-1alpha/CXCL13), and IL-4 was observed. The validity of this method was supported by an additional ELISA analysis of the expression profile of CXCL16 and IGFBP3, which was identical to that observed by protein array. In conclusion, the use of protein array technology led to an extension of the current picture of protein expression in pneumococcal meningitis. Most important, new factors that might play a role in pneumococcal meningitis were identified.


Assuntos
Encéfalo/metabolismo , Quimiocinas C/biossíntese , Proteínas Inflamatórias de Macrófagos/biossíntese , Meningite Pneumocócica/metabolismo , Proteínas Quimioatraentes de Monócitos/biossíntese , Monocinas/biossíntese , Fator Plaquetário 4/biossíntese , Streptococcus pneumoniae , Animais , Antibacterianos/uso terapêutico , Encéfalo/imunologia , Ceftriaxona/uso terapêutico , Quimiocina CXCL10 , Quimiocina CXCL13 , Quimiocinas CC , Quimiocinas CXC/biossíntese , Citocinas/biossíntese , Modelos Animais de Doenças , Interleucina-4/biossíntese , Meningite Pneumocócica/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Análise Serial de Proteínas , Fatores de Tempo
9.
Cancer Res ; 51(1): 341-8, 1991 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-1824823

RESUMO

A new human leukemia cell line with megakaryocytic features, designated UT-7, was established from the bone marrow of a patient with acute megakaryoblastic leukemia. Surface marker analysis revealed that the majority of the cells reacted with monoclonal antibodies against platelet glycoprotein Ib (CD42b), glycoprotein IIb/IIIa (CD41a), MY 7 (CD13), MY 9 (CD33), and glycophorin A antigens. Cytogenetic analysis showed a human male near-tetraploid karyotype with a modal chromosome number of 92-96. Flow cytometry-derived DNA histograms demonstrated that the majority of the cells spontaneously contained 4 N DNA ploidy levels. Ultrastructural study showed that platelet peroxidase activity was weakly positive but myeloperoxidase activity was negative. Ferritin and theta-granule, which have been used as ultrastructural markers for the erythroid lineage, could not be detected. In response to phorbol myristate acetate, platelet factor 4 and beta-thromboglobulin, which were specifically synthesized in the process of megakaryocyte maturation, dramatically increased in UT-7 cells. This was accompanied by an increase in cell size, ploidy level, platelet peroxidase activity, and the surface density of glycoprotein IIb/IIIa antigen. These findings suggest that UT-7 is a new leukemic cell line with megakaryocytic features and with the potential to differentiate into cells with more mature megakaryocytic properties in response to phorbol myristate acetate. This cell line showed strict dependency on interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor, or erythropoietin. The maximal effective doses of IL-3, granulocyte-macrophage colony-stimulating factor, and erythropoietin for proliferation in liquid culture were 10 units/ml, 1 ng/ml, and 1 unit/ml, respectively. These concentrations were comparable to the doses that maximally stimulate the clonal growth of normal hemopoietic cells. IL-6 could stimulate the proliferation of UT-7 cells but not maintain the line in long-term culture. UT-7 cells may be a useful model for (a) the analysis of gene regulation of megakaryocytic maturation-associated proteins expressed in the process of megakaryocytic differentiation and (b) the study of signal transduction of hemopoietic factors associated with megakaryocytopoiesis.


Assuntos
Trombocitemia Essencial/patologia , Antígenos CD/análise , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Sinergismo Farmacológico , Eritropoetina/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Hematopoese/efeitos dos fármacos , Humanos , Interleucina-3/farmacologia , Interleucina-6/farmacologia , Cariotipagem , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Fator Plaquetário 4/biossíntese , Acetato de Tetradecanoilforbol/farmacologia , Trombocitemia Essencial/fisiopatologia , Células Tumorais Cultivadas , beta-Tromboglobulina/biossíntese
10.
Eur Cytokine Netw ; 16(4): 289-92, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16464743

RESUMO

Since cytokines and chemokines are important actors in rheumatoid arthritis (RA), the aim of this study was to compare the gene expression profiles in cultured fibroblast-like synoviocytes (FLS) obtained from patients with either RA, or osteoarthritis (OA), focusing our analysis on genes for cytokines and chemokines, and their respective receptors. Gene expression in cultured FLS (third passage) from eight patients with RA (RA-FLS) were compared with gene expression in cultured FLS from nine patients with OA (OA-FLS) using Affymetrix Human Genome U133 Plus 2.0 Array microarray, allowing analysis of over 54,000 transcripts. Among the 171 genes studied (241 probes), limiting the selection of differentially expressed genes to a significant value (p < 0.05), and a differential ratio of expression > 1.6, only four genes, namely IL-32, CCL2, PF4F1 and GDF10 were found to be differentially expressed. Out of these four genes, only higher expression of CCL2 has been reported previously in RA. The newly described cytokine IL-32 was the most prominently differentially expressed gene in the present study, with higher expression in RA-FLS than in OA-FLS (p < 0.0073). IL-32 might have a previously unidentified pivotal role in RA.


Assuntos
Artrite Reumatoide/genética , Proteína Morfogenética Óssea 3/genética , Quimiocina CCL2/genética , Fator 10 de Diferenciação de Crescimento/genética , Interleucinas/genética , Osteoartrite/genética , Fator Plaquetário 4/genética , Membrana Sinovial/citologia , Artrite Reumatoide/metabolismo , Proteína Morfogenética Óssea 3/biossíntese , Células Cultivadas , Quimiocina CCL2/biossíntese , Perfilação da Expressão Gênica , Fator 10 de Diferenciação de Crescimento/biossíntese , Humanos , Interleucinas/biossíntese , Análise de Sequência com Séries de Oligonucleotídeos , Osteoartrite/metabolismo , Fator Plaquetário 4/biossíntese , Membrana Sinovial/metabolismo
11.
Anticancer Res ; 25(2A): 847-51, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15868918

RESUMO

Tumor growth and metastasis depend on angiogenesis, which is triggered by a chemical signal from the tumor cells to resting endothelial cells which then enter into a phase of rapid growth. Platelet Factor 4 (PF4) inhibits endothelial proliferation in vitro and angiogenesis in vivo. PF4 also inhibits tumor growth, however, as with other angiogenesis inhibitors, sustained tumor growth inhibition requires prolonged exposure to the recombinant protein. In this study, Lewis lung carcinoma (LLH) cells were transfected with the human PF4 via mammalian expression vectors and the ability of the transfected cells to form tumors and metastasis in vivo was evaluated. To evaluate the tumor growth rate of PF4-transfected (LLH/PF4) or control (LLH/neo) cells in vivo, we injected LLH/PF4 or LLH/neo cells subcutaneously (s.c.) or intravenously (i.v.). In the s.c. assay, LLH/PF4 had no significant effect on tumor growth. Conversely, in the i.v. assay, PF4 significantly reduced the number of lung metastasis (p=0.019) and weight (p=0.056). The inhibition of lung metastasis suggests that PF4 may inhibit tumor-associated neovascularization, and may prevent the affinity of tumor cells for the normal lung tissue.


Assuntos
Carcinoma Pulmonar de Lewis/irrigação sanguínea , Carcinoma Pulmonar de Lewis/terapia , Neovascularização Patológica/genética , Neovascularização Patológica/terapia , Fator Plaquetário 4/genética , Animais , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/patologia , Processos de Crescimento Celular/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células NIH 3T3 , Transplante de Neoplasias , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Fator Plaquetário 4/biossíntese , Transfecção
12.
Leukemia ; 9(2): 341-9, 1995 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-7869773

RESUMO

We have established a novel human megakaryoblastic cell line, designated as MEG-A2, from a patient with megakaryoblastic crisis of Philadelphia (Ph) chromosome positive chronic myelogenous leukemia. MEG-A2 cells showed positive phenotypes for periodic acid Schiff and alpha-naphthylbutyrate esterase reactions, but were negative for myeloperoxidase and naphthol ASD chloroacetate esterase reactions. Flow cytometric analyses of cell surface markers revealed that MEG-A2 cells had a low level of GP IIb/IIIa expression as well as apparent expressions of CD4, CD7, CD13, CD33 and CD34 antigens, but no expression of GP Ib nor glycophorin A. Stimulation with phorbol 12-myristate 13-acetate (PMA) dramatically increased the expression of megakaryocyte-related markers such as HPL-3, J15, Pit-1, Y2/51 and AN51 in MEG-A2 cells. The PMA-stimulation also induced expression of platelet peroxidase (PPO) in MEG-A2 cells on electromicroscopic observation. Proliferative responses to granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) or erythropoietin were observed, and the expression of GP IIb/IIIa was increased by stimulation with GM-CSF, IL-3, erythropoietin and interleukin-6 (IL-6). Protein S mRNA expression was seen in cultured cells on Northern blot analysis. Expression of platelet factor 4 mRNA was induced in PMA-stimulated cells, and a marked accumulation of protein was observed in the culture medium. In conclusion, a new cell line, MEG-A2, belongs to the relatively immature megakaryocytic lineage and has markedly increased megakaryocytic characteristics with PMA stimulation.


Assuntos
Células-Tronco Hematopoéticas/patologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Megacariócitos , Células-Tronco Neoplásicas/patologia , Receptores de Citocinas , Células Tumorais Cultivadas , Adulto , Aneuploidia , Antígenos CD/análise , Antígenos de Diferenciação/análise , Sequência de Bases , Biomarcadores Tumorais/análise , Crise Blástica/patologia , Hidrolases de Éster Carboxílico , Divisão Celular/efeitos dos fármacos , Evolução Fatal , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Fatores de Crescimento de Células Hematopoéticas/farmacologia , Células-Tronco Hematopoéticas/química , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Cariotipagem , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Masculino , Dados de Sequência Molecular , Proteínas de Neoplasias/análise , Células-Tronco Neoplásicas/química , Células-Tronco Neoplásicas/efeitos dos fármacos , Fator Plaquetário 4/biossíntese , Fator Plaquetário 4/genética , Glicoproteínas da Membrana de Plaquetas/análise , Proteína S/biossíntese , Proteína S/genética , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Receptores Imunológicos/biossíntese , Receptores Imunológicos/genética , Receptores de Trombopoetina , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas/química , Células Tumorais Cultivadas/efeitos dos fármacos , beta-Tromboglobulina/biossíntese , beta-Tromboglobulina/genética
13.
Clin Cancer Res ; 10(13): 4527-37, 2004 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-15240545

RESUMO

PURPOSE: We investigated the ability of the combinatorial administration of different inhibitors with activities on glioma angiogenesis, migration, and proliferation to produce a prolonged inhibition of glioma growth. EXPERIMENTAL DESIGN: We combined inhibitors affecting solely tumor angiogenesis (PF-4/CTF, cyclo-VEGI) or inhibitors affecting both angiogenesis and invasion together (PEX, PF-4/DLR). RESULTS: When administered in combination, these drugs produced a prolonged and increased inhibition of glioma growth independently from the type of inhibitor used. The combinatory administration was more effective than the administration of a single inhibitor alone, and a strong therapeutic response was reached with a significantly lower amount of protein. The strongest inhibition was observed when human PEX and PF-4/DLR, which affect both glioma angiogenesis and invasion by separate mechanisms, were combined. CONCLUSIONS: This supports the concept that prolonged glioma growth inhibition can be achieved by simultaneous delivery of molecules that target both tumor and endothelial cells and acting by separate mechanisms.


Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Glioma/tratamento farmacológico , Neovascularização Patológica , Animais , Apoptose , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Colágeno , Modelos Animais de Doenças , Combinação de Medicamentos , Fatores de Crescimento Endotelial/biossíntese , Endotélio Vascular/patologia , Glioma/patologia , Humanos , Laminina , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microcirculação , Microscopia de Fluorescência , Invasividade Neoplásica , Transplante de Neoplasias , Neoplasias/patologia , Endopeptidase Neutra Reguladora de Fosfato PHEX , Peptídeos Cíclicos/biossíntese , Fator Plaquetário 4/biossíntese , Proteínas/metabolismo , Proteoglicanas , Proteínas Recombinantes/química , Fatores de Tempo
14.
Exp Hematol ; 23(5): 397-401, 1995 May.
Artigo em Inglês | MEDLINE | ID: mdl-7720812

RESUMO

We have examined the effect of recombinant murine interleukin-6 (rmIL-6) on megakaryocytopoiesis in a liquid rat bone marrow culture system. At concentrations of IL-6 up to 100 ng/mL, no stimulation of megakaryocyte ploidy was observed. However, transient expression studies revealed that IL-6 did have a significant effect on the transcriptional activity of the platelet factor four (PF4) gene, a platelet alpha-granule protein gene uniquely expressed in megakaryocytes. Furthermore, when the amount of PF4 message was directly measured in megakaryocytes, it was increased three-fold in response to IL-6. We also note that the PF4 promoter contains a hexamer, CTGGGA, described as the IL-6-responsive element in other genes. Our results suggest that the platelet progeny of IL-6-stimulated megakaryocytes may have altered alpha-granule constituents.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-6/farmacologia , Fator Plaquetário 4/genética , Proteínas Recombinantes de Fusão/farmacologia , Transcrição Gênica/efeitos dos fármacos , Animais , Sequência de Bases , Células da Medula Óssea , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Citometria de Fluxo , Genes , Hormônio do Crescimento/biossíntese , Hormônio do Crescimento/genética , Humanos , Megacariócitos/efeitos dos fármacos , Megacariócitos/ultraestrutura , Camundongos , Dados de Sequência Molecular , Plasmídeos , Fator Plaquetário 4/biossíntese , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Transfecção
15.
PLoS One ; 10(3): e0118551, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25738304

RESUMO

Deregulated expression of oncogenes or transcription factors such as specificity protein 1 (Sp1) is observed in many human cancers and plays a role in tumor maintenance. Paradoxically in untransformed cells, Sp1 overexpression induces late apoptosis but the early intrinsic response is poorly characterized. In the present work, we studied increased Sp1 level consequences in untransformed cells and showed that it turns on an early innate immune transcriptome. Sp1 overexpression does not activate known cellular stress pathways such as DNA damage response or endoplasmic reticulum stress, but induces the activation of the OAS-RNase L pathway and the generation of small self-RNAs, leading to the upregulation of genes of the antiviral RIG-I pathway at the transcriptional and translational levels. Finally, Sp1-induced intrinsic innate immune response leads to the production of the chemokine CXCL4 and to the recruitment of inflammatory cells in vitro and in vivo. Altogether our results showed that increased Sp1 level in untransformed cells constitutes a novel danger signal sensed by the OAS-RNase L axis leading to the activation of the RIG-I pathway. These results suggested that the OAS-RNase L-RIG-I pathway may be activated in sterile condition in absence of pathogen.


Assuntos
2',5'-Oligoadenilato Sintetase/metabolismo , RNA Helicases DEAD-box/metabolismo , Endorribonucleases/metabolismo , Transdução de Sinais , Fator de Transcrição Sp1/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular , Transformação Celular Neoplásica , Proteína DEAD-box 58 , Expressão Gênica , Humanos , Imunidade Inata/genética , Fator Regulador 3 de Interferon/genética , Camundongos , Fator Plaquetário 4/biossíntese , Regiões Promotoras Genéticas/genética , Receptores Imunológicos , Transdução de Sinais/imunologia , Fator de Transcrição Sp1/metabolismo , Transcrição Gênica , Transcriptoma , Regulação para Cima , Vesiculovirus/fisiologia
16.
J Clin Invest ; 125(5): 1857-72, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25822018

RESUMO

Myelodysplastic syndromes and chronic myelomonocytic leukemia (CMML) are characterized by mutations in genes encoding epigenetic modifiers and aberrant DNA methylation. DNA methyltransferase inhibitors (DMTis) are used to treat these disorders, but response is highly variable, with few means to predict which patients will benefit. Here, we examined baseline differences in mutations, DNA methylation, and gene expression in 40 CMML patients who were responsive or resistant to decitabine (DAC) in order to develop a molecular means of predicting response at diagnosis. While somatic mutations did not differentiate responders from nonresponders, we identified 167 differentially methylated regions (DMRs) of DNA at baseline that distinguished responders from nonresponders using next-generation sequencing. These DMRs were primarily localized to nonpromoter regions and overlapped with distal regulatory enhancers. Using the methylation profiles, we developed an epigenetic classifier that accurately predicted DAC response at the time of diagnosis. Transcriptional analysis revealed differences in gene expression at diagnosis between responders and nonresponders. In responders, the upregulated genes included those that are associated with the cell cycle, potentially contributing to effective DAC incorporation. Treatment with CXCL4 and CXCL7, which were overexpressed in nonresponders, blocked DAC effects in isolated normal CD34+ and primary CMML cells, suggesting that their upregulation contributes to primary DAC resistance.


Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Azacitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Genes Neoplásicos , Leucemia Mielomonocítica Crônica/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/farmacologia , Azacitidina/uso terapêutico , Medula Óssea/patologia , Metilação de DNA/efeitos dos fármacos , Análise Mutacional de DNA , DNA Intergênico/genética , Decitabina , Elementos Facilitadores Genéticos/genética , Feminino , Humanos , Leucemia Mielomonocítica Crônica/genética , Leucemia Mielomonocítica Crônica/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Fator Plaquetário 4/biossíntese , Fator Plaquetário 4/genética , Fator Plaquetário 4/fisiologia , Resultado do Tratamento , beta-Tromboglobulina/biossíntese , beta-Tromboglobulina/genética , beta-Tromboglobulina/fisiologia
17.
Thromb Haemost ; 52(1): 4-6, 1984 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-6238444

RESUMO

Platelet rich plasma (PRP) exposed in vitro to 200 mm Hg above atmospheric pressure showed a significant increase in malondialdehyde (MDA) formation compared to PRP at atmospheric pressure. This difference is also evident when platelets are incubated with arachidonic acid. The increase of MDA demonstrates that the increased beta-thromboglobulin and platelet factor 4 in plasma and the shape changes of platelets after pressure stimulation in vitro that were described in a previous paper result from the release reaction. Pressure-induced effects in vivo are discussed.


Assuntos
Plaquetas/metabolismo , Malonatos/sangue , Malondialdeído/sangue , Pressão , Humanos , Técnicas In Vitro , Fator Plaquetário 4/biossíntese , Prostaglandinas/sangue , beta-Tromboglobulina/biossíntese
18.
Thromb Haemost ; 54(4): 842-8, 1985 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-2935968

RESUMO

Heparin is known to affect platelet function in vitro, but little is known about the effect of heparin on the interaction of platelets with polymer surfaces in general, and vascular graft materials in particular. For this reason, the effect of heparin vs. citrate anticoagulation on the interaction of platelets with the vascular graft materials expanded polytetrafluoroethylene (ePTFE), Dacron Bionit (DB) and preclotted Dacron Bionit (DB/PC) was studied in a recirculating, in vitro perfusion system. Platelet activation, as shown by a decrease in platelet count, an increase in platelet release and a decrease in platelet aggregation, was observed for all vascular graft materials tested using heparin and was greater for Dacron and preclotted Dacron than for ePTFE. Significant differences between heparin and citrate anticoagulation were seen for platelet release, platelet aggregation and the relative ranking of material platelet-reactivity. However, the trends and time course of platelet activation were similar with both heparin and citrate for the materials tested.


Assuntos
Plaquetas/efeitos dos fármacos , Prótese Vascular , Citratos/farmacologia , Heparina/farmacologia , Adulto , Materiais Biocompatíveis , Plaquetas/fisiologia , Ácido Cítrico , Fibrinopeptídeo A/metabolismo , Humanos , Técnicas In Vitro , Agregação Plaquetária/efeitos dos fármacos , Contagem de Plaquetas , Fator Plaquetário 4/biossíntese , Polietilenotereftalatos , Politetrafluoretileno , beta-Tromboglobulina/metabolismo
19.
Thromb Haemost ; 83(2): 327-33, 2000 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10739394

RESUMO

Thrombin plays a central role in the genesis of thrombotic events and is the most potent known platelet agonist. This enzyme activates platelets by cleaving G-protein coupled protease activated receptors (PARs) and by binding to glycoprotein (GP) Ib. Thrombin also cleaves platelet GPV to liberate a soluble 69 kDa fragment (GPVf1), leaving a 20 kDa fragment (GPVf2) attached to the membrane. The aim of this study was to assess the value of GPV as an in vivo marker of the activation of platelets by thrombin. Newly developed monoclonal and polyclonal antibodies recognizing rat GPVf1 and GPVf2 respectively were used to detect soluble GPV by ELISA and the new NH2-terminus exposed by thrombin using flow cytometry. These assays were employed in a rat thrombosis model designed to trigger thrombin formation in vivo. When thromboplastin (4.8 ml/kg/h) was infused for 30 min, thrombin generation was reflected by a rapid increase in thrombin-antithrombin (TAT) complexes in plasma and by the appearance of GPVf2 at the surface of circulating platelets. Simultaneously, GPVf1 disappeared from the surface of platelets and accumulated as a soluble fragment in plasma, where it was detected by GPV ELISA. These effects were inhibited by pretreatment of the rats with hirudin. Levels of plasma PF4 also increased in this model, but unlike GPV levels which returned slowly (> 2 hours) to baseline, PF4 had a very short half-life. In conclusion, GPV is cleaved by thrombin in vivo, circulates and is a reliable in vivo marker of the activation of platelets by thrombin. Monitoring of GPV levels in rats should be useful to evaluate the effects of antithrombotic and antiplatelet drugs, while further studies will be required to confirm the potential interest of GPV as a marker of thrombotic states in humans.


Assuntos
Ativação Plaquetária , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Trombose/sangue , Animais , Anticorpos Monoclonais , Antitrombina III/biossíntese , Antitrombina III/efeitos dos fármacos , Biomarcadores/sangue , Plaquetas/efeitos dos fármacos , Coagulantes/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Epitopos , Citometria de Fluxo , Elastase Pancreática/metabolismo , Peptídeo Hidrolases/biossíntese , Peptídeo Hidrolases/efeitos dos fármacos , Fator Plaquetário 4/biossíntese , Fator Plaquetário 4/efeitos dos fármacos , Complexo Glicoproteico GPIb-IX de Plaquetas/imunologia , Complexo Glicoproteico GPIb-IX de Plaquetas/fisiologia , Glicoproteínas da Membrana de Plaquetas/imunologia , Glicoproteínas da Membrana de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/fisiologia , Ratos , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Receptores de Superfície Celular/fisiologia , Sensibilidade e Especificidade , Solubilidade , Trombina/biossíntese , Trombina/metabolismo , Trombina/farmacologia , Tromboplastina/administração & dosagem , Tromboplastina/farmacologia , Trombose/metabolismo
20.
Biomaterials ; 10(3): 147-55, 1989 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-2524222

RESUMO

A recirculating in vitro perfusion system was used to assess the effect of albumin precoating on the thrombogenicity of Dacron vascular grafts. A complete analysis of platelet activation was carried out, involving platelet count, release, adhesion and aggregation. Fibrin formation was assessed by measuring fibrinogen levels and fibrinopeptide A production; leucocyte interaction was analysed by measuring total leucocyte count as well as an analysis of cell adhesion to the surface by scanning electron microscopy. The platelet count decreased progressively with perfusion time for Dacron until by 30 min, it had declined to 69% +/- 2% of baseline. The platelet count did not, however, change significantly from baseline when albumin-coated Dacron was tested. Release of platelet factor 4 and beta-thromboglobulin at 180 min for Dacron was 37.8 +/- 29.8 times and 66.9 +/- 18.2 times baseline, respectively, while albumin coating caused significantly less (P less than 0.03) platelet release. Albumin coating diminished coagulation activation and fibrinopeptide A formation. The total leucocyte concentration decreased significantly for Dacron by 180 min, while that for albumin-coated Dacron did not change significantly from baseline levels. Albumin coating produced a film-like covering over the Dacron. For Dacron, there were numerous leucocytes and platelets adherent to the surface, whilst cellular deposition was minimal upon the albumin-coated surface. Thus, albumin coating improved the short-term blood compatibility of Dacron by all of the methods employed in this study.


Assuntos
Albuminas , Materiais Biocompatíveis , Prótese Vascular , Polietilenotereftalatos , Coagulação Sanguínea , Adesão Celular , Humanos , Técnicas In Vitro , Contagem de Leucócitos , Contagem de Plaquetas , Fator Plaquetário 3/biossíntese , Fator Plaquetário 4/biossíntese , Propriedades de Superfície , beta-Tromboglobulina/biossíntese
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