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1.
J Cell Mol Med ; 21(7): 1280-1291, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28004483

RESUMO

A growing body of evidence points towards smoking-related phenotypic differences in chronic obstructive pulmonary disease (COPD). As COPD is associated with systemic inflammation, we determined whether smoking status is related to serum levels of matrix metalloproteinase-9 (pro- and active MMP-9), neutrophil gelatinase-associated lipocalin (NGAL) and the proMMP-9/NGAL complex in patients with COPD. Serum samples were collected in 100 stable-phase COPD patients (82 smokers, 18 never-smokers) and 28 healthy adults (21 smokers, 7 never-smokers). Serum levels of studied factors were measured in ELISA. Our data provide the first evidence of simultaneously elevated serum levels of MMP-9, NGAL and proMMP-9/NGAL in COPD smokers. While the triad discriminated between smokers and non-smokers in the COPD group, MMP-9 and proMMP-9/NGAL (but not NGAL) discriminated between smokers with and without COPD. Adjustment for age and smoking pack-years did not alter the findings. Serum MMP-9, NGAL and proMMP-9/NGAL levels were not correlated with the GOLD stage or FEV1 decline. Furthermore, serum levels of neutrophil elastase (NE) and MMP-3 (but not of IL-6 and MMP-12) were also higher in COPD smokers than in healthy smokers before and after adjustment for age and pack-years. Among COPD smokers, levels of MMP-9, NGAL and proMMP-9/NGAL were positively correlated with NE (P < 0.0001) but not with the remaining factors. Gelatin zymography detected proMMP-9 in serum samples of healthy and COPD smoking groups. Our results suggest that associated serum levels of proMMP-9, NGAL, proMMP-9/NGAL and NE may reflect the state of systemic inflammation in COPD related to cigarette smoking.


Assuntos
Elastase de Leucócito/sangue , Lipocalina-2/sangue , Metaloproteinase 9 da Matriz/sangue , Doença Pulmonar Obstrutiva Crônica/sangue , Adulto , Idoso , Precursores Enzimáticos/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Complexos Multiproteicos/sangue , Doença Pulmonar Obstrutiva Crônica/patologia , Fumantes , Fumar/efeitos adversos , Fumar/sangue
2.
Crit Care Med ; 45(9): e954-e962, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28445239

RESUMO

OBJECTIVES: Vascular mineralocorticoid receptors play a role in vascular tone and blood pressure regulation, might participate in the pathophysiology of circulatory failure during sepsis, and represent a potential therapeutic target in this disease. We aimed to study the effects of mineralocorticoids and the involvement of vascular mineralocorticoid receptors in murine endotoxic and human septic shock. DESIGN: Experimental study. SETTING: Translational investigation including animal research and in vitro experiments using human vascular cells and plasma from septic patients. SUBJECTS: Adult male C57Black 6 mice, adult patients with septic shock. INTERVENTIONS: Mice were injected with lipopolysaccharide and/or aldosterone. Human endothelial and smooth muscle cells were treated with pro-inflammatory cytokines with or without aldosterone, nuclear factor-κB inhibitor BAY 11-7082, or plasma from septic patients. MEASUREMENTS AND MAIN RESULTS: Aldosterone improved 5-day survival, invasive arterial pressure, and in vivo and ex vivo arterial response to phenylephrine at 18 hours after induction of murine endotoxic shock. Both α1-adrenoceptor and mineralocorticoid receptor expressions studied in mouse aortas were down-regulated at 6 and 18 hours in endotoxemic mice and restored in aldosterone-treated mice. Furthermore, tumor necrosis factor-α decreased both mineralocorticoid receptor and α1-adrenoceptor expressions within 5 hours in human vascular cells in a nuclear factor-κB pathway-dependent manner. Mineralocorticoid receptor expression was also blunted in human cells treated with plasma from septic patients. CONCLUSION: We found a beneficial effect of mineralocorticoids on survival, blood pressure, and vascular reactivity, associated with a restoration of α1-adrenoceptor expression in endotoxic shock. Furthermore, blunted vascular mineralocorticoid receptor expression might participate in hemodynamic failure during sepsis.


Assuntos
Aldosterona/farmacologia , Nitrilas/farmacologia , Receptores de Mineralocorticoides/biossíntese , Choque Séptico/tratamento farmacológico , Choque Séptico/fisiopatologia , Sulfonas/farmacologia , Animais , Pressão Sanguínea , Citocinas/farmacologia , Modelos Animais de Doenças , Regulação para Baixo , Endotoxinas , Humanos , Mediadores da Inflamação/farmacologia , Lipopolissacarídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/antagonistas & inibidores , Choque Séptico/mortalidade
3.
Biochim Biophys Acta ; 1833(6): 1316-28, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23481040

RESUMO

Acute myeloid leukemia (AML) is a deadly disease characterized by the clonal expansion and accumulation of hematopoietic stem cells arrested at various stages of development. Clinical research efforts are currently focusing on targeted therapies that induce apoptosis in AML cells. Herein, the effects and mechanisms of the novel flavone 3,3'-diamino-4'-methoxyflavone (DD1) on AML cell dysfunction were investigated in AML cells (monoblast U937, myelomonocyte OCI-AML3, promyelocyte NB4, myeloblast HL-60) and blood samples from patients with AML. The administration of DD1 inhibited proliferation and induced death of AML cell lines and reduced the clonogenic activity of AML, but not normal, blood cells. The flavone's apoptotic action in U937 cells was associated with recruitment of mitochondria, Bax activation, Bad dephosphorylation (at Ser(136)), activation of caspases -8, -9, and -3 and cleavage of the caspase substrate PARP-1. DD1 induced a marked decrease in (i) Thr(389)-phosphorylation and (ii) protein levels of the caspase-3 substrate P70 ribosomal S6 kinase (P70S6K, known for its ability to phosphorylate Bad). Caspase-dependent apoptosis and P70S6K degradation were simultaneously prevented by the caspase inhibitors. Importantly, DD1 was shown to directly inhibit the proteasome's chymotrypsin-like activity in U937 cells. Apoptotic activity of the proteasome inhibitor bortezomib was also related to Bax activation and P70S6K downregulation. Accordingly, DD1 failed to induce P70S6K cleavage, Bax stimulation and apoptosis in K562 cells resistant to bortezomib. These results indicate that DD1 has the potential to eradicate AML cells and support a critical role for Bax and P70S6K in DD1-mediated proteasome inhibition and apoptosis of leukemia cells.


Assuntos
Apoptose/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Flavonoides/farmacologia , Leucemia Mieloide Aguda/patologia , Inibidores de Proteassoma/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/antagonistas & inibidores , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica , Western Blotting , Ácidos Borônicos/farmacologia , Bortezomib , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Flavonoides/química , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Pessoa de Meia-Idade , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pirazinas/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Células Tumorais Cultivadas , Proteína X Associada a bcl-2/metabolismo
4.
Cell Immunol ; 253(1-2): 16-22, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18639869

RESUMO

The interactions between mesenchymal stem cells (MSCs) and immune system are currently being explored. Leukemia inhibitory factor (LIF) is linked to regulatory transplantation tolerance. Our aim was to study the expression of LIF on human MSCs at both gene and protein level in mixed lymphocyte reaction (MSC/MLR), and its implication in MSC immunosuppressive effect. There was a 7-fold increase (611pg/ml) in LIF in MSC/MLR as compared to MSCs alone. Using LIF neutralizing antibody, a significant restoration of up to 91% of CD3+ lymphocyte proliferation in MSC/MLR was observed (p=0.021). LIF was implicated in the generation of regulatory lymphocytes, as demonstrated by decrease of Foxp3+ regulatory cells after using LIF neutralizing antibody in MSC/MLR (p=0.06) by flow cytometry. A positive correlation between LIF and human leukocyte antigen (HLA-G) gene expression by MSCs was found (R(2)=0.74). Our findings provide evidence supporting the immunomodulatory effect of MSCs.


Assuntos
Tolerância Imunológica/imunologia , Terapia de Imunossupressão , Fator Inibidor de Leucemia/imunologia , Células-Tronco Mesenquimais/imunologia , Anticorpos/imunologia , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Separação Celular , Células Cultivadas , Fatores de Transcrição Forkhead/imunologia , Antígenos HLA/imunologia , Antígenos HLA-G , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Células-Tronco Mesenquimais/citologia
5.
Cell Death Differ ; 25(5): 983-1001, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29323266

RESUMO

Mitochondrial metabolism is a tightly regulated process that plays a central role throughout the lifespan of hematopoietic cells. Herein, we analyze the consequences of the mitochondrial oxidative phosphorylation (OXPHOS)/metabolism disorder associated with the cell-specific hematopoietic ablation of apoptosis-inducing factor (AIF). AIF-null (AIF-/Y ) mice developed pancytopenia that was associated with hypocellular bone marrow (BM) and thymus atrophy. Although myeloid cells were relatively spared, the B-cell and erythroid lineages were altered with increased frequencies of precursor B cells, pro-erythroblasts I, and basophilic erythroblasts II. T-cell populations were dramatically reduced with a thymopoiesis blockade at a double negative (DN) immature state, with DN1 accumulation and delayed DN2/DN3 and DN3/DN4 transitions. In BM cells, the OXPHOS/metabolism dysfunction provoked by the loss of AIF was counterbalanced by the augmentation of the mitochondrial biogenesis and a shift towards anaerobic glycolysis. Nevertheless, in a caspase-independent process, the resulting excess of reactive oxygen species compromised the viability of the hematopoietic stem cells (HSC) and progenitors. This led to the progressive exhaustion of the HSC pool, a reduced capacity of the BM progenitors to differentiate into colonies in methylcellulose assays, and the absence of cell-autonomous HSC repopulating potential in vivo. In contrast to BM cells, AIF-/Y thymocytes compensated for the OXPHOS breakdown by enhancing fatty acid ß-oxidation. By over-expressing CPT1, ACADL and PDK4, three key enzymes facilitating fatty acid ß-oxidation (e.g., palmitic acid assimilation), the AIF-/Y thymocytes retrieved the ATP levels of the AIF +/Y cells. As a consequence, it was possible to significantly reestablish AIF-/Y thymopoiesis in vivo by feeding the animals with a high-fat diet complemented with an antioxidant. Overall, our data reveal that the mitochondrial signals regulated by AIF are critical to hematopoietic decision-making. Emerging as a link between mitochondrial metabolism and hematopoietic cell fate, AIF-mediated OXPHOS regulation represents a target for the development of new immunomodulatory therapeutics.


Assuntos
Fator de Indução de Apoptose/deficiência , Linfócitos B/metabolismo , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Fosforilação Oxidativa , Timócitos/metabolismo , Animais , Linfócitos B/citologia , Células-Tronco Hematopoéticas/citologia , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Timócitos/citologia
6.
Transplantation ; 84(2): 231-7, 2007 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-17667815

RESUMO

INTRODUCTION: Mesenchymal stem cells (MSCs) possess unique immunomodulatory properties. They are able to suppress allogenic T-cell response and modify maturation of antigen-presenting cells. Their role in the treatment of severe graft versus host disease has been reported. The underlying molecular mechanisms of immunosuppression are currently being investigated. Histocompatibility locus antigen (HLA)-G is a nonclassical major histocompatibility complex class I antigen with strong immune-inhibitory properties. METHODS: We studied the role of HLA-G on MSC-induced immunosuppression. The expression of HLA-G on human MSCs cultured alone and in mixed lymphocytes reaction (MSC/MLR) was analyzed. RESULTS: We found that HLA-G can be detected on MSCs by real-time reverse-phase polymerase chain reaction, immunofluorescence, flow cytometry (52.4+/-3.6%), and enzyme-linked immunosorbent assay in the supernatant (38.7+/-5.2 ng/mL). HLA-G protein expression is constitutive and the level is not modified upon stimulation by allogenic lymphocytes in MSC/MLR. The functional role of HLA-G protein expressed by MSCs was analyzed using the 87G anti-HLA-G blocking antibody in a MSC/MLR. We found that blocking HLA-G molecule significantly raised lymphocyte proliferation in MSC/MLR (35.5%, P=0.01). CONCLUSION: Our findings provide evidences supporting involvement of HLA-G in the immunosuppressive properties of MSCs. These results emphasize the potential application of MSCs as a relevant therapeutic candidate in transplantation.


Assuntos
Expressão Gênica , Antígenos HLA/genética , Antígenos HLA/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Terapia de Imunossupressão/métodos , Células-Tronco Mesenquimais/imunologia , RNA Mensageiro/genética , Células da Medula Óssea/citologia , Proliferação de Células , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Antígenos HLA/metabolismo , Antígenos HLA-G , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Transplante de Células-Tronco Mesenquimais/efeitos adversos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologia
7.
Gene Expr ; 13(4-5): 217-26, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17605296

RESUMO

Mesenchymal stem cells (MSC) inhibit the response of allogeneic T lymphocytes in culture. Because the mechanisms of this effect may differ according to the existence of cell contact, we investigated the differences in gene expression of inhibitory molecules during MSC-T lymphocyte coculture when cell contact does and does not occur. Human MSC and T lymphocytes were cultured together in standard and transwell cultures. MSC gene expression was analyzed by semiquantitative real-time RT-PCR. MSC elicited a high dose-dependent inhibition of T lymphocytes in cultures with cell contact, but inhibition occurred even without cell contact. In both cases, we observed significant upregulation of IDO, LIF, and HLA-G, along with downregulation of HGF and SDF1. In cultures with cell contact, IL-10 and TGF-beta transcripts were expressed in a significantly higher level than in cultures without this contact. Furthermore, in the latter, the increased inhibition of T-cell proliferation was positively correlated with IDO gene expression and negatively correlated with SDF1 gene expression. MSC appear to induce T-cell tolerance by two distinct mechanisms. The first of these, which does not require cell contact, induces expression of the tolerogenic genes IDO, LIF, and HLA-G. The second mechanism, which is contact dependent, modulates IL-10 and TGF-beta gene expression. These two mechanisms probably play separate roles in MSC-induced tolerance in allogeneic hematopoietic stem cell transplantation.


Assuntos
Comunicação Celular/fisiologia , Regulação da Expressão Gênica , Interleucina-10/metabolismo , Células-Tronco Mesenquimais/fisiologia , Linfócitos T/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Moléculas de Adesão Celular/metabolismo , Técnicas de Cultura de Células , Proliferação de Células , Células Cultivadas , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , Técnicas de Cocultura , Humanos , Terapia de Imunossupressão , Interleucina-10/genética , Células-Tronco Mesenquimais/citologia , Linfócitos T/citologia , Fator de Crescimento Transformador beta/genética
8.
Leuk Lymphoma ; 47(7): 1340-7, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16923566

RESUMO

This study evaluated the feasibility of using dendritic cells (DCs) to generate, ex vivo, anti-tumor cytotoxic T lymphocytes (CTL) in patients with stage III multiple myeloma (MM). Nucleated cells from eight patients who had received chemotherapy (three of whom had undergone autologous hemopoeitic stem cell transplantation) were collected by apheresis. Their monocytes were enriched using counter-current centrifugation, differentiated into DCs which were further co-cultured with autologous CD8 lymphocytes to induce CTL. The DCs were pulsed either with the idiotypic paraprotein (regarded as a tumor-specific antigen) or with autologous MM cell lysate before co-culture. Specific T-cell responses were measured in IFNgamma enzyme-linked immunospot and chromium release assays of autologous plasmocyte targets. A slight increase in IFNgamma secretion by T-cells was observed for two patients (DCs pulsed with idiotypic paraprotein for one, MM cell lysate for the other). No or weak specific lysis of plasmocyte targets was observed in the chromium release assays. In conclusion, the T-cell response to pulsed DCs was very weak or absent. There are clinical and technical reasons that could explain, in part, this lack of response.


Assuntos
Transplante de Células/métodos , Células Dendríticas/citologia , Idiótipos de Imunoglobulinas , Mieloma Múltiplo/terapia , Linfócitos T/metabolismo , Adulto , Idoso , Remoção de Componentes Sanguíneos , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular , Cromo/metabolismo , Técnicas de Cocultura , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo , Mieloma Múltiplo/imunologia , Fator de Necrose Tumoral alfa/biossíntese
9.
Oncotarget ; 7(15): 19445-67, 2016 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-26655501

RESUMO

The CD13 antigen's binding site for the Asn-Gly-Arg (NGR) motif enables NGR-containing chemotherapeutic drugs to be delivered to CD13-positive tumours. Human CD13-positive acute myeloid leukemia (AML) cells proliferate abnormally and escape death. Here, we show that the CNGRC-GG-D(KLAKLAK)2 peptide induces death in AML cell lines (U937, THP-1, NB4, HL-60) and primary blood cells from AML patients. Cell death was characterized as a caspase-independent mechanism, without DNA fragmentation, but phosphatidylserine externalization and membrane disruption. Our results demonstrate in U937 cells that (i) the NGR-peptide triggers the loss of mitochondrial potential(ΔΨm) and generates superoxide anion (O2-), (ii) N-acetyl-L-cysteine (NAC) and extra/intracellular Ca2+ chelators (BAPTA) prevent both O2- production and cell death, (iii) the Ca2+-channel blocker nifedipine prevents cell death (indicating that Ca2+ influx is the initial death trigger), and (iv) BAPTA, but not NAC, prevents ΔΨm loss (suggesting O2- is a mitochondrial downstream effector). AML cell lines and primary blasts responding to the lethal action of NGR-peptide express promatrix metalloproteinase-12 (proMMP-12) and its substrate progranulin (an 88 kDa cell survival factor). A cell-free assay highlighted proMMP-12 activation by O2-. Accordingly, NGR-peptide's downregulation of 88 kDa progranulin protein was prevented by BAPTA and NAC. Conversely, AML blast resistance to NGR-peptide is associated with the expression of a distinct, 105 kDa progranulin isoform. These results indicate that CNGRC-GG-D(KLAKLAK)2 induces death in AML cells through the Ca2+-mitochondria-O2.-pathway, and support the link between proMMP-12 activation and progranulin cleavage during cell death. Our findings may have implications for the understanding of tumour biology and treatment.


Assuntos
Apoptose/efeitos dos fármacos , Antígenos CD13/metabolismo , Cálcio/metabolismo , Peptídeos/farmacologia , Doença Aguda , Sequência de Aminoácidos , Caspases/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Células HL-60 , Humanos , Immunoblotting , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , Metaloproteinase 12 da Matriz/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Progranulinas , Superóxidos/metabolismo , Células U937
10.
J Clin Endocrinol Metab ; 101(1): 293-304, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26583585

RESUMO

CONTEXT: Extracellular matrix (ECM) in sc adipose tissue (scAT) undergoes pathological remodeling during obesity. However, its evolution during weight loss remains poorly explored. OBJECTIVE: The objective of the investigation was to study the histological, transcriptomic, and physical characteristics of scAT ECM remodeling during the first year of bariatric surgery (BS)-induced weight loss and their relationships with metabolic and bioclinical improvements. DESIGN, SETTING, PATIENTS, AND INTERVENTIONS: A total of 118 morbidly obese candidates for BS were recruited and followed up during 1 year after BS. MAIN OUTCOME MEASURES: scAT surgical biopsy and needle aspiration as well as scAT stiffness measurement were performed in three subgroups before and after BS. Fourteen nonobese, nondiabetic subjects served as controls. RESULTS: Significantly increased picrosirius-red-stained collagen accumulation in scAT after BS was observed along with fat mass loss, despite metabolic and inflammatory improvements and undetectable changes of scAT stiffness. Collagen accumulation positively associated with M2-macrophages (CD163(+) cells) before BS but negatively afterward. Expression levels of genes encoding ECM components (eg, COL3A1, COL6A1, COL6A2, ELN), cross-linking enzymes (eg, lysyl oxidase [LOX], LOXL4, transglutaminase), metalloproteinases, and their inhibitors were modified 1 year after BS. LOX expression and protein were significantly decreased and associated with decreased fat mass as well as other cross-linking enzymes. Although total collagen I and VI staining decreased 1 year after BS, we found increased degraded collagen I and III in scAT, suggesting increased degradation. CONCLUSIONS: After BS-induced weight loss and related metabolic improvements, scAT displays major collagen remodeling with an increased picrosirius-red staining that relates to increased collagen degradation and importantly decreased cross-linking. These features are in agreement with adequate ECM adaptation during fat mass loss.


Assuntos
Cirurgia Bariátrica , Colágeno/metabolismo , Gordura Subcutânea/metabolismo , Adulto , Composição Corporal , Técnicas de Imagem por Elasticidade , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Feminino , Humanos , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Obesidade Mórbida/metabolismo , Obesidade Mórbida/cirurgia , Transcriptoma , Rigidez Vascular , Redução de Peso
11.
Zhonghua Yi Xue Za Zhi ; 85(39): 2780-4, 2005 Oct 19.
Artigo em Chinês | MEDLINE | ID: mdl-16324321

RESUMO

OBJECTIVE: To evaluate whether mesenchymal stem cells (MSCs) obtained from human proximal femurs possess immunosuppressive effect so as to look for ideal bank of MSCs for clinical prophylaxis and treatment of graft versus host disease (GVHD). METHODS: Human marrows were collected from the proximal femurs of patients undergoing hip replacement to isolate MSCs. The puncture materials obtained from the iliac bone marrow of 12 healthy donors were used as controls. Peripheral blood lymphocytes (PBLs) were obtained from the peripheral blood of healthy persons. 1 x 10(5) PBLs were mixed with allogeneic PBLs radiated by 60 cobalt and put into the wells of a 96-well plate. MSCs of the concentrations of 1 x 10(5), 3 x 10(4), 1 x 10(4), and 3 x 10(3), were added into the culture fluid of the mixed PBLs to be co-cultivated for 5 days. 1 microCi/well [(3)H] TdR was added in the last 18 hours. The cells were collected and the counts per minute (cpm) was detected. 3 x 10(4) and 1 x 10(4) MSCs were put into the wells. When the MSCs adhered to the wall, a membrane with micropores was inserted value of 1 x 10(5) PBLs and radiated allo-PBLs were put onto the top of which. Five days after cultivation 1 microCi/well [(3)H] TdR was added and the cpm was tested. MSCs were cultured in RPMI-1640 culture fluid and then contacted MLR constructed by 1 x 10(5) PBLs and 1 x 10(5) allo-PBL directly or via Transwell membrane with micropores. Five days after the supernatant was collected. ELISA was used to detect the content of TGF-beta(1). 0.1 microg/ml, 1 microg/ml, or 10 microg/ml anti-human TGF-beta1 antibody was added to the co-cultivation system. Five days after [(3)H] TdR was added so as to test the value cpm. RESULTS: 3 x 10(3) - 1 x 10(5) MSCs from proximal femurs inhibited the PBLs proliferation to 62 +/- 18% - 28 +/- 12% of maximal response, however, not significantly different from that observed in MSCs collected from bone marrow of healthy donors via iliac crest aspiration (58 +/- 12% - 27 +/- 6%, P > 0.05). When these cells were separated physically from MLR system via a membrane with micropores 0.2 microm in diameter, 3 x 10(4) and 1 x 10(4) cells still markedly suppressed the PBLs proliferation to 36 +/- 8% and 53 +/- 13% of maximal response. ELISA showed that TGF-beta1 was measured in the supernatants of MSCs, MSCs + MLR and MSCs + MLR in Transwell system, without significant differences among these experimental conditions. Furthermore, the presence of increasing amounts of neutralizing anti- TGF-beta1 antibody did not reverse the inhibitory effect. CONCLUSION: MSCs obtained from the proximal femurs possess immunosuppressive activity. A soluble factor/factors was/were involved in such effect, however, TGF-beta1 was not a candidate.


Assuntos
Fêmur/citologia , Tolerância Imunológica , Linfócitos/citologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Adulto , Idoso , Células da Medula Óssea/citologia , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
12.
Int J Radiat Oncol Biol Phys ; 57(2): 500-7, 2003 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-12957263

RESUMO

PURPOSE: To evaluate the residual hematopoiesis at different levels of total body irradiation (TBI) dose in bone marrow (BM) and peripheral blood (PB), and to study the dose-effect relationship on hematopoietic immature and mature progenitors. We also investigated the possibility of expanding ex vivo the residual progenitors exposed to different dose levels of TBI. METHODS AND MATERIALS: Eight patients treated for AML (n = 3) and myeloma (n = 5) were included. BM and PB samples were harvested before TBI and after doses of: 5 Gy. Mononuclear cells (MNCs) were assayed for burst-forming unit erythroid (BFU-E), granulocyte-forming unit macrophage (CFU-GM), and long-term culture initiating cells (LTC-ICs). Ex vivo expansion: MNCs (after irradiation and controls) were suspended in long-term cultures and expanded with a combination of five cytokines. RESULTS: CD34+ cells were detectable at 10 Gy. We observed a significant decrease of CFU-GM and BFU-E, respectively, to 13.5% and 8.5% of baseline values for doses

Assuntos
Células Precursoras Eritroides/efeitos da radiação , Granulócitos/efeitos da radiação , Monócitos/efeitos da radiação , Irradiação Corporal Total/efeitos adversos , Adulto , Medula Óssea/efeitos da radiação , Relação Dose-Resposta à Radiação , Feminino , Citometria de Fluxo , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos da radiação , Humanos , Leucemia Mieloide Aguda/sangue , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/radioterapia , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Mieloma Múltiplo/sangue , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/radioterapia , Liberação Nociva de Radioativos
13.
Cancers (Basel) ; 6(2): 796-812, 2014 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-24713998

RESUMO

Matrix metalloproteinase (MMP)-9 and neutrophil gelatinase-associated lipocalin (NGAL) have gained attention as cancer biomarkers. The inactive zymogen form of MMP-9 (pro-MMP-9) also exists as a disulphide-linked heterodimer bound to NGAL in humans. Leukaemias represent a heterogeneous group of neoplasms, which vary in their clinical behavior and pathophysiology. In this review, we summarize the current literature on the expression profiles of pro-MMP-9 and NGAL as prognostic factors in leukaemias. We also report the expression of the pro-MMP-9/NGAL complex in these diseases. We discuss the roles of (pro)-MMP-9 (active and latent forms) and NGAL in tumour development, and evaluate the mechanisms by which pro-MMP-9/NGAL may influence the actions of (pro)-MMP-9 and NGAL in cancer. Emerging knowledge about the coexpression and the biology of (pro)-MMP-9, NGAL and their complex in cancer including leukaemia may improve treatment outcomes.

14.
Oncotarget ; 5(18): 8211-22, 2014 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-25246708

RESUMO

Secreted matrix metalloproteinases (MMP)-2 and MMP-9 and membrane-anchored aminopeptidase-N/CD13 are abnormally expressed in human acute myeloid leukaemia (AML). We previously showed that CD13 ligation by anti-CD13 monoclonal antibodies can induce apoptosis in AML cells. Here, we assessed ADAM17 expression in primary blood blasts CD13+CD33+ from patients with AML. Primary AML cells expressed ADAM17 transcript and its surface expression was higher in subtype M4 (myelomonocytic) and M5 (monocytic) AML specimens than in M0 and M1/M2 (early and granulocytic) specimens. In AML cell lines defining distinct AML subfamilies (HL-60/M2, NB4/M3, THP-1/M5, U937/M5) and primary AML cells cultured ex vivo, anti-CD13 antibodies downregulated surface CD13 and ADAM17 without affecting MMP-2/-9 release. Knockdown of CD13 by siRNA prevented anti-CD13-mediated ADAM17 downregulation, indicating that CD13 is required for ADAM17 downregulation. Soluble ADAM17 was not detected in the medium of anti-CD13 treated cells, suggesting that ADAM17 was not shed. After ligation by anti-CD13, CD13 and ADAM17 were internalized. Subsequently, we found that ADAM17 interacts with CD13. We postulate that the interaction of ADAM17 with CD13 and its downregulation following CD13 engagement has important implications in AML for the known roles of ADAM17 in tumour-associated cell growth, migration and invasion.


Assuntos
Proteínas ADAM/metabolismo , Antígenos CD13/metabolismo , Endocitose , Leucemia Mieloide Aguda/enzimologia , Proteína ADAM17 , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD13/genética , Regulação para Baixo , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HL-60 , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Fenótipo , Interferência de RNA , Fatores de Tempo , Transfecção , Células U937 , Adulto Jovem
15.
Curr Pharm Biotechnol ; 14(9): 842-8, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24372262

RESUMO

Bone marrow stroma is damaged by chemotherapy and irradiation protocol. Bone marrow microenvironment supports haematopoiesis and comprises Mesenchymal Stem Cells (MSCs). Coinfusion of MSCs with hematopoietic stem cells (HSC) improves engraftment and accelerates haematopoietic recovery. Stroma-derived factor-1 (SDF-1) is a chemotactic factor which plays a crucial role in stem cell transplantation by enhancing the ability of HSC to engraft. In this study expression of SDF-1 in bone marrow MSCs and the level of Colony Forming Unit Fibroblast (CFU-F) were evaluated in 8 patients with Acute Myeloid leukemia (AML). Evaluation was done at diagnosis and after induction/consolidation chemotherapy before the onset of haematopoietic stem cell transplantation (HSCT). CFU-F frequency increases from diagnosis to remission. Nevertheless level of stromal derived factor-1 (SDF-1) transcripts in bone marrow MSCs of patients with AML stays low. Considering the role of SDF-1 in the homing of HSC, the consequences of SDF-1 deficiency observed in this study might be deleterious on the engraftment after HSCT and haematopoietic recovery. The whole result of this clinical study is an argument for MSC infusion to restore a normal level of SDF1 in the bone marrow microenvironment that could reduce hematopoietic toxicity of chemotherapy and improve HSC engraftment after HSCT.


Assuntos
Quimiocina CXCL12/metabolismo , Leucemia Mieloide Aguda/metabolismo , Células-Tronco Mesenquimais/metabolismo , Terapia Baseada em Transplante de Células e Tecidos , Quimiocina CXCL12/genética , Quimiorradioterapia/efeitos adversos , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Humanos , RNA Mensageiro/metabolismo
17.
Stem Cells ; 24(4): 1020-9, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16339642

RESUMO

Mesenchymal stem cells (MSCs) have been shown to migrate to various tissues. There is little information on the fate and potential therapeutic efficacy of the reinfusion of MSCs following total body irradiation (TBI). We addressed this question using human MSC (hMSCs) infused to nonobese diabetic/ severe combined immunodeficient (NOD/SCID) mice submitted to TBI. Further, we tested the impact of additional local irradiation (ALI) superimposed to TBI, as a model of accidental irradiation. NOD/SCID mice were transplanted with hM-SCs. Group 1 was not irradiated before receiving hMSC infusion. Group 2 received only TBI at a dose of 3.5 Gy, group 3 received local irradiation to the abdomen at a dose of 4.5 Gy in addition to TBI, and group 4 received local irradiation to the leg at 26.5 Gy in addition to TBI. Fifteen days after irradiation, quantitative and spatial distribution of the hMSCs were studied. Histological analysis of mouse tissues confirmed the presence of radio-induced lesions in the irradiated fields. Following their infusion into nonirradiated animals, hMSCs homed at a very low level to various tissues (lung, bone marrow, and muscles) and no significant engraftment was found in other organs. TBI induced an increase of engraftment levels of hMSCs in the brain, heart, bone marrow, and muscles. Abdominal irradiation (AI) as compared with leg irradiation (LI) increased hMSC engraftment in the exposed area (the gut, liver, and spleen). Hind LI as compared with AI increased hMSC engraftment in the exposed area (skin, quadriceps, and muscles). An increase of hMSC engraftment in organs outside the fields of the ALI was also observed. Conversely, following LI, hMSC engraftment was increased in the brain as compared with AI. This study shows that engraftment of hMSCs in NOD/ SCID mice with significantly increased in response to tissue injuries following TBI with or without ALI. ALI induced an increase of the level of engraftment at sites outside the local irradiation field, thus suggesting a distant (abscopal) effect of radiation damage. This work supports the use of MSCs to repair damaged normal tissues following accidental irradiation and possibly in patients submitted to radiotherapy.


Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/efeitos da radiação , Animais , Movimento Celular/efeitos da radiação , Expressão Gênica , Globinas/genética , Sobrevivência de Enxerto/genética , Sobrevivência de Enxerto/efeitos da radiação , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Especificidade de Órgãos , Lesões Experimentais por Radiação/patologia , Lesões Experimentais por Radiação/terapia , Transplante Heterólogo , Irradiação Corporal Total , Microglobulina beta-2/metabolismo
18.
Blood ; 103(9): 3313-9, 2004 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-14715641

RESUMO

The Stro-1 antigen potentially defines a mesenchymal stem cell (MSC) progenitor subset. We here report on the role of human ex vivo-expanded selected Stro-1(+) or Stro-1(-) MSC subsets on the engraftment of human CD34(+) cord blood cells in the nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse model. The data show that cotransplantation of expanded Stro-1(-) cells with CD34(+) cells resulted in a significant increase of human CD45, CD34, CD19, and CD11b cells detected in blood or in bone marrow (BM) and spleen as compared with the infusion of CD34(+) cells alone. Infusion into mice of expanded Stro-1(+) and Stro-1(-) cells (without CD34(+) cells) showed that the numbers of Stro-1(+)-derived (as assessed by DNA analysis of human beta-globin with quantitative polymerase chain reaction [PCR]) were higher than Stro-1(-)-derived cells in spleen, muscles, BM, and kidneys, while more Stro-1(-)-derived than Stro-1(+)-derived cells were found in lungs. The transduction of expanded Stro-1(+) cells with an enhanced green fluorescent protein (eGFP) gene did not modify their cytokine release and their homing in NOD/SCID mouse tissues. The difference between the hematopoietic support and the homing capabilities of expanded Stro-1(+) and Stro-1(-) cells may be of importance for clinical therapeutic applications: Stro-1(+) cells may rather be used for gene delivery in tissues while Stro-1(-) cells may rather be used to support hematopoietic engraftment.


Assuntos
Movimento Celular , Transplante de Células-Tronco de Sangue do Cordão Umbilical/métodos , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Proteínas Nucleares , Animais , Células da Medula Óssea , Células Cultivadas , Sobrevivência de Enxerto , Hematopoese , Humanos , Células-Tronco Mesenquimais/imunologia , Camundongos , Camundongos SCID , Modelos Animais , Especificidade de Órgãos , Baço/citologia , Transplante Heterólogo
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