RESUMO
Traditionally, when antibody to the Hepatitis B core antigen (anti-HBc) and antibody to the Hepatitis B surface antigen (anti-HBs) are positive, the donor is considered suitable. However, the literature contains cases with this profile and circulating hepatitis B virus DNA. The aim of the study is to analyze the incidence of occult hepatitis B virus infection (OBI). Retrospective data were evaluated for deceased tissue donors in ten Tissue Establishments (Spain) during 2017. The data included demographic data and the serological markers for hepatitis B that each tissue establishment performed. A total number of 1933 tissue donors were evaluated. A total of 180 donors were excluded: 6 (0.3%) with Hepatitis B surface antigen (HBs positive), and 174 in which DNA testing was not performed. Anti-HBc was positive in 175 donors (10%), in which anti-HBs was negative in 30 (17.1%) and positive in 145 (82.9%). In total, 27 donors with DNA positive (1.5%) were found, of which 3 of 117 donors (1.7%) showed anti-HBc negative and anti-HBs positive (> 10 IU/ml), 4 of 30 donors (13.3%) showed anti-HBc positive and anti-HBs negative and 20 of 145 donors (13.8%) showed both anti-HBc and anti-HBs positive. The highest probability of finding DNA occurs when anti-HBc is positive, regardless of the presence of anti-HBs. In our study, the probability of OBI was 1.5%. The classic concept that when anti-HBc and anti-HBs are positive (even with a titer of over 100 IU/ml) the donor can be accepted should, therefore, be reconsidered, and DNA testing should be mandatory.
Assuntos
DNA Viral/análise , Seleção do Doador , Anticorpos Anti-Hepatite B/análise , Vírus da Hepatite B/isolamento & purificação , Hepatite B/diagnóstico , Idoso , DNA Viral/genética , Hepatite B/epidemiologia , Hepatite B/imunologia , Hepatite B/virologia , Anticorpos Anti-Hepatite B/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Humanos , Pessoa de Meia-Idade , Sangue Oculto , Estudos Retrospectivos , Espanha/epidemiologia , Doadores de TecidosRESUMO
BACKGROUND: The need for high-cellular-content cord blood units (CBUs) for allogenic transplantation is evident to improve clinical outcomes. In our environment and with current donation programs, very few collected units meet suggested clinical thresholds, making collection programs highly inefficient. To increase the clinical conversion rate, we have assessed factors influencing the cellular content of the cord blood collection and established the estimated fetal weight percentile (EFWp) as a tool to predict which deliveries will obtain higher cellular counts. STUDY DESIGN AND METHODS: We conducted a retrospective analysis of 11,349 collected CBUs. An analysis of diagnostic efficiency (receiver operating characteristic [ROC] curve) was performed to establish the cutoffs of several obstetric and perinatal variables from which we would obtain more than 1500 × 106 total nucleated cells and 4 × 106 CD34 cells. We then calculated the optimal EFWp cutoff to increase efficiency. RESULTS: In the univariate analysis, factors positively and significantly associated were a greater neonatal and placental weight and longer weeks of gestation. In the multivariate analysis only neonatal and placental weight remain significant (p < 0.001). The ROC curve analysis showed that the optimal EFWp cutoff is 60, which has the maximum area under the curve. Applying this, donations meeting clinical cellular numbers will increase more than 30% with respect to not using any threshold. CONCLUSION: The EFWp predicts the quality of the collected CBUs and can be used to make a prenatal selection of the donors, therefore increasing the efficiency of umbilical cord blood collection programs.
Assuntos
Armazenamento de Sangue/métodos , Coleta de Amostras Sanguíneas/métodos , Sangue Fetal/citologia , Peso Fetal , Doadores de Sangue , Feminino , Humanos , Recém-Nascido , Masculino , Gravidez , Estudos RetrospectivosRESUMO
It is widely agreed that the best source of nutrition for the newborn is the milk of their own mothers. In those cases where it is not available, especially in very premature and/or very low birth weight infants, as well as other sick newborns, the preferred choice before formula is the human milk provided by selected donors. This indication is supported by the highest international bodies dedicated to the health of the child population, including the World Health Organisation as well as the main national and international scientific societies in the field of Paediatrics. Milk banks are health institutions responsible for the collection, processing and distribution of donated human milk. Currently, there are 14 human milk banks operating in Spain, grouped in the Spanish Association of Human Milk Banks, created in September 2008. In order to homogenise the criteria and to unify the working methods of the different milk banks, the Spanish Association of Human Milk Banks has developed standards to harmonise the protocols, and to serve as a guide for the start-up of new milk banks in the Spanish territory. These standards, set out in the present article, range from the donor selection and the evaluation process to the collection, processing, storage, and distribution of donor human milk.
Assuntos
Bancos de Leite Humano/organização & administração , Humanos , EspanhaRESUMO
Mesenchymal stem cells (MSCs) are self-renewing, multipotent cells that could potentially be used to repair injured cartilage in diseases such as osteoarthritis (OA). In this study we used bone marrow, adipose tissue from articular and subcutaneous locations, and synovial fluid samples from 18 patients with knee OA to find a suitable alternative source for the isolation of MSCs with high chondrogenic potential. MSCs from all tissues analysed had a fibroblastic morphology, but their rates of proliferation varied. Subcutaneous fat-derived MSCs proliferated faster than bone marrow- and Hoffa's fat pad-derived MSCs, while synovial fluid-derived MSCs grew more slowly. CD36 and CD54 expression was similar across all groups of MSCs with several minor differences. High expression of these surface markers in subcutaneous fat-derived MSCs was correlated with poor differentiation into hyaline cartilage. Synovial fluid-derived MSCs presented a relatively small chondrogenic differentiation capacity while Hoffa's fat pad-derived MSCs had strong chondrogenic potential. In conclusion, MSCs from elderly patients with OA may still display significant chondrogenic potential, depending on their origin.
Assuntos
Antígenos CD36/metabolismo , Condrogênese/fisiologia , Molécula 1 de Adesão Intercelular/metabolismo , Células-Tronco Mesenquimais/patologia , Osteoartrite do Joelho/patologia , Adipócitos/citologia , Adipócitos/fisiologia , Idoso , Antígenos de Superfície/metabolismo , Biomarcadores/metabolismo , Cartilagem Articular/citologia , Cartilagem Articular/fisiologia , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Feminino , Citometria de Fluxo , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Osteoartrite do Joelho/cirurgia , Líquido Sinovial/citologiaRESUMO
Drugs containing the quinone group were tested on hyperproliferative leukemia T cells (HLTC: Jhp and Jws) and parental Jurkat cells. Doxorubicin, menadione and adaphostin produced different effects on these cell lines. Rapid doxorubicin-induced cell death in Jurkat cells was mediated by caspase activation. Doxorubicin-induced cell death of HLTCs was delayed due to the absence of caspase-3 and -8 expression. Delayed HLTC cell death was mediated and triggered by the generation of reactive oxygen species (ROS). Other drugs containing quinone groups, such as menadione and adaphostin, were also tested on HLTC and both were toxic by a caspase-independent mechanism. The toxicity of these drugs correlated with the generation of the superoxide anion, which increased and was more effective in HLTCs than in parental Jurkat cells. Accordingly, SOD1 activity was much lower in HLTCs than in Jurkat cells. This lower SOD1 activity in HLTCs was associated not only with the absence of the wild-type (16 kDa) SOD1 monomer but also with the presence of a shortened (14 kDa) SOD1 monomer isoform. Moreover, the cytotoxicity of drugs containing the quinone group was prevented by incubation with manganese(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), a cell-permeable superoxide dismutase mimetic and a potent inhibitor of oxidation. These findings could explain the sensitivity of HLTCs to drugs containing the quinone group using a mechanism dependent on oxidative stress. These observations can also be useful to target hyperproliferative leukemias that are resistant to the classical caspase-dependent apoptotic pathway.
Assuntos
Proliferação de Células/efeitos dos fármacos , Isoenzimas/metabolismo , Leucemia/patologia , Quinonas/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Antineoplásicos/farmacologia , Apoptose , Western Blotting , Caspases/metabolismo , Doxorrubicina/farmacologia , Humanos , Células Jurkat , Leucemia/enzimologia , Leucemia/metabolismo , Proteínas de Neoplasias/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Superóxido Dismutase-1RESUMO
This study analyzed the phenotype and the chondrogenic differentiation of bone marrow-derived MSCs from old patients undergoing knee osteoarthritis or femoral fracture surgery. Twenty patients (12 females), with a mean age of 77.35±8.76 years, were studied. Ten patients suffered of knee osteoarthritis (OA) pathology and underwent surgery for arthroplasty, and the other 10 patients suffered femoral fracture. A comparative study of bone marrow-derived cultured human MSCs was carried out, and the main morphological parameters, proliferative activity and expression of surface markers were characterized. Bone marrow was obtained from the femur in all cases. The χ2-test, Mann-Whitney U-test, correlation coefficient and the Spearman test were applied. Bone marrow MSCs from old patients were able to differentiate into chondrocytic lineages. Proliferation and flow cytometry data showed no difference associated to the gender. No significant differences between the knee arthroplasty group or the femoral fracture group were found, except for higher CD49d % in MSC from fracture, and higher CD49f % in MSC from knee OA patients at passage one. MSCs from old patients suffering knee OA can be differentiated into chondrocytic lineages, and these present no differences with MSCs from femoral fracture patients.
Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular , Condrócitos/citologia , Células-Tronco Mesenquimais/citologia , Idoso , Idoso de 80 Anos ou mais , Medula Óssea , Diferenciação Celular/genética , Linhagem da Célula , Células Cultivadas , Condrogênese/genética , Feminino , Fraturas do Fêmur/patologia , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/patologia , FenótipoRESUMO
OBJECTIVE: We previously observed that T lymphocytes present in synovial fluid (SF) from patients with rheumatoid arthritis (RA) were sensitive to APO2L/TRAIL. In addition, there was a drastic decrease in the amount of bioactive APO2L/TRAIL associated with exosomes in SF from RA patients. This study was undertaken to evaluate the effectiveness of bioactive APO2L/TRAIL conjugated with artificial lipid vesicles resembling natural exosomes as a treatment in a rabbit model of antigen-induced arthritis (AIA). METHODS: We used a novel Ni(2+)-(N-5-amino-1-carboxypentyl)-iminodiacetic acid)-containing liposomal system. APO2L/TRAIL bound to liposomes was intraarticularly injected into the knees of animals with AIA. One week after treatment, rabbits were killed, and arthritic synovial tissue was analyzed. RESULTS: Tethering APO2L/TRAIL to the liposome membrane increased its bioactivity and resulted in more effective treatment of AIA compared with soluble, unconjugated APO2L/TRAIL, with substantially reduced synovial hyperplasia and inflammation in rabbit knee joints. The results of biophysical studies suggested that the increased bioactivity of APO2L/TRAIL associated with liposomes was due to the increase in the local concentration of the recombinant protein, augmenting its receptor crosslinking potential, and not to conformational changes in the protein. In spite of this increase in bioactivity, the treatment lacked systemic toxicity and was not hepatotoxic. CONCLUSION: Our findings indicate that binding APO2L/TRAIL to the liposome membrane increases its bioactivity and results in effective treatment of AIA.
Assuntos
Artrite Experimental/terapia , Artrite Reumatoide/terapia , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêutico , Animais , Artrite Experimental/metabolismo , Artrite Reumatoide/metabolismo , Citometria de Fluxo , Hiperplasia/metabolismo , Hiperplasia/terapia , Inflamação/metabolismo , Inflamação/terapia , Lipossomos/uso terapêutico , Coelhos , Membrana Sinovial/metabolismo , Resultado do TratamentoRESUMO
Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into several mesoderm lineages. They have been isolated from different tissues, such as bone marrow, adult peripheral blood, umbilical cord blood, and adipose tissue. The aim of this study was to analyze the differences in proliferation and phenotype of adipose tissue-derived MSCs from three different species, and to evaluate their capacity to differentiate into chondrocytes in vitro. A comparative study of cultured human, rabbit, and sheep mesenchymal cells from adipose tissue was carried out, and the main morphological parameters, proliferative activity, and expression of surface markers were characterized. Proliferation and flow cytometry data showed species-related differences between animal and human MSCs. Histological staining suggested that rabbit and sheep mesenchymal cells were able to differentiate into chondrocytic lineages. Human mesenchymal cells, though they could also differentiate, accomplished it with more difficulty than animal MSCs. These results could help to explain the differences in the chondrogenic capacity of sheep and rabbit MSCs when they are used as animal models compared to human mesenchymal cells in a clinical assay.
Assuntos
Diferenciação Celular , Condrócitos/citologia , Células-Tronco Mesenquimais/citologia , Tecido Adiposo/citologia , Animais , Antígenos CD/biossíntese , Linhagem da Célula , Células Cultivadas , Humanos , Fenótipo , Coelhos , OvinosRESUMO
Presenilin 1-associated protein (PSAP) was first identified as a protein that interacts with presenilin 1. It was later reported that PSAP is a mitochondrial protein that induces apoptosis when overexpressed in cultured cells. PSAP is also known as mitochondrial carrier homolog 1 (Mtch1). In this study, we show that there are two proapoptotic PSAP isoforms generated by alternative splicing that differ in the length of a hydrophilic loop located between two predicted transmembrane domains. Using RT-PCR and Western blot assays, we determined that both isoforms are expressed in human and rat tissues as well as in culture cells. Our results indicate that PSAP is an integral mitochondrial outer membrane protein, although it contains a mitochondrial carrier domain conserved in several inner membrane carriers, which partially overlaps one of the predicted transmembrane segments. Deletion of this transmembrane segment impairs mitochondrial import of PSAP. Replacement of this segment with each of two transmembrane domains, with opposite membrane orientations, from an unrelated protein indicated that one of them allowed mitochondrial localization of the PSAP mutant, whereas the other one did not. Our interpretation of these results is that PSAP contains multiple mitochondrial targeting motifs dispersed along the protein but that a transmembrane domain in the correct position and orientation is necessary for membrane insertion. The amino acid sequence within this transmembrane domain may also be important. Furthermore, two independent regions in the amino terminal side of the protein are responsible for its proapoptotic activity. Possible implications of these findings in PSAP function are discussed.
Assuntos
Apoptose/fisiologia , Proteínas de Membrana/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Sinais Direcionadores de Proteínas/fisiologia , Processamento Alternativo , Sequência de Aminoácidos , Animais , Linhagem Celular , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Mutação , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sinais Direcionadores de Proteínas/genética , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
The improved recombinant form of the death ligand Apo2L/TRAIL (Apo2L/TRAIL.0) is not cytotoxic for normal human cells and is a good candidate for the therapy of multiple myeloma (MM), a B-cell neoplasia that remains incurable. We have analyzed the molecular determinants of myeloma sensitivity to Apo2L/TRAIL.0 in a number of MM cell lines, the mechanisms of resistance and a possible way of overcoming it. Expression of one death receptor for Apo2L/TRAIL (DR4 or DR5) is sufficient to transduce death signals, though DR5 was more efficient when both receptors were present. Membrane expression of decoy receptors (DcR1, DcR2) and intracellular levels of c-FLIP(L), XIAP and Mcl-1 were not predictive of resistance to Apo2L/TRAIL. Inhibition of Mcl-1 degradation did not prevent Apo2L/TRAIL-induced apoptosis. In IM-9 cells, resistance was associated to a reduced caspase-8 expression. U266 cells, though expressing significant levels of DR4 and caspase-8, were nevertheless resistant to Apo2L/TRAIL. This resistance could be overcome by co-treatment with valproic acid (VPA), a histone deacetylase inhibitor. VPA caused the redistribution of DR4 to plasma membrane lipid rafts and restored DR4 signaling. Overexpression of Mcl-1 in U266 cells did not prevent Apo2L/TRAIL cytotoxicity in VPA-sensitized cells. These results, taken together, support the possible use of Apo2L/TRAIL.0 in the treatment of MM.
Assuntos
Caspase 8/metabolismo , Resistencia a Medicamentos Antineoplásicos , Mieloma Múltiplo/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Apoptose , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 8/análise , Linhagem Celular Tumoral , Membrana Celular/química , Membrana Celular/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas Ligadas por GPI , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/análise , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/análise , Membro 10c de Receptores do Fator de Necrose Tumoral , Proteínas Recombinantes/farmacologia , Receptores Chamariz do Fator de Necrose Tumoral/análise , Receptores Chamariz do Fator de Necrose Tumoral/metabolismo , Ácido Valproico/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
Apo2L/TRAIL is a member of the TNF family, with its receptors DR4 and DR5 containing a death domain. Multiple tumors are sensitive to Apo2L/TRAIL-induced apoptosis, while normal cells are not, so it constitutes a promising new antitumoral therapy. In this review we deal rather with the physiological role of Apo2L/TRAIL, which, in one hand, is clearly related with immune antitumoral surveillance. However, a role of Apo2L/TRAIL as a fine-tuning regulator of the immune system, especially in the regulation of CD8+ T cell activation and memory, has been also demonstrated. In fact, Apo2L/TRAIL can be considered as an additional mechanism needed to prevent the development of autoimmune disease. Indeed, recent developments indicate that Apo2L/TRAIL can be also useful as a treatment against certain chronic autoimmune diseases.
Assuntos
Linfócitos T/imunologia , Ligante Indutor de Apoptose Relacionado a TNF/fisiologia , Animais , Apoptose , Doenças Autoimunes/imunologia , Humanos , Tolerância Imunológica , Ativação Linfocitária , CamundongosRESUMO
The infiltration and accumulation of T cells in the rheumatoid arthritis (RA) synovial fluid (SF) are hallmarks of disease. We aimed to assess the functional relevance of FasL and of APO2L/TRAIL in the persistence of T cells in the rheumatoid SF. We have analyzed the expression of the activation markers HLA-DR and CD69 and also of the death receptor Fas/CD95 and death ligands FasL or APO2L/TRAIL in CD3+ lymphocytes from SF of 62 RA patients, together with their sensitivity to anti-Fas mAb or to rAPO2L/TRAIL, using as controls T lymphocytes present in SF of 20 patients with traumatic arthritis. T lymphocytes infiltrated in SF of RA patients have a chronically activated phenotype, but they are resistant to Fas-induced toxicity. However, they are more susceptible to rAPO2L/TRAIL than T cells in the SF of traumatic arthritis patients. In addition, we found very low amounts of bioactive FasL and APO2L/TRAIL associated with exosomes in SF from RA patients as compared with SF from traumatic arthritis patients. The observation on the sensitivity of RA SF T cells to rAPO2L could have therapeutic implications because bioactive APO2L/TRAIL could be beneficial as a RA treatment.
Assuntos
Artrite Reumatoide/imunologia , Líquido Sinovial/citologia , Linfócitos T/imunologia , Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos de Diferenciação de Linfócitos T/metabolismo , Complexo CD3/imunologia , Complexo CD3/metabolismo , Células Cultivadas , Proteína Ligante Fas/antagonistas & inibidores , Proteína Ligante Fas/imunologia , Proteína Ligante Fas/metabolismo , Feminino , Citometria de Fluxo , Antígenos HLA-DR/imunologia , Antígenos HLA-DR/metabolismo , Humanos , Células Jurkat , Lectinas Tipo C , Masculino , Pessoa de Meia-Idade , Fenótipo , Líquido Sinovial/imunologia , Linfócitos T/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/antagonistas & inibidores , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Receptor fas/imunologia , Receptor fas/metabolismoRESUMO
Tendon xanthomas (TX) are pathognomonic lipid deposits commonly found in familial hypercholesterolemia (FH) patients. The aim of this study was to determine whether macrophages from FH patients with TX (TX+) have higher predisposition to foam cells formation after oxidized LDL (oxLDL) overload than those from FH patients without TX (TX-), and if their differential gene expression profile could explain these different phenotypes. Total RNA pools from macrophages from FH patients TX+ and TX- were analyzed using Affymetrix oligonucleotide arrays to evaluate the gene expression profile in presence and absence of oxLDL. Also, the intracellular lipid content was measured by fluorescence flow cytometry. Results of these studies suggest that macrophages from FH subjects TX+ compared to those TX- have a differential response to oxLDL, since they show higher intracellular cholesterol ester accumulation and a differential gene expression profile. The gene array data were validated by relative quantitative real-time RT-PCR and quantitative ELISA in culture media and plasma samples. FH subjects TX+ showed increased plasma tryptase, TNF-alpha, IL-8 and IL-6 concentrations. We propose that TX formation are associated with higher intracellular lipid content, and higher inflammatory response of macrophages in response to oxLDL.
Assuntos
Células Espumosas/metabolismo , Hiperlipoproteinemia Tipo II/complicações , Lipoproteínas LDL/farmacologia , Tendões , Xantomatose/metabolismo , Ésteres do Colesterol/análise , LDL-Colesterol/farmacologia , Células Espumosas/química , Células Espumosas/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Hiperlipoproteinemia Tipo II/genética , Hiperlipoproteinemia Tipo II/metabolismo , Mediadores da Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Xantomatose/genética , Xantomatose/imunologiaRESUMO
A systematic study was undertaken to characterize the role of APO 2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (APO2L/TRAIL) and Fas ligand (FasL) together with the expression of several anti- or proapoptotic proteins in the down-regulation of normal human T cell responses. We have observed for the first time that the higher sensitivity of normal human T cell blasts to apoptosis and activation-induced cell death (AICD) as compared with naive T cells correlates with the increased expression of Bcl-x short (Bcl-xS) and Bim. T cell blasts die in the absence of interleukin 2 (IL-2) with no additional effect of death receptor ligation. In the presence of IL-2, recombinant APO2L/TRAIL or cytotoxic anti-Fas monoclonal antibodies induce rather inhibition of IL-2-dependent growth and not cell death on normal human T cell blasts. This observation is of physiological relevance, as supernatants from T cell blasts, pulse-stimulated with phytohemagglutinin (PHA) or through CD3 or CD59 ligation and containing bioactive APO2L/TRAIL and/or FasL expressed on microvesicles or direct CD3 or CD59 ligation, had the same effect. Cell death was only observed in the presence of cycloheximide or after a pulse through CD3 or CD59, correlating with a net reduction in cellular Fas-associated death domain-like IL-1beta-converting enzyme-inhibitory protein long (c-FLIPL) and c-FLIPS expression. We also show that death receptor and free radical generation contribute, at least partially, to AICD induced by PHA and also to the inhibition of IL-2-dependent cell growth by CD3 or CD59 ligation. Finally, we have also shown that T cell blasts surviving PHA-induced AICD are memory CD44high cells with increased c-FLIPS and Bcl-xL expression.
Assuntos
Proteínas de Transporte/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas/genética , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/genética , Antígenos CD/imunologia , Apoptose , Proteínas Reguladoras de Apoptose , Proteína 11 Semelhante a Bcl-2 , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Morte Celular , Proteína Ligante Fas , Regulação da Expressão Gênica/imunologia , Humanos , Ligante Indutor de Apoptose Relacionado a TNF , Proteína bcl-XRESUMO
Tumor cells have developed multiple mechanisms to evade control by the immune system. Tumoral cells expressing Fas ligand (FasL) have been proposed to "counterattack" against activated antitumoral effector immune cells, although some authors have indicated that FasL is not expressed on the surface of the same tumors, such in the case of melanoma cells. However, other factors could be implicated, such as the balance of soluble versus membrane-bound forms or the secretion of death ligands on the surface of microvesicles, as described previously by our group in human T cells. In the present study, we analyzed the expression and secretion of FasL and APO2 ligand (APO2L)/TRAIL in the human melanoma cell line MelJuSo. We have observed the expression of preformed FasL and APO2L/TRAIL in these cells, their secretion associated with microvesicles upon melanoma activation with PHA or with alpha-melanocyte stimulating hormone (alpha-MSH), and the toxicity of these microvesicles against normal human T cell blasts. We have also observed that the mechanism of secretion of FasL and APO2L/TRAIL from melanoma cells is depending both on microtubules and actin filaments. From these data, it can be concluded that the MelJuSo melanoma cell line has the possibility to "counterattack" against activated immune effector cells. However, the in vivo outcome seems more complex since it has been also described that FasL expressed in tumors has a proinflammatory effect.
Assuntos
Melanoma/metabolismo , Glicoproteínas de Membrana/metabolismo , Vesículas Secretórias/metabolismo , Linfócitos T/citologia , Fator de Necrose Tumoral alfa/metabolismo , Receptor fas/metabolismo , Actinas/metabolismo , Proteínas Reguladoras de Apoptose , Divisão Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Técnicas de Cocultura , Testes Imunológicos de Citotoxicidade , Humanos , Membranas Intracelulares/fisiologia , Células Jurkat , Ligantes , Ativação Linfocitária , Melanoma/imunologia , Melanoma/patologia , Potenciais da Membrana/efeitos dos fármacos , Microtúbulos/metabolismo , Mitocôndrias/fisiologia , Fito-Hemaglutininas/farmacologia , Vesículas Secretórias/efeitos dos fármacos , Vesículas Secretórias/imunologia , Vesículas Secretórias/ultraestrutura , Linfócitos T/imunologia , Ligante Indutor de Apoptose Relacionado a TNF , alfa-MSH/farmacologia , Receptor fas/imunologiaRESUMO
CD8(+) cytotoxic T lymphocyte (CTL) clones are able to exert both perforin- and Fas-dependent cytotoxicity. We show in the present work that phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 prevent TCR/CD3-induced functional Fas ligand (FasL) expression, but not perforin-dependent cytotoxicity. The specific inhibitor of classical protein kinase C (PKC) isoforms, Gö6976, completely inhibited perforin-dependent cytotoxicity and only affected slightly TCR/CD3-induced FasL expression, while the opposite was observed using rottlerin, an inhibitor with higher specificity for PKCtheta. To address further the dependence of FasL expression on PI3K, a luciferase reporter controlled by the FasL promoter was used. Reporter gene induction by anti-CD3 mAb was abolished in cells transfected with dominant-negative PI3K (PI3K-DN) and increased in cells transfected with constitutively active PI3K (PI3K*). Transfection with constitutively active mutants (A/E) of PKCepsilon, and especially of PKCtheta, improved anti-CD3 mAb-induced reporter expression and completely abolished inhibition by wortmannin, while transfection with dominant-negative (K/R) PKCtheta prevented the induction of the reporter. Finally, transfection with PKCalpha A/E, but not with PKCtheta A/E, cooperated with ionomycin to induce degranulation in the CTL line 1.3E6SN. Altogether, the results suggest that TCR/CD3-induced FasL gene transcription is controlled by PI3K and PKCtheta activation, while this signaling pathway is not implicated in CTL degranulation, which is rather dependent on the activation of classical PKC isoforms.
Assuntos
Degranulação Celular/fisiologia , Glicoproteínas de Membrana/fisiologia , Proteína Quinase C/metabolismo , Receptores de Antígenos de Linfócitos T/fisiologia , Linfócitos T Citotóxicos/fisiologia , Acetofenonas/farmacologia , Androstadienos/farmacologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Benzopiranos/farmacologia , Carbazóis/farmacologia , Degranulação Celular/imunologia , Linhagem Celular , Cromonas/farmacologia , Cicloeximida/farmacologia , Citotoxicidade Imunológica/efeitos dos fármacos , Dactinomicina/farmacologia , Proteína Ligante Fas , Humanos , Indóis/farmacologia , Ionomicina/farmacologia , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Células Jurkat , Luciferases/genética , Luciferases/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Glicoproteínas de Membrana/efeitos dos fármacos , Glicoproteínas de Membrana/genética , Camundongos , Morfolinas/farmacologia , Perforina , Ésteres de Forbol/farmacologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Citotóxicas Formadoras de Poros , Proteína Quinase C/análise , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteína Quinase C-alfa , Proteína Quinase C-épsilon , Proteína Quinase C-theta , Complexo Receptor-CD3 de Antígeno de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/fisiologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/enzimologia , Transfecção , WortmaninaRESUMO
Fas/CD95 is a type-I membrane glycoprotein, which inducesapoptotic cell death when ligated by its physiological ligand. We generated previously hyperproliferative sublines derived from the human T-cell leukemia Jurkat, Jurkat-ws and Jurkat-hp, which lost Fas/CD95 surface expression. We have now observed that the total amount of Fas protein is similar in the sublines and in the parental cells, indicating that in the sublines Fas remains in an intracellular compartment. We have found that the protein is directed toward lysosomes in the sublines, where it is degraded. This defect in the secretory pathway correlates with loss of polyunsaturated fatty acids from cellular lipids, and with the lack of expression of endophilin-I and CtBP/BARS, enzymes that regulate vesicle fission by catalyzing the acylation of arachidonate into lysophosphatidic acid. In addition, great multillamer bodies, which contained acid phosphatase activity, absent in the parental Jurkat cells, were observed by transmission electron microscopy in the sublines.