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1.
Int J Mol Sci ; 25(11)2024 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-38891959

RESUMO

The tyrosine kinase domain of the FMS-Like tyrosine kinase 3 (FLT3-TKD) is recurrently mutated in acute myeloid leukemia (AML). Common molecular techniques used in its detection include PCR and capillary electrophoresis, Sanger sequencing and next-generation sequencing with recognized sensitivity limitations. This study aims to validate the use of droplet digital PCR (ddPCR) in the detection of measurable residual disease (MRD) involving the common FLT3-TKD mutations (D835Y, D835H, D835V, D835E). Twenty-two diagnostic samples, six donor controls, and a commercial D835Y positive control were tested using a commercial Bio-rad® ddPCR assay. All known variants were identified, and no false positives were detected in the wild-type control (100% specificity and sensitivity). The assays achieved a limit of detection suitable for MRD testing at 0.01% variant allelic fraction. Serial samples from seven intensively-treated patients with FLT3-TKD variants at diagnosis were tested. Five patients demonstrated clearance of FLT3-TKD clones, but two patients had FLT3-TKD persistence in the context of primary refractory disease. In conclusion, ddPCR is suitable for the detection and quantification of FLT3-TKD mutations in the MRD setting; however, the clinical significance and optimal management of MRD positivity require further exploration.


Assuntos
Leucemia Mieloide Aguda , Mutação , Neoplasia Residual , Reação em Cadeia da Polimerase , Tirosina Quinase 3 Semelhante a fms , Humanos , Tirosina Quinase 3 Semelhante a fms/genética , Neoplasia Residual/diagnóstico , Neoplasia Residual/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/diagnóstico , Reação em Cadeia da Polimerase/métodos , Feminino , Masculino , Pessoa de Meia-Idade , Idoso , Adulto , Sequenciamento de Nucleotídeos em Larga Escala/métodos
2.
Blood Adv ; 4(19): 4593-4604, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-32986791

RESUMO

CD8+CD57+ terminal effector T (TTE) cells are a component of marrow-infiltrating lymphocytes and may contribute to the altered immune responses in multiple myeloma (MM) patients. We analyzed TTE cells in the bone marrow (BM) and peripheral blood (PB) of age-matched controls and patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering MM (SMM), and newly diagnosed (ND) MM using flow cytometry, mass cytometry, and FlowSOM clustering. TTE cells are heterogeneous in all subjects, with BM containing both CD69- and CD69+ subsets, while only CD69- cells are found in PB. Within the BM-TTE compartment, CD69- and CD69+ cells are found in comparable proportions in controls, while CD69- cells are dominant in MGUS and SMM and predominantly either CD69- or CD69+ cells in NDMM. A positive relationship between CD69+TTE and CD69-TTE cells is observed in the BM of controls, lost in MGUS, and converted to an inverse relationship in NDMM. CD69-TTE cells include multiple oligoclonal expansions of T-cell receptor/Vß families shared between BM and PB of NDMM. Oligoclonal expanded CD69-TTE cells from the PB include myeloma-reactive cells capable of killing autologous CD38hi plasma cells in vitro, involving degranulation and high expression of perforin and granzyme. In contrast to CD69-TTE cells, oligoclonal expansions are not evident within CD69+TTE cells, which possess low perforin and granzyme expression and high inhibitory checkpoint expression and resemble T resident memory cells. Both CD69-TTE and CD69+TTE cells from the BM of NDMM produce large amounts of the inflammatory cytokines interferon-γ and tumor necrosis factor α. The balance between CD69- and CD69+ cells within the BM-TTE compartment may regulate immune responses in NDMM and contribute to the clinical heterogeneity of the disease.


Assuntos
Gamopatia Monoclonal de Significância Indeterminada , Mieloma Múltiplo , Mieloma Múltiplo Latente , Medula Óssea , Humanos , Plasmócitos
3.
Front Immunol ; 10: 1596, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31428081

RESUMO

Multiple Myeloma (MM) is preceded by the clinically stable condition monoclonal gammopathy of undetermined significance (MGUS). Critical immune events that discriminate MGUS from newly diagnosed MM (ND)MM patients remain unknown, but may involve changes in the regulatory T cell (Treg) compartment that favor myeloma growth. To address this possibility, we used mass cytometry and the unsupervised clustering algorithm Flow self-organizing map (FlowSOM) to interrogate the distribution of multiple subsets within CD25+CD127low/negTreg in matched bone marrow (BM) and peripheral blood (PB) of MGUS and NDMM patients. Both mass cytometry and flow cytometry confirmed a trend toward prevalence of CD39-Treg within the Treg compartment in BM and PB of NDMM patients compared to CD39-Treg in MGUS patients. FlowSOM clustering displayed a phenotypic organization of Treg into 25 metaclusters that confirmed Treg heterogeneity. It identified two subsets which emerged within CD39-Treg of NDMM patients that were negligible or absent in CD39-Treg of MGUS patients. One subset was found in both BM and PB which phenotypically resembled activated Treg based on CD45RO, CD49d, and CD62L expression; another subset resembled BM-resident Treg based on its tissue-resident CD69+CD62L-CD49d- phenotype and restricted location within the BM. Both subsets co-expressed PD-1 and TIGIT, but PD-1 was expressed at higher levels on BM-resident Treg than on activated Treg. Within BM, both subsets had limited Perforin and Granzyme B production, whilst activated Treg in PB acquired high Perforin and Granzyme B production. In conclusion, the use of mass cytometry and FlowSOM clustering discovered two discrete subsets of CD39-Treg which are discordant in MGUS and NDMM patients and may be permissive of myeloma growth which warrants further study. Understanding the regulatory properties of these subsets may also advance MGUS and MM diagnosis, prognosis, and therapeutic implications for MM patients.


Assuntos
Apirase/imunologia , Gamopatia Monoclonal de Significância Indeterminada/imunologia , Mieloma Múltiplo/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Medula Óssea/imunologia , Feminino , Citometria de Fluxo/métodos , Humanos , Imunofenotipagem/métodos , Antígenos Comuns de Leucócito/imunologia , Masculino , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/imunologia , Receptores Imunológicos/imunologia
4.
Onco Targets Ther ; 10: 1585-1601, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28352191

RESUMO

Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia (AML) with a unique morphological appearance, associated coagulopathy and canonical balanced translocation of genetic material between chromosomes 15 and 17. APL was first described as a distinct subtype of AML in 1957 by Dr Leif Hillestad who recognized the pattern of an acute leukemia associated with fibrinolysis, hypofibrinogenemia and catastrophic hemorrhage. In the intervening years, the characteristic morphology of APL has been described fully with both classical hypergranular and variant microgranular forms. Both are characterized by a balanced translocation between the long arms of chromosomes 15 and 17, [t(15;17)(q24;q21)], giving rise to a unique fusion gene PML-RARA and an abnormal chimeric transcription factor (PML-RARA), which disrupts normal myeloid differentiation programs. The success of current treatments for APL is in marked contrast to the vast majority of patients with non-promyelocytic AML. The overall prognosis in non-promyelocytic AML is poor, and although there has been an improvement in overall survival in patients aged <60 years, only 30%-40% of younger patients are still alive 5 years after diagnosis. APL therapy has diverged from standard AML therapy through the empirical discovery of two agents that directly target the molecular basis of the disease. The evolution of treatment over the last 4 decades to include all-trans retinoic acid and arsenic trioxide, with chemotherapy limited to patients with high-risk disease, has led to complete remission in 90%-100% of patients in trials and rates of overall survival between 86% and 97%.

5.
J Cardiopulm Rehabil Prev ; 34(2): 98-105, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24531203

RESUMO

PURPOSE: We tested the hypothesis that higher-intensity interval training (HIIT) could be deployed into a standard cardiac rehabilitation (CR) setting and would result in a greater increase in cardiorespiratory fitness (ie, peak oxygen uptake, (·)VO2) versus moderate-intensity continuous training (MCT). METHODS: Thirty-nine patients participating in a standard phase 2 CR program were randomized to HIIT or MCT; 15 patients and 13 patients in the HIIT and MCT groups, respectively, completed CR and baseline and followup cardiopulmonary exercise testing. RESULTS: No patients in either study group experienced an event that required hospitalization during or within 3 hours after exercise. The changes in resting heart rate and blood pressure at followup testing were similar for both HIIT and MCT. (·)VO2 at ventilatory-derived anaerobic threshold increased more (P < .05) with HIIT (3.0 ± 2.8 mL·kg⁻¹·min⁻¹) versus MCT (0.7 ± 2.2 mL·kg⁻¹·min⁻¹). During followup testing, submaximal heart rate at the end of stage 2 of the exercise test was significantly lower within both the HIIT and MCT groups, with no difference noted between groups. Peak (·)VO2 improved more after CR in patients in HIIT versus MCT (3.6 ± 3.1 mL·kg⁻¹·min⁻¹ vs 1.7 ± 1.7 mL·kg⁻¹·min⁻¹; P < .05). CONCLUSIONS: Among patients with stable coronary heart disease on evidence-based therapy, HIIT was successfully integrated into a standard CR setting and, when compared to MCT, resulted in greater improvement in peak exercise capacity and submaximal endurance.


Assuntos
Ponte de Artéria Coronária/reabilitação , Terapia por Exercício/métodos , Infarto do Miocárdio/reabilitação , Consumo de Oxigênio/fisiologia , Intervenção Coronária Percutânea/reabilitação , Pressão Sanguínea/fisiologia , Teste de Esforço , Feminino , Frequência Cardíaca/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Esforço Físico/fisiologia
6.
J Immunol ; 172(6): 3527-34, 2004 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-15004153

RESUMO

Activation of the phosphatidylinositol-3 kinase (PI 3-K) pathway is associated with the proliferation of many cell types, including T lymphocytes. However, recent studies in cell lines stably expressing deletion mutants of IL-2R that fail to activate PI 3-K have questioned the requirement for this pathway in cell cycle regulation. In this study with IL-2 and IL-7, we show in primary T cells that, unlike IL-2, IL-7 fails to induce the early activation of PI 3-K seen within minutes and normally associated with cytokine signaling. However, kinetic experiments showed that both of these T cell growth factors induce a distinct and sustained phase of PI 3-K activity several hours after stimulation. This delayed activation correlates with cell cycle induction and from studies using inhibitors of PI 3-K signaling, we show that this later phase, unlike the early activation within minutes, is required for cell cycle induction. The data presented here will have major implications for our understanding of the mechanism of T cell proliferation as well as the regulation of PI 3-K activity.


Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/enzimologia , Animais , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/imunologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/imunologia , Linhagem Celular , Cromonas/farmacologia , Ciclina E/antagonistas & inibidores , Ciclina E/biossíntese , Cicloeximida/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/imunologia , Inibidores do Crescimento/farmacologia , Imidazóis/farmacologia , Interleucina-2/fisiologia , Interleucina-7/fisiologia , Ativação Linfocitária/imunologia , Camundongos , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/fisiologia , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Proteína do Retinoblastoma/antagonistas & inibidores , Proteína do Retinoblastoma/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Fatores de Tempo
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