RESUMO
Interleukin 4 (IL-4), an essential mediator of B cell development, plays a role in survival of chronic lymphocytic leukemia (CLL) cells. To obtain new insights into the function of the IL-4 pathway in CLL, we analyzed the gene expression response to IL-4 in CLL and in normal B cells (NBC) by oligonucleotide microarrays, resulting in the identification of 232 non-redundant entities in CLL and 146 in NBC (95 common, 283 altogether), of which 189 were well-defined genes in CLL and 123 in NBC (83 common, 229 altogether) (p<0.05, 2-fold cut-off). To the best of our knowledge, most of them were novel IL-4 targets for CLL (98%), B cells of any source (83%), or any cell type (70%). Responses were significantly higher for 54 and 11 genes in CLL and NBC compared to each other, respectively. In CLL, ZAP-70 status had an impact on IL-4 response, since different sets of IL-4 targets correlated positively or negatively with baseline expression of ZAP-70. In addition, the NFκB inhibitor 6-Amino-4-(4-phenoxyphenethylamino)quinazoline, which reversed the anti-apoptotic effect of IL-4, preferentially blocked the response of genes positively correlated with ZAP-70 (e.g. CCR2, SUSD2), but enhanced the response of genes negatively correlated with ZAP-70 (e.g. AUH, BCL6, LY75, NFIL3). Dissection of the gene expression response to IL-4 in CLL and NBC contributes to the understanding of the anti-apoptotic response. Initial evidence of a connection between ZAP-70 and NFκB supports further exploration of targeting NFκB in the context of the assessment of inhibition of the IL-4 pathway as a therapeutic strategy in CLL, especially in patients expressing bad prognostic markers.
Assuntos
Biomarcadores Tumorais/genética , Perfilação da Expressão Gênica , Proteínas I-kappa B/genética , Interleucina-4/farmacologia , Leucemia Linfocítica Crônica de Células B/genética , NF-kappa B/antagonistas & inibidores , Proteína-Tirosina Quinase ZAP-70/genética , Apoptose , Western Blotting , Estudos de Casos e Controles , Proliferação de Células , Células Cultivadas , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/patologia , Linfócitos/citologia , Linfócitos/metabolismo , NF-kappa B/genética , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The density gradient centrifugation method was originally designed for the isolation of mononuclear peripheral blood cells and rapidly adapted to fractionate bone marrow (BM) cells. This method involves the use of gradient density solutions with low viscosity and low osmotic pressure that allows erythrocytes and more mature cells gravitate to the bottom at a density fraction superior to 1.080 g/dL; mononuclear cells (MNCs) held in the plasma-solution to interphase at a density between 1.053 and 1.073 g/dL; plasma, dilution medium and anticoagulant to occupy a density less than 1.050 g/dL and the fat cells to float due to their very low density. BM-mesenchymal stem cells (MSCs) are usually obtained after the separation and cultures of BM-MNCs from the plasma-solution interphase, which is traditionally considered the only source of progenitor cells (hematopoietic and nonhematopoietic). In this study evidences that MSCs could be isolated from the very low-density cells of the fat layer are presented. In addition, we demonstrated that the MSCs obtained from these cells have similar immunophenotypic characteristics, and similar proliferative and differentiation potential to those obtained from the MNCs at plasma-solution interphase. The method represents a simple and cost effective way to increase the MSCs yield from each BM donor, without the need to look for other sources, additional manipulation of cells, and risks of contamination or disturbances of the potential of differentiation. These cells might serve as a complementary source of MSCs to facilitate preclinical and clinical application in tissue engineering and cell therapy.
Assuntos
Células da Medula Óssea/citologia , Separação Celular/métodos , Células-Tronco Mesenquimais/citologia , Adolescente , Adulto , Células da Medula Óssea/fisiologia , Técnicas de Cultura de Células , Diferenciação Celular , Proliferação de Células , Centrifugação com Gradiente de Concentração , Criança , Feminino , Humanos , Imunofenotipagem , Masculino , Células-Tronco Mesenquimais/fisiologia , Pessoa de Meia-IdadeRESUMO
BACKGROUND: The aim of this study was to analyze the impact of cryopreservation in series of peripheral blood progenitor cells stratified by diagnosis, mobilization treatments, and cell concentration, as well as the accuracy of the control aliquots. STUDY DESIGN AND METHODS: Viability and colony-forming unit-granulocyte-macrophage (CFU-GM), CD34+ cell, lymphocyte, monocyte, and granulocyte counts and recovery were analyzed in 397 leukapheresis procedures before freezing and after thawing. Data from control cryotubes were compared to those from infusing bags. RESULTS: Cell viability decreased after thawing. Viability recovery was lower in cryotubes than in bags in non-Hodgkin's lymphoma (NHL), in cyclophosphamide plus granulocyte-colony-stimulating factor (Cy+G-CSF) mobilization, and in cell concentration of median or greater. Viability recovery in cryotube was higher in NHL (92.1%) than in Hodgkin's disease (HD; 87.3%) and in G-CSF (95.9%) than Cy+G-CSF mobilization (91.3%). The number of CD34+ cells decreased after thawing in total group, Cy+G-CSF mobilization, and cell concentration less than median subgroups. CD34+ cell recovery was higher in cryotubes (111.3%) than in bags (99.6%) in multiple myeloma (MM; p = 0.015). CFU-GM decreased after thawing in all groups. CFU-GM recovery was lower in cryotubes than in bags in MM (26.0% vs. 59.3%) and in Cy+G-CSF mobilization (49.8% vs. 76.3%). CFU-GM recovery in cryotubes was lower in MM compared with NHL (61.5%), HD (45.1%), and breast cancer (84.0%). Lymphocytes, monocytes, and granulocytes showed differences in the subgroups. CONCLUSION: Cryopreservation negatively impacts in cell viability, CD34+ cell recovery, granulocytes, and CFU-GM, although slight differences between the groups were observed. Cryotubes satisfactorily reflected the quality of the infused cells.
Assuntos
Células Sanguíneas/citologia , Preservação de Sangue/métodos , Criopreservação/métodos , Células-Tronco/citologia , Adolescente , Adulto , Idoso , Células Sanguíneas/metabolismo , Preservação de Sangue/efeitos adversos , Sobrevivência Celular/fisiologia , Criança , Pré-Escolar , Feminino , Células Progenitoras de Granulócitos e Macrófagos/citologia , Células Progenitoras de Granulócitos e Macrófagos/metabolismo , Granulócitos/citologia , Granulócitos/metabolismo , Humanos , Leucaférese , Linfócitos/citologia , Linfócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Células-Tronco/metabolismo , Adulto JovemRESUMO
Splenic rupture (SR) is a rare adverse event observed in patients treated with G-CSF as a peripheral hematopoietic stem cell (PHSC) mobilizing agent, mostly in myeloma multiple and amiloidosis; to date, to our knowledge, it has not been previously described in plasma-cell leukemia (PCL). We report a case of a woman with PCL, who presented a SR after PHSC mobilization with Cyclophosphamide+G-CSF. The spleen removed showed hematopoietic foci and amiloid material. In the course of a second mobilization, 2 months after, the patient died from sepsis. We considered it important to report this case, in order to keep in mind the possibility of SR in patients with malignant gammopathy.
Assuntos
Fator Estimulador de Colônias de Granulócitos/efeitos adversos , Leucemia Plasmocitária/complicações , Ruptura Esplênica/etiologia , Evolução Fatal , Feminino , Mobilização de Células-Tronco Hematopoéticas/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Pessoa de Meia-Idade , Esplenomegalia/induzido quimicamente , Transplante AutólogoRESUMO
Given that pre-apheresis CD34(+) cell count (PA-CD34) predicts the apheresis' yield, a minimum of 5 to 20 PA-CD34/microl is required in many institutions to initiate cell collection. The aim of this study was to clarify whether large-volume-apheresis (LVA) could facilitate progenitor cell transplantation in patients with low PA-CD34. Apheresis was initiated in 226 patients, disregarding PA-CD34, at days: +5 in G-CSF, +10 in cyclophosphamide+G-CSF, and +15 to +20 in other chemotherapy+G-CSF mobilization, when leucocytes >2.5 x 10(9)/L. Four times the blood volume was processed. Patients were grouped according to their PA-CD34: >or=10/microl (group-A, n = 143); <10/microl but >or=5/microl (group-B, n = 40) and <5/microl (group-C, n = 43). No differences were found in diagnoses, gender, age, previous treatments or mobilization regimen between groups. Enough CD34(+) cells (>1.9 x 10(6)/kg) were obtained in 31 patients (72%) from group-C, although in this group two mobilizations were needed in 20 patients (46.5%), compared to 5 (3.5%) and 1 (2.5%) in groups A and B, respectively (P < 0.01). Evenly three apheresis or more were required in 28 patients (65.1%) from group-C, compared to 8 (5.6%) and 6 (15.0%) in groups A and B, respectively (P < 0.01). In conclusion LVA can facilitate autologous transplantation in poor-mobilizer-patients, low PA-CD34 should not be an inflexible exclusion factor.