Chronic Lymphocytic Leukemia Articles and Brief Reports Enhancement of fludarabine sensitivity by all-trans-retinoic acid in chronic lymphocytic leukemia cells

Background A subset of patients with fludarabine-resistant chronic lymphocytic leukemia has previously been shown to express elevated intracellular levels of the concentrative high-affinity fludarabine transporter hCNT3, without any detectable related activity. We have recently shown that all-trans-retinoic acid is capable of inducing hCNT3 trafficking to plasma membrane in the MEC1 cell line. We, therefore, evaluated the effect of all-trans-retinoic acid on hCNT3 in primary chronic lymphocytic leukemia cells as a suitable mechanism to improve fludarabinebased therapy of chronic lymphocytic leukemia. Design and Methods Cells from 23 chronic lymphocytic leukemia patients wild-type for P53 were analyzed for ex vivo sensitivity to fludarabine. hCNT3 activity in chronic lymphocytic leukemia cell samples was evaluated by measuring the uptake of H]-fludarabine. The amounts of transforming growth factor-b1 and hCNT3 messenger RNA were analyzed by real-time polymerase chain reaction. The effect of all-trans-retinoic acid on hCNT3 subcellular localization was analyzed by confocal microscopy and its effect on fludarabine-induced apoptosis was evaluated by flow cytometry analysis using annexin V staining. Results Chronic lymphocytic leukemia cases showing higher ex vivo basal sensitivity to fludarabine also had a greater basal hCNT3-associated fludarabine uptake capacity compared to the subset of patients showing ex vivo resistance to the drug. hCNT3 transporter activity in chronic lymphocytic leukemia cells from the latter patients was either negligible or absent. Treatment of the fludarabine-resistant subset of chronic lymphocytic leukemia cells with all-trans-retinoic acid induced increased fludarabine transport via hCNT3 which was associated with a significant increase in fludarabine sensitivity. Conclusions Improvement of ex vivo fludarabine sensitivity in chronic lymphocytic leukemia cells is associated with increased hCNT3 activity after all-trans-retinoic acid treatment

Signaling capacity of FcγRII isoforms in B-CLL cells

Two main isoforms of Fcγ receptor II (CD32) have been described in humans: activatory FcγRIIA and inhibitory FcγRIIB. We have previously reported that B cells from a subset of chronic lymphocytic leukemia (B-CLL) patients express not only FcγRIIB, as normal B lymphocytes, but also the myeloid FcγRIIA. The aim of this study was to evaluate the signaling capacity of both FcγRII isoforms in B-CLL cells. We found that FcγRIIA expressed by leukemic cells failed to induce Ca2+ mobilization or protein tyrosine phosphorylation, suggesting that the receptor is not functional. By contrast, FcγRIIB effectively diminished BCR-triggered ERK1 phosphorylation, which indicates that it is able to transduce inhibitory signals in B-CLL cells. Moreover, we found that FcγRIIB homoaggregation in either B-CLL or non-malignant tonsillar B cells did not result in apoptosis as was reported for murine B splenocytes. Together, these results show that FcγRIIB, but not FcγRIIA is biologically active in B-CLL cells and might influence leukemic cell physiology in vivo. © 2005 Elsevier Ltd. All rights reserved.Fil: Gamberale, Romina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Fernández Calotti, Paula. Academia Nacional de Medicina de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Sanjurjo, Julieta. Academia Nacional de Medicina de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Arrossagaray, Guillermo. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: Avalos, Julio Sánchez. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Geffner, Jorge Raúl. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Giordano, Mirta Nilda. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentin

The Effect Of Fludarabine On Interferon-Gamma Production By Lymphoid Cells From Healthy Donors And Patients With B-Cell Chronic Lymphocytic Leukemia

Fludarabine treatment in patients with B-cell chronic lymphocytic leukemia (B-CLL) can trigger or exacerbate the development of autoimmune hemolytic anemia (AHA) through a currently illdefined mechanism. We here show that exposure of peripheral blood lymphocytes from healthy donors and B-CLL patients to fludarabine increases in vitro production of interferon-g, a key cytokine in the pathogenesis of AHA.Fil: Gamberale, Romina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: Fernández Calotti, Paula. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: Sánchez Ávalos, Julio César Américo. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Alberto, Maria Fabiana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: Geffner, Jorge Raúl. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: Giordano, Mirta Nilda. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; Argentin

Downregulation of Mac-1 Expression in Monocytes by Surface-Bound IgG

Physical and functional association between the β2-integrin Mac-1 (CD11b/ CD18) and receptors of immunoglobulin G (IgG) (FcγRs) has been previously reported. In this study, we examined the modulation of Mac-1 expression by IgG in different leucocyte populations. Our data show that human monocytes, but not neutrophils, macrophages, dendritic or natural killer cells, downregulate the expression of Mac-1 after overnight exposure to surface-bound IgG. This effect, which requires at least 6 h of incubation, is not associated with a general downmodulation of membrane antigens, and is selectively induced by immobilized IgG (iIgG), as the stimulation of monocytes with N-formyl-methionyl-leucyl-phenylalanine, lipopolysaccharide, tumour necrosis factor-α (TNF-α) or soluble IgG did not modify the Mac-1 expression after 18 h in culture. The loss of Mac-1 was completely prevented by blocking antibodies (Abs) directed to FcγRII (CD32) or CD 18. On the other hand, the serine protease inhibitor, phenyl methyl sulphonyl fluoride, but not inhibitors of cysteine proteases or neutral endopeptidases, partially prevented the downregulation of Mac-1 by iIgG. Monocytes cultured overnight on iIgG exhibited a dramatic decrease in their capacity to ingest zymosan particles that could be attributed to the reduced expression of Mac-1. However, there was no inhibition of TNF-α production induced by zymosan, suggesting that Mac-1-dependent responses require different levels of its expression to be fully activated.Fil: Fernández Calotti, Paula X.. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: Salamone, Gabriela Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: Gamberale, Romina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: Trevani, Analía Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: Vermeulen, Elba Monica. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: Geffner, Jorge Raúl. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: Giordano, Mirta Nilda. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; Argentin

In vitro susceptibility of CD4+ and CD8+ T cell subsets to fludarabine

Administration of the adenosine analogue fludarabine (FLU) in vivo induces a profound and prolonged T lymphopenia which mainly affects CD4+ cells. To better understand the mechanistic basis underlying this preferential depletion, we analyzed the in vitro susceptibility of T cell subsets to FLU-induced apoptosis. Contrasting with observations in vivo, our results showed that treatment of peripheral blood mononuclear cells with FLU induced a higher level of apoptosis in CD8+ than in CD4+ T lymphocytes. This increased sensitivity of CD8+ T cells to FLU was observed in samples from both, healthy donors and B cell chronic lymphocytic leukemia patients, and resulted in higher CD4:CD8 ratios in FLU-treated than in untreated cultures (P<0.01). Expression of factors involved in FLU transport and metabolism was then evaluated by quantitative real time-PCR in normal T cell subsets. It was found that mRNA levels of human equilibrative nucleoside transporter-1 nucleoside transporter were higher whereas deoxycytidine kinase and IMP/GMP selective 5′-nucleotidase mRNA levels were lower in CD4 + cells. However the dCK/cN-II ratio was 2-fold greater in CD8 + than in CD4+ T lymphocytes, which could account for the higher apoptosis levels observed in the CD8+ subset. These results favor the view that decreased CD4:CD8 ratios in FLU-treated patients should be attributed to differences in cell recovery and/or homing between T cell subsets. © 2003 Elsevier Inc. All rights reserved.Fil: Gamberale, Romina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Invest. Hematologicas "Mariano R. Castex". Departamento de Inmunologia y Medicina Experimental. Div.de Inmunologia Oncologica; ArgentinaFil: Galmarini, Carlos Maria. Faculté de Médécine Rockefeller; FranciaFil: Fernández Calotti, Paula. Academia Nacional de Medicina de Buenos Aires. Instituto de Invest. Hematologicas "Mariano R. Castex". Departamento de Inmunologia y Medicina Experimental. Div.de Inmunologia Oncologica; ArgentinaFil: Jordheim, Lars. Faculté de Médécine Rockefeller; FranciaFil: Sánchez Ávalos, Julio César Américo. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Dumontet, Charles. Faculté de Médécine Rockefeller; FranciaFil: Geffner, Jorge Raúl. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Invest. Hematologicas "Mariano R. Castex". Departamento de Inmunologia y Medicina Experimental. Div.de Inmunologia Oncologica; ArgentinaFil: Giordano, Mirta Nilda. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Invest. Hematologicas "Mariano R. Castex". Departamento de Inmunologia y Medicina Experimental. Div.de Inmunologia Oncologica; Argentin

Acidosis Improves Uptake of Antigens and MHC Class I-Restricted Presentation by Dendritic Cells

It is widely appreciated that inflammatory responses in peripheral tissues are usually associated to the development of acidic microenvironments. Despite this, there are few studies aimed to analyze the effect of extracellular pH on immune cell functions. We analyzed the impact of acidosis on the behavior of dendritic cells (DCs) derived from murine bone marrow. We found that extracellular acidosis (pH 6.5) markedly stimulated the uptake of FITC-OVA, FITC-dextran, and HRP by DCs. In fact, to reach similar levels of endocytosis, DCs cultured at pH 7.3 required concentrations of Ag in the extracellular medium almost 10-fold higher compared with DCs cultured at pH 6.5. Not only the endocytic capacity of DCs was up-regulated by extracellular acidosis, but also the expression of CD11c, MHC class II, CD40, and CD86 as well as the acquisition of extracellular Ags by DCs for MHC class I-restricted presentation. Importantly, DCs pulsed with Ag under acidosis showed an improved efficacy to induce both specific CD8+ CTLs and specific Ab responses in vivo. Our results suggest that extracellular acidosis improves the Ag-presenting capacity of DCs.Fil: Vermeulen, Elba Monica. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología; ArgentinaFil: Giordano, Mirta Nilda. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología; Argentina. Academia Nacional de Medicina de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Trevani, Analía Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología; Argentina. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: Sedlik, Christine. Institut National de la Santé et de la Recherche Médicale; FranciaFil: Gamberale, Romina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología; Argentina. Academia Nacional de Medicina de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fernández Calotti, Paula. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: Salamone, Gabriela Veronica. Academia Nacional de Medicina de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Raiden, Silvina Claudia. Academia Nacional de Medicina de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sanjurjo, Julieta. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: Geffner, Jorge Raúl. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología; Argentina. Academia Nacional de Medicina de Buenos Aires; Argentin