Dynamics of human erythroblast enucleation.

How human erythroblasts enucleate remains obscure, and some investigators suspect the effect of mechanical forces on enucleation in vitro. We determined the dynamics of the enucleation process of highly purified human erythroblasts and whether enucleation can occur without external mechanical forces. Highly purified human CD34(+) cells were cultured in liquid phase with interleukin-3, stem cell factor and erythropoietin (EPO) for 7 days and the generated erythroblasts were replaced in the same medium with EPO alone. In some experiments, the enucleating cells were processed without centrifugation and pipette aspiration to avoid physical stress and were directly observed by differential interference contrast (DIC) microscopy. Enucleation initiated at day 12 and the enucleation ratio (percent of enucleated reticulocytes in total cells) reached a maximum at day 14 with a value of 63 +/- 7%. The direct observation by DIC microscopy showed 61 +/- 9% of enucleation ratio at day 14. The human erythroblasts enucleated without contact with macrophage. The time required for enucleation was 8.4 +/- 3.4 min. The enucleation rate was 1.16 +/- 0.42%/h at day 12 and then decreased with a time dependent manner. The expelled nucleus was connected to the reticulocyte through plasma membrane and associated cytoskeletal elements, and spontaneous separation of the extruded nucleus from reticulocyte was extremely rare. In conclusion, human erythroblasts enucleate in a relatively short period without contact with macrophages, but nascent reticulocytes fail to completely separate from nuclei in the absence of macrophages, unless some physical force is applied to them.

Phagocytosis of co-developing neutrophil progenitors by dendritic cells in a culture of human CD34(+) cells with granulocyte colony-stimulating factor and tumor necrosis factor-alpha.

Tumor necrosis factor-alpha (TNF-alpha) has been shown to induce the differentiation of CD34(+) cells toward dendritic cells (DCs). We have previously shown that DCs are co-generated from human CD34(+) cells during erythroid or megakaryocytic differentiation in the presence of TNF-alpha, and those DCs are able to stimulate autologous T cell proliferation. The aim of this study was to learn whether the co-stimulation of granulocyte colony-stimulating factor (G-CSF) and TNF-alpha would generate neutrophil progenitors and DCs together from human CD34(+) cells, and if this was the case, to clarify the phenotypic and functional characteristics of these DCs. When highly purified human CD34(+) cells were cultured for 7 days with G-CSF alone, the generated cells predominantly expressed a granulocyte marker, CD15, and then differentiated into neutrophils after 14 days of culture. The addition of TNF-alpha with G-CSF markedly decreased the number of CD15(+) cells without affecting the total number of cells during 7 days of culture. Almost one third of the generated cells were positive for CD11c and CD123. Furthermore, CD11c(+) cells were found to phagocytose CD15(+) cells and were able to induce allogeneic, but not autologous, T cell proliferation in the mixed lymphocyte reaction (MLR). On the other hand, the CD11c(+) cells generated by TNF-alpha and cytokines capable of inducing erythroid differentiation were able to stimulate autologous T cells. There was a difference in the expression of CD80, CD83 and CD86 among CD11c(+) cells induced by G-CSF plus TNF-alpha and those generated by interleukin-3, stem cell factor, and erythropoietin plus TNF-alpha. These results indicate that the co-stimulation of human CD34(+) cells with G-CSF and TNF-alpha induces the phagocytosis of co-developing neutrophil progenitors by DCs, and the stimulatory effects of these DCs on autologous T cells is different from that of DCs generated from CD34(+) cells during erythroid differentiation.

Itraconazole oral solution enhanced vincristine neurotoxicity in five patients with malignant lymphoma.

To prevent fungal infections in patients undergoing treatment for hematological malignancies, we investigated the use of oral itraconazole solution as opposed to itraconazole or fluconazole capsules. Herein, we report five lymphoma patients with severe vincristine neurotoxicity in strong association with oral itraconazole solution. Four patients suffered from severe myalgia with or without arthralgia which clinically resembled polymyalgia rheumatica. Two patients suffered from constipation due to subileus and one patient had a severe paralytic ileus. Appropriate management of the above symptoms, which included discontinuation of oral itraconazole solution, resulted in rapid recovery from neurotoxicity. Given the more consistent plasma concentrations of oral itraconazole solution when compared to itraconazole capsules and the ability of itraconazole to interfere with hepatic vincristine metabolism, we strongly recommend avoiding the combined administration of oral itraconazole solution and vincristine.

Phagocytosis of codeveloping megakaryocytic progenitors by dendritic cells in culture with thrombopoietin and tumor necrosis factor-alpha and its possible role in hemophagocytic syndrome.

Tumor necrosis factor-alpha (TNF-alpha) and thrombopoietin (TPO) have been shown to induce the differentiation and proliferation of CD34+ cells toward dendritic cells (DCs) in the presence of multiacting cytokines. We hypothesized that the costimulation of TPO and TNF-alpha generates megakaryocytic progenitors and DCs together from human CD34+ cells and that the interaction of these cells may indicate a physiologic and/or a pathologic role of DCs in megakaryopoiesis. When highly purified human CD34+ cells were cultured for 7 days with TPO alone, the generated cells expressed megakaryocytic markers, such as CD41, CD42b, and CD61. The addition of TNF-alpha with TPO remarkably decreased the number of megakaryocytic progenitor cells without affecting the cell yield. Almost half of the cells thus generated expressed CD11c, and most of them were positive for CD4 and CD123. Furthermore, CD11c+ cells were found to capture damaged CD61+ cells and to induce autologous T-cell proliferation, although the cytokine productions were low. We also confirmed an engulfment of CD61+ cells and their fragment by CD11c+ cells in bone marrow cells from patients with hemophagocytic syndrome. These findings suggest that DCs generated under megakaryocytic and inflammatory stimuli are involved in megakaryopoiesis and the subsequent immune responses to self-antigens.