High spontaneous colony growth in chronic myelomonocytic leukemia correlates with increased disease activity and is a novel prognostic factor for predicting short survival.

We have originally shown that spontaneous granulocyte/macrophage colony (CFU-GM) formation in semisolid medium is a characteristic in vitro feature of chronic myelomonocytic leukemia (CMML). However, the clinical significance of spontaneous CFU-GM growth in CMML is unknown so far. CFU-GM growth characteristics were studied in semisolid cultures in the absence of exogenous cytokines using peripheral blood mononuclear cells in 30 patients with CMML at first presentation. The median number of CFU-GM/10(5) MNC of all patients was 48.5 (range 0-622) with 18 patients having colony numbers below 100 (low CFU-GM growth) and 12 patients above 100 (high CFU-GM growth). Kaplan-Meier analysis revealed that patients with high CFU-GM growth had a significantly shorter survival than patients with low CFU-GM growth (median 6.5 vs. 44.5 months, p<0.00002). The probability of survival after 2 years was 60.5% for patients with low colony growth but 0% in those with high colony formation. Patients with CFU-GM >100 had a significantly higher WBC count, a higher LDH, and a higher number of blast cells in blood and bone marrow than patients with low colony growth. Moreover, patients with high colony growth had more often splenomegaly and lower platelet counts. In seven patients, in whom semisolid in vitro cultures were performed after transformation into RAEBT/AML, spontaneous colony growth was significantly increased as compared to CFU-GM growth in patients before transformation (median number/10(5) MNC 533, range 212-4553, p<0.005). This study demonstrates that high (>100) spontaneous CFU-GM formation in CMML at presentation correlates with increased disease activity and represents a novel and important prognostic factor predicting for short survival of CMML patients.

Circulating myeloid colony-forming cells predict survival in myelodysplastic syndromes.

The growth characteristics and the prognostic value of cytokine-stimulated myeloid colony formation from peripheral blood mononuclear cells (PBMC) of patients with myelodysplastic syndromes (MDS) are largely unknown. In this study we have determined the number of myeloid colony-forming units (mCFUs) in semisolid medium from 112 MDS patients and correlated them with French-American-British (FAB) type, the international prognostic scoring system (IPSS), karyotype, peripheral blood (PB) and bone marrow (BM) blast cells, cytopenias, lactate dehydrogenase (LDH), and survival data. Concerning the FAB classification, lower median mCFUs were found in patients with refractory anemia (RA) and refractory anemia with ringed sideroblasts (RARS) compared to refractory anemia with excess of blast cells (RAEB) and refractory anemia with excess of blasts cells in transformation (RAEB-T). In vitro growth in MDS clearly correlated with the cytogenetic risk groups defined by the IPSS (30.5/10(5) PBMCs with favorable karyotypes, 191 in the intermediate prognostic group, 677 with unfavorable cytogenetics, p=0.015 favorable vs unfavorable). BM blast cells >5% (60.5 vs 255 colonies, p=0.032) as well as LDH levels above the normal limit (64.5 vs 425 colonies, p=0.045) were also associated with higher colony formation. Patients were stratified according to the number of circulating mCFUs into a low growth, intermediate growth and high growth group. Median survival was 343 days in the high growth, 1119 days in the low growth, and 2341 days in the intermediate growth group ( p=0.0002). Multivariate analyses revealed colony growth ( p=0.0056), PB blast cells ( p=0.0069), cytogenetic risk group ( p=0.024), and platelet count ( p=0.018) to predict survival in our patients. After inclusion of the IPSS risk categories, mCFU levels remained a highly predictive parameter for survival ( p=0.0056) and acute myeloblastic leukemia (AML) transformation ( p=0.0003).

CD20 antibody (C2B8)-induced apoptosis of lymphoma cells promotes phagocytosis by dendritic cells and cross-priming of CD8+ cytotoxic T cells.

C2B8 (Rituximab, MabThera) is a chimeric mouse/human monoclonal antibody (mAb) directed against the human B cell-restricted cell surface antigen CD20 which is used as an alternative medication in the treatment of B cell non-Hodgkin lymphomas (NHL). Treatment of CD20+ B cells with C2B8 triggers different cell damaging effects including complement-dependent lysis of tumor cells, antibody-dependent cellular cytotoxicity and induction of apoptosis. Dendritic cells (DC) have recently been shown to ingest cell debris and to present associated antigens even on MHC class I molecules, a mechanism called cross-presentation. In this study, we investigated whether C2B8 treatment of lymphoma promotes the induction of CD8+ T cell responses against lymphoma cell-associated antigens via, cross-presentation. We used Daudi lymphoma cells as a model system in our studies and could demonstrate, that C2B8-treated Daudi cells undergo apoptosis, are phagocytosed by DC and induce in DC typical features of maturation; among them, the induction of CD83 expression as well as the up-regulation of prominent accessory molecules (CD40, CD86) and MHC molecules. Importantly, upon co-culture of such lymphoma cell-pulsed DC with autologous T cells, we could induce efficient cytotoxic T cell (CTL) responses against Daudi cell-associated antigens. These findings suggest that antibody treatment of tumor cells can, in addition to its direct cell damaging effects, under certain conditions, contribute to an induction of potentially protective cytotoxic T cell responses.

Calcium ionophore: a single reagent for the differentiation of primary human acute myelogenous leukaemia cells towards dendritic cells.

Blood monocytes and CD34(+) haemopoietic progenitor cells, as well as certain leukaemic cell lines, acquire characteristics of mature dendritic cells (DC) after stimulation with calcium ionophore (CI). We studied whether the in vitro treatment of primary human acute myelogenous leukaemia (AML) cells with CI leads to differentiation towards DC. Blast cells derived from nine AML patients were cultured in the presence of either CI or an established differentiation cocktail consisting of granulocyte-macrophage colony-stimulating factor plus interleukin 4 and tumour necrosis factor-alpha for 5-7 d. Microscopic examination revealed that under both conditions, AML cells were shifted along the DC pathway. In seven out of nine cases, CI-cultivation led to a higher proportion of cells with dendritic morphology. The percentage of CD40 and CD86 expressing cells was significantly increased upon CI treatment compared with cytokine-cultured cells. DC molecules as CD80 and CD83 were up-regulated upon calcium mobilization of AML cells in four out of nine samples. In four cases, CI-treated stimulator cells induced an enhanced proliferative allogeneic T-cell response compared with cytokine-treated stimulator cells. In conclusion, these data demonstrate that CI treatment is an alternative in vitro strategy to differentiate human AML cells into DC.

Cytogenetic risk groups in acute myeloblastic leukaemia differ greatly in their semi-solid colony growth.

We have analysed the results of semi-solid bone marrow cultures in 296 patients with de novo acute myeloblastic leukaemia (AML) and correlated them with the leukaemic karyotype. A favourable prognostic karyotype was found in 52 patients (group A, 18.3%), an intermediate karyotype in 163 patients (group B, 57.4%), and unfavourable cytogenetics were observed in 69 patients (group C, 24.3%). Median colony growth according to the three risk groups was 2 (range 0--344) in group A, 14.5 (range 0--5000) in group B and 50.0 (0--3000) in group C (A vs. B, P < 0.001; A vs. C, P < 0.001; B vs. C, P < 0.01). Among the patients treated with chemotherapy (n = 257), median colony growth was 10 (range 0-5000) in those who achieved complete remission (CR) compared with 56.5 (range 0-1000) in patients without remission (NR) (P = 0.002). The median colony growth of all patients [13/10(5) bone marrow mononuclear cells (BMMCs); range 0--5000] significantly discriminated between patients regarding survival (OS 11 vs. 7 months, P = 0.044). However, multiple Cox regression analysis revealed cytogenetic risk groups as the most important predictor for achieving CR, disease-free and overall survival, with colony growth adding no additional prognostic information. In 64 patients, colony growth was also investigated without the addition of exogenous cytokines. Interestingly, none of the patients with a favourable karyotype exhibited autonomous growth, whereas 50% with an intermediate and 73% of patients with an unfavourable karyotype displayed either partial or full autonomous growth in vitro (P = 0.0004). Our data suggest that the growth potential of the leukaemic clone seems to be critically influenced by the molecular changes emerging from chromosomal abnormalities.

Kell is not restricted to the erythropoietic lineage but is also expressed on myeloid progenitor cells.

Anti-Kell antibodies have been shown to suppress fetal erythropoiesis, but little is known about their effect on myelopoiesis. We analysed the effect of Kell-related antibodies on granulocyte-macrophage colony-forming units (CFU-GM) growth in semisolid medium using peripheral blood mononuclear cells (PBMNCs) from haematologically normal individuals. In addition to its inhibitory effect on erythroid burst-forming units (BFU-E) growth, anti-Kell antibodies significantly reduced CFU-GM colony formation from Kell-positive individuals but not from Kell-negative donors. Moreover, anti-cellano and anti-Kpb antibodies also inhibited the growth of CFU-GM from antigen-positive MNCs. These data indicate that Kell is not restricted to erythroid blood cells, but is also expressed on myeloid progenitor cells.

Generation of dendritic cells from human chronic myelomonocytic leukemia cells in fetal calf serum-free medium.

It is generally believed that the immune system plays a role not only in the acquisition of malignant diseases but also in the rejection of microscopic as well as established tumor cells. Failure of the immune system to eliminate tumor cells may be, among other factors, due to an insufficient presentation of tumor antigens. Dendritic cells (DCs), as professional antigen-presenting cells, therefore, may be therapeutically used to initiate or enhance immune responses in patients with malignancies. In this study we demonstrate that peripheral blood cells of patients with chronic myelomonocytic leukemia (CMML) can be induced to acquire DC characteristics. Upon culture with granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin-4 (IL-4) plus tumor necrosis factor alpha (TNFalpha), CMML cells develop DC morphology and acquire the phenotypic characteristics of DCs. When a CD14+ cell population is used for DC generation, a homogeneous differentiation towards the DC lineage occurs similar to that, observed in normal peripheral blood monocytes. CMML-derived DCs are potent stimulators of the allogeneic mixed lymphocyte reaction (MLR) when compared with uncultivated cells. The demonstration of a deletion of the long arm of chromosome 7, del(7)(q22), in 86% of highly enriched CD1a+ cells by fluorescence in situ hybridization (FISH) indicates the leukemic origin of generated DCs. In addition, we present data that generation of CMML-derived DCs is also possible under fetal calf serum-free conditions for a potential clinical use. These DCs may be used as a cellular vaccine to induce anti-tumor immunity in patients with CMML.

Culture requirements for induction of dendritic cell differentiation in acute myeloid leukemia.

Peripheral blood mononuclear cells (PBMCs) from 15 newly diagnosed acute myeloid leukemia (AML) patients were cultured in fetal calf serum-free media supplemented with either granulocyte/macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-4 and tumor necrosis factor alpha (TNFalpha), or GM-CSF, stem cell factor (SCF), TNFalpha and transforming growth factor beta (TGFbeta) in order to generate leukemia-derived dendritic cells (DCs). Cultured cells were analyzed by flow cytometry with respect to DC-associated surface molecules (CD1a, CD83, CD40, CD80, CD86, HLA-DR) when they showed significant DC morphology in culture (14 cases). After cultivation, neo-expression or upregulation of CD1a antigen was found in 8 samples, CD83 in 2, CD40 in 14, CD80 in 7, and CD86 in 9. Twelve of 14 AMLs, in which DC morphology could be induced upon cultivation, showed upregulation of at least 2 DC-associated molecules. For induction of DC differentiation. GM-CSF, IL-4 plus TNFalpha was superior in 11 cases, and better results were obtained with GM-CSF, SCF, TNFalpha plus TGFbeta in 3 cases. In 7 of 14 samples tested, a marked increase of the T-cell stimulatory capacity could be demonstrated in the allogeneic mixed lymphocyte reaction. The leukemic origin of in vitro-generated DCs was demonstrated by fluorescence in situ hybridization in a patient with translocation t(15;17). Our results suggest that the use of different culture conditions may extend the number of AML patients in which a differentiation towards the DC lineage can be induced in vitro.

1,25-Dihydroxyvitamin D(3) inhibits dendritic cell differentiation and maturation in vitro.

OBJECTIVE: Because of its potent immunosuppressive properties in vitro as well as in vivo, we studied the effect of 1,25-dihydroxyvitamin D(3) (calcitriol) on differentiation, maturation, and function of dendritic cells (DC). MATERIALS AND METHODS: Monocyte-derived DCs were generated with GM-CSF plus IL-4, and maturation was induced by a 2-day exposure to TNFalpha. DCs were derived from CD34(+) progenitors using SCF plus GM-CSF plus TNFalpha. For differentiation studies, cells were exposed to calcitriol at concentrations of 10(-)(9)- 10(-7) M at days 0, 6, and 8, respectively. The obtained cell populations were evaluated by morphology, phenotype, and function. RESULTS: When added at day 0, calcitriol blocked DC differentiation from monocytes and inhibited the generation of CD1a(+) cells from progenitor cells while increasing CD14(+) cells. Exposure of immature DCs to calcitriol at day 6 resulted in a loss of the DC-characteristic surface molecule CD1a, downregulation of the costimulatory molecules CD40 and CD80, and MHC class II expression, whereas the monocyte/macrophage marker CD14 was clearly reinduced. In addition, calcitriol hindered TNFalpha-induced DC maturation, which is usually accompanied with induction of CD83 expression and upregulation of costimulatory molecules. In contrast, the mature CD83(+) DCs remained CD1a(+)CD14(-) when exposed to calcitriol. The capacity of cytokine-treated cells to stimulate allogeneic and autologous T cells and to take up soluble antigen was inhibited by calcitriol. CONCLUSION: The potent suppression of DC differentiation, the reversal of DC phenotype, and function in immature DCs, as well as the inhibition of DC maturation by calcitriol, may explain some of its immunosuppressive properties.

Long-term improvement of hematopoiesis following cyclosporine treatment in a patient with myelodysplastic syndrome.

Current treatment of patients with myelodysplastic syndrome (MDS) is unsatisfactory. Very recently, immunosuppressive treatment strategies have been gaining interest. We report a patient with transfusion-dependent MDS who achieved significant hematopoietic improvement following cyclosporine (CsA) therapy and who is now transfusion independent for more than 5 years. This single observation supports the view that CsA, among other immunosuppressive agents, could play an important role in future treatment concepts in MDS and may lead to clinically relevant and sustained improvement of hematopoiesis in a subset of patients.

Interleukin-10 inhibits burst-forming unit-erythroid growth by suppression of endogenous granulocyte-macrophage colony-stimulating factor production from T cells.

Numerous cytokines released from accessory cells have been shown to exert either stimulatory or inhibitory growth signals on burst-forming unit-erythroid (BFU-E) growth. Because of its cytokine synthesis-inhibiting effects on T cells and monocytes, interleukin-10 (IL-10) may be a potential candidate for indirectly affecting erythropoiesis. We investigated the effects of IL-10 on BFU-E growth from normal human peripheral blood mononuclear cells (PBMC) using a clonogenic progenitor cell assay. The addition of recombinant human IL-10 to cultures containing recombinant human erythropoietin suppressed BFU-E growth in a dose-dependent manner (by 55.2%, range 47.3-63.3%, p < 0.01, at 10 ng/mL). In contrast, no inhibitory effect of IL-10 was seen when cultivating highly enriched CD34+ cells. BFU-E growth from PBMC also was markedly suppressed in the presence of a neutralizing anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibody (by 48.7%, range 32.9-61.2% inhibition,p < 0.01), but not by neutralizing antibodies against granulocyte colony-stimulating factor and interleukin-3. This suggests a stimulatory role of endogenously released GM-CSF on BFU-E formation. Also, the addition of exogenous GM-CSF completely restored IL-10-induced suppression of BFU-E growth. To determine the cellular source of GM-CSF production, we analyzed GM-CSF levels in suspension cultures containing PBMC that were either depleted of monocytes or T cells. Monocyte-depleted PBMC showed spontaneous production of increasing amounts of GM-CSF on days 3, 5, and 7, respectively, which could be suppressed by IL-10, whereas GM-CSF levels did not increase in cultures containing T-cell-depleted PBMC. Our data indicate that IL-10 inhibits the growth of erythroid progenitor cells in vitro, most likely by suppression of endogenous GM-CSF production from T cells.

[Ways and means for maintaining the thermal balance of pilots cosmonauts]. / Sposoby i sredstva podderzhaniia teplovogo balansa letchikov i kosmonavtov.

Different principles used in systems ensuring thermal balance of pilots and cosmonauts are discussed. Physiological and hygienic characteristics of the passive thermal insulation, ventilation systems and liquid cooling (heating) suits are presented. A classification of the means and methods is proposed.