EFFECT OF DIHYDROARTEMISININ (DHA) ON HUMAN ERYTHROID CELL DIFFERENTIATION : IMPLICATIONS FOR MALARIA TREATMENT IN PREGNANCY

Objectives: WHO does not recommend the use of Artemisinin Combination Therapy (ACT) to treat malaria during pregnancy, because animal studies showed a depletion of embryonic erythrocytes. We investigated the effect of Dihydroartemisinin (DHA), the metabolite of artemisinins, on an in vitro model reproducing human erythropoiesis. Methods: CD34+ cells differentiate towards erythroblasts under erythropoietin stimulus in 14 days. DHA, 0,5 or 2 \uf06dM, was added on different erythroid stages. At different time cell growth, morphology, Glycophorin A expression as well as globin genes have been evaluated. Results: DHA added on stem cells or on early progenitors caused a transient inhibitory effect, which was then fully restored. On the contrary, DHA added on more differentiated erythroblasts significantly blocked the erythroid differentiation. This indicates that DHA specifically affects the primitive erythropoiesis, occurring in the yolk sac. Therefore, during the first trimester of pregnancy, ACT must be avoided. EU Antimal Project 18834 is acknowledge

Effect of dihydroartemisinin (DHA) on human erythroid cell differentiation : implications for malaria treatment in pregnancy

BACKGROUND: Severe malaria in pregnancy causes maternal anemia, low birth weight increased mortality of both mother and infants. WHO recommends few antimalarials due to safety problems. Artemisinin combination therapy is the first line treatment, however artemisinin derivatives showed animal embryotoxicity with a reduction of embryonic erythrocytes when treatment is performed on certain days of gestation. AIMS: To investigate the effect of Dihydroartemisinin (DHA), the metabolite of artemisinins, on an in vitro model reproducing human erythropoiesis and to characterize the erythroid target stage, in order to predict the window of susceptibility to DHA in human pregnancy. METHODS: The mononuclear cells derived from pheripheral blood of healthy volunteers were enriched for CD34+ cells by positive selection using anti-CD34-tagged magnetic beads. CD34+ cells were cultured for 14 days with a specific medium containing erythropoietin to induce erythroid differentiation. DHA at 0,5 or 2 \uc2\ub5M, according to the dosages of previous animal experiments, was added for the first time at day 0 (on isolated stem cell), at day 2 (on early erythroid progenitors), at day 4 (in presence of both early progenitors and pro-erythroblasts), at day 7 (on basophilic erythroblasts) or at day 11 (polychromatic erythroblasts) then continuously every 3 days up to 14 days, because of its short half life. Cells growth and viability were evaluated by trypan blue exclusion; erythroid differentiation was investigated by cytofluorimetric analysis of Glycophorin A (GPA) expression, by morphological analysis on benzidine-May-Grunwald-Giemsa stained smears and by erythroid specific gene expression analysis with real-time PCR. RESULTS: DHA was added on stem cells or early erythroid progenitors caused a transient inhibition of both cell growth and differentiation up to day 7, but then the treated cells started growing and completed their erythroid differentiation at day 14 of culture. When DHA was added on basophilic erythroblasts, a significant and long lasting effect decrease in proliferation as well as a delay in erythroid differentiation was observed. Up to day 14. DHA added on mature stages i.e. polychromatic erythroblasts, only a small reduction of cell growth has been observed without any consequence for the erythroid cell differentiation. CONCLUSIONS: These data suggest that DHA\ue2 s specific target is the basophilic erythroblast, since DHA added at this stage causes a significant inhibition of erythroid differentiation. Based on these in vitro results, we hypothesize that DHA could affect human primitive erythropoiesis, which occurs during the late phase of human secondary yolk sac erythropoiesis (weeks 4-8 of gestation), when foetal blood is formed of only primitive erythroblasts. This means that if the treatment with DHA or artemisinin derivatives is performed during the first trimester of human pregnancy, toxic effects on embryo could be expected

Effect of dihydroartemisinin on human Erythroid cell differentiation

Women in their first pregnancy are at the very high-risk of developing severe malaria which includes maternal anemia, low birth weight of newborns and increased mortality of both mother and infants. The WHO recommends the Intermittent Preventive Treatment to cure malaria during gestation, but drug safety in pregnancy is an issue. Artemisinin combination therapy is the first line treatment for uncomplicated malaria, but artemisinin derivatives carry a potential toxic effect on embryos. In animal studies they affect embryonic erythroid precursors only on certain days of gestation. This suggests that the target of DHA toxicity could be the primitive erythropoiesis. Our aim was to study the effect of artemisinin and 4-aminoquinoline derivatives on in vitro models which reproduce human erythropoiesis: K562 leukemia cells and CD34+ from human peripheral blood. Cells switch from fetal and embryonic to adult hemoglobin in presence of hemin or butyric acid (K562) or erythropoietin (CD34+). We found that artemisinins inhibit both cell growth and erythroid differentiation (P<0.05), measured as adult hemoglobin synthesis and erythroblasts count. The effect is dose and time-dependent. DHA, which is the active metabolite of artemisinins, has the strongest effect. As expected chloroquine and amodiaquine did not affect cell differentiation, confirming the suitability of the models for studying drug toxicity on developmental erythropoiesis. Moreover, as in animal studies, our results show that a toxic effect of DHA could occur if administered during first trimester of pregnancy, when fetal blood consists mostly of primitive erythroblasts. The support of EU Antimal Project 18834 is acknowledge

Effect of dihydroartemisinin of human erythroid cell differentiation : implications for malaria treatment in pregnancy

BACKGROUND: Severe malaria in pregnancy causes maternal anemia, low birth weight increased mortality of both mother and infants. WHO recommends few antimalarials due to safety problems. Artemisinin combination therapy is the first line treatment, however artemisinin derivatives showed animal embryotoxicity with a reduction of embryonic erythrocytes when treatment is performed on certain days of gestation. AIMS: To investigate the effect of Dihydroartemisinin (DHA), the metabolite of artemisinins, on an in vitro model reproducing human erythropoiesis and to characterize the erythroid target stage, in order to predict the window of susceptibility to DHA in human pregnancy. METHODS: The mononuclear cells derived from pheripheral blood of healthy volunteers were enriched for CD34+ cells by positive selection using anti-CD34-tagged magnetic beads. CD34+ cells were cultured for 14 days with a specific medium containing erythropoietin to induce erythroid differentiation. DHA at 0,5 or 2 µM, according to the dosages of previous animal experiments, was added for the first time at day 0 (on isolated stem cell), at day 2 (on early erythroid progenitors), at day 4 (in presence of both early progenitors and pro-erythroblasts), at day 7 (on basophilic erythroblasts) or at day 11 (polychromatic erythroblasts) then continuously every 3 days up to 14 days, because of its short half life. Cells growth and viability were evaluated by trypan blue exclusion; erythroid differentiation was investigated by cytofluorimetric analysis of Glycophorin A (GPA) expression, by morphological analysis on benzidine-May-Grunwald-Giemsa stained smears and by erythroid specific gene expression analysis with real-time PCR. RESULTS: DHA was added on stem cells or early erythroid progenitors caused a transient inhibition of both cell growth and differentiation up to day 7, but then the treated cells started growing and completed their erythroid differentiation at day 14 of culture. When DHA was added on basophilic erythroblasts, a significant and long lasting effect decrease in proliferation as well as a delay in erythroid differentiation was observed. Up to day 14. DHA added on mature stages i.e. polychromatic erythroblasts, only a small reduction of cell growth has been observed without any consequence for the erythroid cell differentiation. CONCLUSIONS: These data suggest that DHAâ s specific target is the basophilic erythroblast, since DHA added at this stage causes a significant inhibition of erythroid differentiation. Based on these in vitro results, we hypothesize that DHA could affect human primitive erythropoiesis, which occurs during the late phase of human secondary yolk sac erythropoiesis (weeks 4-8 of gestation), when foetal blood is formed of only primitive erythroblasts. This means that if the treatment with DHA or artemisinin derivatives is performed during the first trimester of human pregnancy, toxic effects on embryo could be expected

In vitro ferroportin expression in thalassemia intermedia during erythroid differentiation

INTRODUCTION: Ferroportin (FPN) is the sole mammalian iron exporter protein known and it plays a critical role in iron metabolism. It is expressed in various types of cells including duodenal enterocytes, hepatocytes, erythroblasts cells, syncytiotrophoblasts and reticuloendothelial macrophages. Ferroportin is expressed in multiple alternative transcripts: with (FPN1A) or without (FPN1B) an iron-responsive element (IRE). The expression of one form rather than the other depends on cell type and iron availability. The expression of ferroportin in thalassemia intermedia (TI), characterized by iron overload, is not yet fully elucidated. AIM: To investigate the different expression profile of ferroportin isoforms during erythroid differentiation in control and TI cell cultures. METHODS: After informed consent, the CD34+ cells were obtained from peripheral blood of healthy volunteers and from patients with thalassemia intermedia by positive selection using anti-CD34-tagged magnetic beads and cultured for 14 days with a medium containing stem cell factor (SCF), interleukin 3 (IL-3) and erythropoietin to induce erythroid differentiation. The expression profiling of FPN1A and FPN1B was evaluated at baseline, day 7 and day 14 of culture by real-time PCR (-dCt). RESULTS: In control cultures, FPN1A isoform was highly expressed at erythroid progenitors stage (day 0 of culture), decreased at early erythroblasts stage (day 7) and increased again at late erythroblasts stage (day 14). In TI cultures, the FPN1A isoform expression remained high even in early erythroblasts (Table 1). In control cultures the FPN1B isoform expression was very low at any stage of erythroid differentiation, whereas in TI cultures it was highly expressed at baseline and, althoug decreased during differentiation, remained always higher than control (Table 2). CONCLUSIONS: In thalassemic conditions the FPN1B is the major expressed ferroportin isoform, possibly contributing to iron overload. In control cultures, FPN1A was mainly expressed in undifferentiated erythroid progenitors and in mature erythroblasts, suggesting a functional role at these stages of erythroid differentiation. In TI cultures, the persistent expression of FPN1A at early erythroblasts stage was probably due to thalassemic erythropoiesis. These data suggest that in TI condition other signals, such as the erythropoiesis status, can override iron overload in regulating ferroportin expressio