Hematopoietic and endothelial progenitor cell trafficking in patients with myeloproliferative diseases.

BACKGROUND AND OBJECTIVES: The presence of circulating hematopoietic progenitor cells in patients with myeloproliferative diseases (MPD) has been described. However, the exact nature of such progenitor cells has not been specified until now. The aim of this work was to investigate the presence of endothelial precursor cells in the blood of patients with MPD and to assess the role of the endothelial cell lineage in the pathophysiology of this disease. DESIGN AND METHODS: Endothelial progenitor cell marker expression (CD34, prominin (CD133), kinase insert domain receptor (KDR) or vascular endothelial growth factor receptor 2 (VEGFR2), and von Willebrand factor) was assessed in the blood of 53 patients with MPD by quantitative polymerase chain reaction. Clonogenic stem cell assays were performed with progenitor cells and monocytes to assess differentiation towards the endothelial cell lineage. The patients' were divided according to whether they had essential thrombocythemia (ET, n=17), polycythemia vera (PV, n=21) or chronic idiopathic myelofibrosis (CIMF, n=15) and their data compared with data from normal controls (n=16) and patients with secondary thrombo- or erythrocytosis (n=17). RESULTS: Trafficking of CD34-positive cells was increased above the physiological level in 4/17 patients with ET, 5/21 patients with PV and 13/15 patients with CIMF. A subset of patients with CIMF co-expressed the markers CD34, prominin (CD133) and KDR, suggesting the presence of endothelial precursors among the circulating progenitor cells. Clonogenic stem cell assays confirmed differentiation towards both the hematopoietic and the endothelial cell lineage in 5/10 patients with CIMF. Furthermore, the molecular markers trisomy 8 and JAK2 V617F were found in the grown endothelial cells of patients positive for trisomy 8 or JAK2 V617F in the peripheral blood, confirming the common clonal origin of both hematopoietic and endothelial cell lineages. INTERPRETATION AND CONCLUSIONS: Endothelial precursor cells are increased in the blood of a subset of patients with CIMF, and peripheral endothelial cells bear the same molecular markers as hematopoietic cells, suggesting a primary role of pathological endothelial cells in this disease.

Progenitor cell recruitment during individualized high-flow, very-large-volume apheresis for autologous transplantation improves collection efficiency.

BACKGROUND: Individual adaptation of processed patient's blood volume (PBV) should reduce number and/or duration of autologous peripheral blood progenitor cell (PBPC) collections. STUDY DESIGN AND METHODS: The durations of leukapheresis procedures were adapted by means of an interim analysis of harvested CD34+ cells to obtain the intended yield of CD34+ within as few and/or short as possible leukapheresis procedures. Absolute efficiency (AE; CD34+/kg body weight) and relative efficiency (RE; total CD34+ yield of single apheresis/total number of preapheresis CD34+) were calculated, assuming an intraapheresis recruitment if RE was greater than 1, and a yield prediction models for adults was generated. RESULTS: A total of 196 adults required a total of 266 PBPC collections. The median AE was 7.99 x 10(6), and the median RE was 1.76. The prediction model for AE showed a satisfactory predictive value for preapheresis CD34+ only. The prediction model for RE also showed a low predictive value (R2 = 0.36). Twenty-eight children underwent 44 PBPC collections. The median AE was 12.13 x 10(6), and the median RE was 1.62. Major complications comprised bleeding episodes related to central venous catheters (n = 4) and severe thrombocytopenia of less than 10 x 10(9) per L (n = 16). CONCLUSION: A CD34+ interim analysis is a suitable tool for individual adaptation of the duration of leukapheresis. During leukapheresis, a substantial recruitment of CD34+ was observed, resulting in a RE of greater than 1 in more than 75 percent of patients. The upper limit of processed PBV showing an intraapheresis CD34+ recruitment is higher than in a standard large-volume leukapheresis. Therefore, a reduction of individually needed PBPC collections by means of a further escalation of the processed PBV seems possible.

cIAP-2 and survivin contribute to cytokine-mediated delayed eosinophil apoptosis.

The exact molecular mechanisms leading to delayed apoptosis, a phenomenon frequently observed in eosinophil inflammatory responses, remain largely unknown. Here, we show that cultured eosinophils purified from blood of hypereosinophilic syndrome (HES) patients exhibit delayed spontaneous death and relative resistance towards ceramide- but not CD95-mediated death. The subsequent investigation of members of the inhibitor of apoptosis (IAP) family revealed that HES but not normal eosinophils expressed high levels of cellular IAP-2 (cIAP-2) and survivin. The eosinophil hematopoietins IL-3, IL-5, and GM-CSF increased the expression of cIAP-2 and survivin in normal eosinophils in vitro. In the blood of HES patients, we observed increased concentrations of IL-3 and/or IL-5, suggesting that these cytokines are, at least partially, responsible for the elevated levels of cIAP-2 and survivin in the eosinophils of these patients. Utilizing a cell-free system in which caspase-3 was activated in eosinophil cytosolic extracts by addition of cytochrome c and immunodepletion of cIAP-2 or survivin resulted in accelerated caspase activation. These data suggest that some members of the IAP family including survivin are regulated by survival cytokines and inhibit the caspase cascade in HES eosinophils. The cytokine-dependent mechanism of delayed eosinophil apoptosis described here may also apply to other eosinophilic diseases.

Progenitor cell trafficking in physiologic conditions and in myeloproliferative diseases: quantification of CD34+ cells by polymerase chain reaction.

BACKGROUND AND OBJECTIVES: Previous studies using flow cytometry have shown that CD34+ cell trafficking is increased in patients with chronic idiopathic myelofibrosis. Few data exist on physiologic CD34 + cell trafficking and the quantification of very low cell ranges requires reliable and sensitive measurement techniques. The aim of this study was to establish a quantitative polymerase chain reaction (PCR) technique for studying CD34+ cell trafficking in physiologic conditions, and in patients with myeloproliferative diseases. DESIGN AND METHODS: CD34+ cell trafficking was measured in 56 controls [(healthy controls (n=21), patients with ischemic cardiopathy (n=21), patients with secondary thrombocytosis or erythrocytosis (n=14)], and in 37 untreated patients with myeloproliferative diseases diagnosed according to the WHO-criteria [(essential thrombocythemia (n=10), polycythemia vera (n=14) and chronic idiopathic myelofibrosis (n=13)]. Quantitative PCR was used to determine CD34 mRNA expression in peripheral blood samples. RESULTS: Physiologic CD34 mRNA expression ranges were determined in the healthy control group. Mean CD34 mRNA expression was within the physiologic range in patients with ischemic cardiopathy, secondary thrombocytosis or erythrocytosis, essential thrombocythemia and polycythemia vera (p=0.146), but was significantly increased in patients with chronic idiopathic myelofibrosis (p<0.001). When analyzed individually, 12/13 patients with chronic idiopathic myelofibrosis and 3/14 patients with polycythemia vera showed CD34 mRNA expression above the physiologic range. INTERPRETATION AND CONCLUSIONS: This is a first report about CD34+ cell trafficking measured by quantitative PCR. Quantitative PCR is a reliable method suitable for the quantification of very low cell populations. Our study confirms the significant increase of CD34+ cell trafficking in patients with chronic idiopathic myelofibrosis, and in a subset of patients with polycythemia vera. Prospective studies are underway to characterize these circulating CD34+ cells and to investigate their role in the pathophysiology of myeloproliferative diseases.

Splenectomy in relapsing and plasma-refractory acquired thrombotic thrombocytopenic purpura.

BACKGROUND AND OBJECTIVES: Acquired thrombotic thrombocytopenic purpura (TTP) is often due to autoantibodies inhibiting ADAMTS-13 activity resulting in impaired processing of very large von Willebrand factor multimers. TTP usually presents with an acute onset and a fulminant, sometimes fatal course. With appropriate treatment including plasma exchange, and fresh frozen plasma replacement, often supplemented by immuno-suppressive therapy, the acute episode generally resolves within days to weeks. DESIGN AND METHODS: We describe the clinical course of 3 patients with acquired TTP. One was refractory to PE, the other 2 relapsed after this treatment. All three were treated with splenectomy. ADAMTS-13 activity and inhibitor levels were monitored. RESULTS: ADAMTS-13 activity was initially < 5% in all 3 patients. After splenectomy the inhibitor against ADAMTS-13 disappeared rapidly in 2 patients and there was full recovery of ADAMTS-13 activity in all 3 patients. INTERPRETATION AND CONCLUSIONS: Splenectomy, by eliminating a source of pathogenic autoantibody production, can be a successful treatment for patients with relapsing or plasma-refractory acquired TTP due to autoantibody-mediated ADAMTS-13 deficiency.

The predictive value of clonogenic stem cell assays for the diagnosis of polycythaemia vera.

Spontaneous growth of erythroid progenitor cells is one of the hallmarks of polycythaemia vera (PV). In vitro clonogenic assays can be used to support the diagnosis of PV. However, clonogenic assays are difficult to standardize, and their diagnostic value is accepted as a minor criteria for PV only in the case of positive results. Clonogenic assays need to be validated and normal ranges established carefully before they can be used for diagnostic purposes. In a retrospective study, we analysed the results of 146 consecutive clonogenic assays performed in patients with erythrocytosis between January 1992 and December 2000. Normal ranges were established on the basis of the results from 81 controls. The charts of the 146 patients were reviewed and the patients were allocated to the following diagnostic groups: PV (n = 38) and non-primary erythrocytosis (NPE, n = 65) according to the criteria of Pearson & Messinezy. In total, 43 patients were excluded from the study because of missing data. The clonogenic assay results were correlated with the final clinical diagnosis of the patients. At a cut-off value of 5 erythroid colony-forming unit colonies per well, 29 out of 38 patients with PV showed positive results, and 9 out of 38 patients showed false-negative results. In the NPE group, there were 64 out of 65 real-negative results and 1 out of 65 false-positive result. Thus the positive predictive value of the clonogenic assay for the diagnosis of PV was 97%. We conclude that clonogenic assays, in the case of positive results, and if carefully validated, are very useful and reliable tools for the diagnosis of PV.