Missense mutation of the erythropoietin receptor is a rare event in human erythroid malignancies

Human erythroid malignancies (polycythemia vera [PV] and erythroleukemia) are associated with erythropoietin (Epo)-independent growth and differentiation. Missense or nonsense mutations in the Epo receptor (Epo-R) have been recently described in experimental erythroleukemia in mice and in cases of erythrocytosis in humans. To search for a similar genetic alteration in erythroleukemia and PV, we entirely sequenced the exons of the Epo-R gene as well as the intron- exon junctions in these disorders using polymerase chain reaction. In 1 of 10 cases of erythroleukemia, a single allele mutation was found in the 8th Epo-R gene exon that changed asparagine 487 into a serine. No Epo-r gene mutation was found in 12 PV cases studied, but the same mutation (N487S) was found in 1 patient with polycythemia that did not fulfill the criteria of PV (polycythemia of unknown origin). We did not detect this mutation after sequencing part of the 8th exon of the Epo-R gene from 21 other patients with polycythemia of unknown origin and 51 normal controls. The Epo-R mutation was also found in Epstein-Barr virus-derived cell lines from both cases, suggesting that it is not related to the malignant clone. Therefore, this mutation does not appear to be somatic, although no familial cases were found. The biologic effect of this mutation was subsequently studied. Erythroid progenitors from the polycythemic patient normally responded to Epo, whereas those from the erythroleukemic patient were Epo-independent due to autocrine stimulation by Epo. The normal and the mutated Epo-R were transfected into the murine Ba/F3 cell line. Both types of cells displayed the same response to Epo for proliferation, differentiation, and inhibition of apoptosis. Although this mutation may destroy a consensus binding site for Grb2, no obvious differences either in the pattern of Epo-induced tyrosine phosphorylated proteins or in the binding of Grb2 to the Epo-R were observed. In conclusion, a somatic Epo-R missense mutation does not appear to be a molecular mechanism involved in the abnormal growth of human erythroleukemia and PV. However, the Epo-R mutation (N487S) that we describe is located in the same tyrosine sequence beginning at AA 485 as the one previously observed (P488S) in as case of polycythemia (Sokol et al, Exp Hematol 22:447, 1994). These results suggest that this phosphopeptide sequence may play an important role in Epo signalling.     

The Tie receptor tyrosine kinase is expressed by human hematopoietic progenitor cells and by a subset of megakaryocytic cells

Growth factor receptors in human hematopoietic progenitor cells have become the focus of intense interest, because they may provide tools for the monitoring, enrichment, and expansion of stem cells. We have shown earlier that the Tie receptor tyrosine kinase is expressed in erythroid and megakaryoblastic human leukemia cell lines, in the blood islands of the yolk sac, and in endothelial cells starting from day 8.0 of mouse development. Here, the expression of Tie was studied in human hematopoietic cells of various sources. Peripheral blood mononuclear cells were Tie-. However, a large fraction of CD34+ cells from umbilical cord blood (UCB) and bone marrow (BM) expressed tie protein and mRNA. On average, 64% of the fluorescence-activated cell sorting- gated UCB CD34+ cells including CD38- cells and a fraction of cells expressing low levels of c-Kit were Tie+. Also, 30% to 60% of BM CD34+ cells were Tie+, including most of the BM CD34+CD38-, CD34+Thy-1+, and CD34+HLA-DR- cells. Under culture conditions allowing myeloid, erythroid, and/or megakaryocytic differentiation, purified UCB CD34+ cells lost Tie mRNA and protein expression concomitantly with that of CD34; however, a significant fraction of cells expressed Tie during megakaryocytic differentiation. These data suggest that, in humans, the Tie receptor and presumably its ligand may function at an early stage of hematopoietic cell differentiation.     

Intracranial circulation: pulse-sequence considerations in three- dimensional (volume) MR angiography

The technique and feasibility of magnetic resonance (MR) angiography of intracranial vessels were studied in 35 healthy volunteers. Variations in image orientation, repetition time (TR), and flip angle were evaluated to determine their effects on flow-related enhancement. Gradient modifications--including echo time (TE), motion compensation, bandwidth, and field of view--were also studied in an effort to reduce motion-induced phase shifts. Results indicated that a FISP (fast imaging with steady precession) sequence with a TR of 50 msec, TE of 15 msec, velocity compensation in the read and section-select directions, acceleration compensation in the read direction, anisotropic volume, and a 1.25-mm partition thickness produced three-dimensional angiographic MR images that were accurate and reproducible in the depiction of the major intracranial vessels. Difficulties with field of view, persistent signal void secondary to higher-order motion, and spatial resolution remain major problems requiring additional study.     

Acute experimental cerebral ischemia: MR enhancement using Gd-DTPA

The effects of a paramagnetic contrast agent, gadolinium-DTPA (Gd-DTPA), on magnetic resonance (MR) imaging of acute cerebral ischemia was investigated in a feline model of middle cerebral artery occlusion. Imaging was performed both before and after administration of an intravenous dose of 0.2 mmol/kg of Gd-DTPA. The animals were then sacrificed for pathologic correlation. No changes in intensity or relaxation times were noted before or after Gd-DTPA administration in two animals with 2 hours of occlusion. Infarcts were noted before and after contrast enhancement in all six cats with ischemia of greater than 16-hours duration. Gd-DTPA caused significant increase in intensity of infarct but not in that of normal cerebral tissue. Rapid enhancement was visible in infarcts of 16-24 hours, but such enhancement was slower in infarcts of 72-168 hours, presumably owing to slowed inflow caused by increased vasogenic edema in the latter group. Contrast enhancement of acute cerebral ischemic lesions with Gd-DTPA offers no improvement in sensitivity of MR imaging, although the conspicuity of the lesion may be improved. Additionally, contrast media may provide potential temporal and pathophysiological data for better characterization of cerebral ischemia.