Erythropoietin receptor transcription is neither elevated nor predictive of surface expression in human tumour cells.

Erythropoietin receptor (EpoR) has been reported to be overexpressed in tumours and has raised safety concerns regarding the use of erythropoiesis-stimulating agents (ESAs) to treat anaemia in cancer patients. To investigate the potential for EpoR to be overexpressed in tumours, we have evaluated human tumours for amplification of the EPOR locus, levels of EPOR transcripts, and expression of surface EpoR protein. Gene amplification analysis of 1083 solid tumours found that amplification of the EPOR locus was rare with frequencies similar to other non-oncogenes. EPOR transcript levels in tumours and tumour cell lines were low in comparison with bone marrow and were equivalent to, or lower than, levels in normal tissues of tumour origin. Although EpoR mRNA was detected in some tumour lines, no EpoR could be detected on the cell surface using (125)I-Epo binding studies. This may be due to the lack of EpoR protein expression or lack of cell-surface-trafficking factors, such as Jak2. Taken together, we have found no evidence that EpoR is overexpressed in tumours or gets to the surface of tumour cells. This suggests that there is no selective advantage for tumours to overexpress EpoR and questions the functional relevance of EpoR gene transcription in tumours.

Lymphoid apoptosis and myeloid hyperplasia in CCAAT displacement protein mutant mice.

CCAAT displacement protein (cux/CDP) is an atypical homeodomain protein that represses expression of several developmentally regulated lymphoid and myeloid genes in vitro, including gp91-phox, immunoglobulin heavy chain, the T-cell receptor beta and gamma chains, and CD8. To determine how this activity affects cell development in vivo, a hypomorphic allele of cux/CDP was created by gene targeting. Homozygous mutant mice (cux/CDP(Delta HD/Delta HD)) demonstrated a partial neonatal lethality phenotype. Surviving animals suffered from a wasting disease, which usually resulted in death between 2 and 3 weeks of age. Analysis of T lymphopoiesis demonstrated that cux/CDP(Delta HD/Delta HD) mice had dramatically reduced thymic cellularity due to enhanced apoptosis, with a preferential loss of CD4(+)CD8(+) thymocytes. Ectopic CD25 expression was also observed in maturing thymocytes. B lymphopoiesis was also perturbed, with a 2- to 3-fold reduction in total bone marrow B-lineage cells and a preferential loss of cells in transition from pro-B/pre-BI to pre-BII stages due to enhanced apoptosis. These lymphoid abnormalities were independent of effects related to antigen receptor rearrangement. In contrast to the lymphoid demise, cux/CDP(Delta HD/Delta HD) mice demonstrated myeloid hyperplasia. Bone marrow reconstitution experiments identified that many of the hematopoietic defects were linked to microenvironmental effects, suggesting that underexpression of survival factors or overexpression of death-inducing factors accounted for the phenotypes observed. Tumor necrosis factor (TNF) levels were elevated in several tissues, especially thymus, suggesting that TNF may be a target gene for cux/CDP-mediated repression. These data suggest that cux/CDP regulates normal hematopoiesis, in part, by modulating the levels of survival and/or apoptosis factors expressed by the microenvironment.

Connection between B lymphocyte and osteoclast differentiation pathways.

Osteoclasts differentiate from the hemopoietic monocyte/macrophage cell lineage in bone marrow through cell-cell interactions between osteoclast progenitors and stromal/osteoblastic cells. Here we show another osteoclast differentiation pathway closely connected with B lymphocyte differentiation. Recently the TNF family molecule osteoclast differentiation factor/receptor activator of NF-kappaB ligand (ODF/RANKL) was identified as a key membrane-associated factor regulating osteoclast differentiation. We demonstrate that B-lymphoid lineage cells are a major source of endogenous ODF/RANKL in bone marrow and support osteoclast differentiation in vitro. In addition, B-lymphoid lineage cells in earlier developmental stages may hold a potential to differentiate into osteoclasts when stimulated with M-CSF and soluble ODF/RANKL in vitro. B-lymphoid lineage cells may participate in osteoclastogenesis in two ways: they 1) express ODF/RANKL to support osteoclast differentiation, and 2) serve themselves as osteoclast progenitors. Consistent with these observations in vitro, a decrease in osteoclasts is associated with a decrease in B-lymphoid cells in klotho mutant mice (KL(-/-)), a mouse model for human aging that exhibits reduced turnover during bone metabolism, rather than a decrease in the differentiation potential of osteoclast progenitors. Taken together, B-lymphoid lineage cells may affect the pathophysiology of bone disorders through regulating osteoclastogenesis.

Analysis of vertebrate SCL loci identifies conserved enhancers.

The SCL gene encodes a highly conserved bHLH transcription factor with a pivotal role in hemopoiesis and vasculogenesis. We have sequenced and analyzed 320 kb of genomic DNA composing the SCL loci from human, mouse, and chicken. Long-range sequence comparisons demonstrated multiple peaks of human/mouse homology, a subset of which corresponded precisely with known SCL enhancers. Comparisons between mammalian and chicken sequences identified some, but not all, SCL enhancers. Moreover, one peak of human/mouse homology (+23 region), which did not correspond to a known enhancer, showed significant homology to an analogous region of the chicken SCL locus. A transgenic Xenopus reporter assay was established and demonstrated that the +23 region contained a new neural enhancer. This combination of long-range comparative sequence analysis with a high-throughput transgenic bioassay provides a powerful strategy for identifying and characterizing developmentally important enhancers.

An SCL 3' enhancer targets developing endothelium together with embryonic and adult haematopoietic progenitors.

The SCL gene encodes a basic helix-loop-helix transcription factor which is expressed in early haematopoietic progenitors throughout ontogeny and is essential for the normal development of blood and blood vessels. Transgenic studies have characterised spatially distinct 5' enhancers which direct lacZ expression to subdomains of the normal SCL expression pattern, but the same elements failed to produce appropriate haematopoietic expression. We now describe an SCL 3' enhancer with unique properties. It directed lacZ expression in transgenic mice to extra-embryonic mesoderm and subsequently to both endothelial cells and to a subset of blood cells at multiple sites of embryonic haematopoiesis including the yolk sac, para-aortic splanchnopleura and AGM region. The 3' enhancer also targeted expression to haematopoietic progenitors in both foetal liver and adult bone marrow. Purified lacZ(+ )cells were highly enriched for clonogenic myeloid and erythroid progenitors as well as day-12 spleen colony forming units (CFU-S). Within the total gated population from bone marrow, 95% of the myeloid and 90% of the erythroid colony-forming cells were contained in the lacZ(+) fraction, as were 98% of the CFU-S. Activation of the enhancer did not require SCL protein. On the contrary, transgene expression in yolk sacs was markedly increased in an SCL-/- background, suggesting that SCL is subject to negative autoregulation. Alternatively the SCL-/- environment may alter differentiation of extra-embryonic mesoderm and result in an increased number of cells capable of expressing high levels of the transgene. Our data represents the first description of an enhancer that integrates information necessary for expression in developing endothelium and early haematopoietic progenitors at distinct times and sites throughout ontogeny. This enhancer provides a potent tool for the manipulation of haematopoiesis and vasculogenesis in vivo.

Distinct 5' SCL enhancers direct transcription to developing brain, spinal cord, and endothelium: neural expression is mediated by GATA factor binding sites.

The SCL gene encodes a basic helix-loop-helix transcription factor with a pivotal role in the development of endothelium and of all hematopoietic lineages. SCL is also expressed in the central nervous system, although its expression pattern has not been examined in detail and its function in neural development is unknown. In this article we present the first analysis of SCL transcriptional regulation in vivo. We have identified three spatially distinct regulatory modules, each of which was both necessary and sufficient to direct reporter gene expression in vivo to three different regions within the normal SCL expression domain, namely, developing endothelium, midbrain, and hindbrain/spinal cord. In addition we have demonstrated that GATA factor binding sites are essential for neural expression of the SCL constructs. The midbrain element was particularly powerful and axonal lacZ expression revealed the details of axonal projections, thus implicating SCL in the development of occulomotor, pupillary, or retinotectal pathways. The neural expression pattern of the SCL gene was highly conserved in mouse, chicken, and zebrafish embryos and the 5' region of the chicken SCL locus exhibited a striking degree of functional conservation in transgenic mice. These data suggest that SCL performs critical functions in neural development. The regulatory elements identified here provide important tools for analyzing these functions.

The hochschild cohomology problem for von neumann algebras.

In 1967, when Kadison and Ringrose began the development of continuous cohomology theory for operator algebras, they conjectured that the cohomology groups Hn(M, M), n >/= 1, for a von Neumann algebra M, should all be zero. This conjecture, which has important structural implications for von Neumann algebras, has been solved affirmatively in the type I, IIinfinity, and III cases, leaving open only the type II1 case. In this paper, we describe a positive solution when M is type II1 and has a Cartan subalgebra and a separable predual.

Interaction of vesicular stomatitis virus-G pseudotyped retrovirus with CD34+ and CD34+ CD38- hematopoietic progenitor cells.

Retroviral vectors have had limited success in mediating gene transfer to hematopoietic stem cells, particularly in primates, due in part to low or absent expression of the amphotropic receptor (RAM-1). We have been interested in determining whether retrovirus pseudotyped with vesicular stomatitis virus G protein (VSV-G) would allow more efficient gene delivery to hematopoietic stem cells as the VSV-G receptors appear to be ubiquitously present phospholipids. However, we previously found that completion of retroviral vector reverse transcription does not occur in CD34+ CD38- hematopoietic stem cells that were exposed to VSV-G pseudotyped retrovirus. To determine at which stage the block to infection of CD34+ CD38- cells occurs, we confirmed by FACS analysis that VSV-G pseudotyped viral particles could bind to CD34+ CD38- cells. Virus binding to CD34+ cells was saturable at 4 degrees C but nonsaturable at 37 degrees C, up to a multiplicity of infection of 1080. This suggests that surface levels of phospholipid receptors available for viral binding are limiting on CD34+ cells. Cytokine stimulation increased virus binding to CD34+ cells. However, no increase in the level of surface phosphatidylserine (PS), a strong candidate for the VSV-G receptor, was seen as detected by the PS-specific reagent, annexin V. This suggests that another molecule is serving as the VSV-G receptor on CD34+ cells. Here, we show that once virus binding to cytokine-stimulated CD34+ CD38- cells has occurred, virus fusion proceeds efficiently as determined by octadecyl rhodamine (R18) fusion assays. Taken together with our previous observation that reverse transcription does not occur in CD34+ CD38- cells, we suggest that there are intracellular mechanisms leading to blockage of complete reverse transcription of the retrovirus in CD34+ CD38- cells. This has important implications for retrovirus-mediated gene transfer to quiescent stem cells.

Cell-cycle kinetics and VSV-G pseudotyped retrovirus-mediated gene transfer in blood-derived CD34+ cells.

As hematopoietic stem and progenitor cells have a low mitotic index, we have quantitated the impact of cytokine combinations on cell cycling of CD34+ cells and, using VSV-G pseudotyped retroviral vectors, correlated our findings with ex vivo gene transfer. We tested nine different combinations of cytokines for induction of human peripheral blood CD34+ cells into cell cycle over 72 hours. Using the 5-bromodeoxyuridine-Hoechst 33258 (BrdU-Hoechst) assay, we measured the cell-cycle kinetics. The combinations of cytokines tested that were most efficient in inducing the CD34+ cells into cycle were stem cell factor (SCF) plus one of the following: interleukin-1 (IL-1), IL-3, granulocyte colony-stimulating factor (G-CSF). The maximum numbers of cells in S+G2M phase were observed after 48 hours of culture. At least 35 +/- 5% of the CD34+ cells remained quiescent in the first G0/G1 phase, however, no matter which cytokine combination was used. Cell-cycle analysis of the CD34+CD38- subset by 7-amino actinomycin D staining did not detect cycling cells during 72 hours of culture with any of the cytokines tested. To investigate whether the cells could be infected by the VSV-G pseudotyped virus containing the neomycin phospho-transferase gene (neo), we exposed CD34+ cells to the virus for 7-8 hours after 0, 36, and 48 hours of cytokine stimulation. Total CD34+ cells and the CD34+CD38- subset were analyzed by polymerase chain reaction (PCR) for reverse-transcribed viral DNA of the neomycin resistance gene (RT-neoDNA). Immediately after exposure to the virus, RT-neoDNA was detectable in CD34+ cells that have been cultured with or without cytokines for 36 to 48 hours. Forty-eight hours postinfection, however, RT-neoDNA could be detected only with cytokine combinations that induced mitosis of the CD34+ cells, consistent with the requirement for mitotic activity for retroviral integration. Similar experiments performed with the 34+CD38- subset showed that RT-reoDNA could not be detected at any time point. Thus, postinfection RT-neoDNA could be immediately detected in noncycling CD34+ cells but not in CD34+CD38- cells. These results suggest during short-term liquid culture, there may be blocks for reverse transcription of retroviral RNA in CD34+CD38-cells in addition to the lack of mitotic activity.

The effects of antihypertensive therapy on carotid vascular structure in man.

OBJECTIVE: An increased carotid intima-media thickness (IMT) has been found to be associated with a number of cardiovascular risk factors such as age, hypertension, cigarette smoking, hypercholesterolaemia and left ventricular hypertrophy. Our objective was to assess whether carotid intima-media thickness in hypertensive patients could be reduced by antihypertensive therapy. METHODS: 13 hypertensive patients, 10 previously untreated, were examined using carotid ultrasonography and echocardiography at baseline and then at 8 weeks and 39 weeks after commencement of antihypertensive therapy with ramipril and the second-line addition of felodipine. RESULTS: By the end of the study significant regression of IMT (0.1(0.05-0.16) mm, F-value 10.2, P < 0.01) and left ventricular mass index had occurred (25(10.7-39.3) g/m2, F-value 9.7, P < 0.01). The reduction in IMT was significantly related to the reduction in mean arterial pressure, r = 0.55, P = 0.05). CONCLUSION: Antihypertensive therapy with ramipril and felodipine causes regression of IMT in hypertensive patients, probably chiefly through blood pressure reduction. Large prospective studies are required to assess whether a reduction in IMT results in a reduction in morbidity and mortality.

Structural changes in the cardiovascular system of untreated essential hypertensives.

Arterial hypertension is associated with structural changes in the cardiovascular system. This study has examined the effect of hypertension on the carotid artery wall and examined the relation between changes in the structure of carotid artery wall and left ventricle in untreated hypertensives. The carotid artery wall was visualized using a high resolution ultrasound technique in 37 untreated hypertensive patients (25 males, 12 females) and 37 age and sex matched normotensive individuals and carotid intima-media thickness (IMT) and carotid artery diameter measured. IMT and intima-media cross sectional area was significantly greater in the hypertensive group compared with the normotensive group, though the carotid artery diameter did not differ significantly. There was a significant association between age and IMT in both groups. In the hypertensive group there was also a significant association between left ventricular mass index, ventricular septal or posterior wall thickness and IMT. This study indicates that there is an association between cardiac and carotid arterial structure in hypertension. Such a relationship may be important in understanding the associated risks of high blood pressure.

Short arm plaster cast for distal pediatric forearm fractures.

Ten years' clinical experience with below-elbow plaster cast treatment of distal one third pediatric forearm fractures was subjected to an independent retrospective radiographic review. In the study population of 761 fractures, no significant displacement occurred while the forearm remained in plaster. The average angulation change was 4.5 degrees (SD +/- 2.2 degrees). In each angulation change > 5 degrees, poor cast molding was evident, as reflected by a high "cast index" (p < 0.01). Although this technique is technically demanding, excellent results are obtained in all distal pediatric forearm fractures if proper cast molding is used.

Cloning of the complete Ly-6E.1 gene and identification of DNase I hypersensitive sites corresponding to expression in hematopoietic cells.

The Sca-1 antibody recognizes antigens encoded by members of the Ly-6 multigene family. These antigens are expressed on fetal and adult hematopoietic stem cells, progenitor cells, mature activated T cells, and some nonhematopoietic cells and are most likely encoded by the Ly-6E.1 and Ly-6A.2 genes. Characterization and isolation of regulatory elements of Ly-6E.1 and A.2 genes that govern tissue-specific and high levels of expression in the cells of the hematopoietic system (particularly stem cells) are of considerable interest. To characterize the control elements of this gene, we have cloned a 30-kb fragment encoding a fully functional Ly-6E.1 gene and 13 kb of 5' and 13 kb of 3' flanking sequence. Transfection studies in murine erythroleukemia (MEL) cells show that a 14-kb BamHI fragment from this clone is sufficient to confer Ly-6E.1 gene expression at levels equivalent to those of the endogenous gene. By mapping regions of chromatin sensitive to DNase I digestion, we have located hypersensitive sites in the 5' and 3' regions of the gene in FDCP-1 cells, MEL cells, and various T-cell lines. The appearance of two 5' hypersensitive sites in hematopoietic cells correlates with Ly-6E.1 expression after gamma-interferon induction. We show that the presence of hypersensitive sites in the 5' and 3' regions corresponds to Sca-1 expression, and we also discuss the localization of putative regulatory control elements.

Structural changes in the heart and carotid arteries associated with hypertension in humans.

Hypertension is associated with structural changes in the vascular system and in the heart. This study has examined the relationships between carotid artery intima-media thickness and other risk factors in 52 untreated patients (20 hypertensive). Carotid intima-media thickness was measured bilaterally using a Duplex doppler ultrasonic scanner. In the hypertensive individuals the left ventricle was examined by echo-cardiography and the left ventricular mass index determined. There was a significant association between age and IMT, and both SBP and DBP and IMT. The IMT in the hypertensive group was significantly larger than in the normotensive group and in the hypertensive subjects there was a positive association between left ventricular mass index and IMT. There was no significant difference in calculated media stress between the normotensive and hypertensive groups, probably due to a small increase in carotid intima-media area combined with a small reduction in carotid lumen diameter. Hypertension is associated with a thickening of the intima-media of the carotid artery and an increase in left ventricular mass. Whether these changes in cardiac and arterial structure are in response to similar influences remains to be established.

Effect of nifedipine and glyceryl trinitrate on retinal blood flow in normal subjects.

The retinal vasculature is an accessible region of the microcirculation in humans which undergoes change in essential hypertension. In this study we have examined the effect of nifedipine and glyceryl trinitrate (GTN) on retinal blood flow velocities. Retinal blood flow was measured in 11 subjects using colour Doppler ultrasound. Nifedipine (10 mg orally) or glyceryl trinitrate (0.3 mg, GTN) was given after 30 minutes recumbancy. Systolic retinal arterial blood flow velocity (SRV), diastolic retinal arterial blood flow velocity (DRV), retinal resistive index (RI), SBP, DBP were measured at -10, 0, 5, 10, 15, 20 and 25 minutes following drug administration. Nifedipine significantly increased SRV (peak effect at 15 minutes) but by 25 minutes SRV did not differ significantly from pre-drug values. Nifedipine did not significantly affect DRV, RI, SBP or DBP. GTN significantly increased both SRV and DRV, although both tended to fall towards pre-drug values by 25 minutes. RI, SBP nor DBP were not significantly altered by GTN. Both nifedipine and GTN transiently increase blood flow velocity in the central retinal artery although at the doses used neither agent affected BP or RI. Doppler ultrasound measurement of retinal arterial blood velocity may be a useful technique in the investigation of the retinal microcirculation in essential hypertension.

Secondary hypertension in a blood pressure clinic.

The prevalence, reversibility, and mortality of secondary hypertension among 3783 patients with moderately severe nonmalignant hypertension attending the Glasgow (Scotland) Blood Pressure Clinic were assessed. Underlying causes of hypertension were found in 297 patients (7.9%). Eighty-seven patients (2.3%) were considered to have a potentially reversible cause for their hypertension, including the oral contraceptive pill (38 patients), renovascular disease (27 patients), and primary hyperaldosteronism (ten patients), but of these only 33 patients (0.9% of total clinic population) were cured by specific intervention. Two hundred ten patients (5.6%) had irreversible renal parenchymal disease and significantly higher mortality than men and women with other causes of hypertension. Excess deaths in the renal group were attributed to renal failure (International Classification of Diseases [ICD] 580 to 589) and vascular causes (ICD 390 to 458) but not to cancer (ICD 140 to 208; 235 to 239) or other nonvascular disease. These results suggest that investigation of hypertension for an underlying cause will reveal a small number of patients with treatable disorders, of whom only a few will be cured by specific intervention, and a moderate number with irreversible disease who are at high risk of myocardial infarction and stroke.

Posterior sternoclavicular dislocations.

Twelve cases of posterior sternoclavicular dislocation were seen over 15 years at the Victoria General Hospital and the Izaak Walton Kiliam Hospital for Children in Halifax, NS. Two patients required open reduction of the dislocation but the rest were treated by closed reduction. One of the former group required threaded K wires for stability, but all other dislocations were deemed stable after reduction and application of a figure-of-eight bandage and an arm sling. There were no failures of reduction and no recurrent dislocations; the authors have been successful even in late closed reduction (up to 5 days) and therefore recommend it highly as the primary treatment. Open reduction is much more difficult and hazardous.