Effects of host habitat quality on the viability of Cryptocotyle lingua (Trematoda: Digenea) cercariae.

In an earlier study it was shown that Cryptocotyle lingua cercariae, matured in Littorina littorea from a polluted marine lagoon, displayed slower horizontal swimming rates, and reduced longevity compared to cercariae released by periwinkles from a cleaner environment. This work investigated whether the pollution-induced reduction in swimming rates was due to an inefficient swimming action or the adoption of a less direct swimming path. In addition, cercariae from L. littorea that had been transferred from an 'unpolluted' to a 'polluted' site for 1 month provided information on the speed with which pollutants affect cercariae through their intermediate hosts. Results indicated that, in general, horizontal swimming rates were reduced due to slower swimming rather than disorientation and longer swimming pathways. Effects of host transplantation to a polluted site were clearly evident after 1 month. Evidence suggested that the pollutants accumulated by the cercariae via their first intermediate host affected the neuromusculature associated with swimming performance rather than sensory structures. Bearing in mind the reduced viability of C. lingua cercariae in polluted sites it is assumed that high prevalence of this digenean in gastropods (at such sites) must be due to their continual introduction by infected birds attracted to these habitats from other areas.

A new method to account for the depth distribution of 137Cs in soils in the calculation of external radiation dose-rate.

This work reports a new method for calculating the external dose-rate as a function of height above land that has been contaminated with a surface deposition of (137)Cs. Unlike previous work this method accounts for vertical migration of (137)Cs using the Advection Dispersion Equation (ADE) with appropriate parameters. The results have been successfully verified with field measurements from the (137)Cs contaminated regions within the Republic of Belarus. The method also correctly predicts the observed variation of dose-rate with elevation above the soil surface and it is shown how this method can be used to predict the reduction in surface dose-rate after remediation measures such as deep ploughing have taken place.

An analysis of the environmental mobility of radiostrontium from weapons testing and Chernobyl in Finnish river catchments.

The mobility of radiostrontium within the Arctic environment and surrounding area has been studied by analysing the mobility of 90Sr in river catchments that are within Finland. The environmental mobility of 90Sr deposited by both nuclear weapons testing and the Chernobyl accident has been investigated in five Finnish river catchments. Different models assessing the time-dependent mobility of 90Sr have been evaluated. No significant differences were found between the mobility of 90Sr from nuclear weapons tests and from the Chernobyl accident. Model parameters obtained by fitting to the measurements of the deposition and runoff rates of the nuclear weapons test fallout gave predictions which were consistent with the mid- and long-term contamination by the Chernobyl fallout. A comparison of 90Sr with 137Cs showed that they had similar mobility on deposition but, as time passed, the relative mobility of 90Sr increased with respect to 137Cs over a period of 5-8 years. Once the relative migration of 90Sr with respect to 137Cs reached equilibrium, its runoff rate was, on average, approximately an order of magnitude greater than 137Cs.

Effects of heavy metal pollution on swimming and longevity in cercariae of Cryptocotyle lingua (Digenea: Heterophyidae).

Effects of heavy metal pollution on the cercariae of the marine trematode Cryptocotyle lingua (Creplin) were studied by measuring horizontal swimming rate (HSR) and longevity. These factors are important for transmission to the next host, a fish. Cercariae released by Littorina littorea (L.) collected from polluted and unpolluted sites were compared. Both HSR and longevity were significantly reduced in cercariae from the polluted environment. Cercarial quality was therefore reduced, directly or indirectly, by development within a metal-accumulating host. Cercariae released by hosts from a clean environment were subjected to nominal concentrations of 2 and 3 mg/l copper, 1 and 2 mg/l zinc, 2 and 5 mg/l iron and 2 and 4 mg/l manganese in artificial seawater. In all cases the HSRs and longevity were reduced. The effect was more pronounced in the higher concentrations. The significant HSR tests indicate that the absorption and effect of metals occurred within 1 min. The cercarial tegument, specialized for absorption in endoparasitic environments, is possibly responsible. Cercariae may therefore be excellent indicator organisms for pollution. The pollution-induced reductions in cercarial quality seem capable of producing transmission failure. Heavy metal pollution could therefore alter parasite populations and communities.

A minimal serpin promoter with high activity in haematopoietic progenitors and activated T cells.

INTRODUCTION: The serine protease inhibitor Serpin 2A is highly expressed in ex vivo bipotent granulocyte/macrophage progenitor cells and in cultured myeloid stem cells. The gene undergoes rapid down-regulation as these cells are induced to differentiate, and constitutive expression in cultured myeloid stem cells retards maturation. Serpin 2A is also expressed in T cells as a consequence of activation. We now report analysis of the upstream regulatory elements that control Serpin 2A transcription. MATERIALS AND METHODS: Using primer extension and rapid amplification of cDNA ends the transcription start site of the Serpin 2A gene was mapped, and a 1.2 Kb genomic upstream fragment cloned and sequenced. Promoter activity and protein binding of deletion and site-directed mutant constructs were analysed by transient transfection and by electrophoretic mobility shift assays. RESULTS: A minimal promoter fragment was identified with high activity dependent on NF-kappa and Moloney murine leukaemia enhancer factor LVa binding sites in both myeloid stem cells and activated T cells. NF-kappa was shown to be the main DNA binding protein in T cells, whereas that in haematopoietic stem cells appears to be novel. CONCLUSION: Serpin 2A promoter activity in T cells is due predominantly to NF-kappa binding to its consensus site. Activity in haematopoietic stem cells appears to be mediated by a novel protein, which recognises the NF-kappa consensus only in the context of flanking sequences. This concise regulatory element may be of potential value in gene therapeutic applications.

A transient assay for regulatory gene function in haemopoietic progenitor cells.

This work aimed to provide a means of assaying directly the effects of transient expression of introduced genes on the survival, proliferation, lineage commitment and differentiation of haemopoietic progenitor cells. For this purpose, we have developed a system that allows isolation of productively transfected, mulitipotent haemopoietic cells within a few hours of the introduction of test genes. We have shown that FDCP-mix cells productively transfected with expression plasmids encoding green fluorescent protein (GFP) differentiate normally and retain colony-forming potential. We constructed an expression vector consisting of a bicistronic cassette in which a GFP marker gene and a test gene are driven from the same promoter. The vector design has been optimized for co-expression and the test gene was shown to be biologically active. The expression profile from a transiently transfected template under different growth conditions reveals that active expression continues for at least 2 d after transfection. The transient transfection of FDCP-mix cells with the vectors described provides a powerful tool for analysis of the immediate early effects of test gene overexpression during haemopoietic differentiation.

Prevention of increases in blood pressure and left ventricular mass and remodeling of resistance arteries in young New Zealand genetically hypertensive rats: the effects of chronic treatment with valsartan, enalapril and felodipine.

The relative efficacy of three antihypertensive drugs in the prevention of further elevation of blood pressure (BP) and cardiovascular structural remodeling in 4-week-old genetically hypertensive (GH) rats was studied by means of two complementary methods, stereology and myography. Four to 10-week-old GH rats were treated with valsartan (10 mg/kg/day), enalapril (10 mg/kg/day) or felodipine (30 mg/kg/day). Untreated GH and normotensive control rats of Wistar origin served as controls. Tail-cuff systolic SBP was measured weekly and left ventricular (LV) mass determined at the end of the experiment. Mesenteric resistance arteries (MRA) were either fixed by perfusion, embedded in Technovit and sections stained for stereological analysis, or mounted on a wire myograph for structural and functional measurements. BP and LV mass were significantly reduced by all drugs; decreases in BP and LV mass were smaller after felodipine treatment. Valsartan and enalapril caused a decrease in BP to normotensive control values. Felodipine kept BP at the 4-week level and prevented further rise with age. Valsartan caused hypotrophic outward remodeling of MRA, enalapril eutrophic outward remodeling and felodipine hypotrophic remodeling. Myograph measurements showed remodeling of the same order. While all drugs lowered the media/lumen ratio in GH to normal, the outward remodeling after valsartan and enalapril indicates that valsartan and enalapril might be more effective in reversing the inward remodeling of resistance arteries found in essential hypertension.

Expression of the imprinted tumour-suppressor gene H19 is tightly regulated during normal haematopoiesis and is reduced in haematopoietic precursors of patients with the myeloproliferative disease polycythaemia vera.

cDNA subtraction was employed to uncover differences in gene expression between myeloproliferative polycythaemia vera (PV) and normal haematopoietic precursors. Following cDNA subtraction using mRNAs isolated from PV and normal CD34+/CD33- bone-marrow cells, expression of the tumour suppressor H19 was found to be low or absent in the PV sample. Low levels of H19 expression in PV patients were confirmed by in situ hybridization. Using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to examine expression in the pluripotent haematopoietic cell line FDCP-mix and single bone-marrow precursors, unambiguous IGF2 and H19 expression was demonstrated in normal haematopoietic precursors. Examination of individual bone-marrow precursors revealed that all IGF2-expressing haematopoietic precursors also co-expressed H19, indicating that H19 and IGF2 may be co-ordinately regulated during haematopoiesis. Analysis of FDCP-mix undergoing differentiation and single pluripotent and committed bone-marrow precursors revealed that the pattern of H19 expression coincided with the commitment to a single lineage. Taken together, these observations demonstrate that H19 and IGF2 are specifically expressed during haematopoiesis and that low levels of H19 expression are associated with PV and may contribute to the pathology of the disease.

Def-2, -3, -6 and -8, novel mouse genes differentially expressed in the haemopoietic system.

To identify developmentally regulated genes during myeloid differentiation, a self-inactivating retroviral gene-trap vector carrying a beta-galactosidase-neomycin (SA/lacZ/neo) fusion gene was constructed and used to infect myeloid progenitor cells (FDCP-Mix A4). G418-resistant and beta-galactosidase positive cell lines (gene-trap integration [GTI] clones) were established and induced to differentiate in vitro into either macrophages or granulocytes. Expression of the trapped loci was monitored at a single-cell level by analysing the mature cell types for beta-galactosidase activity. All 37 GTI clones tested showed down-regulation either during granulocyte or both granulocytic and macrophage differentiation. The endogenous coding regions fused to the SA/lacZ/neo reporter gene were isolated from eight clones. Molecular analysis revealed that half of them represented novel mouse genes (def-2, -3, -6 and -8) which we confirmed to be differentially expressed in primary haemopoietic tissues. Database searches revealed no significant similarities for def-2 (associated with haemopoietic progenitors) and def-8 (expressed most strongly in peripheral leucocytes). Def-6, which is down-regulated upon the differentiation into myeloid as well as erythroid lineages, was found to be closely related but not identical with the recently described B-cell-specific switch recombinase SWAP-70. Def-3, which is down-regulated upon differentiation into granulocytes but expressed in progenitor cells and macrophages, defines a novel family of RNA binding proteins.

Upstream elements bestow T-cell and haemopoietic progenitor-specific activity on the granzyme B promoter.

Cytotoxic T cells and early haemopoietic progenitors share the expression of a number of specific genes. Of these, granzyme B has attracted particular interest because of its role in inducing apoptosis during cytotoxic T cell-mediated target cell killing, and its potential role in the mobilisation and homeostasis of haemopoietic stem cells. Studies of granzyme B regulation should therefore yield valuable information concerning the molecular control of these processes, and also identify elements capable of directing gene expression to two cell types of relevance to gene therapy. Here we show that proximal regulatory elements already known to direct promoter activity in T cells are similarly active in haemopoietic progenitors. However, this activity is not strictly specific, since the promoter regions also direct low levels of reporter gene expression in fibroblasts. More importantly, we also report the presence of two previously unidentified clusters of DNaseI hypersensitive sites upstream from the murine granzyme B gene, and show that these regions impart both increased transcriptional activity and the appropriate cell type specificity on the granzyme B promoter. These upstream regulatory regions are therefore likely to play a key role in the coordination of granzyme B expression in vivo.

Expression of macrophage inflammatory protein-1 receptors in human CD34(+) hematopoietic cells and their modulation by tumor necrosis factor-alpha and interferon-gamma.

Macrophage inflammatory protein-1alpha (MIP-1alpha) can stimulate growth inhibitory and potent chemotactic functions in hematopoietic cells. To investigate whether the action of MIP-1alpha may be regulated at the cellular receptor level, we studied the expression and modulation of MIP-1alpha receptors on CD34(+) cells isolated from normal bone marrow (NBM), umbilical cord blood (CB), and leukapheresis products (LP). Expression of MIP-1alpha receptors on CD34(+) cells was analyzed by two-color flow cytometry using a biotinylated MIP-1alpha molecule. The mean percentage of LP CD34(+) cells expressing the MIP-1alpha receptors was 67.7 +/- 7.2% (mean +/- SEM; n = 22) as compared with 89.9 +/- 2.6% (n = 10) and 74.69 +/- 7.04% (n = 10) in CB and NBM, respectively (P = .4). The expression of the MIP-1alpha receptor subtypes on LP CD34(+) cells was studied by indirect immunofluorescence using specific antibodies for the detection of CCR-1, CCR-4, and CCR-5. Microscopical examination revealed a characteristic staining of the cytoplasmic cell membrane for all three receptor subtypes. Detailed analysis of two LP samples showed that 65.8%, 4.4%, and 30.5% of CD34(+) cells express CCR-1, CCR-4, and CCR-5, respectively. Culture of LP CD34(+) cells for 24 to 36 hours in the presence of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) resulted in a significant increase in MIP-1alpha receptor expression. TNF-alpha induced MIP-1alpha receptor upregulation in a time- and concentration-dependent manner. Our results suggest that inhibitory cytokines produced by the bone marrow microenvironment are likely to be involved in the regulation of MIP-1alpha receptor expression on hematopoietic cells.

Differential response of CD34+ cells isolated from cord blood and bone marrow to MIP-1 alpha and the expression of MIP-1 alpha receptors on these immature cells.

Macrophage inflammatory protein-1 alpha (MIP-1alpha) has been shown to have a role in the control of myeloid stem and progenitor cell proliferation. Recent evidence suggests that MIP-1alpha also has a stimulatory effect on proliferation of mature progenitors as well as an inhibitory effect on immature progenitors in vitro. We have compared the effect of MIP-1alpha on myeloid and erythroid colony formation of CD34+ cells isolated from bone marrow and cord blood. In the presence of MIP-1alpha, bone marrow granulocyte-macrophage-colony forming cells (GM-CFC) were inhibited over a dose range of 15 ng/ml to 500 ng/ml, and GM-CFC from cord blood CD34+ cells were stimulated over the same dose range. MIP-1alpha suppressed BFU-E colonies in both bone marrow and cord blood. Using thymidine suicide assays, the influence of MIP-1alpha on the cycling status of the cells was assessed. A good correlation between the effect of MIP-1alpha on colony formation and cell cycle progression was observed. These results suggest that there is a differential response to MIP-1alpha when bone marrow and cord blood CD34+ cells are compared. Using flow cytometry and a biotinylated human MIP-1alpha/avidin fluorescein conjugate, the expression of MIP-1alpha receptors on CD34+ cells was assessed. The data indicated that there was little quantitative difference in overall expression of receptors (82.9% versus 93%) from bone marrow or cord blood, respectively. However, when Northern blot analysis was used, mRNA for two different MIP-1alpha receptors CCR1 and CCR5 could be detected in bone marrow, but only CCR1 mRNA was seen in cord blood CD34+ samples. Therefore, the expression of different receptor subtypes on CD34+ cells may be responsible for the difference in MIP-1alpha responsiveness observed.

Tips for recruiting I.T. professionals in tough times.

Health care organizations are struggling to recruit information technology staff in a fiercely competitive market. Providers and insurers need more technology personnel to manage the growing number of systems their organizations are demanding. But finding qualified staff is not easy at a time when technology is changing rapidly and experienced specialists are scarce. Some CIOs, however, are devising creative strategies for attracting the talented technicians that they need.

Accelerated telomere shortening in young recipients of allogeneic bone-marrow transplants.

BACKGROUND: The establishment of donor-derived haemopoiesis in the recipients of allogeneic bone-marrow transplants (BMT) involves extensive proliferation of haemopoietic stem cells. The biological consequences of this replicative stress are ill defined, but any "ageing" effect would carry the risk of an increased frequency of clonal disorders during later life. We compared blood-cell mean telomere lengths in donor/recipient pairs. METHODS: Mean telomere length was calculated by in-gel hybridisation to leucocyte DNA from 56 normal individuals aged 0-96 years, and from 14 consecutive BMT recipients (aged 2-14 years) plus their respective donors (aged 2-46 years). Engraftment was confirmed by variable numbers of tandem repeats (VNTR) or gender analysis. FINDINGS: On average, blood-cell telomeres of transplant recipients were 0.4 kb (95% CI -0.2 to -0.6) shorter than those of their respective donors. This degree of telomere loss is equivalent to a median of 15 years' (range 0-40) ageing in the healthy controls. INTERPRETATION: The kinetics of haemopoietic engraftment impose replicative stress on the haemopoietic stem cells, resulting in a pronounced ageing effect, which may be sufficient to accelerate the onset of clonal haemopoietic disorders usually associated with later life. Monitoring of haemopoietic status in BMT recipients as time since BMT increases will be important. Assessment of transplant protocols under development in terms of their effects on telomere shortening is also indicated.

The lineage commitment of haemopoietic progenitor cells.

Multipotent haemopoietic progenitor cells appear to be 'primed' for commitment by co-expression of a multiplicity of genes characteristic of different lineages. Lineage commitment proceeds as the consolidation of a distinct pattern of gene expression out of this milieu.

Planning for disaster recovery.

Planning for restoring information systems in the event of a disaster is rapidly changing and growing in importance. As health care organizations become more reliant on information technology, they're taking extra steps to safeguard their investments and to ensure reliable access to data.