Developing principles of sustainability and stakeholder engagement for "gentle" remediation approaches: the European context.

Gentle Remediation Options (GRO) are risk management strategies or techniques for contaminated sites that result in no gross reduction in soil functionality (or a net gain) as well as risk management. Intelligently applied GROs can provide: (a) rapid risk management via pathway control, through containment and stabilisation, coupled with a longer term removal or immobilisation/isolation of the contaminant source term; and (b) a range of additional economic (e.g. biomass generation), social (e.g. leisure and recreation) and environmental (e.g. CO2 sequestration) benefits. In order for these benefits to be optimised or indeed realised, effective stakeholder engagement is required. This paper reviews current sector practice in stakeholder engagement and its importance when implementing GRO and other remediation options. From this, knowledge gaps are identified, and strategies to promote more effective stakeholder engagement during GRO application are outlined. Further work is required on integrating stakeholder engagement strategies into decision support systems and tools for GRO (to raise the profile of the benefits of effective stakeholder engagement and participation, particularly with sector professionals), and developing criteria for the identification of different stakeholder profiles/categories. Demonstrator sites can make a significant contribution to stakeholder engagement via providing evidence on the effectiveness of GRO under varying site contexts and conditions. Effective and sustained engagement strategies however will be required to ensure that site risk is effectively managed over the longer-term, and that full potential benefits of GRO (e.g. CO2 sequestration, economic returns from biomass generation and "leverage" of marginal land, amenity and educational value, ecosystem services) are realised and communicated to stakeholders.

Microbial community structure and activity in trace element-contaminated soils phytomanaged by Gentle Remediation Options (GRO).

Gentle remediation options (GRO) are based on the combined use of plants, associated microorganisms and soil amendments, which can potentially restore soil functions and quality. We studied the effects of three GRO (aided-phytostabilisation, in situ stabilisation and phytoexclusion, and aided-phytoextraction) on the soil microbial biomass and respiration, the activities of hydrolase enzymes involved in the biogeochemical cycles of C, N, P, and S, and bacterial community structure of trace element contaminated soils (TECS) from six field trials across Europe. Community structure was studied using denaturing gradient gel electrophoresis (DGGE) fingerprinting of Bacteria, α- and ß-Proteobacteria, Actinobacteria and Streptomycetaceae, and sequencing of DGGE bands characteristic of specific treatments. The number of copies of genes involved in ammonia oxidation and denitrification were determined by qPCR. Phytomanagement increased soil microbial biomass at three sites and respiration at the Biogeco site (France). Enzyme activities were consistently higher in treated soils compared to untreated soils at the Biogeco site. At this site, microbial biomass increased from 696 to 2352 mg ATP kg-1 soil, respiration increased from 7.4 to 40.1 mg C-CO2 kg-1 soil d-1, and enzyme activities were 2-11-fold higher in treated soils compared to untreated soil. Phytomanagement induced shifts in the bacterial community structure at both, the total community and functional group levels, and generally increased the number of copies of genes involved in the N cycle (nirK, nirS, nosZ, and amoA). The influence of the main soil physico-chemical properties and trace element availability were assessed and eventual site-specific effects elucidated. Overall, our results demonstrate that phytomanagement of TECS influences soil biological activity in the long term.

A novel method for direct and fluorescence independent determination of drug efflux out of leukemic blast cells.

Multi drug resistance (MDR) is often due to an increased efflux of anti cancer drugs out of leukemic blast cells. Efflux assays are used to get an impression of functional resistance in those cells. Dyes like rhodamine 123 or 3'3'-diethyloxocarbocyanine iodide are commonly used for this purpose. A major known disadvantage is that dyes do not behave like cytotoxic drugs in efflux experiments. Assays using the self fluorescence of drugs like anthracyclines can not reveal a real impression of intracellular or effluxed drug due to quenching of the drug fluorescence in the nuclei of the cells. We have developed a reproducible and sensitive assay for direct and quantitative determination of drug efflux out of blast cells. This was done by a novel double radioactive labelling using a 3H-labelled drug and 14C-labelled sucrose as extracellular marker. So this assay can be applied to every drug of interest. Quenching of fluorescence is also by-passed with this technique as well as protracting washing or silicon oil procedures. As a model system we used the T-lymphoblastoid cell line CCRF CEM and its resistant sublines vincristine 100 and adriamycin 5000. The results were also transferable to clinical specimens of leukemic patients. In conclusion our assay may be used for precise and direct efflux measurement of a broad range of anti-cancer drugs in clinical MDR evaluation.

Isolation and phenotypic characterization of CD117-positive cells.

Mononuclear cells derived from cord blood were stained using the CD1 17-specific, fluorochrome-labeled monoclonal mouse antibody 95C3. Additional staining was performed using an isotype-specific rat-anti-mouse antibody, labeled with supermagnetic microparticles. Target cells were enriched by the technique of magnetic cell separation, MACS. The resulting cell population contained 96.5% (+/-1.7% S.D.) CD1 17-expressing cells (n = 12) with different levels of CD117 antigen expression. Using flow cytometry, two cell populations differing in size were found. A majority (93%) of cells with high forward scatter revealed a phenotype positive for CD117 and CD34. Isolated cells revealed a high fraction of hematopoietic progenitors (16%). The technique presented allows for an alternative approach of stem cell enrichment and might be useful in autologous transplantation of cells with hematopoietic properties.

Phenotypic and functional characterization of mobilized peripheral blood CD34+ cells coexpressing different levels of c-Kit.

In this report we evaluated the exact expression pattern of c-Kit on mobilized peripheral blood (PB) CD34+ cells. Using a monoclonal antibody against CD117 antigen (95C3), flow cytometric analysis revealed that approximately 25% of the mobilized PB CD34+ cells coexpress c-Kit. This cell fraction showed a considerable heterogeneity with respect to c-Kit expression, consisting of a small fraction with high levels of c-Kit (4.2%) (CD34+/CD117high fraction) and a larger proportion of cells expressing low levels of this antigen (21.0%) (CD34+/CD117low fraction). Clonogenic assays showed that CD34+/CD117high cell fraction consisted almost exclusively of erythroid progenitors, in contrast to CD34+/CD117low cell subset which gave rise mostly to granulocyte-monocyte colonies. The majority of CFU-GEMM and the most primitive week 6 cobblestone area forming cells (CAFCs) segregated in the CD34+/CD117low cell subset, suggesting the highest content of multipotential progenitors within this cell fraction. None of the sorted cell subsets was able to produce reactive oxygen intermediates (ROI). However, ex vivo expansion of the sorted subsets with interleukin 3, stem cell factor and FLT3 ligand for 2 weeks resulted in a significant production of O2- and H2O2/HOCl by CD34+/CD117low cell fraction, compared to the same sorted but not expanded counterparts. According to the major content of multipotential hematopoietic progenitors and highest capacity to generate sufficient amounts of ROI after ex vivo expansion, we suggest that CD34+/CD117low cell subset would be one of the most potential candidates for transplantation in patients with acute lymphoblastic leukemia, which lack c-Kit antigen expression.

Isolation and transplantation of autologous peripheral CD34+ progenitor cells highly purified by magnetic-activated cell sorting.

Peripheral stem cells were mobilized and collected in 26 pediatric patients with malignant diseases. A total of 47 leukaphereses were performed in the 26 patients. The mean number of nucleated cells collected was 4.5 +/- 2.6 x 10(8)/kg and the number of CD34+ progenitors collected was 6.7 +/- 6.8 x 10(6)/kg. CD34-positive selection was performed using a two-step method of magnetic-activated cell sorting (MACS) in 24 patients or a combination of an immunoaffinity column and MACS in two patients. The purity of the positively selected CD34+ progenitors was 98.8 +/- 0.7% and the number of isolated CD34+ cells was 6.5 +/- 5.9 x 10(6)/kg. Thus, the mean recovery of CD34+ cells was 93 +/- 10%. In 22 of the 26 patients, high-dose chemotherapy was performed with subsequent reinfusion of the highly purified CD34+ cells. In all 22 patients, a normal hematopoietic reconstitution was seen with a mean time of 12.4 +/- 2.7 days to reach >0.5 x 10(9)/l neutrophils (range 8-19 days). The time to reach independence from platelet transfusion was 31.6 +/- 17.0 days (range 16-78 days). There were no transplant-related deaths. In summary, we have shown that mobilized peripheral CD34+ progenitors can be highly purified with a good recovery, and that reinfusion of these cells after high-dose chemotherapy results in a rapid, complete and sustained engraftment. We conclude that this method can be used for purging in any CD34-negative malignancies and for autologous T and B cell depletion in the treatment of autoimmune diseases with high-dose immunoablative therapy.

Expression of CD44 isoforms by highly enriched CD34-positive cells in cord blood, bone marrow and leukaphereses.

CD34-positive cells were isolated from cord blood (n = 8), bone marrow (n = 4) and leukapheresed material (n = 7), using an immunomagnetic isolation technique, MACS (Miltenyi Biotec, Bergisch Gladbach, Germany). In flow cytometric analysis, cell populations after enrichment revealed a fraction of 96.1% (cord blood), 96.2% (bone marrow) and 98.6% (leukapheresis material) CD34-positive cells. Cells were further stained with antibodies specific for CD44 isoforms: CD44s (SFF-2), CD44v5 (VFF-8) and CD44v6 (VFF-18). CD44-positive cells were detected by directly (FITC, fluorescein isothiocyanate) or indirectly (streptavidin-PE, phycoerythrin)-conjugated fluorochromes in flow cytometric analysis. Analysis was restricted to CD34-positive cells. A high expression of CD44s was noted in all kinds of material under investigation with mean values in the range of 98.6-100%. There was little expression of CD44v6 (mean values in the range of 1.5-3.6%) and very slight expression of CD44v5 (mean values in the range of 0.6-1.4%). The finding that CD34-positive hematopoietic stem cells express CD44v5 and CD44v6 to a very small extent offers the possibility of using antibodies specific to CD44v5 and CD44v6 in immunopurging in the course of autologous stem cell transplantation.

Clinical scale isolation of highly purified peripheral CD34+progenitors for autologous and allogeneic transplantation in children.

We present our experience with three clinical scale isolation methods for positive selection of CD34+ progenitors from peripheral blood for autologous and allogeneic transplantation in children. A combination of the CellPro device and the Magnetic Activated Cell Sorting system (MACS), as well as two different combinations of MACS systems were used (VarioMACS-SuperMACS and SuperMACS-SuperMACS). With the CellPro-MACS combination (16 separations), a median purity of 96.2% and a median recovery of 42% CD34+ cells could be achieved, whereas the two step MACS systems (55 and 29 separations) showed a median purity of 97.6% and 98.0% and a median recovery of 96.5% and 97%, respectively. Depletion of T cells was profound (4-5 log). A total of 34 patients in the autologous and 18 patients in the allogeneic setting have been transplanted with highly enriched CD34+ cells, obtained by these methods. Only one patient failed to engraft, all other patients showed a rapid and sustained hematological engraftment with the longest follow-up of 3 years. In summary, especially the two step MACS systems have proven to be appropriate tools for enrichment of CD34+ cells, yielding both high purity and good recovery, and can thus be used for tumor cell purging in the autologous setting and for effective T cell depletion in the allogeneic setting.

Megadose transplantation of purified peripheral blood CD34(+) progenitor cells from HLA-mismatched parental donors in children.

We performed HLA-mismatched stem cell transplantation with megadoses of purified positively selected mobilized peripheral blood CD34(+) progenitor cells (PBPC) from related adult donors in 39 children lacking an otherwise suitable donor. The patients received a mean number of 20.7 +/- 9.8 x 10(6)/kg purified CD34(+) and a mean number of 15.5 +/- 20.4 x 10(3)/kg CD3(+) T lymphocytes. The first seven patients received short term (<4 weeks) GVHD prophylaxis with cyclosporin A, whereas in all the following 32 patients no GVHD prophylaxis was used. In 38 evaluable patients, five patients experienced primary acute GVHD grade I and one patient grade II. In 32 patients, no signs of primary GVHD were seen and GVHD only occurred after T cell add backs. T cell reconstitution was more rapid if the number of transplanted CD34(+) cells exceeded 20 x 10(6)/kg. Of the 39 patients, 15 are alive and well, 13 died due to relapse and 10 transplant-related deaths occurred. We conclude that the HLA barrier can be overcome by transplantation of megadoses of highly purified mismatched CD34(+) stem cells. GVHD can be prevented without pharmacological immunosuppression by the efficient T cell depletion associated with the CD34(+) positive selection procedure. This approach offers a promising therapeutic option for every child without an otherwise suitable donor.

Prophylactic effects of sucralfate and geraniin on ethanol-induced gastric mucosal damage in rats.

The aim is to study the protective effects of topical sucralfate and geraniin on acidified ethanol (EtOH dissolved in 100 mM HCI plus 54 mM NaCl)-induced gastric acid back-diffusion, mucus production and mucosal ulcerations in the rat. After irrigation the stomach with acidified EtOH (10-40% v/v) for 3 hrs, a concentration-dependent increase in acid back-diffusion and in mucosal lesions, and -dependent reduction in mucus production was found. These ulcerogenic effects of EtOH were dose-relatedly inhibited by pretreatment of intragastric sucralfate (10-200 mg/kg). A high correlation (r = -0.9572) between sucralfate-induced inhibition in acid back-diffusion and -induced reduction in mucosal ulceration provoked by 30% EtOH was observed. Geraniin (10-100 mg/kg), given 30 min prior to EtOH perfusion, produced potent inhibitory effects on those ulcerogenic parameters provoked by EtOH in a dose-dependent manner. The correlation (r = -0.8638) between geraniin-induced inhibitions in acid back-diffusion and in mucosal ulceration produced by EtOH was achieved. These cytoprotective effects of sucralfate and geraniin were further confirmed by morphological and histological studies. The results suggested that the protective effects of sucralfate and geraniin on gastric mucosa against acidified EtOH-induced damage are at least partly through the inhibition in acid back-diffusion and the elevation of gastric mucus production.

Postoperative pulmonary edema after cervical spine surgery--a case report.

Injury of the cervical spine may cause serious complications and neurological sequelae. Recently, a patient with C1-2 spinal cord compression developed pulmonary edema postoperatively associated with unstable hemodynamics, which might result from overzealous fluid administration in order to correct neurogenic shock during anesthesia. Therefore, early recognition and timely use of vasoconstrictors, together with judicious fluid replacement are important in the anesthetic management of patients with cervical spine injury undergoing surgery. In addition, the placement of pulmonary artery catheter is crucial for assessing the cardiac function and fluid status.

Postoperative right atrial and pulmonary embolism after prolonged spinal surgery.

Perioperative pulmonary thromboembolism can proceed rapidly with grave prognosis, in which immediate or accurate diagnosis and management is not easy. According to the literatures, patients receiving spinal surgery are at relatively lower risk of developing thromboembolism. We would like to present a case of postoperative pulmonary thromboembolism which developed after a prolonged lumbar spinal surgery. Tachycardia and unstable hemodynamics were noted postoperatively. Pulmonary and right atrial thrombi were disclosed by transesophageal echocardiography. Although cardiotomy and thrombectomy were immediately performed, the patient finally died 3 days after the operation. The pathogenesis of venous thromboembolism (VTE) in the surgical patients, the risk factors which predispose a patient to VTE, diagnosis, and treatment as well as the prophylactic measures of VTE are herein reviewed and discussed.

Acid-induced gastric damage in rats is aggravated by starvation and prevented by several nutrients.

The aggravation of acid-induced gastric damage and its prevention by glucose, ascorbate or glutathione precursors was studied in fed and food-deprived rats. The stomachs of fed rats and those starved for 1, 3 or 5 d were vagotomized just before irrigating for 3 h with solutions containing 0-150 mmol HCI/L. Mucosal glutathione, mucus, lipid peroxides and acid back-diffusion were measured. Stomach ulcers were evaluated by morphological and histological examination. The preventive effects of glucose, ascorbate and a mixture of L-glutamine, L-glycine and L-cysteine were evaluated in the stomachs of rats that were starved for 5 d, vagotomized, then perfused for 3 h with 100 mmol HCI/L. Greater acid back-diffusion and ulcer formation, and lower glutathione and mucus levels in starved rats were dependent on the duration of starvation and luminal acidity. Increased acid back-diffusion and decreased glutathione and mucus production were negatively correlated (r < -0.80, P < 0.05) with ulcer formation. A significant enhancement in mucosal lipid peroxide concentration and serious damage of forestomach and corpus mucosal cells were observed in starved rats exposed to 100 mmol HCI/L. These ulcerogenic factors were effectively inhibited in acid-perfused stomachs of food-deprived rats by daily intraperitoneal injection of the amino acid mixture (150 mg/kg) or by an average daily consumption via drinking water of glucose (10 g) or ascorbate (1.2 g). Starvation aggravated acid-induced gastric damage and was associated with greater acid back-diffusion and oxygen radical generation, and lower mucosal glutathione and mucus production.

Stopping long-term dialysis. An empirical study of withdrawal of life-supporting treatment.

Dialysis was discontinued in 155 (9 percent) of 1766 patients being treated for end-stage renal disease, accounting for 22 percent of all deaths. Treatment was withdrawn more frequently in older than in younger non-diabetic patients, and more often in young diabetic patients than in young nondiabetic patients. Withdrawal was twice as common in nondiabetic patients with other degenerative disorders (P less than 0.005); in patients receiving intermittent peritoneal dialysis (P less than 0.025); and in patients living in nursing homes (P less than 0.025). Half the patients were competent when the decision to withdraw was made, and 39 percent of this group had no new preceding medical complications. Among incompetent patients, the physician initiated the decision for withdrawal in 73 percent, and the patient's family in 27 percent; all patients had recent medical complications. In the early 1970s the physician initiated the decision in 66 percent of all patients; in the early 1980s this figure had decreased to 30 percent (P less than 0.0005). We conclude that stopping treatment is a common mode of death in patients receiving long-term dialysis, particularly in those who are old and those who have complicating degenerative diseases. Because of the increasing age of patients on dialysis, withdrawal of treatment will probably become more common in the future.

The spreading of focal brain edema induced by ultraviolet irradiation.

Focal brain edema limited to one cerebral hemisphere was produced by ultraviolet irradiation of the exposed cortex. Tissue water content was determined by the gravimetric method which allows microsampling. Therefore, the spread of edema around the small necrotic area could be mapped more precisely than by determination of dry weight which calls for larger samples. As early as 30 min following irradiation, hyperemia and swelling of the brain are observed under the operating microscope. This correlates with venous stasis, hyperemia, and broadened perivascular spaces around venules and large capillaries accompanied by a marked rise in the specific weight of the tissue. After 4 h an edema front can be observed spreading from the perinecrotic zone in which there is a marked rise in endothelial cell vesicular activity. Edema reaches maximum levels in the deep white matter at 48 h post irradiation with normalisation of the tissue water content after 96 h. The velocity at which the edema front spreads from the cortex to the periventricular area lies in the range of 0.25 mm/hr. Edema reabsorption coincides with signs of retrograde micropinocytosis in endothelial cells.

Granulocyte colony-stimulating factor receptor expression on neutrophils of term and preterm neonates with and without signs of infection.

UNLABELLED: Neutrophils are an essential component of the human host defence system against infection. Recombinant human granulocyte colony-stimulating factor induces neutrophilia and enhances effector functions of mature neutrophils. Since the biological effects of granulocyte colony-stimulating factor (G-CSF) are mediated by its receptor, we investigated the expression of G-CSF receptor on the surface of neutrophils of term and preterm neonates (n = 22) with and without signs of infection and of healthy adults (n = 13) by flow cytometry. In healthy adults, the percentage of neutrophils expressing G-CSF receptor was higher compared to cord blood of term and preterm neonates (87% vs 53%, P < 0.05). Between 2 and 32 h of life, neonates with signs of infection showed lower values of G-CSF receptor expression compared to neonates without signs of infection (32% vs 54%, P < 0.05). No correlation was detectable between expression of G-CSF receptor and gestational age. CONCLUSION: Expression of granulocyte colony-stimulating factor receptor on neutrophils is lower than in adults. This may adversely affect granulopoiesis and neutrophil function during the neonatal period. Moreover, granulocyte colony-stimulating factor receptor expression seems to be down-regulated during neonatal infection.