The impact of smoke-free policies on smoking at outdoor sports clubs: a qualitative study.

OBJECTIVES: Smoking may still occur at sports clubs with an outdoor smoke-free policy (SFP). This study aims to map the occurrence of smoking at various sports clubs in the Netherlands and to understand why smoking occurs at some clubs but not at others. STUDY DESIGN: This was a qualitative design in the form of semistructured interviews. METHODS: Semistructured interviews (n = 34) were held online with smoking and non-smoking members of 17 Dutch outdoor sports clubs (in field hockey, korfball, football, and tennis) with an outdoor SFP. Data were analyzed using content analysis. RESULTS: We identified four situations where smoking still occurred: (1) directly at the entrance, (2) at some distance from the entrance, (3) in particular places on the premises, and (4) in various places or on occasions when alcohol is consumed. Smoking directly at the entrance was most often perceived as a bothersome situation that was difficult to avoid. The occurrence of these situations differed per sports club depending on the scope of the SFP (the comprehensiveness of the SFP and the presence or absence of a smoking area) and factors influencing policy compliance (physical characteristics of the sports club's premises, the presence or absence of children, and several enforcement difficulties). CONCLUSION: In some sports clubs, smoking remained common on the premises despite an outdoor SFP. Exposure to second-hand smoke might be reduced by formulating a comprehensive SFP, improving policy compliance also in situations where children are absent, and organizing the enforcement of the policy.

Cell aggregation enhances bone formation by human mesenchymal stromal cells.

The amount of bone generated using current tissue engineering approaches is insufficient for many clinical applications. Previous in vitro studies suggest that culturing cells as 3D aggregates can enhance their osteogenic potential, but the effect on bone formation in vivo is unknown. Here, we use agarose wells to generate uniformly sized mesenchymal stromal cell (MSC) aggregates. When combined with calcium phosphate ceramic particles and a gel prepared from human platelet-rich plasma, we generated a tissue engineered construct which significantly improved in vivo bone forming capacity as compared to the conventional system of using single cells seeded directly on the ceramic surface. Histology demonstrated the reproducibility of this system, which was tested using cells from four different donors. In vitro studies established that MSC aggregation results in an up-regulation of osteogenic transcripts. And finally, the in vivo performance of the constructs was significantly diminished when unaggregated cells were used, indicating that cell aggregation is a potent trigger of in vivo bone formation by MSCs. Cell aggregation could thus be used to improve bone tissue engineering strategies.

Relevance of an academic GMP Pan-European vector infra-structure (PEVI).

In the past 5 years, European investigators have played a major role in the development of clinical gene therapy. The provision of substantial funds by some individual member states to construct GMP facilities makes it an opportune time to network available gene therapy GMP facilities at an EU level. The integrated coordination of GMP production facilities and human skills for advanced gene and genetically-modified (GM) cell therapy, can dramatically enhance academic-led "First-in-man" gene therapy trials. Once proof of efficacy is gathered, technology can be transferred to the private sector which will take over further development taking advantage of knowledge and know-how. Complex technical challenges require existing production facilities to adapt to emerging technologies in a coordinated manner. These include a mandatory requirement for the highest quality of production translating gene-transfer technologies with pharmaceutical-grade GMP processes to the clinic. A consensus has emerged on the directions and priorities to adopt, applying to advanced technologies with improved efficacy and safety profiles, in particular AAV, lentivirus-based and oncolytic vectors. Translating cutting-edge research into "First-in-man" trials require that pre-normative research is conducted which aims to develop standard assays, processes and candidate reference materials. This research will help harmonise practices and quality in the production of GMP vector lots and GM-cells. In gathering critical expertise in Europe and establish conditions for interoperability, the PEVI infrastructure will contribute to the demands of the advanced therapy medicinal products* regulation and to both health and quality of life of EU-citizens.

Association study of serotonin-2A receptor gene polymorphism and panic disorder in patients from Canada and Germany.

The T102C serotonin-2A (5-HT2A) receptor gene polymorphism has been studied extensively in a number of complex psychiatric conditions with mixed results. Recently a genetic association has been described between this polymorphism and panic disorder in a Japanese sample. To evaluate the impact of the T102C polymorphism in panic disorder we genotyped triad families (panic disorder patient and parents), and cases with controls in Canadian and German samples. No significant transmission disequilibrium was observed between the alleles of the T102C 5-HT2A receptor gene polymorphism and panic disorder, nor was a significant excess of either allele found in the case control analysis. Our data suggest thus that this polymorphism is unlikely to play a major role in the pathogenesis of panic disorder.

Autologous red cells derived from cord blood: collection, preparation, storage and quality controls with optimal additive storage medium (Sag-mannitol).

To investigate whether packed red cells (PRCs) prepared from autologous cord blood-packed red cells (AC-PRCs) could be used as an alternative for homologous-packed red cells (H-PRCs), we developed a system to collect and prepare AC-PRCs and determined standard storage parameters during 35 days of storage in extended storage medium (Sag-mannitol). We collected and fractionated cord blood from 390 newborns. The amount and quality of the AC-PRCs were analysed. The bacterial contamination rate was 1.84%. Twelve AC-PRCs were stored for 35 days, and standard laboratory parameters were measured at day 1 and day 35. The initial laboratory parameters of the AC-PRCs were similar to the parameters of the H-PRCs. After 35 days, the AC-PRCs displayed an increased haemolysis rate compared to H-PRCs (1.1 versus 0.2%) and also a significant decreased adenosine triphosphate value (1.2 versus 2.3 micromol L(-1)). Haemoglobin, haematocrit and pH were comparable in both groups. AC-PRCs meet the quality criteria for H-PRCs after 35 days. Utilizing a closed collection system for cord blood and an extended storage medium will increase safety and quality and facilitate the routine transfusion of autologous red cells derived from cord blood.

Platelet glycoprotein complex Ia/IIa antibodies cause neonatal alloimmune thrombocytopenia but do not inhibit megakaryopoiesis and platelet recovery after allogeneic cord blood stem cell transplantation.

A sibling cord blood (CB) transplantation was performed in a boy with Wiskott-Aldrich syndrome. The CB (31 x 10(6) CD34(+) cells) derived from a newborn sister with neonatal alloimmune thrombocytopenia (NAIT) with 40,000 platelets/microl, caused by a maternal anti-HPA-5b and HLA-A2 antibody. Maternal serum did not inhibit clonogenicity after in vitro testing of megakaryopoiesis. Accordingly, this CB was accepted for sibling transplantation. The transplantation showed a good course with fast and sustained hematopoietic reconstitution (granulocytes >500/microl on day +16, platelets >50,000/microl on day +30). This case demonstrates a successful CB transplantation from a donor suffering from NAIT.

Volume-dependent collection of peripheral blood progenitor cells during large-volume leukapheresis for patients with solid tumours and haematological malignancies.

We investigated the efficacy of peripheral blood progenitor cell (PBPC) collection during large-volume leukapheresis (LVL) in patients with solid tumours and haematological malignancies (n = 18). The time- and volume-dependent harvest of leucocytes (WBC), mononuclear cells (MNC), CD34+ cells and colony-forming cells (CFU-GM) during LVL was analysed in six sequentially filled collection bags processing four times the patient's blood volumes. The amounts of leucocytes (WBC) and the purity of mononuclear cells (MNC%) did not show any significant changes during LVL. The percentage of CD34+ cells remained constant for the first three bags but consecutively decreased from initially 1.71% CD34+ cells in the beginning of LVL to finally 1.34% CD34+ cells (P = 0.02). The mean numbers of colony-forming cells (CFU-GM) decreased from 74 microL-1 to 59 microL-1 during LVL (P = 0.16). Furthermore, the comparison of volume-dependent PBPC collection for patients with high, medium and low total yields of CD34+ cells showed similar kinetics on different levels for the three groups. We concluded that - relative to the initial total amount of PBPC harvested - comparable numbers of progenitor cells can be collected during all stages of LVL with a slight decreasing trend processing four times the patient's blood volumes.

Quantitative immunophenotypic characterization, cryopreservation, and enrichment of second- and third-trimester human fetal cord blood hematopoietic stem cells (progenitor cells).

OBJECTIVE: The aims of this study were (1) to assess the hematopoietic stem cell (progenitor cell) contents of umbilical cord blood samples from second-trimester and early-third-trimester fetuses versus term fetuses and (2) to determine the feasibility of cryopreservation and enrichment of cord blood from fetuses of different gestational ages. STUDY DESIGN: Cord blood between 13 and 42 weeks' gestation (n = 31) was sampled after delivery or fetal expulsion. Fluorescence-activated cell sorting was used to measure CD34(+) and CD34(+)CD38(-) cell numbers. Samples were cryopreserved with 10% dimethylsulfoxide, and CD34(+) enrichment was performed by magnetically activated cell sorting with the MiniMACS system (Miltenyi Biotech, Bergisch Gladbach, Germany). Kruskal-Wallis analysis of variance and the Mann-Whitney U test were used for analysis of data. RESULTS: CD34(+) and CD34(+)CD38(-) cell contents were significantly higher in second- and early third-trimester fetuses than in term fetuses (CD34(+) 2.57% +/- 0.38%, 1.48% +/- 0. 31%, and 0.7% +/- 0.13%, respectively, P =.0067; CD34(+)CD38(-) 0. 72% +/- 0.26%, 0.18% +/- 0.05%, and 0.06% +/- 0.02%, respectively, P =.0132). Mononuclear cell recovery, viability, and CD34(+) cell purity after cryopreservation and enrichment were similar among different gestational ages. CONCLUSION: Cord blood stem cell content decreases significantly from the second trimester to term. Cryopreservation and enrichment of these cells from earlier gestational ages is feasible. This might be especially useful for allogeneic stem cell transplantation and for in utero gene therapy.

[Peripheral blood stem cell transplantation as an interdisciplinary challenge--theory and practice]. / Die periphere Blutstammzelltransplantation als interdisziplinäre Herausforderung--Theorie und Praxis.

High dose chemotherapy with consecutive autologous peripheral blood stem cell transplantation becomes increasingly important for the treatment of hematological diseases and solid tumors. A complete remission or at least a prolonged survival can be achieved for numerous malignant diseases by an intensification of chemo- and radiotherapy. Therefore, the autologous peripheral blood stem cell transplantation (PBSCT) represents an elementary precaution to reduce the therapy-associated aplasia by administration of hematopoietic precursor cells. Both, high dose chemotherapy with consecutive PBSCT demands great clinical experience and the collection, processing and positive selection of blood stem cells is a challenge for the Transfusion Medicine. Correct handling and utilization of blood stem cells for clinical and laboratory purposes (e.g. positive selection) must be guaranteed, since each restriction of the function of processed blood stem cells may lead to an insufficient engraftment after PBSCT. Therefore, the clinical divisions of the University Hospital Münster are planning and practising peripheral blood stem cell transplantations in cooperation with the Department of Transfusion Medicine. The collection, processing and quality control are performed by the Department of Transfusion Medicine in close contact with the other clinical departments, who subsequently perform high dose chemotherapy and peripheral blood stem cell transplantations.

The influence of different erythrocyte lysing procedures on flow cytometric determination of CD34+ cells in umbilical cord blood transplants.

Since the correct determination of CD34+ cells is of great clinical importance for successful transplantation with haematopoietic progenitor cells (HPCs) from cord blood, we investigated the influence of different erythrocyte lysing techniques on the quantification of CD34+ cells in umbilical cord blood. Flow cytometric determinations of CD34+ cells were performed from 20 cord blood samples, using three different erythrocyte lysing procedures and two monoclonal CD34 antibodies (n = 360). Flow cytometric analysis showed characteristic patterns of the forward (FSC) and side (SSC) scatter light properties for the leucocyte subsets for each of the investigated erythrocyte lysing procedures, indicating that these reagents cause different morphological changes on leucocytes. Furthermore, significant differences of CD34+ cell counts were obtained for identical samples using different lysing techniques (P = 0.001 and P = 0.002). In some cases, a more than 100% difference was found comparing different erythrocyte lysing procedures. In contrast, the determination of CD34+ cells by two CD34 antibodies showed a good reproducibility without significant differences between both antibodies for each of the erythrocyte lysing techniques. We conclude that the erythrocyte lysing procedure represents a very critical and important step for accurate determination of CD34+ cells in whole blood samples. Especially for the quantification of HPCs in cord blood transplants, this influence may be of high clinical relevance.

Efficacy and kinetics of bone marrow processing and enrichment of haematopoietic progenitor cells (HPC) by a large-volume apheresis procedure.

We investigated the efficacy of bone marrow (BM) processing by an automated large-volume apheresis procedure (6 x original BM volume) in 10 paediatric and adult patients undergoing BM harvesting before myeloablative therapy. Volume-dependent kinetics during apheresis were analyzed by sequential collection of processed cells into a six-fold collection bag system with consecutive analysis of the single bags. BM processing resulted in an 83.3% (+/- 21) recovery of mononuclear cells (MNC), a 97.9% (+/- 1.1) reduction of erythrocytes (RBC) and a 87.7% (+/- 2.9) volume reduction. To determine volume-dependent kinetics of haematopoietic progenitor cell (HPC) enrichment during apheresis, leukocytes (WBC), mononuclear cells (MNC), CD34 cells and colony-forming cells (CFU-GM) were serially quantitated in subsequent collection bags. Large-volume BM processing significantly enhanced absolute yields of CD34+ cells (mean: 4.01 (+/- 2.81) x 10(6)/kg bw) and CFU-GM (mean: 1.92 (+/- 1.47) x 10(4)/kg bw) compared with the standard procedure (3 x BM volume) by 26.9% (+/- 10.9) and 27.2% (+/- 11.6), respectively. We concluded that large-volume apheresis for BM processing is an efficient technique significantly improving the yields of haematopoietic progenitor cells (HPC) without any relevant changes in the purity of the final product. Moreover, sequential collection and analysis of HPC represents a good model to investigate the volume-dependent kinetics and efficacy of BM processing.

Improved automated bone marrow processing and enrichment of CD34+ cells by a large-volume apheresis procedure.

We investigated the efficacy of bone marrow (bm) processing by a large-volume apheresis procedure using a self constructed sixfold collection bag system for sequential cell collection and analysis for 5 pediatric patients. Quantitation of leukocytes (WBC), CD34+ cells and colony-forming cells (CFU-GM) within the single bags showed a relative time-dependent decrease of all cell fractions during leukapheresis, whereas the relative amount of mononuclear cells (MNC) droped only slightly. At the same time the large volume apheresis (6 x original bm-volume) clearly enhanced the absolute yield of CD34+ cells compared to the standard procedure (3 x bm-volume) for more than 20%. We conclude that large-volume apheresis for bm processing is an efficient technique to improve the yields of progenitor cells.

Carotenoids located in human lymphocyte subpopulations and natural killer cells by Raman microspectroscopy.

The presence and subcellular location of carotenoids in human lymphocyte subpopulations (CD4+, CD8+, T-cell receptor-gamma delta+, and CD19+) and natural killer cells (CD16+) were studied by means of Raman microspectroscopy. In CD4+ lymphocytes a high concentration (approximately 10(-3) M) of carotenoids was found in the Gall body. In CD8+ lymphocytes, T-cell-receptor-gamma delta+ lymphocytes and in natural killer cells carotenoids appeared to be concentrated (approximately 10(-4) M) in the Golgi complex. The concentration of carotenoids in CD19+ lymphocytes was found to be below the present detection limit, which is approximately 10(-6) to 10(-5) M. The results provide new possibilities to investigate the mechanism(s) behind the suggested protective role of carotenoids against the development of cancers.