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.

High sensitivity and specificity of a new functional flow cytometry assay for clinically significant heparin-induced thrombocytopenia antibodies.

INTRODUCTION: Heparin-induced thrombocytopenia (HIT) is a life-threatening condition, in which the anticoagulant heparin, platelet factor 4 (PF4), and platelet-activating antibodies form complexes with prothrombotic properties. Laboratory tests to support clinical diagnosis are subdivided into functional, platelet activation assays, which lack standardization, or immunological assays, which have moderate specificity toward HIT. In this study, clinical performance of HITAlert, a novel in vitro diagnostic (IVD) registered platelet activation assay, was tested in a large cohort of HIT-suspected patients and compared with immunological assays. METHODS: From 346 HIT-suspected patients (single center), clinical data including 4T pretest probability results, citrated platelet-poor plasmas, and sera were collected, allowing direct comparison of clinical observations with HITAlert results. HITAlert performance was compared with PF4 IgG ELISA (246 patients, three centers) and PF4 PaGIA (298 patients, single center). RESULTS: HITAlert showed high sensitivity (88.2%) and specificity (99.1%) when compared with clinical diagnosis. Agreement of HITAlert with PF4 ELISA- and PF4 PaGIA-positive patients is low (52.7 and 23.2%, respectively), while agreement with PF4 IgG ELISA- and PF4 PaGIA-negative patients is very high (98.1 and 99.1%, respectively). CONCLUSION: HITAlert performance is excellent when compared with clinical HIT diagnosis, making it a suitable assay for rapid testing of platelet activation due to anticoagulant therapy.

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.

Identification of a novel HLA-DRB3*01 variant, HLA-DRB3*0114, containing a DRB1 sequence motif by micro-temperature gradient gel electrophoresis confirmed by sequence-based typing.

A novel human leukocyte antigen (HLA)-DRB3*01 allele carrying a HLA-DRB1-specific sequence motive in exon 2 is described.

GARP: a key receptor controlling FOXP3 in human regulatory T cells.

Recent evidence suggests that regulatory pathways might control sustained high levels of FOXP3 in regulatory CD4(+)CD25(hi) T (T(reg)) cells. Based on transcriptional profiling of ex vivo activated T(reg) and helper CD4(+)CD25(-) T (T(h)) cells we have identified GARP (glycoprotein-A repetitions predominant), LGALS3 (lectin, galactoside-binding, soluble, 3) and LGMN (legumain) as novel genes implicated in human T(reg) cell function, which are induced upon T-cell receptor stimulation. Retroviral overexpression of GARP in antigen-specific T(h) cells leads to an efficient and stable re-programming of an effector T cell towards a regulatory T cell, which involves up-regulation of FOXP3, LGALS3, LGMN and other T(reg)-associated markers. In contrast, overexpression of LGALS3 and LGMN enhance FOXP3 and GARP expression, but only partially induced a regulatory phenotype. Lentiviral down-regulation of GARP in T(reg) cells significantly impaired the suppressor function and was associated with down-regulation of FOXP3. Moreover, down-regulation of FOXP3 resulted in similar phenotypic changes and down-regulation of GARP. This provides compelling evidence for a GARP-FOXP3 positive feedback loop and provides a rational molecular basis for the known difference between natural and transforming growth factor-beta induced T(reg) cells as we show here that the latter do not up-regulate GARP. In summary, we have identified GARP as a key receptor controlling FOXP3 in T(reg) cells following T-cell activation in a positive feedback loop assisted by LGALS3 and LGMN, which represents a promising new system for the therapeutic manipulation of T cells in human disease.

Quality, stability, and safety data of packed red cells and plasma processed by gravity separation using a new fully integrated hollow-fibre filter device.

Background. We developed a completely closed system based on gravity separation without centrifugation steps for separation of whole blood. With this new system we compared quality and stability of the processed blood components (PRC and plasma) with respect to classical preparation. Furthermore the cost-effectiveness of this hollow fibre system was evaluated. Study Design and Methods. Whole blood collections of 15 regular blood donors were used for component preparation using the U shaped hollow fibre filter device. Results were compared to 15 whole blood preparations using centrifugation. The following parameters were evaluated: total hemoglobin, leukocyte counts, the serum concentration of total protein, lactate dehydrogenase (LDH) and potassium. Furthermore ATIII, vWF and F VIII were analyzed at different timepoints. Results. packed red cells: the data directly after separation and after 42 days of storage are in line with the guidelines of the council of Europe. Plasma. all plasma quality data are in line with the guidelines of the council of Europe for quality assurance of plasma, except for a low protein amount (factor 0.75). Conclusion. Separation of whole blood on a clinical scale in this new closed system is feasible, however the plasma protein content must be optimized.

[Legal aspects of preparation and handling of concentrated red cells derived from placental blood in Germany]. / Juristische Aspekte bei der Herstellung und Verwendung von Plazentablut-Erythrozytenkonzentraten in Deutschland.

Legal aspects of collection, preparation and storage of autologous red cells derived from cord blood according to German law are presented and discussed.

Cholecystokinin- and cholecystokinin-B-receptor gene polymorphisms in panic disorder.

Panic disorder like other neuropsychiatric disorders is believed to be caused by multiple psychosocial and biological factors. Several lines of evidence point to a role for the peptide neurotransmitter cholecystokinin in the pathogenesis of panic disorder. We therefore determined the allele and genotype frequencies of a single nucleotide polymorphism in the CCK gene (-36C>T) and one CT repeat polymorphism in the CCK-B-receptor gene in a German panic disorder sample (n = 115 for CCK gene polymorphism, n = 111 for CCK-B-receptor polymorphism) and compared them with gender and age matched controls. The length of the polymorphic CT repeat alleles varies between 146 bp and 180 bp. We first analysed the results by a permutation test which provided evidence for heterogeneity between patients and controls (p=0.002). We then analysed the data as a di-allelic polymorphism with a short (146-162bp) and a long (164-180bp) allele and as a tetra-allelic polymorphism with 4 alleles (146-154bp, 156-162bp, 164-170bp, 172-180bp). In the di-allelic analysis as well as in the tetra-allelic analysis there was an excess of the longer allele (p = 0.001) or the two longer alleles (p = 0.041) respectively in patients with panic disorder. No difference between groups was observed for the -36C > T polymorphism. Our findings are consistent with the notion that genetic variation in the CCK neurotransmitter system contributes to the pathogenesis of panic disorder.

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.

Human nuclear transcription factor gene CREM: genomic organization, mutation screening, and association analysis in panic disorder.

Panic disorder is an anxiety disorder with an estimated heritability of 48%. Variation in the gene of the nuclear transcription factor "cAMP-responsive element modulator" (CREM) might contribute to its pathogenesis. CREM knock-out mice exhibit significantly less anxiety behavior than wild-type mice and the alternative CREM gene product "inducible cAMP early repressor" (ICER) plays a pivotal role in the hypothalamo-pituitary-adrenal (HPA) axis, which is disturbed in panic disorder. We characterized the genomic organization of the human CREM gene and performed a systematic mutation screening by means of single stranded conformational analysis (SSCA) in a sample of 40 German patients with panic disorder (DSM-III-R). Four novel single nucleotide polymorphisms in CREM promoters P 1 and P 4, one trinucleotide (ATT)-repeat polymorphism in CREM promoter P 2-generating the ICER isoform-and a rare amino acid substitution in CREM exon glut 2 were identified. Association analysis in an extended sample of German patients (n = 88) revealed a significant excess of the shorter CREM P 2 promoter eight-repeat trinucleotide allele and of genotypes containing the eight-repeat trinucleotide allele in panic disorder (P = 0.02), in particular in panic disorder without agoraphobia (P = 0.001). A replication study in independent Italian (n = 76) and Spanish (n = 62) samples, however, failed to confirm this observation. This suggests that the CREM P 2 promoter trinucleotide polymorphism is not a major susceptibility factor in the pathogenesis of panic disorder. Functional analysis of the observed CREM P 2 promoter polymorphism as well as studies in independent panic disorder samples are necessary.

Polymorphisms in the non-coding region of the human mitochondrial genome in unrelated plateletapheresis donors.

Human mitochondrial DNA polymorphisms are unique targets to discriminate nucleated cells and platelets between donor and recipient in the setting of transplantation or transfusion. We have previously used this approach to discriminate allogeneic platelets from autologous platelets after transfusion. In the present study, we used DNA sequencing to investigate polymorphisms present in two of the hypervariable segments (HVR1 and HVR2) found within the non-coding region of the mitochondrial genome among 100 plateletapheresis donors. Alignments were made with the Cambridge Reference Sequence (CRS) for human mitochondrial DNA (mtDNA). Combining the sequencing information of HVR1 and HVR2 we could demonstrate that, of the 100 investigated mtDNA samples, none was identical to the CRS. We found a total of 2-17 polymorphisms per donor in the investigated regions, most of them were basepair substitutions (563) and insertions (151). No deletions were found. Sixty-six of the 110 detected polymorphisms were detected in more than one sample. Seven polymorphisms are newly described and have not been published in the Mitomap database. Our results demonstrate that polymerase chain reaction analysis of the many polymorphisms found in the hypervariable region of mitochondrial DNA represents a more informative target than previously described mitochondrial polymorphisms for discriminating donor-recipient cells after transfusion or transplantation.

Identifying allogeneic platelets by resolution of point mutations in mitochondrial DNA using single-stranded conformational polymorphism PCR.

BACKGROUND: The purpose of this study was to evaluate single-stranded conformational polymorphism (SSCP)-PCR utilizing two different regions of mitochondrial DNA (mtDNA) as a method to discriminate between donor platelets and recipient cells. STUDY DESIGN AND METHODS: Twenty-eight mixtures of platelets (1:1 ratio) were prepared from eight randomly selected persons to simulate donor-recipient combinations after allogeneic platelet transfusion. The mtDNA was extracted from each donor and each prepared mixture. Four primer pairs were designed to amplify two regions of mtDNA, hypervariable region (HVR) 1 and 2. An SSCP-PCR method was developed to analyze the four different amplicons. In addition, the amplified DNA samples containing HVR1 and HVR2 mtDNA of the eight persons were sequenced by using dye-terminator cycle sequencing to determine mtDNA polymorphisms. RESULTS: With four different primer pairs and SSCP-PCR, it was possible to discriminate between donor and recipient DNA in all 28 combinations. DNA sequencing confirmed that the suspected differences were localized within the amplicons examined by SSCP-PCR. CONCLUSION: SSCP-PCR analysis targeting the HVR1 and HVR2 mtDNA is a promising new method to potentially identify donor cells on the basis of mtDNA polymorphisms. The method does not require prior knowledge of sequence differences between donor and recipient and can be optimized to quantify the amount of residual transfused allogeneic platelets.

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.

Enrichment of fetal nucleated red blood cells from the maternal circulation for prenatal diagnosis: experiences with triple density gradient and MACS based on more than 600 cases.

OBJECTIVE: We wanted to obtain statistically relevant data about the efficiency of our method for the isolation of fetal nucleated red blood cells (NRBCs) from the maternal circulation. METHODS: More than 600 samples were investigated using a triple density gradient followed by magnetic separation of anti-CD71-labeled cells, and yields and purities of recovered NRBCs were determined. RESULTS: The enrichment effectivity as well as the morphological condition of cells was reproducibly good, if blood samples were enriched within 48 h after sampling. The efficacy was independent of various methodological parameters and our technique was superior to other magnetic cell-sorting techniques. Mean yields and purities of NRBCs increased with increasing gestational age, ranging from 100 to 1,000 cells per 40-ml blood sample and from 0.1 to 1%, respectively, from the 6th week of gestation to term. In pregnancies with preeclampsia NRBCs were increased by a factor of 10. CONCLUSION: Our enrichment technique proved to be optimized with respect to various methodological parameters, which were compared in the present study, and it is efficient and reproducible for the enrichment of NRBCs from the maternal circulation in all three gestational trimesters.

[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.

Suppression of panel-reactive antibodies by treatment with mycophenolate mofetil.

"Panel Reactive Antibody" (PRA) testing is commonly used to assess the pretransplant antibody status in order to estimate the risk of an adverse humoral response following transplantation. We report on a female patient with end-stage cardiac failure suffering from acute myocarditis who underwent implantation of a left-ventricular assist device (Novacor, Baxter Healthcare Corp. Oakland, CA). During evaluation for heart transplantation, a PRA level of 50-70% was detected. After treatment with mycophenolate mofetil at a dosage of 2 g daily, PRA levels declined within one week to 0-5%, and remained low after discontinuation of the immunosuppressive drug. We feel that pretreatment of patients with elevated PRA levels with mycophenolate mofetil is well justified.