Evaluation of West-Austrian junior athletes' knowledge regarding doping in sports.

BACKGROUND: An important factor while developing efficient doping prevention strategies is to identify relevant target groups, to evaluate the state of knowledge about this topic as well as to evaluate motivations behind using prohibited substances. Measures to prevent doping substances abuse have to be supported in early stages of childhood. PURPOSE: The aim of this prospective study was to evaluate the knowledge of Tyrolean junior athletes about doping in sport. Next to the knowledge, their attitudes in regard to doping practices have also been a focus of this project. METHODS: Within a prospective cross-sectional study, Tyrolean junior athletes aged between 14 and 19 years (n = 408) were anonymously questioned by distributing questionnaires in three Tyrolean sport schools as well as two Tyrolean sport-training centers. To collect the data, an anonymous questionnaire with close-ended questions was used. Next to sociodemographic data, questions also evaluated the knowledge about prohibited substances as well as attitudes and behaviors towards doping. The concept was set up based on contents of comparable studies and publications. RESULTS: The knowledge about doping among junior athletes was moderate. The consumer behavior of the young athletes on the other hand has turned out to be satisfactory. Nevertheless, the overall knowledge especially regarding potential negative side effects of doping agents is poor. CONCLUSIONS: To incorporate an effective doping-prevention strategy, improved education, particularly in terms of side effects, is clearly needed. To achieve sustainable doping-prevention effects, focus has to be generally set on education within the frame of junior competitive sport.

Isolation and characterization of CD133+CD34+VEGFR-2+CD45- fetal endothelial cells from human term placenta.

The phenotypes and functions of endothelial cells (EC), a heterogeneous cell population, vary along the vascular tree and even in the same organ between different vessels. The placenta is an organ with abundant vessels. To enhance further knowledge concerning placenta derived EC, we develop a new method for isolation, purification and culture of these EC. Moreover, in order to investigate the peculiarity of placenta derived EC we compare their phenotypic and functional characteristics with human dermal lymphatic endothelial cells (HDLEC) and human umbilical vein endothelial cells (HUVEC). Freshly isolated placenta derived EC displayed an elongated shape with pale cytoplasm and showed the typical cobblestone pattern of EC but also a swirling pattern when confluent. FISH-analyses of the isolated EC from placentae of male fetus revealed an XY genotype strongly indicating their fetal origin. Characterisation of placenta derived fetal EC (fEC) underlined their blood vessel phenotype by the expression of vWF, Ulex europaeus lectin-1, HLA-class I molecules, CD31, CD34, CD36, CD51/61, CD54, CD62E, CD105, CD106, CD133, CD141, CD143, CD144, CD146, VEGFR-1, VEGFR-2, EN-4, PAL-E, BMA120, Tie-1, Tie-2 and α-Tubulin. In contrast to previous reports the expression of lymphatic markers, like VEGFR-3, LYVE-1, Prox-1 and Podoplanin was consistently negative. Haematopoietic surface markers like CD45 and CD14 were also always negative. Various functional tests (Dil-Ac-LDL uptake, Matrigel assay and TNF-α induced upregulation of CD62E and CD54) substantiated the endothelial nature of propagated fEC. At the ultrastructural level, fEC harboured numerous microvilli, micropinocytic vesicles at their basis, were rich in intermediate filaments and possessed typical Weibel - Palade bodies. In conclusion, the placenta is a plentiful source of fetal, microvascular, blood EC with an expression profile (CD34+, CD133+, VEGFR-2+, CD45-) suggestive of an endothelial progenitor phenotype.

Endothelial cells from cord blood CD133+CD34+ progenitors share phenotypic, functional and gene expression profile similarities with lymphatics.

The existence of endothelial progenitor cells (EPC) with high cell-cycle rate in human umbilical cord blood has been recently shown and represents a challenging strategy for therapeutic neovascularization. To enhance knowledge for future cellular therapy, we compared the phenotypic, functional and gene expression differences between EPC-derived cells generated from cord blood CD34(+) cells, and lymphatic and macrovascular endothelial cells (EC) isolated from human foreskins and umbilical veins, respectively. Under appropriate culture conditions, EPC developed into fully matured EC with expression of similar endothelial markers as lymphatic and macrovascular EC, including CD31, CD36, von Willebrand factor FVIII, CD54 (ICAM-1), CD105 (endoglin), CD144 (VE-cadherin), Tie-1, Tie-2, VEGFR-1/Flt-1 and VEGFR-2/Flk-1. Few EPC-derived cells became positive for LYVE-1, indicating their origin from haematopoietic stem cells. However they lacked expression of other lymphatic cell-specific markers such as podoplanin and Prox-1. Functional tests demonstrated that the cobblestone EPC-derived cells up-regulated CD54 and CD62E expression in response to TNF-alpha, incorporated DiI-acetylated low-density liproprotein and formed cord- and tubular-like structures with capillary lumen in three-dimensional collagen culture--all characteristic features of the vascular endothelium. Structures compatible with Weibel-Palade bodies were also found by electron microscopy. Gene microarray profiling revealed that only a small percentage of genes investigated showed differential expression in EPC-derived cells and lymphatic EC. Among them were adhesion molecules, extracellular matrix proteins and cytokines. Our data point to the close lineage relationship of both types of vascular cells and support the theory of a venous origin of the lymphatic system.

Infantile hemangioma is a proliferation of LYVE-1-negative blood endothelial cells without lymphatic competence.

Infantile hemangiomas are common benign vascular tumors that exhibit a characteristic history of rapid proliferation in the first year of life and slow spontaneous involution during early childhood. The causative pathogenic event responsible for the abnormal endothelial proliferation remains elusive. The recent discovery of an immature phenotype of proliferating hemangioma endothelial cells due to the exclusive expression of the lymphatic endothelial hyaluronan receptor LYVE-1 led to the proposal that infantile hemangiomas are the result of a primary defect in endothelial cell maturation. To test this hypothesis, we looked for the expression of the lymphatic endothelial cell-specific markers LYVE-1, Prox-1, podoplanin and D2-40 in beta4 integrin-negative proliferating and beta4 integrin-positive involuting infantile hemangiomas. As beta4 integrin proved to be a suitable marker for staging infantile hemangiomas, we used it in combination with clinical and histological criteria to objectively determine the proliferative and involutional phases. In immunohistochemical and immunofluorescent stains, hemangioma vessels were negative for all lymphatic endothelial cell-specific markers tested during both proliferation and involution. LYVE-1 immunoreactivity, however, was found in the dense network of perivascular HLA-DR-positive cells with dendritic cell morphology that are supposed to play a role in hemangiogenesis by releasing pro- and antiangiogenic factors. Notably, this LYVE-1 staining failed to correlate with the growth status of infantile hemangiomas. Our results do not support the notion that LYVE-1 expression was restricted to the proliferative phase and downregulated during involution. Thus, LYVE-1 does not seem to be a reliable marker for proliferating infantile hemangiomas. We conclude that the suggested intrinsic defect in endothelial cell maturation is unlikely the cause for the post-natal rapid growth in infantile hemangiomas. In addition, the lack of lymphatic endothelial cell-specific markers implies that infantile hemangiomas are tumors of blood vessels without lymphatic competence.

Dendritic cells regulate T-cell deattachment through the integrin-interacting protein CYTIP.

When T cells are primed by dendritic cells (DCs) to initiate antigen-specific immune responses screening for matching antigen receptor-MHC/peptide pairs takes place in DC-T-cell conjugates. For an immune response DC-T-cell conjugates formed during priming events need to dissolve. Although detailed knowledge on molecules involved in the conjugate formation is available, dissolving of them has not been considered to be an active process. Here, we identify CYTIP (cytohesin-interacting protein) to mediate DC-T-cell deattachment. CYTIP, which is induced during maturation of DCs, shortly accumulates to the contact zones with T cells within the first hour of coculture. Specific silencing of CYTIP results in stronger adhesion of DCs to T cells and to fibronectin. When a need for deattachment is created in a T-cell priming assay by only partially loading DCs with antigen, CYTIP silencing causes reduced priming capacity. Thus, CYTIP allows DCs to actively control DC-T-cell interactions.

Infantile hemangioma is a proliferation of beta 4-negative endothelial cells adjacent to HLA-DR-positive cells with dendritic cell morphology.

Although hemangioma is referred as to the most common tumor in infancy, the underlying pathogenetic events and the biologic origin of this benign vascular neoplasm have remained obscure. By using immunohistochemistry on frozen sections of infantile hemangiomas, we show here that proliferating endothelial cells abundantly expressed alpha(v)beta(3) but lacked beta(4) integrins. Instead, regressing and involuting infantile hemangiomas due to treatment with IFN-alpha showed positive staining of beta(4) integrin, which might point to the angiogenic significance of beta(4) integrin in infantile hemangiomas. Moreover, immunofluorescence analysis revealed the existence of HLA-DR(+), mostly CD68(+) and partly DC-SIGN/CD209(+) cells with dendritic cell morphology in the intimate vicinity of hemangiomatous vessels. Such cells were also detected in the dermal microvascular unit in normal skin. The coupled occurrence of vascular structures and perivascular cells that were stained positive with markers of monocyte or macrophage or dendritic cells might suggest that the development of infantile hemangioma is a result of vasculogenesis, that is, the formation of primitive blood vessels from angioblasts, rather than of angiogenesis, that is, the sprouting of capillaries from preexisting vessels.

Adhesive interactions between CD34(+)-derived dendritic cell precursors and dermal microvascular endothelial cells studied by scanning electron microscopy.

Dendritic cells are migratory cells. Before they extravasate from the circulation into the skin across capillary blood vessel walls, they have to interact with endothelial cells. Using a fluorimetric adhesion assay, we have recently shown that CD34(+)-derived dendritic cell precursors are able to bind to resting and stimulated dermal microvascular endothelial cells. In the present study, we attempted to visualize this process at an ultrastructural level. CD34(+) progenitor cells were purified from human cord blood samples by means of immunomagnetic beads, and dendritic cells were generated by culture in the presence of GM-CSF, TNF-alpha and hSCF for 5 days. Immature CD83(-) CD86(low) dendritic cells were added to human dermal microvascular endothelial cells grown to confluence on membrane chambers. After 2 h, unbound dendritic cell precursors were removed, and bound cells were prepared for routine scanning electron microscopy. We found that (1). dendritic cell precursors firmly adhere to microvascular endothelial cells, enveloping them with their surface processes; (2). dendritic cell precursors are extremely deformable as they squeeze through the dense network of microvascular endothelial cells; (3). microvascular endothelial cells form, in part, a multi-layered network rather than the typical cobblestone pattern as seen by phase-contrast microscopy. The morphology of dendritic cell precursors and of human dermal microvascular endothelial cells was examined here, for the first time, by scanning electron microscopy. These data further emphasize that CD34(+)-derived dendritic cells efficiently adhere to dermal microvascular endothelial cells.

A novel role for IL-3: human monocytes cultured in the presence of IL-3 and IL-4 differentiate into dendritic cells that produce less IL-12 and shift Th cell responses toward a Th2 cytokine pattern.

Dendritic cells (DC) derived from plasmacytoid precursors depend on IL-3 for survival and proliferation in culture, and they induce preferentially Th2 responses. Monocytes express not only GM-CSF receptors, but also IL-3Rs. Therefore, we examined whether IL-3 had an effect on the functional plasticity of human monocyte-derived DC generated in a cell culture system that is widely used in immunotherapy. DC were generated with IL-3 (instead of GM-CSF) and IL-4. Yields, maturation, phenotype (surface markers and Toll-like receptors), morphology, and immunostimulatory capacity were similar. Only CD1a was differentially expressed, being absent on IL-3-treated DC. In response to CD40 ligation DC generated in the presence of IL-3 secreted significantly less IL-12 p70 and more IL-10 compared with DC grown with GM-CSF. Coculture of naive allogeneic CD4(+) T cells with DC generated in the presence of IL-3 induced T cells to produce significantly more IL-5 and IL-4 and less IFN-gamma compared with stimulation with DC generated with GM-CSF. These data extend the evidence that different cytokine environments during differentiation of monocyte-derived DC can modify their Th cell-inducing properties. A hitherto unrecognized effect of IL-3 on DC was defined, namely suppression of IL-12 secretion and a resulting shift from Th1 toward Th2.

Adhesion of dendritic cells derived from CD34+ progenitors to resting human dermal microvascular endothelial cells is down-regulated upon maturation and partially depends on CD11a-CD18, CD11b-CD18 and CD36.

DC are sentinels of the immune system. In order to reach the skin, bone-marrow-derived DC precursors need to bind and migrate through microvascular endothelial cells. Binding of DC toprimary endothelial cells of the skin has not been investigated. We therefore determined adhesion of DC at different stages of development to human dermal microvascular endothelial cells (HDMEC). DC were derived from CD34+ progenitors in cord blood. To enhance DC maturation, a defined cocktail of IL-1beta+IL-6+TNF-alpha+PGE2 was applied. Adhesion was quantified by fluorimetric and phase-contrast microscopical assays. Significantly more DC precursors (tested on day 5 after isolation) than mature DC (spontaneously matured or cytokine-cocktail-matured and tested on day 13) bound to unstimulated HDMEC. In contrast, the maturation stage of DC had no influence on their binding to human umbilical vein endothelial cells. Pretreatment of HDMEC with TNF-alpha and IFN-gamma resulted in an enhanced attachment of both DC precursors and mature DC. Mature DC lacked expression of CD31, CD36, CD45RA and CLA, and expressed lower levels of CD11a, CD11b and CD49d as compared with precursors tested on day 5. mAb against CD18, CD11a, CD11b, and CD36 markedly inhibited DC binding, whereas anti-CLA, anti-DC-SIGN, anti-CD29 and anti-CD49 mAb did not. Our data support the hypothesis of immunosurveillance with selective recruitment of blood DC precursors to resting and, more so, to inflamed skin. The data have potential relevance for anti-cancer immunotherapy strategies favoring the intracutaneous application of mature DC.