Assisted Reproductive Technologies in Europe. Usage and Regulation in the Context of Cross-Boarder Reproductive Care

This chapter reviews assisted reproductive technologies (ART) usage and policies across European countries, and scrutinizes emerging issues related to cross-border reproductive care (or “reproductive tourism”). Although Europe is currently the largest market for ART, the extent of usage varies widely across countries, largely because of differences in the laws, the affordability, the types of reimbursement, and the norms surrounding childbearing and conception. Since 2009, the regulation of ART has been expanding in Europe, and all countries now have some form of ART legislation. Countries where the treatments are completely covered by national health plans have the highest level of ART utilization. Being in a legal marriage or a stable union is often a prerequisite for access to ART. Currently, only half of European countries allow single women to use ART, and even fewer grant access to lesbian women. Surrogate motherhood is strictly prohibited in many countries in Europe, and where it is allowed, strong restrictions against commercial surrogacy are in place. While restrictive national legislation can be easily circumvented by crossing national boundaries for ART treatments, questions of equity of access have been raised, as not all prospective parents can afford to travel for treatment

Wycena zamówień publicznych na roboty budowlane w Polsce i Republice Czeskiej

Estimating the value of public construction works in Poland and the Czech Republic. The article outlines the legislation concerning the methodology of estimating the value of works in Poland and the Czech Republic. In both countries it is necessary for the public investor to respect the law governing public procurement, which defines the structure of compulsory documents needed for the tender documentation, but not directly the way of their preparation. In both countries, though, there exist model proceeding schedules for the calculation of the value of a public procurement for construction works. To illustrate and compare the calculation methods a sample calculation of the procurement value is presented for a selected thermal efficiency improvement project.Wycena zamówień publicznych na roboty budowlane w Polsce i Republice Czeskiej. Roboty budowlane są realizowane w dwóch podstawowych systemach. Pierwszy to system tradycyjny, gdzie zamawiający rozdziela projektowanie od wykonania (tzw. system Design-Bid-Build). Drugi system to Zaprojektuj i Buduj (Design & Build), gdzie wykonawca zarówno projektuje, jak i zgodnie z własnym projektem realizuje roboty budowlane. W obydwu przypadkach zamawiający publiczny jest zobligowany do wyznaczenia wartości zamówienia zgodnie z obowiązującymi przepisami prawnymi. W artykule przedstawiono porównanie przepisów prawnych dotyczących metodologii szacowania wartości zamówienia na roboty budowlane w Polsce i Czechach. Szczególną uwagę zwrócono na metody stosowane w przypadku zamówień realizowanych w systemie tradycyjnym. Zaprezentowany przykład obliczeniowy pozwala na określenie podobieństw i różnic w sposobach kalkulacji w obydwu krajach

Phylogenetic relationships of rock-inhabiting black fungi belonging to the widespread genera Lichenothelia and Saxomyces

Rock-inhabiting fungi (RIF) are adapted to thrive in oligotrophic environments and to survive under conditions of abiotic stress. Under these circumstances, they form biocoenoses with other tolerant organisms, such as lichens, or with less specific phototrophic consortia of aerial algae or cyanobacteria. RIF are phylogenetically diverse, and their plastic morphological characters hamper the straightforward species delimitation of many taxa. Here, we present a phylogenetic study of two RIF genera, Lichenothelia and Saxomyces. Representatives of both genera inhabit rather similar niches on rocks, but their phylogenetic relationships are unknown so far. The cosmopolitan genus Lichenothelia is recognized by characters of fertile ascomata and includes species with different life strategies. In contrast, Saxomyces species were described exclusively by mycelial characters found in cultured isolates from rock samples collected at high alpine elevations. Here, we use an extended taxon sampling of Dothideomycetes to study the phylogenetic relationships of both Lichenothelia and Saxomyces. We consider environmental samples, type species, and cultured isolates of both genera and demonstrate their paraphyly, as well as the occurrence of teleomorphs in Saxomyces. We applied three species delimitation methods to improve species recognition based on molecular data. We show the distinctiveness of the two main lineages of Lichenothelia (Lichenotheliales s. str.) and Saxomyces and discuss differences in species delimitation depending on molecular markers or methods. We revise the taxonomy of the two genera and describe three new taxa, Lichenothelia papilliformis, L. muriformis, and Saxomyces americanus, and the teleomorph of S. penninicus

<i>Bordetella</i> Adenylate Cyclase Toxin Differentially Modulates Toll-Like Receptor-Stimulated Activation, Migration and T Cell Stimulatory Capacity of Dendritic Cells

<div><p>Adenylate cyclase toxin (CyaA) is a key virulence factor of the whooping cough agent <i>Bordetella pertussis</i>. The toxin targets CD11b-expressing phagocytes and delivers into their cytosol an adenylyl cyclase (AC) enzyme that subverts cellular signaling by increasing cAMP levels. In the present study, we analyzed the modulatory effects of CyaA on adhesive, migratory and antigen presenting properties of Toll-like receptor (TLR)-activated murine and human dendritic cells (DCs). cAMP signaling of CyaA enhanced TLR-induced dissolution of cell adhesive contacts and migration of DCs towards the lymph node-homing chemokines CCL19 and CCL21 <i>in vitro</i>. Moreover, we examined in detail the capacity of toxin-treated DCs to induce CD4⁺ and CD8⁺ T cell responses. Exposure to CyaA decreased the capacity of LPS-stimulated DCs to present soluble protein antigen to CD4⁺ T cells independently of modulation of co-stimulatory molecules and cytokine production, and enhanced their capacity to promote CD4⁺CD25⁺Foxp3⁺ T regulatory cells <i>in vitro</i>. In addition, CyaA decreased the capacity of LPS-stimulated DCs to induce CD8⁺ T cell proliferation and limited the induction of IFN-γ producing CD8⁺ T cells while enhancing IL-10 and IL-17-production. These results indicate that through activation of cAMP signaling, the CyaA may be mobilizing DCs impaired in T cell stimulatory capacity and arrival of such DCs into draining lymph nodes may than contribute to delay and subversion of host immune responses during <i>B. pertussis</i> infection.</p></div

CyaA decreases the capacity of TLR-stimulated DCs to present soluble antigen to CD4⁺ T cells.

<p>BMDCs were left untreated, incubated with LPS (100 ng/ml) alone or in combination with CyaA or CyaA-AC⁻ at 10 ng/ml in the presence of OVA protein at 2.5 µg/ml or OVA_323–339 peptide (5 µg/ml) for 4 h prior to washing and co-cultivation with naïve CFSE-labeled OT-II CD4⁺ T cells. T cell proliferation was determined by flow cytometry after 72 h as a dilution of CFSE. (A) Histograms are representative of n = 4. (B) Quantitative analysis of A where the percentage of undivided LPS-treated cells (medium) was set to 100% (* <i>p</i><0.05). (C) Expansion of adoptively transferred CFSE-labeled CD4⁺ T cells <i>in vivo</i> was determined after 72 h by flow cytometry as a fold of expansion of 2×10⁶ counted spleen cells where 1 represents the non-stimulated adoptively transferred CD4⁺ T cells (control). Dot plots are representative of n = 3. (D, E) CyaA inhibits macropinocytosis but not receptor-mediated endocytosis and antigen (Ag) degradation in LPS-treated DCs. DCs were left untreated, incubated with LPS alone or in combinantion with 10 ng/ml of toxins or chloroquine (100 µM) for 30 min. (D) Lucifer Yellow (500 µg/ml), transferrin-Alexa647 or OVA-Alexa647 (both 5 µg/ml) were subsequently added for 30 min. The Ag uptake in living CD11c⁺ cells was determined by flow cytometry. (E) A mixture of OVA-Alexa647 (5 µg/ml, marker for Ag uptake) and OVA-DQ (5 µg/ml, marker for Ag uptake and degradation) were added for 30 min. The processed OVA-DQ was determined from gated CD11c⁺OVA-DQ⁺OVA-Alexa647⁺ DCs and calculated as a ratio of MFI OVA-DQ/OVA-Alexa647. Values represent means ± SEM of n = 5 where Ags taken up by LPS-treated DC (medium) was set to 100% of MFI (ratio 1) (* <i>p</i><0.05).</p

CyaA accelerates cell detachment and migration of TLR-activated DCs.

<p>(A) Impedance measurements using the real-time cell electronic sensing system xCelligence were used to determine MDDC adhesion and spreading. MDDCs were seeded on fibronectin-coated sensors and were left untreated (medium), or treated with LPS (1 µg/ml) alone, or in combination with CyaA or CyaA-AC⁻ at 10 ng/ml for 24 h. The representative experiment is shown (A) as well as quantitative analysis of 4 donors at time point of 12 h (B) where cell index (CI) of LPS-treated DCs at 12 h was normalized to 1.0. (C) Migration of DCs treated with toxins and LPS (for 24 h) towards CCL19 or CCL21 (both 200 ng/ml) in transwell plates was determined by flow cytometry after additional 14 h (MDDCs) or 4 h (BMDCs) of incubation at 37°C. Values represent the means ± SEM of n = 4 or 5 donors, respectively (* <i>p</i><0.05) where the number of transmigrated LPS-treated DCs (medium) was set to 1. (D) CCR7 expression on DCs was determined by flow cytometry after 24 h and 48 h. Values represent the means ± SEM of n = 3–5 or 5 donors, respectively (* <i>p</i><0.05).</p

CyaA commits TLR-stimulated DCs to expand CD4⁺CD25⁺Foxp3⁺ T regulatory cells <i>in vitro</i>.

<p>(A) BMDCs were left untreated, incubated with LPS (100 ng/ml) alone or in combination with CyaA or CyaA-AC⁻ at 10 ng/ml in the presence of OVA at 2.5 µg/ml for 4 h prior to washing and co-cultivation with naïve CFSE-labeled OT-II CD4⁺ T cells. After 72 h the number of CD4⁺CD25⁺Foxp3⁺ T cells was determined by flow cytometry. Dot plots show one representative experiment and quantitative analysis represent means ± SEM of n = 4 (* <i>p</i><0.05). (B) MDDCs were incubated with LPS (1 µg/ml) alone or in combination with CyaA or CyaA-AC⁻ at 10 ng/ml. After 24 h cells were used as stimulators of naïve allogeneic T cells at DC : T cell ratio of 1 : 10. The expansion of human CD4⁺CD25⁺Foxp3⁺ T regulatory cells was determined after 7 days. The representative experiment is shown and quantitative analysis represent means ± SEM of n = 5 (* <i>p</i><0.05).</p