A simple and sensitive flow cytometric assay for the determination of the cytotoxic activity of human natural killer cells

A new, simple and sensitive flow cytometric assay for the determination of the cytotoxic activity of human natural killer cells is described. The assay is based on the use of two fluorochromes. The target cell population is stained with one fluorochrome (octadecylamine-fluorescein isothiocyanate, F-18) prior to incubation with the effector cells. F-18 remains in the membrane of the target cells even when they are killed thereby permitting a clear separation between effector and target cells. Dead cells are determined by staining with a second fluorochrome (propidium iodide) after incubation of effector and target cells.\ud F-18 is not toxic and does not decrease the cytotoxic activity of human natural killer cells. It is also stable (exchange between labeled and non-labeled cells is negligible in a period of at least 4 h at 37°C) and it remains in the membrane of the killed cells. A clear distinction between unlabeled effector and labeled target cells is obtained, even after incubation of target and effector cells for 4 h at 37°C and using a high effector cell-target cell ratio (75:1). A good correlation with the 51Cr release assay was obtained.\ud A potential application of the flow cytometric cytotoxicity assay using whole blood instead of isolated lymphocytes is presented.\ud \u

A rapid and accurate approach to identify single nucleotide polymorphisms of mitochondrial DNA using MALDI-TOF mass spectrometry

Background: Single nucleotide polymorphisms (SNPs) of mitochondrial DNA (mtDNA) are involved in physiological and pathological conditions. We developed a rapid, accurate, highly sensitive and high-throughput approach with low cost to identify mtDNA SNPs. Methods: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to detect 18 SNPs of mtDNA by uniplex and multiplex assays. The sensitivity and specificity of the MALDI-TOF MS were evaluated. The accuracy of the approach was validated by the comparison of using the robust sequencing analysis. Results: The detection limit achieved with the assays corresponded to the identification of five-genome equivalence of mtDNA per reaction after first round PCR amplification. The testing system enabled the discrimination of as little as 5% of mtDNA polymorphism in the predominating background of mtDNA not containing the SNP. No false positive and false negative results were obtained using the uniplex and multiplex MALDI-TOF MS assays for the analysis of the 18 SNPs compared with those obtained by sequencing analysis. Conclusions: Possible fields which could benefit from this powerful and sensitive tool include forensic medicine, tracing of matrilineage, transplantation immunology, transfusion medicine, the diagnosis of mtDNA mutation related disorders, and the research regarding aging, apoptosis and carcinogenesis based on physiologic and pathogenic alterations of mtDNA for the analysis of large-scale samples, multiple SNPs or rare mtDNA. Clin Chem Lab Med 2008;46:299-30

Smoke-free policies at sports clubs and other venues:Adoption, implementation, and impact

Dit proefschrift onderzocht de adoptie, implementatie en impact van rookvrij beleid. Deel I onderzocht de impact op rookgedrag in verschillende omgevingen. De resultaten laten zien dat zowel in horecagelegenheden, op ziekenhuisterreinen en in appartementencomplexen een rookvrij beleid bijdraagt aan een vermindering van roken en blootstelling aan meeroken. Deel II onderzocht de adoptie en implementatie van een rookvrij beleid op sportverenigingen. De resultaten laten zien dat jongeren voornamelijk negatieve attitudes hebben tegenover roken in combinatie met sport. Ook steunen zij over het algemeen een rookvrij beleid. Stakeholders op sportverenigingen waren positief, maar zagen ook uitdagingen. Besluitvorming bij sportverenigingen werd beïnvloed door bestuur, clubleden en rokers. Implementatie bleek haalbaar maar kende uitdagingen, vooral in kritische situaties. De verspreiding van rookvrij beleid op sportverenigingen in Nederland neemt toe, vooral bij hockey- en korfbalverenigingen, verenigingen met veel (jeugd-)leden en in stedelijke gebieden. Gemeentelijk tabaksontmoedigingsbeleid kan het aantal rookvrije sportverenigingen doen toenemen. Tot slot pleiten we voor een bredere invoering van rookvrij beleid op sportverenigingen in Europa, waarbij vijf toekomstige uitdagingen zijn geïdentificeerd

Smoke-free policies at sports clubs and other venues:Adoption, implementation, and impact

Dit proefschrift onderzocht de adoptie, implementatie en impact van rookvrij beleid. Deel I onderzocht de impact op rookgedrag in verschillende omgevingen. De resultaten laten zien dat zowel in horecagelegenheden, op ziekenhuisterreinen en in appartementencomplexen een rookvrij beleid bijdraagt aan een vermindering van roken en blootstelling aan meeroken. Deel II onderzocht de adoptie en implementatie van een rookvrij beleid op sportverenigingen. De resultaten laten zien dat jongeren voornamelijk negatieve attitudes hebben tegenover roken in combinatie met sport. Ook steunen zij over het algemeen een rookvrij beleid. Stakeholders op sportverenigingen waren positief, maar zagen ook uitdagingen. Besluitvorming bij sportverenigingen werd beïnvloed door bestuur, clubleden en rokers. Implementatie bleek haalbaar maar kende uitdagingen, vooral in kritische situaties. De verspreiding van rookvrij beleid op sportverenigingen in Nederland neemt toe, vooral bij hockey- en korfbalverenigingen, verenigingen met veel (jeugd-)leden en in stedelijke gebieden. Gemeentelijk tabaksontmoedigingsbeleid kan het aantal rookvrije sportverenigingen doen toenemen. Tot slot pleiten we voor een bredere invoering van rookvrij beleid op sportverenigingen in Europa, waarbij vijf toekomstige uitdagingen zijn geïdentificeerd

Thiopurine metabolite levels in patients with atopic dermatitis and/or chronic hand/foot eczema treated with azathioprine

Background: Azathioprine is frequently used in severe eczema. It is converted in the liver into active metabolites, including 6-thioguanine nucleotide (6-TGN) and methylated 6-methylmercaptopurine (6-MMP). In the past, the therapeutic potential of azathioprine may have not been fully utilized. Recent investigations on inflammatory bowel disease have led to a better understanding of azathioprine metabolism and optimizing treatment. Objective: To investigate whether measuring thiopurine metabolites in circulation can improve the effectiveness and safety of azathioprine treatment in patients with atopic dermatitis and/or chronic hand/foot eczema. Methods: Azathioprine metabolite levels were measured in eczema patients during maintenance treatment (Part I) and dose escalation (Part II). Clinical effectiveness, hepatotoxicity, and bone marrow suppression were analyzed and TPMT genotype was assessed. Results: A wide variation in metabolite levels in all dose groups was observed. In Part I (32 patients), there were no significant differences in 6-TGN levels between clinical responders and non-responders (p = .806). No hepatoxicity or myelotoxicity was observed. In Part II, all 6-TGN and 6-MMP levels increased during dose escalation. Hypermethylation was observed in 2/8 patients. Conclusion: For individual eczema patients treated with azathioprine, routinely measuring 6-TGN and 6-MMP can be helpful in optimizing azathioprine dose, improving clinical effectiveness, and preventing side effects

Healthcare Governance Design in Blockchain

As a technology, Blockchain will make an important contribution to organizing differently within profit, non-profit organizations and society. The aim of this reorganization is to reduce the pressure of regulation and work by digitally regulating the trust that is now often given by third parties. In addition, Blockchain goes over and through various processes, as a result of which third parties are no longer directly necessary due to the different organization of trust. What does organizing differently now mean for the use of coordination as a mechanism to steer organizations? This article, based on a thorough literature study will give an answer to this question. We looked at how coordination problems can be minimized by organizing it differently. Organizing differently here means no longer working from a central network but collaborating within a decentralized network with Blockchain as the technology. The interaction between the type of organization, the organizational policy, the processes, applications and infrastructure is essential in this respect, in which interoperability plays an important role. Blockchain ultimately contributes to reducing the response time of organizations, which in turn increases their adaptive capacity and therefore makes it possible to respond more quickly to changing market conditions, which are currently also occurring in the healthcare sector

Willingness to pay for shared mobility hubs: a stated choice joint-survey in four European cities

This paper aims to assess the relative importance of different shared mobility hub design elements. A standardised survey was developed and applied among users and residents in different areas, including a stated preference experiment, to examine trade-offs between integration dimensions and the willingness-to-pay for different attributes of hubs. The results underscore a disparity between the attributes most valued by respondents and their willingness-to-pay for them, which holds significance in the planning and functioning of mobility hubs. Although elements such as information and digital integration were indicated as being prerequisites for a successful shared mobility hub, respondents are not willing to pay for it, indicating that these would have to be arranged by the government and/or transport operators. Respondents are more willing to pay for shared mobility and public transport within walking distance from one another or for placemaking strategies (such as services or landscaping), which are more evident elements related to the physical integration (and design) of hubs