Legislating Patient Representation: A Comparison Between Austrian and German Regulations on Self-Help Organizations as Patient Representatives

Governments are increasingly inviting patient organizations (POs) to participate in healthcare policymaking. By inviting POs that claim to represent patients, representation comes into being. However, little is known about the circumstances under which governments accept POs as patient representatives. Based on the analysis of relevant legislation, this article investigates the criteria that self-help organizations (SHOs), a special type of PO, must fulfil in order to be accepted as patient representatives by governments in Austria and Germany. Thereby, it aims to contribute to the discussion on the role of governments in steering SHOs. There are different degrees of regulation (very little in Austria, more in Germany). Governments in both countries not only formulate explicit criteria for SHOs with respect to patient representation but also guide SHOs representing patients through implicit criteria for associations. We discuss the findings against concepts of responsiveness, authorization, and accountability. Our findings indicate that governmental steering is not negative per se as indicated by previous research but—depending on legislative criteria—can promote transparency and democratic quality in patient representation.     

SGD: Saccharomyces Genome Database.

The Saccharomyces Genome Database (SGD) provides Internet access to the complete Saccharomyces cerevisiae genomic sequence, its genes and their products, the phenotypes of its mutants, and the literature supporting these data. The amount of information and the number of features provided by SGD have increased greatly following the release of the S.cerevisiae genomic sequence, which is currently the only complete sequence of a eukaryotic genome. SGD aids researchers by providing not only basic information, but also tools such as sequence similarity searching that lead to detailed information about features of the genome and relationships between genes. SGD presents information using a variety of user-friendly, dynamically created graphical displays illustrating physical, genetic and sequence feature maps. SGD can be accessed via the World Wide Web at http://genome-www.stanford.edu/Saccharomyces/     

The role of loop F residues in determining differential d-tubocurarine potencies in mouse and human 5-hydroxytryptamine 3A receptors

The competitive antagonist d-tubocurarine (curare) has greater potency at mouse than at human 5-hydroxytryptamine 3A (5-HT3A) receptors, despite 84% amino acid sequence identity between the receptors. Within the ligand binding domain of this receptor are six loops (A-F). A previous report demonstrated that loop C of the 5-HT3A receptor contributed to differential potency between the receptors [Hope, A. G. et al. (1999) Mol. Pharmacol. 55, 1037-1043]. The present study tested the hypothesis that loop F plays a significant role in conferring interspecies curare potency differences. Wild-type, chimeric, and point mutant 5-HT3A receptors were expressed in Xenopus oocytes, and two-electrode voltage clamp electrophysiological recordings were performed. Our data suggest that loops C and F contribute to curare potency, given that the curare IC50's (concentration of drug that produces 50% inhibition of the response) for chimeric human receptors with substitutions of mouse residues in loop C (40.07 +/- 2.52 nM) or loop F (131.8 +/- 5.95 nM) were intermediate between those for the mouse (12.99 +/- 0.77 nM) and human (1817 +/- 92.36 nM) wild-type receptors. Two human point mutant receptors containing mouse receptor substitutions in loop F (H-K195E or H-V202I) had significantly lower curare IC50's than that of the human receptor. The human double mutant receptor, H-K195E,V202I, had the same curare IC50 (133.8 +/- 6.38 nM) as that of the human receptor containing all six loop F mouse substitutions. These results demonstrate that two loop F residues make a significant contribution in determining curare potency at the 5-HT3A receptor.     

CodY of Streptococcus pneumoniae: link between nutritional gene regulation and colonization

CodY is a nutritional regulator mainly involved in amino acid metabolism. It has been extensively studied in Bacillus subtilis and Lactococcus lactis. We investigated the role of CodY in gene regulation and virulence of the human pathogen Streptococcus pneumoniae. We constructed a codY mutant and examined the effect on gene and protein expression by microarray and two-dimensional differential gel electrophoresis analysis. The pneumococcal CodY regulon was found to consist predominantly of genes involved in amino acid metabolism but also several other cellular processes, such as carbon metabolism and iron uptake. By means of electrophoretic mobility shift assays and DNA footprinting, we showed that most of the targets identified are under the direct control of CodY. By mutating DNA predicted to represent the CodY box based on the L. lactis consensus, we demonstrated that this sequence is indeed required for in vitro DNA binding to target promoters. Similar to L. lactis, DNA binding of CodY was enhanced in the presence of branched-chain amino acids, but not by GTP. We observed in experimental mouse models that codY is transcribed in the murine nasopharynx and lungs and is specifically required for colonization. This finding was underscored by the diminished ability of the codY mutant to adhere to nasopharyngeal cells in vitro. Furthermore, we found that pcpA, activated by CodY, is required for adherence to nasopharyngeal cells, suggesting a direct link between nutritional regulation and adherence. In conclusion, pneumococcal CodY predominantly regulates genes involved in amino acid metabolism and contributes to the early stages of infection, i.e., colonization of the nasopharynx.     

Activation of the Liver X Receptor Stimulates Trans-intestinal Excretion of Plasma Cholesterol

Recent studies have indicated that direct intestinal secretion of plasma cholesterol significantly contributes to fecal neutral sterol loss in mice. The physiological relevance of this novel route, which represents a part of the reverse cholesterol transport pathway, has not been directly established in vivo as yet. We have developed a method to quantify the fractional and absolute contributions of several cholesterol fluxes to total fecal neutral sterol loss in vivo in mice, by assessing the kinetics of orally and intravenously administered stable isotopically labeled cholesterol combined with an isotopic approach to assess the fate of de novo synthesized cholesterol. Our results show that trans-intestinal cholesterol excretion significantly contributes to removal of blood-derived free cholesterol in C57Bl6/J mice (33% of 231 μmol/kg/day) and that pharmacological activation of LXR with T0901317 strongly stimulates this pathway (63% of 706 μmol/kg/day). Trans-intestinal cholesterol excretion is impaired in mice lacking Abcg5 (−4%), suggesting that the cholesterol transporting Abcg5/Abcg8 heterodimer is involved in this pathway. Our data demonstrate that intestinal excretion represents a quantitatively important route for fecal removal of neutral sterols independent of biliary secretion in mice. This pathway is sensitive to pharmacological activation of the LXR system. These data support the concept that the intestine substantially contributes to reverse cholesterol transport.Reverse cholesterol transport (RCT)³ is defined as the flux of excess cholesterol from peripheral tissues toward the liver followed by biliary secretion and subsequent disposal via the feces (1). Accumulation of cholesterol in macrophages in the vessel wall is considered a primary event in the development of atherosclerosis and, therefore, removal of excess cholesterol from these cells is of crucial importance for prevention and/or treatment of atherosclerotic cardiovascular diseases. It is generally accepted that HDL is the obligate transport vehicle in RCT and that plasma HDL levels reflect the capacity to accommodate this flux. In line herewith, HDL-raising therapies are currently considered as a promising strategy for prevention and treatment of atherosclerotic cardiovascular diseases (2). In the “classical” scenario, the liver has a central role in RCT (3). Biliary secretion of free cholesterol, facilitated by the heterodimeric ABC-transporter ABCG5/ABCG8 (4), and hepatic conversion of cholesterol into bile acids followed by fecal excretion are referred to as the main routes for quantitatively important elimination of cholesterol from the body. Fecal excretion of sterols is stimulated upon whole body activation of the liver X receptor (LXR, NR1H2/3), a member of the nuclear receptor family for which oxysterols have been identified as natural ligands (5). LXR regulates expression of several genes involved in RCT and activation of LXR by synthetic agonists leads to elevated plasma HDL-cholesterol levels, increased hepatobiliary cholesterol secretion, reduced fractional intestinal cholesterol absorption and increased fecal sterol loss (6). LXR is thus considered an attractive target for therapeutic strategies aimed at stimulation of RCT, which, however, will require approaches to circumvent potential detrimental consequences of LXR activation such as induction of lipogenesis.Recent studies indicate that the classical concept of RCT may require reconsideration. Studies in apoA-I-deficient mice revealed that the magnitude of the centripetal cholesterol flux from the periphery to the liver is not related to the concentration of HDL-cholesterol or apoA-I in plasma (7). Furthermore, Abca1^−/− mice that completely lack plasma HDL show unaffected rates of hepatobiliary cholesterol secretion and fecal sterol loss (8). Additionally, mice lacking both Abcg5 and Abcg8 do not show a reduction in fecal neutral sterol excretion to the extent expected on the basis of their strongly reduced hepatobiliary cholesterol secretion (9). Recent studies by Plösch et al. (6) have revealed that increased fecal neutral sterol loss upon general LXR activation cannot be attributed to the increased hepatobiliary cholesterol secretion only, suggesting a major contribution of the intestine in excretion of cholesterol. This potential role of the intestine in cholesterol removal from the body has been corroborated by Kruit et al. (10), who showed that fecal sterol loss is not affected in Mdr2^−/− (Abcb4^−/−) mice that have a dramatic reduction in biliary cholesterol secretion (11). Moreover, intravenously administered [³H]cholesterol could be recovered in the neutral sterol fraction of the feces in these mice and fecal excretion of neutral sterols was stimulated upon treatment with an LXR agonist (10). However, the exact quantitative contribution of the direct intestinal pathway under physiological conditions has not directly been determined so far. Very recently, intestinal perfusion studies in mice revealed that, in the presence of mixed micelles as cholesterol acceptors in the intestinal lumen, murine enterocytes indeed have a high capacity to secrete cholesterol via a specific process that is most active in the proximal part of the small intestine (12). In addition, it was shown that direct trans-intestinal cholesterol excretion (TICE) could be stimulated by a high fat diet. The existence of a non-biliary route for fecal neutral sterol excretion is further supported by very recent studies by Brown et al. (13) in mice with targeted deletion of hepatic ACAT2.The present study provides insight into the relative and absolute contributions of several cholesterol fluxes relevant to total fecal sterol loss in mice, making use of a panel of stable isotope tracers. Our results show that TICE is a major route for removal of blood-derived free cholesterol and that pharmacological LXR activation strongly stimulates this arm of the reverse cholesterol transport pathway.     

Keratinocyte-derived Chemokine in Obesity: EXPRESSION, REGULATION, AND ROLE IN ADIPOSE MACROPHAGE INFILTRATION AND GLUCOSE HOMEOSTASIS*

Obese adipose tissue (AT) is associated with chronic inflammation, and we hypothesized that the keratinocyte-derived chemokine (KC), the mouse ortholog of human interleukin-8, plays a role in obesity-mediated AT inflammation and the subsequent manifestation of insulin resistance. KC expression is increased in the AT and plasma of genetically (ob/ob) and high fat diet-induced obese mouse models, and this increase may be mediated by the elevated leptin and tumor necrosis factor-α levels associated with obesity. Obesity-induced KC expression occurs primarily in stromal vascular cells and not in adipocytes, and it is high in preadipocytes and decreases during adipogenesis. Although KC has no effect on adipogenesis, it induces adipocyte expression of inflammatory factors and the insulin resistance mediator, suppressor of cytokine signaling 3. Using chimeric mice deficient in the KC receptor CXCR2 in their bone marrow, we show that the lack of CXCR2 in hematopoietic cells is sufficient to protect from adipose and skeletal muscle macrophage recruitment and development of insulin resistance in diet-induced obese mice. These studies suggest that KC and its receptor CXCR2 are potential targets for the development of new therapeutic approaches for treatment of obesity-related insulin resistance, type II diabetes, and related cardiovascular diseases.Obesity is characterized by systemic low grade inflammation that appears to contribute to the genesis of insulin resistance (IR),³ type 2 diabetes, and increased risk for cardiovascular diseases (reviewed in Ref. 1). Furthermore, adipose tissue (AT) produces a variety of inflammatory factors, and its excessive development in obesity is associated with accumulation of AT macrophages (ATMs) (1), whose recruitment and proinflammatory activation are required for the development of IR in obese mice (reviewed in Ref. 2). An important question concerning ATMs is/are the trigger(s) driving the recruitment of these cells in obesity.Efforts at identifying factors that attract and recruit ATMs have mostly focused on the CC chemokine MCP-1 (monocyte chemoattractant protein-1) and its receptor CCR2. These studies have led to contradicting results with several publications showing that MCP-1 and CCR2 are important for ATM recruitment and the subsequent development of IR (3–5), whereas others show no involvement of this chemokine and its receptor in these processes (6–8). Furthermore, the studies that claim a role for MCP-1 and CCR2 in ATM recruitment and IR show that deficiency of the ligand or the receptor did not result in normalization of ATM content, indicating that other factors also participate in ATM recruitment. These findings suggest that the precise role of the MCP-1/CCR2 axis in ATM recruitment and IR is unclear, and that other chemokines and their receptors could also play a role in these processes. One such chemokine is interleukin 8 (IL-8), the prototypical CXC chemokine known to recruit and activate monocytes and to attract polymorphonuclear leukocytes to sites of inflammation (9). IL-8 is elevated in plasma of obese subjects (10, 11) and correlates with adiposity and insulin sensitivity, suggesting an involvement of this chemokine in obesity-related health complications (12–14). Additionally, IL-8 is implicated in the pathogenesis of atherosclerosis and cardiovascular disease, two obesity-associated disorders (15). Finally, IL-8 is an angiogenic factor, and angiogenesis is a hallmark of AT expansion in obesity (16). Although these findings suggest an important role for IL-8 in AT biology and pathology, little is known regarding the mechanism of regulation of IL-8 in obesity and its role in AT biology and pathology. This is probably due, in part, to the absence of suitable animal models because mice and rats do not have a clear-cut IL-8 ortholog (17).Although rodent keratinocyte-derived chemokine (KC) shows the highest homology with human growth-related oncogene (GRO-α), it appears to be the closest equivalent to IL-8, as judged by its pattern of expression and putative function (18). Monocytes express the KC receptor (CXCR2), and KC triggers monocyte arrest on early atherosclerotic endothelium, one of the first steps in the invasion of tissues by macrophages (19). Interaction of monocyte CXCR2 with its ligand KC leads to up-regulation of α₄β₁ integrin affinity and firm adhesion to the endothelium (19). Furthermore, both KC and its receptor play a central role in macrophage infiltration and accumulation in atherosclerotic lesions in mice (20, 21). However, no information is currently available regarding the role of KC in macrophage recruitment in obese AT or its role in AT biology and pathology.In this study, we show that KC expression is elevated in AT and plasma of genetically (ob/ob) and diet-induced obese (DIO) mice, probably as the result of increased leptin and tumor necrosis factor α (TNF-α) levels associated with obesity. We also show that obesity-induced KC is mostly derived from nonadipocyte sources in AT and that KC does not affect adipocyte differentiation but does increase pro-inflammatory cytokine expression in adipocytes. Finally, we show in a DIO model in chimeric mice lacking CXCR2 on their macrophages that the KC receptor plays an important role in macrophage accumulation in adipose and skeletal muscle tissue and subsequent development of IR.     

In silico prioritisation of candidate genes for prokaryotic gene function discovery: an application of phylogenetic profiles

Background  

In silico candidate gene prioritisation (CGP) aids the discovery of gene functions by ranking genes according to an objective relevance score. While several CGP methods have been described for identifying human disease genes, corresponding methods for prokaryotic gene function discovery are lacking. Here we present two prokaryotic CGP methods, based on phylogenetic profiles, to assist with this task.     

Comparison of Comparative Genomic Hybridization Technologies Across Microarray Platforms

In the 2007 Association of Biomolecular Resource Facilities Microarray Research Group project, we analyzed HL-60 DNA with five platforms: Agilent, Affymetrix 500K, Affymetrix U133 Plus 2.0, Illumina, and RPCI 19K BAC arrays. Copy number variation was analyzed using circular binary segmentation (CBS) analysis of log ratio scores from four independently assessed hybridizations of each platform. Data obtained from these platforms were assessed for reproducibility and the ability to detect formerly reported copy number variations in HL-60. In HL-60, all of the tested platforms detected genomic DNA amplification of the 8q24 locus, trisomy 18, and monosomy X; and deletions at loci 5q11.2~q31, 9p21.3~p22, 10p12~p15, 14q22~q31, and 17p12~p13.3. In the HL-60 genome, at least two of the five platforms detected five novel losses and five novel gains. This report provides guidance in the selection of platforms based on this wide-ranging evaluation of available CGH platforms.