Direct costs of warfarin treatment among patients with atrial fibrillation in a Finnish health care setting.

OBJECTIVE: The main objective was to estimate the mean direct costs of warfarin treatment for atrial fibrillation (AF) patients. Secondly, the costs of initiating warfarin treatment during a 60-day period and the impact of International Normalized Ratio (INR) and co-morbidities on costs were estimated. DESIGN AND DATA: The study was performed as a retrospective cohort study over a 12-month period in a Finnish communal health care setting. All AF patients aged 65 years or older (n = 250) with warfarin treatment were identified from the database of the health service district of an urban area. Patient specific information related to comorbidities, INR-control, complications and health care resource use were collected. Cost information was obtained from the Finnish national health care unit cost list. METHODS: The effect of treatment balance and other background variables on treatment costs were evaluated using ordinary least squares regression (OLS), log-transformed OLS and generalized linear model (GLM). The mean costs were calculated on the basis of the different models and bias corrected and accelerated (BCa) bootstrap confidence intervals (CIs) were calculated for the mean costs. RESULTS: The best fitting cost model was log-transformed OLS. The costs of warfarin treatment on the basis of the log-transformed model were 589.82 Euros (BCa 95% CI: 586.68-591.99) per patient compared to 616.00 Euros (BCa 95% CI: 579.98-652.96) obtained with the OLS-model. For the treatment initiation period, the mean costs were 263 Euros (BCa 95% CI: 218.90-314.71). Depending on the way that INR-control was defined, the mean costs were 95.27 Euros or 166.92 Euros higher for patients who were not in the defined INR-balance. CONCLUSIONS: The INR-control has a significant impact on the warfarin treatment costs. The choice of model influences the estimated mean costs. In addition, different models identify statistically significant effects between different background variables and costs.

Description of two biovars in the Rhizobium galegae species: biovar orientalis and biovar officinalis.

Twenty-six Rhizobium galegae strains, representing the center of origin of the host plants Galega orientalis and G. officinalis as well as other geographic regions, were used in a polyphasic analysis of the relationships of R. galegae strains. Phage typing, lipopolysaccharide (LPS) profiling, pulsed field gel electrophoresis (PFGE) profiling and rep-PCR (use of repetitive sequences as PCR primers for genomic fingerprinting) with REP and ERIC primers investigated nonsymbiotic properties, whereas plasmid profiling and hybridisation with a nif gene probe, and with nodB, nodD, nod box and an IS sequence from the symbiotic region as probes, were used to reveal the relationships of symbiotic genes. The results were used in pairwise calculations of distances between the strains, and the distances were visualised as a dendrogram. Indexes of association were compared for all tests pooled, and for chromosomal tests and symbiotic markers separately, to display the input of the different categories of tests on the grouping of the strains. Our study shows that symbiosis related genetic traits in R. galegae divide strains belonging to the species into two groups, which correspond to strains forming an effective symbioses with G. orientalis and G. officinalis respectively. We therefore propose that Rhizobium galegae strains forming an effective symbiosis with Galega orientalis are called R. galegae bv. orientalis and strains forming an effective symbiosis with Galega officinalis are called R. galegae bv. officinalis.

Identification and structure of the Rhizobium galegae common nodulation genes: evidence for horizontal gene transfer.

Rhizobia are soil bacteria able to fix atmospheric nitrogen in symbiosis with leguminous plants. In response to a signal cascade coded by genes of both symbiotic partners, a specific plant organ, the nodule, is formed. Rhizobial nodulation (nod) genes trigger nodule formation through the synthesis of Nod factors, a family of chitolipooligosaccharides that are specifically recognized by the host plant at the first stages of the nodulation process. Here, we present the organization and sequence of the common nod genes from Rhizobium galegae, a symbiotic member of the RHIZOBIACEAE: This species has an intriguing phylogenetic position, being symbiotic among pathogenic agrobacteria, which induce tumors instead of nodules in plant shoots or roots. This apparent incongruence raises special interest in the origin of the symbiotic apparatus of R. galegae. Our analysis of DNA sequence data indicated that the organization of the common nod gene region of R. galegae was similar to that of Sinorhizobium meliloti and Rhizobium leguminosarum, with nodIJ downstream of nodABC and the regulatory nodD gene closely linked to the common nod operon. Moreover, phylogenetic analyses of the nod gene sequences showed a close relationship especially between the common nodA sequences of R. galegae, S. meliloti, and R. leguminosarum biovars viciae and trifolii. This relationship in structure and sequence contrasts with the phylogeny based on 16S rRNA, which groups R. galegae close to agrobacteria and separate from most other rhizobia. The topology of the nodA tree was similar to that of the corresponding host plant tree. Taken together, these observations indicate that lateral nod gene transfer occurred from fast-growing rhizobia toward agrobacteria, after which the symbiotic apparatus evolved under host plant constraint.

Evaluation of the Galega-Rhizobium galegae system for the bioremediation of oil-contaminated soil.

The bioremediation potential of a nitrogen-fixing leguminous plant, Galega orientalis, and its microsymbiont Rhizobium galegae was evaluated in BTX (benzene, toluene, xylene)-contaminated soils in microcosm and mesocosm scale. To measure the intrinsic tolerance of the organisms to m-toluate, a model compound representing BTX, G. orientalis and R. galegae were cultivated under increasing concentrations of m-toluate alone and in association with Pseudomonas putida pWWO, a bacterial strain able to degrade toluene-derived compounds. The test plants and rhizobia remained viable in m-toluate concentrations as high as 3000 ppm. Plant growth was inhibited in concentrations higher than 500 ppm, but restituted when plants were transferred into m-toluate-free medium. Nodulation was blocked under the influence of m-toluate, but was restored after the plants were transferred into the non-contaminated media. In the mesocosm assay the Galega plants showed good growth, nodulation and nitrogen fixation, and developed a strong rhizosphere in soils contaminated with oil or spiked with 2000 ppm m-toluate. Thus, this legume system has good potential for use on oil-contaminated sites

Identification of nodulation promoter (nod-box) regions of Rhizobium galegae.

A hybridisation analysis of a genomic clone library of Rhizobium galegae HAMBI 1174 located four EcoRI fragments homologous to the nod-box promoter sequence of Sinorhizobium meliloti in two separate gene regions. Two of the five nod-boxes detected in the R. galegae genome were carried on a single cosmid clone, pRg30, upstream from the nodABCIJ and nodF genes, whereas the other three nod-boxes were carried on a different cosmid clone, pRg10. Hybridisations with various nod gene probes from S. meliloti and Rhizobium leguminosarum species detected a nodD homolog in pRg10. The sequence data obtained from regions adjacent to each nod-box in pRg10 confirmed the presence of a second nodD in the R. galegae genome and, in addition, revealed the presence of nodN, nodU, dctA nifH and nifQ-like genes in pRg10. Thus, by using a promoter-specific nod-box probe we could identify a new region carrying genes involved in nitrogen fixation and host specificity functions.

Expression of Rhizobium galegae common nod clones in various backgrounds.

The cosmid clone pRg30, carrying common nodulation genes of Rhizobium galegae HAMBI 1174, and pRg33, a subclone of pRg30 that contains a 5.7-kb ClaI insert carrying nodDABC were conjugated into various Rhizobium nod- mutant strains and into a Ti plasmid-cured Agrobacterium tumefaciens. Complementation and expression of the nodABC genes of R. galegae were studied by following microscopically the infection process and the nodulation on different test plants. The nodABC genes of R. galegae complemented the nod- strains of other Rhizobium species. The presence of extra copies of common nod genes in the homologous R. galegae nodABC- strain induced an increased nodulation on Galega orientalis. However, the inserts of R. galegae in pRg30 and pRg33 do not carry sufficient genetic information for normal nodulation of test plants in an Agrobacterium background, because the Agrobacterium transconjugants induced root hair deformation on Galega plants, but no infection threads were detected and nodulelike structures developed only at low frequency. The Agrobacterium carrying the nodDABC of R. galegae did not cause the root hairs of Medigo sativa to deform.

Differentiation-associated decrease in muscarinic receptor sensitivity in human neuroblastoma cells.

Muscarinic receptor-linked increases in intracellular free Ca2+ as measured with quin-2 and Ca2+ release from monolayers of cells have been measured in the human neuroblastoma cell line SH-SY5Y. Induction of differentiation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) leads to a decrease in the sensitivity of the cells to low concentrations of agonists with respect to the induced increase in cytosolic free Ca2+ and stimulation of Ca2+ efflux. No decrease in agonist binding affinity was observed when the displacement of a labelled antagonist, 3H-NMS, by a non-labelled agonist was studied.

Accumulation and mobilization of cholesteryl esters in cultured human fibroblasts exposed to free cholesterol-rich phospholipid vesicles.

The uptake of free cholesterol (FC) into cells by surface transfer and its esterification to cholesteryl esters (CE) has been studied with a system of FC-egg phosphatidylcholine (EPC) vesicles and human lung fibroblasts in serum-free growth medium. The influx of FC was dependent on the molar ratio of FC to EPC in the donor vesicles. The FC incorporated by surface transfer was available for esterification in the cells. Incubations with FC/EPC vesicles with a FC/EPC molar ratio of 0.5: 1 gave a small net decrease in cellular CE, while 1:1 vesicles gave a mild increase. When the cells were incubated with 2:1 FC/EPC vesicles an extensive accumulation of CE was demonstrated, which was accentuated if albumin was present in the medium. The CE accumulated in the form of lipid droplets within the cells. The largest of these droplets exhibited positive birefringence with formée crosses, that is typical for liquid crystals of cholesteryl esters. If cells loaded with CE were incubated with vesicles with low FC/EPC ratios a net efflux of CE was noted. The present study demonstrates that the uptake of FC from lipid vesicles by surface transfer can reproduce typical features of foam cells in early atherosclerosis.

Physicochemical transfer of [3H]cholesterol from plasma lipoproteins to cultured human fibroblasts.

The transfer of free cholesterol from [3H]cholesterol-labelled plasma lipoproteins to cultured human lung fibroblasts was studied in a serum-free medium. The uptake of [3H]cholesterol depended upon time of incubation, concentration of lipoprotein in the medium, and temperature. Modified (reduced and methylated) low-density lipoprotein (LDL), which did not enter the cells by the receptor pathway, gave a somewhat lower transfer rate than unmodified LDL, but if the transfer values for native LDL were corrected for the receptor-mediated uptake of cholesterol the difference was eliminated. The initial rates of transfer of [3H]cholesterol from LDL and high-density lipoprotein (HDL) were of the same order of magnitude (0.67 +/- 0.05 and 0.75 +/- 0.06 nmol of cholesterol/h per mg of cell protein, respectively) while that from very-low-density lipoprotein (VLDL) was much lower (0.23 +/- 0.02 nmol of cholesterol/h per mg) (means +/- S.D., n = 5). The activation energy for transfer of cholesterol from reduced, methylated LDL to fibroblasts was determined to be 57.5 kJ/mol. If albumin was added to the incubation medium the transfer of [3H]cholesterol was enhanced, while that of [14C]dipalmitoyl phosphatidylcholine was decreased compared with the protein-free system. The results demonstrate that, in spite of its low water solubility, free cholesterol can move from lipoproteins to cellular membranes, probably by aqueous diffusion. We propose that physicochemical transfer of free cholesterol may be a significant mechanism for net uptake of the sterol into the artery during atherogenesis.

Preparation of biologically active analogs of serum low density lipoprotein.

A method for the preparation of stable and water-soluble analogs of low density lipoprotein (LDL) is presented. The experimental protocols start with the preparation of a cholesteryl ester/phospholipid microemulsion by a combined injection-sonication procedure and delipidation of apoprotein B (apoB) with sodium deoxycholate (NaDOC). The association of lipid microemulsion and NaDOC-solubilized apoB is achieved by incubation and sonication of the components above the melting point of the cholesteryl ester. The reconstituted model LDL (m-LDL) proved to be quite homogeneous both with respect to particle size and composition. Negative-stain electron microscopy shows spherical particles with a mean diameter of 21 nm. The mean density of the reconstituted LDL was 1.07 g/ml as determined by sucrose density gradient centrifugation. The reconstituted LDL retained its beta-mobility on agarose gel electrophoresis, and sodium dodecyl sulfate (SDS)-gel electrophoresis showed no degradation of apoB during the reconstitution procedures. Studies of biological activity showed that the m-LDL particles are bound, incorporated, and degraded by human fibroblasts in a way similar to native LDL. The reconstituted m-LDL has potential use for metabolic, physiochemical, and enzymatic studies of lipoproteins.

Incorporation of cholesterol into serum high density lipoprotein apoprotein and recombinants.

The uptake of cholesterol (CHL) by serum high density lipoprotein (HDL) delipidated apoproteins and phospholipid-apoprotein recombinants has been studied with two methods; by incubation with Celite-dispersed cholesterol or with cholesterol crystals. The apoproteins bind very small amounts of cholesterol with a maximum of about 6 micrograms/mg apoprotein. Recombinants with dimyristoyl phosphatidylcholine (DMPC) or egg phosphatidylcholine (EPC) as phospholipid component gave similar values for cholesterol uptake. The initial rate for uptake from Celite-cholesterol by recombinants was high (0.1 mol cholesterol/mol phospholipid/h) and somewhat higher than that for phospholipid vesicles. The maximal uptake was by gel filtration shown to depend on the size of the complexes with values about 0.95 mol cholesterol per phospholipid for vesicular complexes, 0.75 for discoidal complexes and between 0.5 and 0.2 for small 'protein-rich' complexes. During the incubation of recombinants with cholesterol there was considerable decomposition of discoidal complexes and formation of larger ones. The results show that phospholipid-apoprotein complexes are efficient acceptors for cholesterol but also that about 25% of the phospholipid in the discoidal complexes is excluded from interaction with cholesterol by interaction with apoprotein.

Incorporation of cholesterol into serum high and low density lipoproteins.

Excess cholesterol (CHL) was incorporated into serum lipoproteins by incubation with Celite-dispersed CHL or CHL crystals. The initial rates of uptake were 15 and 7 mol CHL/ mol lipoprotein X h-1 for low (LDL) and high (HDL) density lipoprotein, respectively. Saturation values were obtained after 48 h and were 90 and 65 mol CHL/mol LDL, and 42 and 32 mol/mol HDL using CHL on Celite and CHL crystals, respectively. Characterization of the lipoproteins showed a small change in electrophoretic mobility and an increase in molecular masses, especially after incubation with CHL on Celite. Spectroscopic methods showed only minor effects on the protein moieties.