Preventing microalbuminuria in patients with diabetes: rationale and design of the Randomised Olmesartan and Diabetes Microalbuminuria Prevention (ROADMAP) study

Diabetic nephropathy has developed into a worldwide epidemic and is responsible for the majority of end-stage renal disease in most countries. Antihypertensive treatment slows the progression of the disease. In addition, blockade of the renin-angiotensin system reduces the degree of albuminuria and angiotensin II receptor blockers (ARBs) have been shown to delay the progression from microalbuminuria to overt proteinuria in patients with diabetes. However, few studies have examined whether the initial stage of diabetic nephropathy (i.e. the development of microalbuminuria) in patients with type 2 diabetes can be slowed or prevented by ARB treatment. The Randomised Olmesartan And Diabetes MicroAlbuminuria Prevention (ROADMAP) study is a placebo-controlled, multicentre, double-blind, parallel group study investigating the effect of the ARB, olmesartan medoxomil, on the incidence of microalbuminuria. A total of 4400 type 2 diabetes patients with normoalbuminuria will be randomized to treatment with 40 mg of olmesartan medoxomil once daily or placebo. Goal blood pressure will be 130/80 mmHg. The primary endpoint of the study is the occurrence of microalbuminuria. In ROADMAP, we will also assess as secondary endpoints the effects of olmesartan on fatal and non-fatal cardiovascular events in patients with diabetes. In addition, within subgroups of the ROADMAP patients, the effects of olmesartan on retinopathy and other microvascular circulations will be analysed. The study is expected to last a median of 5 years. The ROADMAP study will answer the question whether an ARB can prevent or delay the onset of microalbuminuria and whether this translates into protection against cardiovascular events and renal disease.     

Splitting high-dose oral methotrexate improves bioavailability: a pharmacokinetic study in patients with rheumatoid arthritis

OBJECTIVE: To study the bioavailability of a divided higher oral dose of methotrexate (MTX), in comparison to a single dose, in adult patients with rheumatoid arthritis (RA). METHODS: A pharmacokinetic analysis was performed in 10 patients with RA taking a stable dose (25-35 mg weekly) of MTX. Separated by one week, a pharmacokinetic analysis was performed in each patient after an oral single dose, and after an equal but split dose separated by 8 hours. MTX serum concentrations were measured by a fluorescence polarization immunoassay technique. Analysis was performed by calculation of the area under the curve (AUC) by the trapezoidal rule and by means of an iterative 2-stage Bayesian population procedure, obtaining population and individual pharmacokinetic parameters. For the population analysis, data from 15 patients in our previous study comparing oral and subcutaneous administration of MTX were also used. RESULTS: The median MTX dose was 30 mg weekly (range 25-35 mg). The bioavailability of the split dose was 28% higher compared to the single dose (p = 0.007). In the population pharmacokinetic modeling, a 2-compartment model best described the serum MTX concentration versus time curves. The mean bioavailability after single-dose and split-dose MTX was 0.76 and 0.90, respectively, compared to subcutaneous administration. There was a statistically significant difference in the bioavailability of the 2 oral administration regimens (p = 0.008). CONCLUSION: The bioavailability of oral higher dose MTX in adult patients with RA can be improved by splitting the dose.     

A probabilistic finite element analysis of the stresses in the augmented vertebral body after vertebroplasty

Fractured vertebral bodies are often stabilized by vertebroplasty. Several parameters, including fracture type, cement filling shape, cement volume, elastic moduli of cement, cancellous bone and fractured region, may all affect the stresses in the augmented vertebral body and in bone cement. The aim of this study was to determine numerically the effects of these input parameters on the stresses caused. In a probabilistic finite element study, an osteoligamentous model of the lumbar spine was employed. Seven input parameters were simultaneously and randomly varied within appropriate limits for >110 combinations thereof. The maximum von Mises stresses in cancellous and cortical bone of the treated vertebral body L3 and in bone cement were calculated. The loading cases standing, flexion, extension, lateral bending, axial rotation and walking were simulated. In a subsequent sensitivity analysis, the coefficients of correlation and determination of the input parameters on the von Mises stresses were calculated. The loading case has a strong influence on the maximum von Mises stress. In cancellous bone, the median value of the maximum von Mises stresses for the different input parameter combinations varied between 1.5 (standing) and 4.5 MPa (flexion). The ranges of the stresses are large for all loading cases studied. Depending on the loading case, up to 69% of the maximum stress variation could be explained by the seven input parameters. The fracture shape and the elastic modulus of the fractured region have the highest influence. In cortical bone, the median values of the maximum von Mises stresses varied between 31.1 (standing) and 61.8 MPa (flexion). The seven input parameters could explain up to 80% of the stress variation here. It is the fracture shape, which has always the highest influence on the stress variation. In bone cement, the median value of the maximum von Mises stresses varied between 3.8 (standing) and 12.7 MPa (flexion). Up to 75% of the maximum stress variation in cement could be explained by the seven input parameters. Fracture shape, and the elastic moduli of bone cement and of the fracture region are those input parameters with the highest influence on the stress variation. In the model with no fracture, the maximum von Mises stresses are generally low. The present probabilistic and sensitivity study clearly showed that in vertebroplasty the maximum stresses in the augmented vertebral body and in bone cement depend mainly on the loading case and fracture shape. Elastic moduli of cement, fracture region and cancellous bone as well as cement volume have sometimes a moderate effect while number and symmetry of cement plugs have virtually no effect on the maximum stresses.     

Work Experience and Style Explain Variation Among Pediatricians in the Detection of Children With Psychosocial Problems

ObjectiveTo assess whether variation in the proportion of children identified as having psychosocial problems by individual preventive pediatricians can be explained by pediatrician characteristics, over and above variations in the mix of children. Furthermore, to assess whether the characteristics of preventive pediatricians were related to the quality of problem identification.MethodsWe used data from approximately 3070 children ages 5 to 6 years who were assessed during a routine well-child visit by a preventive pediatrician in the Netherlands (response rate 85.2%). We obtained data about parent-reported child problems by using the Child Behavior Checklist (CBCL), sociodemographic background of the family, and characteristics of the preventive pediatrician. After each assessment, preventive pediatricians reported whether they had identified any psychosocial problem in the child. Multilevel logistic regression analyses were used to assess whether variation in the proportion of children identified by preventive pediatricians as having a psychosocial problem could be explained by the characteristics of preventive pediatricians and whether these characteristics were related to the quality of problem identification.ResultsPreventive pediatricians varied widely in the proportion of children identified as having psychosocial problems. Pediatrician characteristics such as work experience and work style (for example, on indication use of behavior questionnaires like the CBCL in routine care) explained about a quarter of this inter-pediatrician variation; child characteristics did not explain this variation even though characteristics like gender and parental education level were associated with likelihood of problem identification. More use of the CBCL and less use of the Teacher Report Form in routine care resulted in a better problem identification by preventive pediatricians. Work experience was not related to better problem identification.ConclusionsPreventive pediatricians identify psychosocial problems in children in a standardized way, but important inter-pediatrician variation remains. This variation may be reduced further and quality improved by changing their work style and targeted training.     

Changes in gene expression profiles in developing B cells of murine bone marrow.

Gene expression profiles of five consecutive stages of mouse B cell development were generated with high-density oligonucleotide arrays from as few as 2 x 10(4) ex vivo isolated and flow-cytometrically purified cells. Between 2.8% and 6.8% of all genes change on differentiation from one cellular stage to the next by at least twofold. The entire pathway involves differential expression of 10.7% of all genes. Previously known expression patterns of 15 genes (like surrogate light chain, RAG-1/2, MHC class II, mel-14 antigen) are confirmed. The gene expression patterns of the proliferating pre-BI and large pre-BII cells on the one hand, and the resting immature and mature B cells on the other hand, are most similar to each other. Small pre-BII cells display a pattern that is transitional between these two groups. Most of the genes expressed in early precursors are involved in general processes, like protein folding or cell cycle regulation, whereas more mature precursors express genes involved in more specific molecular programs (cell surface receptors, secreted factors, and adhesion molecules, among others). Between 19 and 139 genes share a given expression pattern. Combining knowledge about gene function and expression pattern allows identification of novel candidate genes potentially involved in self-maintenance of pre-BI cells, allelic exclusion and pre-B cell receptor signaling in large pre BII cells, cell-cycle arrest of small pre-BII cells, propensity toward apoptosis or anergization in immature B cells, propensity toward cell division and activation in mature B cells, and stage-specific interactions with stromal cells in the bone marrow.     

The B lineage potential of thymus settling progenitors is critically dependent on mouse age

The nature and lineage potential, particularly that for B cells, of thymus settling progenitors (TSP) in the adult mouse has been the subject of considerable debate. Lack of B cell potential would suggest pre-thymic, whereas its presence would suggest intra-thymic loss of B cell potential. Using limiting dilution analysis (LDA) in vitro and transfer experiments in vivo, we show that the B cell potential of TSP is critically dependent on mouse age, reaching a maximum of about 1 in 20 cells at birth, decreasing 50-fold in adult mice. Cells with a TSP phenotype can be found in the neonatal blood. Furthermore, using LDA, we show that Notch ligand signaling of TSP results in the loss of B cell potential with a half-life of approximately 12 h. Taken together, these results indicate that loss of B cell potential by TSP is an intra-thymic event and highlight the developmental pressure acting on the immune system to rapidly colonize primary lymphoid organs with functional progenitors. This critical time coincides with birth in the mouse. In the adult mouse, we estimate than only about 5 TSP cells/day would be required to maintain steady-state thymopoiesis.     

Motifs within the CLN3 protein: modulation of cell growth rates and apoptosis

Juvenile Batten disease (JNCL) is an autosomal recessive disease that results from mutations in the CLN3 gene. The wild-type CLN3 gene coding sequence has 15 exons, and the translated protein consists of 438 amino acids. The most commonly observed mutation is a 1.02 kb deletion in the genomic DNA. This deletion results in a truncated protein due to the loss of amino acids 154-438, and the introduction of 28 novel amino acids at the c-terminus. We demonstrate that, compared to normal controls, CLN3-deficient immortalization of lymphoblasts homozygous for this deletion grow at a slower rate, and show increased sensitivity to etoposide-induced apoptosis, supporting the notion that CLN3 may negatively regulate apoptosis. Using immortalized JNCL lymphoblast cell lines as a model system, we assess the effects of specific CLN3 mutations on cell growth rates and protection from etoposide-induced apoptosis. Protection from etoposide-induced apoptosis occurs and the cell growth rate is restored following transfection of JNCL lymphoblasts with mutant CLN3 cDNA that includes exons 11 or 13. We show that deletion of the glycosylation sites 71NQSH74 and 310NTSL313, and also mutations within the highly conserved amino acid stretches 184WSSGTGGAGLLG195, 291VYFAE295 and 330VFASRSSL337, result in slowed growth and susceptibility to apoptosis.     

Development of lymphocyte subpopulations in preterm infants

In preterm neonates the immune system is thought to be less developed at birth, but very little is known about the actual size of lymphocyte subpopulations, and even less about the maturation of these subpopulations during the first months after a premature birth. To evaluate the development of lymphocyte subpopulations in preterm infants during the first 3 months after birth, we performed a prospective longitudinal study in two hospitals in the Netherlands. Preterm neonates (n = 38) of all post-menstrual ages were included and blood samples were taken from cord blood, and at 1 week, 6 weeks, and 3 months. Lymphocyte subpopulations were measured by four-colour flow cytometry. The data were compared with follow-up data obtained in healthy term neonates (n = 8), and with single samples from school age children (n = 5) and adults (n = 5). Overall, we found a similar pattern of post-natal development of lymphocyte subpopulations in the term and preterm infants. Both B lymphocytes and helper and cytotoxic T lymphocytes mainly consist of naive cells at birth and during the 3 months of follow-up in all neonatal age groups. So, the preterm immune system seems to be able to generate an outburst of naive T and B lymphocytes from the thymus and bone marrow within the same time span after the start of post-natal antigenic stimulation from the environment as the term immune system, but, with lower post-menstrual age, the absolute counts of naive helper T lymphocytes are lower.     

Intraarticular ferritin-bound iron in rheumatoid arthritis: A factor that increases oxygen free radical-induced tissue destruction

Iron mobilized from ferritin is able to convert superoxide and hydrogen peroxide, which are produced in large amounts in rheumatoid arthritis (RA), to the extremely toxic hydroxyl radical. We have found that synovial fluid ferritin is increased significantly in RA patients compared with levels in controls. The high synovial fluid:serum ferritin ratio is compatible with the hypothesis that synovial fluid ferritin is derived from the synovial membrane. We found no difference in ferritin concentrations in the synovial membranes of RA patients compared with those of controls. Quantitative data on the amount of iron bound to ferritin showed that the level was 2.9 times higher in RA synovial membranes than in those of controls. Moreover, RA synovial fluid contained considerable amounts of iron bound to ferritin. Calculation of the iron saturation of ferritin revealed that RA synovial membranes contained a mean of 2,210 moles of iron per mole of ferritin: a significant elevation when compared with the mean value of 1,500 moles found in the synovial membranes of the controls. The decreased saturation of ferritin in RA synovial fluid, compared with that in the synovial membrane, could be caused by an uncompensated release of iron from ferritin, which has been induced by superoxide that is produced by stimulated granulocytes. The results demonstrate that in the joints of RA patients, sufficient ferritin loaded with iron is available to stimulate oxygen free radical damage.