Nadroparin therapy in pediatric patients with venous thromboembolic disease

Low-molecular-weight heparins are increasingly used for treatment of pediatric venous thromboembolic disease (VTE). Pediatric data about therapeutic doses of nadroparin are not available. To evaluate pharmacodynamics and safety of therapeutic doses of nadroparin, consecutive patients (age 0 to 18 y) with objectively diagnosed VTE and treated with nadroparin were included in this single center study over a 12-year period. All patients started with 85.5 IU/kg of nadroparin twice daily. The target therapeutic range (TTR) was set at 0.5 to 1.0 anti-Xa IU/mL 4 hours postdose. Safety end points were major bleeding and therapy-related death. A total of 84 patients were enrolled, of whom 8 patients did not undergo measurement of anti-Xa levels. Fifty-four (71%) of 76 patients achieved TTR. The maintenance dose (mean+/-SE) was 448+/-42 IU/kg/d in neonates (<2 mo, n=6), 253+/-22 IU/kg/d in infants (2 mo to 1 y, n=10), 214+/-8 IU/kg/d in children (2 to 11 y, n=13), and 183+/-5 IU/kg/d in adolescents (12 to 18 y, n=25). Neonates required significantly more dose adjustments and time to achieve TTR than adolescents. No major bleeding or therapy-related death occurred. In summary, an age-dependent response to nadroparin exists in pediatric patients. Nadroparin therapy seems to be safe for treatment of pediatric VTE.     

Aberrant regulation of polymorphonuclear phagocyte responsiveness in multitrauma patients

A systemic inflammatory response often follows severe trauma. Priming (preactivation) of polymorphonuclear phagocytes (PMNs) is an essential first step in the processes that lead to damage caused by the systemic activation of innate immune response. Until recently, priming could only accurately be measured by functional assays, which require isolation of cells, thereby potentially inducing artificial activation. The aim of this study was to identify primed PMNs in response to trauma by using a whole blood analysis with a broad detection range. Twenty-two trauma patients were analyzed for PMN priming with novel developed antibodies recognizing priming epitopes by flow cytometric analysis. Expression of priming epitopes on PMNs was analyzed with respect to time, injury, and disease severity. Expression of priming epitopes in the circulation was compared with expression profiles of PMNs obtained from lung fluid. Fourteen healthy volunteers served as controls. Expression of priming epitopes on peripheral blood PMNs of injured patients was similar, as found in healthy controls, whereas highly primed cells were found in the lung fluid of injured patients (increase of >50 times as compared with peripheral blood cells). In fact, the responsiveness of PMNs toward the bacteria-derived stimulus N-formyl-methionyl-leucyl-phenylalanine was markedly decreased in trauma patients. Lack of expression of priming epitopes and the unresponsiveness to N-formyl-methionyl-leucyl-phenylalanine demonstrates the presence of partially refractory cells in the circulation of trauma patients. An increased expression of epitopes found on pulmonary PMNs suggests that optimal (pre)activation of these cells only occurs in the tissues.     

Lack of arginine vasopressin-induced phosphorylation of aquaporin-2 mutant AQP2-R254L explains dominant nephrogenic diabetes insipidus

Water homeostasis in humans is regulated by vasopressin, which induces the translocation of homotetrameric aquaporin-2 (AQP2) water channels from intracellular vesicles to the apical membrane of renal principal cells. For this process, phosphorylation of AQP2 at S256 by cAMP-dependent protein kinase A is thought to be essential. Mutations in the AQP2 gene cause recessive and dominant nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin. Here, a family in which dominant NDI was caused by an exchange of arginine 254 by leucine in the intracellular C terminus of AQP2 (AQP2-R254L), which destroys the protein kinase A consensus site, was identified. Expressed in oocytes, AQP2-R254L appeared to be a functional water channel but was impaired in its transport to the cell surface to the same degree as AQP2-S256A, which mimics nonphosphorylated AQP2. In polarized renal cells, AQP2-R254L was retained intracellularly and was distributed similarly as AQP2-S256A or wild-type AQP2 in unstimulated cells. Upon co-expression in MDCK cells, AQP2-R254L interacted with and retained wild-type AQP2 in intracellular vesicles. Furthermore, AQP2-R254L had a low basal phosphorylation level, which was not increased with forskolin, and mimicking constitutive phosphorylation in AQP2-R254L with the S256D mutation shifted its expression to the basolateral and apical membrane. These data indicate that dominant NDI in this family is due to a R254L mutation, resulting in the loss of arginine vasopressin-mediated phosphorylation of AQP2 at S256, and illustrates the in vivo importance of phosphorylation of AQP2 at S256 for the first time.     

The tetraspanin CD63 enhances the internalization of the H,K-ATPase beta-subunit

The tetraspanin CD63 resides in late endosomes, lysosomes, secretory vesicles, and at the plasma membrane, and it moves among these compartments. We find that CD63 is present also in tubulovesicular elements, the intracellular compartments that contain the H,K-ATPase in unstimulated gastric parietal cells. The H,K-ATPase beta-subunit and CD63 colocalize in parietal cells and form a complex that can be coprecipitated. The beta-subunit and CD63 also interact when they are coexpressed in COS-7 cells. Furthermore, expression with CD63 induces the redistribution of the beta-subunit from the cell surface to CD63+ intracellular compartments. Immunofluorescence and biochemical experiments reveal that this redistribution occurs by enhanced endocytosis of H,K-ATPase beta-subunit complexed with CD63. Coexpression of the beta-subunit with mutant CD63 polypeptides demonstrates that the enhanced internalization of the beta-subunit depends on the capacity of CD63 to interact with adaptor protein complexes 2 and 3. These data indicate that CD63 serves as an adaptor protein that links its interaction partners to the endocytic machinery of the cell and suggest a previously uncharacterized protein-trafficking role for the tetraspanins.     

Resistance of ovarian carcinoma cells to docetaxel is XIAP dependent and reversible by phenoxodiol

Although several pathways have been proposed to explain chemoresistance, all lead to some specific defect in the mechanism of apoptosis. The objective of this study was to characterize the molecular mechanisms of drug resistance to docetaxel in epithelial ovarian cancer cells (EOC) and the use of phenoxodiol as a chemosensitizer. Four established and 12 primary cultures of ovarian carcinoma cell lines (EOC) were treated with docetaxel (5-500 ng/ml) for 24 and/or 48 h. In all the studied cell lines, the best response was seen using 500 ng/ml of docetaxel. Sensitive cell lines were identified as those with IC50 < 100 ng/ml for 48 h while resistant cell lines were identified as those with IC50 > 100 ng/ml. The morphological features of apoptosis and the activation of caspases were seen only in the sensitive cell lines determined by Hoechst staining and Caspase Glo assay. Although X-linked inhibitor of apoptosis protein (XIAP) was expressed in all EOC cells, it was only inactivated in chemosensitive cells. We confirmed the role of XIAP in docetaxel resistance by downregulation of XIAP expression using RNA interference (RNAi) as well as by pretreatment with phenoxodiol. Our results indicate that 1) docetaxel induces its cytotoxic effect through the activation of apoptosis; 2) caspase activation relies on the removal of XIAP; and 3) phenoxodiol restores sensitivity in docetaxel-resistant EOC cells. We demonstrate that phenoxodiol, by interfering with XIAP activity, functions as a chemosensitizer to docetaxel and could provide a more effective treatment for refractory ovarian cancer.     

Fluorescence in situ hybridization for the detection of malignant bile duct strictures: has FISH found a new pond?

Routine cytology for the diagnosis of biliary and pancreatic malignancies has exceptionally poor sensitivity and has spurred a search for new technology in the evaluation of these diseases. The recent study by Kipp et al. in this issue of the Journal, compares one of these novel techniques (fluorescence in situ hybridization to standard cytology) for the detection of malignant biliary strictures. Although larger studies are indicated, the modality shows considerable promise for future clinical use.     

A prospective cohort study on sustained effects of low-dose ecstasy use on the brain in new ecstasy users.

It is debated whether ecstasy use has neurotoxic effects on the human brain and what the effects are of a low dose of ecstasy use. We prospectively studied sustained effects (>2 weeks abstinence) of a low dose of ecstasy on the brain in ecstasy-naive volunteers using a combination of advanced MR techniques and self-report questionnaires on psychopathology as part of the NeXT (Netherlands XTC Toxicity) study. Outcomes of proton magnetic resonance spectroscopy (1H-MRS), diffusion tensor imaging (DTI), perfusion-weighted imaging (PWI), and questionnaires on depression, impulsivity, and sensation seeking were compared in 30 subjects (12M, 21.8+/-3.1 years) in two sessions before and after first ecstasy use (1.8+/-1.3 tablets). Interval between baseline and follow-up was on average 8.1+/-6.5 months and time between last ecstasy use and follow-up was 7.7+/-4.4 weeks. Using 1H-MRS, no significant changes were observed in metabolite concentrations of N-acetylaspartate (NAA), choline (Cho), myo-inositol (mI), and creatine (Cr), nor in ratios of NAA, Cho, and mI relative to Cr. However, ecstasy use was followed by a sustained 0.9% increase in fractional anisotropy (FA) in frontoparietal white matter, a 3.4% decrease in apparent diffusion (ADC) in the thalamus and a sustained decrease in relative regional cerebral blood volume (rrCBV) in the thalamus (-6.2%), dorsolateral frontal cortex (-4.0%), and superior parietal cortex (-3.0%) (all significant at p<0.05, paired t-tests). After correction for multiple comparisons, only the rrCBV decrease in the dorsolateral frontal cortex remained significant. We also observed increased impulsivity (+3.7% on the Barratt Impulsiveness Scale) and decreased depression (-28.0% on the Beck Depression Inventory) in novel ecstasy users, although effect sizes were limited and clinical relevance questionable. As no indications were found for structural neuronal damage with the currently used techniques, our data do not support the concern that incidental ecstasy use leads to extensive axonal damage. However, sustained decreases in rrCBV and ADC values may indicate that even low ecstasy doses can induce prolonged vasoconstriction in some brain areas, although it is not known whether this effect is permanent. Additional studies are needed to replicate these findings.