The recommended dose of idarucizumab may not always be sufficient for sustained reversal of dabigatran.

Essentials Reversal of anticoagulant effects of dabigatran may occur despite application of idarucizumab. Monitoring of dabigatran level after antidote application is crucial to detect rebound. Repeated doses of idarucizumab may be necessary in cases of massive dabigatran accumulation. Combination of antidote application and renal replacement therapy may offer additional benefit. SUMMARY: Idarucizumab is a monoclonal antibody fragment designed for reversing the anticoagulant effects of dabigatran. Administration is recommended as two intravenous boluses of 2.5 g within 15 min of each other or as a single 5 g bolus. However, in certain situations a second dose of the drug could be necessary. We report the case of a 77-year-old man, treated with dabigatran for paroxysmal atrial fibrillation. He presented at our department with acute renal failure, concomitant massive dabigatran accumulation and subsequent acute gastrointestinal bleeding. Fifty minutes after the administration of idarucizumab, the dabigatran plasma concentration decreased from a peak of 1630 ng ml-1 to a level below the detection limit of 30 ng ml-1 and bleeding stopped. Eight hours after administration, the dabigatran plasma level started to increase up to 1560 ng ml-1 (96% of the maximum value obtained), accompanied by a further drop in hemoglobin. Concomitant hemodialysis and hemofiltration led to a continuous decrease in dabigatran plasma levels. However, sepsis and multiorgan failure ensued, which led to death. With this case report we raise the question of whether massive dabigatran accumulation requires repeated doses of idarucizumab, or alternatively, if the combination of antidote with hemodialysis/renal replacement therapy is advisable in order to remove circulating levels of dabigatran.

An inosine 5'-monophosphate dehydrogenase 2 single-nucleotide polymorphism impairs the effect of mycophenolic acid.

Mycophenolic acid (MPA) is a selective inhibitor of inosine 5'-monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme of de novo synthesis of guanine nucleotides. The isoenzyme IMPDH2 predominates in activated lymphocytes, and its inhibition by MPA is part of standard immunosuppressive regimens. Yet, there are significant unexplained differences in efficacy and tolerability among patients. The objective of this study was to analyze whether frequent variants in the IMPDH2 gene lead to changes in IMPDH activity and to differences in responsiveness to MPA therapy. All 14 exons and intron-exon boundary regions of IMPDH2 were sequenced from genomic DNA probes from 100 healthy individuals. Two novel exonic single-nucleotide polymorphisms were identified in 1% and one intronic polymorphism (rs11706052) in 19% of the study population. Lymphocyte IMPDH activity and proliferation under three MPA concentrations (2.5, 10 and 25 micromol l(-1)) were compared in rs11706052 carriers and wild-type individuals. The presence of rs11706052 polymorphism reduced the antiproliferative effect of MPA on lymphocytes by approximately 50% compared with the IMPDH2 wild-type form at therapeutic relevant concentrations of 10 micromol l(-1) and 25 micromol l(-1). We conclude that a poorer response to MPA therapy can be explained in some individuals by the presence of the rs11706052 polymorphism.

Anemia in patients with Wegener's granulomatosis.

AIMS: Anemia is commonly observed among patients with chronic kidney disease (CKD). No such information is available for patients with a history of systemic vasculitis. METHODS: We examined the prevalence of anemia, the response to therapy with erythropoiesis-stimulating agents (ESA), and the association of anemia with the kidney function in clinically stable patients with Wegener's granulomatosis in a retrospective, single-center study. RESULTS: The mean hemoglobin concentration of 36 patients (mean age: 58 years; 15 female, 21 male; mean duration of disease: 4.6 years) was 13.0+/-2.1 g/dl, and the mean estimated glomerular filtration rate (eGFR) was 41+/-21 ml/min/1.73 m(2). 14 of 36 patients (38.8%) presented with anemia (hemoglobin concentration < 12 g/dl in women, < 13 g/dl in men, or ESA therapy). In patients with a CKD Stage 3 or 4, anemia was present about twice as much as compared to the Third National Health and Nutrition Examination Survey (NHANES III) population. The hemoglobin concentration, however, was not associated with a change of kidney function (p = 0.1578). CONCLUSIONS: We found a higher prevalence of anemia in patients with Wegener's granulomatosis, as compared to the NHANES III population. The hemoglobin concentrations showed no association with changes of kidney function.

Surface antigens of human mesangial cells: impact of growth surface or IL-1alpha.

The interactions of mesangial cells (MC) with their environment are important events in glomerular physiology and pathology, yet a detailed characterization of the MC-surface antigens mediating these interactions is still lacking. In this study, a comparative phenotype analysis of primary human MC in culture using 191 monoclonal antibodies directed against 108 antigens was performed by flow-cytometry. The MC were grown on three different surfaces (human matrix, fibronectin, polystyrene) and cultured in the presence or absence of IL-1alpha. Seventy-one antibodies recognizing 35 different antigens (integrins: CD29, 49b, 49c, 49e, 51, 61; immunoglobulin gene family: CD54, 58, 90, 106, 146, 147, 166; growth factor receptors: CD105, 140b; apoptosis related: CD95; hemostatis related: CD141, 142; miscellaneous: CD44, 109, 138, 151, 157, 165, and 11 nonclustered antigens) reacted with mesangial cells. CD58, 109, 146, 147, 151, 157, 165, and 166 are reported for the first time to be present on human mesangial cells. In comparison to growth on polystyrene, CD44, 54, 95, 105, 109, 140b, 146, 147, 157, 165 and 166, were up-regulated on fibronectin, and CD44, 54, 90, 95, 105, 106, 109, 138, 140b, 141, 142, 146, 147, 151, 157, 165 and 166 were up-regulated on human matrix. The stimulation by IL-1alpha up-regulated CD44, 49e, 51, 54, 61, 106 on MC on polystyrene; CD49e, 51, 61, 106, 146, 165 on MC on fibronectin, and CD49e, 51, 54 on MC grown on human matrix. This analysis of surface antigen expression provides new information to enable a better understanding of the role of mesangial cells in glomerular pathophysiology.

Erythropoietin-inducible immediate-early genes in human vascular endothelial cells.

BACKGROUND: Patients receiving recombinant human erythropoietin (rHuEPO) may experience side effects arising from the vascular system. The underlying mechanisms, however, are largely unknown. METHODS: To elucidate downstream events following erythropoietin receptor triggering, a differential display analysis of human vascular endothelial cell mRNA was performed. RESULTS: We identified eight genes that were upregulated by rHuEPO as confirmed in two further independent cell culture experiments using a semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) protocol. The genes coded for proteins that may be assigned to four different groups: 1) proteins implicated in the regulation of vascular functions (thrombospondin-1, 20 kDa myosin regulatory light chain; relative increase of rHuEPO induced mRNA levels: 155.2%, P = 0.043; 137.6%, P = 0.046, respectively); 2) gene products involved in gene transcription and/or translation (c-myc purine-binding transcription factor PuF, tryptophanyl-tRNA synthetase, S19 ribosomal protein; increase of mRNA levels: 126.4%, P = 0.032; 150.9%, P = 0.012; 134.9%, P = 0.038); 3) subunits of mitochondrial enzymes related to energy transfer (NADH dehydrogenase subunit 6, cytochrome C oxidase subunit 1; increase of mRNA concentrations: 141.7%, P = 0.007; 140.3%, P = 0.01); and 4) regulators of signal transduction (protein tyrosine phosphatase G1, increase of transcript level: 160.3%, P = 0.016). CONCLUSIONS: We report on novel molecular downstream events following rHuEPO receptor triggering of human vascular endothelial cells. We identified EPO-responsive immediate-early genes, coding for proteins involved in vascular functions, gene transcription, and/or translation, energy transfer, and signal transduction. Thus, our data provide new insights into the molecular changes induced by EPO in human vascular endothelial cells.

Endothelial cell adhesion molecule and PMNL response to inflammatory stimuli and AGE-modified fibronectin.

BACKGROUND: Atherosclerotic vascular disease is the leading cause of death in patients with diabetes mellitus and end-stage renal disease. Advanced glycation end products (AGEs) are strongly suggested to be involved in the pathogenesis of atherosclerosis in these patients who also frequently experience infectious complications. We hypothesized that the interaction of AGEs and inflammatory mediators contributes to the up-regulation of endothelial cell activation. METHODS: We investigated the effect of advanced glycated fibronectin in the presence or absence of inflammatory stimuli on the endothelial cell surface and mRNA expression of cell adhesion molecules. Furthermore, the influence of advanced glycated fibronectin on the transendothelial migration pattern of polymorphonuclear cells was analyzed. RESULTS: Exposure to advanced glycated fibronectin together with inflammatory stimuli such as interleukin (IL)-1alpha, tumor necrosis factor-alpha (TNF-alpha) or lipopolysaccharide (LPS) led to a significant increase in the surface expression of the cell adhesion molecules E-selectin, ICAM-1, VCAM-1 and PECAM-1 on endothelial cells. Soluble AGEs in combination with advanced glycated fibronectin significantly enhanced the endothelial cell surface expression of ICAM-1, VCAM-1 and PECAM-1, whereas this was not the case for E-selectin. At the transcriptional level short-time exposure of endothelial cells to advanced glycated fibronectin and inflammatory mediators resulted in an increased expression of E-selectin, ICAM-1 and VCAM-1 mRNA levels, whereas PECAM-1 repeatedly showed a significant decrease of gene transcript levels. An increase of mRNA levels was also observed for E-selectin, ICAM-1, VCAM-1 and PECAM-1 following incubation with a combination of advanced glycated fibronectin and soluble advanced glycation end-products. Furthermore, polymorphonuclear cells responded with a sevenfold increase in transendothelial migration following exposure of endothelial cells to advanced glycated fibronectin and inflammatory mediators. CONCLUSIONS: These results suggest that the combination of matrix glycation and inflammation up-regulates the activation of the endothelial cell adhesion cascade, a mechanism that might contribute to the increased burden of atherosclerotic morbidity and mortality in patients suffering from diabetes mellitus or chronic renal failure.

Paracetamol catalyzes myeloperoxidase-initiated lipid oxidation in LDL.

The oxidative modification of LDL may play a significant role in atherogenesis. Myeloperoxidase (MPO) expressed in human atherosclerotic plaques has been suggested to be operative in vivo, making LDL atherogenic. Tyrosyl radicals generated by MPO have been shown to act as physiological pro-oxidants of lipid peroxidation in LDL. Assuming that a variety of phenolic compounds are able to form phenoxyl radicals when exposed to peroxidases, we tested the ability of paracetamol, a known analgesic drug with a tyrosine-like monophenolic structure, to act as a pro-oxidant of lipid peroxidation in LDL. Spectroscopic analyses indicated that paracetamol, similar to tyrosine, could undergo peroxidase-induced phenoxyl radical formation, which was inhibited by the radical scavenger ascorbic acid as well as by heme poisons and catalase. Measurement of conjugated dienes and lipid hydroperoxides in LDL preparations exposed to MPO/H2O2 in the absence or presence of paracetamol revealed that the drug could act as a catalyst of lipid oxidation in LDL. Similar results were found when LDL oxidation was performed with activated human neutrophils, which use MPO to promote lipid peroxidation. In conclusion, the results suggest that paracetamol could act, via a phenoxyl radical, as a catalyst of LDL oxidative modification by MPO.

Effects of aminoguanidine on adhesion molecule expression of human endothelial cells.

The effect of aminoguanidine (AG) on the expression of adhesion molecules on nonactivated human umbilical vein endothelial cells (HUVEC) was investigated in vitro. Nonactivated HUVEC cultivated on long-term glycated fibronectin (FN) as compared to native FN showed a significant upregulation of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and CD31 which could be further promoted by long-term glycated bovine serum albumin. AG, at a concentration of 0.01 mol/l, caused an upregulation of ICAM-1 of 48 +/- 17.4% in HUVEC cultivated on gelatin. In contrast, VCAM-1 and E-selectin remained unaffected. At this concentration, formation of advanced glycation end products (AGE) was inhibited by 57%, as determined immunologically, and by 50%, as verified by AGE-specific fluorescence. A hypothesis concerning the upregulation of ICAM-1 by AG as compared to VCAM-1 is proposed relating to its relative redox insensitivity. Our results demonstrate that the beneficial effect of AG in reducing the risk of accelerated development of atherosclerosis in diabetic patients by inhibiting formation of AGE on matrix proteins such as FN might be hampered by its tendency to upregulate ICAM-1 on endothelial cells.

Thrombin augments vascular cell-dependent migration of human mast cells: role of MGF.

Recent data suggest that auricular thrombosis is associated with accumulation of mast cells (MC) in the upper endocardium (where usually no MC reside) and local expression of MGF (mast cell growth factor) (25). In this study, the role of vascular cells, thrombin-activation and MGF, in MC-migration was analyzed. For this purpose, cultured human auricular endocardial cells (HAUEC), umbilical vein endothelial cells (HUVEC) and uterine- (HUTMEC) and skin-derived (HSMEC) microvascular endothelial cells were exposed to thrombin or control medium, and the migration of primary tissue MC (lung, n = 6) and HMC-1 cells (human MC-line) against vascular cells (supernatants) measured. Supernatants (24 h) of unstimulated vascular cells (monolayers of endocardium or endothelium) as well as recombinant (rh) MGF induced a significant migratory response in HMC-1 (control: 3025 +/- 344 cells [100 +/- 11.4%] vs. MGF, 100 ng/ml: 8806 +/- 1019 [291 +/- 34%] vs. HAUEC: 9703 +/- 1506 [320.8 +/- 49.8%] vs. HUTMEC: 8950 +/- 1857 [295.9 +/- 61.4%] vs. HSMEC: 9965 +/- 2018 [329.4 +/- 66.7%] vs. HUVEC: 9487 +/- 1402 [313.6 +/- 46.4%], p < 0.05) as well as in primary lung MC. Thrombin-activation (5 U/ml, 12 h) of vascular cells led to an augmentation of the directed migration of MC as well as to a hirudin-sensitive increase in MGF synthesis and release. Moreover, a blocking anti-MGF antibody was found to inhibit MC-migration induced by unstimulated or thrombin-activated vascular cells. Together, these data show that endocardial and other vascular cells can induce migration of human MC. This MC-chemotactic signal of the vasculature is associated with expression and release of MGF, augmentable by thrombin, and may play a role in the pathophysiology of (auricular) thrombosis.

Quantification of leukocyte migration: improvement of a method.

Eighteen different permeable membrane supports with and without confluent endothelial cell monolayers were incubated with normal donor derived neutrophils in the upper chambers of a 24 multiwell double chamber system. In order to study transmembrane or transendothelial leukocyte migration leukocytes were stimulated by chemoattractants, or endothelial cells were activated by IL-1. After coincubation the membrane supports building the upper chambers were discarded. Using this technique, leukocytes that had migrated into the lower chamber were exposed to the fluorescent dye calcein AM without additional washing or transfer steps. Absolute cell counts were determined computer assisted using dilution series of calcein AM labeled leukocytes as standards. Serial dilutions of neutrophils exposed to calcein AM showed reproducible linear fluorescence intensity, and relative fluorescence intensity correlated significant with cell counts (r2 = 0.974, p < 0.0001). Out of 18 membrane supports only one was suitable for our assay set up. Best technical and optical performance was achieved with a membrane made of polyethylene terephtalate with a pore size of 3 mm at a pore density of 0.8 x 10(6)/cm2. Stimulation of leukocytes or endothelium by FMLP or IL-1 revealed an increase of transendothelial migration to 7.2 +/- 1.8 x 10(5) PMN and 5.1 +/- 0.7 x 10(5) PMN respectively if compared with medium (0.6 +/- 0.2 x 10(5) PMN). IL-1 induced migration of neutrophils was inhibited by anti IL-1 autoantibodies derived from chronic renal failure patients (IL-1: 100% of PMN migrated, anti IL-1 antibody: 39% of PMN migrated, control antibody: 84% of PMN migrated). In summary, a simple fluorimetric assay was established for the quantification of transmembrane and transendothelial leukocyte migration.

Ibuprofen inhibits pyrogen-dependent expression of VCAM-1 and ICAM-1 on human endothelial cells.

Leukocyte adhesion and transmigration through the endothelial cell (EC) layer plays a crucial role in inflammation. IL-1 alpha and TNF alpha increase EC-adhesiveness for leukocytes by stimulating surface expression of ICAM-1 (intercellular adhesion molecule 1, CD54), VCAM-1 (vascular cell adhesion molecule 1, CD106) and E-selectin (CD62E). In this study, the effects of ibuprofen on IL-1 alpha and TNF alpha-induced expression of ICAM-1, VCAM-1 and E-selectin on cultured human umbilical vein EC (HUVEC) were analyzed. Exposure to IL-1 alpha or TNF alpha resulted in an increased expression of VCAM-1, ICAM-1, and E-selectin. Ibuprofen was identified as a potent inhibitor of IL-1 alpha and TNF alpha-induced surface expression of VCAM-1 and a less potent inhibitor of pyrogen-induced expression of ICAM-1, whereas no effect on E-selectin was found. The effects of ibuprofen on VCAM-1 expression were dose-dependent (IC50 [IL-1 alpha]: 0.5 mM; IC50 [TNF alpha]: 0.5 mM) and time-dependent with maximum responses observed after 18 h. Moreover, ibuprofen abrogated pyrogen-dependent adhesion of leukocytes to HUVEC. Ibuprofen also inhibited VCAM-1 mRNA expression in pyrogen activated EC. VCAM-1-downregulation on EC by ibuprofen may contribute to the anti-inflammatory actions of the drug.

Modulation of pyrogen-induced upregulation of endothelial cell adhesion molecules (CAMs) by interleukin-4: transcriptional mechanisms and CAM-shedding.

The pyrogens interleukin 1 (IL-1), tumor necrosis factor (TNF), and bacterial lipopolysaccharides (LPS) are known to increase endothelial cell (EC) adhesiveness for leukocytes by stimulating surface expression of various adhesion molecules. IL-4, a product of activated T-cells, was shown to affect pyrogen-mediated regulation of EC adhesion molecule surface expression. In the present study, we investigated the effect of IL-4 on pyrogen-induced upregulation of the cell adhesion molecules (CAMs) ICAM-1 (intercellular cell adhesion molecule-1), ELAM-1 (endothelial leucocyte adhesion molecule-1), and VCAM-1 (vascular cell adhesion molecule-1) in cultured human umbilical vein EC (HUVEC). Surface expression of adhesion molecules was quantified by flow cytometry, HUVEC mRNA content was estimated by Northern blot analysis, and ICAM-1 antigen in conditioned media was measured by ELISA. Incubation of HUVEC with IL-1 (100 U/ml), TNF (500 U/ml), and LPS (10 micrograms/ml) caused significant increase in ICAM-1, ELAM-1, and VCAM-1 surface expression; IL-1 caused about an eightfold increase in ICAM-1 expression, about a 13-fold increase in ELAM-1 surface expression, and about a fourfold increase in VCAM-1 expression. Coincubation of pyrogens with IL-4 (500 U/ml) differentially influenced their proadhesive effects on the HUVEC surface. In the presence of IL-4, IL-1-induced ICAM-1 upregulation was reduced, ELAM-1 upregulation was not significantly influenced by IL-4, and induction of VCAM-1 was enhanced by IL-4.(ABSTRACT TRUNCATED AT 250 WORDS)