Nanopore device and a method for nucleic acid analysis

A nanopore device is described wherein is provided a sample input (110), an input chamber (120), and first and second sample chambers (130, 140) connected to the input chambers (120) via first and second nanopores (135, 145)

HDL cholesterol levels are an important factor for determining the lifespan of erythrocytes

Scavenger receptor class B, type I (SR-BI) is a multifunctional receptor that promotes the selective uptake of cholesteryl esters from high-density lipoprotein (HDL). Disruption of SR-BI in mice results in a dramatic increase in HDL cholesterol. Interestingly, mice lacking SR-BI also develop anemia, as evidenced by accumulation of reticulocytes in the circulation. The objective of the current study was to delineate the mechanism underlying development of anemia in the absence of SR-BI. Expression of important mediators of erythropoiesis, as well as key enzymes in the degradation of erythrocytes, were analyzed using real-time polymerase chain reaction in SR-BI wild-type and SR-BI knockout mice. In addition, in vivo studies were performed using biotinylated erythrocytes to determine erythrocyte survival. mRNA expression of TAL-1, GATA-1, FOG-1, erythropoietin receptor, and ferrochelatase, important mediators of erythropoiesis, was increased in spleens of SR-BI-deficient mice. In addition, the relative amount of early Ter119(high)CD71(high) -expressing erythroblasts was increased in SR-BI-deficient spleens. Interestingly, also expression of hemeoxygenase 1 and biliverdin reductase, enzymes involved in the degradation of erythrocytes, was increased. Furthermore, an elevated amount of conjugated bilirubin, the breakdown product of hemoglobin, was found in bile. Using biotinylated erythrocytes, we show that survival of erythrocytes was decreased in SR-BI-deficient mice. Thus, the observed increased erythropoiesis in the SR-BI-deficient mice is most likely a direct response to the reduced erythrocyte lifespan. Finally, we show that increased HDL cholesterol levels due to SR-BI deficiency induce erythrocyte cholesterol:phospholipid ratios, resulting in decreased deformability and increased osmotic fragility, thereby providing an explanation for the observed reduced lifespan. SR-BI is not only essential for HDL cholesterol homeostasis and atherosclerosis susceptibility, but also for maintaining normal erythrocyte lifespa

Repopulation of apolipoprotein E knockout mice with CCR2-deficient bone marrow progenitor cells does not inhibit ongoing atherosclerotic lesion development

OBJECTIVE: Using bone marrow transplantation, we have previously demonstrated the critical role that hematopoietic CCR2 plays in early atherogenesis. Reconstitution of irradiated apolipoprotein (apo) E3-Leiden mice with CCR2-deficient bone marrow progenitor cells resulted in 86% reduction on overall atherosclerotic lesion development. However, no data on CCR2 in the cause of established atherosclerosis have been reported so far. METHODS AND RESULTS: To study the role of CCR2 in established atherosclerotic lesions, bone marrow progenitor cells harvested from apoE-/- and apoE-/-/CCR2-/- mice were transplanted into lethally irradiated 16-week-old apoE-/- mice with established atherosclerotic lesions. No significant differences were found in serum total cholesterol and triglycerides levels at different time points after transplantation. At age 16 weeks, lesion size in control apoE-/- mice was 3.28+/-1.06x10(5) microm2. At 9 weeks after transplantation, apoE-/---> apoE-/- and apoE-/-/CCR2-/---> apoE-/- mice had developed significantly larger atherosclerotic lesions (4.49+/-0.92x10(5) microm2, P <0.02 and 4.15+/-0.62x10(5) microm2, P <0.04 compared with controls, respectively). However, no significant effect of disruption of hematopoietic CCR2 was observed on the progression of lesions. Furthermore, the macrophage positive area (78+/-4% versus 72+/-9%) and collagen content (11+/-6% versus 15+/-3%) of the lesions were similar as well. CONCLUSIONS: In contrast to the critical role of CCR2 in the initiation of atherogenesis, bone marrow progenitor cell-derived CCR2 does not influence the progression of established atherosclerotic lesions, pointing to additional mechanisms for recruitment of monocytes at later stages of lesion developmen

Vaccination using oxidized low-density lipoprotein-pulsed dendritic cells reduces atherosclerosis in LDL receptor-deficient mice

Aims Modification of lipoproteins plays an important role in the development of atherosclerosis. Oxidatively modified low-density lipoprotein (oxLDL) has a number of pro-inflammatory effects, whereas immunization with various forms of oxLDL is able to reduce atherosclerosis. The uptake of modified LDL by dendritic cells (DCs) and the presentation of epitopes thereof may form an important step in the immunomodulatory effects of LDL. In this study, we transferred oxLDL-pulsed mature DCs (mDCs) to LDL receptor-null (LDLr-/-) mice and examined the effects on atherosclerosis. Methods and results Bone marrow-derived DCs were cultured for 10 days in the presence of granulocyte-macrophage colony-stimulating factor. Immature DCs were matured by lipopolysaccharide and pulsed with copper-oxidized LDL. These mDCs were transferred three times to LDLr-/- mice before the induction of atherosclerosis by Western-type diet feeding. The transfer of oxLDL-pulsed mDCs resulted in an 87% reduction in carotid artery lesion size (P < 0.001) with a concurrent increase in plaque stability, whereas treatment using mDCs pulsed with the atherosclerosis-irrelevant antigen, ovalbumin, did not influence lesion size or stability. Furthermore, the vaccination procedure resulted in the induction of oxLDL-specific T cells with a reduced Th1 profile and an increase in oxLDL-specific IgG levels, which contributed to a reduction in foam cell formation. Conclusion These data indicate that vaccination with oxLDL-pulsed mDCs provides a novel and powerful strategy for the immunomodulation of atherosclerosis.Pathophysiology and treatment of rheumatic disease

Comparison of lumen-apposing metal stents versus double-pigtail plastic stents for infected necrotising pancreatitis

Objective Lumen-apposing metal stents (LAMS) are believed to clinically improve endoscopic transluminal drainage of infected necrosis when compared with double-pigtail plastic stents. However, comparative data from prospective studies are very limited. Design Patients with infected necrotising pancreatitis, who underwent an endoscopic step-up approach with LAMS within a multicentre prospective cohort study were compared with the data of 51 patients in the randomised TENSION trial who had been assigned to the endoscopic step-up approach with double-pigtail plastic stents. The clinical study protocol was otherwise identical for both groups. Primary end point was the need for endoscopic transluminal necrosectomy. Secondary end points included mortality, major complications, hospital stay and healthcare costs. Results A total of 53 patients were treated with LAMS in 16 hospitals during 27 months. The need for endoscopic transluminal necrosectomy was 64% (n=34) and was not different from the previous trial using plastic stents (53%, n=27)), also after correction for baseline characteristics (OR 1.21 (95% CI 0.45 to 3.23)). Secondary end points did not differ between groups either, which also included bleeding requiring intervention-5 patients (9%) after LAMS placement vs 11 patients (22%) after placement of plastic stents (relative risk 0.44; 95% CI 0.16 to 1.17). Total healthcare costs were also comparable (mean difference -euro6348, bias-corrected and accelerated 95% CI -euro26 386 to euro10 121). Conclusion Our comparison of two patient groups from two multicentre prospective studies with a similar design suggests that LAMS do not reduce the need for endoscopic transluminal necrosectomy when compared with double-pigtail plastic stents in patients with infected necrotising pancreatitis. Also, the rate of bleeding complications was comparable