A novel method for high-throughput detection and quantification of neutrophil extracellular traps reveals ROS-independent NET release with immune complexes

AbstractA newly-described first-line immune defence mechanism of neutrophils is the release of neutrophil extracellular traps (NETs). Immune complexes (ICxs) induce low level NET release. As such, the in vitro quantification of NETs is challenging with current methodologies. In order to investigate the role of NET release in ICx-mediated autoimmune diseases, we developed a highly sensitive and automated method for quantification of NETs. After labelling human neutrophils with PKH26 and extracellular DNA with Sytox green, cells are fixed and automatically imaged with 3-dimensional confocal laser scanning microscopy (3D-CLSM). NET release is then quantified with digital image analysis whereby the NET amount (Sytox green area) is corrected for the number of imaged neutrophils (PKH26 area). A high sensitivity of the assay is achieved by a) significantly augmenting the area of the well imaged (11%) as compared to conventional assays (0.5%) and b) using a 3D imaging technique for optimal capture of NETs, which are topologically superimposed on neutrophils. In this assay, we confirmed low levels of NET release upon human ICx stimulation which were positive for citrullinated histones and neutrophil elastase. In contrast to PMA-induced NET release, ICx-induced NET release was unchanged when co-incubated with diphenyleneiodonium (DPI). We were able to quantify NET release upon stimulation with serum from RA and SLE patients, which was not observed with normal human serum. To our knowledge, this is the first semi-automated assay capable of sensitive detection and quantification of NET release at a low threshold by using 3D CLSM. The assay is applicable in a high-throughput manner and allows the in vitro analysis of NET release in ICx-mediated autoimmune diseases

Clinical outcomes from a randomised comparison of Microwave Endometrial Ablation with Thermal Balloon endometrial ablation for the treatment of heavy menstrual bleeding

The definitive version is available at www3.interscience.wiley.comPeer reviewedPostprin

Ube2j2 ubiquitinates hydroxylated amino acids on ER-associated degradation substrates

An E2–E3 complex can ubiquitinate substrates via either an isopeptide bond (to a lysine) or an ester bond (to a serine or threonine) and preferentially uses the latter to induce ERAD

Low Mannose-Binding Lectin (MBL) genotype is associated with future cardiovascular events in type 2 diabetic South Asians. A prospective cohort study

Background: South Asians have a high burden of type 2 diabetes and vascular complications. Vascular inflammation is considered central in the pathophysiology of atherosclerosis, and the complement system is thought to play an important role. Mannose-Binding Lectin (MBL), which activates the lectin pathway of complement activation, has been introduced as a risk marker of vascular damage. The present study explores the association of MBL levels, genotype and cardiovascular events in type 2 diabetic South Asians.Methods: We conducted a prospective observational study. A cohort consisting of 168 type 2 diabetic South Asians was followed for a median duration of 7.66 years. At baseline, MBL levels and genotype were determined. The association with future cardiovascular events was assessed by Cox proportional hazard regression.Results: During follow-up, 31 cardiovascular events occurred in 22 subjects (11 men, 11 women). The O/O genotype was significantly associated with the occurrence of cardiovascular events (hazard ratio 3.42, 95%CI 1.24-9.49, P = 0.018). However, log MBL levels were not associated with the occurrence of cardiovascular events (hazard ratio 0.93, 95% CI 0.50-1.73).Conclusions: In type 2 diabetic South Asians, the O/O MBL genotype is associated with cardiovascular events, although single serum MBL levels are not

Global endometrial ablation: A modern day solution to an age‐old problem

Menorrhagia remains a significant health issue for women worldwide. Traditionally hysterectomy has been the treatment of choice when excessive menstrual bleeding remains unresolved by hormonal manipulation. In an attempt to provide a less invasive alternative to hysterectomy, traditional techniques such as rollerball endometrial ablation were developed 20 years ago. Although extremely effective, they possessed the potential of significant intra‐operative risks and their success depended on high technical proficiency of the surgeon. As surgery and technology evolved, second generation endometrial ablation devices were developed which demonstrated improved safety and efficacy rates that paralleled traditional treatments. Since 1997, the Food and Drug Administration (FDA) has approved five such devices for use in the United States. Each possesses a unique technology profile with supporting level I evidence that allows for the treatment of a wide variety of uterine anatomy.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/135279/1/ijgo156.pd

17β-Estradiol inhibits proliferation and migration of human vascular smooth muscle cells: similar effects in cells from postmenopausal females and in males

Objectives: Cardiovascular disease is rare in premenopausal women, but increases after the menopause when hormone replacement therapy reduces coronary events. Vascular smooth muscle cell (SMC) proliferation and migration occur in atherosclerosis, restenosis and venous graft disease. We studied the effects of 17β-estradiol on SMC proliferation and migration. Methods: SMC were cultured from saphenous veins of postmenopausal women and age-matched men. Cell growth was determined by 3H-thymidine incorporation and cell counting. Migration of SMC was assessed in 4-well chambers. SMC were seeded in one corner and PDGF-BB in filter paper glued onto the opposite wall. Results: PDGF-BB (5 ng/ml for 24 h) similarly stimulated 3H-thymidine incorporation in female (511 ± 57%; n = 8) and male (528 ± 62%; n = 12) SMC. This was reduced by 17β-estradiol (10−8-10−6 M; female 313 ± 52%; male 337 ± 54%; P < 0.05). PDGF-BB increased the number of SMC (P < 0.0001 at 10 days) obtained from females (153 ± 3%; n = 5) and males (150 ± 4%; n = 5), which was inhibited by 17 β-estradiol (10−6 M; female 134 ± 7%; male 128 ± 5%; P < 0.05). Similar results were obtained with basic fibroblast growth factor. In contrast to 17β-estradiol, another steroid (dexamethasone) had no effects on 3H-thymidine incorporation in these cells stimulated with PDGF-BB. PDGF-BB (0.01-1 ng) stimulated SMC migration (P < 0.05) which was inhibited by 17β-estradiol (10−10-10−6 M; n = 5; P < 0.005). Conclusion: 17β-Estradiol inhibits growth-factor-induced SMC proliferation and migration regardless of gender. These effects of 17β-estradiol may contribute to its cardiovascular protective properties in postmenopausal women during replacement therap

Imaging the renal microcirculation in cell therapy

Renal microvascular rarefaction plays a pivotal role in progressive kidney disease. Therefore, modalities to visualize the microcirculation of the kidney will increase our understanding of disease mechanisms and consequently may provide new approaches for evaluating cell-based therapy. At the moment, however, clinical practice is lacking non-invasive, safe, and efficient imaging modalities to monitor renal microvascular changes over time in patients suffering from renal disease. To emphasize the importance, we summarize current knowledge of the renal microcirculation and discussed the involvement in progressive kidney disease. Moreover, an overview of available imaging techniques to uncover renal microvascular morphology, function, and behavior is presented with the associated benefits and limitations. Ultimately, the necessity to assess and investigate renal disease based on in vivo readouts with a resolution up to capillary level may provide a paradigm shift for diagnosis and therapy in the field of nephrology.Nephrolog