Editor's Choice - The National Norwegian Carotid Study: Time from Symptom Onset to Surgery is too Long, Resulting in Additional Neurological Events.

OBJECTIVE/BACKGROUND: The objective was to observe for 1 year all patients in Norway operated on for symptomatic carotid stenosis with respect to (i) the time from the index event to surgery and neurological events during this time; (ii) the level in the healthcare system causing delay of surgical treatment; and (iii) the possible relationship between peri-operative use of platelet inhibitors and neurological events while awaiting surgery. METHODS: This was a prospective national multicentre study of a consecutive series of symptomatic patients. Patients were eligible for inclusion when referred for surgery. An index event was defined as the neurological event prompting contact with the healthcare system. All 15 departments in Norway performing carotid endarterectomy (CEA) participated. RESULTS: Three hundred and seventy one patients were eligible for inclusion between 1 April 2014 and 31 March 2015, and 368 patients (99.2%) were included. Fifty-four percent of the patients contacted their general practitioner on the day of the index event. Primary healthcare referred 84.2% of the patients to hospital on the same day as examined. In hospital median time from admission to referral for vascular surgery was 3 days. Median time between referral to the operating unit and actual CEA was 5 days. Overall, 61.7% of the patients were operated on within 2 weeks of the index event. Twelve patients (3.3%) suffered a new neurological event while awaiting surgery. The percentage of patients on dual antiplatelet therapy was lower (25.0%) in this group than among the other patients (62.6%) (p = .008). The combined 30 day mortality and stroke rate was 3.8%. CONCLUSION: This national study with almost complete inclusion and follow-up shows that the delays occur mainly at patient level and in hospital. The delay is associated with new neurological events. Dual antiplatelet therapy is associated with reduced risk of having a new neurological event before surgery.

HNA antibody-mediated neutrophil aggregation is dependent on serine protease activity.

BACKGROUND AND OBJECTIVES: Transfusion-related acute lung injury (TRALI) is often caused by antibodies against human neutrophil alloantigen-2 (HNA-2) and HNA-3a. Neutrophil aggregation is considered as a major cause of TRALI, but little is known about how HNA antibodies initiate this process. We explored mechanisms involved in neutrophil aggregation induced by HNA-2 and HNA-3a antibodies. MATERIALS AND METHODS: Isolated neutrophils were pretreated with broad-spectrum or specific inhibitors against different cell functions or proteases. Granulocyte agglutination test (GAT) was performed with serially diluted anti-HNA-2 and anti-HNA-3a plasmas or control plasma, and reactivity was evaluated microscopically. Reactive oxygen species (ROS) production in neutrophils was investigated using a lucigenin-based chemiluminescence assay. RESULTS: HNA-2 and HNA-3a antibody-mediated neutrophil aggregation was inhibited by pretreatment with formaldehyde, iodoacetamide and the serine protease inhibitors Pefabloc-SC, N-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and Nα-tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK). In contrast, inhibition of actin polymerization, respiratory burst, cysteine proteases, metalloproteases or aspartic proteases did not affect neutrophil aggregation. Furthermore, HNA-3a antibodies did not directly cause ROS production in neutrophils. CONCLUSION: Aggregation of neutrophils induced by HNA-2 and HNA-3a antibodies is an active process and depends on trypsin- or chymotrypsin-like serine proteases but is not dependent on the production of ROS. These findings may open new prospects for the pharmacologic prevention of neutrophil-associated acute lung injury.

RSK2 regulates endocytosis of FGF receptor 1 by phosphorylation on serine 789.

FGFR1 (fibroblast growth factor receptor 1) regulates many key cellular responses including proliferation, migration and differentiation through activation of signaling pathways. Irregularities in FGFR1 signaling have been implicated in several pathological conditions, including human cancer. In order to discover novel regulators of FGFR1 signaling, we performed yeast two-hybrid screens and identified RSK2 (p90 ribosomal S6 kinase 2) as a potential FGFR1 interaction partner. RSK2 belongs to the family of serine/threonine kinases that are activated through the Ras-MAPK signal transduction pathway. Both in vitro and in vivo experiments confirmed the interaction and we show that phosphorylated RSK2 binds to and phosphorylates serine 789 in the C-terminal tail of FGFR1. Inhibition of RSK2 activity led to prolonged tyrosine transphosphorylation of FGFR1. Furthermore, prevention of FGFR1 phosphorylation by inhibition of RSK2 activity or mutation of serine 789 to alanine reduced FGFR1 endocytosis and ubiquitination explaining mechanistically the prolonged signaling activity. We propose a novel regulatory mechanism whereby activated RSK2 directly interacts with and phosphorylates FGFR1, thereby modulating receptor signaling through regulation of endocytosis.

Does the great saphenous vein stripping improve arterial leg blood flow during exercise?

OBJECTIVES: It has been shown that the leg muscle pump increases arterial leg blood flow during upright exercise in healthy subjects, and that this effect is reduced in patients with incompetence of the great saphenous vein (GSV). In this study, patients with GSV reflux causing varicose veins were investigated after GSV stripping, to see whether the muscle pump effect on arterial leg blood flow is improved. DESIGN: Prospective case study. METHODS: Nine patients with GSV incompetence resulting in symptomatic varicose veins, but without peripheral artery disease were included in this study. Patients exercised in the supine and 30° head up tilted positions by rhythmically pressing down a pedal with one foot. Blood flow was measured in the femoral artery using Doppler ultrasound. The Exercise-induced rise in femoral artery blood flow was compared in the supine and 30° head up tilted positions. Patients were investigated both before and after undergoing saphenofemoral ligation and GSV stripping as a treatment for their varicose veins. The arterial blood flow response to exercise was compared between the pre and postoperative observations. RESULTS: Prior to GSV stripping the immediate rise in femoral flow was 0.25 l min(-1) above rest in both supine and tilted positions. After GSV stripping however, the rise in flow was 30% larger in the tilted position than in the supine position (0.26 vs. 0.20 l min(-1), P < 0.05). CONCLUSIONS: GSV stripping modestly improves arterial leg blood flow at the onset of exercise in patients with GSV insufficiency, because of an improved effect of the leg muscle pump.

Encapsulation of islets in rough surface, hydroxymethylated polysulfone capillaries stimulates VEGF release and promotes vascularization after transplantation.

The transplantation of encapsulated islets of Langerhans is one approach to treat type 1 diabetes without the need of lifelong immunosuppression. Capillaries have been used for macroencapsulation because they have a favorable surface-to-volume ratio and because they can be refilled. It is unclear at present whether the outer surface of such capillaries should be smooth to prevent, or rough to promote, cell adhesions. In this study we tested a new capillary made of modified polysulfone (MWCO: 50 kDa) with a rough, open-porous outer surface for islet transplantation. Compared with free-floating islets, encapsulation of freshly isolated rat islets affected neither the kinetics nor the efficiency of glucose-induced insulin release in perifusion experiments. Free-floating islets maintained insulin secretion during cell culture but encapsulated islets gradually lost their glucose responsiveness and released VEGF. This indicated hypoxia in the capillary lumen. Transplantation of encapsulated rat islets into diabetic rats significantly reduced blood glucose concentrations from the first week of implantation. This hypoglycaemic effect persisted until explantation 4 weeks later. Transplantation of encapsulated porcine islets into diabetic rats reduced blood glucose concentrations depending on the islet purity. With semipurified islets a transient reduction of blood glucose concentrations was observed (2, 8, 18, 18 days) whereas with highly purified islets a sustained normoglycaemia was achieved (more than 28 days). Explanted capillaries containing rat islets were covered with blood vessels. Vascularization was also observed on capillaries containing porcine islets that were explanted from normoglycaemic rats. In contrast, on capillaries containing porcine islets that were explanted from hyperglycemic rats a fibrous capsule and lymphocyte accumulations were observed. No vascularization on the surface of transplanted capillaries was observed in the absence of islets. In conclusion, encapsulated islets can release VEGF, which appears to be an important signal for the vascularization of the capillary material. The rough, open-porous outer surface of the polysulfone capillary provides a site well suited for vascular tissue formation and may allow a prolonged islet function after transplantation.

Three-dimensional ultrasound-based navigation combined with preoperative CT during abdominal interventions: a feasibility study.

PURPOSE: Three-dimensional (3D) intraoperative ultrasound may be easier to interpret when used in combination with less noisy preoperative image data such as CT. The purpose of this study was to evaluate the use of preoperative image data in a 3D ultrasound-based navigation system specially designed for minimally invasive abdominal surgery. A prototype system has been tested in patients with aortic aneurysms undergoing clinical assessment before and after abdominal aortic stent-graft implantation. METHODS: All patients were first imaged by spiral CT followed by 3D ultrasound scanning. The CT volume was registered to the patient using fiducial markers. This enabled us to compare corresponding slices from 3D ultrasound and CT volumes. The accuracy of the patient registration was evaluated both using the external fiducial markers (artificial landmarks glued on the patient's skin) and using intraoperative 3D ultrasound as a measure of the true positioning of anatomic landmarks inside the body. RESULTS: The mean registration accuracy on the surface was found to be 7.1 mm, but increased to 13.0 mm for specific landmarks inside the body. CT and ultrasound gave supplementary information of surrounding structures and position of the patient's anatomy. Fine-tuning the initial patient registration of the CT data with a multimodal CT to intraoperative 3D ultrasound registration (e.g., mutual information), as well as ensuring no movements between this registration and image guidance, may improve the registration accuracy. CONCLUSION: Preoperative CT in combination with 3D ultrasound might be helpful for guiding minimal invasive abdominal interventions.

Endovascular treatment of type B aortic dissections.

The purpose is to describe our experience with endovascular treatment of type B aortic dissections. Five patients were treated for complications following type B dissections like, false channel aneurysm formation, rupture and arterial obstruction. They were treated in general anaesthesia using a 'homemade' endoprosthesis or a commercially available endoprosthesis (Excluder) deployed during fluoroscopy. The patients have been followed at regular intervals with a median observation time of 18 months (range 12--36). One patient needed a secondary intervention due to dislodgement of the proximal stentgraft with haemorrhage into both the false and the true lumen. Otherwise there have been no early or late mortality or major complications in this series. Even if our experience with endovascular treatment of type B dissections is rather limited, the results so far are promising. Open surgery in many of these cases is complicated with high morbidity and mortality rate and the endovascular technique offers great advantages. A longer follow-up period is necessary to define the place of endovascular treatment.

Ability of the Tat basic domain and VP22 to mediate cell binding, but not membrane translocation of the diphtheria toxin A-fragment.

A number of proteins are able to enter cells from the extracellular environment, including protein toxins, growth factors, viral proteins, homeoproteins, and others. Many such translocating proteins, or parts of them, appear to be able to carry with them cargo into the cell, and a basic sequence from the HIV-1 Tat protein has been reported to provide intracellular delivery of several fused proteins. For evaluating the efficiency of translocation to the cytosol, this TAT-peptide was fused to the diphtheria toxin A-fragment (dtA), an extremely potent inhibitor of protein synthesis which normally is delivered efficiently to the cytosol by the toxin B-fragment. The fusion of the TAT-peptide to dtA converted the protein to a heparin-binding protein that bound avidly to the cell surface. However, no cytotoxicity of this protein was detected, indicating that the TAT-peptide is unable to efficiently deliver enzymatically active dtA to the cytosol. Interestingly, the fused TAT-peptide potentiated the binding and cytotoxic effect of the corresponding holotoxin. We made a fusion protein between VP22, another membrane-permeant protein, and dtA, and also in this case we detected association with cells in the absence of a cytotoxic effect. The data indicate that transport of dtA into the cell by the TAT-peptide and VP22 is inefficient.

Dependence of ricin toxicity on translocation of the toxin A-chain from the endoplasmic reticulum to the cytosol.

Ricin acts by translocating to the cytosol the enzymatically active toxin A-chain, which inactivates ribosomes. Retrograde intracellular transport and translocation of ricin was studied under conditions that alter the sensitivity of cells to the toxin. For this purpose tyrosine sulfation of mutant A-chain in the Golgi apparatus, glycosylation in the endoplasmic reticulum (ER) and appearance of A-chain in the cytosolic fraction was monitored. Introduction of an ER retrieval signal, a C-terminal KDEL sequence, into the A-chain increased the toxicity and resulted in more efficient glycosylation, indicating enhanced transport from Golgi to ER. Calcium depletion inhibited neither sulfation nor glycosylation but inhibited translocation and toxicity, suggesting that the toxin is translocated to the cytosol by the pathway used by misfolded proteins that are targeted to the proteasomes for degradation. Slightly acidified medium had a similar effect. The proteasome inhibitor, lactacystin, sensitized cells to ricin and increased the amount of ricin A-chain in the cytosol. Anti-Sec61alpha precipitated sulfated and glycosylated ricin A-chain, suggesting that retrograde toxin translocation involves Sec61p. The data indicate that retrograde translocation across the ER membrane is required for intoxication.

Gradual increase in leg oxygen uptake during repeated submaximal contractions in humans.

We examine whether muscle oxygen consumption (VO2) increases gradually during repeated submaximal isometric contractions. Six subjects made two-legged isometric quadriceps contractions at 30% maximal voluntary contraction for 6 s with 4 s of rest between until exhaustion (58 +/- 8 min). Blood samples were taken from the femoral vein and artery, and blood velocity was recorded by ultrasound-Doppler technique in the femoral artery. Blood flow was calculated from velocity and artery diameter values. Leg VO2 increased sixfold within the 1st min of exercise. A further doubling of the VO2 was seen during the remainder of the exercise, reaching 307 +/- 22 ml/min at exhaustion. This latter increase was due to a 54% increase in blood flow and a 34% increase in oxygen extraction. After 20 min of recovery VO2 was still 75% higher than preexercise values. The results show a twofold increase in energy demand of the working muscle during repeated constant-force isometric contractions. The increased energy cost of contraction is probably localized at the cellular level, and it parallels fatigue determined as decreased force-generating capacity.