GLUT2 surface expression and intracellular transport via the constitutive pathway in pancreatic beta cells and insulinoma: evidence for a block in trans-Golgi network exit by brefeldin A.

The biosynthesis, intracellular transport, and surface expression of the beta cell glucose transporter GLUT2 was investigated in isolated islets and insulinoma cells. Using a trypsin sensitivity assay to measure cell surface expression, we determined that: (a) greater than 95% of GLUT2 was expressed on the plasma membrane; (b) GLUT2 did not recycle in intracellular vesicles; and (c) after trypsin treatment, reexpression of the intact transporter occurred with a t1/2 of approximately 7 h. Kinetics of intracellular transport of GLUT2 was investigated in pulse-labeling experiments combined with glycosidase treatment and the trypsin sensitivity assay. We determined that transport from the endoplasmic reticulum to the trans-Golgi network (TGN) occurred with a t1/2 of 15 min and that transport from the TGN to the plasma membrane required a similar half-time. When added at the start of a pulse-labeling experiment, brefeldin A prevented exit of GLUT2 from the endoplasmic reticulum. When the transporter was first accumulated in the TGN during a 15-min period of chase, but not following a low temperature (22 degrees C) incubation, addition of brefeldin A (BFA) prevented subsequent surface expression of the transporter. This indicated that brefeldin A prevented GLUT2 exit from the TGN by acting at a site proximal to the 22 degrees C block. Together, these data demonstrate that GLUT2 surface expression in beta cells is via the constitutive pathway, that transport can be blocked by BFA at two distinct steps and that once on the surface, GLUT2 does not recycle in intracellular vesicles.

Disparate activation of the coronary sinus and inferior left atrium during atrial tachycardia after persistent atrial fibrillation ablation: prevalence, pitfalls, and impact on mapping.

INTRODUCTION: Persistent atrial fibrillation (AF) ablation may lead to partial disconnection of the coronary sinus (CS). As a result, disparate activation sequences of the local CS versus contiguous left atrium (LA) may be observed during atrial tachycardia (AT). We aimed to evaluate the prevalence of this phenomenon and its impact on activation mapping. METHODS: AT occurring after persistent AF ablation were investigated in 74 consecutive patients. Partial CS disconnection during AT was suspected when double potentials with disparate activation sequences were observed on the CS catheter. Endocardial mapping facing CS bipoles was performed to differentiate LA far-field from local CS potentials. RESULTS: A total of 149 ATs were observed. Disparate LA-CS activations were apparent in 20 ATs after magnifying the recording scale (13%). The most common pattern (90%) was distal to proximal endocardial LA activation against proximal to distal CS activation, the latter involving the whole CS or its distal part. Perimitral macroreentry was more common when disparate LA-CS activations were observed (67% vs 29%; P = 0.002). Partial CS disconnection also resulted in "pseudo" mitral isthmus (MI) block during LA appendage pacing in 20% of patients as local CS activation was proximal to distal despite distal to proximal activation of the contiguous LA. CONCLUSION: Careful analysis of CS recordings during AT following persistent AF ablation often reveals disparate patterns of activation. Recognizing when endocardial LA activation occurs in the opposite direction to the more obvious local CS signals is critical to avoid misleading interpretations during mapping of AT and evaluation of MI block.

IVA in addition to BMD can change the osteoporosis management in 25% of clinical routine patients

Introduction: Vertebral fracture is one of the major osteoporotic fractures which are unfortunately very often undetected. In addition, it is well known that prevalent vertebral fracture increases dramatically the risk of future additional fracture. Instant Vertebral Assessment (IVA) has been introduced in DXA device couple years ago to ease the detection of such fracture when routine DXA are performed. To correctly use such tool, ISCD provided clinical recommendation on when and how to use it. The aim of our study was to evaluate the ISCD guidelines in clinical routine patients and see how often it may change of patient management. Methods: During two months (March and April 2010), a medical questionnaire was systematically given to our clinical routine patient to check the validity of ISCD IVA recommendations in our population. In addition, all women had BMD measurement at AP spine, Femur and 1/3 radius using a Discovery A System (Hologic, Waltham, USA). When appropriate, IVA measurement had been performed on the same DXA system and had been centrally evaluated by two trained Doctors for fracture status according to the semi-quantitative method of Genant. The reading had been performed when possible between L5 and T4. Results: Out of 210 women seen in the consultation, 109 (52%) of them (mean age 68.2 ± 11.5 years) fulfilled the necessary criteria to have an IVA measurement. Out of these 109 women, 43 (incidence 39.4%) had osteoporosis at one of the three skeletal sites and 31 (incidence 28.4%) had at least one vertebral fracture. 14.7% of women had both osteoporosis and at least one vertebral fracture classifying them as "severe osteoporosis" while 46.8% did not have osteoporosis nor vertebral fracture. 24.8% of the women had osteoporosis but no vertebral fracture while 13.8% of women did have osteoporosis and vertebral fracture (clinical osteoporosis). Conclusion: In conclusion, in 52% of our patients, IVA was needed according to ISCD criteria. In half of them the IVA test influenced of patient management either by changing the type of treatment of simply by classifying patient as "clinical osteoporosis". IVA appears to be an important tool in clinical routine but unfortunately is not yet very often used in most of the centers.

Respiratory self-navigated postcontrast whole-heart coronary MR angiography: initial experience in patients.

PURPOSE: To assess the diagnostic performance of respiratory self-navigation for whole-heart coronary magnetic resonance (MR) angiography in a patient cohort referred for diagnostic cardiac MR imaging. MATERIALS AND METHODS: Written informed consent was obtained from all participants for this institutional review board-approved study. Self-navigated coronary MR angiography was performed after administration of a contrast agent in 78 patients (mean age, 48.5 years ± 20.7 [standard deviation]; 53 male patients) referred for cardiac MR imaging because of coronary artery disease (n = 40), cardiomyopathy (n = 14), congenital anomaly (n = 17), or "other" (n = 7). Examination duration was recorded, and the image quality for each coronary segment was assessed with consensus reading. Vessel sharpness, length, and diameter were measured. Quantitative values in proximal, middle, and distal segments were compared by using analysis of variance and t tests. A double-blinded comparison with the results of x-ray angiography was performed when such results were available. RESULTS: When patients with different indications for cardiac MR imaging were examined with self-navigated postcontrast coronary MR angiography, whole-heart data sets with 1.15-mm isotropic spatial resolution were acquired in an average of 7.38 minutes ± 1.85. The main and proximal coronary segments could be visualized in 92.3% of cases, while the middle and distal segments could be visualized in 84.0% and 55.8% of cases, respectively. Subjective scores and vessel sharpness were significantly higher in the proximal segments than in the middle and distal segments (P < .05). Anomalies of the coronary arteries could be confirmed or excluded in all cases. Per-vessel sensitivity and specificity for stenosis detection were 64.7% and 85.0%, respectively, in the 31 patients for whom reference standard x-ray coronary angiography results were available. CONCLUSION: The self-navigated coronary MR angiography sequence shows promise for coronary imaging. However, technical improvements are needed to improve image quality, especially in the more distal coronary segments.

IVA in addition to BMD can change the osteoporosis management in 25% of clinical routine patients.

Vertebral fracture is one of the major osteoporotic fractures which are unfortunately very often undetected. In addition, it is well known that prevalent vertebral fracture increases dramatically the risk of future additional fracture. Instant Vertebral Assessment (IVA) has been introduced in DXA device couple years ago to ease the detection of such fracture when routine DXA are performed. To correctly use such tool, ISCD provided clinical recommendation on when and how to use it. The aim of our study was to evaluate the ISCD guidelines in clinical routine patients and see how often it may change of patient management. During two months (March and April 2010), a medical questionnaire was systematically given to our clinical routine patient to check the validity of ISCD IVA recommendations in our population. In addition, all women had BMD measurement at AP spine, Femur and 1/3 radius using a Discovery A System (Hologic, Waltham, USA). When appropriate, IVA measurement had been performed on the same DXA system and had been centrally evaluated by two trained Doctors for fracture status according to the semi-quantitative method of Genant. The reading had been performed when possible between L5 and T4. Out of 210 women seen in the consultation, 109 (52%) of them (mean age 68.2±11.5 years) fulfilled the necessary criteria to have an IVA measurement. Out of these 109 women, 43 (incidence 39.4%) had osteoporosis at one of the three skeletal sites and 31 (incidence 28.4%) had at least one vertebral fracture. 14.7% of women had both osteoporosis and at least one vertebral fracture classifying them as "severe osteoporosis" while 46.8% did not have osteoporosis not vertebral fracture. 24.8% of the women had osteoporosis but no vertebral fracture while 13.8% of women did have osteoporosis but vertebral fracture (Clinical osteoporosis). In conclusion, in 52% of our patients, IVA was needed according to ISCD criteria. In half of them the IVA test influenced of patient management either my changing the type of treatment of simply by classifying patient as "clinical osteoporosis". IVA appears to be an important tool in clinical routine but unfortunately is not yet very often use in most of the centers.

The effect of COX-2 inhibitors on the formation of heterotopic bone in a rat model : O1323

Aims: 1) to create a new and reproducible animal model to produce heterotopic ossification (HO) 2) to be able to exactly quantify the amount of HO using a microCT scan and 3) to prove the hypothesis that COX-2 inhibitors are efficacious in the prevention of HO. Methods: We developed a IACUC-approved Lewis rat model, in which the ventral side of the right femur was scraped to mechanically disrupt the periosteum. By clamping the vastus intermedius ischemic injury to the muscle was produced to enhance HO. Finally homologous bone marrow from a donor rat was placed on the anterior surface of the femur. Half of the study group (8 rats) received chow mixed with a COX-2 inhibitor, while the other half received normal chow. After 6 weeks the animals were sacrificed, the femurs removed and imaged by microCT. Grading of HO was based on the thickness of ectopic bone as evaluated in a blinded fashion by 3 independent observers. Results: All animals developed bilateral HO. Rats treated with COX-2 inhibitors developed significantly less ectopic bone than the control group rats. Conclusions: The results suggest that we have created a very reliable, reproducible model to form ectopic bone in rats. Using the microCT we can precisely quantify the amount of HO. We have been able to show that COX-2 inhibitors significantly decrease the amount of HO formation and are thus a good alternative to non-specific NSAIDs with their potential serious side effects on the gastrointestinal tract and on hemo-stastis.

La nappe du Lebendun entre Alte Kaserne et le Val Cairasca (massif du Simplon): nouvelles observations et interprétations

The lithostratigraphic description of the covers of three Lower Penninic nappes (Monte Leone, Lebendun and Antigorio) allows the comparison of their sedimentary content and their thickness. It has been established that the Lebendun nappe is formed by an ante-Triassic paragneissic core (Valgrande gneiss), and a Mesozoic sedimentary cover in reversed position. The cover series shows a continuous detritic sedimentation, off which the material comes from a continental erosion related to the early Lias rifting phase of the Alpine Tethys. The erosion has reached the basement, resedimented as pebbles and sandstones. This can be observed in both Lebendun and Antigorio covers. The definition of a unit named <<serie intermediaire>> between the Lebendun and the Antigorio covers has important palinspastic implications for both nappes. The unit is composed of a banded marble, a garnet bearing gneiss and a calcschist with great blocks. The comparison between the thickness of Antigorio and Lebendun covers suggests a shoulder position for Antigorio. and a proximal rift basin position tor Lebendun. The general thickness decrease of the series towards the SW points to a NE origin for the Lebendun clastics, taking into account the increase of tectonic deformation in the region trending from east to west. The detritic sedimentation ends with the basin drowning during the Malm, represented by a pure marble sealing the erosive disconformity of the Antigorio cover, and the clastic deposits of Lebendun. Three hypotheses are proposed for the calcschists age and attribution of the <<serie intermediaire>>: A: they belong entirely or partially to the Lebendun cover and correspond to a conglomeratic deposit of Cretaceous-Tertiary Niesen flysch type, of proximal facies. The tectonic limit could be situated in the middle of the calcschists at the level of the huge blocks encountered. B: they belong to Antigorio and correspond to an upper Lias-Dogger synrift deposit, then the marble is liassic. C: they belong to Antigorio and have been deposited following the Lebendun basin inversion (Cretaceous-Tertiary). that generates Tertiary wildflysch deposits, coming from the South for the ultrahelvetic and from the North for the Niesen.

Human CD8(+) T cells engineered with supraphysiological TCRs are functionally inhibited via PD-1 and SHP-1 phosphatase

Purpose/Objective: Protective CD8+ T cell responses rely on TCRdependent recognition of immunogenic peptides presented by MHC I. Cytolytic T lymphocytes directed against self/tumor antigens express TCRs of lower affinity/avidity than pathogen-derived T lymphocytes and elicit less protective immune responses due to mechanisms of central and peripheral tolerance. Anti-tumor T cell reactivity can be improved by increasing the TCR-pMHC affinity within physiological limits, while intriguingly further increase in the supraphysiological range (KD < 1 lM) leads to drastic functional declines. We aim at identifying the molecular mechanisms underlying the loss of T cell responsiveness associated with supraphysiological TCRpMHC affinities in order to improve effectiveness of TCR-engineered T cells used in adoptive cell transfer (ACT) cancer immunotherapy. Materials and methods: Using a panel of human CD8+ T cells engineered with TCRs of incremental affinity for the HLA-A2-resticted tumor cancer testis antigen NY-ESO-1, we performed comparative gene expression microarray and TCR-mediated signaling analysis together with membrane receptors level analysis. Results: As compared to cells expressing TCR affinities generating optimal function (KD from 5to 1 lM), those with supraphysiological affinity (KD from 1 lM to 15 nM) had an overall reduced expression of genes implied in signaling, cell activation and proliferation, and showed impaired proximal and distal TCR signaling capacity. This correlated with a decline in surface expression of CD8b, CD28 and activatory TNFR superfamily members. Importantly, expression of inhibitory receptor PD-1 and SHP-1 phosphatase was upregulated in a TCR affinity-dependent manner. Consequently, PD-L1 and SHP-1 blockade restored the function of T cells with high TCRs affinity. Moreover, SHP-1 inhibition also augmented functional efficacy of T cells with TCRs of optimal affinity. Conclusions: Our findings indicate that TCR affinity-associated regulatory mechanisms control T cells responsiveness at various levels to limit potential auto-reactive cytotoxic effects. They also support the development of ACT therapies combined with blockade of inhibitory molecules such as SHP-1 to enhance effectiveness of T cell immunotherapy.

Long gamma nail in the treatment of subtrochanteric fractures.

INTRODUCTION: The purpose of our study was to retrospectively evaluate the clinical and radiological results of subtrochanteric fractures treated with a long gamma nail (LGN). The LGN has been the implant of choice at our level-1 trauma center since 1992. MATERIALS AND METHODS: Over a period of 7 years, we have treated 90 consecutive patients with subtrochanteric fractures. In order to evaluate the clinical and radiological outcomes, we reviewed the clinical and radiographic charts of these patients followed for a mean time of 2 years (range 13-36 months). RESULTS: We found no intra- or perioperative complications nor early or late infection. Clinical and radiological union was achieved at a mean of 4.3 months in all of the patients (range 3-9 months); in 24 cases (30%) the distal locking bolts were retrieved in order to enhance callus formation and remodeling as a planned secondary surgery. Three patients (3.3%) needed unplanned secondary surgery for problems related to the nailing technique. Two mechanical failures with breakage of the nail were encountered due to proximal varus malalignment, of which one was treated with exchange nailing and grafting and the other one by removal of the broken hardware, blade-plating, and bone grafting. One fracture below a short LGN was treated by exchange nailing. CONCLUSIONS: The minimally invasive technique and simple application of the LGN lead to a low percentage of complications in these difficult fractures after a relatively short learning curve. The biomechanical properties of this implant allow early mobilization and partial weight-bearing even in patients with advanced osteoporosis.

Peroxisome proliferator-activated receptor beta/delta exerts a strong protection from ischemic acute renal failure.

Ischemic acute renal failure is characterized by damages to the proximal straight tubule in the outer medulla. Lesions include loss of polarity, shedding into the tubule lumen, and eventually necrotic or apoptotic death of epithelial cells. It was recently shown that peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) increases keratinocyte survival after an inflammatory reaction. Therefore, whether PPARbeta/delta could contribute also to the control of tubular epithelium death after renal ischemia/reperfusion was tested. It was found that PPARbeta/delta+/- and PPARbeta/delta-/- mutant mice exhibited much greater kidney dysfunction and injury than wild-type counterparts after a 30-min renal ischemia followed by a 36-h reperfusion. Conversely, wild-type mice that were given the specific PPARbeta/delta ligand L-165041 before renal ischemia were completely protected against renal dysfunction, as indicated by the lack of rise in serum creatinine and fractional excretion of Na+. This protective effect was accompanied by a significant reduction in medullary necrosis, apoptosis, and inflammation. On the basis of in vitro studies, PPARbeta/delta ligands seem to exert their role by activating the antiapoptotic Akt signaling pathway and, unexpectedly, by increasing the spreading of tubular epithelial cells, thus limiting potentially their shedding and anoikis. These results point to PPARbeta/delta as a remarkable new target for preconditioning strategies.

Tibio-talar arthrodesis with long anterograde nail after complex open fracture-dislocation of the ankle : P17

Introduction: Posttraumatic painful osteoarthritis of the ankle joint after fracture-dislocation often has to be treated with arthrodesis. In the presence of major soft tissue lesions and important bone loss the technique to achieve arthrodesis has to be well chosen in order to prevent hardware failure, infection of bulky implants or non-union. Methods: We present the case of a 53 year-old biker suffering of a fracture-dislocation of the ankle associated with a mayor degloving injury of the heel. After initial immobilization of the lesion by external fixation in Spain the patient was transferred to our hospital for further treatment. The degloving injury of the heel with MRSA infection was initially treated by repeated débridement, changing of the configuration of the Ex Fix and antibiotic therapy with favourable outcome. Because of the bony lesions reconstruction of the ankle-joint was juged not to be an option and arthrodesis was planned. Due to bad soft-tissue situation standard open fixtion with plate and/or screws was not wanted but an option for intramedullary nailing was taken. However the use of a standard retrograde arthrodesis nail comes with two problems: 1) Risk of infection of the heel-part of the calaneus/nail in an unstable soft tissue situation with protruding nail. And 2) talo-calcaneal arthrodesis of an initially healthy subtalar joint. Given the situation of an unstable plantar/heel flap it was decided to perform anklearthrodesis by means of an anterograde nail with static fixation in the talus and in the proximal tibia. Results:This operation was performed with minimal opening at the ankle-site in order to remove the remaining cartilage and improve direct bone to bone contact. Arthrodesis was achieved by means of an anterograde T2 Stryker tibial nail.One year after the anterograde nailing the patient walks without pain for up to 4 hours with a heel of good quality and arthrodesis is achieved. Conclusion: Tibiotalar arthrodesis in the presence of mayor soft tissue lesions and bone loss can be successfully achieved with antegrade nailing.

Medial femoral condyle fracture as a complication of antegrade intramedullary nailing.

A 49-year-old man suffered a closed oblique fracture of the middle third of his left femur. Closed reduction and internal fixation by intramedullary (IM) nailing were performed. Per-operative fluoroscopic imaging and initial postoperative X-rays were judged normal and the patient followed the usual rehabilitation protocol. At 3-month follow-up the patient still demonstrated poor knee function and pain. A plain X-ray and a CT scan of the left knee revealed a displaced fracture of the medial femoral condyle. Analysis of the postoperative imaging suggests that the fracture occurred during the insertion of the IM nail. The nail possibly hit the Steinmann traction pin in the distal femur causing the medial condyle fracture. The patient was reoperated; open reduction and internal plate and screw fixation were performed with satisfactory clinical progress postoperatively. The description and illustration of this case is intended to make trauma surgeons aware of this rare but serious complication of IM femoral nailing.

Early Cretaceous life, climate and anoxia

Early Cretaceous life and the environment were strongly influenced by the accelerated break up of Pangaea, which was associated with the formation of a multitude of rift basins, intensified spreading, and important volcanic activity on land and in the sea. These processes likely interacted with greenhouse conditions, and Early Cretaceous climate oscillated between "normal" greenhouse, predominantly arid conditions, and intensified greenhouse, predominantly humid conditions. Arid conditions were important during the latest Jurassic and early Berriasian, the late Barremian, and partly also during the late Aptian. Humid conditions were particularly intense and widespread during shorter episodes of environmental change (EECs): the Valanginian Weissert, the latest Hauterivian Faraoni, the latest Barremian earliest Aptian Taxy, the early Aptian Selli, the early late Aptian Fallot and the late Aptian-early Albian Paquier episodes. Arid conditions were associated with evaporation, low biogeochemical weathering rates, low nutrient fluxes, and partly stratified oceans, leading to oxygen depletion and enhanced preservation of laminated, organic-rich mud (LOM). Humid conditions enabled elevated biogeochemical weathering rates and nutrient fluxes, important runoff and the buildup of freshwater lids in proximal basins, intensified oceanic and atmospheric circulation, widespread upwelling and phosphogenesis, important primary productivity and enhanced preservation of LOM in expanded oxygen-minimum zones. The transition of arid to humid climates may have been associated with the net transfer of water to the continent owing to the infill of dried-out groundwater reservoirs in internally drained inland basins. This resulted in shorter-term sea-level fall, which was followed by sea-level rise. These sea-level changes and the influx of freshwater into the ocean may have influenced oxygen-isotope signatures. Climate change preceding and during the Early Cretaceous EECs may have been rapid, but in general, the EECs had a "pre"-history, during which the stage was set for environmental change. Negative feedback on the climate through increased marine LOM preservation was unlikely, because of the low overall organic-carbon accumulation rates during these episodes. Life and climate co-evolved during the Early Cretaceous. Arid conditions may have affected continental life, such as across the Tithonian/Berriasian boundary. Humid conditions and the corresponding tendency to develop dys- to anaerobic conditions in deeper ocean waters led to phases of accelerated extinction in oceans, but may have led to more luxuriant vegetation cover on continents, such as during the Valanginian, to the benefit of herbivores. During Early Cretaceous EECs, reef systems and carbonate platforms in general were particularly vulnerable. They were the first to disappear and the last to recover, often only after several million years. (C) 2011 Elsevier Ltd. All rights reserved.

Causal mechanisms underlying host specificity in bat ectoparasites.

In parasites, host specificity may result either from restricted dispersal capacity or from fixed coevolutionary host-parasite adaptations. Knowledge of those proximal mechanisms leading to particular host specificity is fundamental to understand host-parasite interactions and potential coevolution of parasites and hosts. The relative importance of these two mechanisms was quantified through infection and cross-infection experiments using mites and bats as a model. Monospecific pools of parasitic mites (Spinturnix myoti and S. andegavinus) were subjected either to individual bats belonging to their traditional, native bat host species, or to another substitute host species within the same bat genus (Myotis). The two parasite species reacted differently to these treatments. S. myoti exhibited a clear preference for, and had a higher fitness on, its native host, Myotis myotis. In contrast, S. andegavinus showed no host choice, although its fitness was higher on its native host M. daubentoni. The causal mechanisms mediating host specificity can apparently differ within closely related host-parasite systems.

LDLs stimulate p38 MAPKs and wound healing through SR-BI independently of Ras and PI3 kinase.

Intracellular signals elicited by LDLs are likely to play a role in the pathogenesis associated with increased LDL blood levels. We have previously determined that LDL stimulation of human skin fibroblasts, used as a model system for adventitial fibroblasts, activates p38 mitogen-activated protein kinases (MAPKs), followed by IL-8 production and increased wound-healing capacity of the cells. The proximal events triggering these responses had not been characterized, however. Here we show that MAPK kinases MKK3 and MKK6, but not MKK4, are the upstream kinases responsible for the activation of the p38 MAPKs and stimulation of wound closure in response to LDLs. Phosphoinositide 3 kinases (PI3Ks) and Ras have been suggested to participate in lipoprotein-induced MAPK activation. However, specific PI3K inhibitors or expression of a dominant-negative form of Ras failed to blunt LDL-induced p38 MAPK activation. The classical LDL receptor does not participate in LDL signaling, but the contribution of other candidate lipoprotein receptors has not been investigated. Using cells derived from scavenger receptor class B type I (SR-BI) knockout mice or the BLT-1 SR-BI inhibitor, we now show that this receptor is required for LDLs to stimulate p38 MAPKs and to promote wound healing. Identification of MKK3/6 and SR-BI as cellular relays in LDL-mediated p38 activation further defines the signaling events that could participate in LDL-mediated pathophysiological responses.