Biosynthesis and expression of VE-cadherin is regulated by the PI3K/mTOR signaling pathway

Vascular endothelial (VE)-cadherin is an essential protein of adherens junctions of endothelial cells and plays a pivotal role in vascular homeostasis. Mammalian target of rapamycin complex 2 (mTORC2) deficient mice display defects in fetal vascular development. Blocking mTOR or the upstream kinase phosphoinositide 3-kinase (PI3K) led to a dose-dependently decrease of the VE-cadherin mRNA and protein expression. Immunofluorescent staining showed a strongly decreased expression of VE-cadherin at the interface of human umbilical endothelial cells (HUVECs) followed by intercellular gap formation. Herewith, we demonstrated that the expression of VE-cadherin is dependent on mTOR and PI3K signaling.

Transdermal nitroglycerin in patients with subarachnoid hemorrhage

Delayed ischemic neurological deficit (DIND) following cerebral vasospasm remains a cause for high morbidity and mortality in patients with subarachnoid hemorrhage (SAH). There is experimental and clinical evidence of positive effects of nitric oxide (NO) donors on cerebral vasospasm. We therefore analysed the effect of transdermal nitroglycerin in patients with SAH measuring transcranial Doppler velocities (TCD), cerebral blood flow (CBF) and DIND. Nitroglycerin was used in a target dose of 14 microg/kg/h. TCD assessment was performed daily. CBF measurements were done using the perfusion CT-technique. Blood pressure, volume intake and vasopressor administration, were registered. Nine patients were randomly assigned either to the nitroglycerin group (N-group) and eight patients in the control group (C-group). Mean TCD values in the extracranial portion of the internal carotid artery (ICA) were lower in the N-group (p<0.005). Mean TCD in the middle cerebral arteries (MCA) showed no difference. The Lindegaard ratio was higher in the N-group (p<0.04). CBF in the N-group was higher than in the C-group (p<0.03). Even though nitroglycerin reduces blood pressure and lowers ICA TCD-values and increases the Lindegaard ratio, a higher CBF was measured in the N-group. Thus, nitroglycerin influences the cerebral vascular tone and increases CBF. SAH therapy with nitroglycerin is possible without increasing the risk of DIND. The exact timing of onset, duration and reduction of nitroglycerin administration in respect to the appearance of vasospasm may have a strong impact on the success of such a therapy.

Influence of inspired oxygen on glucose-lactate dynamics after subdural hematoma in the rat

The mechanisms causing brain damage after acute subdural hematoma (SDH) are poorly understood. A decrease in cerebral blood flow develops immediately after the hematoma forms, thus reducing cerebral oxygenation. This in turn may activate mitochondrial failure and tissue damage leading to ionic imbalance and possibly to cellular breakdown. The purpose of this study was to test whether a simple therapeutic measure, namely increased fraction of inspired oxygen (FiO2 100), and hence increased arterial and brain tissue oxygen tension, can influence brain glucose and lactate dynamics acutely after subdural hematoma in the rat. Twenty-five male Sprague-Dawley anesthetized rats were studied before, during and after induction of the SDH in two separate groups. The Oxygen group (n = 10) was ventilated with 100% oxygen immediately after induction of the SDH. The Air group (n = 10) was ventilated during the entire study with 21% oxygen. Brain microdialysate samples were analyzed for glucose and lactate. All rats were monitored with femoral arterial blood pressure catheters, arterial blood gas analysis, arterial glucose, lactate and end tidal CO2 (EtCO2). Five male Sprague-Dawley rats were sham operated to measure the effect of oxygen challenge on glucose-lactate dynamics without injury. Arterial oxygen tension in the Oxygen group was 371 +/- 30 mmHg and was associated with significantly greater increase in dialysate lactate in the first 30 min after induction of SDH. Dialysate glucose initially dropped in both groups, after SDH, but then reverted significantly faster to values above baseline in the Oxygen group. Changes in ventilatory parameters had no significant effect on dialysate glucose and lactate parameters in the sham group. Extracellular dialysate lactate and glucose are influenced by administration of 100% O2 after SDH. Dialysate glucose normalizes significantly quicker upon 100% oxygen ventilation. We hypothesize that increased neural tissue oxygen tension, in presence of reduced regional CBF, and possibly compromised mitochondrial function, after acute SDH results in upregulation of rate-limiting enzyme systems responsible for both glycolytic and aerobic metabolism. Similar changes have been seen in severe human head injury, and suggest that a simple therapeutic measure, such as early ventilation with 100% O2, may improve cerebral energy metabolism, early after SDH. Further studies to measure the generation of adenosine triphosphate (ATP) are needed to validate the hypothesis.

Clinical trials in head injury

Secondary brain damage, following severe head injury is considered to be a major cause for bad outcome. Impressive reductions of the extent of brain damage in experimental studies have raised high expectations for cerebral neuroprotective treatment, in the clinic. Therefore multiple compounds were and are being evaluated in trials. In this review we discuss the pathomechanisms of traumatic brain damage, based upon their clinical importance. The role of hypothermia, mannitol, barbiturates, steroids, free radical scavengers, arachidonic acid inhibitors, calcium channel blockers, N-methyl-D-aspartate (NMDA) antagonists, and potassium channel blockers, will be discussed. The importance of a uniform strategic approach for evaluation of potentially interesting new compounds in clinical trials, to ameliorate outcome in patients with severe head injury, is proposed. To achieve this goal, two nonprofit organizations were founded: the European Brain Injury Consortium (EBIC) and the American Brain Injury Consortium (ABIC). Their aim lies in conducting better clinical trials, which incorporate lessons learned from previous trials, such that the succession of negative, or incomplete studies, as performed in previous years, will cease.

Peptide-based probes for cancer imaging

Receptors for regulatory peptides are overexpressed in a variety of human cancers. They represent the molecular basis for in vivo imaging with radiolabeled peptide probes. Somatostatin-derived tracers, designed to image the sst2-overexpressing neuroendocrine tumors, have enjoyed almost 2 decades of successful development and extensive clinical applications. More recent developments include second- and third-generation somatostatin analogs, with a broader receptor subtype profile or with antagonistic properties. Emerging tracers for other peptide receptors, including cholecystokinin/gastrin and GLP-1 analogs for neuroendocrine tumors, bombesin and neuropeptide-Y analogs for prostate or breast cancers, or Arg-Gly-Asp peptides for neoangiogenesis labeling, are also in current development. Application fields include both SPECT/CT and PET/CT.

Bombesin receptor antagonists may be preferable to agonists for tumor targeting

Two bombesin analogs, Demobesin 4 and Demobesin 1, were characterized in vitro as gastrin-releasing peptide (GRP) receptor agonist and antagonist, respectively, and were compared as (99m)Tc-labeled ligands for their in vitro and in vivo tumor-targeting properties. METHODS: N(4)-[Pro(1),Tyr(4),Nle(14)]Bombesin (Demobesin 4) and N(4)-[d-Phe(6),Leu-NHEt(13),des-Met(14)]bombesin(6-14) (Demobesin 1) were characterized in vitro for their binding properties with GRP receptor autoradiography using GRP receptor-transfected HEK293 cells, PC3 cells, and human prostate cancer specimens. Their ability to modulate calcium mobilization in PC3 and transfected HEK293 cells was analyzed as well as their ability to trigger internalization of the GRP receptor in transfected HEK293 cells, as determined qualitatively by immunofluorescence microscopy and quantitatively by enzyme-linked immunosorbent assay (ELISA). Further, their internalization properties as (99m)Tc-labeled radioligands were tested in vitro in both cell lines. Finally, their biodistribution was analyzed in PC3 tumor-bearing mice. RESULTS: A comparable binding affinity with the 50% inhibitory concentration (IC(50)) in the nanomolar range was measured for Demobesin 4 and Demobesin 1 in all tested tissues. Demobesin 4 behaved as an agonist by strongly stimulating calcium mobilization and by triggering GRP receptor internalization. Demobesin 1 was ineffective in stimulating calcium mobilization and in triggering GRP receptor internalization. However, in these assays, it behaved as a competitive antagonist as it reversed completely the agonist-induced effects in both systems. (99m)Tc-Labeled Demobesin 1 was only weakly taken up by PC3 cells or GRP receptor-transfected HEK293 cells (10% and 5%, respectively, of total added radioactivity) compared with (99m)Tc-labeled Demobesin 4 (45% of total added radioactivity in both cell lines). Remarkably, the biodistribution study revealed a much more pronounced uptake at 1, 4, and 24 h after injection of (99m)Tc-labeled Demobesin 1 in vivo into PC3 tumors than (99m)Tc-labeled Demobesin 4. In vivo competition experiments demonstrated a specific uptake in PC3 tumors and in physiologic GRP receptor-expressing tissues. The tumor-to-kidney ratios were 0.7 for Demobesin 4 and 5.2 for Demobesin 1 at 4 h. CONCLUSION: This comparative in vitro/in vivo study with Demobesin 1 and Demobesin 4 indicates that GRP receptor antagonists may be superior targeting agents to GRP receptor agonists, suggesting a change of paradigm in the field of bombesin radiopharmaceuticals.

Sensitization to wasp venom does not induce autoantibodies leading to infertility

Antigenic cross-reactivity has been described between the venom allergen (antigen 5) and mammalian testis proteins. Based on an allergen database we have previously shown that allergens can be represented by allergen motifs. A motif group was found containing venom antigen 5 sequences from different vespids. Using an optimized amino acid profile based on antigen 5 sequences for searching cross-reactive proteins, three human semen proteins belonging to the family of cysteine-rich secretory proteins (hCRISP) were found in the Swiss Protein database. To analyze antigenic cross-reactivity between antigen 5 and hCRISPs, antigen 5 from yellow jacket venom (Ves v 5) and two hCRISPs (CRISP-2 and -3) were chosen and produced as recombinant proteins in E. coli. A correlation was found between antibodies reacting with rVes v 5 and rhCRISP-2, -3 in a small human sera population indicating the presence of cross-reactive antibodies in human serum. Using intravenous immunoglobulin (IVIg), a therapeutic multidonor IgG preparation, cross-reactive antibodies were isolated that recognize rVes v 5, hCRISP-2 and -3 suggesting the presence of common epitopes between Ves v 5 and hCRISPs. However this cross-reactivity seems not to be linked to allergy to wasp venom as we could show no correlation between increasing CAP-class IgE level to wasp venom and IgG to sperm extract and hCRISPs. These data suggest that higher sensitization to wasp venom does not induce more antibodies against autoantigens and might not represent a higher risk to develop autoantibodies leading to infertility.

Monomerization of dimeric IgG of intravenous immunoglobulin (IVIg) increases the antibody reactivity against intracellular antigens

Intravenous immunoglobulin (IVIg) preparations are derived from pooled plasma from up to 60,000 healthy human donors and reflect the immunologic experience of the donor population. IVIg contains monomeric and dimeric IgG populations which are in a dynamic equilibrium depending on concentration, pH, temperature, donor pool size, time and stabilizers added in order to keep the portion of dimeric IgG below a certain level. In the present study, monomeric and dimeric fractions were isolated by size exclusion chromatography. The dimeric fractions, however, showed a dynamic instability and tended to dissociate. Both dimeric and monomeric IgG fractions were acid treated (pH 4) in order to dissociate the dimeric IgG. Western-blot analysis identified a sub-population of SDS resistant IgG dimers. Furthermore, the reactivities of the fractions were tested against a panel of self- and exo-antigens. There was a marked increase in activity of the dimeric compared to the monomeric IgG fraction against various intracellular self-antigens. Our data indicates that the increased reactivities of pH 4-treated fractions can mainly be attributed to dimer dissociation, as pH 4-treated monomers do not show significantly increased activities against a range of antigens.

Low molecular weight dextran sulfate as complement inhibitor and cytoprotectant in solid organ and islet transplantation

Complement is an essential part of the innate immune system and plays a crucial role in organ and islet transplantation. Its activation, triggered for example by ischemia/reperfusion (I/R), significantly influences graft survival, and blocking of complement by inhibitors has been shown to attenuate I/R injury. Another player of innate immunity are the dendritic cells (DC), which form an important link between innate and adaptive immunity. DC are relevant in the induction of an immune response as well as in the maintenance of tolerance. Modulation or inhibition of both components, complement and DC, may be crucial to improve the clinical outcome of solid organ as well as islet transplantation. Low molecular weight dextran sulfate (DXS), a well-known complement inhibitor, has been shown to prevent complement-mediated damage of the donor graft endothelium and is thus acting as an endothelial protectant. In this review we will discuss the evidence for this cytoprotective effect of DXS and also highlight recent data which show that DXS inhibits the maturation of human DC. Taken together the available data suggest that DXS may be a useful reagent to prevent the activation of innate immunity, both in solid organ and islet transplantation.

Highly efficient in vivo agonist-induced internalization of sst2 receptors in somatostatin target tissues

The successful peptide receptor imaging of tumors, as exemplified for somatostatin receptors, is based on the overexpression of peptide receptors in selected tumors and the high-affinity binding to these tumors of agonist radioligands that are subsequently internalized into the tumor cells in which they accumulate. Although in vitro studies have shown ample evidence that the ligand-receptor complex is internalized, in vivo evidence of agonist-induced internalization of peptide receptors, such as somatostatin receptors, is missing. METHODS: Rats subcutaneously transplanted with the somatostatin receptor subtype 2 (sst(2))-expressing AR42J tumor cells were treated with intravenous injections of various doses of the sst(2) agonist [Tyr(3), Thr(8)]-octreotide (TATE) or of the sst(2) antagonist 1,4,7,10-tetraazacyclododecane-N,N',N'',N''',-tetraacetic acid (DOTA)-Bass and were sacrificed at various times ranging from 2.5 min to 24 h after injection. The tumors and pancreas were then removed from each animal. All tissue samples were processed for sst(2) immunohistochemistry using sst(2)-specific antibodies. RESULTS: Compared with the sst(2) receptors in untreated animals, which localized at the plasma membrane in pancreatic and AR42J tumor cells, the sst(2) receptors in treated animals are detected intracellularly after an intravenous injection of the agonist TATE. Internalization is fast, as the receptors are already internalizing 2.5 min after TATE injection. The process is extremely efficient, as most of the cell surface receptors internalize into the cell and are found in endosomelike structures after TATE injection. The internalization is most likely reversible, because 24 h after injection the receptors are again found at the cell surface. The process is also agonist-dependent, because internalization is seen with high-affinity sst(2) agonists but not with high-affinity sst(2) antagonists. The same internalization properties are seen in pancreatic and AR42J tumor cells. They can further be confirmed in vitro in human embryonic kidney-sst(2) cells, with an immunofluorescence microscopy-based sst(2) internalization assay. CONCLUSION: These animal data strongly indicate that the process of in vivo sst(2) internalization after agonist stimulation is fast, extremely efficient, and fully functional under in vivo conditions in neoplastic and physiologic sst(2) target tissues. This molecular process is, therefore, likely to be responsible for the high and long-lasting uptake of sst(2) radioligands seen in vivo in sst(2)-expressing tumors.

Biomechanical analysis of torsion and shear forces in lumbar and lumbosacral spine segments of nonchondrodystrophic dogs

OBJECTIVE: To determine stiffness and load-displacement curves as a biomechanical response to applied torsion and shear forces in cadaveric canine lumbar and lumbosacral specimens. STUDY DESIGN: Biomechanical study. ANIMALS: Caudal lumbar and lumbosacral functional spine units (FSU) of nonchondrodystrophic large-breed dogs (n=31) with radiographically normal spines. METHODS: FSU from dogs without musculoskeletal disease were tested in torsion in a custom-built spine loading simulator with 6 degrees of freedom, which uses orthogonally mounted electric motors to apply pure axial rotation. For shear tests, specimens were mounted to a custom-made shear-testing device, driven by a servo hydraulic testing machine. Load-displacement curves were recorded for torsion and shear. RESULTS: Left and right torsion stiffness was not different within each FSU level; however, torsional stiffness of L7-S1 was significantly smaller compared with lumbar FSU (L4-5-L6-7). Ventral/dorsal stiffness was significantly different from lateral stiffness within an individual FSU level for L5-6, L6-7, and L7-S1 but not for L4-5. When the data from 4 tested shear directions from the same specimen were pooled, level L5-6 was significantly stiffer than L7-S1. CONCLUSIONS: Increased range of motion of the lumbosacral joint is reflected by an overall decreased shear and rotational stiffness at the lumbosacral FSU. CLINICAL RELEVANCE: Data from dogs with disc degeneration have to be collected, analyzed, and compared with results from our chondrodystrophic large-breed dogs with radiographically normal spines.