The Effectiveness of Dexmedetomidine as a Prophylactic Treatment for Emergence Delirium Among Combat Veterans With High Anxiety: A Randomized Placebo-Controlled Trial.

INTRODUCTION: Emergence delirium (ED) is characterized by agitation, confusion, and violent physical and verbal behavior associated with awakening from general anesthesia. Combat exposure among U.S. military veterans has been identified as a risk factor for ED. Preoperative baseline anxiety was shown to be a predictor of ED, and combat veterans are known to be at high risk for anxiety as well as depression and PTSD. Dexmedetomidine is an alpha-2 receptor agonist proven to mitigate ED in several patient populations. Perioperative use of dexmedetomidine demonstrated promising benefits in pediatric ED but has not been evaluated in combat veterans. MATERIALS AND METHODS: This study was a multi-site, prospective, randomized controlled investigation of 369 patients with a history of military combat exposure who were scheduled for elective surgery with a general anesthetic as the primary means of anesthesia. The trial was funded by the Tri-Service Nursing Research Program Grant HU0001-14-TS05 (N14-PO3) and approved by the Institutional Review Boards at the Naval Medical Center San Diego, Womack Army Medical Center, Walter Reed National Military Medical Center, and the Uniformed Services University of the Health Sciences, Bethesda, MD. All subjects were administered the State-Trait Anxiety Inventory (STAI) to evaluate baseline anxiety. Those enrolled subjects with a low anxiety level (STAI < 39) (n = 215) were placed in the observational arm of the study. Those with a high anxiety level (STAI ≥ 39) were placed in the experimental arm (n = 153) and were further randomized to treatment with intraoperative dexmedetomidine infusion (1 µg/kg bolus at induction, followed by a 0.6 µg/kg/h infusion continued until emergence) (n = 75) or a placebo intraoperative infusion (n = 75). Following the delivery of the prescribed anesthetic, all subjects were observed for signs of ED using the Pediatric Anesthesia Emergence Delirium (PAED) Scale. The patient and data recorder remained blinded to the randomization results. RESULTS: The central tendencies of demographics and clinical characteristics are reported. PAED among those randomized to dexmedetomidine (median 7, interquartile interval (IQI) 5.2-9.2) tended to be less (P < .0001) than that of those randomized to control (median 12, IQI 10-13). Dexmedetomidine was found to be the most important predictor of PAED (35% relative importance), followed by Patient Health Questionnaire (14%), STAI-Trait (9%), and PTSD Checklist-Military Version (8%); the overall rankings are featured. Randomization to receipt of dexmedetomidine was associated with a 3.7-unit reduction (95% CI 2.5-4.9) in PAED (P < .001) in a linear model controlling for several variables, and the directionality of the effect persisted upon regularization in a penalized linear model. CONCLUSIONS: Dexmedetomidine was effective at reducing PAED among combat veterans who were experiencing symptoms of pre-operative anxiety (i.e., STAI-State ≥39). Although psychological morbidity is not unique to the military population, combat veterans carry some of the highest rates of anxiety, PTSD and depression compared to the general population. Dexmedetomidine can be safety employed by anesthesia providers to reduce symptoms of ED in the perioperative period. The double-blind randomized, controlled study design strengthens our analyses; however, this study did not control for the type of surgical procedure or the duration of anesthetic. Furthermore, we only enrolled patients with combat exposure experiencing symptoms of anxiety and did not investigate the role of dexmedetomidine in combat veterans with less anxiety. Further study of the relationship between psychological comorbidities, ED, and dexmedetomidine is warranted.

The morphogen Sonic hedgehog is an indirect angiogenic agent upregulating two families of angiogenic growth factors.

Sonic hedgehog (Shh) is a prototypical morphogen known to regulate epithelial/mesenchymal interactions during embryonic development. We found that the hedgehog-signaling pathway is present in adult cardiovascular tissues and can be activated in vivo. Shh was able to induce robust angiogenesis, characterized by distinct large-diameter vessels. Shh also augmented blood-flow recovery and limb salvage following operatively induced hind-limb ischemia in aged mice. In vitro, Shh had no effect on endothelial-cell migration or proliferation; instead, it induced expression of two families of angiogenic cytokines, including all three vascular endothelial growth factor-1 isoforms and angiopoietins-1 and -2 from interstitial mesenchymal cells. These findings reveal a novel role for Shh as an indirect angiogenic factor regulating expression of multiple angiogenic cytokines and indicate that Shh might have potential therapeutic use for ischemic disorders.

Structure of CD40 ligand in complex with the Fab fragment of a neutralizing humanized antibody.

BACKGROUND: CD40 ligand (CD40L or CD154), a member of the tumor necrosis factor (TNF) family, plays a critical role in both humoral and cellular immune responses and has been implicated in biological pathways involving epithelial cells, fibroblasts, and platelets. Such a pathway is T cell-mediated B cell activation, a process that occurs through the interaction of CD40L with CD40 receptor expressed on B cells. It results in various B cell responses, including immunoglobulin isotype switching and B cell differentiation and proliferation. These responses can be inhibited by the monoclonal antibody 5c8, which binds with high affinity to CD40L. RESULTS: To understand the structural basis of the inhibition, we determined the crystal structure of the complex of the extracellular domain of CD40L and the Fab fragment of humanized 5c8 antibody. The structure shows that the complex has the shape of a three-bladed propeller with three Fab fragments bound symmetrically to a CD40L homotrimer. To further study the nature of the antibody-antigen interface, we assessed the ability of 23 site-directed mutants of CD40L to bind to 5c8 and CD40 and analyzed the results in the context of the crystal structure. Finally, we observed via confocal microscopy that 5c8 binding to CD40L on the cell surface results in the formation of patches of clustered complexes. CONCLUSIONS: The structure reveals that 5c8 neutralizes CD40L function by sterically blocking CD40 binding. The antigenic epitope is localized in a region of the surface that is likely to be structurally perturbed as a result of genetic mutations that cause hyper-IgM syndrome. The symmetric trimeric arrangement of the Fab fragments in the complex results in a geometry that facilitates the formation of large clusters of complexes on the cell surface.

Triaglycerol metabolism in Saccharomyces cerevisiae. Relation to phospholipid synthesis.

The acylglycerol content of Saccharomyces cerevisiae has been examined during cellular growth. The cells maintained a constant amount of phospholipid and diacylglycerol throughout growth. Triacylglycerol content fell in the early exponential phase of growth and then increased sharply upon entry of the culture into the stationary growth phase. Pulse-chase experiments with [1-14C]oleic acid and [2-3H]- and [1-14C]glycerol indicated that the triacylglycerol molecule was utilized for phospholipid synthesis in early exponential phase probably through a diacylglycerol intermediate. A substantial turnover of phospholipid during growth was also apparent. No role for the triacylglycerol could be found in regulating the fatty acid species of the phospholipid nor in the storage of fatty acid for energy metabolism.

Regular articles: conditional disruption of hedgehog signaling pathway defines its critical role in hair development and regeneration.

Members of the vertebrate hedgehog family (Sonic, Indian, and Desert) have been shown to be essential for the development of various organ systems, including neural, somite, limb, skeletal, and for male gonad morphogenesis. Sonic hedgehog and its cognate receptor Patched are expressed in the epithelial and/or mesenchymal cell components of the hair follicle. Recent studies have demonstrated an essential role for this pathway in hair development in the skin of Sonic hedgehog null embryos. We have further explored the role of the hedgehog pathway using anti-hedgehog blocking monoclonal antibodies to treat pregnant mice at different stages of gestation and have generated viable offspring that lack body coat hair. Histologic analysis revealed the presence of ectodermal placode and primodium of dermal papilla in these mice, yet the subsequent hair shaft formation was inhibited. In contrast, the vibrissae (whisker) development appears to be unaffected upon anti-hedgehog blocking monoclonal antibody treatment. Strikingly, inhibition of body coat hair morphogenesis also was observed in mice treated postnatally with anti-hedgehog monoclonal antibody during the growing (anagen) phase of the hair cycle. The hairless phenotype was reversible upon suspension of monoclonal antibody treatment. Taken together, our results underscore a direct role of the Sonic hedgehog signaling pathway in embryonic hair follicle development as well as in subsequent hair cycles in young and adult mice. Our system of generating an inducible and reversible hairless phenotype by anti-hedgehog monoclonal antibody treatment will be valuable for studying the regulation and mechanism of hair regeneration.

Biochemical analysis of retroviral structural proteins to identify and quantify retrovirus expressed by an NS0 murine myeloma cell line.

A subclone of the NS0 murine myeloma cell line, frequently used to produce recombinant monoclonal antibodies, was found by a transmission electron microscopy method to express a surprisingly high titer of 10(11) retroviral particles per ml of culture supernatant. Infectivity assays showed a very low infectious titer with the restricted host range expected for a murine amphotropic retrovirus. A Western blot assay for the viral capsid protein was developed to confirm the high titer values and provide a means for monitoring batch consistency and virus removal during the purification process. Mass spectrometry of several of the viral Gag proteins demonstrated that the cell line appeared to produce at least two closely related retroviruses. N-terminal sequencing of three of the Gag proteins demonstrated that these retroviruses were members of the murine leukemia retroviral family. Western blot detection with an antibody for the capsid protein gave a linear standard curve over the range of 0.1-3 ng per lane. This allows the detection of viral titers as low as 6x10(7) virions per ml without the need to concentrate the sample. The Western blot method has higher throughput and less variability than transmission electron microscopy methods and has potential for monitoring viral titer and clearance during development of manufacturing processes.

Nebivolol vs atenolol and placebo in essential hypertension: a double-blind randomised trial.

A double-blind, randomised, parallel-group trial was conducted in patients with essential hypertension in British general practices, of nebivolol 5 mg, atenolol 50 mg, and placebo each given once daily. Both active drugs, in comparison with placebo, caused highly significant and similar reductions in systolic and diastolic pressures without orthostatic effect, and small significant falls in heart rate. Both active drugs were well tolerated, nebivolol marginally more so. Nebivolol, a long-acting, cardioselective, vasodilating beta-blocker which acts partly via the l-arginine/nitric oxide mechanism, appears potentially valuable for the treatment of hypertension.

A screening programme for hypertension in general practice.

It is recommended that hypertensive patients with a diastolic blood pressure of 100 mmHg be treated. However, it has been proposed that only 50% of hypertensives are diagnosed, and of these, 50% are not treated. We therefore set out to identify hypertensive patients in our practice, who were then actively treated, some in a clinical trial of ketanserin. Of the 3,384 patients (1,667 males and 2,707 females) who were invited to our surgery for a blood pressure measurement, 2,606 patients (77%) attended. The overall prevalence of hypertension in the patients, aged 35-65 years was 2.9%. This prevalence increased with age: from 1.0% in the 35-44 year age group to 3.5% at 45-54 years, and 4.4% at 55-64 years. The mean systolic but not diastolic blood pressure increased with age. There were more male hypertensives than females, except over 55 years of age. Of the 84 hypertensives identified after one visit, the majority remained hypertensive after an additional visit, and 31 agreed to participate in a clinical trial. After a four-week placebo run-in, 22 patients had a diastolic blood pressure above 95 mmHg and were randomly allocated to receive ketanserin 40 mg twice daily or metoprolol 100 mg twice daily. Treatment continued double-blind for three months. There were no significant differences in blood pressure reduction between the treatment groups. The heart rate was reduced more by metoprolol. There were no withdrawals for side effects and no major differences in subjective complaints between the two treatment groups.

Oxysterol binding protein.

A binding protein is described for certain oxygenated derivatives of cholesterol which suppress 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol synthesis in cultured mammalian cells. This protein is found in the cytosolic fraction of many cell types and is distinct from cytosolic proteins which bind cholesterol. The relative binding affinity of a wide variety of oxysterols correlates with their ability to suppress reductase and it is proposed that the binding protein functions as a receptor for endogenous regulatory oxysterols. The binding protein from cultured mouse fibroblasts (L cells) has been partially purified and characterized. Changes in its molecular form occur when a ligand is bound and further changes in form and binding kinetics occur at acid pH and in the presence of urea. Based on these changes a subunit model for the binding protein is presented.

A facile synthesis of lanost-8-en-3 beta-ol-24-one (24-ketolanosterol). An inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

A facile chemical synthesis of lanost-8-en-3 beta-ol-24-one (24-ketolanosterol) is described. This compound was found to be a potent inhibitor of 3-hydroxy-3-methylglutaryl (HMG) CoA reductase activity in cultured mouse L cells. The synthetic scheme developed in this study utilizes commercial lanosterol as a starting material and involves selective hydroboration of the C-24 double bond followed by oxidation of the carbon-boron bond at C-24 by pyridinium chlorochromate (PCC).

Chemical synthesis of 4,4'-dimethyl-7-oxygenated sterols. Inhibitors of 3-hydroxy-3-methylglutaryl reductase.

The chemical syntheses of 4,4'-dimethylcholest-5-en-3 beta-ol-7-one, 4,4'-dimethylcholest-5-ene-3 beta, 7 beta-diol and 4,4'-dimethylcholest-5-ene-3 beta, 7 alpha-diol are described. All of these compounds were found to be potent inhibitors of 3-hydroxy-3-methylglutaryl (HMG-CoA) reductase activity in cultured mouse L cells. The synthetic scheme developed in this study utilizes commercial cholesterol as the starting material and provides a simplified method for the preparation of 4,4'-dimethyl-7-oxygenated steroids.

Sterols in yeast subcellular fractions.

Yeast is the most primitive organism synthesizing substantial amounts of sterols. Because of this eucaryotic organism's versatility in growth conditions, ease of culture, well-defined genetic mechanism, and characteristic subcellar architecture, it is readily applied to studies of the role of sterols in the general economy of the cell. Sterols exist in two major forms, as the free sterol, or esterified with long chain fatty acids. The importance of sterols for this organism can be demonstrated using a naturally occurring antimycotic azasterol. This agent inhibits yeast growth. Three effects are seen on sterol synthesis: inhibition of the enzymes delta14-reductase, sterol methyltransferase, and methylene reductase. Cells cultured on respiratory substrates are more sensitive to inhibition than are cells growing on glucose. We have demonstrated a relationship between respiratory competency and sterol biosynthesis in this organism. Many mutants altered in sterol synthesis are respirationally defective and must grow fermentatively. One clone has temperature conditional respiration. Experiments with purified mitochondria, prepared from this mutant and its isogenic wildtype, show that the mutant organism is able to respire at the higher temperature but lacks the ability to couple respiration to phosphorylation. No similar loss is seen in the wild-type clones. Data are given which support the proposal that, for inclusion in mitochondrial structures, yeast cells may discriminate among sterols available from the total sterol pool in favor of ergosterol.

Oxysterols: chemical synthesis, biosynthesis and biological activities.

As a class of compounds, oxysterols have demonstrated a wide variety of biological properties. Due to the general interest in these compounds, new methods of chemical synthesis have been developed to provide them for biological investigation. The specific inhibition by oxysterols of cholesterol biosynthesis in mammalian cells has been shown to result primarily from a decrease in cellular levels of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity. Recent evidence suggests these cellular responses may be mediated by an oxysterol binding protein found in the cytosol of many lines of cultured cells. In certain instances, oxysterols have been shown to be produced in biological systems. These results support the supposition that oxysterols may regulate sterol biosynthesis at the cellular level. Included herein are the inhibitory effects of 9 alpha, 11 alpha-epoxycholest-7-en-3 beta-ol cholest-8-en-3 beta-ol-7-one and cholest-8-en-3 beta-ol-11-one on HMG-CoA reductase activity and their relative affinities for a cytosolic binding protein.

Activation of adult rat CNS endothelial cells by opioid-induced toll-like receptor 4 (TLR4) signaling induces proinflammatory, biochemical, morphological, and behavioral sequelae.

CNS immune signaling contributes to deleterious opioid effects including hyperalgesia, tolerance, reward, and dependence/withdrawal. Such effects are mediated by opioid signaling at toll-like receptor 4 (TLR4), presumptively of glial origin. Whether CNS endothelial cells express TLR4 is controversial. If so, they would be well positioned for activation by blood-borne opioids, contributing to opioid-induced pro-inflammatory responses. These studies examined adult primary rat CNS endothelial cell responses to (-)-morphine or its mu opioid receptor (MOR)-inactive metabolite morphine-3-glucuronide (M3G), both known TLR4 agonists. We demonstrate that adult rat CNS endothelial cells express functional TLR4. M3G activated nuclear factor kappaB (NF-κB), increased tumor necrosis factor-α (TNFα) and cyclooxygenase-2 (COX2) mRNAs, and released prostaglandin E2 (PGE2) from these cells. (-)-Morphine-induced upregulation of TNFα mRNA and PGE2 release were unmasked by pre-treatment with nalmefene, a MOR antagonist without TLR4 activity (unlike CTAP, shown to have both MOR- and TLR4-activity), suggestive of an interplay between MOR and TLR4 co-activation by (-)-morphine. In support, MOR-dependent Protein Kinase A (PKA) opposed TLR4 signaling, as PKA inhibition (H-89) also unmasked (-)-morphine-induced TNFα and COX2 mRNA upregulation. Intrathecal injection of CNS endothelial cells, stimulated in vitro with M3G, produced TLR4-dependent tactile allodynia. Further, cortical suffusion with M3G in vivo induced TLR4-dependent vasodilation. Finally, endothelial cell TLR4 activation by lipopolysaccharide and/or M3G was blocked by the glial inhibitors AV1013 and propentofylline, demonstrating endothelial cells as a new target of such drugs. These data indicate that (-)-morphine and M3G can activate CNS endothelial cells via TLR4, inducing proinflammatory, biochemical, morphological, and behavioral sequelae. CNS endothelial cells may have previously unanticipated roles in opioid-induced effects, in phenomena blocked by presumptive glial inhibitors, as well as TLR4-mediated phenomena more broadly.

Spinal upregulation of glutamate transporter GLT-1 by ceftriaxone: therapeutic efficacy in a range of experimental nervous system disorders.

Glutamate neurotransmission is highly regulated, largely by glutamate transporters. In the spinal cord, the glutamate transporter GLT-1 is primarily responsible for glutamate clearance. Downregulation of GLT-1 can occur in activated astrocytes, and is associated with increased extracellular glutamate and neuroexcitation. Among other conditions, astrocyte activation occurs following repeated opioids and in models of chronic pain. If GLT-1 downregulation occurs in these states, GLT-1 could be a pharmacological target for improving opioid efficacy and controlling chronic pain. The present studies explored whether daily intrathecal treatment of rats with ceftriaxone, a beta-lactam antibiotic that upregulates GLT-1 expression, could prevent development of hyperalgesia and allodynia following repeated morphine, reverse pain arising from central or peripheral neuropathy, and reduce glial activation in these models. Ceftriaxone pre-treatment attenuated the development of hyperalgesia and allodynia in response to repeated morphine, and prevented associated astrocyte activation. In a model of multiple sclerosis (experimental autoimmune encephalomyelitis; EAE), ceftriaxone reversed tactile allodynia and halted the progression of motor weakness and paralysis. Similarly, ceftriaxone reversed tactile allodynia induced by chronic constriction nerve injury (CCI). EAE and CCI each significantly reduced the expression of membrane-bound, dimerized GLT-1 protein in lumbar spinal cord, an effect normalized by ceftriaxone. Lastly, ceftriaxone normalized CCI- and EAE-induced astrocyte activation in lumbar spinal cord. Together, these data indicate that increasing spinal GLT-1 expression attenuates opioid-induced paradoxical pain, alleviates neuropathic pain, and suppresses associated glial activation. GLT-1 therefore may be a therapeutic target that could improve available treatment options for patients with chronic pain.

Correlation among oxysterol potencies in the regulation of the degradation of 3-hydroxy-3-methylglutaryl CoA reductase, the repression of 3-hydroxy-3-methylglutaryl CoA synthase and affinities for the oxysterol receptor.

25-Hydroxycholesterol regulates cholesterol biosynthesis by two mechanisms: repression of the transcription of the genes for several cholesterogenic enzymes and acceleration of the degradation of the enzyme 3-hydroxy-3-methylglutaryl CoA reductase. In the present work the structural features which govern oxysterol potency were determined separately for each regulatory mechanism. Regulation of degradation was tested using a 3-hydroxy-3-methylglutaryl CoA reductase-beta-galactosidase fusion protein. Repression of enzyme synthesis was tested by measuring 3-hydroxy-3-methylglutaryl CoA synthase activity since this protein is not regulated by a degradative mechanism. Oxysterol activities were highly correlated between the two assays (R = .959) demonstrating that the degradative and repressor mechanisms share an element which determines oxysterol regulatory potency. Correlation of these results with previous data for the affinity of these oxysterols for the oxysterol receptor suggests that the receptor is the element involved in both these regulatory pathways.

Metabolic interconversion of free sterols and steryl esters in Saccharomyces cerevisiae.

The interconversion of free and esterified sterols was followed radioisotopically with [U-14C]acetate and [methyl-14C]methionine. In pulse-chase experiments, radioactivity first appeared mainly in unesterified sterols in exponential-phase cells. Within one generation time, the label equilibrated between the free and esterified sterol pools and subsequently accumulated in steryl esters in stationary-phase cells. When the sterol pools were prelabeled by growing cells aerobically to the stationary phase and the cells were diluted into unlabeled medium, the prelabeled steryl esters returned to the free sterol form under several conditions. (i) During aerobic growth, the prelabeled sterols decreased from 80% to 45% esters in the early exponential phase and then returned to 80% esters as the culture reached the stationary phase. (ii) Under anaerobic conditions, the percentage of prelabeled steryl esters declined continuously. When growth stopped, only 15% of the sterols remained esterified. (iii) In the presence of an inhibitor of sterol biosynthesis, which causes accumulation of a precursor to ergosterol, prelabeled sterols decreased to 40% steryl esters while the precursor was found preferentially in the esterified form. These results indicate that the bulk of the free sterol and steryl ester pools are freely interconvertible, with the steryl esters serving as a supply of free sterols. Furthermore, there is an active cellular control over what types of sterol are found in the free and esterified sterol pools.