Current practice of rapid sequence induction in adults: A national survey among anesthesiologists in Spain. / Práctica actual de la inducción de secuencia rápida en adultos: una encuesta nacional entre anestesiólogos en España.

BACKGROUND: Rapid sequence induction (RSI) in adults has undergone changes in recent years due to pharmacological and technological advances. The objective of this survey was to evaluate current practice among Spanish anesthesiologists. METHODS: A 31-item questionnaire regarding RSI practice was sent to anesthesiologists working in Spanish public hospitals. Differences in responses according to the type of hospital or experience of the respondent were compared for all data using the chi-square and Fisher's exact test. RESULTS: Approximately 15.89% of Spanish anesthesiologists participated in the survey (1002 questionnaires). The results show considerable heterogeneity in most aspects of RSI. Less than 20% of respondents administer sodium citrate. Sixty-four percent place a nasogastric tube in patients with intestinal obstruction. Gastric residue is assessed by ultrasound in 6% of cases. Only 25% of respondents measure ETO2 to check the effectiveness of preoxygenation, and 22% use nasal oxygen insufflation with nasal prongs or THRIVE. Sixty two percent of respondents apply cricoid pressure, but only 50% release the pressure when encountering intubation difficulty. Up to 40% of respondents reported cases of aspiration despite applying cricoid pressure. Propofol was the most commonly used hypnotic (97.6%), but there was no clear preference in the choice of neuromuscular relaxant (suxamethonium versus rocuronium ratio of approximately 1:1). Only 44% of respondents calculated the dose of sugammadex that would be required for emergency reversal of rocuronium. CONCLUSIONS: The survey showed significant variation in RSI practice, similar to that of other countries. Quality prospective studies are needed to standardize clinical practice.

Bmi1 limits dilated cardiomyopathy and heart failure by inhibiting cardiac senescence.

Dilated cardiomyopathy (DCM) is the most frequent cause of heart failure and the leading indication for heart transplantation. Here we show that epigenetic regulator and central transcriptional instructor in adult stem cells, Bmi1, protects against DCM by repressing cardiac senescence. Cardiac-specific Bmi1 deletion induces the development of DCM, which progresses to lung congestion and heart failure. In contrast, Bmi1 overexpression in the heart protects from hypertrophic stimuli. Transcriptome analysis of mouse and human DCM samples indicates that p16(INK4a) derepression, accompanied by a senescence-associated secretory phenotype (SASP), is linked to severely impaired ventricular dimensions and contractility. Genetic reduction of p16(INK4a) levels reverses the pathology of Bmi1-deficient hearts. In parabiosis assays, the paracrine senescence response underlying the DCM phenotype does not transmit to healthy mice. As senescence is implicated in tissue repair and the loss of regenerative potential in aging tissues, these findings suggest a source for cardiac rejuvenation.

Fractal aggregates evolution of methyl red in liquid crystal.

The spontaneous formation of dendritic aggregates is observed in a two-dimensional confined layered system consisting of a film composed of liquid crystal, dye and solvent cast above a polymer substrate. The observed aggregates are promoted by phase separation processes induced by dye diffusion and solvent evaporation. The growth properties of the aggregates are studied through the temporal evolution of their topological properties (surface, perimeter, fractal dimension). The fractal dimension of the completely formed structures, when they are coexistent with different types of structures, is consistent with theoretical and experimental values obtained for Diffusion-Limited Aggregates. Under different experimental conditions (temperature and local dye concentration) the structure forms without interactions with other kinds of structures, and its equilibrium fractal dimension is smaller. The fractal dimension is thus not a universal property of the observed structures, but rather depends on the experimental conditions.

In vivo upregulation of CD95 and CD95L causes synergistic inhibition of angiogenesis by TSP1 peptide and metronomic doxorubicin treatment.

Antiangiogenic thrombospondin-1 (TSP1) induces endothelial cell death via a CD95-mediated cascade. We used this signaling pathway, where CD95/Fas is a rate-limiting intermediate, as a target to optimize the efficacy of TSP1 active peptide, DI-TSP. Like TSP1, DI-TSP upregulated endothelial CD95L in vivo. To modulate CD95 levels, we chose chemotherapy agent doxorubicin (DXR). DXR caused sustained upregulation of CD95 in the activated endothelium at 1/100 of the maximal tolerated dose. DI-TSP and DXR synergistically induced endothelial apoptosis in vitro, and in vivo, in developing murine vessels. Fas decoy, TSP1 receptor antibody and Pifithrin, a p53 inhibitor, severely decreased apoptosis and restored angiogenesis by DXR-DI-TSP combination, evidencing critical roles of CD95 and TSP1. Combined therapy synergistically blocked neovascularization and progression of the bladder and prostate carcinoma. Such informed design of a complex antiangiogenic therapy based on the rate-limiting molecular targets is a novel concept, which may yield new approaches to cancer treatment.

Selective inhibition of vascular endothelial growth factor-mediated angiogenesis by cyclosporin A: roles of the nuclear factor of activated T cells and cyclooxygenase 2.

Cyclosporin A (CsA) is an immunosuppressive drug that inhibits the activity of transcription factors of the nuclear factor of activated T cells (NFAT) family, interfering with the induction of cytokines and other inducible genes required for the immune response. Here we show that CsA inhibits migration of primary endothelial cells and angiogenesis induced by vascular endothelial growth factor (VEGF); this effect appears to be mediated through the inhibition of cyclooxygenase (Cox)-2, the transcription of which is activated by VEGF in primary endothelial cells. Consistent with this, we show that the induction of Cox-2 gene expression by VEGF requires NFAT activation. Most important, the CsA-mediated inhibition of angiogenesis both in vitro and in vivo was comparable to the Cox-2 inhibitor NS-398, and reversed by prostaglandin E(2). Furthermore, the in vivo corneal angiogenesis induced by VEGF, but not by basic fibroblast growth factor, was selectively inhibited in mice treated with CsA systemically. These findings involve NFAT in the regulation of Cox-2 in endothelial cells, point to a role for this transcription factor in angiogenesis, and may provide a novel mechanism underlying the beneficial effects of CsA in angiogenesis-related diseases such as rheumatoid arthritis and psoriasis.

Identification of phosphorylation sites in proteins by nanospray quadrupole ion trap mass spectrometry.

A method is described for identifying serine phosphorylation sites in proteins, based on conventional (32)P labeling followed by electrophoretic separation, 'in-gel' digestion with a protease, peptide extraction, reversed-phase high-performance liquid chromatographic separation and collection and off-line analysis of the radioactive fractions by nanospray ion trap mass spectrometry. The method was successfully applied to the identification of three phosphorylation sites in two proteins which were subjected to in vitro phosphorylation under physiological conditions. Different combinations of the various scanning modes of the ion trap, including high-resolution, multiple subfragmentation (or MS(n)) and fast scan analysis, were employed to identify the phosphopeptides, determine their sequence and localize the exact site of phosphorylation. 'Blind' fragmentation using fast scans was used to analyze a phosphopeptide which was undetectable in other scanning modes. The sequence, phosphorylation site and double cysteine modification of the potassium adduct of a peptide containing 35 residues were also determined by multiple fragmentation. The results not only support the validity of the proposed method for routine identification of phosphorylation sites, but also demonstrate the exceptional capability of off-line ion trap mass spectrometry in combination with nanospray ionization for performing very detailed studies on the structure of peptides.

An essential role of the nuclear factor of activated T cells in the regulation of the expression of the cyclooxygenase-2 gene in human T lymphocytes.

We have previously reported that transcriptional induction of cyclooxygenase-2 (COX-2) isoenzyme occurs early after T cell receptor triggering, suggesting functional implications of cyclooxygenase activity in this process. Here, we identify the cis-acting elements responsible for the transcriptional activation of this gene in human T lymphocytes. COX-2 promoter activity was induced upon T cell activation both in primary resting T lymphocytes and in Jurkat cells. This induction was abrogated by inhibition of calcineurin phosphatase with the immunosuppressive drug cyclosporin A, whereas expression of an active calcineurin catalytic subunit enhanced COX-2 transcriptional activation. Moreover, cotransfection of nuclear factor of activated T cells (NFAT) wild type protein transactivated COX-2 promoter activity. Conversely, dominant negative mutants of NFATc or c-Jun proteins inhibited COX-2 induction. Electrophoretic mobility shift assays and site-directed mutagenesis allowed the identification of two regions of DNA located in the positions -117 and -58 relative to the transcriptional start site that serves as NFAT recognition sequences. These results emphasize the central role that the Ca(2+)/calcineurin pathway plays in COX-2 transcriptional regulation in T lymphocytes pointing to NFAT/activator protein-1 transcription factors as essential for COX-2 promoter regulation in these cells.

A role for the p38 MAP kinase pathway in the nuclear shuttling of NFATp.

Calcium signals lead to the translocation of nuclear factor of activated T cells (NFAT) from the cytoplasm to the nucleus. This process is regulated by the calcium-activated phosphatase calcineurin, which can be cotransported with NFAT to the nucleus to maintain it transcriptionally active for the duration of calcium signaling. When the calcium signal ceases, NFAT is exported to the cytoplasm, and different NFAT kinases have been reported to oppose calcineurin activities and regulate the nuclear export of NFAT. Here we show that p38 MAPK phosphorylates in vitro and interacts in vivo with NFATp. Furthermore, the activation of this pathway in HeLa cells by cotransfection with activated MKK6 and p38 counteracts the calcium-induced nuclear accumulation of NFATp but not that of NFATc. By contrast, activation of JNK or ERK pathways failed to modify the nuclear shuttling of NFATp. Consistently, activation of p38, but not the JNK MAPK pathway, results in the inhibition of NFATp-driven transcription. In addition, the inhibition of the nuclear accumulation of NFATp by p38 appears to be mediated through the activation of NFATp nuclear export and takes place in a Leptomycin B-sensitive fashion, suggesting the involvement of the exportin CRM1 in this process. Thus, the p38 signal transduction pathway appears to play an important role in the regulation of the nuclear shuttling of NFATp and in cellular homeostasis.

Pyrrolidine dithiocarbamate protects mice from lethal shock induced by LPS or TNF-alpha.

Although important advances have been made in the development of antibiotics and medical intensive care technology in recent years, systemic response to infection remains a major health problem, with growing incidence and high mortality rates. Here we demonstrate the ability of the antioxidant agent pyrrolidine dithiocarbamate (PDTC) to inhibit the in vivo activation of NF-kappaB in lung and liver tissues, as well as the systemic release of TNF-alpha in lipopolysaccharide (LPS)-treated mice. The in vivo effect of PDTC on NF-kappaB activation in liver tissues involved the inhibition of both LPS-induced I kappaB-alpha degradation and the translocation of the p50 and p65 NF-kappaB subunits to the nucleus. In addition to protecting mice against lethal LPS doses, PDTC curtailed TNF-alpha-induced lethal shock. This effect was observed even after LPS injection, and when PDTC was administered at a time when TNF-alpha was already at maximum levels in serum. PDTC-treated mice survived despite high IL-1beta and IL-6 levels, induction of VCAM-1 and ICAM-1 expression or leukocyte infiltration in tissues known to be associated with LPS-induced shock, indicating that PDTC does not act by modifying these responses. Taken together, these results indicate that PDTC interferes with the production as well as the action of TNF-alpha, and points to a possible approach toward the treatment of septic shock.

Vascular endothelial growth factor activates nuclear factor of activated T cells in human endothelial cells: a role for tissue factor gene expression.

Vascular endothelial growth factor (VEGF) is a potent angiogenic inducer that stimulates the expression of tissue factor (TF), the major cellular initiator of blood coagulation. Here we show that signaling triggered by VEGF induced DNA-binding and transcriptional activities of nuclear factor of activated T cells (NFAT) and AP-1 in human umbilical vein endothelial cells (HUVECs). VEGF also induced TF mRNA expression and gene promoter activation by a cyclosporin A (CsA)-sensitive mechanism. As in lymphoid cells, NFAT was dephosphorylated and translocated to the nucleus upon activation of HUVECs, and these processes were blocked by CsA. NFAT was involved in the VEGF-mediated TF promoter activation as evidenced by cotransfection experiments with a dominant negative version of NFAT and site-directed mutagenesis of a newly identified NFAT site within the TF promoter that overlaps with a previously identified kappaB-like site. Strikingly, this site bound exclusively NFAT not only from nuclear extracts of HUVECs activated by VEGF, a stimulus that failed to induce NF-kappaB-binding activity, but also from extracts of cells activated with phorbol esters and calcium ionophore, a combination of stimuli that triggered the simultaneous activation of NFAT and NF-kappaB. These results implicate NFAT in the regulation of endothelial genes by physiological means and shed light on the mechanisms that switch on the gene expression program induced by VEGF and those regulating TF gene expression.

The hepatitis B virus X protein activates nuclear factor of activated T cells (NF-AT) by a cyclosporin A-sensitive pathway.

The X gene product of the human hepatitis B virus (HBx) is a transcriptional activator of various viral and cellular genes. We recently have determined that the production of tumor necrosis factor-alpha (TNF-alpha) by HBV-infected hepatocytes is transcriptionally up-regulated by HBx, involving nuclear factor of activated T cells (NF-AT)-dependent activation of the TNF-alpha gene promoter. Here we show that HBx activates NF-AT by a cyclosporin A-sensitive mechanism involving dephosphorylation and nuclear translocation of the transcription factor. Luciferase gene expression assays demonstrated that HBx transactivates transcription through NF-AT-binding sites and activates a Gal4-NF-AT chimeric protein. DNA-protein interaction assays revealed that HBx induces the formation of NF-AT-containing DNA-binding complexes. Immunofluorescence analysis demonstrated that HBx induces the nuclear translocation of NF-AT, which can be blocked by the immunosuppressive drug cyclosporin A. Furthermore, immunoblot analysis showed that the HBx-induced activation and translocation of NF-AT are associated with its dephosphorylation. Thus, HBx may play a relevant role in the intrahepatic inflammatory processes by inducing locally the expression of cytokines that are regulated by NF-AT.

Pentoxifylline inhibits adhesion and activation of human T lymphocytes.

We have herein studied the effect of pentoxifylline (PTX) on the adhesion and activation of human T lymphocytes. We found that PTX inhibited the adhesion of T cells to the beta1 and beta2 integrin ligands VCAM-1 and ICAM-1; this inhibitory activity was dose dependent, with a maximal effect from 12 to 24 h. We also found that PTX was able to interfere with the activation of beta1 integrins induced by intracellular signals; however, the conformational change of beta1 integrins induced by extracellular stimuli (e.g., activating mAbs, or Mn2+) was not significantly affected by this drug. In addition, the homotypic aggregation of T cells induced by anti-beta1 and -beta2 integrin chain mAbs was also inhibited by PTX. PTX had a significant inhibitory effect on the T lymphocyte expression of the activation Ags CD25 (IL-2R alpha-chain), CD69 (activation-inducer molecule), and CD98 (4F2) induced by PHA. Accordingly, PTX also interfered with early cell activation events such as the rise in intracellular Ca2+ and the activation of the Na+/H+ antiporter induced by PHA and phorbol esters, respectively. Furthermore, this drug inhibited both the cell cycle progression and cell proliferation of T cells induced through the CD3/TCR complex. However, this drug did not show any effect on the cell activation/proliferation induced by PMA plus ionomycin. Our results indicate that PTX interferes efficiently with the activation and cell adhesion of human T lymphocytes. These effects may be of relevance for the clinical uses of this drug.

Blockade of T-cell activation by dithiocarbamates involves novel mechanisms of inhibition of nuclear factor of activated T cells.

Dithiocarbamates (DTCs) have recently been reported as powerful inhibitors of NF-kappaB activation in a number of cell types. Given the role of this transcription factor in the regulation of gene expression in the inflammatory response, NF-kappaB inhibitors have been suggested as potential therapeutic drugs for inflammatory diseases. We show here that DTCs inhibited both interleukin 2 (IL-2) synthesis and membrane expression of antigens which are induced during T-cell activation. This inhibition, which occurred with a parallel activation of c-Jun transactivating functions and expression, was reflected by transfection experiments at the IL-2 promoter level, and involved not only the inhibition of NF-kappaB-driven reporter activation but also that of nuclear factor of activated T cells (NFAT). Accordingly, electrophoretic mobility shift assays (EMSAs) indicated that pyrrolidine DTC (PDTC) prevented NF-kappaB, and NFAT DNA-binding activity in T cells stimulated with either phorbol myristate acetate plus ionophore or antibodies against the CD3-T-cell receptor complex and simultaneously activated the binding of AP-1. Furthermore, PDTC differentially targeted both NFATp and NFATc family members, inhibiting the transactivation functions of NFATp and mRNA induction of NFATc. Strikingly, Western blotting and immunocytochemical experiments indicated that PDTC promoted a transient and rapid shuttling of NFATp and NFATc, leading to their accelerated export from the nucleus of activated T cells. We propose that the activation of an NFAT kinase by PDTC could be responsible for the rapid shuttling of the NFAT, therefore transiently converting the sustained transactivation of this transcription factor that occurs during lymphocyte activation, and show that c-Jun NH2-terminal kinase (JNK) can act by directly phosphorylating NFATp. In addition, the combined inhibitory effects on NFAT and NF-KB support a potential use of DTCs as immunosuppressants.

Antioxidants and AP-1 activation: a brief overview.

Activity of the transcription factor AP-1 is controlled by different MAPK cascades that regulate the different AP-1 components at the transcriptional and posttranscriptional level. Recently, AP-1 has been shown to behave as a redox-sensitive transcription factor that can be induced under both pro-oxidative and antioxidative conditions. In this overview we summarize the signaling pathways that converge on the activation of AP-1 and the components of these pathways that have been shown to be targets of antioxidants. The activation of AP-1 by antioxidants may account for the expression of a number of genes that mediate important functions under physiological conditions.

Pyrrolidine dithiocarbamate inhibits the production of interleukin-6, interleukin-8, and granulocyte-macrophage colony-stimulating factor by human endothelial cells in response to inflammatory mediators: modulation of NF-kappa B and AP-1 transcription factors activity.

Endothelial cells (EC) play a key role in the inflammatory response, both by the production of proinflammatory cytokines and by their interaction with leukocytes. Molecular genetic analysis has demonstrated that functional NF-kappa B sites are involved in the transcription of interleukin-6 (IL-6), IL-8, and granulocyte-macrophage colony-stimulating factor (GM-CSF) genes in response to inflammatory mediators. Thus, we have explored the effect of two inhibitors of the NF-kappa B activation, pyrrolidine dithiocarbamate (PDTC) and N-acetylcysteine (NAC), on the production of these cytokines by EC. Both PDTC and NAC inhibited, in a dose-dependent manner, the synthesis of IL-6, IL-8, and GM-CSF induced by tumor necrosis factor (TNF)-alpha or bacterial lipopolysaccharides (LPS) in human umbilical vein endothelial cells (HUVEC). PDTC appeared to prevent IL-6, IL-8, and GM-CSF gene transcription, as it blocked the induction of specific mRNA by TNF-alpha or LPS. The TNF-alpha mediated transcriptional activation of a chloramphenicol acetyltransferase (CAT) plasmid containing three copies of the -72 kappa B binding site from the IL-6 promoter was abrogated by PDTC. According to transfection experiments, electrophoretic mobility shift assays (EMSA) demonstrated that the antioxidant prevented the induction of NF-kappa B DNA-binding activity by TNF-alpha. Under the same conditions, PDTC by itself or in combination with TNF-alpha, enhanced the DNA-binding activity of AP-1, as well as c-fos and c-jun mRNA levels. Altogether, these results indicate that the antioxidant PDTC specifically inhibits the transcription of IL-6, IL-8, and GM-CSF genes through the inhibition of the NF-kappa B activation, while increasing the expression of AP-1. Our data make evident the antiinflammatory and immunoregulatory potential of the pharmacological inhibition of the NF-kappa B activation. In addition, PDTC and related molecules may be a useful tool to explore the expression of genes involved in the inflammatory response.