Linker for Activation of T-cell Family Member2 (LAT2) a Lipid Raft Adaptor Protein for AKT Signaling, Is an Early Mediator of Alkylphospholipid Anti-leukemic Activity

Lipid rafts are highly ordered membrane domains rich in cholesterol and sphingolipids that provide a scaffold for signal transduction proteins; altered raft structure has also been implicated in cancer progression. We have shown that 25 mu M 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC), an alkylphospholipid, targets high cholesterol domains in model membranes and induces apoptosis in leukemia cells but spares normal hematopoietic and epithelial cells under the same conditions. We performed a quantitative (SILAC) proteomic screening of ODPC targets in a lipid-raft-enriched fraction of leukemic cells to identify early events prior to the initiation of apoptosis. Six proteins, three with demonstrated palmitoylation sites, were reduced in abundance. One, the linker for activation of T-cell family member 2 (LAT2), is an adaptor protein associated with lipid rafts in its palmitoylated form and is specifically expressed in B lymphocytes and myeloid cells. Interestingly, LAT2 is not expressed in K562, a cell line more resistant to ODPC-induced apoptosis. There was an early loss of LAT2 in the lipid-raft-enriched fraction of NB4 cells within 3 h following treatment with 25 mu M ODPC. Subsequent degradation of LAT2 by proteasomes was observed. Twenty-five mu M ODPC inhibited AKT activation via myeloid growth factors, and LAT2 knockdown in NB4 cells by shRNA reproduced this effect. LAT2 knockdown in NB4 cells also decreased cell proliferation and increased cell sensitivity to ODPC (7.5X), perifosine (3X), and arsenic trioxide (8.5X). Taken together, these data indicate that LAT2 is an early mediator of the anti-leukemic activity of alkylphospholipids and arsenic trioxide. Thus, LAT2 may be used as a target for the design of drugs for cancer therapy. Molecular & Cellular Proteomics 11: 10.1074/mcp.M112.019661, 1898-1912, 2012.

HYDROGEN SULFIDE AS A CRYOGENIC MEDIATOR OF HYPDXIA-INDUCED ANAPYREXIA

Hypoxia causes a regulated decrease in body temperature (Tb), a response that has been aptly called anapyrexia, but the mechanisms involved are not completely understood. The roles played by nitric oxide (NO) and other neurotransmitters have been documented during hypoxia-induced anapyrexia, but no information exists with respect to hydrogen sulfide (H(2)S), a gaseous molecule endogenously produced by cystathionine beta-synthase (CBS). We tested the hypothesis that HA production is enhanced during hypoxia and that the gas acts in the anteroventral preoptic region (AVPO; the most important thermosensitive and thermointegrative region of the CNS) modulating hypoxia-induced anapyrexia. Thus, we assessed CBS and nitric oxide synthase (NOS) activities [by means of H2S and nitrite/nitrate (NO(x)) production, respectively] as well as cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine 3',5'-monophosphate (cGMP) levels in the anteroventral third ventricle region (AV3V; where the AVPO is located) during normoxia and hypoxia. Furthermore, we evaluated the effects of pharmacological modifiers of the H2S pathway given i.c.v. or intra-AVPO. I.c.v. or intra-AVPO microinjection of CBS inhibitor caused no change in Tb under normoxia but significantly attenuated hypoxia-induced anapyrexia. During hypoxia there were concurrent increases in H2S production, which could be prevented by CBS inhibitor, indicating the endogenous source of the gas. cAMP concentration, but not cGMP and NOR, correlated with CBS activity. CBS inhibition increased NOS activity, whereas H2S donor decreased NO. production. In conclusion, hypoxia activates H2S endogenous production through the CBS-H(2)S pathway in the AVPO, having a cryogenic effect. Moreover, the present data are consistent with the notion that the two gaseous molecules, H(2)S and NO, play a key role in mediating the drop in Tb caused by hypoxia and that a fine-balanced interplay between NOS-NO and CBS-H(2)S pathways takes place in the AVPO of rats exposed to hypoxia. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Divergent Roles of the CRH Receptors in the Control of Gonadotropin Secretion Induced by Acute Restraint Stress at Proestrus

CRH has been implicated as a mediator of stress-induced effects on the hypothalamus-pituitary-gonad axis, acting via CRH receptors in various brain regions. We investigated whether the effects of restraint stress on the secretion of gonadotropins on the morning of proestrus are mediated by the CRH-R1 or CRH-R2 receptors in the oval subdivision of the anterolateral BST, the central amygdala, the locus coeruleus (LC), or the A1 and A2 neuron groups in the medulla. At proestrus morning, rats were injected with antalarmin (a CRH-R1 antagonist), asstressin2-B (a CRH-R2 antagonist) or vehicles. Thirty minutes after the injection, the animals were placed into restraints for 30 min, and blood was sampled for 2 h. At the end of the experiment, the brains were removed for immunofluorescence analyses. Restraint stress increased the levels of FSH and LH. Antalarmin blocked the stress-induced increases in FSH and LH secretion, but astressin2-B only blocked the increase in FSH secretion. LC showed intense stress-induced neuronal activity. FOS/tyrosine-hydroxylase coexpression in LC was reduced by antalarmin, but not astressin2-B. The CRH-R1 receptor, more than CRH-R2 receptor, appears to be essential for the stimulation of the hypothalamus-pituitary-gonad axis by acute stress; this response is likely mediated in part by noradrenergic neurons in the LC. We postulate that the stress-induced facilitation of reproductive function is mediated, at least in part, by CRH action through CRH-R1 on noradrenaline neurons residing in the LC that trigger GnRH discharge and gonadotropin secretion. (Endocrinology 153: 4838-4848, 2012)

Physical and psychosocial risk factors for musculoskeletal disorders in Brazilian and Italian nurses

As part of the international CUPID investigation, we compared physical and psychosocial risk factors for musculoskeletal disorders among nurses in Brazil and Italy. Using questionnaires, we collected information on musculoskeletal disorders and potential risk factors from 751 nurses employed in public hospitals. By fitting country-specific multiple logistic regression models, we investigated the association of stressful physical activities and psychosocial characteristics with site-specific and multisite pain, and associated sickness absence. We found no clear relationship between low back pain and occupational lifting, but neck and shoulder pain were more common among nurses who reported prolonged work with the arms in an elevated position. After adjustment for potential confounding variables, pain in the low back, neck and shoulder, multisite pain, and sickness absence were all associated with somatizing tendency in both countries. Our findings support a role of somatizing tendency in predisposition to musculoskeletal disorders, acting as an important mediator of the individual response to triggering exposures, such as work-load.

The nurse and the care to the elderly women: a social phenomenological approach

Objective: To understand how nurses see care delivery to elderly women. Methods: In this phenomenological study, ten nurses working at Primary Health Care Units were interviewed between September 2010 and January 2011. Results: In care delivery, nurses consider the elderly women's knowledge background and biographical situation, and also value the family's participation as a care mediator. These professionals have the acuity to capture these women's specific demands, but face difficulties to deliver care to these clients. Nurses expect to deliver qualified care to these women. Conclusion: The theoretical and methodological approach of social phenomenology permitted revealing that the nurse designs qualified care to elderly women, considering the possibilities in the context. This includes the participation of different social actors and health sectors, assuming collective efforts in action strategies and professional training, in line with the particularities and care needs of elderly women nurses identify.

Endothelin-1 induces neutrophil recruitment in adaptive inflammation via TNF alpha and CXCL1/CXCR2 in mice

Endothelin mediates neutrophil recruitment during innate inflammation. Herein we address whether endothelin-1 (ET-1) is involved in neutrophil recruitment in adaptive inflammation in mice, and its mechanisms. Pharmacological treatments were used to determine the role of endothelin in neutrophil recruitment to the peritoneal cavity of mice challenged with antigen (ovalbumin) or ET-1. Levels of ET-1, tumour necrosis factor a (TNF alpha), and CXC chemokine ligand 1 (CXCL1) were determined by enzyme-linked immunosorbent assay. Neutrophil migration and flow cytometry analyses were performed 4 h after the intraperitoneal stimulus. ET-1 induced dose-dependent neutrophil recruitment to the peritoneal cavity. Treatment with the non-selective ETA/ETB receptor antagonist bosentan, and selective ETA or ETB receptor antagonists BQ-123 or BQ-788, respectively, inhibited ET-1- and ovalbumin-induced neutrophil migration to the peritoneal cavity. In agreement with the above, the antigen challenge significantly increased levels of ET-1 in peritoneal exudates. The ET-1- and ovalbumin-induced neutrophil recruitment were reduced in TNFR1 deficient mice, and by treatments targeting CXCL1 or CXC chemokine receptor 2 (CXCR2); further, treatment with bosentan, BQ-123, or BQ-788 inhibited ET-1- and antigen-induced production of TNFa and CXCL1. Furthermore, ET-1 and ovalbumin challenge induced an increase in the number of cells expressing the Gr1(+) markers in the granulocyte gate, CD11c+ markers in the monocyte gate, and CD4(+) and CD45(+) (B220) markers in the lymphocyte gate in an ETA-and ETB-dependent manner, as determined by flow cytometry analysis, suggesting that ET-1 might be involved in the recruitment of neutrophils and other cells in adaptive inflammation. Therefore, the present study demonstrates that ET-1 is an important mediator for neutrophil recruitment in adaptive inflammation via TNF alpha and CXCL1/CXCR2-dependent mechanism.

Improving the performance of a glucose biosensor using an ionic liquid for enzyme immobilization. On the chemical interaction between the biomolecule, the ionic liquid and the cross-linking agent

The effect of the room temperature ionic liquid (1-butyl-2,3-dimethylimidazolium tetrafluoroborate ([BMMI][BF4])) on the immobilization of glucose oxidase (GOx) was studied. The electrochemical performance of biosensors prepared following different protocols indicated a beneficial effect of the ionic liquid on the analytical parameters. The chemical interaction between GOx, [BMMI][BF4] and glutaraldehyde was investigated using UV-visible spectroscopy (UV-vis) and circular dichroism (CD). Structural changes of the biomolecule were observed to depend on the method used for the immobilization. (C) 2011 Elsevier Ltd. All rights reserved.

Major involvement of mTOR in the PPAR gamma-induced stimulation of adipose tissue lipid uptake and fat accretion

Evidence points to a role of the mammalian target of rapamycin (mTOR) signaling pathway as a regulator of adiposity, yet its involvement as a mediator of the positive actions of peroxisome proliferator-activated receptor (PPAR)gamma agonism on lipemia, fat accretion, lipid uptake, and its major determinant lipoprotein lipase (LPL) remains to be elucidated. Herein we evaluated the plasma lipid profile, triacylglycerol (TAG) secretion rates, and adipose tissue LPL-dependent lipid uptake, LPL expression/activity, and expression profile of other lipid metabolism genes in rats treated with the PPAR gamma agonist rosiglitazone (15 mg/kg/day) in combination or not with the mTOR inhibitor rapamycin (2 mg/kg/day) for 15 days. Rosiglitazone stimulated adipose tissue mTOR complex 1 and AMPK and induced TAG-derived lipid uptake (136%), LPL mRNA/activity (2- to 6-fold), and fat accretion in subcutaneous (but not visceral) white adipose tissue (WAT; 50%) and in brown adipose tissue (BAT; 266%). Chronic mTOR inhibition attenuated the upregulation of lipid uptake, LPL expression/activity, and fat accretion induced by PPAR gamma activation in both subcutaneous WAT and BAT, which resulted in hyperlipidemia. In contrast, rapamycin did not affect most of the other WAT lipogenic genes upregulated by rosiglitazone. Together these findings demonstrate that mTOR is a major regulator of adipose tissue LPL-mediated lipid uptake and a critical mediator of the hypolipidemic and lipogenic actions of PPAR gamma activation.-Blanchard, P-G., W. T. Festuccia, V. P. Houde, P. St-Pierre, S. Brule, V. Turcotte, M. Cote, K. Bellmann, A. Marette, and Y. Deshaies. Major involvement of mTOR in the PPAR gamma-induced stimulation of adipose tissue lipid uptake and fat accretion. J. Lipid Res. 2012. 53: 1117-1125.

Erythropoietin prevents sepsis-related acute kidney injury in rats by inhibiting NF-kappa B and upregulating endothelial nitric oxide synthase

de Souza ACCP, Volpini RA, Shimizu MH, Sanches TR, Camara NOS, Semedo P, Rodrigues CE, Seguro AC, Andrade L. Erythropoietin prevents sepsis-related acute kidney injury in rats by inhibiting nuclear factor-kappa B and upregulating endothelial nitric oxide synthase. Am J Physiol Renal Physiol 302: F1045-F1054, 2012. First published January 11, 2012; doi:10.1152/ajprenal.00148.2011.-The pathophysiology of sepsis involves complex cytokine and inflammatory mediator networks, a mechanism to which NF-kappa B activation is central. Downregulation of endothelial nitric oxide synthase (eNOS) contributes to sepsis-induced endothelial dysfunction. Erythropoietin (EPO) has emerged as a major tissue-protective cytokine in the setting of stress. We investigated the role of EPO in sepsis-related acute kidney injury using a cecal ligation and puncture (CLP) model. Wistar rats were divided into three primary groups: control (sham-operated); CLP; and CLP + EPO. EPO (4,000 IU/kg body wt ip) was administered 24 and 1 h before CLP. Another group of rats received N-nitro-L-arginine methyl ester (L-NAME) simultaneously with EPO administration (CLP + EPO + L-NAME). A fifth group (CLP + EPOtreat) received EPO at 1 and 4 h after CLP. At 48 h postprocedure, CLP + EPO rats presented significantly higher inulin clearance than did CLP and CLP + EPO + L-NAME rats; hematocrit levels, mean arterial pressure, and metabolic balance remained unchanged in the CLP + EPO rats; and inulin clearance was significantly higher in CLP + EPOtreat rats than in CLP rats. At 48 h after CLP, creatinine clearance was significantly higher in the CLP + EPO rats than in the CLP rats. In renal tissue, pre-CLP EPO administration prevented the sepsis-induced increase in macrophage infiltration, as well as preserving eNOS expression, EPO receptor (EpoR) expression, IKK-alpha activation, NF-kappa B activation, and inflammatory cytokine levels, thereby increasing survival. We conclude that this protection, which appears to be dependent on EpoR activation and on eNOS expression, is attributable, in part, to inhibition of the inflammatory response via NF-kappa B downregulation.

Augmented nitric oxide production and up-regulation of endothelial nitric oxide synthase during cecal ligation and perforation

Nitric oxide (NO) has been pointed out as being the main mediator involved in the hypotension and tissue injury taking place during sepsis. This study aimed to investigate the cellular mechanisms implicated in the acetylcholine (ACh)-induced relaxation detected in aortic rings isolated from rats submitted to cecal ligation and perforation (CLP group), 6 h post-CLP. The mean arterial pressure was recorded, and the concentration-effect curves for ACh were constructed for endothelium-intact aortic rings in the absence (control) or after incubation with one of the following NO synthase inhibitors: L-NAME (non-selective), L-NNA (more selective for eNOS), 7-nitroindazole (more selective for nNOS), or 1400W (selective for iNOS). The NO concentration was determined by using confocal microscopy. The protein expression of the NOS isoforms was quantified by Western blot analysis. The prostacyclin concentration was indirectly analyzed on the basis of 6-keto-prostaglandin F-1 alpha (6-keto-PGF(1 alpha)) levels measured by enzyme immunoassay. There were no differences between Sham- and CLP-operated rats in terms of the relaxation induced by acetylcholine. However, the NOS inhibitors reduced this relaxation in both groups, but this effect remained more pronounced in the CLP group as compared to the Sham group. The acetylcholine-induced NO production was higher in the rat aortic endothelial cells of the CLP group than in those of the Sham group. eNOS protein expression was larger in the CLP group, but the iNOS protein was not verified in any of the groups. The basal 6-keto-PGF(1 alpha) levels were higher in the CLP group, but the acetylcholine-stimulated levels did not increase in CLP as much as they did in the Sham group. Taken together, our results show that the augmented NO production in sepsis syndrome elicited by cecal ligation and perforation is due to eNOS up-regulation and not to iNOS. (C) 2012 Elsevier Inc. All rights reserved.

SMALL INTERFERING RNA TARGETING FOCAL ADHESION KINASE PREVENTS CARDIAC DYSFUNCTION IN ENDOTOXEMIA

Sepsis and septic shock are associated with cardiac depression. Cardiovascular instability is a major cause of death in patients with sepsis. Focal adhesion kinase (FAK) is a potential mediator of cardiomyocyte responses to oxidative and mechanical stress. Myocardial collagen deposition can affect cardiac compliance and contractility. The aim of the present study was to determine whether the silencing of FAK is protective against endotoxemia-induced alterations of cardiac structure and function. In male Wistar rats, endotoxemia was induced by intraperitoneal injection of lipopolysaccharide (10 mg/kg). Cardiac morphometry and function were studied in vivo by left ventricular catheterization and histology. Intravenous injection of small interfering RNA targeting FAK was used to silence myocardial expression of the kinase. The hearts of lipopolysaccharide-injected rats showed collagen deposition, increased matrix metalloproteinase 2 activity, and myocyte hypertrophy, as well as reduced 24-h +dP/dt and -dP/dt, together with hypotension, increased left ventricular end-diastolic pressure, and elevated levels of FAK (phosphorylated and unphosphorylated). Focal adhesion kinase silencing reduced the expression and activation of the kinase in cardiac tissue, as well as protecting against the increased collagen deposition, greater matrix metalloproteinase 2 activity, and reduced cardiac contractility that occur during endotoxemia. In conclusion, FAK is activated in endotoxemia, playing a role in cardiac remodeling and in the impairment of cardiac function. This kinase represents a potential therapeutic target for the protection of cardiac function in patients with sepsis.

Protection conferred by heterologous vaccination against tuberculosis is dependent on the ratio of CD4(+)/CD4(+) Foxp3(+) cells

CD4(+) Foxp3(+) regulatory T cells inhibit the production of interferon-?, which is the major mediator of protection against Mycobacterium tuberculosis infection. In this study, we evaluated whether the protection conferred by three different vaccines against tuberculosis was associated with the number of spleen and lung regulatory T cells. We observed that after homologous immunization with the 65 000 molecular weight heat-shock protein (hsp 65) DNA vaccine, there was a significantly higher number of spleen CD4(+) Foxp3(+) cells compared with non-immunized mice. Heterologous immunization using bacillus Calmette Guerin (BCG) to prime and DNA-hsp 65 to boost (BCG/DNA-hsp 65) or BCG to prime and culture filtrate proteins (CFP)-CpG to boost (BCG/CFP-CpG) induced a significantly higher ratio of spleen CD4(+)/CD4(+) Foxp3(+) cells compared with non-immunized mice. In addition, the protection conferred by either the BCG/DNA-hsp 65 or the BCG/CFP-CpG vaccines was significant compared with the DNA-hsp 65 vaccine. Despite the higher ratio of spleen CD4(+)/CD4(+) Foxp3(+) cells found in BCG/DNA-hsp 65-immunized or BCG/CFP-CpG-immunized mice, the lungs of both groups of mice were better preserved than those of DNA-hsp 65-immunized mice. These results confirm the protective efficacy of BCG/DNA-hsp 65 and BCG/CFP-CpG heterologous prime-boost vaccines and the DNA-hsp 65 homologous vaccine. Additionally, the prime-boost regimens assayed here represent a promising strategy for the development of new vaccines to protect against tuberculosis because they probably induce a proper ratio of CD4(+) and regulatory (CD4(+) Foxp3(+)) cells during the immunization regimen. In this study, this ratio was associated with a reduced number of regulatory cells and no injury to the lungs.

Macrophage Dectin-1 Expression Is Controlled by Leukotriene B-4 via a GM-CSF/PU.1 Axis

Pattern recognition receptors for fungi include dectin-1 and mannose receptor, and these mediate phagocytosis, as well as production of cytokines, reactive oxygen species, and the lipid mediator leukotriene B-4 (LTB4). The influence of G protein-coupled receptor ligands such as LTB4 on fungal pattern recognition receptor expression is unknown. In this study, we investigated the role of LTB4 signaling in dectin-1 expression and responsiveness in macrophages. Genetic and pharmacologic approaches showed that LTB4 production and signaling through its high-affinity G protein-coupled receptor leukotriene B4 receptor 1 (BLT1) direct dectin-1-dependent binding, ingestion, and cytokine production both in vitro and in vivo. Impaired responses to fungal glucans correlated with lower dectin-1 expression in macrophages from leukotriene (LT)- and BLT1-deficent mice than their wildtype counterparts. LTB4 increased the expression of the transcription factor responsible for dectin-1 expression, PU.1, and PU.1 small interfering RNA abolished LTB4-enhanced dectin-1 expression. GM-CSF controls PU.1 expression, and this cytokine was decreased in LT-deficient macrophages. Addition of GM-CSF to LT-deficient cells restored expression of dectin-1 and PU.1, as well as dectin-1 responsiveness. In addition, LTB4 effects on dectin-1, PU.1, and cytokine production were blunted in GM-CSF-/- macrophages. Our results identify LTB4-BLT1 signaling as an unrecognized controller of dectin-1 transcription via GM-CSF and PU.1 that is required for fungi-protective host responses. The Journal of Immunology, 2012, 189: 906-915.

Influence of Mid-Trimester Amniotic Fluid on Endogenous and Lipopolysaccharide-Mediated Responses of Mononuclear Lymphoid Cells

Problem We evaluated the influence of amniotic fluid (AF) on immune mediator production by mononuclear leukocytes. Method of study Thirty mid-gestation AFs were incubated with peripheral blood mononuclear cells (PBMCs) in the presence or absence of lipopolysaccharide (LPS). Supernatants were tested for interleukin (IL) -6, 10, 12, 23, tumor necrosis factor-alpha (TNF-alpha) and monocyte chemotactic protein (MCP)-1. Results Endogenous mediator production was minimal or non-detectable. AF stimulated endogenous MCP-1, IL-6 and TNF-alpha release. In the presence of LPS, production of MCP-1 and IL-10 by PBMCs was enhanced eightto ninefold by AF. Release of IL-6 and IL-23 was enhanced less than twofold by the addition of AF while TNF-alpha production was unchanged. AF-stimulated mediator production was similar irrespective of pregnancy outcome. Conclusion Selective AF stimulation of LPS-mediated MCP-1 and IL-10 release may be a mechanism to promote antibody production and the influx of phagocytic cells to engulf pathogens while downregulating the production of pro-inflammatory cytokines.

Diagnostic methods in sepsis: the need of speed

Objective: Sepsis is a common condition encountered in hospital environments. There is no effective treatment for sepsis, and it remains an important cause of death at intensive care units. This study aimed to discuss some methods that are available in clinics, and tests that have been recently developed for the diagnosis of sepsis. Methods: A systematic review was performed through the analysis of the following descriptors: sepsis, diagnostic methods, biological markers, and cytokines. Results: The deleterious effects of sepsis are caused by an imbalance between the invasiveness of the pathogen and the ability of the host to mount an effective immune response. Consequently, the host's immune surveillance fails to eliminate the pathogen, allowing it to spread. Moreover, there is a pro-inflammatory mediator release, inappropriate activation of the coagulation and complement cascades, leading to dysfunction of multiple organs and systems. The difficulty achieve total recovery of the patient is explainable. There is an increased incidence of sepsis worldwide due to factors such as aging population, larger number of surgeries, and number of microorganisms resistant to existing antibiotics. Conclusion: The search for new diagnostic markers associated with increased risk of sepsis development and molecules that can be correlated to certain steps of sepsis is becoming necessary. This would allow for earlier diagnosis, facilitate patient prognosis characterization, and prediction of possible evolution of each case. All other markers are regrettably constrained to research units.