The role of NF-kappaB in the angiogenic response of coronary microvessel endothelial cells.

The activation of nuclear factor (NF)-kappaB by 12(R)-hydroxyeicosatrienoic acid [12(R)-HETrE], an arachidonic acid metabolite with potent stereospecific proinflammatory and angiogenic properties, was examined and its role in the angiogenic response was determined in capillary endothelial cells derived from coronary microvessels. Electrophoretic mobility-shift assay of nuclear protein extracts from cells treated with 12(R)-HETrE demonstrated a rapid and stereospecific time- and concentration-dependent increase in the binding activity of NF-kappaB, which was inhibitable by the antioxidants N-acetylcysteine, butylated hydroxyanisole, and pyrrolidine dithiocarbamate and was partially attenuated by the protein kinase C inhibitors, staurosporine and calphostin C. Neither 12(S)-HETrE nor other related eicosanoids--e.g., 12(R)-HETE, 12(S)-HETE, and leukotriene B4--stimulated the activation of NF-kappaB relative to 12(R)-HETrE, substantiating the claim for a specific receptor-mediated mechanism. 12(R)-HETrE stimulated the formation of capillary-like cords of microvessel endothelial cells distinguishable from a control; this effect was comparable to that observed with basic fibroblast growth factor (bFGF). Inhibition of NF-kappaB activation resulted in inhibition of capillary-like formation of endothelial cells treated with 12(R)-HETrE by 80% but did not affect growth observed with bFGF. It is suggested that 12(R)-HETrE's angiogenic activity involves the activation of NF-kappaB, possibly via protein kinase C stimulation and the generation of reactive oxygen intermediates for downstream signaling.

Aspirin triggers previously undescribed bioactive eicosanoids by human endothelial cell-leukocyte interactions.

Aspirin [acetylsalicylic acid (ASA)], along with its analgesic-antipyretic uses, is now also being considered for cardiovascular protection and treatments in cancer and human immunodeficiency virus infection. Although many of ASA's pharmacological actions are related to its ability to inhibit prostaglandin and thromboxane biosynthesis, some of its beneficial therapeutic effects are not completely understood. Here, ASA triggered transcellular biosynthesis of a previously unrecognized class of eicosanoids during coincubations of human umbilical vein endothelial cells (HUVEC) and neutrophils [polymorphonuclear leukocytes (PMN)]. These eicosanoids were generated with ASA but not by indomethacin, salicylate, or dexamethasone. Formation was enhanced by cytokines (interleukin 1 beta) that induced the appearance of prostaglandin G/H synthase 2 (PGHS-2) but not 15-lipoxygenase, which initiates their biosynthesis from arachidonic acid in HUVEC. Costimulation of HUVEC/PMN by either thrombin plus the chemotactic peptide fMet-Leu-Phe or phorbol 12-myristate 13-acetate or ionophore A23187 leads to the production of these eicosanoids from endogenous sources. Four of these eicosanoids were also produced when PMN were exposed to 15R-HETE [(15R)-15-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid] and an agonist. Physical methods showed that the class consists of four tetraene-containing products from arachidonic acid that proved to be 15R-epimers of lipoxins. Two of these compounds (III and IV) were potent inhibitors of leukotriene B4-mediated PMN adhesion to HUVEC, with compound IV [(5S,6R,15R)-5,6,15-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoi c acid; 15-epilipoxin A4] active in the nanomolar range. These results demonstrate that ASA evokes a unique class of eicosanoids formed by acetylated PGHS-2 and 5-lipoxygenase interactions, which may contribute to the therapeutic impact of this drug. Moreover, they provide an example of a drug's ability to pirate endogenous biosynthetic mechanisms to trigger new mediators.

Plasma cytokine changes in relation to exercise intensity and muscle damage

The purpose of this study was to compare the effects of exercise intensity and exercise-induced muscle damage on changes in anti-inflammatory cytokines and other inflammatory mediators. Nine well-trained male runners completed three different exercise trials on separate occasions: ( 1) level treadmill running at 60% VO2max (moderate-intensity trial) for 60 min; (2) level treadmill running at 85% VO2max (high-intensity trial) for 60 min; (3) downhill treadmill running ( - 10% gradient) at 60% VO2 max (downhill running trial) for 45 min. Blood was sampled before, immediately after and 1 h after exercise. Plasma was analyzed for interleukin-1 receptor antagonist (IL-1ra), IL-4, IL-5, IL-10, IL-12p40, IL-13, monocyte chemotactic protein-1 (MCP-1), prostaglandin E-2, leukotriene B-4 and heat shock protein 70 (HSP70). The plasma concentrations of IL-1ra, IL-12p40, MCP-1 and HSP70 increased significantly (P< 0.05) after all three trials. Plasma prostaglandin E-2 concentration increased significantly after the downhill running and high-intensity trials, while plasma IL-10 concentration increased significantly only after the high-intensity trial. IL-4 and leukotriene B4 did not increase significantly after exercise. Plasma IL-1ra and IL-10 concentrations were significantly higher ( P< 0.05) after the high-intensity trial than after both the moderate-intensity and downhill running trials. Therefore, following exercise up to 1 h duration, exercise intensity appears to have a greater effect on anti-inflammatory cytokine production than exercise-induced muscle damage.

Neutrophil Chemotaxis in Multiple Chemoattractant Gradients

Chemotaxis, the phenomenon in which cells move in response to extracellular chemical gradients, plays a prominent role in the mammalian immune response. During this process, a number of chemical signals, called chemoattractants, are produced at or proximal to sites of infection and diffuse into the surrounding tissue. Immune cells sense these chemoattractants and move in the direction where their concentration is greatest, thereby locating the source of attractants and their associated targets. Leading the assault against new infections is a specialized class of leukocytes (white blood cells) known as neutrophils, which normally circulate in the bloodstream. Upon activation, these cells emigrate out of the vasculature and navigate through interstitial tissues toward target sites. There they phagocytose bacteria and release a number of proteases and reactive oxygen intermediates with antimicrobial activity. Neutrophils recruited by infected tissue in vivo are likely confronted by complex chemical environments consisting of a number of different chemoattractant species. These signals may include end target chemicals produced in the vicinity of the infectious agents, and endogenous chemicals released by local host tissues during the inflammatory response. To successfully locate their pathogenic targets within these chemically diverse and heterogeneous settings, activated neutrophils must be capable of distinguishing between the different signals and employing some sort of logic to prioritize among them. This ability to simultaneously process and interpret mulitple signals is thought to be essential for efficient navigation of the cells to target areas. In particular, aberrant cell signaling and defects in this functionality are known to contribute to medical conditions such as chronic inflammation, asthma and rheumatoid arthritis. To elucidate the biomolecular mechanisms underlying the neutrophil response to different chemoattractants, a number of efforts have been made toward understanding how cells respond to different combinations of chemicals. Most notably, recent investigations have shown that in the presence of both end target and endogenous chemoattractant variants, the cells migrate preferentially toward the former type, even in very low relative concentrations of the latter. Interestingly, however, when the cells are exposed to two different endogenous chemical species, they exhibit a combinatorial response in which distant sources are favored over proximal sources. Some additional results also suggest that cells located between two endogenous chemoattractant sources will respond to the vectorial sum of the combined gradients. In the long run, this peculiar behavior could result in oscillatory cell trajectories between the two sources. To further explore the significance of these and other observations, particularly in the context of physiological conditions, we introduce in this work a simplified phenomenological model of neutrophil chemotaxis. In particular, this model incorporates a trait commonly known as directional persistence - the tendency for migrating neutrophils to continue moving in the same direction (much like momentum) - while also accounting for the dose-response characteristics of cells to different chemical species. Simulations based on this model suggest that the efficiency of cell migration in complex chemical environments depends significantly on the degree of directional persistence. In particular, with appropriate values for this parameter, cells can improve their odds of locating end targets by drifting through a network of attractant sources in a loosely-guided fashion. This corroborates the prediction that neutrophils randomly migrate from one chemoattractant source to the next while searching for their end targets. These cells may thus use persistence as a general mechanism to avoid being trapped near sources of endogenous chemoattractants - the mathematical analogue of local maxima in a global optimization problem. Moreover, this general foraging strategy may apply to other biological processes involving multiple signals and long-range navigation.

Karta öfver Finland (Sektionen B4)

1 kartta :, vär. ;, 51,3 x 43,1 cm, lehti 58 x 50,2 cm

Respuesta inflamatoria y de fase aguda en el absceso hepático amibiano mediante el estudio de leucotrienos B4, C4, E4 y D4 e interleucina1 beta, interleucina 6 y factor de necrosis tumoral alfa

Tesis (Doctorado en Ciencias con Especialidad en Química Biomédica) UANL

Patent DE10 2004 037 519 B4 “Sensor Process and Device for Determining a Physical Value“. A Proof of Concept

At present, a fraction of 0.1 - 0.2% of the patients undergoing surgery become aware during the process. The situation is referred to as anesthesia awareness and is obviously very traumatic for the person experiencing it. The reason for its occurrence is mostly an insufficient dosage of the narcotic Propofol combined with the incapability of the technology monitoring the depth of the patient’s anesthetic state to notice the patient becoming aware. A solution can be a highly sensitive and selective real time monitoring device for Propofol based on optical absorption spectroscopy. Its working principle has been postulated by Prof. Dr. habil. H. Hillmer and formulated in DE10 2004 037 519 B4, filed on Aug 30th, 2004. It consists of the exploitation of Intra Cavity Absorption effects in a two mode laser system. In this Dissertation, a two mode external cavity semiconductor laser, which has been developed previously to this work is enhanced and optimized to a functional sensor. Enhancements include the implementation of variable couplers into the system and the implementation of a collimator arrangement into which samples can be introduced. A sample holder and cells are developed and characterized with a focus on compatibility with the measurement approach. Further optimization concerns the overall performance of the system: scattering sources are reduced by re-splicing all fiber-to-fiber connections, parasitic cavities are eliminated by suppressing the Fresnel reflexes of all one fiber ends by means of optical isolators and wavelength stability of the system is improved by the implementation of thermal insulation to the Fiber Bragg Gratings (FBG). The final laser sensor is characterized in detail thermally and optically. Two separate modes are obtained at 1542.0 and 1542.5 nm, tunable in a range of 1nm each. Mode Full Width at Half Maximum (FWHM) is 0.06nm and Signal to Noise Ratio (SNR) is as high as 55 dB. Independent of tuning the two modes of the system can always be equalized in intensity, which is important as the delicacy of the intensity equilibrium is one of the main sensitivity enhancing effects formulated in DE10 2004 037 519 B4. For the proof of concept (POC) measurements the target substance Propofol is diluted in the solvents Acetone and DiChloroMethane (DCM), which have been investigated for compatibility with Propofol beforehand. Eight measurement series (two solvents, two cell lengths and two different mode spacings) are taken, which draw a uniform picture: mode intensity ratio responds linearly to an increase of Propofol in all cases. The slope of the linear response indicates the sensitivity of the system. The eight series are split up into two groups: measurements taken in long cells and measurements taken in short cells.

Differential kinase requirement for enhancement of Fc gamma R-mediated phagocytosis in alveolar macrophages by leukotriene B(4) vs. D(4)

In alveolar macrophages, leukotriene (IT) B(4) and cysteinyl LTs (LTC(4), LTD(4) and LTE(4)) both enhance Fc gamma receptor (Fc gamma R)-mediated phagocytosis. In the present study we investigated the role of specific PKC isoforms (PKC-alpha and -delta), the MAP kinases p38 and ERK 1/2, and PI3K in mediating the potentiation of Fc gamma R-mediated phagocytosis induced by addition of leukotrienes to the AMs. It was found that exogenously added LTB(4) and LTD(4) both enhanced PKC-delta and -alpha phosphorylation during Fc gamma R engagement. Studies with isoform-selective inhibitors indicated that exogenous LTB(4) effects were dependent on both PKC-alpha and -delta, while LTD(4) effects were exclusively due to PKC-delta activation. Although both exogenous LTB(4) and LTD(4) enhanced p38 and ERK 1/2 activation, LTB(4) required only ERK 1/2, while LTD(4) required only p38 activation. Activation by both LTs was dependent on PI3K activation. Effects of endogenous LTs on kinase activation were also investigated using selective LT receptor antagonists. Endogenous LTB(4) contributed to Fc gamma R-mediated activation of PKC-alpha, ERK 1/2 and PI3K, while endogenous cysLTs contributes to activation of PKC-delta, p38 and PI3K. Taken together, our data show that the capacities of LTB(4) and LTD(4) to enhance Fc gamma R-mediated phagocytosis reflect their differential activation of specific kinase programs. (C) 2008 Elsevier Ltd. All rights reserved.

Connective tissue mast cells are the target of formaldehyde to induce tracheal hyperresponsiveness in rats: Putative role of leukotriene B(4) and nitric oxide

Formaldehyde (FA) exposure induces upper airways irritation and respiratory abnormalities, but its mechanisms are not understood. Since mast cells are widely distributed in the airways, we hypothesized that FA might modify the airways reactivity by mechanism involving their activation. Tracheal rings of rats were incubated with Dulbecco`s modified medium culture containing FA (0.1 ppm) in 96-well plastic microplates in a humid atmosphere. After 30 min, 6 h, and 24-72 h, the rings were suspended in an organ bath and dose-response curve to methacholine (MCh) were determined. incubation with FA caused a transient tracheal hyperresponsiveness to MCh that was independent from tracheal epithelium integrity. Connective tissue mast cell depletion caused by compound 48/80 or mast cell activation by the allergic reaction, before exposure of tracheal rings to FA prevented the increased responsiveness to MCh. LTB(4) concentrations were increased in the culture medium of tracheas incubated with FA for 48 h, whereas the LTB(4)-receptor antagonist MK886 (1 mu M) added before FA exposure rendered the tracheal rings normoreactive to MCh. In addition, FA exposure did not cause hyperresponsiveness in tracheal segments incubated with L-arginine (1 mu M). We suggest that airway connective tissue mast cells constitute the target and may provide the increased LTB(4) generation as well as an elevated consumption of NO leading to tracheal hyperresponsiveness to MCh. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Effect of a leukotriene inhibitor (MK886) on nitric oxide and hydrogen peroxide production by macrophages of acutely and chronically stressed mice

We evaluated the effect of a leukotriene inhibitor (MK886) on nitric oxide (NO) and hydrogen peroxide (H2O2) production by peritoneal macrophages of mice subjected to acute and chronic stress. Acute stress was induced by keeping mice immobilized in a tube for 2 h. Chronic stress was induced over a 7-day period by the same method, but with increasing duration of immobilization. The effects of MK886 were investigated in-vitro after incubation with peritoneal macrophages, and in-vivo by submitting mice to stress and treating them daily with MK886. Supernatants of macrophage cultures were collected for NO determination and adherent cells were used for H2O2 determination. Macrophages from mice submitted to acute or chronic stress showed no alterations in H2O2 production. However, macrophages of acutely and chronically stressed mice showed inhibition of NO after incubation with MK886 in-vitro. Administration of MK886 to chronically stressed mice increased generation of H2O2 and inhibited production of NO. Our data suggest an important role of leukotrienes in NO synthesis, which is important in controlling replication of several infectious agents, mainly in stressed and immunosuppressed animals.

The Leukotriene B-4/BLT1 Axis Is a Key Determinant in Susceptibility and Resistance to Histoplasmosis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)