No description available.

In humans, washing stored blood products prior to transfusion reduces storage lesions and the potential for transfusion reaction, but the effectiveness of washing units of canine whole blood is unknown. The objective of this study was to determine if a manual method of washing of stored whole blood units reduced storage lesions without adversely affecting erythrocytes. Units of canine whole blood were stored for 28 days and manually washed three times with sterile .9% NaCl. Following the first wash, there was a decrease in serum potassium (P<.0001), lactate (P<.0001), pH (P=.0110), pCO2 (P<.0001), TCO2 (P<.0001), arachidonic acid (P<.0001), and thromboxane B2 (P=.0417), and increases in iCa (P=.0494), iMg (P=.0024), MCV (P<.0001), MCHC (P=.0093), RDW (P=.0009), hemoglobin (P=.0011), and MCF (P=.0006). No bacterial growth was identified on the post-transfusion samples. Manual washing of stored blood significantly reduces storage lesions after a single wash and additional washing may cause in vitro hemolysis.

In the presented study, we demonstrate that the interaction of group IVA cytosolic phospholipase A2 and ceramide-1-phosphate is crucial for production of eicosanoid synthesis in inflammation. Inflammation is a critical component of many disease states including anaphylaxis, cancer, cardiovascular disease, rheumatoid arthritis, diabetes and asthma. Eicosanoids are well established mediators of inflammation, and the initial rate limiting step in the production of eicosanoids is the liberation of arachidonic acid (AA) from membrane phospholipids by a phospholipase A2 (PLA2). The major phospholipase involved in this liberation of AA during the inflammatory response is group IVA cytosolic phospholipase A2 (cPLA2α). Previous studies from our laboratory demonstrated that the bioactive sphingolipid, ceramide-1-phosphate (C1P), binds cPLA2α at a three amino acid sequence, which is located in the cationic β-groove of the C2 domain of cPLA2α. In this study we examined the effects of the genetic ablation of ceramide kinase (CERK) on eicosanoid synthesis, as CERK is the only known enzyme to produce C1P in mammalian systems. We utilized primary mouse fibroblasts (MEFs) and macrophages isolated from CERK-/- and +/+ mice. The ceramide-1-phosphate and eicosanoid profiles were investigated, and both ceramide-1-phosphate and eicosanoid levels in CERK-/- MEFs were found to be dysregulated. This study also presents the development of a global eicosanoid method to analyze eicosanoids via LC-ESI-MS/MS. Using this new analysis method, we demonstrated that there are significant differences in eicosanoid levels in ex vivo CERK-/- cells when compared to wild type counterparts, but the effect of the genetic ablation of CERK on eicosanoid synthesis and the serum levels of C1P was not apparent in vivo.

A participação da COX-1 na inflamação sistêmica tem sido questionada. Nós investigamos os mecanismos pelos quais a COX-1 participa da inflamação sistêmica mais grave. O inibidor da COX-1 (SC-560) atenuou a hipotermia, o hipometabolismo e a hipotensão induzidos por LPS. Este efeito atenuante teve duas fases: 30-60 min e 60-100 min. Em animais esplenectomizados, o efeito do SC-560 foi observado apenas na primeira fase. O SC-560 não alterou o nível plasmático das citocinas TNF-&#945;, IL-1&#946; e IL-10 em ambas as fases. No entanto, reduziu a expressão de IL-10 no baço, com tendência a aumentar IL-1&#946; (80 min). Eicosanoides (PGE2, PGD2, PGF2, TXB2 e LTC4) foram detectados no baço e na circulação (80 min). Nossos resultados indicam que o mecanismo da COX-1 no choque endotóxico tem duas fases: (i) na fase inicial a COX-1 não é proveniente do baço e age independentemente de citocinas; (ii) na fase tardia da resposta a COX-1 parece agir de forma dependente do baço e através da produção de eicosanoides, independentemente de TNF-&#945;, mas modulando a síntese de IL-10 e IL-1&#946;. / The participation of COX-1 in systemic inflammation has been questioned. We investigated the mechanisms by which COX-1 participates in the most severe systemic inflammation. The COX-1 inhibitor (SC-560) attenuated the hypothermia, hypometabolism and hypotension induced by LPS. This attenuating effect had two phases: 30-60 min and 60-100 min. In splenectomized animals, the effect of SC-560 was observed only in the first phase. SC-560 did not alter the plasma levels of cytokines TNF-&#945;, IL-1&#946; and IL-10 in both phases. However, it reduced IL-10 expression in the spleen, with tendency to increase IL-1&#946; (80 min). Eicosanoids (PGE2, PGD2, PGF2, TXB2 and LTC4) were detected in spleen and circulation (80 min). Our results indicate that the mechanism of COX-1 in endotoxic shock has two phases: (i) COX-1 does not originate from the spleen and acts independently of cytokines; (ii) in the late phase the COX-1 it seems to act in a spleen-dependent manner and through the production of eicosanoids, independently of TNF-&#945;, but modulating the synthesis of IL-10 and IL-1&#946;.

No / The effects of a conjugated linoleic acid (CLA) mixture of single isomers (50:50, w/w, cis9,trans11:trans10,cis12) and the individual isomers on (a) the production of resting and calcium ionophore stimulated 14C-eicosanoids and (b) the incorporation of 14C-arachidonic acid (AA) into membrane phospholipids of human saphenous vein endothelial cells were investigated. The CLA mixture and the individual isomers were found to inhibit resting production of 14C-prostaglandin F2a by 50, 43 and 40%, respectively. A dose dependent inhibition of stimulated 14C-prostaglandins was observed with the CLA mixture (IC50 100 ¿M). The cis9,trans11 and trans10,cis12 (50 ¿M) isomers individually inhibited the overall production of stimulated 14C-prostaglandins (between 35 and 55% and 23 and 42%, respectively). When tested at a high concentration (100 ¿M), cis9,trans11 was found to inhibit eicosanoid production in contrast to trans10,cis12 that caused stimulation. The overall degree of 14C-AA incorporation into membrane phospholipids of the CLA (mixture and individual isomers) treated cells was found to be lower than that of control cells and the cis9,trans11 isomer was found to increase the incorporation of 14C-AA into phosphatidylcholine. Docosahexaenoic acid, eicosapentaenoic acid and linoleic acid did not alter the overall degree of incorporation of 14C-AA. The results of this study suggest that both isomers inhibit eicosanoid production, and although trans10,cis12 exhibits pro-inflammatory activity at high concentrations, the CLA mixture maintains its beneficial anti-inflammatory action that contributes to its anti-carcinogenic and anti-atherogenic properties.

No / Eicosanoids play an integral part in homeostatic mechanisms related to skin health and structural integrity. They also mediate inflammatory events developed in response to environmental factors, such as exposure to ultraviolet radiation, and inflammatory and allergic disorders, including psoriasis and atopic dermatitis. This review article discusses biochemical aspects related to cutaneous eicosanoid metabolism, the contribution of these potent autacoids to skin inflammation and related conditions, and considers the importance of nutritional supplementation with bioactives such as omega-3 and omega-6 polyunsaturated fatty acids and plant-derived antioxidants as means of addressing skin health issues. / The Wellcome Trust and BBSRC-DRINC

No description available.

No description available.

Human 5-lipoxygenase (5-LOX) oxidizes arachidonic acid to 5S-hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-HpETE) and leukotriene (LT) A4. In neutrophils, LTA4 is further converted to the potent chemoattractant LTB4. These cells also contain the heme enzyme myeloperoxidase (MPO), which produces several potent oxidants such as hypochlorous acid (HOCl), which are involved in pathogen defense and immune regulation. Here, we addressed the question whether MPO-derived oxidants are able to affect the activity of 5-LOX and the product profile of this enzyme. Human 5-LOX was incubated with increasing amounts of HOCl or HOBr. Afterward, arachidonic acid metabolites of 5-LOX were analyzed by reverse-phase high-performance liquid chromatography as well as by liquid chromatography-electrospray ionization-tandem mass spectrometry. The incubation of 5-LOX with the MPO-derived oxidants significantly changed the product profile of 5-LOX. Thereby, HOCl and HOBr increased the ratio of 5-H(p)ETE to 6-trans-LTB4 in a concentration-dependent manner. At low oxidant concentrations, there was a strong decrease in the yield of 6-trans-LTB4, whereas 5-HpETE did not change or increased. Additionally, the formation of 8-HpETE and 12-HpETE by 5-LOX rose slightly with increasing HOCl and HOBr. Comparable results were obtained with the MPO-H2O2-Cl– system when glucose oxidase and glucose were applied as a source of H2O2. This was necessary because of a strong impairment of 5-LOX activity by H2O2. In summary, MPO-derived oxidants showed a considerable impact on 5-LOX, impairing the epoxidation of 5-HpETE, whereas the hydroperoxidation of arachidonic acid was unaffected. Apparently, this was caused by an oxidative modification of critical amino acid residues of 5-LOX. Further work is necessary to assess the specific type and position of oxidation in the substrate-binding cavity of 5-LOX and to specify whether this interaction between 5-LOX and MPO-derived oxidants also takes place in stimulated neutrophils.

Preliminary studies from our laboratory showed that cPLA2α may be responsible for approximately 50-60% of the PGE2 production in response to inflammatory cytokines. Thus, we hypothesized that a closely-related PLA2 is responsible for 40-50% of the PGE2 produced in response to inflammatory cytokines. To this end, we utilized RNAi technology, extensively optimized, to down regulate the expression of closely-related isoforms of phospholipase A2 in A549 cells and used an enzyme linked immuno sorbent assay (ELISA) to quantitate the PGE2 produced. These studies found that cytosolic phospholipase A2α (cPLA2α) regulated 97.7% of the prostaglandin E2 (PGE2) produced in response to inflammatory cytokines (e.g. IL-1β or TNFα), as well as regulating the basal levels of this prostanoid. Furthermore, cPLA2γ, cPLA2δ, and iPLA2 were found to also to regulate the basal levels of PGE2 production. On the other hand, cPLA2β was not involved in prostanoid synthesis in A549 cells either in the presence or absence of inflammatory cytokines. Thus, our studies show that cPLA2α plays the pivotal role in the production of PGE2 in response to inflammatory cytokines, and suggests that cPLA2α may be a possible drug target in diseases such as asthma, inflammation, and cancer.