Plasma eicosanoid profiles determined by high-performance liquid chromatography coupled with tandem mass spectrometry in stimulated peripheral blood from healthy individuals and sickle cell anemia patients in treatment.

Eicosanoids play an important role in homeostasis and in the pathogenesis of various human diseases. Pharmacological agents such as Ca(2+) ionophores and Ca(2+)-ATPase inhibitors, as well as natural agonists such as formylmethionine-leucyl-phenylalanine (fMLP), can stimulate eicosanoid biosynthesis. The aims of this work were to develop a method to determine the eicosanoid profile of human plasma samples after whole blood stimulation and to assess differences between healthy and sick individuals. For this purpose, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was partially validated for the quantification of 22 eicosanoids using human plasma from healthy volunteers. In addition, we optimized a method for the stimulation of eicosanoids in human whole blood. LC-MS/MS analyses were performed by negative electrospray ionization and multiple reaction monitoring. An assumption of linearity resulted in a regression coefficient ≥0.98 for all eicosanoids tested. The mean intra-assay and inter-assay accuracy and precision values had relative standard deviations and relative errors of ≤15%, except for the lower limit of quantification, where these values were ≤20%. For whole blood stimulation, four stimuli (fMLP, ionomycin, A23187, and thapsigargin) were tested. Results of the statistical analysis showed that A23187 and thapsigargin were potent stimuli for the production or liberation of eicosanoids. We next compared the eicosanoid profiles of stimulated whole blood samples of healthy volunteers to those of patients with sickle cell anemia (SCA) under treatment with hydroxyurea (HU) or after chronic red blood cell (RBC) transfusion. The results indicate that the method was sufficient to find a difference between lipid mediators released in whole blood of SCA patients and those of healthy subjects, mainly for 5-HETE, 12-HETE, LTB4, LTE4, TXB2, and PGE2. In conclusion, our analytical method can detect significant changes in eicosanoid profiles in stimulated whole blood, which will contribute to establishing the eicosanoid profiles associated with different inflammatory and infectious diseases.

Erythropoietin Exacerbates Inflammation and Increases the Mortality of Histoplasma capsulatum-Infected Mice.

Erythropoietin (EPO) is a key hormone involved in red blood cell formation, but its effects on nonerythroid cells, such as macrophages, have not been described. Macrophages are key cells in controlling histoplasmosis, a fungal infection caused by Histoplasma capsulatum (Hc). Considering that little is known about EPO's role during fungal infections and its capacity to activate macrophages, in this study we investigated the impact of EPO pretreatment on the alveolar immune response during Hc infection. The consequence of EPO pretreatment on fungal infection was determined by evaluating animal survival, fungal burden, activation of bronchoalveolar macrophages, inflammatory mediator release, and lung inflammation. Pretreatment with EPO diminished mononuclear cell numbers, increased the recruitment of F4/80(+)/CD80(+) and F4/80(+)/CD86(+) cells to the bronchoalveolar space, induced higher production of IFN-γ, IL-6, MIP-1α, MCP-1, and LTB4, reduced PGE2 concentration, and did not affect fungal burden. As a consequence, we observed an increase in lung inflammation with extensive tissue damage that might account for augmented mouse mortality after infection. Our results demonstrate for the first time that EPO treatment has a deleterious impact on lung immune responses during fungal infection.

Lipoxin A4 encapsulated in PLGA microparticles accelerates wound healing of skin ulcers.

Lipoxin A4 (LXA4) is involved in the resolution of inflammation and wound healing; however, it is extremely unstable. Thus, to preserve its biological activities and confer stability, we encapsulated LXA4 in poly-lactic-co-glycolic acid (PLGA) microparticles (LXA4-MS) and assessed its application in treating dorsal rat skin lesions. Ulcers were sealed with fibrin adhesive and treated with either LXA4-MS, unloaded microparticles (Un-MS), soluble LXA4, or PBS/glue (vehicle). All groups were compared at 0, 2, 7, and 14 days post-lesions. Our results revealed that LXA4-MS accelerated wound healing from day 7 and reduced initial ulcer diameters by 80%. Soluble LXA4, Un-MS, or PBS closed wounds by 60%, 45%, and 39%, respectively. LXA4-MS reduced IL-1ß and TNF-α, but increased TGF-ß, collagen deposition, and the number of blood vessels. Compared to other treatments, LXA4-MS reduced inflammatory cell numbers, myeloperoxidase (MPO) concentration, and metalloproteinase-8 (MMP8) mRNA in scar tissue, indicating decreased neutrophil chemotaxis. In addition, LXA4-MS treatment increased macrophages and IL-4, suggesting a positive impact on wound healing. Finally, we demonstrated that WRW4, a selective LXA4 receptor (ALX) antagonist, reversed healing by 50%, indicating that LXA4 must interact with ALX to induce wound healing. Our results show that LXA4-MS could be used as a pharmaceutical formulation for the treatment of skin ulcers.