Enhancement of the inhibitory effect of an IL-15 antagonist peptide by alanine scanning.

IL-15 is a proinflammatory cytokine that acts early in the inflammatory response and has been associated with several autoimmune diseases including rheumatoid arthritis, where it had been proposed as a therapeutic target. We recently reported an IL-15 antagonist peptide corresponding to sequence 36-45 of IL-15 (KVTAMKCFLL) named P8, which specifically binds to IL-15Rα and inhibits IL-15 biological activity with a half maximal inhibitory concentration (IC50) of 130 µ m in CTLL-2 proliferation assay. In order to improve binding of peptide P8 to the receptor IL-15Rα, we used an Ala scan strategy to study contribution of each individual amino acid to the peptide's antagonist effect. Here, we found that Phe and Cys are important for peptide binding to IL-15Rα. We also investigated other single site mutations and replaced the second Lys in the sequence by the polar non-charged amino acid threonine. The resulting peptide [K6T]P8 exhibited a higher activity than P8 with an IC50 of 24 µm. We also found that this peptide was more active than peptide P8 in the inhibition of TNFα secretion by synovial cells from rheumatoid arthritis patients. The peptide [K6T]P8 described in this work is a new type of IL-15 antagonist and constitutes a potential therapeutic agent for rheumatoid arthritis.

Imbalanced low-grade inflammation and endothelial activation in patients with type 2 diabetes mellitus and erectile dysfunction.

INTRODUCTION: Erectile dysfunction (ED) is highly prevalent among type 2 diabetes mellitus patients (T2DM). Although a link among systemic inflammation, endothelial dysfunction, and ED is described in clinical situations mainly related with coronary heart disease (CHD) risk, evidences of this link in T2DM patients are rather limited. AIMS: To evaluate the association between endothelial dysfunction and balance of pro-/anti-inflammatory mediators with ED presence and severity in T2DM. METHODS: We conducted a cross-sectional study of 190 T2DM patients without symptomatic CHD, 150 out of them with ED and 40 without ED. Serum levels of E-selectin, intercellular adhesion molecule-1, tumor necrosis factor-α (TNF-α), and interleukin (IL)-10 were measured using specific enzyme-linked immunosorbent assays (ELISAs). ED presence and severity were tested by the five-item version of the International Index of Erectile Function questionnaire. MAIN OUTCOME MEASURES: Differences in circulating levels of endothelial dysfunction (ICAM-1, E-selectin) and inflammatory/anti-inflammatory (TNF-α, IL-10, TNF-α : IL-10 ratio) markers between T2DM patients with and without ED, and assessment of biomarkers ED predictive value while adjusting for other known ED risk factors. RESULTS: Patients with ED were older and had longer duration of diabetes than patients without ED. E-selectin serum levels were significantly increased, while IL-10 were lower in patients with ED; because TNF-α levels tend to be higher, TNF-α : IL-10 ratio was more elevated in ED patients. No significant differences of ICAM-1 levels were observed between study groups. Endothelial activation markers and TNF-α, as well as diabetes duration, were negatively correlated with erectile function. On multivariate analysis including age, duration of diabetes, insulin treatment, hypertension, insulin resistance, fair-to-poor glycemic control, and metabolic syndrome, increments in E-selectin levels and TNF-α : IL-10 ratio predicted independently ED presence, while IL-10 increases were associated with lower risk of ED in T2DM patients. CONCLUSIONS: ED in T2DM patients without symptomatic CHD is associated with systemic endothelial dysfunction and a predominant, imbalanced low-grade inflammatory response.

Dialyzable leukocyte extract differentially regulates the production of TNFalpha, IL-6, and IL-8 in bacterial component-activated leukocytes and endothelial cells.

OBJECTIVE: To investigate i) whether the Dialyzable Leukocyte Extract (DLE) modulates the production of proinflammatory cytokines in leukocytes activated by the bacterial cell wall components lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PGN); ii) the effect of DLE on LPS-stimulated endothelial cells; and iii) whether the regulatory effect of DLE on inflammatory mediators is related to the modulation of Toll-like receptors (TLRs), NF-kappaB and cAMP signaling pathways. METHODS: Leukocytes were stimulated with LPS, LTA, and PGN in the presence of DLE. Endothelial cells were stimulated with LPS and treated with DLE. The levels of Tumor Necrosis Factor-alpha(TNFalpha), Interleukin-6 (IL-6), and IL-8 in culture supernatants were evaluated by ELISA. The expression of Toll-like receptor 2 (TLR2) and 4 (TLR4), NF-kappaB activity and cAMP levels were evaluated by flow cytometry, EMSA, and EIA, respectively. RESULTS: The addition of DLE to leukocytes stimulated with cell wall constituents suppressed the production of TNFalpha. However, DLE induced IL-8 release in monocytes and enhanced IL-6 and IL-8 production by activated monocytes and endothelial cells. Also, DLE induced TLR2 and TLR4 expression, and increased cAMP levels, whereas NF-kappaB activity was inhibited. CONCLUSIONS: The present data indicate the differential regulation by DLE of the production of TNFalpha, IL-6, and IL-8 cytokines, associated with effects on TLR2 and TLR4 expression and NF-kappaB and cAMP activities. We suggest a putative mechanism for the biological effects of DLE in activated leukocytes and endothelial cells.