Miriam Ojeda

Prophylactic Use of Epidermal Growth Factor Reduces Ischemia/Reperfusion Intestinal Damage

Ischemia/reperfusion of mesenteric vessels is a useful model for acute vascular insufficiency and the early stages of multiorgan failure, conditions associated with high morbidity and mortality. Epidermal growth factor (EGF) is a potent mitogen that shows potential for use in intestinal injury. We therefore examined its influence on this model. Male Sprague-Dawley rats received human recombinant EGF (2 mg/kg i.p., n = 14) or saline (n = 16); 25 minutes before arterial clamping of the superior mesenteric artery (ischemic period) for 60 minutes followed by a final 60-minute reperfusion period. Additional rats were not operated on (controls, n = 7) or had sham operation (laparotomy only, n = 10). Ischemia/reperfusion caused macroscopic damage affecting 56%, 51 to 67% (median, interquartile range), of small intestinal length and intraluminal bleeding. Malondialdehyde levels (free radical marker) increased eightfold compared to nonoperated animals (2400, 2200 to 2700 micro mol/mg protein versus 290, 250 to 350 micro mol/mg protein, P < 0.01) and myeloperoxidase levels (marker for inflammatory infiltrate) increased 15-fold (3150, 2670 to 4180 U/g tissue versus 240, 190 to 250 U/g tissue, P < 0.01). Pretreatment with EGF reduced macroscopic injury to 11%, 0 to 15%; prevented intraluminal bleeding; and reduced malondialdehyde and myeloperoxidase levels by approximately 60% and 90% (all P < 0.01 versus non-EGF-treated). Mesenteric ischemia/reperfusion also damaged the lungs and kidneys and increased serum tumor necrosis factor-alpha levels (circulating cytokine activity marker). EGF pretreatment also reduced these changes. These studies provide preliminary evidence that EGF is a novel therapy for the early treatment or prevention of intestinal damage and multiorgan failure resulting from mesenteric hypoperfusion.

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.

Abstract.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-κB 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-α(TNFα), 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-κB 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 TNFα. 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-κB activity was inhibited.Conclusions: The present data indicate the differential regulation by DLE of the production of TNFα, IL-6, and IL-8 cytokines, associated with effects on TLR2 and TLR4 expression and NF-κB and cAMP activities. We suggest a putative mechanism for the biological effects of DLE in activated leukocytes and endothelial cells.

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 peptides 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. Copyright

Dialysable leucocyte extract (DLE) reduces lipopolysaccharide- induced tumour necrosis factor secretion in human leucocytes

Dialysable leucocyte extract (DLE), obtained from lysed leucocytes, provide clinical effectiveness in a broad spectrum of diseases. Tumour necrosis factor (TNF) is raised in AIDS patients leading to an increase in human immunodeficiency virus (HIV) replication in vitro [1,2], whereas progression to AIDS in asymptomatic HIV infected individuals is retarded under treatment with DLE. In the present study we tested the DLE effect in vitro on both TNF biological activity (cytotoxicity) in L929 cells and its induction by lipopolysaccharide (LPS) in human monocytes as well as in whole blood from healthy donors. When monocytic cells were simultaneously exposed to LPS and DLE during a period of 5 1/2 hours, the induction of TNF was strongly diminished. The same inhibitory effect of DLE on TNF induction was observed when LPS was added to the culture medium prior to DLE. No significant effect of DLE on TNF-mediated cytotoxicity, even in the presence of the highest concentrations of DLE tested, was detected. DLE treatment of whole human blood regulates responses to LPS: simultaneous in vitro exposure to endotoxin provokes a remarkable decrease (4- and 1.6-fold) of TNF release. In pre-incubation experiments, TNF production was largely reduced or completed abrogated. These results could, in part, explain the in vivo observed effect, when under treatment with this extract, the progression to AIDS of HIV-infected individuals was retarded. The results suggest that ‘natural’ substances like DLE may be important immunomodulators in inflammatory diseases.

Inhibition of in vitro HIV infection by dialysable leucocyte extracts

Dialysable Leucocyte Extract (DLE) is a low molecular weight dialysable material of disrupted peripheral human leucocytes with widespread effects on the immune system. We described thein vitro anti-HIV activity of DLE as well as its three chromatographic fractions (Fa, Fb and Fc). To determine the levels of inhibition on HIV replication by DLE we infected MT-4 cell cultures, using the Bru viral isolate at 0.05, 0.1, 0.5 and 1 m.o.i. Previously, MT-4 cells cultures were treated with DLE or fractions at non-toxic concentrations. Reverse transcriptase (RT) activity and p24 antigen were evaluated in culture supernatants at seven days postinfection. No effect was observed when MT-4 cells were incubated with DLE for 3 h. Whereas inhibition of HIV production was observed when MT-4 cells were pre-treated for a longer period of time. DLE inhibited p24 production and RT activity more than 50% at 0.1 m.o.i. More than 80% of inhibition was observed for all doses of DLE tested at 0.05 m.o.i. Higher viral doses (m.o.i. 0.5 and 1) were used to assess the antiviral activity of DLE fractions. Fraction Fb inhibits viral production more than 80%. Otherwise, fractions Fa and Fc did not show inhibitory effect for any viral dose used. These results indicate that DLE is able to modulate cell susceptibility to viral infectionin vitro.