Inhibition of ROS production in peripheral blood mononuclear cells from type 2 diabetic patients by autologous plasma depends on Akt/PKB signaling pathway.

OBJECTIVE: To compare the role of Akt/PKB signaling pathway in the modulation of reactive oxygen species (ROS) production by autologous plasma in peripheral blood mononuclear cells (PBMNC) from type 2 diabetic patients and healthy subjects. MATERIALS AND METHODS: This study was approved by Santa Casa Ethical Committee and has included patients diagnosed with diabetes type 2 (DM2) and control group (non-diabetic) (ND). PBMNC were purified utilizing Ficoll-hypaque gradient. ROS was quantified by luminol-dependent chemiluminescence. The Akt/PKB phosphorylation was measured using a CASE kit. Statistical analyses were made with t Student test and chi-square (chi(2)). p<0.05 was considered significant. RESULTS: 12, 13-Phorbol dibutyrate (PDB) stimulated the production of higher levels of ROS in PBMNC from type 2 diabetic patients than that from healthy subjects. Autologous plasma, however, inhibited induced or not ROS production in PBMNC in both groups. The inhibition of PBMNC-ROS derived by autologous plasma from healthy subjects was higher than that from type 2 diabetic patients. Plasma phosphorylated (activated) Akt/PKB. The percentage of phosphorylation induced by autologous plasma in PBMNC from patients and healthy control were 14% and 93%, respectively. Inhibition of ROS production in PBMNC from DM2 were similar for PBMNC+plasma; PBMNC+Akti; and PBMNC+plasma+Akti. However, in ND control, plasma showed a higher ROS inhibition than Akti or plasma plus Akti. CONCLUSIONS: Our results suggest that the low antioxidant capacity observed in autologous plasma from DM2 patients in conjunction with the decreased activation of PKB may cause an imbalance in the oxidizing/reducing responses, possible inducing an oxidative stress state, which could be associated with tissular damage.

Retrospective analysis of patients who developed pulmonary hypertension during the early postoperative period after liver transplantation.

INTRODUCTION: Pulmonary hypertension (PH) (mean pulmonary arterial pressure [mPAP] > 25 mm Hg) is frequently observed during the postoperative period after liver transplantation (LT). OBJECTIVE: The objective was to compare respiratory function, intensive care unit (ICU) length of stay (LOS), and 30-day survival rates among patients evolving with PH with those who do versus do not develop it during the postoperative period after LT. METHODS: Fifty-seven patients undergoing LT from January 1999 to December 2000 were divided into 2 groups: Group 1 (G1; n = 26), without PH; and Group 2 (G2; n = 31), with moderate PH. Preoperative parameters were Child-Pugh's classification, pulmonary function tests, mPAP, and P(A-a)O(2). During the intraoperative period, warm and cold ischemic times and the amount of blood transfusion were evaluated, whereas mPAP, PaO(2)/FiO(2) ratio, weaning time, ICU LOS, and 30-day survival rates were evaluated postoperatively. RESULTS: mPAP in early postoperative period was 21 +/- 13 mm Hg and 32 +/- 4 mm Hg in G1 and G2, respectively (P <.0001). PaO(2)/FiO(2) was 310 +/- 82 mm Hg in G1 and 272 +/- 84 mm Hg in G2 (P =.48). In G1 and G2, 77% and 74% of patients, respectively, were successfully weaned in the first 24 hours postoperative (P =.10). ICU LOS was 111 hours (range, 45-1098 hours) in G1 and 102 hours (range, 59-284 hours) in G2 (P =.36). The 30-day survival rate was 20 of 26 (77%) in G1 and 26 of 31 (84%) in G2 (P =.44). CONCLUSION: Our data suggest that moderate PH during the early postoperative phases of LT cannot be considered an additional risk factor for pulmonary dysfunction, and for an increased ICU LOS or 30-day mortality rate.

TLR4 and RAGE: similar routes leading to inflammation in type 2 diabetic patients.

AIM: The present study investigates the interaction of TLR4 and RAGE with their respective ligands as inducers of the inflammatory markers IL-6 and TNF-α. Also, the reactivity of peripheral blood mononuclear cells (PBMNC) from type 2 diabetic (T2D) patients and non-diabetic healthy controls (ND) were comparatively studied. METHODS: Concentrations of IL-6 and TNF-α were measured by sandwich Elisa, using kits supplied by Assay Designs (Ann Arbor, MI, USA). PBMNC from T2D and ND were incubated in the presence or absence of LPS, anti-TLR4 or anti-RAGE for 72 hours at 37°C under 5% CO(2). The final volume was adjusted to 300 µL in DMEM supplemented with 10% fetal bovine serum. After incubation, the cells were centrifuged, the supernatant collected and the cytokines measured. RESULTS: PBMNC from T2D were more sensitive to innate immune stimulation with LPS and monoclonal agonist anti-TLR4 than were cells from ND. The actions of LPS, anti-TLR4 and anti-RAGE potentiated the production of IL-6 and TNF-α in both groups. The simultaneous activation of monoclonal anti-RAGE and anti-TLR4 suggests that both antibodies used different receptors on the cell surface, but converged on the same PBMNC signaling metabolic pathways. This simultaneous activation induced a higher production of IL-6 and TNF-α in PBMNC from the T2D patients than from the ND subjects. CONCLUSION: Our results clearly show an exacerbation of innate immunity in PBMNC with T2D that was possibly hyperglycaemia-induced. These data, when analyzed together, suggest the importance of innate immunity in the pathogenesis of T2D.