Differential membrane expression of Toll-like receptors and intracellular cytokine induction in peripheral blood monocytes of patients with chronic kidney disease and diabetic nephropathy.

BACKGROUND: Toll-like receptors (TLRs) are key players in the innate immune system whose activation leads to an inflammatory response. Inflammation plays an important role in the pathogenesis of chronic kidney disease (CKD) and diabetes mellitus. The aim of our study was to assess the proinflammatory state of nondialysis CKD patients by evaluating the membrane expression of TLR2 and TLR4 and the intracellular IL-1ß and IL-6 production in response to the ligand Pam3Cys-Ser-(Lys)4 (Pam3CSK4). METHODS: 85 nondialysis CKD patients [mean estimated glomerular filtration rate: 34 (17-90) ml/min/1.73 m(2)] were divided in 2 groups: 55 nondiabetic CKD patients (CKD group) and 30 patients with diabetic nephropathy (DN group). The two groups were compared with 36 healthy subjects (control group). TLR2 and TLR4 membrane expression in monocytes and Pam3CSK4-induced intracellular production of IL-1ß and IL-6 were assessed by flow cytometry. RESULTS: Both patient groups showed increased TLR2 membrane expression compared with the control group, both at baseline (p < 0.05 for both) and after Pam3CSK4 stimulation (p < 0.05 for both). The DN group exhibited significantly higher TLR4 expression at baseline compared to the CKD and control groups (p < 0.04 and p < 0.02, respectively). Intracellular IL-1ß and IL-6 levels at baseline were significantly lower in CKD patients compared to the DN and control groups. After Pam3CSK4 stimulation, intracellular IL-1ß and IL-6 increased in all groups, but were lower in the CKD group versus the control group or DN group, which exhibited higher levels than the controls. CONCLUSIONS: Nondialysis CKD patients showed significant alterations in TLR2 and TLR4 membrane expression, and impaired Pam3CSK4-induced cytokine production in monocytes, a phenomenon that is markedly influenced by the presence of diabetes.

Effect of simvastatin or its combination with ezetimibe on Toll-like receptor expression and lipopolysaccharide - induced cytokine production in monocytes of hypercholesterolemic patients.

OBJECTIVES: Toll-like receptors (TLRs) are key players in the innate immune system. Recently, a pivotal role of TLR2 and TLR4 has been recognized in atherogenesis. We investigated the effect of simvastatin monotherapy or its combination with ezetimibe on TLR2 and TLR4 membrane expression and on lipopolysaccharide (LPS)-induced interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) production in peripheral blood monocytes of patients with primary hypercholesterolemia. METHODS: This was a prospective, randomized, open-label, blinded endpoint study. After a 3-month period of lifestyle changes patients (n = 60) (mean age 55 ± 13) with LDL-cholesterol levels above those recommended by the NCEP ATP III, were randomly allocated to open-label simvastatin 40 mg (n = 30) or simvastatin/ezetimibe 10/10 mg (n = 30) daily. Both groups were similar with regard to demographics, risk factors, medications and baseline lipid values. TLR2 and TLR4 membrane expression in monocytes, LPS-induced intracellular production of IL-1ß and IL-6 were assessed by flow cytometry at baseline and 3 months post-treatment in both patient groups, as well as in 30 age- and sex-matched normolipidemic controls. RESULTS: Hypercholesterolemic patients exhibited higher TLR2 and TLR4 membrane expression compared with controls (p < 0.02). LPS induced a significant increase in the intracellular levels of IL-1ß and IL-6 in all groups however both patient groups exhibited significantly lower levels compared with controls. Three months of treatment with either simvastatin or its combination with ezetimibe resulted in a significant reduction of TLR2 and TLR4 expression (p < 0.01 compared with baseline values) with no intergroup differences. Furthermore, in both groups the post-treatment values of LPS-induced IL-1ß and IL-6 production were significantly lower compared with baseline (p < 0.05 for all comparisons). CONCLUSIONS: A high simvastatin dose or the combination of a low-dose simvastatin with ezetimibe reduce to a similar extent TLR2, TLR4 membrane expression and LPS-induced IL-6 and IL-1ß production in monocytes of hypercholesterolemic patients. The pathophysiological significance of these effects regarding atherosclerosis, reserves further investigation.

A highly constrained cyclic (S,S)-CDC- peptide is a potent inhibitor of carotid artery thrombosis in rabbits.

Inhibition of platelet aggregation is indispensable for the treatment of acute arterial thrombotic episodes. We have previously reported the synthesis of a highly constrained cyclic peptide, that incorporates the -CDC- sequence, (S,S) PSRCDCR-NH(2), which potently inhibits aggregation and fibrinogen binding to human platelets in vitro. We have tested the safety and efficacy of the peptide on the electrically induced carotid artery thrombosis experimental rabbit model. The peptide's effects on carotid blood flow, thrombus weight, in vitro and ex vivo platelet aggregation, and bleeding and hemostatic parameters were evaluated. The peptide was administered via the femoral vein. Carotid blood flow was continuously monitored for 90 min after electrical thrombus formation. The peptide, at 12 mg/kg, prevented total artery occlusion and significantly preserved carotid artery's patency compared with placebo and eptifibatide. Furthermore, (S,S) PSRCDCR-NH(2) administration at 12 mg/kg reduced thrombus weight, whereas it inhibited ex vivo ADP, arachidonic acid (AA) and collagen-induced platelet aggregation. Moreover (S,S) PSRCDCR-NH(2) at 12 mg/kg presented significantly higher inhibitory effects on AA and collagen-induced ex vivo platelet aggregation compared to eptifibatide. The peptide at any dose did not affect the coagulation cascade, the bleeding times or the hemostatic response of the animals. Thus highly constrained cyclic peptides like (S,S) PSRCDCR-NH(2) that incorporate the -CDC- motif and fulfil certain conformational criteria represent novel compounds that potently inhibit thrombus formation, ex vivo platelet aggregation and carotid artery occlusion superiorly to other non-RGD peptides, such as YMESRADR, without causing hemorrhagic complications in a rabbit model of arterial thrombosis.