Interaction between Advanced Glycation End Products Formation and Vascular Responses in Femoral and Coronary Arteries from Exercised Diabetic Rats

Background: The majority of studies have investigated the effect of exercise training (TR) on vascular responses in diabetic animals (DB), but none evaluated nitric oxide (NO) and advanced glycation end products (AGEs) formation associated with oxidant and antioxidant activities in femoral and coronary arteries from trained diabetic rats. Our hypothesis was that 8-week TR would alter AGEs levels in type 1 diabetic rats ameliorating vascular responsiveness. Methodology/Principal Findings: Male Wistar rats were divided into control sedentary (C/SD), sedentary diabetic (SD/DB), and trained diabetic (TR/DB). DB was induced by streptozotocin (i.p.: 60 mg/kg). TR was performed for 60 min per day, 5 days/week, during 8 weeks. Concentration-response curves to acetylcholine (ACh), sodium nitroprusside (SNP), phenylephrine (PHE) and tromboxane analog (U46619) were obtained. The protein expressions of eNOS, receptor for AGEs (RAGE), Cu/Zn-SOD and Mn-SOD were analyzed. Tissues NO production and reactive oxygen species (ROS) generation were evaluated. Plasma nitrate/nitrite (NOx-), superoxide dismutase (SOD), catalase (CAT), thiobarbituric acid reactive substances (TBARS) and N-epsilon-(carboxymethyl) lysine (CML, AGE biomarker). A rightward shift in the concentration-response curves to ACh was observed in femoral and coronary arteries from SD/DB that was accompanied by an increase in TBARS and CML levels. Decreased in the eNOS expression, tissues NO production and NOx- levels were associated with increased ROS generation. A positive interaction between the beneficial effect of TR on the relaxing responses to ACh and the reduction in TBARS and CML levels were observed without changing in antioxidant activities. The eNOS protein expression, tissues NO production and ROS generation were fully re-established in TR/DB, but plasma NOx- levels were partially restored. Conclusion: Shear stress induced by TR fully restores the eNOS/NO pathway in both preparations from non-treated diabetic rats, however, a massive production of AGEs still affecting relaxing responses possibly involving other endothelium-dependent vasodilator agents, mainly in coronary artery.

Advanced Glycation in macrophages induces intracellular accumulation of 7-ketocholesterol and total sterols by decreasing the expression of ABCA-1 and ABCG-1

Abstract Background Advanced glycation end products (AGE) alter lipid metabolism and reduce the macrophage expression of ABCA-1 and ABCG-1 which impairs the reverse cholesterol transport, a system that drives cholesterol from arterial wall macrophages to the liver, allowing its excretion into the bile and feces. Oxysterols favors lipid homeostasis in macrophages and drive the reverse cholesterol transport, although the accumulation of 7-ketocholesterol, 7alpha- hydroxycholesterol and 7beta- hydroxycholesterol is related to atherogenesis and cell death. We evaluated the effect of glycolaldehyde treatment (GAD; oxoaldehyde that induces a fast formation of intracellular AGE) in macrophages overloaded with oxidized LDL and incubated with HDL alone or HDL plus LXR agonist (T0901317) in: 1) the intracellular content of oxysterols and total sterols and 2) the contents of ABCA-1 and ABCG-1. Methods Total cholesterol and oxysterol subspecies were determined by gas chromatography/mass spectrometry and HDL receptors content by immunoblot. Results In control macrophages (C), incubation with HDL or HDL + T0901317 reduced the intracellular content of total sterols (total cholesterol + oxysterols), cholesterol and 7-ketocholesterol, which was not observed in GAD macrophages. In all experimental conditions no changes were found in the intracellular content of other oxysterol subspecies comparing C and GAD macrophages. GAD macrophages presented a 45% reduction in ABCA-1 protein level as compared to C cells, even after the addition of HDL or HDL + T0901317. The content of ABCG-1 was 36.6% reduced in GAD macrophages in the presence of HDL as compared to C macrophages. Conclusion In macrophages overloaded with oxidized LDL, glycolaldehyde treatment reduces the HDL-mediated cholesterol and 7-ketocholesterol efflux which is ascribed to the reduction in ABCA-1 and ABCG-1 protein level. This may contribute to atherosclerosis in diabetes mellitus.

Fibronectin glycation increases IGF-I induced proliferation of human aortic smooth muscle cells

The advanced glycation end products, namely AGEs, contribute to long-termed complications of diabetes mellitus, including macroangiopathy, where smooth muscle cells (SMC) proliferation stimulated by platelet-derived growth factor (PDGF) isoforms and insulin-like growth factor-I (IGF-I) plays an important role. The objective of the present study was to investigate the effect of an AGE-modified extracellular matrix protein on IGF-I induced SMC proliferation and on the IGF-I-IGF binding protein 4 (IGFBP-4) axis under basal conditions and after stimulation with PDGF-BB. IGF-I resulted in significantly higher thymidine incorporation in SMC seeded on AGE-modified fibronectin (AGE-FN) in comparison to cells seeded on fibronectin (FN). This augmented proliferation could not be accounted for by increased expression of IGF-IR, by decreased secretion of IGFBP-4, a binding protein that inhibits IGF-I mitogenic effects or by increased IGF-IR autophosphorylation. PDGF-BB did not modulate IGF-IR and IGFBP-4 mRNA expression in any of the substrata, however, this growth factor elicited opposite effects on the IGFBP-4 content in the conditioned media, increasing it in cells plated on FN and diminishing it in cells plated on AGE-FN. These findings suggest that one mechanism by which AGE-modified proteins is involved in the pathogenesis of diabetes-associated atherosclerosis might be by increasing SMC susceptibility to IGF-I mitogenic effects.

Inhibition of Macrophage Oxidative Stress Prevents the Reduction of ABCA-1 Transporter Induced by Advanced Glycated Albumin

We investigated the role of aminoguanidine and benfotiamine on the inhibition of reactive oxygen species (ROS) generation in macrophages induced by advanced glycated albumin (AGE-albumin) and its relationship with cell cholesterol homeostasis, emphasizing the expression of the ATP binding cassette transporter A-1 (ABCA-1). AGE-albumin was made by incubating fatty acid-free albumin with 10 mM glycolaldehyde. ROS production and ABCA-1 protein level were determined by flow cytometry in J774 macrophages treated along time with control (C) or AGE-albumin alone or in the presence of aminoguanidine or benfotiamine. Mitochondrial function was evaluated by oxygraphy. Compared to C-albumin, AGE-albumin increased ROS production in macrophages, which was ascribed to the activities of NADPH oxidase and of the mitochondrial system. Mitochondrial respiratory chain activity was reduced in cells incubated with AGE-albumin. ROS generation along time was associated with the reduction in macrophage ABCA-1 protein level. Aminoguanidine prevented ROS elevation and restored the ABCA-1 content in macrophages; on the other hand, benfotiamine that promoted a lesser reduction in ROS generation was not able to restore ABCA-1 levels. Inhibition of oxidative stress induced by AGE-albumin prevents disturbances in reverse cholesterol transport by curbing the reduction of ABCA-1 elicited by advanced glycation in macrophages and therefore may contribute to the prevention of atherosclerosis in diabetes mellitus.

N-Acetylcysteine protects the peritoneum from the injury induced by hypertonic dialysis solution

Background: Oxidative stress has been implicated in the development of peritoneal damage. The aim of this study was to evaluate the effects of N-acetylcysteine (NAC) in a rat peritoneal infusion model. Methods: Eighteen male Wistar rats were divided in 3 groups: (i) control group; (ii) HDS group, receiving peritoneal dialysis solution (PDS); and (iii) HDS+NAC group, receiving PDS and oral NAC. Six weeks later they were evaluated for dialysate to plasma urea ratio (D/P), ratio of glucose concentration in peritoneal fluid (G1/G0), thiobarbituric acid reactive substances in plasma and urine and histology of peritoneal membrane. Results: The HDS+NAC group presented a lower increase in solute transport (D/P 0.51 +/- 0.1, and G1/GO 0.35 +/- 0.06) in comparison with the HDS group (D/P 0.67 +/- 0.1; p=0.03, and G1/G0 0.27 +/- 0.07; p=0.01). The HDS+NAC group showed lower thiobarbituric acid reactive substance concentrations compared with the HDS group. In the treated group, the peritoneal membrane presented lower thickness. Conclusions: Functional and histological peritoneal changes were significantly reduced by the treatment with NAC.

Experimental diabetes modulates collagen remodelling of joints in rats

The aim of this study was to evaluate extracellular matrix components in articular cartilage, ligaments and synovia in an experimental model of diabetes. Young Wistar rats were divided into a streptozotocin-induced (STZ; 35 mg/kg) diabetic group (DG; n=15) and a control group (CG; n=15). Weight, blood glucose and plasma anti-carboxymethyllysine were measured 70 days after STZ infusions. Knee joints, patellar ligaments, and lateral and medial collateral ligaments were isolated and stained with hematoxylineosin and Picrosirius. The total collagen content was determined by morphometry. Immunofluorescence was employed to evaluate types I, III, and V collagen in ligaments and synovial tissues and types II and XI collagen in cartilage. Results: Higher blood glucose levels and plasma anti-carboxymethyllysine were observed in DG rats when compared to those in CG rats. The final weight was significantly lower in the DG rats than in the CG rats. Histomorphometric evaluation depicted a small quantity of collagen fibers in ligaments and articular cartilage in DG rats, as well as increased collagen in synovial tissue. There was a decrease in cartilage proteoglycans in DG rats when compared with CG rats. Immunofluorescence staining revealed an increase of collagen III and V in ligaments, collagen XI in cartilage, and collagen I in synovial tissue of DG rats compared with CG rats. Conclusion: The ligaments, cartilage and synovia are highly affected following STZ-induced diabetes in rats, due the remodeling of collagen types in these tissues. This process may promote the degradation of the extracellular matrix, thus compromising joint function. Our data may help to better understand the pathogenesis of joint involvement related to diabetes.

Advanced glycated albumin impairs HDL anti-inflammatory activity and primes macrophages for inflammatory response that reduces reverse cholesterol transport

Objective: We investigated the effect of advanced glycated albumin (AGE-albumin) on macrophage sensitivity to inflammation elicited by S100B calgranulin and lipopolysaccharide (LPS) and the mechanism by which HDL modulates this response. We also measured the influence of the culture medium, isolated from macrophages treated with AGE-albumin, on reverse cholesterol transport (RCT). Methods and results: Macrophages were incubated with control (C) or AGE-albumin in the presence or absence of HDL, followed by incubations with S100B or LPS. Also, culture medium obtained from cells treated with C- or AGE-albumin, following S100B or LPS stimulation was utilized to treat naive macrophages in order to evaluate cholesterol efflux and the expression of HDL receptors. In comparison with C-albumin, AGE-albumin, promoted a greater secretion of cytokines after stimulation with S100B or LPS. A greater amount of cytokines was also produced by macrophages treated with AGE-albumin even in the presence of HDL Cytokine-enriched medium, drawn from incubations with AGE-albumin and S100B or LPS impaired the cholesterol efflux mediated by apoA-I (23% and 37%, respectively), HDL2 (43% and 47%, respectively) and HDL3 (20% and 8.5%, respectively) and reduced ABCA-1 protein level (16% and 26%, respectively). Conclusions: AGE-albumin primes macrophages for an inflammatory response impairing the RCT. Moreover, AGE-albumin abrogates the anti-inflammatory role of HDL, which may aggravate the development of atherosclerosis in DM. (C) 2012 Elsevier BM. All rights reserved.

Depletion of Langerhans cells in the tongue from patients with advanced-stage acquired immune deficiency syndrome: relation to opportunistic infections

Aims: To quantify and compare the expression of Langerhans cells (LCs) in the tongue mucosa of AIDS patients with different opportunistic infections, and from acquired immune deficiency syndrome (AIDS) and non-AIDS patients with normal tongues, using autopsy material. Methods and results: Human leucocyte antigen D-related (HLA-DR), CD1a and CD83 antibodies were used to identify and quantify LCs by immunohistochemistry in tongue tissue of 40 AIDS patients (10 with lingual candidiasis, 10 with lingual herpes, 10 with oral hairy leukoplakia and 10 with no lesions) and 23 tongues from human immunodeficiency virus (HIV)negative control patients. Quantification was performed by means of conventional morphometry in four different regions (anterior, middle, posterior and lateral) of the tongue. The results were expressed as positive cells per area of epithelium. The AIDS patients presented a lower density of CD1a(+) cells (P < 0.001), HLA-DR (P < 0.003) and CD83 (P < 0.001) in all regions of the tongue compared to the non-AIDS control group. However, no differences in any of the markers were found when AIDS patients with different opportunistic infections were compared with AIDS patients without tongue infection. Conclusions: Advanced stage AIDS patients showed a depletion of LCs in the tongue mucosa. HIV infection induces cytopathic changes in LCs, contributing to their depletion regardless of the presence of oral infections.

Sorafenib in Combination With Capecitabine: An Oral Regimen for Patients With HER2-Negative Locally Advanced or Metastatic Breast Cancer

Purpose Sorafenib is a multikinase inhibitor with antiangiogenic/antiproliferative activity. A randomized, double-blind, placebo-controlled phase IIB trial assessed sorafenib with capecitabine for locally advanced or metastatic human epidermal growth factor receptor 2 (HER2) -negative breast cancer. Patients and Methods Patients were randomly assigned to first-or second-line capecitabine 1,000 mg/m(2) orally twice a day for days 1 to 14 of every 21-day cycle with sorafenib 400 mg orally twice a day or placebo. The primary end point was progression-free survival (PFS). Results In total, 229 patients were enrolled. The addition of sorafenib to capecitabine resulted in a significant improvement in PFS versus placebo (median, 6.4 v 4.1 months; hazard ratio [HR], 0.58; 95% CI, 0.41 to 0.81; P = .001) with sorafenib favored across subgroups, including first-line (HR, 0.50; 95% CI, 0.30 to 0.82) and second-line (HR, 0.65; 95% CI, 0.41 to 1.04) treatment. There was no significant improvement for overall survival (median, 22.2 v 20.9 months; HR, 0.86; 95% CI, 0.61 to 1.23; P = .42) and overall response (38% v 31%; P = .25). Toxicities (sorafenib v placebo) of any grade included rash (22% v 8%), diarrhea (58% v 30%), mucosal inflammation (33% v 21%), neutropenia (13% v 4%), hypertension (18% v 12%), and hand-foot skin reaction/hand-foot syndrome (HFSR/HFS; 90% v 66%); grade 3 to 4 toxicities were comparable between treatment arms except HFSR/HFS (44% v 14%). Reasons for discontinuation in the sorafenib and placebo arms included disease progression (63% v 82%, respectively), adverse events (20% v 9%, respectively), and death (0% v 1%, respectively). Conclusion Addition of sorafenib to capecitabine improved PFS in patients with HER2-negative advanced breast cancer. The dose of sorafenib used in this trial resulted in unacceptable toxicity for many patients. A phase III confirmatory trial has been initiated with a reduced sorafenib dose.

Surrogate endpoints for prostate cancer-specific mortality after radiotherapy and androgen suppression therapy in men with localised or locally advanced prostate cancer: an analysis of two randomised trials

Background Androgen suppression therapy and radiotherapy are used to treat locally advanced prostate cancer. 3 years of androgen suppression confers a small survival benefit compared with 6 months of therapy in this setting, but is associated with more toxic effects. Early identification of men in whom radiotherapy and 6 months of androgen suppression is insufficient for cure is important. Thus, we assessed whether prostate-specific antigen (PSA) values can act as an early surrogate for prostate cancer-specific mortality (PCSM). Methods We systematically reviewed randomised controlled trials that showed improved overall and prostate cancer-specific survival with radiotherapy and 6 months of androgen suppression compared with radio therapy alone and measured lowest PSA concentrations (PSA nadir) and those immediately after treatment (PSA end). We assessed a cohort of 734 men with localised or locally advanced prostate cancer from two eligible trials in the USA and Australasia that randomly allocated participants between Feb 2, 1996, and Dec 27, 2001. We used Prentice criteria to assess whether reported PSA nadir or PSA end concentrations of more than 0.5 ng/mL were surrogates for PCSM. Findings Men treated with radiotherapy and 6 months of androgen suppression in both trials were significantly less likely to have PSA end and PSA nadir values of more than 0.5 ng/mL than were those treated with radiotherapy alone (p<0.0001). Presence of candidate surrogates (ie, PSA end and PSA nadir values >0.5 ng/mL) alone and when assessed in conjunction with the randomised treatment group increased risk of PCSM in the US trial (PSA nadir p=0.0016; PSA end p=0.017) and Australasian trial (PSA nadir p<0.0001; PSA end p=0.0012). In both trials, the randomised treatment group was no longer associated with PCSM (p >= 0.20) when the candidate surrogates were included in the model. Therefore, both PSA metrics satisfied Prentice criteria for surrogacy. Interpretation After radiotherapy and 6 months of androgen suppression, men with PSA end values exceeding 0.5 ng/mL should be considered for long-term androgen suppression and those with localised or locally advanced prostate cancer with PSA nadir values exceeding 0.5 ng/mL should be considered for inclusion in randomised trials investigating the use of drugs that have extended survival in castration-resistant metastatic prostate cancer.

Interferon-gamma and interleukin-10 production by mononuclear cells from patients with advanced head and neck cancer

OBJECTIVE: This study aims to evaluate the production of interferon-gamma and interleukin-10 by stimulated peripheral blood mononuclear cells isolated from patients with supraglottic laryngeal cancer before and after surgical treatment. METHODS: Fourteen patients with advanced supraglottic laryngeal cancer were studied. Cultures of peripheral blood mononuclear cells isolated during the preoperative and late postoperative periods were stimulated with concanavalin A and Bacille Calmette-Guerin, and the supernatant concentrations of interferon-gamma and interleukin-10 were measured. RESULTS: For non-stimulated cultures, the interferon-gamma levels produced by the preoperative period and the late postoperative period cultures were lower than the levels produced by the control group cultures. The interferon-gamma levels after stimulation with concanavalin A were higher in the late postoperative period cultures than in the preoperative evaluation cultures. Stimulation with Bacille Calmette-Guerin led to the production of similar levels of interferon-gamma and interleukin-10 by all cultures; thus, stimulation increased the levels of interferon-gamma produced by both the preoperative and postoperative cultures relative to the levels produced by the corresponding unstimulated cultures. CONCLUSION: Patients with advanced supraglottic laryngeal cancer exhibit an in vitro deficiency in interferongamma secretion by mononuclear cells. Stimulated cells seem to recover this function during the postoperative period.

Delineation of vertebral area on the coronal plane using three-dimensional ultrasonography advanced volume contrast imaging (VCI) Omni view: intrarater reliability and agreement using standard mouse, high definition mouse, and pen-tablet

Objective: To assess the fetal lumbosacral spine by three-dimensional (3D) ultrasonography using volume contrast imaging (VCI) omni view method and compare reproducibility and agreement between three different measurement techniques: standard mouse, high definition mouse and pen-tablet. Methods: A comparative and prospective study with 40 pregnant women between 20 and 34+6 weeks was realized. 3D volume datasets of the fetal spine were acquired using a convex transabdominal transducer. Starting scan plane was the coronal section of fetal lumbosacral spine by VCI-C function. Omni view manual trace was selected and a parallel plane of fetal spine was drawn including interest region. Intraclass correlation coefficient (ICC) was used for reproducibility analysis. The relative difference between three used techniques was compared by chi-square test and Fischer test. Results: Pen-tablet showed better reliability (ICC = 0.987). In the relative proportion of differences, this was significantly higher for the pen-tablet (82.14%; p < 0.01). In paired comparison, the relative difference was significantly greater for the pen-tablet (p < 0.01). Conclusion: The pen-tablet showed to be the most reproductive and concordant method in the measurement of body vertebral area of fetal lumbosacral spine by 3D ultrasonography using the VCI.

Advanced oxidation process H2O2/UV combined with anaerobic digestion to remove chlorinated organics from bleached kraft pulp mill wastewater

This study investigated the application of an advanced oxidation process combining hydrogen peroxide with ultraviolet radiation (H2O2/UV) to remove recalcitrant compounds from Kraft bleaching effluent. Anaerobic pre-treatment was performed to remove easily degraded organics using a horizontal-flow anaerobic immobilized biomass (HAIB) reactor. Bleaching plant effluent was treated in the HAIB reactor processed over 19 h of hydraulic retention time (HRT), reaching the expected removal efficiencies for COD (61 +/- 3%), TOC (69 +/- 9%), BOD5 (90 +/- 5%) and AOX (55 +/- 14%). However, the anaerobic treatment did not achieve acceptable removal of UV254 compounds. Furthermore, there was an increase of lignin, measured as total phenols. The H2O2/UV post-treatment provided a wide range of removal efficiencies depending on the dosage of hydrogen peroxide and UV irradiation: COD ranged from 0 to 11%, UV254 from 16 to 35%, lignin from 0 to 29% and AOX from 23 to 54%. All peroxide dosages applied in this work promoted an increase in the BOD5/COD ratio of the wastewater. The experiments demonstrate the technical feasibility of using H2O2/UV for post-treatment of bleaching effluents submitted to anaerobic pre-treatment.

Advanced QSAR Studies on PPAR delta Ligands Related to Metabolic Diseases

PPAR delta is a nuclear receptor that, when activated, regulates the metabolism of carbohydrates and lipids and is related to metabolic syndrome and type 2 diabetes. To understand the main interactions between ligands and PPAR delta, we have constructed 2D and 3D QSAR models and compared them with HOMO, LUMO and electrostatic potential maps of the compounds studied, as well as docking results. All QSAR models showed good statistical parameters and prediction outcomes. The QSAR models were used to predict the biological activity of an external test set, and the predicted values are in good agreement with the experimental results. Furthermore, we employed all maps to evaluate the possible interactions between the ligands and PPAR delta. These predictive QSAR models, along with the HOMO, LUMO and MEP maps, can provide insights into the structural and chemical properties that are needed in the design of new PPAR delta ligands that have improved biological activity and can be employed to treat metabolic diseases.

Advanced prostate cancer as a cause of oncogenic osteomalacia: an underdiagnosed condition

Tumor-induced osteomalacia (TIO) is a paraneoplastic bone mineral disturbance related to fibroblast growth factor 23 (FGF23) overproduction by the tumor, usually from mesenchymal origin. Such condition leads to high phosphate renal wasting and, consequently, to cumbersome symptoms as weakness, bone pain, and fractures. Case report. We report a case of an advanced castration-refractory prostate cancer patient, which developed severe hypophosphatemia with elevated phosphate excretion fraction. TIO was suspected, and increased levels of FGF23 reinforced such diagnosis. The patient died 4 months after being diagnosed with TIO. This case suggests that TIO has a dismal prognosis in prostate cancer patients. The clinical oncology community must be aware about such disturbance that can be present in those patients with weakness, bone pain, and hypophosphatemia.