Oxidative stress and inflammation in renal patients and healthy subjects

The first goal of this study was to measure the oxidative stress (OS) and relate it to lipoprotein variables in 35 renal patients before dialysis (CKD), 37 on hemodialysis (HD) and 63 healthy subjects. The method for OS was based on the ratio of cholesteryl esters (CE) containing C18/C16 fatty acids (R2) measured by gas chromatography (GC) which is a simple, direct, rapid and reliable procedure. The second goal was to investigate and identify a triacylglycerol peak on GC, referred to as TG48 (48 represents the sum of the three fatty acids carbon chain lengths) which was markedly increased in renal patients compared to healthy controls. We measured TG48 in patients and controls. Mass spectrometry (MS) and MS twice in tandem were used to analyze the fatty acid composition of TG48. MS showed that TG48 was abundant in saturated fatty acids (SFAs) that were known for their pro-inflammatory property. TG48 was significantly and inversely correlated with OS. Renal patients were characterized by higher OS and inflammation than healthy subjects. Inflammation correlated strongly with TG, VLDL-cholesterol, apolipoprotein (apo) C-III and apoC-III bound to apoB-containing lipoproteins, but not with either total cholesterol or LDL-cholesterol.In conclusion, we have discovered a new inflammatory factor, TG48. It is characterized with TG rich in saturated fatty acids. Renal patients have increased TG48 than healthy controls.     

Anti-HIV activity mediated by natural killer and CD8+ cells after toll-like receptor 7/8 triggering

We previously found that triggering TLR7/8 either by single stranded HIV RNA or synthetic compounds induced changes in the lymphoid microenvironment unfavorable to HIV. In this study, we used selective TLR7 and 8 agonists to dissect their contribution to the anti-HIV effects. While triggering TLR7 inhibited efficiently HIV replication in lymphoid suspension cells from tonsillar origin, its effect was inconsistent in peripheral blood mononuclear cells (PBMC). In contrast, triggering TLR8 showed a very prominent and overall very consistent effect in PBMC and tonsillar lymphoid suspension cells. Depletion of dendritic cells (DC), Natural killer cells (NK) and CD8+ T-cells from PBMC resulted in the reversal of TLR8 induced anti-HIV effects. Especially noteworthy, depletion of either NK or CD8+ T-cells alone was only partially effective. We interpret these findings that DC are the initiator of complex changes in the microenvironment that culminates in the anti-HIV active NK and CD8+ effector cells. The near lack of NK and the low number of CD8+ T-cells in tonsillar lymphoid suspension cells may explain the lower TLR8 agonist's anti-HIV effects in that tissue. However, additional cell-type specific differences must exist since the TLR7 agonists had a very strong inhibitory effect in tonsillar lymphoid suspension cells. Separation of effector from the CD4+ target cells did not abolish the anti-HIV effects pointing to the critical role of soluble factors. Triggering TLR7 or 8 were accompanied by major changes in the cytokine milieu; however, it appeared that not a single soluble factor could be assigned for the potent effects. These results delineate the complex effects of triggering TLR7/8 for an efficient antiviral defense. While the ultimate mechanism(s) remains unknown, the potent effects described may have therapeutic value for treating chronic viral diseases. Notably, HIV replication is blocked by TLR triggering before HIV integrates into the host chromosome which would prevent the establishment or maintenance of the latent reservoir.     

Oxidative stress in subjects affected by celiac disease

In order to study the role of oxidative stress in celiac disease, protein carbonyl groups, thiobarbituric acid-reactive substance and pentosidine were evaluated in the plasma of nine patients with asymptomatic celiac disease and in a control group (n = 25). Plasma alpha-tocopherol, retinol and lipids were determined in the same samples. The levels of markers of oxidative stress derived from both protein (carbonyl groups) and lipids (thiobarbituric acid-reactive substances) were significantly higher in celiac disease patients, whereas lipoproteins and alpha-tocopherol were significantly lower. These data indicate that in celiac disease, even when asymptomatic, a redox imbalance persists; this is probably caused by an absorption deficiency, even if slight. Dietary supplementation with antioxidant molecules may offer some benefit and deserves further investigation.     

Placental toll-like receptor 3 and toll-like receptor 7/8 activation contributes to preeclampsia in humans and mice

Preeclampsia (PE) is a pregnancy-specific hypertensive syndrome characterized by excessive maternal immune system activation, inflammation, and endothelial dysfunction. Toll-like receptor (TLR) 3 activation by double-stranded RNA (dsRNA) and TLR7/8 activation by single-stranded RNA (ssRNA) expressed by viruses and/or released from necrotic cells initiates a pro-inflammatory immune response; however it is unknown whether viral/endogenous RNA is a key initiating signal that contributes to the development of PE. We hypothesized that TLR3/7/8 activation will be evident in placentas of women with PE, and sufficient to induce PE-like symptoms in mice. Placental immunoreactivity and mRNA levels of TLR3, TLR7, and TLR8 were increased significantly in women with PE compared to normotensive women. Treatment of human trophoblasts with the TLR3 agonist polyinosine-polycytidylic acid (poly I:C), the TLR7-specific agonist imiquimod (R-837), or the TLR7/8 agonist CLO97 significantly increased TLR3/7/8 levels. Treatment of mice with poly I:C, R-837, or CLO97 caused pregnancy-dependent hypertension, endothelial dysfunction, splenomegaly, and placental inflammation. These data demonstrate that RNA-mediated activation of TLR3 and TLR7/8 plays a key role in the development of PE.     

Neutrophil elastase up-regulates interleukin-8 via toll-like receptor 4

Cystic fibrosis is characterised in the lungs by high levels of neutrophil elastase (NE). NE induces interleukin-8 (IL-8) expression via an IL-1 receptor-associated kinase signalling pathway. Here, we show that these events involve the cell surface membrane bound toll-like receptor 4 (TLR4). We demonstrate that human embryonic kidney (HEK)293 cells transfected with a TLR4 cDNA (HEK-TLR4) express TLR4 mRNA and protein and induce IL-8 promoter activity in response to NE. Treatment of both HEK-TLR4 and human bronchial epithelial cells with NE decreases TLR4 protein expression. Furthermore, a TLR4 neutralising antibody abrogates NE-induced IL-8 production, and induces tolerance to a secondary lipopolysaccharide stimulus. These data implicate TLR4 in NE induced IL-8 expression in bronchial epithelium.     

Activation of toll-like receptor 2 in acne triggers inflammatory cytokine responses

One of the factors that contributes to the pathogenesis of acne is Propionibacterium acnes; yet, the molecular mechanism by which P. acnes induces inflammation is not known. Recent studies have demonstrated that microbial agents trigger cytokine responses via Toll-like receptors (TLRs). We investigated whether TLR2 mediates P. acnes-induced cytokine production in acne. Transfection of TLR2 into a nonresponsive cell line was sufficient for NF-kappa B activation in response to P. acnes. In addition, peritoneal macrophages from wild-type, TLR6 knockout, and TLR1 knockout mice, but not TLR2 knockout mice, produced IL-6 in response to P. acnes. P. acnes also induced activation of IL-12 p40 promoter activity via TLR2. Furthermore, P. acnes induced IL-12 and IL-8 protein production by primary human monocytes and this cytokine production was inhibited by anti-TLR2 blocking Ab. Finally, in acne lesions, TLR2 was expressed on the cell surface of macrophages surrounding pilosebaceous follicles. These data suggest that P. acnes triggers inflammatory cytokine responses in acne by activation of TLR2. As such, TLR2 may provide a novel target for treatment of this common skin disease.     

Oxidative stress and transferrin receptor recycling

Perturbation of the oxidative balance in biological systems plays an important role in numerous pathological states as well as in many physiological processes such as receptor activity. In order to evaluate if oxidative stress induced by menadione influences membrane receptor processes, a study was conducted on the transferrin receptor. Consequently, biochemical, biophysical and ultrastructural studies were carried out on different cell lines. The results obtained seem to indicate that oxidative stress is able of inducing a rapid and specific down-modulation of membrane transferrin receptor due to a block of receptor recycling on the cell surface without affecting binding affinity.     

Oxidative stress augments the secretion of atrial natriuretic peptide in isolated rat atria

Reactive oxygen species (ROS) play a role in cardiovascular diseases such as hypertension and heart failure. The objective of the present study was to investigate the role of endogenous ROS in atrial hemodynamics and ANP secretion in isolated perfused beating rat atria. Pyrogallol (a generator of superoxide anion, 0.1, 1 mM) or hydrogen peroxide (0.1, 1, 10, 30 mM) was perfused into atria paced at 1.2 Hz. Pyrogallol and hydrogen peroxide stimulated ANP secretion and concentration in a dose-dependent manner and dramatically decreased atrial contractility and translocation of extracellular fluid. The stimulatory effect of pyrogallol and hydrogen peroxide on ANP secretion was attenuated by the pretreatment with ascorbic acid (an antioxidant; 1 mM) and cariporide (an inhibitor of the Na⁺/H⁺ exchanger; 1 μM) but negative inotropic effect was not changed. U120 (a MAPK^erk pathway inhibitor; 10 μM) attenuated the stimulatory effect of hydrogen peroxide on ANP secretion. However, U120 augmented negative inotropic effect and stimulatory effect of ANP concentration induced by pyrogallol. Antioxidant such as N-acetyl cystein, gallate, propyl gallate, or ellagic acid did not cause any significant changes in atrial parameters. These results suggest that intracellular - formed ROS stimulates ANP secretion partly through activation of MAPK^erk pathway and Na⁺/H⁺ exchanger.     

Oxidative stress and transferrin receptor recycling

Perturbation of the oxidative balance in biological systems plays an important role in numerous pathological states as well as in many physiological processes such as receptor activity. In order to evaluate if oxidative stress induced by menadione influences membrane receptor processes, a study was conducted on the transferrin receptor. Consequently, biochemical, biophysical and ultrastructural studies were carried out on different cell lines. The results obtained seem to indicate that oxidative stress is able of inducing a rapid and specific down-modulation of membrane transferrin receptor due to a block of receptor recycling on the cell surface without affecting binding affinity.     

Oxidative stress and antioxidant defence in a healthy nonagenarian population

Results on oxidative markers during ageing are not consistent throughout the scientific literature; however, successful ageing may depend on better ability to cope with oxidative stress. A previous study of ours showed that successful ageing could actually be related to enhanced response to oxidatively modified proteins. In this study, a healthy nonagenarian population (OVER-90) was examined for various blood oxidative biomarkers and compared with a healthy population of blood donors (age range, 23-66 years). Blood glutathione, both total (tGSH) and oxidised (GSSG), and total plasmatic antioxidant status were maintained in the OVER-90 at a level similar to the control population. Sulphydryl (sulfhydryl) groups and glutathione peroxidase (GPx) were instead decreased. The results are discussed in a possible unifying view: the OVER-90 population could possess a globally preserved antioxidant ability, though some signs of oxidative damage are present and some structures could be 'sacrificed' in order to keep the redox equilibrium.     

Oxidative stress responses during cassava post-harvest physiological deterioration

A major constraint to the development of cassava (Manihot esculenta Crantz) as a crop to both farmers and processors is its starchy storage roots' rapid post-harvest deterioration, which can render it unpalatable and unmarketable within 24–72 h. An oxidative burst occurs within 15 min of the root being injured, that is followed by the altered regulation of genes, notably for catalase and peroxidase, related to the modulation of reactive oxygen species, and the accumulation of secondary metabolites, some of which show antioxidant properties. The interactions between these enzymes and compounds, in particular peroxidase and the coumarin, scopoletin, are largely confined to the vascular tissues where the visible symptoms of deterioration are observed. These, together with other data, are used to develop a tentative model of some of the principal events involved in the deterioration process. Abbreviations: ACMV, African cassava mosaic virus; AFLP, amplified fragment length polymorphism; CAT, catalase; cDNA, complementary deoxyribonucleic acid; CIAT, International Centre for Tropical Agriculture; Cu/ZnSOD, copper/zinc superoxide dismutase; DAB, 3,3-diaminobenzidine tetrahydrochloride; DPPH, 1,1-diphenyl-2-picrylhydrazyl; FeSOD, iron superoxide dismutase; FW, fresh weight; GUS, -glucuronidase; HPTLC, high-performance thin-layer chromatography; HR, hypersensitive response; IEF-PAGE, isoelectric focusing polyacrylamide gel electrophoresis; MAS, marker-assisted selection; MeJa, methyl jasmonate; MnSOD, manganese superoxide dismutase; NADPH, nicotinamide adenine dinucleotide phosphate (reduced form); NBT, nitroblue tetrazolium; PAL, phenylalanine ammonia-lyase; PCD, programmed cell death; PCR, polymerase chain reaction; POX, peroxidase; PPD, post-harvest physiological deterioration; QTL, quantitative trait loci; ROS, reactive oxygen species; RT, room temperature; SAR, systemic acquired resistance; SDS, sodium dodecyl sulfate; SOD, superoxide dismutase     

Oxidative stress responses during cassava post-harvest physiological deterioration

A major constraint to the development of cassava (Manihot esculenta Crantz) as a crop to both farmers and processors is its starchy storage roots’ rapid post-harvest deterioration, which can render it unpalatable and unmarketable within 24–72 h. An oxidative burst occurs within 15 min of the root being injured, that is followed by the altered regulation of genes, notably for catalase and peroxidase, related to the modulation of reactive oxygen species, and the accumulation of secondary metabolites, some of which show antioxidant properties. The interactions between these enzymes and compounds, in particular peroxidase and the coumarin, scopoletin, are largely confined to the vascular tissues where the visible symptoms of deterioration are observed. These, together with other data, are used to develop a tentative model of some of the principal events involved in the deterioration process.     

Oxidative stress responses in Escherichia coli and Salmonella typhimurium.

Oxidative stress is strongly implicated in a number of diseases, such as rheumatoid arthritis, inflammatory bowel disorders, and atherosclerosis, and its emerging as one of the most important causative agents of mutagenesis, tumorigenesis, and aging. Recent progress on the genetics and molecular biology of the cellular responses to oxidative stress, primarily in Escherichia coli and Salmonella typhimurium, is summarized. Bacteria respond to oxidative stress by invoking two distinct stress responses, the peroxide stimulon and the superoxide stimulon, depending on whether the stress is mediated by peroxides or the superoxide anion. The two stimulons each contain a set of more than 30 genes. The expression of a subset of genes in each stimulon is under the control of a positive regulatory element; these genes constitute the OxyR and SoxRS regulons. The schemes of regulation of the two regulons by their respective regulators are reviewed in detail, and the overlaps of these regulons with other stress responses such as the heat shock and SOS responses are discussed. The products of Oxy-R- and SoxRS-regulated genes, such as catalases and superoxide dismutases, are involved in the prevention of oxidative damage, whereas others, such as endonuclease IV, play a role in the repair of oxidative damage. The potential roles of these and other gene products in the defense against oxidative damage in DNA, proteins, and membranes are discussed in detail. A brief discussion of the similarities and differences between oxidative stress responses in bacteria and eukaryotic organisms concludes this review.     

Innate immunity at the mucosal surface: role of toll-like receptor 3 and toll-like receptor 9 in cervical epithelial cell responses to microbial pathogens

Toll-like receptors (TLRs) are a family of pattern recognition receptors that recognize distinct molecular patterns shared by a broad range of pathogens, including nucleic acids. TLR9, for example, recognizes unmethylated deoxycytidyl-phosphate-deoxyguanosine (CpG) dinucleotides that are common in bacterial and some viral nucleic acids, whereas TLR3 recognizes double-stranded RNA and TLR7/TLR8 recognize single-stranded RNA, which would be found during viral replication. We were interested in whether TLR3, TLR9, and the related TLR9 family members TLR7/TLR8 might play a role in antiviral immune defense at the mucosal epithelial surface of the lower female reproductive tract. We studied cervical epithelial cells and found that they expressed mRNA for TLR3, TLR9, and TLR7, but had only a weak signal for TLR8. For TLR3 and TLR9, protein expression was confirmed to be intracellular. When epithelial cells were incubated with polyinosine-polycytidylic acid and CpG oligodinucleotides, we observed dose-dependent upregulation of interleukin-8 secretion. However, cells failed to respond to a variety of TLR7/TLR8 ligands. Polyinosine-polycytidylic acid also induced production of interferon-beta and chemokine C-C motif ligand 5, whereas CpG DNA did not. Cell activation by synthetic oligodinucleotides occurred only in response to the B class sequences, and required the presence of human-specific CpG motifs. In addition, responses to CpG oligodinucleotides could be inhibited by chloroquine, demonstrating the requirement for endosomal maturation. These data demonstrate that mucosal epithelial cells express functional TLR3 and TLR9, and suggest that these receptors play a role in regulating the proinflammatory cytokine and antiviral environment of the lower female reproductive tract during infection with viral and bacterial pathogens.     

Coxsackievirus B4-induced cytokine production in pancreatic cells is mediated through toll-like receptor 4

Coxsackievirus B4 (CBV4), a member of the Picornavirus genus, has long been implicated in the development of insulin-dependent diabetes mellitus (IDDM) caused by virus-induced pancreatic cell damage. The progressive destruction of pancreatic beta cells is responsible for the development of IDDM. It has recently been suggested that CBV4 infection can induce the production of proinflammatory cytokines, and these cytokines seem to be involved in the damage to the insulin-producing cells. In this study we investigated whether toll-like receptors (TLRs) are responsible for triggering the proinflammatory cytokine production in human pancreatic cells in response to CBV4. Here we demonstrate that CBV4 triggers cytokine production through a TLR4-dependent pathway. This interaction seems to be independent of virus attachment and cell entry.     

Neonatal B cells suppress innate toll-like receptor immune responses and modulate alloimmunity

It has been known for decades that neonates are susceptible to transplant tolerance, but the immunological mechanisms involved remain to be fully elucidated. Recent evidence indicates that the maturation state of DCs responding to an allograft may have a profound impact on whether immunity or tolerance ensues. Given that TLR activation is a key process leading to DC maturation, we hypothesized that DCs from neonates have defective TLR immune responses. Contrary to our hypothesis, we found that murine neonatal DCs demonstrated enhanced TLR responses in comparison to adult counterparts in vitro. However, we found that neonatal B cells possess unique immunoregulatory functions as they impaired DC responses to TLR activation in an IL-10-dependent fashion. Functionally, we demonstrated that TLR-activated neonatal, but not adult, B cells impaired Th1, but not Th2, T cell alloimmune responses in vitro and in vivo, in models of alloimmune priming and allotransplantation. We conclude that neonatal B cells possess unique immunoregulatory properties that inhibit DC function and modulate alloimmunity in our murine experimental systems.