miR-1226 detection in GCF as potential biomarker of chronic periodontitis: A pilot study.

BACKGROUND: The study and identification of new biomarkers for periodontal disease, such as microRNAs (miRNAs), may give us more information about the location and severity of the disease and will serve as a basis for treatment planning and disease-monitoring. miRNAs are a group of small RNAs which are involved in gene regulation by binding to their messenger RNA target (mRNA). In this pilot study, the procedure for purifying miRNAs from gingival crevicular fluid (GCF) was, for the first time, described. In addition, the concentration of miRNAs in GCF was analyzed and compared between patients with moderate or severe chronic periodontitis (CP) and healthy controls. MATERIAL AND METHODS: GCF samples were collected from single-rooted teeth of patients with moderate or severe CP (n=9) and of healthy individuals (n=9). miRNAs were isolated from GCF using miRNeasy Serum/Plasma kit (Qiagen, CA. USA). Reverse transcription polymerase chain reaction (qRT-PCR) was used to determine the expression of a series of miRNAs candidates that are related to bone metabolism. The significance of differences in miRNA levels between both groups was determined using Mann-Whitney U test. RESULTS: The results from this pilot study indicate that miRNAs can be isolated from GCF. Six different miRNAs were analyzed (miR-671, miR-122, miR-1306, miR-27a, miR-223, miR-1226), but only miR-1226 showed statically significant differences between the CP group and healthy controls (p<0.05). This miRNA was downregulated in patients with CP. CONCLUSIONS: Within the limitations of the present study, it may be concluded that miR-1226 can be a promising biomarker for periodontal disease, adding relevant information to common clinical parameters used for diagnosis and prognosis of periodontitis.

A new mass spectrometry-based method for the quantification of histones in plasma from septic shock patients.

The aim of this study was to develop a novel method to detect circulating histones H3 and H2B in plasma based on multiple reaction monitoring targeted mass spectrometry and a multiple reaction monitoring approach (MRM-MS) for its clinical application in critical bacteriaemic septic shock patients. Plasma samples from 17 septic shock patients with confirmed bacteraemia and 10 healthy controls were analysed by an MRM-MS method, which specifically detects presence of histones H3 and H2B. By an internal standard, it was possible to quantify the concentration of circulating histones in plasma, which were significantly higher in patients, and thus confirmed their potential as biomarkers for diagnosing septic shock. After comparing surviving patients and non-survivors, a correlation was found between higher levels of circulating histones and unfavourable outcome. Indeed, histone H3 proved a more efficient and sensitive biomarker for septic shock prognosis. In conclusion, these findings suggest the accuracy of the MRM-MS technique and stable isotope labelled peptides to detect and quantify circulating plasma histones H2B and H3. This method may be used for early septic shock diagnoses and for the prognosis of fatal outcomes.

Circulating miR-323-3p is a biomarker for cardiomyopathy and an indicator of phenotypic variability in Friedreich's ataxia patients.

MicroRNAs (miRNAs) are noncoding RNAs that contribute to gene expression modulation by regulating important cellular pathways. In this study, we used small RNA sequencing to identify a series of circulating miRNAs in blood samples taken from Friedreich's ataxia patients. We were thus able to develop a miRNA biomarker signature to differentiate Friedreich's ataxia (FRDA) patients from healthy people. Most research on FDRA has focused on understanding the role of frataxin in the mitochondria, and a whole molecular view of pathological pathways underlying FRDA therefore remains to be elucidated. We found seven differentially expressed miRNAs, and we propose that these miRNAs represent key mechanisms in the modulation of several signalling pathways that regulate the physiopathology of FRDA. If this is the case, miRNAs can be used to characterize phenotypic variation in FRDA and stratify patients' risk of cardiomyopathy. In this study, we identify miR-323-3p as a candidate marker for phenotypic differentiation in FRDA patients suffering from cardiomyopathy. We propose the use of dynamic miRNAs as biomarkers for phenotypic characterization and prognosis of FRDA.

Epigenetic biomarkers: A new perspective in laboratory diagnostics.

Epigenetics comprises the study of chemical modifications in the DNA and histones that regulates the gene expression or cellular phenotype. However, during the last decade this term has evolved after the elucidation of different mechanisms (microRNAs and nuclear organization of the chromosomes) involved in regulating gene expression. Epigenetics and the new designed technologies capable to analyze epigenetic changes (e.g., methylated DNA, miRNAs expression, post-translational modifications on histones among others) have disclosed an appealing scenario that will offer for the biomedical sciences new biomarkers for the study of neurodegenerative diseases, multifactorial complex diseases, rare diseases and cancer. Moreover, new technologies adapted for epigenetic studies will offer promising applications that in the next years will be common technologies in clinical laboratories. In this review we discuss epigenetic modifications used as possible biomarkers in several diseases. We also present the potential of methodologies to purify histones, and high throughput technologies as candidates to be set in clinical laboratories for their high potential analyzing epigenetic processes.

Increased plasma xanthine oxidase activity is related to nuclear factor kappa beta activation and inflammatory markers in familial combined hyperlipidemia.

BACKGROUND AND AIMS: Xanthine oxidase (XO) has been described as one of the major enzymes producing free radicals in blood. Oxidative stress and inflammatory processes have been implicated in the pathogenesis of endothelial dysfunction and the progression of atherosclerosis but until now, there is little data about the influence of vascular prooxidant systems and inflammation in familial combined hyperlipidemia (FCH). Our goal was to evaluate whether XO activity was altered in FCH and if it was related to the inflammatory process represented by NFkB, IL-6 and hsCRP, and assessing the correlation between XO activity and insulin resistance (IR). METHOD AND RESULTS: 40 Non-related subjects with FCH and 30 control subjects were included, all of them non-diabetic, normotensive and non-smokers. We measured lipid profile, glucose, insulin, uric acid, XO activity, malondialdehyde (MDA), IL-6 and hsCRP in plasma and NFkB activity in circulating mononuclear cells. Patients with FCH showed significantly higher levels of uric acid, XO activity, MDA, NFkB activity, IL-6 and hsCRP than controls. XO activity was independently related to NFkB activity with an odds ratio of 4.082; to IL-6 with an odds ratio of 4.191; and to IR with an odds ratio of 3.830. Furthermore, mean NFkB activity, IL-6 levels, and IR were highest in the highest percentile of XO activity. CONCLUSIONS: Subjects with FCH showed increased XO and NFkB activities and low grade inflammatory markers related to atherosclerosis. XO activity was correlated with higher inflammatory activity and IR. These data could explain, in part, the high cardiovascular disease risk present in these patients.

Circulating mononuclear cells nuclear factor-kappa B activity, plasma xanthine oxidase, and low grade inflammatory markers in adult patients with familial hypercholesterolaemia.

BACKGROUND: Few data are available on circulating mononuclear cells nuclear factor-kappa B (NF-kB) activity and plasma xanthine oxidase (XO) activity in heterozygous familial hypercholesterolaemia (FH). The goal of the study was to analyse circulating mononuclear cells NF-kB and plasma XO activities in FH patients. MATERIALS AND METHODS: Thirty FH index patients and 30 normoglycaemic normocholesterolaemic controls matched by age, gender, body mass index, abdominal circumference and homeostasis model assessment index were studied. Plasma XO and inflammatory markers were measured by standard methods. NF-kB was assayed in circulating mononuclear cells. RESULTS: Familial hypercholesterolaemia patients showed a significantly higher NF-kB (75.0 +/- 20.7 vs. 42.7 +/- 16.8 relative luminiscence units) and XO (0.44 +/- 0.13 vs. 0.32 +/- 0.09 mU mL(-1)) activities than controls. In addition, interleukin-1, interleukin-6, high sensitivity C reactive protein (hsCRP) and oxidized LDL (LDL-ox) were also significantly higher in FH patients. In the total group (FH and controls), XO was significantly associated with LDL-cholesterol (LDL-C), apolipoprotein B (apoB), NF-kB and hsCPR, and NF-kB activity was significantly associated with XO, hsCPR, LDL-ox, LDL-C and apoB plasma values. Using multiple regression analysis, XO was independently associated with hsCPR and NF-kB, and NF-kB activity in circulating mononuclear cells was independently associated with apoB and LDL-ox plasma values. CONCLUSION: Familial hypercholesterolaemia patients show increased activities of NF-kB and XO, and higher values of low grade inflammatory markers related to atherosclerosis. NF-kB activity was independently associated with apoB plasma values. These data could explain in part the high cardiovascular disease risk present in these patients.

Effect of intermittent hypoxia on hematological parameters after recombinant human erythropoietin administration.

Recent publications reflect the anti-doping authorities' concern about the use of altitude simulator systems as violating the spirit of sport criterion (Levine 2006; Loland and Murray 2007; Spriggs 2005). The aim of our study was to determine whether intermittent hypoxic treatments could modify the hemoglobin, hematocrit, reticulocytes, and erythropoietic stimulation index (OFF-Hr Score) values after administration of rHuEPO-alpha. Although these hematological parameters are of secondary nature some international sport federations currently exclude athletes who show aberrant values of these parameters from competition. Ten young male Wistar rats were treated, three times a week for 2 weeks, with 500 IU of rHuEPO-alpha. After the treatment, the animals were randomly divided into two groups: normoxic and hypoxic. The normoxic group was maintained at 21% O(2) 24 h a day for 23 days. The hypoxic group was maintained 12 h at 21% O(2) and 12 h at 12% O(2) (~4,000 m) the same time period. After the rHuEPO-alpha treatment, the hypoxic group of animals had a faster recovery rate in the reticulocyte count, elevated concentrations of hemoglobin and hematocrit and a significant increase in the endogenous EPO levels when compared with the normoxic group of animals. These changes led to significant modifications in the OFF-Hr Score between the hypoxic and normoxic animals. Intermittent hypoxic treatments after rHuEPO administration can significantly modify the main hematological parameters tested by the anti-doping authorities. Our results in an animal model suggest checking the described phenomena in humans in order to reach major conclusions.

Vitamins C and E prevent AZT-induced leukopenia and loss of cellularity in bone marrow. Studies in mice.

A major limitation in the use of AZT for AIDS treatment is the occurrence of side effects, such as leukopenia. The effects of antioxidant vitamins C and E on AZT-induced leukopenia were investigated in mice. Mice were divided into four groups: (1) controls; (2) AZT-treated; (3) treated with AZT plus vitamins C and E; and (4) pre-treated with vitamins and then treated with AZT plus vitamins. Our results demonstrate that AZT causes leukopenia in mice, which was abrogated by administration of vitamins C and E in the pre-treated group. These vitamins prevented the decrease in cellular content induced by AZT in bone marrow and diminished peroxide levels in myeloid precursors in bone marrow. AZT also caused an increase in plasma malondialdehyde and blood oxidized glutathione levels, which was prevented by the administration of antioxidant vitamins. In conclusion, oxidative stress is involved in AZT-induced leukopenia which may be prevented by antioxidant treatment.

Oxidative stress as a signal to up-regulate gamma-cystathionase in the fetal-to-neonatal transition in rats.

Hepatic gamma-cystathionase, a rate-limiting enzyme for the synthesis of L-cysteine from L-methionine in the trans-sulphuration pathway, exhibits significantly higher activity in the newly born infant as compared to the fetus. The aim of this work was: 1) To determine whether the increase in gamma-cystathionase activity occurring in the fetal-to-neonatal transition is due to up-regulation of its mRNA and protein, 2) To elucidate the mechanisms responsible for this increase in gamma-cystathionase activity. Our results show that expression of gamma-cystathionase at both the mRNA and protein levels was higher in newborn than in fetal liver. gamma-Cystathionase activity in fetal hepatocytes in vitro increased when incubated with tert-butyl-hydroperoxide at low concentration (0.01 mM). Hence, moderate oxidative stress would act as a signal to up-regulate gamma-cystathionase in the fetal to neonatal transition. Stress hormones, such as phenylephrine or glucagon also increased gamma-cystathionase activity in fetal hepatocytes. We also report a competitive inhibition of purified gamma-cystathionase by L-cysteine, which would help to maintain physiological low L-cysteine levels in hepatocytes. In conclusion, our results show that increased hepatic gamma-cystathionase activity in the fetal-to-neonatal transition is due to up-regulation of its gene expression mediated by stress hormones together with the physiological oxidative stress that occurs at birth.

Role of mitochondrial oxidative stress to explain the different longevity between genders: protective effect of estrogens.

Females live longer than males. Work from our laboratory has shown that this may be due to the up-regulation of longevity-associated genes by estrogens. Estrogens bind to the estrogen receptors and subsequently activate the mitogen activated protein kinase and nuclear factor kappa B signalling pathways, resulting in an up-regulation of antioxidant enzymes. Estrogen administration, however, has serious undesirable effects and of course, cannot be administered to males because of its powerful feminizing effects. Thus, we tested the effect of genistein, a phytoestrogen of high nutritional importance whose structure is similar to estradiol, on the regulation of the expression of antioxidant, longevity-related genes and consequently on oxidant levels in mammary gland tumour cells in culture. Phytoestrogens mimic the protective effect of oestradiol using the same signalling pathway. The critical importance of up-regulating antioxidant genes, by hormonal and dietary manipulations, to increase longevity is discussed.

Role of GSH in the modulation of NOS-2 expression in the weaned mammary gland.

GSH delivery to the lactating mammary gland is essential for the maintenance of lactation as its decrease leads to apoptosis and involution of the mammary gland. In fact, it has already been demonstrated that some of the changes in gene expression found in the lactating mammary gland after forced weaning are reproduced in rats treated with buthionine sulphoximine to deplete GSH levels. An oligonucleotide microarray experiment would give us a better knowledge of the mRNA expression patterns during lactation and after weaning and the possible functions of GSH in the modulation of these events.

Na+ dependent glutamate transporters (EAAT1, EAAT2, and EAAT3) in primary astrocyte cultures: effect of oxidative stress.

The Na+ -dependent L-glutamate transporters EAAT1(GLAST), EAAT2 (GLT-1) and EAAT3 (EAAC1) are expressed in primary astrocyte cultures, showing that the EAAT3 transporter is not neuron-specific. The presence of these three transporters was evaluated by RT-PCR, immunoblotting, immunocytochemical techniques, and transport activity. When primary astrocyte cultures were incubated with L-buthionine-(S,R)-sulfoximine (BSO), a selective inhibitor of gamma-glutamylcysteine synthetase, the GSH concentration was significantly lower than in control cultures, but the expression and amount of protein of EAAT1, EAAT2 and EAAT3 and transport of L-glutamate was unchanged. Oxidative stress was created by adding H(2)O(2) or tert.-butyl hydroperoxide (t-bOOH) to the primary astrocyte cultures and cell damage was evaluated by measuring activity of lactate dehydrogenase. Under oxidative stress, GSH levels were significantly lower than in control astrocytes; but the expression and the amount of protein of the three transporters remained unchanged. However, L-glutamate uptake was significantly lower in astrocytes under oxidative conditions when compared to controls. L-Glutamate uptake was not changed in the presence of ascorbate, but was partially recovered in the presence of DTT and GSH ethyl ester. This report emphasizes that oxidative stress and not GSH depletion alters transporter activity without changing transporter expression.

Resuscitation with room air instead of 100% oxygen prevents oxidative stress in moderately asphyxiated term neonates.

BACKGROUND: Traditionally, asphyxiated newborn infants have been ventilated using 100% oxygen. However, a recent multinational trial has shown that the use of room air was just as efficient as pure oxygen in securing the survival of severely asphyxiated newborn infants. Oxidative stress markers in moderately asphyxiated term newborn infants resuscitated with either 100% oxygen or room air have been studied for the first time in this work. METHODS: Eligible term neonates with perinatal asphyxia were randomly resuscitated with either room air or 100% oxygen. The clinical parameters recorded were those of the Apgar score at 1, 5, and 10 minutes, the time of onset of the first cry, and the time of onset of the sustained pattern of respiration. In addition, reduced and oxidized glutathione concentrations and antioxidant enzyme activities (superoxide dismutase, catalase, and glutathione peroxidase) were determined in blood from the umbilical artery during delivery and in peripheral blood at 72 hours and at 4 weeks' postnatal age. RESULTS: Our results show that the room-air resuscitated (RAR) group needed significantly less time to first cry than the group resuscitated with 100% oxygen (1.2 +/- 0.6 minutes vs 1.7 +/- 0.5). Moreover, the RAR group needed less time undergoing ventilation to achieve a sustained respiratory pattern than the group resuscitated with pure oxygen (4.6 +/- 0.7 vs 7.5 +/- 1.8 minutes). The reduced-to-oxidized-glutathione ratio, which is an accurate index of oxidative stress, of the RAR group (53 +/- 9) at 28 days of postnatal life showed no differences with the control nonasphyxiated group (50 +/- 12). However, the reduced-to-oxidized-glutathione ratio of the 100% oxygen-resuscitated group (OxR) (15 +/- 5) was significantly lower and revealed protracted oxidative stress. Furthermore, the activities of superoxide dismutase and catalase in erythrocytes were 69% and 78% higher, respectively, in the OxR group than in the control group at 28 days of postnatal life. Thus, this shows that these antioxidant enzymes, although higher than in controls, could not cope with the ongoing generation of free radicals in the OxR group. However, there were no differences in antioxidant enzyme activities between the RAR group and the control group at this stage. CONCLUSIONS: There are no apparent clinical disadvantages in using room air for ventilation of asphyxiated neonates rather than 100% oxygen. Furthermore, RAR infants recover more quickly as assessed by Apgar scores, time to the first cry, and the sustained pattern of respiration. In addition, neonates resuscitated with 100% oxygen exhibit biochemical findings reflecting prolonged oxidative stress present even after 4 weeks of postnatal life, which do not appear in the RAR group. Thus, the current accepted recommendations for using 100% oxygen in the resuscitation of asphyxiated newborn infants should be further discussed and investigated.

Cyanoside chloride and chromocarbe diethylamine are more effective than vitamin C against exercise-induced oxidative stress.

Exercise generates free radicals only when it is exhaustive. Free radicals are involved in tissue damage caused by exercise. Antioxidant vitamins (vitamin C and E) and other antioxidants such as coenzyme Q, and N-acetyl cysteine prevent muscle damage and decrease muscle fatigue. The main aim of this paper was to test the possible protective effect of two new antioxidants, cyanoside chloride and chromocarbe diethylamine, on the oxidative stress generated by exhaustive exercise. The antioxidants were given to rats daily (50 mg/kg) in drinking water for 30 days. Blood oxidized glutathione/ reduced glutathione ratio, and plasma malondialdehyde levels were determined as indexes of oxidative stress. Plasma creatine kinase, alanine-aminotransferase and lactate dehydrogenase activities were used as markers of muscle damage. Both cyanoside chloride and chromocarbe diethylamine were more effective than vitamin C in the prevention of glutathione oxidation in blood. Furthermore, cyanoside chloride and chromocarbe diethylamine partially prevented muscle damage. Chromocarbe diethylamine was the most effective compound in the prevention of exercise-induced lipid peroxidation (malondialdehyde) in plasma.

Mechanism of free radical production in exhaustive exercise in humans and rats; role of xanthine oxidase and protection by allopurinol.

Exhaustive exercise generates free radicals. However, the source of this oxidative damage remains controversial. The aim of this paper was to study further the mechanism of exercise-induced production of free radicals. Testing the hypothesis that xanthine oxidase contributes to the production of free radicals during exercise, we found not only that exercise caused an increase in blood xanthine oxidase activity in rats but also that inhibiting xanthine oxidase with allopurinol prevented exercise-induced oxidation of glutathione in both rats and in humans. Furthermore, inhibiting xanthine oxidase prevented the increases in the plasma activity of cytosolic enzymes (lactate dehydrogenase, aspartate aminotransferase, and creatine kinase) seen after exhaustive exercise. Our results provide evidence that xanthine oxidase is responsible for the free radical production and tissue damage during exhaustive exercise. These findings also suggest that mitochondria play a minor role as a source of free radicals during exhaustive physical exercise.

Vitamin A deficiency causes oxidative damage to liver mitochondria in rats.

Mitochondrial damage in rat liver induced by chronic vitamin A-deficiency was studied using three different groups of rats: (i) control rats, (ii) rats fed a vitamin A-free diet until 50 d after birth and (iii) vitamin A-deficient rats re-fed a control diet for 30 d. No statistical difference in body weight and food intake was found between control and vitamin A-deficient rats. Liver GSH concentration was similar in both groups. However, in vitamin A-deficient rats, the mitochondrial GSH/GSSG ratio was significantly lower and the levels of malondialdehyde (MDA) and 8-oxo-7, 8-dihydro-2'-deoxyguanosine (oxo8dG) were higher when compared to control rats. These values were partially restored in re-fed rats. The mitochondrial membrane potential of vitamin A-deficient rats was significantly lower than in control rats and returned to normal levels in restored vitamin A rats. Two populations of mitochondria were found in vitamin A-deficient rats according to the composition of membrane lipids. One population showed a similar pattern to the control mitochondria and the second population had a higher membrane lipid content. This report emphasizes the protective role of vitamin A in liver mitochondria under physiological circumstances.

Mitochondrial oxidative stress plays a key role in aging and apoptosis.

Harman first suggested in 1972 that mitochondria might be the biological clock in aging, noting that the rate of oxygen consumption should determine the rate of accumulation of mitochondrial damage produced by free radical reactions. Later in 1980 Miquel and coworkers proposed the mitochondrial theory of cell aging. Mitochondria from postmitotic cells use O2 at a high rate, hence releasing oxygen radicals that exceed the cellular antioxidant defences. The key role of mitochondria in cell aging has been outlined by the degeneration induced in cells microinjected with mitochondria isolated from fibroblasts of old rats, especially by the inverse relationship reported between the rate of mitochondrial production of hydroperoxide and the maximum life span of species. An important change in mitochondrial lipid composition is the age-related decrease found in cardiolipin content. The concurrent enhancement of lipid peroxidation and oxidative modification of proteins in mitochondria further increases mutations and oxidative damage to mitochondrial DNA (mtDNA) in the aging process. The respiratory enzymes containing the defective mtDNA-encoded protein subunits may increase the production of reactive oxygen species, which in turn would aggravate the oxidative damage to mitochondria. Moreover, superoxide radicals produced during mitochondrial respiration react with nitric oxide inside mitochondria to yield damaging peroxynitrite. Treatment with certain antioxidants, such as sulphur-containing antioxidants, vitamins C and E, or the Ginkgo biloba extract EGb 761, protects against the age-associated oxidative damage to mtDNA and the oxidation of mitochondrial glutathione. Moreover, the EGb 761 extract also prevents changes in mitochondrial morphology and function associated with aging of the brain and liver.

Pentoxifylline ameliorates cerulein-induced pancreatitis in rats: role of glutathione and nitric oxide.

Reactive oxygen radicals, nitric oxide, and cytokines have been implicated in the initiation of pancreatic tissue damage and impairment of the pancreatic microcirculation in acute pancreatitis. Pentoxifylline is a methylxanthine derivative with rheologic and marked anti-inflammatory properties and inhibits the production of proinflammatory cytokines. We have examined whether pentoxifylline ameliorates interstitial edema, inflammatory infiltrate, and glutathione depletion associated with cerulein-induced pancreatitis. Cotreatment of animals with pentoxifylline significantly reduced cerulein-induced pancreatic inflammation and edema and attenuated the depletion of pancreatic glutathione and the increase in serum lipase activity, nitrate, and tumor necrosis factor-alpha levels. Pentoxifylline also prevented both mitochondrial swelling and damage to mitochondrial cristae caused by cerulein. Our findings provide an experimental basis for using pentoxifylline to attenuate inflammatory responses within the pancreas in acute pancreatitis and as an adjuvant in the treatment of acute pancreatitis.

Mitochondria, oxidative stress and aging.

In the eighties, Miquel and Fleming suggested that mitochondria play a key role in cellular aging. Mitochondria, and specially mitochondrial DNA (mtDNA), are major targets of free radical attack. At present, it is well established that mitochondrial deficits accumulate upon aging due to oxidative damage. Thus, oxidative lesions to mtDNA accumulate with age in human and rodent tissues. Furthermore, levels of oxidative damage to mtDNA are several times higher than those of nuclear DNA. Mitochondrial size increases whereas mitochondrial membrane potential decreases with age in brain and liver. Recently, we have shown that treatment with certain antioxidants, such as sulphur-containing antioxidants, vitamins C and E or the Ginkgo biloba extract EGb 761, protects against the age-associated oxidative damage to mtDNA and oxidation of mitochondrial glutathione. Moreover, the extract EGb 761 also prevents changes in mitochondrial morphology and function associated with aging of the brain and liver. Thus, mitochondrial aging may be prevented by antioxidants. Furthermore, late onset administration of certain antioxidants is also able to prevent the impairment in physiological performance, particularly motor co-ordination, that occurs upon aging.