Damage-associated molecular patterns and immune activation in bipolar disorder.

OBJECTIVE: Immune activation in bipolar disorder (BD) has been frequently reported. Damage-associated molecular patterns (DAMPs) are key players in the immune activation reaction. The aim of this study was to assess DAMP levels in drug-free patients with BD during acute episodes. METHOD: Serum levels of a predetermined set of DAMPs were assessed in drug-free patients with BD (n = 20) during an acute mood episode. We also included two control groups: healthy subjects, used as a negative control (n = 20); and patients with sepsis, used as a positive control for severe immune activation (n = 20). RESULTS: Multivariate analysis using generalized linear mixed model indicated that all DAMPs differed as a function of group membership after controlling for age and addressing multiplicity (P < 0.0006 for all comparisons). Follow-up analyses showed higher levels in BD subjects of circulating cell-free (ccf) nuclear (n)DNA (P = 0.02), HSP70 (P = 0.03) and HSP90α (P = 0.02) as compared to healthy subjects. Also, patients with BD showed lower levels of ccf nDNA (P = 0.04), HSP60 (P = 0.03), HSP70 (P = 0.01), and HSP90α (P = 0.002) as compared to patients with sepsis and higher levels of ccf mitochondrial DNA (P < 0.0001). CONCLUSION: The present findings may be linked to the inflammatory activity previously described among patients with BD and may help in the development of more targeted and personalized treatments for patients under acute episodes of BD.

Circulating concentrations, cerebral output of the CINC-1 and blood­brain barrier disruption in Wistar rats after pneumococcal meningitis induction.

Pneumococcal meningitis is a severe infectious illness of the central nervous system (CNS), with high rates of lethality and morbidity, being that the microorganism and the host's inflammatory response are responsible for cerebral complications. Moreover, the blood­brain barrier (BBB) itself secretes cytokines and, because of the bipolar nature of the BBB, these substances can be secreted into either the CNS compartment or in the blood, so patients with acute bacterial meningitis frequently develop sepsis. Therefore, the aim of this study was to evaluate the cytokine/chemokine levels in different vessels and the BBB integrity after pneumococcal meningitis induction. Wistar rats were infected with Streptococcus pneumoniae, and the BBB integrity was investigated using Evan's blue dye. Also, blood from the carotid artery and jugular vein was collected in order to perform tumour necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), interleukin-6 (IL-60 and cytokine-induced neutrophil chemoattractant-1 (CINC-1) analyses by enzyme-linked immunosorbent assay (ELISA). CINC-1 levels were increased at 6 h in the arterial plasma and at 3 and 6 h in the jugular plasma. We observed BBB breakdown between 12 and 24 h in the hippocampus and at 12 and 18 h in the cortex after pneumococcal meningitis induction. The increase of CINC-1 occurred prior to the BBB breakdown. CINC-1 is a neutrophil chemoattractant and it may be related to early events in the pneumococcal meningitis pathophysiology.

Antioxidant therapy attenuates oxidative stress in the blood of subjects exposed to occupational airborne contamination from coal mining extraction and incineration of hospital residues.

Coal mining and incineration of solid residues of health services (SRHS) generate several contaminants that are delivered into the environment, such as heavy metals and dioxins. These xenobiotics can lead to oxidative stress overgeneration in organisms and cause different kinds of pathologies, including cancer. In the present study the concentrations of heavy metals such as lead, copper, iron, manganese and zinc in the urine, as well as several enzymatic and non-enzymatic biomarkers of oxidative stress in the blood (contents of lipoperoxidation = TBARS, protein carbonyls = PC, protein thiols = PT, α-tocopherol = AT, reduced glutathione = GSH, and the activities of glutathione S-transferase = GST, glutathione reductase = GR, glutathione peroxidase = GPx, catalase = CAT and superoxide dismutase = SOD), in the blood of six different groups (n = 20 each) of subjects exposed to airborne contamination related to coal mining as well as incineration of solid residues of health services (SRHS) after vitamin E (800 mg/day) and vitamin C (500 mg/day) supplementation during 6 months, which were compared to the situation before the antioxidant intervention (Ávila et al., Ecotoxicology 18:1150-1157, 2009; Possamai et al., Ecotoxicology 18:1158-1164, 2009). Except for the decreased manganese contents, heavy metal concentrations were elevated in all groups exposed to both sources of airborne contamination when compared to controls. TBARS and PC concentrations, which were elevated before the antioxidant intervention decreased after the antioxidant supplementation. Similarly, the contents of PC, AT and GSH, which were decreased before the antioxidant intervention, reached values near those found in controls, GPx activity was reestablished in underground miners, and SOD, CAT and GST activities were reestablished in all groups. The results showed that the oxidative stress condition detected previously to the antioxidant supplementation in both directly and indirectly subjects exposed to the airborne contamination from coal dusts and SRHS incineration, was attenuated after the antioxidant intervention.

Serum-induced macrophage activation is related to the severity of septic shock.

BACKGROUND: It seems that a balance between anti and pro-inflammatory responses must be kept to eliminate the pathogen without inducing inflammatory damage in the host. Thus we determined the relation between macrophage activation and the severity and clinical outcome in septic patients. MATERIAL AND METHODS: This was a prospective study at a tertiary general intensive care unit. Thirty-three patients admitted with sepsis, severe sepsis or septic shock were included. As a control group, healthy volunteers were included matched to septic patients by age and sex. Peritoneal rat macrophages were cultured with 2% serum from healthy volunteers or from septic patients for determination of phagocytic potential or the capacity to produce cytokines. RESULTS: TNF and IL1 secretion by macrophages activated with serum from sepsis and severe sepsis patients was higher than with serum from healthy controls. In addition, proinflammatory cytokines released in vitro from macrophages, but not determined directly in the serum from patients, were lower in non-survivor septic patients when compared to survivors. In contrast, IL-10 secretion by macrophages activated with serum from septic patients was higher in nonsurvivors. In the septic shock group we observed a diminution in the phagocytic index compared to sepsis and severe sepsis groups, and the phagocytic index was higher in sepsis survivors. CONCLUSIONS: Markers of antiinflammation are predominant in more severe types of sepsis suggesting that antiinflammation is related to mortality.

Neuropeptide S produces hyperlocomotion and prevents oxidative stress damage in the mouse brain: a comparative study with amphetamine and diazepam.

Neuropeptide S (NPS) is a recently discovered peptide which induces hyperlocomotion, anxiolysis and wakefulness. This study aimed to compare behavioral and biochemical effects of NPS with amphetamine (AMPH), and diazepam (DZP). To this aim, the effects of NPS (0.01, 0.1 and 1 nmol, ICV), AMPH (2 mg/kg, IP) and DZP (1 mg/kg, IP) on locomotion and oxidative stress parameters were assessed in mouse brain structures. The administration of NPS and AMPH, but not DZP, increased locomotion compared to control. Biochemical analyses revealed that AMPH increased carbonylated proteins in striatum, but did not alter lipid peroxidation. DZP increased lipid peroxidation in the cortex and cerebellum, and increased protein carbonyl formation in the striatum. In contrast, NPS reduced carbonylated protein in the cerebellum and striatum, and also lipid peroxidation in the cortex. Additionally, the treatment with AMPH increased superoxide dismutase (SOD) activity in the striatum, while it did not affect catalase (CAT) activity. DZP did not alter SOD and CAT activity. NPS inhibited the increase of SOD activity in the cortex and cerebellum, but little influenced CAT activity. Altogether, this is the first evidence of a putative role of NPS in oxidative stress and brain injury.

Effects of an antagonist of the bombesin/gastrin-releasing peptide receptor on complete Freund's adjuvant-induced arthritis in rats.

OBJECTIVE: To determine the effects of RC-3095 in clinical and histopathologic parameters and inflammatory mediators on complete Freund's adjuvant-induced arthritis (CFA). METHODS: The arthritis was induced by injection of CFA into the left hind footpad. The animals were divided into control, vehicle injected control, placebo group (saline subcutaneously 50ml/kg, once daily for 8 days after modeling), treatment group (0.3mg/kg of RC-3095 subcutaneously, once daily for 8 days after induction). Clinical evaluation was accomplished daily, through scoring of the paw edema. The animals were sacrificed 15 days after induction for collection of hind foot joints for histology. We used a histological scoring system which was previously described, and interferon (INF)-gamma, interleukin (IL)-1beta, tumor necrosis factor (TNF), interleukin (IL)-6 and interleukin (IL)-10 were measured by ELISA. RESULTS: There was a significant inhibition of joint histological findings in the RC-3095 treated group, including synovial inflammatory infiltration and hyperplasia, cartilage and bone erosion. IFN-gamma, IL-1beta, TNF, IL-6 and IL-10 serum levels were significantly lower in the treated group. Paw swelling and subcutaneous inflammation, evaluated clinically, were not different between CFA-induced groups. CONCLUSIONS: RC-3095 was able to improve experimental arthritis, attenuate joint damage and decrease serum levels of IFN-gamma, IL-1beta, TNF, IL-6 and IL-10. These data indicate that interference with GRP pathway is a potential new strategy for the treatment of RA that needs further investigational studies.

Oxidative stress in chronic obstructive pulmonary disease patients submitted to a rehabilitation program.

Pulmonary rehabilitation (PR) improves physical capacity and health quality in patients with chronic obstructive pulmonary disease (COPD). However, the effect of exercise on oxidative stress markers in COPD patients is only partially known. This study was designed to evaluate the oxidative stress response to long-term exercise in patients with COPD enrolled in a PR program. Fifteen COPD patients (FEV1 < 60%), age between 50 and 60 years, ex-smokers, were separated in two groups: exercise-trained (n=8) and sedentary group (n=7). Exercise consisted of an 8-week conditioning program using a cycle ergometer (three times a week, 1h session). An endurance test (60% of maximal load in an incremental cycle test) was performed before and after PR. Blood samples were obtained at baseline and immediately after each endurance test. We measured the index of lipid peroxidation, thiobarbituric acid reactive species (TBARS), total radical-trapping antioxidant parameter (TRAP) and xanthine oxidase (XO) activity. TRAP was significantly different between the exercise-trained group and sedentary group of COPD patients. Baseline TBARS values were increased after the exercise training program but decreased after the endurance test. XO decrease after effort in the trained and untrained groups. The results suggest that patients with COPD are characterized by increased systemic and pulmonary oxidative stress markers both at rest as well as induced by cardiopulmonary exercise test and that PR program was associated with decreased systemic exercise-induced oxidative damage.

Protective effects of Passiflora alata extract pretreatment on carbon tetrachloride induced oxidative damage in rats.

The leaf extract of Passiflora alata Dryander (P. alata) has been demonstrated to possess antioxidant activity in vitro. The aim of this study was to investigate the effects of P. alata leaf extract pretreatment on carbon tetrachloride-treated rats. Male Wistar rats were randomly allocated into four groups: group 1 (control - vehicle), group 2 and 3 (P. alata extract - 1 and 5mg/kg, respectively) and group 4 (trolox - 0.18mg/kg). Rats received daily pretreatment by oral gavage for 30 days followed by a single dose of CCl(4) (3ml/kg i.p. in vegetable oil) on the 30th day and were killed after 6h. The pretreatment with the P. alata extract provided significant protection to liver, evidenced by lower degree of necrosis, decreased lipid peroxidation (TBARS) and higher catalase and superoxide dismutase activities. Additionally, pretreated-rats with P. alata (5mg/kg) showed significantly decreased cardiac TBARS levels. Our results indicate that a low oral dose of P. alata leaf extract has both hepato and cardioprotective effects on rats treated with CCl(4).

Behavioral deficits in sepsis-surviving rats induced by cecal ligation and perforation.

Sepsis and its complications are the leading causes of mortality in intensive care units, accounting for 10-50% of deaths. Intensive care unit survivors present long-term cognitive impairment, including alterations in memory, attention, concentration, and/or global loss of cognitive function. In the present study, we investigated behavioral alterations in sepsis-surviving rats. One hundred and ten male Wistar rats (3-4 months, 250-300 g) were submitted to cecal ligation and puncture (CLP), and 44 were submitted to sham operation. Forty-four rats (40%) survived after CLP, and all sham-operated animals survived and were used as control. Twenty animals of each group were used in the object recognition task (10 in short-term memory and 10 in long-term memory), 12 in the plus-maze test and 12 in the forced swimming test. Ten days after surgery, the animals were submitted individually to an object recognition task, plus-maze and forced swimming tests. A significant impairment of short- and long-term recognition memory was observed in the sepsis group (recognition index 0.75 vs 0.55 and 0.74 vs 0.51 for short- and long-term memory, respectively (P < 0.05). In the elevated plus-maze test no difference was observed between groups in any of the parameters assessed. In addition, sepsis survivors presented an increase in immobility time in the forced swimming test (180 vs 233 s, P < 0.05), suggesting the presence of depressive-like symptoms in these animals after recovery from sepsis. The present results demonstrated that rats surviving exposure to CLP, a classical sepsis model, presented recognition memory impairment and depressive-like symptoms but not anxiety-like behavior.

Oxidative stress after acute and sub-chronic malathion intoxication in Wistar rats.

Malathion is an insecticide of the group of organophosphate pesticides (OPs), which shows strong insecticidal effects. However, it possesses mutagenic and carcinogenic properties and shows organ-specific toxicity in relation to the heart, kidney and other vertebrate organs. The exact mechanism of the genotoxic effects of malathion is not yet known. Free radical damage is an important direct or indirect factor in several pathological and toxicological processes, including malathion poisoning. The aim of the present study was the evaluation of oxidative damage in different tissues of Wistar rats, administered intra peritoneally at doses of 25, 50, 100 and 150mgmalathion/kg, after acute and sub-chronic malathion exposure. Oxidative stress evaluation was based on lipid peroxidation by levels of thiobarbituric acid reactive substances (TBARS), protein oxidation by levels of carbonyl groups, and also on the activities of superoxide dismutase and catalase, two antioxidant enzymes that detoxity superoxide radical (O(2)(-)) and hydrogen peroxide, respectively. The results showed that the most sensitive targets of oxidative damage were kidney, lung and diaphragm after acute treatment, and liver, quadriceps and serum after sub-chronic treatment. Also, in general, increased lipid peroxidation measured as TBARS levels seems to be a better biomarker of oxidative stress compared to the contents of protein carbonyls after acute and sub-chronic malathion treatments. The present findings reinforce the concept that oxidative stress and particularly lipoperoxidation, are involved in OPs toxicity.

Molecular mechanisms mediating gastrin-releasing peptide receptor modulation of memory consolidation in the hippocampus.

Although the gastrin-releasing peptide-preferring bombesin receptor (GRPR) has been implicated in memory formation, the underlying molecular events are poorly understood. In the present study, we examined interactions between the GRPR and cellular signaling pathways in influencing memory consolidation in the hippocampus. Male Wistar rats received bilateral infusions of bombesin (BB) into the dorsal hippocampus immediately after inhibitory avoidance (IA) training. Intermediate doses of BB enhanced, whereas a higher dose impaired, 24-h IA memory retention. The BB-induced memory enhancement was prevented by pretraining infusions of a GRPR antagonist or inhibitors of protein kinase C (PKC), mitogen-activated protein kinase (MAPK) kinase and protein kinase A (PKA), but not by a neuromedin B receptor (NMBR) antagonist. We next further investigated the interactions between the GRPR and the PKA pathway. BB-induced enhancement of consolidation was potentiated by coinfusion of activators of the dopamine D1/D5 receptor (D1R)/cAMP/PKA pathway and prevented by a PKA inhibitor. We conclude that memory modulation by hippocampal GRPRs is mediated by the PKC, MAPK, and PKA pathways. Furthermore, pretraining infusion of BB prevented beta-amyloid peptide (25-35)-induced memory impairment, supporting the view that the GRPR is a target for the development of cognitive enhancers for dementia.

Imbalance of antioxidant defense in mice lacking cellular prion protein.

Prion diseases are fatal neurodegenerative disorders resulting from conformational changes in the prion protein from its normal cellular isoform, PrPC, to the infectious scrapie isoform, PrP(Sc). In spite of many studies, the physiological function of PrPC remains unknown. Recent work shows that PrPC binds Cu2+, internalizing it into the cytoplasm. Since many antioxidant enzymes depend on Cu2+ (e.g., Cu/ZnSOD), their function could be affected in prion diseases. Here we investigate a possible relationship between PrP(C) and the cellular antioxidant systems in different structures isolated from PrPC knockout and wild-type mice by determining oxidative damage in protein and lipids and activity of antioxidant enzymes (CAT, SOD) and stress-adaptive enzymes (ODC). Our results show that, in the absence of PrPC, there is an increased oxidation of lipid and protein in all structures investigated. Decreased SOD activity and changes in CAT/ODC activities were also observed. Taking into account these results, we suggest that the physiological function of PrP(C) is related to cellular antioxidant defenses. Therefore, during development of prion diseases, the whole organism becomes more sensitive to ROS injury, leading to a progressive oxidative disruption of tissues and vital organs, especially the central nervous system.

Neonatal iron exposure induces oxidative stress in adult Wistar rat.

Oxidative stress and excess of iron in the brain has been implicated in a variety of acute and chronic neurological conditions. The neonatal period is critical for the establishment of normal iron content in the adult brain. In the present study, the long-term oxidative effects of iron exposure during this period were assessed by treating Wistar rats orally with 0, 7.5 or 15 mg Fe(+2)/kg of body weight on postnatal days 10-12. Thiobarbituric acid reactive species, protein carbonyl, superoxide dismutase activity were measured at the age of 3 months. It was found that there was an increase in thiobarbituric acid reactive species and protein carbonyl in the substantia nigra of iron treated rats. In contrast, oxidative stress in the striatum was decreased. Superoxide dismutase activity was decreased in the substantia nigra iron treated rats. There were no differences in cerebellum measures among the groups. Our results demonstrated that iron supplementation in a critical neonatal period induced oxidative stress and modulated SOD activity in the adult life in selective brain regions.

Lipid peroxidation in hippocampus early and late after status epilepticus induced by pilocarpine or kainic acid in Wistar rats.

Oxidative stress has been implicated in a variety of acute and chronic neurologic conditions, including epilepsy. Both the kainic acid and pilocarpine are useful models of temporal lobe epilepsy in rodents. As an index of lipid peroxidation the level thiobarbituric acid reactive substances (TBARS) was measured after the status epileticus induced by pilocarpine or kainic acid. In hippocampus there was a slight enhancement in the TBARS levels measured 12-14 h after the end of status epileticus induced by pilocarpine and kainic acid. The TBARS levels in pilocarpine treated animals was significantly decreased late after status epileticus and in kainic acid model the TBARS returned to basal levels. These results indicating a putative role of reactive oxygen species in kainic acid and pilocarpine induced epilepsy.

Retinol supplementation induces oxidative stress and modulates antioxidant enzyme activities in rat sertoli cells.

Recent intervention studies revealed that supplementation with retinoids resulted in a higher incidence of lung cancer. Recently the causal mechanism has begun to be clarified. We report here that retinol caused cellular oxidative stress and modulated superoxide dismutase, catalase and glutathione peroxidase activities. Retinol (7 microM) significantly increased TBARS, conjugated dienes, and hydroperoxide-initiated chemiluminescence in cultured Sertoli cells. In response to retinol treatment superoxide dismutase, catalase and glutathione peroxidase activities increased. TBARS content and catalase activities were decreased by a free radical scavenger. These findings suggest that retinol may induce oxidative stress and modulate antioxidant enzyme activities in Sertoli cells.

Mitogenic signaling mediated by oxidants in retinol treated Sertoli cells.

Recent intervention studies revealed that supplementation with retinoids resulted in a higher incidence of lung cancer. Recently the causal mechanism has begun to be clarified. We report here that retinol-induced oxidative stress is accompanied by cellular proliferation. Retinol (7 microM) significantly induced thiobarbituric acid reactive species (TBARS) formation, which was inhibited by trolox, superoxide dismutase, N-acetylcysteine and ethanol. This was accompanied by an increase in DNA synthesis and focus formation in cultured rat Sertoli cells. Antioxidants and ethanol inhibited retinol-induced DNA synthesis. Our findings suggest that retinol-induced oxidative stress was associated with cellular proliferation complementing our understanding of the significance of retinol supplementation in neoplastic transformation.

Retinol supplementation induces DNA damage and modulates iron turnover in rat Sertoli cells.

Recent intervention studies revealed that supplementation with retinoids resulted in a higher incidence of lung cancer. Recently the causal mechanism has begun to be clarified. We report here that retinol caused cellular DNA damage probably involving cellular iron accumulation. Retinol (7 microM) significantly induced DNA single strands breaks, DNA fragmentation and production of 8-oxo-7, 8-dihydro-2'-deoxyguanosine in cultured Sertoli cells. In contrast, lower doses seemed not to induce single-strands break in this experimental model. The breaks in DNA were inhibited by an iron scavenger; and 7 microM retinol treatment modulated iron turnover leading to iron accumulation, suggesting that iron ions were required for the retinol cellular effects. These findings suggest that retinol-induced DNA damage was associated with the modulation of iron turnover, and these characteristics could be responsible for the increased incidence of lung cancer associated with retinoids supplementation.

Antioxidant, a pro-oxidant and cytotoxic effects of Achyrocline satureioides extracts.

In this study we compared the antioxidant properties of five different extracts of different composition obtained from Achyrocline satureioides' inflorescences (Compositae), a widely used Brazilian folk medicinal herb. All of the extracts presented significant antioxidant potential identified by TRAP assay, which increased in the presence of human plasma. Characterization of the content of flavonoids in each extract showed that the FDP80 (ethanol 80%) and FFr (enriched flavonoid fraction) extracts contained a higher content of flavonoids. Cytotoxicity of the extracts as determined in Sertoli cell culture showed that FDP80 and FFr were highly toxic at most concentrations tested. The extracts induced a significant increase in lipid peroxidation levels in Sertoli cells. These results suggest that medicinal herb extracts that contain higher flavonoid concentrations and shows higher antioxidant protection in vitro might not always produce the greatest benefit.

Retinol-induced changes in the phosphorylation levels of histones and high mobility group proteins from Sertoli cells.

Chromatin proteins play a role in the organization and functions of DNA. Covalent modifications of nuclear proteins modulate their interactions with DNA sequences and are probably one of the multiple factors involved in the process of switch on/off transcriptionally active regions of DNA. Histones and high mobility group proteins (HMG) are subject to many covalent modifications that may modulate their capacity to bind to DNA. We investigated the changes induced in the phosphorylation pattern of cultured Wistar rat Sertoli cell histones and high mobility group protein subfamilies exposed to 7 microM retinol for up to 48 h. In each experiment, 6 h before the end of the retinol treatment each culture flask received 370 KBq/ml [32P]-phosphate. The histone and HMGs were isolated as previously described [Moreira et al. Medical Science Research (1994) 22: 783-784]. The total protein obtained by either method was quantified and electrophoresed as described by Spiker [Analytical Biochemistry (1980) 108: 263-265]. The gels were stained with Coomassie brilliant blue R-250 and the stained bands were cut and dissolved in 0.5 ml 30% H2O2 at 60oC for 12 h. The vials were chilled and 5.0 ml scintillation liquid was added. The radioactivity in each vial was determined with a liquid scintillation counter. Retinol treatment significantly changed the pattern of each subfamily of histone and high mobility group proteins.