Effects of Electrical Stimulation on Delayed Onset Muscle Soreness (DOMS): Evidences from Laboratory and In-Field Studies.

Intense, long exercise can increase oxidative stress, leading to higher levels of inflammatory mediators and muscle damage. At the same time, fatigue has been suggested as one of the factors giving rise to delayed-onset muscle soreness (DOMS). The aim of this study was to investigate the efficacy of a specific electrical stimulation (ES) treatment (without elicited muscular contraction) on two different scenarios: in the laboratory on eleven healthy volunteers (56.45 ± 4.87 years) after upper limbs eccentric exercise (Study 1) and in the field on fourteen ultra-endurance athletes (age 47.4 ± 10.2 year) after an ultra-running race (134 km, altitude difference of 10,970 m+) by lower exercising limbs (Study 2). Subjects were randomly assigned to two experimental tasks in cross-over: Active or Sham ES treatments. The ES efficacy was assessed by monitoring the oxy-inflammation status: Reactive Oxygen Species production, total antioxidant capacity, IL-6 cytokine levels, and lactate with micro-invasive measurements (capillary blood, urine) and scales for fatigue and recovery assessments. No significant differences (p > 0.05) were found in the time course of recovery and/or pre-post-race between Sham and Active groups in both study conditions. A subjective positive role of sham stimulation (VAS scores for muscle pain assessment) was reported. In conclusion, the effectiveness of ES in treating DOMS and its effects on muscle recovery remain still unclear.

Evaluation of Murine Macrophage Cytokine Production After In Vivo Morphine Treatment.

The discovery of opioid receptor expression on immune cells has originated a large research activity on the possible modulation by opioid drugs of immune system responses. In this chapter we describe an easy methodology useful to obtain information about the potential immunomodulatory activity of opioid drugs. An in vivo treatment schedule is used, and macrophages are studied for their ability to release different cytokines.

Transcription Factors Regulation in Human Peripheral White Blood Cells during Hypobaric Hypoxia Exposure: an in-vivo experimental study.

High altitude is a natural laboratory, within which the clinical study of human physiological response to hypobaric hypoxia (HH) is possible. Failure in the response results in progressive hypoxemia, inflammation and increased tissue oxidative stress (OxS). Thus, investigating temporal changes in key transcription factors (TFs) HIF-1α, HIF-2α, NF-κB and NRF2 mRNA levels, relative to OxS and inflammatory markers, may reveal molecular targets which contrast deleterious effects of hypoxia. Biological samples and clinical data from 15 healthy participants were collected at baseline and after rapid, passive ascent to 3830 m (24 h and 72 h). Gene expression was assessed by qPCR and ROS generation was determined by EPR spectroscopy. Oxidative damage and cytokine levels were estimated by immuno or enzymatic methods. Hypoxia transiently enhanced HIF-1α mRNA levels over time reaching a peak after 24 h. Whereas, HIF-2α and NRF2 mRNA levels increased over time. In contrast, the NF-κB mRNA levels remained unchanged. Plasma levels of IL-1ß and IL-6 also remained within normal ranges. ROS production rate and markers of OxS damage were significantly increased over time. The analysis of TF-gene expression suggests that HIF-1α is a lead TF during sub-acute HH exposure. The prolongation of the HH exposure led to a switch between HIF-1α and HIF-2α/NRF2, suggesting the activation of new pathways. These results provide new insights regarding the temporal regulation of TFs, inflammatory state, and ROS homeostasis involved in human hypoxic response, potentially also relevant to the mediation of diseases that induce a hypoxic state.

Pemetrexed-loaded nanoparticles targeted to malignant pleural mesothelioma cells: an in vitro study.

PURPOSE: Malignant pleural mesothelioma (MPM) is an aggressive tumor characterized by poor prognosis. Its incidence is steadily increasing due to widespread asbestos exposure. There is still no effective therapy for MPM. Pemetrexed (Pe) is one of the few chemotherapeutic agents approved for advanced-stage disease, although the objective response to the drug is limited. The use of gold nanoparticles (GNPs) as a drug delivery system promises several advantages, including specific targeting of malignant cells, with increased intracellular drug accumulation and reduced systemic toxicity, and, in the case of MPM, direct treatment administration into the pleural space. This study aims at exploring CD146 as a potential MPM cell-specific target for engineered Pe-loaded GNPs and to assess their effectiveness in inhibiting MPM cell line growth. METHODS: MPM cell lines and primary cultures obtained by pleural effusions from MPM patients were assayed for CD146 expression by flow cytometry. Internalization by MPM cell lines of fluorescent dye-marked GNPs decorated with a monoclonal anti CD146 coated GNPs (GNP-HC) was proven by confocal microscopy. The effects of anti CD146 coated GNPs loaded with Pe (GNP-HCPe) on MPM cell lines were evaluated by cell cycle (flow cytometry), viability (MTT test), clonogenic capacity (soft agar assay), ROS production (electric paramagnetic resonance), motility (wound healing assay), and apoptosis (flow cytometry). RESULTS: GNP-HC were selectively uptaken by MPM cells within 1 hour. MPM cell lines were blocked in the S cell cycle phase in the presence of GNP-HCPe. Both cell viability and motility were significantly affected by nanoparticle treatment compared to Pe. Apoptotic rate and ROS production were significantly higher in the presence of nanoparticles. Clonogenic capacity was completely inhibited following nanoparticle internalization. CONCLUSION: GNP-HCPe treatment displays in vitro antineoplastic action and is more effective than Pe alone in inhibiting MPM cell line malignant phenotype. The innovative use of specifically targeted GNPs opens the perspective of local intrapleural administration to avoid normal cell toxicity and enhance chemotherapy efficacy.

Effects of Combined Physical and Cognitive Virtual Reality-Based Training on Cognitive Impairment and Oxidative Stress in MCI Patients: A Pilot Study.

The growing elderly population and the increased incidence of mild cognitive impairment (MCI) and Alzheimer's disease (AD) call for the improvement of the quality and the efficacy of the healthcare and social support services. Exercise and cognitive stimulation have been demonstrated to mitigate cognitive impairment and oxidative stress (OxS) has been recognized as a factor that contributes to the advancement of neurodegenerative diseases. Taking these aspects into account, the impact of a novel virtual reality (VR)-based program combining aerobic exercise and cognitive training has been evaluated in the pilot study proposed here. Ten patients (aged 73.3 ± 5.7 years) with MCI (Mini-Mental State Examination, MMSE: 23.0 ± 3.4) were randomly assigned to either 6 weeks physical and cognitive training (EXP) or control (CTR) group. Evaluations of cognitive profile, by a neuropsychological tests battery, and OxS, by collection of blood and urine samples, were performed before and at the end of the experimental period. The assessment of the patients' opinions toward the intervention was investigated through questionnaires. EXP group showed a tendency towards improvements in the MMSE, in visual-constructive test and visuo-spatial tests of attention, while CTR worsened. EXP group showed a greater improvement than CTR in the executive test, memory functions and verbal fluency. No statistical significance was obtained when comparing within and between both the groups, probably due to small number of subjects examined, which amplifies the effect of the slight heterogeneity in scores recorded. Despite a greater worsening of Daily Living Activities tests, all participants reported a better performance in real life, thanks to the elicited self-perceived improvement. After training intervention OxS (i.e., reactive oxygen species (ROS) production, oxidative damage of lipids and DNA) decreased resulting in significantly (range p < 0.05-0.001) lower in EXP vs. CTR group. Although not conclusive, the recorded effects in the present study are promising and suggest that this proposal would be a useful tool in support of cognitive training reducing OxS too. However, further studies on larger scale samples of patients are needed.

R(+)-Thioctic Acid Effects on Oxidative Stress and Peripheral Neuropathy in Type II Diabetic Patients: Preliminary Results by Electron Paramagnetic Resonance and Electroneurography.

OBJECTIVES: Diabetic neuropathy is the most common complication of diabetes. The idea of alterations in energy metabolism in diabetes is emerging. The biogenic antioxidant R(+)-thioctic acid has been successfully used in the treatment of diabetic polyneuropathic (DPN) patients. METHODS: The effects of R(+)-thioctic acid (1 tablet, 1.6 g) administration were evaluated in 12 DPN patients at baseline and at 15, 30, 60, and 120 administration days throughout the assessment of oxidative stress (OxS); ROS production rate by electron paramagnetic resonance (EPR) technique; and oxidative damage biomarkers (thiobarbituric acid reactive substances (TBARS) and protein carbonyls (PC)), electroneurography (ENG) and visual analogue scale. RESULTS: Supplementation induced significant changes (p < 0.05) at 30 and 60 days. ROS production rate up to -16%; TBARS (-31%), PC (-38%), and TAC up to +48%. Motor nerve conduction velocity in SPE and ulnar nerves (+22% and +16%) and sensor conduction velocity in sural and median nerves (+22% and +5%). Patients reported a general wellness sensation improvement (+35%) at 30 days: lower limb pain sensation (-40%) and upper limbs (-23%). CONCLUSION: The results strongly indicate that an increased antioxidant capacity plays an important role in OxS, nerve conduction velocity, pain, and general wellness improvement. Nevertheless, the effects of the antioxidant compound were found positive up to 60 days. Then, a hormesis effect was observed. Novelty of the research would be a challenge for investigators to carefully address issues, including dose range factors, appropriate administration time, and targeting population to counteract possible "boomerang effects." The great number of monitored parameters would firmly stress these conclusions.

Oxidative stress as a biomarker for monitoring treated celiac disease.

INTRODUCTION: High levels of reactive oxygen species (ROS) and impaired antioxidant defense systems lead to oxidative stress (OxS) and tissue injury in different intestinal and extra intestinal conditions, including celiac disease (CD). The aim of the present study was to investigate the role and potential use of ROS and other biomarkers of OxS in the clinical management of CD. METHODS: We collected duodenal specimens and blood samples from naïve patients (N-CD), patients on a gluten free diet (GFD) including responders (CD-GFD) and non-responders (NRCD). We measured plasmatic ROS production (electron paramagnetic resonance, EPR), lipid peroxidation (thiobarbituric acid-reactive substances, TBARS), protein oxidation (protein carbonyl, PC), total antioxidant capacity (TAC), nitric oxides and glutathione (GSH) in erythrocytes. RESULTS: Fifty-four patients affected by CD were enrolled (17 N-CD, 18 CD-GFD and 19 NRCD; 44 F; age 44 ± 13 years). A significant increase of plasmatic OxS biomarkers (ROS, peroxidated lipids, oxidized proteins, and nitrate concentrations) and decrease of antioxidant species (TAC and GSH levels) were found in NRCD and N-CD compared to CD-GFD. Comparably, a significant direct relationship between the severity of duodenal atrophy, ROS production rates and TBARS was found; conversely, TAC and GSH presented an inverse correlation. DISCUSSION: OxS is involved in CD tissue damage and correlates with the degree of duodenal atrophy. These findings suggest the possible role of OxS biomarkers as indicators of CD activity during the clinical follow-up.

Spirometry and oxidative stress after rebreather diving in warm water.

INTRODUCTION: Hyperbaric oxygen (HBO2) therapy and use of enriched air can result in oxidative injury affecting the brain, lungs and eyes. HBO2 exposure during diving can lead to a decrease in respiratory parameters. However, the possible effects of acute exposure to oxygen-enriched diving on subsequent spirometric performance and oxidative state in humans have not been recently described recently. We aim to investigate possible effects of acute (i) hyperbaric and (ii) hyperbaric hyperoxic exposure using scuba or closed-circuit rebreather (CCR) on subsequent spirometry and to assess the role of oxidative state after hyperoxic diving. METHODS: Spirometry and urine samples were obtained from six well-trained divers (males, mean ± SD, age: 43.33 ± 9.16 years; weight: 79.00 ± 4.90 kg; height: 1.77 ± 0.07 meters) before (CTRL) and after a dive breathing air, and after a dive using CCR (PO2 1.4). In the crossover design (two dives separated by six hours) each subject performed a 20-minute session of light underwater exercise at a depth of 15 meters in warm water (31-32°C). We measured urinary 8-isoprostane and 8-OH-2-deoxyguanosine evaluating lipid and DNA oxidative damages. RESULTS: Different breathing conditions (air vs. CCR) did not significantly affect spirometry. A significant increase of 8-OH-dG (1.85 ± 0.66 vs. 4.35 ± 2.12; P ⟨ 0.05) and 8-isoprostane (1.35 ± 0.20 vs. 2.59 ± 0.61; P ⟨ 0.05) levels after CCR dive with respect to the CTRL was observed. Subjects did not have any ill effects during diving. CONCLUSIONS: Subjects using CCR showed elevated oxidative stress, but this did not correlate with a reduction in pulmonary function.

Bioengineered gold nanoparticles targeted to mesenchymal cells from patients with bronchiolitis obliterans syndrome does not rise the inflammatory response and can be safely inhaled by rodents.

The use of gold nanoparticles (GNPs) as drug delivery system represents a promising issue for diseases without effective pharmacological treatment due to insufficient local drug accumulation and excessive systemic toxicity. Bronchiolitis obliterans syndrome (BOS) represents about 70% of cases of chronic lung allograft dysfunction, the main challenge to long-term lung transplantation. It is believed that due to repeated insults to epithelial bronchiolar cells local inflammatory response creates a milieu that favors epithelial-mesenchymal transition and activation of local mesenchymal cells (MCs) leading to airway fibro-obliteration. In a previous work, we engineered GNPs loaded with the mammalian target of rapamycin inhibitor everolimus, specifically decorated with an antibody against CD44, a surface receptor expressed by primary MCs isolated from bronchoalveolar lavage of BOS patients. We proved in vitro that these GNPs (GNP-HCe) were able to specifically inhibit primary MCs without affecting the bronchial epithelial cell. In the present work, we investigated the effect of these bioengineered nanoconstructs on inflammatory cells, given that a stimulating effect on macrophages, neutrophils or lymphocytes is strongly unwanted in graft airways since it would foster fibrogenesis. In addition, we administered GNP-HCe by the inhalatory route to normal mice for a preliminary assessment of their pulmonary and peripheral (liver, spleen and kidney) uptake. By these experiments, an evaluation of tissue toxicity was also performed. The present study proves that our bioengineered nanotools do not rise an inflammatory response and, under the tested inhalatory conditions that were used, are non-toxic.

Oxidative stress response to acute hypobaric hypoxia and its association with indirect measurement of increased intracranial pressure: a field study.

High altitude is the most intriguing natural laboratory to study human physiological response to hypoxic conditions. In this study, we investigated changes in reactive oxygen species (ROS) and oxidative stress biomarkers during exposure to hypobaric hypoxia in 16 lowlanders. Moreover, we looked at the potential relationship between ROS related cellular damage and optic nerve sheath diameter (ONSD) as an indirect measurement of intracranial pressure. Baseline measurement of clinical signs and symptoms, biological samples and ultrasonography were assessed at 262 m and after passive ascent to 3830 m (9, 24 and 72 h). After 24 h the imbalance between ROS production (+141%) and scavenging (-41%) reflected an increase in oxidative stress related damage of 50-85%. ONSD concurrently increased, but regression analysis did not infer a causal relationship between oxidative stress biomarkers and changes in ONSD. These results provide new insight regarding ROS homeostasis and potential pathophysiological mechanisms of acute exposure to hypobaric hypoxia, plus other disease states associated with oxidative-stress damage as a result of tissue hypoxia.

Oxidative Stress Assessment in Response to Ultraendurance Exercise: Thiols Redox Status and ROS Production according to Duration of a Competitive Race.

Purpose. Response to an ultraendurance competitive race on thiols redox status, reactive oxygen species (ROS) production, and oxidative stress (OxS) was investigated according to duration. Methods. Twenty-four elite runners were examined: six completed 50 km and eighteen 100 km. Blood and urine samples were collected before and immediately after the race. Erythrocytes and plasma aminothiols by high-performance liquid chromatography, total antioxidant capacity (TAC), and OxS biomarkers (protein carbonyl (PC), thiobarbituric acid-reactive substances (TBARS), 8-isoprostane (8-iso-PGF2α), and 8-OH-2-deoxyguanosine (8-OH-dG)) by immunoenzymatic assays and ROS production by Electron Paramagnetic Resonance were assessed. Results. Significant increases (P between <0.05 and <0.0001) were recorded in plasma total and oxidized aminothiols concentration and TAC (P < 0.0001) only after 100 km: plasmatic (ROS production (+12 versus +29%), PC (+54 versus +115%), and TBARS (+28 versus +55%)) and urinary (8-OH-dG.creatinine(-1) (+71 versus +158%) and 8-iso-PGF2α.creatinine(-1) (+43 versus +135%)) concentrations for 50 and 100 km (duration 4 h 3' versus 8 h 42'), respectively. Conclusion. Very prolonged ultraendurance exercise causes an increase in ROS production and OxS depending on specific biomarker examined but always linearly and directly related to exercise duration. Redox status of erythrocytes was preserved. A relationship between running performance and both prerace ROS production and antioxidant-redox status was found in 100 km race.

A Novel Application for Cognitive Evaluation in Mountain Ultramarathons: Olfactory Assessment.

OBJECTIVE: Olfactory function, a cognitive impairment biomarker, was evaluated in mountain ultramarathon (MUM) runners during the Tor des Géants race (332.5 km with an overall altitude gain of 24,000 m; altitude range 330-3296 m above the sea). METHODS: An Odor Identification Test was administered before (T0; n = 53), at 148.7 kms (T1; n = 32) and after the race (T2; n = 28). The effect of dehydration and sleep deprivation on olfactory function was assessed. Olfactory function was also assessed in non-MUM athletes and sedentary controls (C) at rest. RESULTS: A majority of the athletes completed the olfactory test at all time intervals. Olfactory function decreased throughout the race (T0: 13.8 ± 1.9, T1: 13.7 ± 1.6, T2: 13.1 ± 1.8; T0 vs T2 P = .01). There was no relationship with race time or sleep deprivation on the sense of smell throughout the competition. However, there was a combined effect with decreased olfaction during the second half of the race, while a poor relationship was seen between olfaction and total body water at midterm (T1: rs = -0.427; P = .019), but not at baseline or after the race. MUM athletes had similar olfactory scores to C (13.8 ± 1.9 vs 13.7 ± 1.4) and non-MUM (13.8 ± 1.9 vs 13.9 ± 1.6) athletes. CONCLUSIONS: This pilot study showed the feasibility of olfactory evaluation as a minimally invasive cognitive impairment assessment. The test can be used in logistically difficult environments, adding scientific value to this promising method. Although olfaction decreased after prolonged physical activity, further studies are warranted to make the relationship between cognition and external factors (eg, sleep deprivation, dehydration) more clear.

Concentration of Cu, Zn, Cr, Ni, Cd, and Pb in soil, sugarcane leaf and juice: residual effect of sewage sludge and organic compost application.

Many researchers have evaluated the effects of successive applications of sewage sludge (SS) on soil plant-systems, but most have not taken into account the residual effect of organic matter remaining from prior applications. Furthermore, few studies have been carried out to compare the effects of the agricultural use of SS and sewage sludge compost (SSC). Therefore, we evaluated the residual effect of SS and SSC on the heavy metal concentrations in soil and in sugarcane (Saccharum spp.) leaves and juice. The field experiment was established after the second harvesting of unburned sugarcane, when the organic materials were applied. The SS and SSC rates were (t ha(-1), dry base): 0, 12.5, 25, and 50; and 0, 21, 42, and 84, respectively. All element concentrations in the soil were below the standards established by São Paulo State environmental legislation. SS promoted small increases in Zn concentrations in soil and Cu concentrations in leaves. However, all heavy metals concentrations in the leaves were lower than the limits established for toxic elements and were in accordance with the limits established for micronutrients. There were reductions in the concentrations of Ni and Cu in soil and the concentration of Pb in juice, with increasing rates of SSC. The heavy metal concentrations were very low in the juice. Under humid tropical conditions and with short-term use, SS and SSC containing low heavy metal concentrations did not have negative effects on plants and soil.

Effects of Mountain Ultra-Marathon Running on ROS Production and Oxidative Damage by Micro-Invasive Analytic Techniques.

PURPOSE: Aiming to gain a detailed insight into the physiological mechanisms involved under extreme conditions, a group of experienced ultra-marathon runners, performing the mountain Tor des Géants® ultra-marathon: 330 km trail-run in Valle d'Aosta, 24000 m of positive and negative elevation changes, was monitored. ROS production rate, antioxidant capacity, oxidative damage and inflammation markers were assessed, adopting micro-invasive analytic techniques. METHODS: Forty-six male athletes (45.04±8.75 yr, 72.6±8.4 kg, 1.76±0.05 m) were tested. Capillary blood and urine were collected before (Pre-), in the middle (Middle-) and immediately after (Post-) Race. Samples were analyzed for: Reactive Oxygen Species (ROS) production by Electron Paramagnetic Resonance; Antioxidant Capacity by Electrochemistry; oxidative damage (8-hydroxy-2-deoxy Guanosine: 8-OH-dG; 8-isoprostane: 8-isoPGF2α) and nitric oxide metabolites by enzymatic assays; inflammatory biomarkers (plasma and urine interleukin-6: IL-6-P and IL-6-U) by enzyme-linked immunosorbent assays (ELISA); Creatinine and Neopterin by HPLC, hematologic (lactate, glucose and hematocrit) and urine parameters by standard analyses. RESULTS: Twenty-five athletes finished the race, while twenty-one dropped out of it. A significant increase (Post-Race vs Pre) of the ROS production rate (2.20±0.27 vs 1.65±0.22 µmol.min-1), oxidative damage biomarkers (8-OH-dG: 6.32±2.38 vs 4.16±1.25 ng.mg-1 Creatinine and 8-isoPGF2α: 1404.0±518.30 vs 822.51±448.91 pg.mg-1Creatinine), inflammatory state (IL-6-P: 66.42±36.92 vs 1.29±0.54 pg.mL-1 and IL-6-U: 1.33±0.56 vs 0.71±0.17 pg.mL1) and lactate production (+190%), associated with a decrease of both antioxidant capacity (-7%) and renal function (i.e. Creatinine level +76%) was found. CONCLUSIONS: The used micro-invasive analytic methods allowed us to perform most of them before, during and immediately after the race directly in the field, by passing the need of storing and transporting samples for further analysis. Considered altogether the investigated variables showed up that exhaustive and prolonged exercise not only promotes the generation of ROS but also induces oxidative stress, transient renal impairment and inflammation.

Chronic and acute effects of endurance training on telomere length.

Telomere shortening is considered a cellular marker of health status and biological ageing. Exercise may influence the health and lifespan of an individual by affecting telomere length (TL). However, it is unclear whether different endurance exercise levels may have beneficial or detrimental effects on biological aging. The aims of the study were to assess both chronic and acute effects of endurance training on TL after an exceptional and extreme trail race. TL was assessed in 20 endurance athletes (17 males; age = 45.4 ± 9.2 years) and 42 age- and gender-matched sedentary controls (32 males; age = 45.9 ± 9.5 years) with quantitative real-time PCR at baseline conditions. Of the 20 runners enrolled in the 'Tor des Géants ®' ultra-distance trail race, 15 athletes (12 males; age = 47.2 ± 8.5 years) were re-evaluated at the intermediate point and 14 athletes (11 males; age = 47.1 ± 8.8 years) completed the competition and were analysed at the final point. Comparison between the two groups (endurance athletes vs. sedentary controls) revealed a significant difference in TL (1.28 ± 0.4 vs. 1.02 ± 0.3, P = 0.005). TL was better preserved in elder endurance runners compared with the same age control group (1.3 ± 0.27 vs. 0.91 ± 0.21, P = 0.003). TL was significantly reduced at the intermediate (0.88 ± 0.36 vs. 1.11 ± 0.34, P = 0.002) and final point compared with baseline measurements (0.86 ± 0.4 vs. 1.11 ± 0.34, P = 0.0006) for athletes engaged in the ultra-marathon race. Our data suggest that chronic endurance training may provide protective effects on TL attenuating biological aging. Conversely, acute exposure to an ultra-distance endurance trail race implies telomere shortening probably caused by oxidative DNA damage.

Exposure of Adolescent Mice to Delta-9-Tetrahydrocannabinol Induces Long-Lasting Modulation of Pro- and Anti-Inflammatory Cytokines in Hypothalamus and Hippocampus Similar to that Observed for Peripheral Macrophages.

Cannabis use is frequent among adolescents. Its main component, delta-9-tetrahydrocannabinol (THC), affects the immune system. We recently demonstrated that chronic exposure of adolescent mice to THC suppressed immunity immediately after treatment but that after a washout period THC induced a long-lasting opposite modulation towards a proinflammatory and T-helper-1 phenotype in adulthood. The main objective of this study was to investigate whether the same effect was also present in brain regions such as the hypothalamus and hippocampus. Thirty-three-day-old adolescent and 80-day-old adult male mice were used. Acute THC administration induced a similar reduction of macrophage proinflammatory cytokines and an IL-10 increase in adult and adolescent mice. THC did not affect brain cytokines in adult mice, but a proinflammatory cytokine decrease was evident in the adolescent brain. A similar effect was present in the hypothalamus and hippocampus after 10 days' THC administration. In contrast, when brain cytokines were measured 47 days after the final THC administration, we observed an inverted effect in adult mice treated as adolescents, i.e., IL-1ß and TNF-α increased and IL-10 decreased, indicating a shift toward neuroinflammation. These data suggest that THC exposure in adolescence has long-lasting effects on brain cytokines that parallel those present in the periphery. This modulation may affect vulnerability to immune and behavioural diseases in adulthood.

Evaluation of murine macrophage cytokine production after in vivo morphine treatment.

The discovery of opioid receptors expression on immune cells has originated a large research activity on the possible modulation by opioid drugs of immune system responses. In the present chapter we describe an easy methodology useful to obtain information about the potential immunomodulatory activity of opioid drugs. An in vivo treatment schedule is used, and macrophages are studied for their ability to release different cytokines.

Δ9-Tetrahydrocannabinol-induced anti-inflammatory responses in adolescent mice switch to proinflammatory in adulthood.

Marijuana abuse is prominent among adolescents. Although Δ(9)-THC, one of its main components, has been demonstrated to modulate immunity in adults, little is known about its impact during adolescence on the immune system and the long-lasting effects in adulthood. We demonstrate that 10 days of Δ(9)-THC treatment induced a similar alteration of macrophage and splenocyte cytokines in adolescent and adult mice. Immediately at the end of chronic Δ(9)-THC, a decrease of proinflammatory cytokines IL- 1ß and TNF-α and an increase of anti-inflammatory cytokine IL-10 production by macrophages were present as protein and mRNA in adolescent and adult mice. In splenocytes, Δ(9)-THC modulated Th1/Th2 cytokines skewing toward Th2: IFN-γ was reduced, and IL-4 and IL-10 increased. These effects were lost in adult animals, 47 days after the last administration. In contrast, in adult animals treated as adolescents, a perturbation of immune responses, although in an opposite direction, was present. In adults treated as adolescents, a proinflammatory macrophage phenotype was observed (IL-1ß and TNF-α were elevated; IL-10 decreased), and the production of Th cytokines was blunted. IgM titers were also reduced. Corticosterone concentrations indicate a long-lasting dysregulation of HPA in adolescent mice. We measured blood concentrations of Δ(9)-THC and its metabolites, showing that Δ(9)-THC plasma levels in our mice are in the order of those achieved in human heavy smokers. Our data demonstrate that Δ(9)-THC in adolescent mice triggers immune dysfunctions that last long after the end of abuse, switching the murine immune system to proinflammatory status in adulthood.

Changes of substance P in the crevicular fluid in relation to orthodontic movement preliminary investigation.

Substance P (SP) is a tachykinin released from both the central and the peripheral endings of primary afferent neurons and functions as a neurotransmitter. As a transmitter signaling pain, substance P is involved in nociception and is an extremely potent vasodilator. We found several studies about this neuropeptide especially in relation to parodontology and a few orthodontic reviews. This is because in the past the importance of this neuropeptide in dental element undergoing periodontal inflammation was observed. The aims of the present pilot study was to investigate whether the substance P was present in gingival crevicular fluid in dental elements undergoing orthodontic treatment with Invisalign technique compared to teeth belonging to the same series but not undergoing orthodontic movement. We analysed gengival crevicular fluid (GCF) collected from four young subjects, using a paper cone for a time of 60 seconds. The results showed that SP is present in the gengival sulcus in elements undergoing orthodontic forces during treatment with Invisalign technique and not in the control teeth. During the literature analysis, we have found a lot of papers describing involvement of SP in periodontitis and inflammatory diseases, but further studies are needed in order to demonstrate the role of this neuropeptide during teeth movement.

Effects of NSAIDs and paracetamol (acetaminophen) on protein kinase C epsilon translocation and on substance P synthesis and release in cultured sensory neurons.

Celecoxib, diclofenac, ibuprofen, and nimesulide are nonsteroidal anti-inflammatory drugs (NSAIDs) very commonly used for the treatment of moderate to mild pain, together with paracetamol (acetaminophen), a very widely used analgesic with a lesser anti-inflammatory effect. In the study reported here, we tested the efficacy of celecoxib, diclofenac, and ibuprofen on preprotachykinin mRNA synthesis, substance P (SP) release, prostaglandin E(2) (PGE(2)) release, and protein kinase C epsilon (PKCɛ) translocation in rat cultured sensory neurons from dorsal root ganglia (DRGs). The efficacy of these NSAIDs was compared with the efficacy of paracetamol and nimesulide in in vitro models of hyperalgesia (investigated previously). While nimesulide and paracetamol, as in previous experiments, decreased the percentage of cultured DRG neurons showing translocation of PKCɛ caused by 100 nM thrombin or 1 µM bradykinin in a dose-dependent manner, the other NSAIDs tested did not have a significant effect. The amount of SP released by peptidergic neurons and the expression level of preprotachykinin mRNA were assessed in basal conditions and after 70 minutes or 36 hours of stimulation with an inflammatory soup (IS) containing potassium chloride, thrombin, bradykinin, and endothelin-1. The release of SP at 70 minutes was inhibited only by nimesulide, while celecoxib and diclofenac were effective at 36 hours. The mRNA basal level of the SP precursor preprotachykinin expressed in DRG neurons was reduced only by nimesulide, while the increased levels expressed during treatment with the IS were significantly reduced by all drugs tested, with the exception of ibuprofen. All drugs were able to decrease basal and IS-stimulated PGE(2) release. Our study demonstrates novel mechanisms of action of commonly used NSAIDS.