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.

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.

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.

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.

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.

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.

Cytokine modulation is necessary for efficacious treatment of experimental neuropathic pain.

Neuropathic pain originates from a damage or disease affecting the somatosensory system. Its treatment is unsatisfactory as it appears refractory to most analgesics. Animal models of neuropathic pain are now available that help to clarify the underlying mechanisms. Recently it has been recognized that inflammatory and immune mechanisms in the peripheral and in the central nervous system play a role in the onset and the maintenance of pain. In response to nervous tissue damage, activation of resident or recruited immune cells leads to the production of inflammatory mediators, as cytokines. In models of neuropathic pain, such as nerve injury and diabetes induced neuropathy, the time course of the expression of the proinflammatory cytokines TNF-α,IL-1ß and IL-6 and of the antiinflammatory cytokine IL-10 has been well characterized both in the peripheral (sciatic nerve, dorsal root ganglia) and the central (spinal cord) nervous system. These cytokines appear activated/modulated in the nervous tissue in parallel with the occurrence of painful behaviour, i.e. allodynia and hyperalgesia. Novel therapeutic approaches efficacious to reduce painful symptoms, for example treatments with the non specific purinergic antagonist PPADS, the phytoestrogen genistein and a cell stem therapy with murine adult neural stem cells also re-established a balance between pro and antinflammatory mediators in the peripheral and central nervous system. These data suggest a pivotal role of immune system and inflammation in neuropathic pain. The modulation of inflammatory molecules appears to be a common trait accomplished throughout different mechanisms by different drugs that might converge in neuropathic pain modulation.

Acute and late changes in intraarticular cytokine levels following anterior cruciate ligament injury.

Surgical reconstruction of the anterior cruciate ligament (ACL) does not necessarily decrease the risk of developing osteoarthritis (OA). The inflammatory response and relative changes in pro- and anti-inflammatory cytokines could participate in triggering the development of OA. To test this hypothesis we measured the concentrations of IL-1ß, IL-1ra, IL-6, IL-8, IL-10, and TNF-α at different times after ACL rupture. The sample population consisted of 48 patients with ACL tear which were assigned to different groups according to the time elapsed from the injury: 22 acute (A), 7 early sub-acute (ESA), 11 late sub-acute (LSA), and 8 chronic (C). In group A, there were high levels of IL-1ß, IL-6, and IL-8, whereas levels of IL-1ra and TNF-α were significantly lower than usually reported. IL-1ß and IL-8 concentrations returned with time to normal levels in the ESA group. Interestingly, IL-1ra levels remained always significantly lower than normally reported levels, and TNF-α levels did not increase after trauma. Our data show increased level of pro-inflammatory cytokines (IL-6 and IL-8) in the acute phase of inflammation which could be responsible for triggering cartilage catabolism and suggest that prompt neutralization of IL-6 and IL-8 accumulations in synovial fluid could help prevent development of OA in ACL-injured knees.