Search

Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating, and degenerative disease that affects the central nervous system. A recent study showed that interaction between the immune system and the gut microbiota plays a crucial role in the development of MS. This review reports the clinical studies carried out in recent years that aimed to evaluate the composition of the microbiota in patients with relapsing–remitting MS (RR-MS). We also report what is available in the literature regarding the effectiveness of fecal microbiota transplantation and the role of the diet in restoring the intestinal bacterial population. Studies report that patients with RR-MS have a microbiota that, compared with healthy controls, has higher amounts of Pedobacteria, Flavobacterium, Pseudomonas, Mycoplana, Acinetobacter, Eggerthella, Dorea, Blautia, Streptococcusand Akkermansia. In contrast, MS patients have a microbiota with impoverished microbial populations of Prevotella, Bacteroides, Parabacteroides, Haemophilus, Sutterella, Adlercreutzia, Coprobacillus, Lactobacillus, Clostridium, Anaerostipesand Faecalibacterium. In conclusion, the restoration of the microbial population in patients with RR-MS appears to reduce inflammatory events and the reactivation of the immune system.

Mesenchymal stem cells (MSCs) are widely used in stem cell therapy for regenerative purposes. Oral-derived MSCs, such as gingival MSCs (GMSCs), deriving from the neural crest seem more suitable to differentiate toward the neuronal lineage. In addition, the preconditioning of MSCs may improve their beneficial effects. Since it is known that hypoxia may influence stem cell properties, we were interested in evaluating the effects of hypoxia preconditioning on the neuronal differentiation of GMSCs. With this aim, we evaluated the transcriptional profile of GMSCs exposed to basal and neuroinductive medium both in normoxia and in cells preconditioned for 48 h in hypoxia. We compared their transcriptional profile using Next Generation Sequencing. At first we observed that hypoxia did not alter cell morphology compared with the GMSCs cultured in a normoxic condition. In order to understand hypoxia preconditioning effects on neuronal differentiation, we screened genes with Log2 fold change ≥2 using the database Cortecon, that collects gene expression data set of in vitro corticogenesis. We observed that hypoxia preconditioning induced the expression of more genes associated with different stages of cortical development. The common genes, expressed both in normoxia and hypoxia preconditioning, were involved in developmental and neuronal processes. Interestingly, a larger number of genes associated with development biology and neuronal process was expressed in GMSCs differentiated after hypoxia preconditioning compared with those in normoxia. In addition, hypoxic-preconditioned differentiated GMSCs showed a higher expression of nestin, PAX6, and GAP43. Our data demonstrated that hypoxia preconditioning enhanced the differentiation potential of GMSCs and induced the activation of a higher number of genes associated with neuronal development. In conclusion, hypoxia may be used to improve MSCs’ properties for stem cell therapy.

Natalizumab (NTZ, Tysabri®; Biogen-Idec, Cambridge, MA, USA) is a humanized anti-α4 integrin monoclonal antibody, largely used in the treatment of Relapsing-Remitting Multiple Sclerosis (RRMS). Although the drug has shown great efficacy in clinical trials (AFFIRM and SENTINEL) and in post-marketing observational studies (TYGRIS), by reducing clinical signs as disability status progression, brain lesions and annual relapse rate, there are numerous papers concerning the associated risk of progressive multifocal leukoencephalopathy (PML). PML is a brain infection caused by John Cunningham virus (JCV) and its incidence is related to intrinsic and extrinsic risk factors, such as long-term natalizumab therapy (more than 24 infusion doses), previous pharmacological immunosuppressive treatment and JVC antibody-positive status. The identification of risk factors provides an instrument to improve treatment decisions and to obtain an accurate benefit-risk evaluation. In order to evaluate the most appropriate natalizumab-MS therapy and to obtain minor incidence of PML, an accurate description of risk factors and a biological markers identification are needed. This article review aims to list some biomarkers that have been proposed to evaluate the safety of natalizumab therapy.

Neuronal cell death is a normal process during central nervous system (CNS) development and is also involved in the death of motor neurons in diverse spinal motor neuron degenerative diseases. Here, we investigated the neuroprotective effect of secretory factors released from human gingival mesenchymal stem cells (hGMSCs) in mechanically injured murine motor-neuron-like NSC-34 cells. The cells were exposed to scratch injury and the markers for apoptosis and oxidative stress were examined. Immunocytochemistry results showed that proapoptotic markers cleaved caspase-3 and Bax were elevated while anti-apoptotic protein Bcl-2 was suppressed in scratch-injured NSC-34 cells. Oxidative stress markers SOD-1, inducible nitric oxide synthase (iNOS), Cox-2, and proinflammatory cytokine tumor necrosis factor alpha (TNF-α) were activated. Conditioned medium (CM) derived from hGMSCs (hGMSC-CM) significantly blocked the cell death by suppressing SOD-1, iNOS, TNF-α, cleaved caspase-3, and Bax. Bcl-2 and anti-inflammatory cytokine anti-interleukin 10 (IL-10) were increased in hGMSC-CM-treated injured cells. Moreover, hGMSC-CM treatment upregulated neurotrophins anti-brain-derived neurotrophic factor (BDNF) and NT3. Western blot data of hGMSC-CM revealed the presence of neurotrophins nerve growth factor (NGF), NT3, anti-inflammatory cytokines IL-10, and transforming growth factor beta (TGF-β), suggesting their positive role to elicit neuroprotection. Our results propose that hGMSC-CM may serve as a simple and potential autologous therapeutic tool to treat motor neuron injury.

Several lines of evidence suggest that the inflammasome activation is involved in the progression of neurodegenerative diseases. However, the relation between Parkinson’s disease (PD) and the inflammasome is still unclear. This study was designed to assess the involvement of inflammasome in acute experimental PD. Specifically, acute PD was induced in C57BL/6 mice by an injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). At seven days from MPTP induction, mice were euthanized and the midbrains were sampled to carry out immunohistochemical evaluations and western blot analysis. Our results show the activation of Nod-like receptor-3 inflammasome in acute MPTP mice, as suggested by the increase of nuclear factor-κB expression, which represents the first signal for inflammasome induction. The Nod-like receptor-3 assembly induces the activation of caspase-1, which in turn activates interleukin-1β and interleukin-18 production, as confirmed by our evaluations. A dysregulation of autophagy system was also found in acute MPTP mice by looking at the expression of Beclin-1, LC-3, and Bcl-2, chosen as markers of autophagy. Thus, in an effort to identify the molecular mechanism underlying the well-known crosstalk between autophagy and the inflammasome, we evaluated the involvement of the phosphoinositide-3 kinase/protein kinase-B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway, which plays a key role in autophagy. Our results showed a clear upregulation of this signaling after MPTP induction. Taken together, our findings suggest that the triggering of inflammasome could be linked to impaired autophagy because of aberrant upstream activation of the PI3K/Akt/mTOR pathway. Finally, our results propose the inflammasome as a new potential therapeutic target in the management of PD.

Spasticity is a common symptom of multiple sclerosis (MS) affecting about 80% of MS patients. Numerous lines of evidence suggest that spasticity due to its complexity is not adequately managed with conventional anti-spastic therapies. Therefore, in order to improve the outcomes for the majority of MS patients, alternative approaches are needed to be discovered. Over the last years, the use of cannabinoid compounds as a potential treatment for MS-related symptoms has aroused great interest, owing to encouraging preclinical and clinical studies. To date, Sativex, an oromucosal spray containing tetrahydrocannabinol and cannabidiol in approximately 1:1 ratio, is the only commercially available formulation containing cannabinoids used as add-on therapy for treatment of spasticity in adult MS patients who are not responding to conventional antispastic therapies.

Research in recent years has largely explored the immunomodulatory effects of mesenchymal stem cells (MSCs) and their secretory products, called “secretome,” in the treatment of neuroinflammatory diseases. Here, we examined whether such immunosuppressive effects might be elicited due to inflammasome inactivation. To this end, we treated experimental autoimmune encephalomyelitis (EAE) mice model of multiple sclerosis (MS) with the conditioned medium or purified exosomes/microvesicles (EMVs) obtained from relapsing-remitting-MS patients human periodontal ligament stem cells (hPDLSCs) and investigated the regulation of NALP3 inflammasome. We noticed enhanced expression of NALP3, Cleaved Caspase 1, interleukin (IL)-1β, and IL-18 in EAE mouse spinal cord. Conversely, hPDLSCs-conditioned medium and EMVs significantly blocked NALP3 inflammasome activation and provided protection from EAE. Reduction in NALP3, Cleaved Caspase 1, IL-1β, and IL-18 level was noticed in conditioned medium and EMVs-treated EAE mice. Pro-inflammatory Toll-like receptor (TLR)-4 and nuclear factor (NF)-κB were elevated in EAE, while hPDLSCs-conditioned medium and EMVs treatment reduced their expression and increased IκB-α expression. Characterization of hPDLSCs-conditioned medium showed substantial level of anti-inflammatory IL-10, transforming growth factor (TGF)-β, and stromal cell–derived factor 1α (SDF-1α). We propose that the immunosuppressive role of hPDLSCs-derived conditioned medium and EMVs in EAE mice may partly attribute to the presence of soluble immunomodulatory factors, NALP3 inflammasome inactivation, and NF-κB reduction.

The detection of low abundant biomarkers is of great interest in different clinical cases, such as the early diagnosis of Alzheimer's disease. Here we present what we call pin-based pyro-electrohydrodynamic jet (p-jet) sensor that can linearly tune the accumulation rate of biomolecules in tiny droplets. In this newly revealed pin-based configuration we demonstrate the ability to detect biomolecules down to sub-picogram concentration. Two different fluorescent probes at a varying number of accumulated droplets, up to 50, have been used to investigate the p-jet sensor. Moreover, a comparative study with a commercial piezo-driven bio-spotter has been performed. The spots of biomolecules exhibit high reproducibility by achieving a coefficient of variation <10%, both in diameter and fluorescence intensity, respectively. Remarkably, the spot intensity increases linearly with the number of accumulated droplets, thus showing the effective inherent additive functionality of this technique. Such accumulation effect allowed us to reach a limit of detection down to sub-picogram level. The p-jet sensor was applied to detect the Aβ1–42 protein, a typical Alzheimer's disease biomarker, through an immunoreaction protocol carried out on the p-jet spots. The results demonstrate the compatibility of the p-jet with immunoassay procedures, opening the route to the development of a highly sensitive device able to detect low abundant biomarkers for early diagnosis applications.

The Wnt canonical or the Wnt/β-catenin pathway has been implicated in the regulation of several physiopathological pathways such as inflammation. Glucocorticoids (GCs) are administered widely to treat inflammation in several diseases, including spinal cord injury (SCI). The aim of this study was to evaluate whether the Wnt canonical pathway is involved in experimental SCI and whether it is implicated in the anti-inflammatory activity of two different GCs: the methylprednisolone sodium succinate (MPSS), considered the standard treatment for acute SCI, and mometasone furoate (MF), mainly administered for the treatment of airway and skin diseases. Experimental SCI was induced in mice by surgical spinal cord compression at the T6–T7 level. Then, mice were treated with MPSS (6 mg/kg) or MF (0.1 mg/kg) for 7 days until they were killed. Both GCs were found to modulate the Wnt canonical pathway, but in particular, the MF treatment was shown to restore completely the downregulated pathway in SCI. The MF treatment also significantly increased peroxisome proliferator-activated receptor-γ, a Wnt target gene with anti-inflammatory properties, compared with MPSS, and it also inhibited the levels of the proinflammatory cytokines interleukin 1β and tumor necrosis factor-α. Here, we suggest that MF has more efficacy than MPSS in inhibiting inflammation in an SCI experimental model and we propose the β-catenin/peroxisome proliferator-activated receptor-γ axis as the mechanism by which MF exerts these beneficial effects.

Glucocorticoids (GC) are widely used as antiinflammatory/immunosuppressive drugs and antitumor agents in several types of lymphoma and leukemia. Therapeutic doses of GC induce growth-suppressive and cytotoxic effects on various leukocytes including B cells. Molecular mechanisms of GC action include induction of GC target genes. Glucocorticoid-induced leucine zipper (GILZ) is a rapidly, potently, and invariably GC-induced gene. It mediates a number of GC effects, such as control of cell proliferation, differentiation, and apoptosis. Here we show that deletion of GILZ in mice leads to an accumulation of B lymphocytes in the bone marrow, blood, and lymphoid tissues. Gilz knockout (KO) mice develop a progressive nonlethal B lymphocytosis, with expansion of B220+cells in the bone marrow and in the periphery, dependent on increased B-cell survival. Decreased B-cell apoptosis in mice lacking GILZ correlates with increased NF-κB transcriptional activity and Bcl-2 expression. B cell–specific gilzKO mice confirmed that the effect of GILZ deletion is B-cell self-intrinsic. These results establish GILZ as an important regulator of B-cell survival and suggest that the deregulation of GILZ expression could be implicated in the pathogenesis of B-cell disorders.

Glucocorticoids (GC) are widely used as antiinflammatory/immunosuppressive drugs and antitumor agents in several types of lymphoma and leukemia. Therapeutic doses of GC induce growth-suppressive and cytotoxic effects on various leukocytes including B cells. Molecular mechanisms of GC action include induction of GC target genes. Glucocorticoid-induced leucine zipper (GILZ) is a rapidly, potently, and invariably GC-induced gene. It mediates a number of GC effects, such as control of cell proliferation, differentiation, and apoptosis. Here we show that deletion of GILZ in mice leads to an accumulation of B lymphocytes in the bone marrow, blood, and lymphoid tissues. Gilz knockout (KO) mice develop a progressive nonlethal B lymphocytosis, with expansion of B220+ cells in the bone marrow and in the periphery, dependent on increased B-cell survival. Decreased B-cell apoptosis in mice lacking GILZ correlates with increased NF-κB transcriptional activity and Bcl-2 expression. B cell–specific gilz KO mice confirmed that the effect of GILZ deletion is B-cell self-intrinsic. These results establish GILZ as an important regulator of B-cell survival and suggest that the deregulation of GILZ expression could be implicated in the pathogenesis of B-cell disorders.

Fingolimod is the first oral disease-modifying drug approved for the treatment of multiple sclerosis. The drug is usually well tolerated, and common adverse effects include bradycardia, headache, influenza, diarrhea, back pain, increased liver enzyme levels, and cough. Fingolimod is thought to provide therapeutic benefit by preventing normal lymphocyte egress from lymphoid tissues, thus reducing the infiltration of autoaggressive lymphocytes into the central nervous system. However, because the drug acts on different sphingosine-1-phosphate receptors, it may induce several biological effects by influencing endothelial cell-cell adhesion, angiogenesis, vascular development, and cardiovascular function. We describe a patient with multiple sclerosis who, after 3 weeks of fingolimod administration, developed purplish blotches over the dorsal surface of the distal phalanges of the second and fifth digits and the middle phalanx of the fourth ray, itching, and edema on his left hand, without other evident clinical manifestations. When fingolimod therapy was discontinued, the clinical picture regressed within a few days but reappeared after a rechallenge test. Physicians should be aware of unexpected peripheral vascular adverse effects due to fingolimod use, and patients with vascular-based acropathies should be carefully screened and monitored when taking this drug.

Rho kinase (ROK) may play an important role in regulating the biological events of cells, including proliferation, differentiation, and survival/death. Blockade of ROK promotes axonal regeneration and neuron survival in vivo and in vitro, thereby exhibiting potential clinical applications in spinal cord damage and stroke. The aim of this experimental study was to determine the role of ROK signaling pathways in the inflammatory response, in particular in the secondary injury associated with the experimental model of spinal cord trauma. The injury was induced by application of vascular clips to the dura via a four-level T5 to T8 laminectomy in mice. Fasudil was administered in mice (10 mg/kg i.p.) 1 and 6 h after the trauma. The treatment with fasudil significantly decreased 1) histological damage; 2) motor recovery; 3) nuclear factor-κB (NF-κB) expression; 4) ROK activity; 5) inflammasome activation (caspase-1 and NOD-like receptor family, pyrin domain-containing 3 expression); 6) production of proinflammatory cytokine such as tumor necrosis factor and interleukin-1β (IL-1β); 7) neutrophil infiltration; 8) nitrotyrosine and poly-ADP-ribose formation; 9) glial fibrillary acidic protein expression; 10) apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling staining, FAS ligand expression, and Bax and Bcl-2 expression); and 11) mitogen-activated protein kinase activation (phospho-extracellular signal-regulated kinase and phospho-c-Jun NH(2)-terminal kinase expression). Our results indicate that inhibition of ROK by fasudil may represent a useful therapeutic perspective in the treatment of inflammation associated with spinal cord trauma.

PEA causes a substantial reduction of intestinal I/R injury in mice by reducing inflammatory mediators and apoptosis. Our primary aim in this study was to test the hypothesis that PEA, a member of the fatty acid ethanolamide family and an endogenous PPAR‐α ligand, exerts anti‐inflammatory effects on SAO shock, causing a severe form of circulatory shock and enhanced formation of ROS. SAO shock was induced by clamping the superior mesenteric artery and the celiac trunk, resulting in a total occlusion of these arteries for 30 min. After this period of occlusion, the clamps were removed. In this study, we demonstrated that the administration of PEA, 5 min before reperfusion, significantly reduced all of the parameters involved during inflammation, such as proinflammatory cytokine production (TNF‐α, IL‐1β), adhesion molecules (ICAM‐1, P‐selectin) expression, NF‐κB expression, and apoptosis (Bax, Bcl‐2, TUNEL assay) activation. In addition, to study whether the protective action of PEA on SAO shock is also related to the activation of PPAR‐α, we have investigated the effect of PEA in PPAR‐α KO mice subjected to SAO shock. Our study clearly demonstrates that PEA significantly attenuated the degree of intestinal injury and inflammation caused by I/R injury. Moreover, the positive effects of PEA were at least in part dependent on the PPAR‐α pathway. The results clearly indicate that PEA exerts an anti‐inflammatory effect, also in a SAO shock model, which could imply a future use of PEA in the treatment of I/R shock.

Microvascular thrombosis occurs in severe acute pancreatitis (AP). Exploiting this knowledge, we used human recombinant activated protein C (Xigris; Eli Lilly, Indianapolis, Ind) known to preserve microvascular patency, in evaluating the role of Xigris in experimental AP.

Extracellular signal–regulated kinase (ERK) influences a number of pathways in all cells. The ERK cascade has long been known to be central to the activation of cellular processes such as proliferation, differentiation, and oncogenic transformation. The mitogen-activated protein (MAP) serinethreonine family of protein kinases, of which ERK is a member, is evolutionarily conserved and is activated by a mechanism that includes protein kinase cascades. The aim of this study was to investigate the effects of PD98059 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one, a highly selective inhibitor of MAPERK kinase 1 (MEK1) activation, on the development of acute pancreatitis.

The aim of this study was to investigate the effect of oleuropein aglycone, an olive oil compound, on the modulation of the inflammatory response in mice subjected to collagen-induced arthritis (CIA). CIA was induced in mice by an intradermal injection of 100 μl of an emulsion containing 100 μg of bovine type II collagen (CII) and complete Freund's adjuvant (CFA) at the base of the tail. On day 21, a second injection of CII in CFA was administered. Mice developed erosive hind paw arthritis when immunized with CII in CFA. Macroscopic clinical evidence of CIA first appeared as periarticular erythema and edema in the hind paws. The incidence of CIA was 100% by day 28 in the CII-challenged mice and the severity of CIA progressed over a 35-day period with resorption of bone. The histopathology of CIA included erosion of the cartilage at the joint. Treatment with oleuropein aglycone starting at the onset of arthritis (day 25) ameliorated the clinical signs at days 26 to 35 and improved histological status in the joint and paw. The degree of oxidative and nitrosative damage was also significantly reduced in oleuropein aglycone-treated mice. Plasma levels of the proinflammatory cytokines were also significantly reduced by oleuropein aglycone. In addition, we have confirmed the beneficial effects of oleuropein aglycone on an experimental model of CIA in a therapeutic regimen of post-treatment, with treatment started at day 28, demonstrating that oleuropein aglycone exerts an anti-inflammatory effect during chronic inflammation and ameliorates the tissue damage associated with CIA.

OBJECTIVE: The aim of our study was to investigate the effect of an adenosine A2A receptor agonist, 2-[p-(2-carboxyethyl)phenylethylamino]-50 ethylcarboxamidoadenosine (CGS 21680), on modulation of the inflammatory response in mice subjected to collagen-induced arthritis (CIA). METHODS: CIA was induced by intradermal injection of 100 µl of emulsion containing 100 µg of bovine type II collagen (CII) and complete Freund’s adjuvant (CFA) at the base of the tail. On Day 21, a second injection of CII in CFA was administered. Immunized mice developed erosive hind paw arthritis. Macroscopic clinical evidence of CIA first appeared as periarticular erythema and edema in the hind paws. The incidence of CIA was 100% by Day 27 in the CII challenged mice and the severity of CIA progressed over a 35-day period, with radiographic evaluation revealing focal resorption of bone. The histopathology of CIA included erosion of cartilage at the joint margins. RESULTS: Treatment of mice with CGS 21680 starting at the onset of arthritis (Day 25) ameliorated the clinical signs at Days 26–35 and improved histological status in the joint and paw. The degree of oxidative and nitrosative damage was significantly reduced in CGS 21680-treated mice as indicated by elevated levels of malondialdehyde, formation of nitrotyrosine, and activation of poly(ADP-ribose) polymerase. Plasma levels of proinflammatory cytokines such as tumor necrosis factor, interleukin 1ss (IL-1ss) and IL-6 were also reduced by CGS 21680. Treatment with CGS 21680 also decreased the expression of inducible nitric oxide synthase and cyclooxygenase-2. CONCLUSION: We demonstrate that CGS 21680 exerts an antiinflammatory effect during chronic inflammation and ameliorates the tissue damage associated with CIA.

Multiple organ dysfunction syndrome (MODS) is a systemic inflammatory event that can result in organ damage, failure, and high risk of mortality. The aim of this study was to evaluate the possible role of glucocorticoid-induced TNFR-related (GITR) on zymosan-induced MODS. Mice were allocated into one GITR knockout (GITR-KO) and two GITR wild-type (GITR-WT) experimental groups. All the animals were treated with zymosan (500 mg/kg, suspended in saline solution, i.p.), and animals of one GITR-WT group received GITR-Fc (6.25 g/mouse; 3 h after zymosan injection) by mini-osmotic pump. Moreover, three control groups were performed (one GITR-KO and two GITR-WT experimental groups), administering saline instead of zymosan and treating one of the GITR-WT group with GITR-Fc (6.25 g/mouse; 3 h after saline injection) by mini-osmotic pump. A number of inflammatory parameters such as edema formation, histological damage, adhesion molecules expression, neutrophil infiltration, proinflammatory cytokines, nitrotyrosine, and iNOS production are significantly reduced in GITR-KO as compared with GITR-WT mice as well as in GITR-WT mice treated with GITR-Fc. We here show that GITR plays a role in the modulation of experimental MODS. In particular, we show that genetic inhibition of GITR expression, in GITR-KO mice, or administration of soluble GITR-Fc receptor in GITR-WT mice, reduces inflammation, organ tissue damage, and mortality. Results, while confirming the proinflammatory role of GITR, extend our observations indicating that GITR plays a role in zymosan-induced inflammation and MODS.

Pharmacological action of Apocynin, used as an inhibitor of NADPH‐oxidase, when applied to an experimental model of SAO shock. SAO shock is a severe form of circulatory shock produced by I/R of the splanchnic organs. SAO causes an enhanced formation of ROS, which contributes to the pathophysiology of shock. Apocynin is a naturally occurring, methoxy‐substituted catechol, experimentally used as an inhibitor of NOX. It can decrease the production of ROS from activated neutrophils and macrophages, inhibiting the assembly of NADPH‐oxidase activity. In this study, we wanted to evaluate the pharmacological action of apocynin in rats subjected to SAO shock, which was induced by clamping the superior mesenteric artery and the celiac trunk, resulting in a total occlusion of these arteries for 45 min. After this period of occlusion, the clamps were removed. Administration of apocynin i.p. (5 mg/kg i.p. 10% DMSO) 5 min before reperfusion significantly reduced the (1) histological evidence of tissue injury, (2) proinflammatory cytokine production (TNF‐α, IL‐1β), (3) adhesion molecules (ICAM‐1, P‐selectin), (4) nitrotyrosine formation, (5) NF‐κB expression, and (6) apoptosis (Bax, Bcl‐2, Fas‐L, and TUNEL). These results could imply a future use of apocynin in the treatment of I/R shock.

GW‐0742 impacts the inflammatory process associated with intestinal ischemia reperfusion. PPARs belong to a subfamily of transcription nuclear factors. Three isoforms of PPARs have been identified: α, β/δ, and γ, encoded by different genes and distributed in various tissues. They play important roles in metabolic processes, such as regulation of glucose and lipid redistribution. They also have antiatherogenic, anti‐inflammatory, as well as antihypertensive functions. There is good evidence that ligands of PPARs reduce tissue injury associated with I/R. This study investigated the effects of GW0742, a potent and selective PPAR‐β/δ agonist, on tissue injury, caused in a mouse model of SAO shock. IRI of the intestine was caused by clamping the superior mesenteric artery and the celiac trunk for 45 min, followed by release of the clamp, allowing reperfusion for 1 or 6 h. Only 10% of the SAO animals survived the entire 6‐h reperfusion period. In a separate set of experiments after 60 min of reperfusion, animals were killed for histological examination and biochemical studies. Administration of GW0742 (0.1 mg/kg, i.p.), 5 min prior to reperfusion, significantly reduced the (1) mortality rate, (2) histological evidence of gut injury, (3) MPO activity, (4) proinflammatory cytokines (TNF‐α, IL‐1β), (5) adhesion molecules (ICAM‐1, P‐selectin), (6) nitrotyrosine formation, (7) NF‐κB expression, (8) PAR formation, and (9) apoptosis (Bax, Bcl‐2, Fas‐L, and TUNEL). Based on these findings, we propose that GW0742 would be useful in the treatment of various I/R diseases.

In the present study, we tested the efficacy of treatment with the selective adenosine A2Areceptor agonist 2-p-(2-carboxyethyl)phenylethylamino-50-ethylcarboxamidoadenosine (CGS 21680) on ischemia and reperfusion injury of the multivisceral organs. Ischemia and reperfusion injury was induced in mice by clamping both the superior mesenteric artery and the celiac artery for 30 min, followed thereafter by reperfusion. Sixty minutes after reperfusion, animals were killed for histological examination and biochemical studies. Injured vehicle-treated mice developed a significant increase of ileum TNF- levels, myeloperoxidase activity, and marked histological injury and apoptosis. Ischemia and reperfusion injury of the multivisceral organs was also associated with significant mortality. Reperfused ileum sections from injured vehicle-treated mice showed positive staining for P-selectin and intercellular adhesion molecule 1. The intensity and degree of P-selectin and intercellular adhesion molecule 1 were markedly reduced in tissue sections from injured CGS 21680-treated mice. Ischemia and reperfusion-injured mice that have been treated with CGS 21680 showed also a significant reduction of neutrophil infiltration into the intestine, a reduction of apoptosis, and improved histological status of the intestine and survival. Taken together, our results clearly demonstrate that selective activation of adenosine A2Areceptors plays an important role in the regulation of ischemia and reperfusion injury and results put forward the hypothesis that selective activation of adenosine A2Areceptors may represent a novel and possible strategy.

Several lines of evidence suggest a biological role for peroxisome proliferator-activated receptor (PPAR)-beta/delta in the pathogenesis many diseases. The aim of the present study was to evaluate the contribution of PPAR-beta/delta in the secondary damage in experimental spinal cord injury (SCI) in mice. To this purpose, we used 4-[[[2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl]methyl]thio]-2-methylphenoxy]acetic acid (GW0742), a high-affinity PPAR-beta/delta agonist. Spinal cord trauma was induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5 to T8 laminectomy. SCI in mice resulted in severe trauma characterized by edema, neutrophil infiltration, production of inflammatory mediators, tissue damage, and apoptosis. GW0742 treatment (0.3 mg kg(-1) i.p.) 1 and 6 h after the SCI significantly reduced 1) the degree of spinal cord inflammation and tissue injury (histological score), 2) neutrophil infiltration (myeloperoxidase activity), 3) nitrotyrosine formation, 4) proinflammatory cytokines expression, 5) nuclear factor-kappaB activation, 6) inducible nitric-oxide synthase expression, and 6) apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling staining, FasL, Bax, and Bcl-2 expression). Moreover, GW0742 significantly ameliorated the recovery of limb function (evaluated by motor recovery score). To elucidate whether the protective effects of GW0742 are related to activation of the PPAR-beta/delta receptor, we also investigated the effect of PPAR-beta/delta antagonist methyl 3-({[2-(methoxy)-4 phenyl]amino}sulfonyl)-2-thiophenecarboxylate (GSK0660) on the protective effects of GW0742. GSK0660 (1 mg/kg i.p. 30 min before treatment with GW0742) significantly blocked the effect of the PPAR-beta/delta agonist and thus abolished the protective effect. Our results clearly demonstrate that GW0742 treatment reduces the development of inflammation and tissue injury associated with spinal cord trauma.

Several lines of evidence suggest a biological role for peroxisome proliferator-activated receptor (PPAR) / in the pathogenesis of a number of diseases. The aim of this study was to investigate the effects of a high-affinity PPAR-/ agonist, GW0742, in a mouse model of carrageenan (CAR)-induced pleurisy. Injection of CAR into the pleural cavity of mice elicited an acute inflammatory response characterized by accumulation of fluid containing a large number of neutrophils (polymorphonuclear leukocytes) in the pleural cavity, infiltration of polymorphonuclear leukocytes in lung tissues and subsequent lipid peroxidation, and increased production of nitrite/nitrate, TNF-, and IL-1. Furthermore, CAR induced lung apoptosis (Bax and Bcl-2 expression), and nitrotyrosine formation was determined by immunohistochemical analysis of lung tissues. Administration of GW0742 (0.3 mg/kg, i.p. bolus) 30 min before and 30 min after a challenge with CAR caused a reduction in all the parameters of inflammation measured. Thus, based on these findings, we propose that a PPAR-/ agonist such as GW0742 may be useful in the treatment of various inflammatory diseases.

T0901317, LXR receptor agonist, reduces the inflammatory response and the ileum injury associated to splanchnic artery occlusion shock. LXR is another member of the superfamily of nuclear hormone receptors that heterodimerizes with RXR and regulates the intracellular levels of cholesterol through gene induction of enzymes and proteins involved in the cholesterol metabolism and transport. LXR ligands inhibit the gene expression of proinflammatory mediators in immunostimulated macrophages; in vivo studies have shown that activation of LXR reduces the inflammatory response in a murine model of contact dermatitis and atherosclerosis. No reports have addressed a role for LXRs in pathophysiology of intestinal ischemia. The aim of this study was to investigate the effects of T0901317, a potent LXR ligand, in a mouse model of SAO shock, which was induced by clamping the superior mesenteric artery and the celiac trunk, resulting in a total occlusion of these arteries for 30 min. After this period of occlusion, the clamps were removed. Mice were killed at 60 min after reperfusion. This study provides the evidence that T0901317, LXR agonist, modulates: the development of SAO shock; the infiltration of the tissue with PMNs; the expression of TNF‐α and IL‐1β; the nitration of tyrosine residues; NF‐κB expression; the MAPK phosphorylation (ERK, JNK, and p38); FasL; apoptosis; Bax and Bcl‐2 expression; and the degree of tissue injury caused by SAO shock. Our results imply that LXR agonists may be useful in the therapy of inflammation.

Permanent functional deficit after spinal cord injury (SCI) arises from both mechanical injury and from secondary tissue reactions involving inflammation. Adenosine is an important regulator of inflammatory mechanisms. Although functional studies indicate a protective effect of adenosine A2Areceptor agonists in SCI, the basic molecular mechanisms accounting for the their protective effects from SCI have to be fully elucidated. In this study, we investigated if the selective A2Areceptor agonist 2-p-(2-carboxyethyl)-phenethylamino-5-N-ethylcarboxamidoadenosine (CGS 21680) administered after SCI has protective effects against tissue damage, motor deficit, and different inflammatory readouts. Spinal cord injury was induced in mice by extradural compression of a section of the SC exposed via a four-level T5-T8 laminectomy. CGS 21680, administered by subcutaneously implanted osmotic minipumps after SCI, clearly reduced motor deficit for up to 19 days after operation. The drug repeatedly administered intraperitoneally after SCI reduced tissue damage, influx of myeloperoxidase-positive leukocytes, nuclear factor-B activation and iNOS expression in injured spinal cord tissue 24 h after SCI. Enhanced immunoreactivity of microglia, astrocytes, and oligodendrocytes (stained by anti-CD11/B, anti-glial fibrillary acidic protein, and anti-Olig2 antibodies, respectively) was also observed 24 h after SCI. Neurons lose immunoreactivity in the nucleus. c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase, quantified by Western blot, was definitely activated in injured tissue. CGS 21680 treatment significantly reduced JNK phosporylation. Phospho-JNK mitogen-activated protein kinase was de novoexpressed selectively in oligodendrocytes. CGS 21680 reduced phospho-JNK immunostaining in oligodendrocytes. Data indicate that protection by the A2Aagonist is secondary to reduced leukocyte recruitment in the damaged area. A reducing effect of JNK activation in oligodendrocytes might account for protective effect of the A2Aagonist against SCI-induced demyelination.

Green tea polyphenols (GTPs) are naturally occurring antioxidants acting through pathways that include reactive oxygen species and nuclear factor kappa B (NF-B). This study investigates the effect of GTPs in a cerulein-induced murine model of acute pancreatitis (AP).

Peritoneal dialysis is an alternative treatment of patients with end-stage renal disease. Sclerosing encapsulating peritonitis is a life-threatening complication of continuous ambulatory peritoneal dialysis. The aim of the present study was to evaluate the effect of thalidomide, which is used for the treatment of various inflammatory and autoimmune diseases, on the development of sclerosing encapsulating peritonitis induced by chlorhexidine gluconate (CG). A peritoneal fibrosis model was established using rats treated intraperitoneally with injections of CG. Thalidomide was administered orally at a dose of 100 mg/kg three times per week. When compared with CG-treated rats, thalidomide (100 mg/kg orally)-treated mice subjected to CG-induced peritoneal fibrosis experienced a significantly lower rate in the extent and severity of histological signs of peritoneal injury. Thalidomide also caused a substantial reduction of 1) the rise in myeloperoxidase activity (mucosa); 2) the expression in the tissue of TNF-, IL-1., transforming growth factor-, and vascular endothelial growth factor; 3) the increase in staining (immunohistochemistry) for nitrotyrosine and for poly(ADP ribose), as well as 4) the nuclear factor-B activation caused by CG in the peritoneum. Thus, thalidomide treatment reduces the degree of peritoneal fibrosis caused by CG. We propose that this evidence may help clarify the potential therapeutic actions of thalidomide in patients with peritoneal fibrosis.

The aim of the present study was to evaluate in a mouse model of spinal cord injury (SCI) the effect of the treatment with ethyl pyruvate (EP). Spinal cord injury was induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy in mice. Treatment with EP (75, 25, or 8.5 mg/kg) 1 and 6 h after the SCI significantly decreased (a) the degree of spinal cord inflammation and tissue injury (histological score), (b) neutrophil infiltration (myeloperoxidase activity), (c) nitrotyrosine formation and iNOS expression, (d) proinflammatory cytokines expression, (e) nuclear factor B activation, (f) extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase phosphorylation, and (g) apoptosis (TUNEL staining, Fas ligand, Bax, and Bcl-2 expression). Moreover, EP (75, 25, or 8.5 mg/kg) significantly ameliorated in a dose-dependent manner the loss of limb function (evaluated by motor recovery score). Taken together, our results demonstrate that EP treatment reduces the development of inflammation and tissue injury associated with spinal cord trauma.

Ceramide is a sphingolipid signaling molecule with powerful proinflammatory and proapoptotic properties. The aim of this study was to investigate the role of altered ceramide metabolism in spinal cord injury. Spinal cord injury was induced by application of vascular clips (force of 24g) to the dura via a four-level T5-T8 laminectomy. Spinal cord injury in mice resulted in severe trauma characterized by edema, neutrophil infiltration, production of a range of inflammatory mediators, tissue damage, and apoptosis. Fumonisin B1, tyclodecan-9-xanthogenate (D609), and (3-carbazol-9-yl-propyl)-2-(3,4-dimethoxy-phenyl)-ethyl-methylamine (NB6) inhibitors of, respectively, ceramide synthase, acid sphingomyelinase, and the secretory form of acid sphingomyelinase significantly reduced the degree of (i) ceramide formation, (ii) tissue injury, (iii) neutrophil infiltration, (iv) nitrotyrosine formation, (v) TNF- and IL-1 production and apoptosis (TUNEL staining and Bax and Bcl-2 expression). Significant improvement of motor function was observed in mice treated with inhibitors of the de novo(fumonisin B1) and sphingomyelin (D609, NB6) pathways. These results implicate ceramide in the pathogenesis of spinal cord injury, providing the rationale for development of candidates for its therapeutic inhibition.

Glycyrrhizin, a major active constituent of liquorice root (Glycyrrhiza glabra), has a free radical scavenging property, and its effects were evaluated on an animal model of spinal cord injury (SCI) induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy. Spinal cord injury in mice resulted in severe trauma characterized by edema, tissue damage, and apoptosis (measured by terminal deoxynucleotidyltransferase-mediated dUTP-biotin end labeling staining, Bax, and Bcl-2 expression). Immunohistochemical examination demonstrated a marked increase in immunoreactivity for nitrotyrosine, iNOS, and poly(adenosine diphosphate-ribose) in the spinal cord tissue. Additionally, we demonstrate that these inflammatory events were associated with the activation of nuclear factor-B. In contrast, the degree of (1) spinal cord inflammation and tissue injury (histological score), (2) nitrotyrosine and poly(adenosine diphosphate ADP ribose) formation, (3) iNOS expression, (4) nuclear factor-B activation, and (5) apoptosis (terminal deoxynucleotidyltransferase-mediated dUTP-biotin end labeling, Bax, and Bcl-2) was markedly reduced in spinal cord tissue obtained from mice treated with glycyrrhizin extract (10 mg/kg, i.p., 30 min before and 1 and 6 h after SCI). In a separate set of experiments, we have clearly demonstrated that glycyrrhizin extract treatment significantly ameliorated the recovery of limb function (evaluated by motor recovery score). Taken together, our results clearly demonstrate that treatment with glycyrrhizin extract reduces the development of inflammation and tissue injury events associated with spinal cord trauma.

Myrtucommulone (MC), a nonprenylated acylphloroglucinol contained in the leaves of myrtle (Myrtus communis), has been reported to suppress the biosynthesis of eicosanoids by inhibition of 5-lipoxygenase and cyclooxygenase-1 in vitro and to inhibit the release of elastase and the formation of reactive oxygen species in activated polymorphonuclear leukocytes. Here, in view of the ability of MC to suppress typical proinflammatory cellular responses in vitro, we have investigated the effects of MC in in vivo models of inflammation. MC was administered to mice intraperitoneally, and paw edema and pleurisy were induced by the subplantar and intrapleural injection of carrageenan, respectively. MC (0.5, 1.5, and 4.5 mg/kg i.p.) reduced the development of mouse carrageenan-induced paw edema in a dose-dependent manner. Moreover, MC (4.5 mg/kg i.p. 30 min before and after carrageenan) exerted anti-inflammatory effects in the pleurisy model. In particular, 4 h after carrageenan injection in the pleurisy model, MC reduced: 1) the exudate volume and leukocyte numbers; 2) lung injury (histological analysis) and neutrophil infiltration (myeloperoxidase activity); 3) the lung intercellular adhesion molecule-1 and P-selectin immunohistochemical localization; 4) the cytokine levels (tumor necrosis factor-alpha and interleukin-1beta) in the pleural exudate and their immunohistochemical localization in the lung; 5) the leukotriene B(4), but not prostaglandin E(2), levels in the pleural exudates; and 6) lung peroxidation (thiobarbituric acid-reactant substance) and nitrotyrosine and poly (ADP-ribose) immunostaining. In conclusion, our results demonstrate that MC exerts potent anti-inflammatory effects in vivo and offer a novel therapeutic approach for the management of acute inflammation.

The effective treatment of pain is typically limited by a decrease in the pain-relieving action of morphine that follows its chronic administration (tolerance). Therefore, restoring opioid efficacy is of great clinical importance. In a murine model of opioid antinociceptive tolerance, repeated administration of morphine significantly stimulated the enzymatic activities of spinal cord serine palmitoyltransferase, ceramide synthase, and acid sphingomyelinase (enzymes involved in the de novo and sphingomyelinase pathways of ceramide biosynthesis, respectively) and led to peroxynitrite-derive nitroxidative stress and neuroimmune activation [activation of spinal glial cells and increase formation of tumor necrosis factor-alpha, interleukin (IL)-1beta, and IL-6]. Inhibition of ceramide biosynthesis with various pharmacological inhibitors significantly attenuated the increase in spinal ceramide production, nitroxidative stress, and neuroimmune activation. These events culminated in a significant inhibition of the development of morphine antinociceptive tolerance at doses devoid of behavioral side effects. Our findings implicate ceramide as a key upstream signaling molecule in the development of morphine antinociceptive tolerance and provide the rationale for development of inhibitors of ceramide biosynthesis as adjuncts to opiates for the management of chronic pain.

Glucocorticoids (GCs) are effective anti-inflammatory agents widely used in therapeutic approach to treatment of inflammatory bowel disease (IBD). Previous results suggest that peroxisome proliferator-activated receptor (PPAR-), an intracellular transcription factor activated by fatty acids, plays a role in control of inflammation. With the aim to characterize the role of PPAR- in GC-mediated anti-inflammatory activity, we tested the efficacy of dexamethasone (DEX), a synthetic GC specific for GR, in an experimental model of IBD induced by dinitrobenzene sulfonic acid, comparing mice lacking PPAR- (PPAR-KO) with wild-type (WT) mice. Results indicate that DEX-mediated anti-inflammatory activity is weakened in PPAR-KO mice as compared with WT controls. In particular, DEX was less effective in PPAR-KO compared with WT mice, as evaluated by inhibition of proinflammatory cytokines production, cell migration, oxidative stress, apoptosis, and colon injury. These results indicate that PPAR- can contribute to the anti-inflammatory activity of GCs in IBD.

Hydrogen sulfide (H2S) is a novel gaseous mediator produced by cystathionine--synthase and cystathionine--lyase in the cardiovascular system, including the heart. Using a rat model of regional myocardial ischemia/reperfusion, we investigated the effects of an H2S donor (sodium hydrogen sulfide NaHS) on the infarct size and apoptosis caused by ischemia (25 min) and reperfusion (2 h). Furthermore, we investigated the potential mechanism(s) of the cardioprotective effect(s) afforded by NaHS. Specifically, we demonstrate that NaHS (1) attenuates the increase in caspase 9 activity observed in cardiac myocytes isolated from the area at risk (AAR) of hearts subjected in vivoto regional myocardial I/R and (2) ameliorates the decrease in expression of Bcl-2 within the AAR obtained from rat hearts subjected to regional myocardial I/R. The cardioprotective effects of NaHS were abolished by 5-hydroxydeconoate, a putative mitochondrial adenosine triphosphate-sensitive potassium channel blocker. Furthermore, NaHS attenuated the increase in the I/R-induced (1) phosphorylation of p38 mitogen-activated protein kinase and Jun N-terminal kinase, (2) translocation from the cytosol to the nucleus of the p65 subunit of nuclear factor-B, (3) intercellular adhesion molecule 1 expression, (4) polymorphonuclear leukocyte accumulation, (5) myeloperoxidase activity, (6) malondialdehyde levels, and (7) nitrotyrosine staining determined in the AAR obtained from rat hearts subjected to regional myocardial I/R. In conclusion, we demonstrate that the cardioprotective effect of NaHS is secondary to a combination of antiapoptotic and anti-inflammatory effects. The antiapoptotic effect of NaHS may be in part due to the opening of the putative mitochondrial adenosine triphosphate-sensitive potassium channels.

Ceramide is a major proapoptotic mediator released in response to numerous stimuli, including oxidative stress and cytokines. The role of ceramide in the pathophysiology of inflammation is just emerging, and the potential relevance of this pathway in nonseptic shock is not known. The aim of this study was to investigate the effects of fumonisin B1 (FB1), a specific inhibitor of ceramide synthase, on the development of nonseptic shock in mice caused by zymosan. CD1 mice received either zymosan (500 mg/kg, administered i.p. as a suspension in saline) or vehicle (0.25 mL per mouse saline). Fumonisin B1 (3 mg/kg, i.p.) was administered 1 and 6 h after zymosan administration. Organ failure and systemic inflammation in mice were assessed 18 h after administration of zymosan and/or FB1. Treatment of mice with FB1 attenuated peritoneal exudate formation and the migration of polymorphonuclear cells caused by zymosan. Fumonisin B1 also attenuated plasma markers of lung, liver and pancreatic injury, and renal dysfunction caused by zymosan and the increase in myeloperoxidase activity in the intestine caused by zymosan. Immunohistochemical analyses for the presence of ceramide and nitrotyrosine revealed positive staining in intestinal tissue obtained from zymosan-injected mice. The degree of staining for ceramide and nitrotyrosine was markedly reduced in tissue sections obtained from zymosan-injected mice that had received FB1. In addition, administration of zymosan caused a severe illness in the mice characterized by a systemic toxicity, significant loss of body weight, and 80% mortality within 12 days. Treatment with FB1 significantly reduced systemic toxicity, weight loss, and mortality caused by zymosan. This study provides evidence that FB1 attenuates the degree of zymosan-induced nonseptic shock in mice.

This study was designed to elucidate the role of peroxisome proliferator-activated receptor (PPAR)-alpha in the development of inflammation after ischemia/reperfusion injury of the kidney. We have evaluated the effects of ischemia/reperfusion on renal dysfunction, injury, and inflammation in wild-type mice or mice in which the gene for PPAR-alpha has been deleted [PPAR-alpha(-/-)] and then treated with the PPAR-alpha agonist fenofibrate. Mice were subjected to bilateral renal ischemia (30 min) and reperfusion (24 h) and received fenofibrate (3 mg/kg i.p.) before reperfusion. Plasma creatinine, urea, and aspartate aminotransferase were all used as indicators of renal dysfunction and injury. Kidneys were used for histological and immunohistochemical analysis and markers of inflammation. Fenofibrate significantly attenuated the degree of renal dysfunction, injury, and inflammation caused by ischemia/reperfusion injury. The degree of renal dysfunction, injury, and inflammation caused by ischemia/reperfusion was also significantly augmented in PPAR-alpha(-/-) mice compared with their wild-type littermates. It is interesting that fenofibrate did not protect PPAR-alpha(-/-) mice against ischemia/reperfusion injury. Therefore, we propose that ligands of PPAR-alpha may be useful in the treatment of renal ischemia/reperfusion injury and that endogenous PPAR-alpha limits the degree of renal dysfunction, injury, and inflammation associated with ischemia/reperfusion injury.

Ceramide is a sphingolipid with potent proinflammatory and proapoptotic properties. This study sought to determine whether pharmacological inhibition of ceramide biosynthesis in the intestine attenuates pathophysiological sequelae of shock induced by splanchnic artery occlusion and reperfusion. Ischemia and reperfusion injury was induced in anesthetized rats by clamping both the superior mesenteric artery and the celiac artery for 45 min followed by reperfusion. Within 6 min after reperfusion, animals developed significant systemic hypotension with 100% of the animals dying during the 4-h period of reperfusion. In parallel experiments, animals were necropsied after 60 min of reperfusion, and the ileum was harvested for histological examination and assessment of biochemical changes. Administration of fumonisin B1 (FB1), a competitive and reversible inhibitor of ceramide synthase (3 mg/kg, 15 min before reperfusion), significantly reduced i) the increased ceramide expression as detected by immunohistochemistry; ii) peroxynitrite-mediated protein nitration; iii) infiltration of the reperfused intestine with polymorphonuclear neutrophils following a decrease in intercellular adhesion molecule-1 expression; iv) production of the proinflammatory cytokine tumor necrosis factor-alpha; and v) apoptosis in the ileum. Overall, tissue-protective effects were clearly observed upon histological examination of the ileum. These beneficial events were ultimately linked to decreases in both the development of hypotension and overall mortality. These results implicate ceramide as a key signaling molecule in splanchnic arterial ischemia and reperfusion-induced shock. The broader implications of our results provide a pharmacological rationale for the development of inhibitors of ceramide biosynthesis as novel therapeutics for ischemia and reperfusion-induced shock of several etiologies.

TNF- seems to play a central role in the inflammatory process of spinal cord injury. We tested the neuroprotective effects of thalidomide, an immunomodulatory agent that inhibits TNF- production, which have not been investigated so far. The aim of our study was to evaluate the therapeutic efficacy of thalidomide in an experimental model of spinal cord trauma, which was induced by the application of vascular clips (force of 24 g) to the dura via a 4-level T5 to T8 laminectomy. Spinal cord injury in mice resulted in severe trauma characterized by edema, neutrophil infiltration, and cytokine production that is followed by recruitment of other inflammatory cells, production of a range of inflammation mediators, tissue damage, apoptosis, and disease. Thalidomide treatment significantly reduced the degree of 1) spinal cord inflammation and tissue injury (histological score); 2) neutrophil infiltration (myeloperoxidase evaluation); 3) iNOS, nitrotyrosine, lipid peroxidation, and cytokine expression (TNF- and IL-1); 4) apoptosis (terminal deoxynucleotidyltransferase-mediated UTP end labeling staining, and Bax and Bcl-2 expression); and 5) nuclear factor-B activation. In a separate set of experiments, we have also clearly demonstrated that thalidomide significantly ameliorated the recovery of limb function (evaluated by motor recovery score). Taken together, our results clearly demonstrate that treatment with thalidomide reduces the development of inflammation and tissue injury events associated with spinal cord trauma.

Peroxisome proliferator-activated receptor (PPAR) is a member of the nuclear-receptor superfamily that binds to DNA with retinoid X receptors as PPAR-retinoid X receptor heterodimers. Recent evidence also suggests that the cyclopentenone prostaglandin 15-deoxy-12,14PGJ2(15d-PGJ2), which is a metabolite of the prostaglandin D2, functions as an endogenous ligand for PPAR- We postulated that 15d-PGJ2would attenuate inflammation, investigating the effects on the degree of experimental spinal cord trauma induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy. Spinal cord injury in mice resulted in severe trauma characterized by edema, neutrophil infiltration, production of a range of inflammatory mediators, tissue damage, and apoptosis. Furthermore, 15d-PGJ2reduced (1) spinal cord inflammation and tissue injury (histological score), (2) neutrophil infiltration (myeloperoxidase activity), (3) nuclear factor-B activation, (4) expression of iNOS, nitrotyrosine and TNF-, and (5) apoptosis (terminal deoxynucleotidyltransferase-mediated uridine triphosphate end labeling staining, Bax, Bcl-2, and FAS-L expression). In a separate set of experiments, 15d-PGJ2significantly ameliorated the recovery of limb function (evaluated by motor recovery score). To elucidate whether the protective effects of 15d-PGJ2are related to activation of the PPAR- receptor, we also investigated the effect of a PPAR- antagonist, GW 9662, on the protective effects of 15d-PGJ2. GW9662 (1 mg/kg administered i.p. 30 min before treatment with 15d-PGJ2) significantly antagonized the effect of the PPAR- agonist and, thus, abolished the protective effect. Taken together, our results clearly demonstrate that treatment with 15d-PGJ2reduces the development of inflammation and tissue injury associated with spinal cord trauma.

Activation of peroxisome proliferator-activated receptor (PPAR)-alpha, a member of the nuclear receptor superfamily, modulates inflammation and tissue injury events associated with spinal cord trauma in mice. Palmitoylethanolamide (PEA), the naturally occurring amide of palmitic acid and ethanolamine, reduces pain and inflammation through a mechanism dependent on PPAR-alpha activation. The aim of the present study was to evaluate the effect of the PEA on secondary damage induced by experimental spinal cord injury (SCI) in mice. SCI was induced by application of vascular clips to the dura mater via a four-level T(5)-T(8) laminectomy. This resulted in severe trauma characterized by edema, neutrophil infiltration, and production of inflammatory mediators, tissue damage, and apoptosis. Repeated PEA administration (10 mg/kg i.p.; 30 min before and 1 and 6 h after SCI) significantly reduced: 1) the degree of spinal cord inflammation and tissue injury, 2) neutrophil infiltration, 3) nitrotyrosine formation, 4) proinflammatory cytokine expression, 5) nuclear transcription factor activation-kappaB activation, 6) inducible nitric-oxide synthase expression, and 6) apoptosis. Moreover, PEA treatment significantly ameliorated the recovery of motor limb function. Together, the results indicate that PEA reduces inflammation and tissue injury associated with SCI and suggest a regulatory role for endogenous PPAR-alpha signaling in the inflammatory response associated with spinal cord trauma.

Glucocorticoid-induced tumor necrosis factor receptor-related (GITR) protein is a costimulatory molecule that plays a role in inflammation so that GITR-Fc fusion protein can exert an anti-inflammatory effect. To investigate the mechanism by which GITR-Fc exerts its effects, we first used GITR knock-out (GITR-/-) mice to verify whether GITR ligand (GITRL)/GITR system played a pro-inflammatory role in the spinal cord injury (SCI) model. It is noteworthy that less pronounced disease was induced in GITR-/-compared with GITR+/+mice. We then evaluated the effect of GITR-Fc fusion protein against SCI-induced injuries in GITR-/-and wild-type (GITR+/+) mice. Administration of GITR-Fc ameliorated SCI-induced inflammation in GITR+/+mice as evaluated through: 1) histological damage and apoptosis, 2) modulation of apoptosis-related transduction factors (Bax and Bcl-2), 3) expression of inflammatory markers [nitrotyrosine, inducible nitric-oxide synthase, interleukin (IL)-2, IL-12, and tumor necrosis factor-α], and 4) T-lymphocyte infiltration. GITR-Fc was effective in GITR+/+but not in GITR-/-, suggesting that in this experimental model, its anti-inflammatory action was due to inhibition of GITR triggering and not to GITRL activation. In conclusion, GITR plays a role in SCI, and administration of GITR-Fc results in amelioration of SCI severity, prompting further studies on the potential anti-inflammatory properties of GITR-Fc.

It has been well demonstrated that TNF-α is integral to the pathogenesis of multiple organ dysfunction syndrome (MODS). In this study, we investigate the effects of etanercept (10 mg/kg, s.c.), a specific TNF-α-soluble inhibitor, on the acute phase and late mortality in a murine model of MODS of nonseptic origin induced by zymosan (500 mg/kg, suspended in saline solution, i.p.). Etanercept was administered 1 h after the injection of zymosan. Animals were killed after 18 h. In another set of experiments, mice were monitored for systemic toxicity, loss of body weight, and mortality for 12 days. Sham-treated and TNF receptor 1 (TNFR1)-deficient animals were used as control. Treatment of mice with Etanercept and TNFR1gene deletion decreased the peritoneal exudation and the migration of neutrophils caused by zymosan. In addition, pharmacological and genetic neutralization of TNF-α attenuated pancreas and ileum injury (histology), the increase in myeloperoxidase activity in the ileum and in the lung, and the formation of TNF-α and IL-1β. Immunohistochemical analysis for TNF-α, transforming growth factor β, and vascular endothelial growth factor revealed a positive staining in pancreas and ileum sections. The degree of immunostaining was markedly reduced after etanercept treatment and in TNFR1 knockout mice. Furthermore, TNF-α neutralization decreased the potent apoptotic stimulus induced by zymosan. All of these findings ultimately led to an amelioration of organ functions at 18 h and to a better survival rate at 12 days. Therefore, we demonstrate that etanercept reduces acute tissue injury and mortality associated to MODS of nonseptic origin in mice.

Mitogen-activated protein kinase (MAPK) signaling pathways involve two closely related MAPKs, known as extracellular signal-regulated kinase (ERK)1 and ERK2. The aim of the present study was to evaluate the contribution of MAPK3/MAPK1 in the secondary damage in experimental spinal cord injury (SCI) in mice. To this purpose, we used 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059), which is an inhibitor of MAPK3/MAPK1. Spinal cord trauma was induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy. SCI in mice resulted in severe trauma characterized by edema, neutrophil infiltration, and production of inflammatory mediators, tissue damage, and apoptosis. PD98059 treatment (10 mg/kg i.p.) at 1 and 6 h after the SCI significantly reduced 1) the degree of spinal cord inflammation and tissue injury (histological score), 2) neutrophil infiltration (myeloperoxidase activity), 3) nitrotyrosine formation, 4) proinflammatory cytokines expression, 5) nuclear factor-kappaB activation, 6) phospho-ERK1/2 expression, and 6) apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling staining, Fas ligand, Bax, and Bcl-2 expression). Moreover, PD98059 significantly ameliorated the recovery of limb function (evaluated by motor recovery score) in a dose-dependent manner. Taken together, our results clearly demonstrate that PD98059 treatment reduces the development of inflammation and tissue injury associated with spinal cord trauma.

Because studies have shown that 17-estradiol (E2) produces anti-inflammatory effects after various adverse circulatory conditions, we examined whether administration of E2 before spinal cord injury (SCI) has any salutary effects in reducing SCI. Spinal cord injury was induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy. To gain a better insight into the mechanism of action of the anti-inflammatory effects of E2, the following end points of the inflammatory process were evaluated (1) spinal cord inflammation and tissue injury (histological score); (2) neutrophil infiltration (myeloperoxidase activity); (3) expression of iNOS, nitrotyrosine, and COX-2; (4) apoptosis (terminal deoxynucleotidyltransferase-mediated UTP end labeling staining and Bax and Bcl-2 expression); and (5) tissue TNF-, IL-6, IL-1, and monocyte chemoattractant protein 1 levels. In another set of experiments, the pretreatment or posttreatment with E2 significantly ameliorates the recovery of limb function (evaluated by motor recovery score). To elucidate whether the protective effects of E2 were mediated via the estrogen receptors, we investigated the effect of an estrogen receptor antagonist, ICI 182,780, on the protective effects of E2. ICI 182,780 (500 g/kg, s.c., 1 h before treatment with E2) significantly antagonized the effect of the E2 and abolished the protective effect against SCI. Taken together, our results clearly demonstrate that administration of E2 before SCI reduces the development of inflammation and tissue injury associated with spinal cord trauma.

Glucocorticoids (GCs) are effective anti-inflammatory agents widely used in the therapeutic approach to treatment of acute and chronic inflammatory diseases. Previous results suggest that peroxisome proliferator-activated receptor-α (PPAR-α), an intracellular transcription factor activated by fatty acids, plays a role in the control of inflammation. With the aim of characterizing the role of PPAR-α in GC-mediated anti-inflammatory activity, we tested the efficacy of dexamethasone (DEX), a synthetic GC specific for glucocorticoid receptor, in an experimental model of lung inflammation, carrageenan-induced pleurisy, comparing mice lacking PPAR-α (PPAR-αKO) with wild-type (WT) mice. We also tested the possible synergism of combined treatment with DEX and clofibrate, a PPAR-α agonist. Results indicate that DEX-mediated anti-inflammatory activity is weakened in PPAR-αKO mice compared with WT controls, and that is increased in WT mice when combined with PPAR-α agonist treatment. In particular, DEX was less effective in PPAR-αKO, compared with WT mice, as evaluated by inhibition of NF-κB, of TNF-α production, of cell migration, of cycloxygenase-2 (COX-2) and inducible nitric-oxide synthase activation. Interestingly enough, macrophages from PPAR-αKO were less susceptible to DEX-induced COX-2 inhibition in vitro compared with WT mice. However, PPAR-α transfection in PPAR-αKO macrophages, with consequent receptor expression, resulted in reconstitution of susceptibility to DEX-induced COX-2 inhibition to levels comparable with that obtained in WT macrophages. It is noteworthy that the DEX effect on macrophages in vitro was significantly increased in WT cells when combined with PPAR-α agonist treatment. These results indicate that PPAR-α can contribute to the anti-inflammatory activity of GCs.

Current knowledge shows that pathophysiology of acute pancreatitis is characterized by intraacinar enzyme activation and subsequent dysregulation in immune response. Interactions between leukocytes, soluble mediators such as cytokines and vascular endothelium contribute to the systemic progression of the inflammatory response, whose entity may-in the end-determine disease severity and outcome. Recently, it has been shown that TNF- may be a novel target for the treatment of acute pancreatitis; but the role of thalidomide, an immunomodulatory agent that inhibits TNF- and angiogenesis, has not been investigated so far. The aim of the present study was to assess the effects of thalidomide in a murine model of necrotizing acute pancreatitis. Necrotizing acute pancreatitis was induced in mice by intraperitoneal injection of cerulein (hourly, ×5, 50 g/kg); in another group of animals, thalidomide was administered (200 mg/kg orally) at 1 h after first cerulein injection. After 24 h, biochemical, histological, and immunohistochemical evidences of acute pancreatitis developed in all cerulein-treated mice. On the contrary, pancreatitis histological features, amylase, lipase, TNF- and IL-1 levels, pancreas edema, and myeloperoxidase activity as well as immunohistochemical staining for inflammatory cytokines, leukocyte adhesion molecules, transforming growth factor , vascular endothelial growth factor, and apoptosis-related proteins were found reduced in thalidomide-treated mice. Therefore, thalidomide treatment attenuates the development of acute pancreatitis caused by cerulein in mice. We propose that this evidence may help to clarify the role of anti-TNF- and immunomodulatory agents in patients with acute pancreatitis.

The aim of our study was to evaluate in vivothe therapeutic efficacy of genetic inhibition of TNF- using TNF-R1 knockout mice in an experimental model of spinal cord trauma. Spinal cord injury was induced by the application of vascular clips to the dura via a four-level T5-T8 laminectomy. To elucidate whether the observed anti-inflammatory status is related to the inhibition of TNF-, we also investigated the effect of infliximab, a TNF--soluble receptor construct, on spinal cord damage. Pharmacological and genetic TNF- inhibition significantly reduced the degree of (1) spinal cord inflammation and tissue injury (histological score), (2) neutrophil infiltration (evaluated by myeloperoxidase activity), (3) cytokine expression (TNF-), (4) and apoptosis (terminal deoxynucleotidyltransferase-mediated uridine triphosphate end labeling staining, Bax, Bcl-2, and Fas-L expression). In a separate set of experiments, we have also demonstrated that TNF- inhibition significantly ameliorated the recovery of limb function (evaluated by motor recovery score). Taken together, our results demonstrate that inhibition of TNF- reduces the development of inflammation and tissue injury associated with spinal cord trauma, suggesting a possible role of TNF- on the pathogenesis of spinal cord injury.