Concetta Crisafulli

Immunomodulatory Effects of Etanercept in an Experimental Model of Spinal Cord Injury

Etanercept is a tumor necrosis factor antagonist with anti-inflammatory effects. The aim of our study was to evaluate for the first time the therapeutic efficacy of in vivo inhibition of tumor necrosis factor-α (TNF-α) in experimental model of spinal cord trauma, which was 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, and cytokine production that it is followed by recruitment of other inflammatory cells, such as production of a range of inflammation mediators, tissue damage, apoptosis, and disease. Treatment of the mice with etanercept significantly reduced the degree of 1) spinal cord inflammation and tissue injury (histological score); 2) neutrophil infiltration (myeloperoxidase evaluation); 3) inducible nitric-oxide synthase, nitrotyrosine, cyclooxygenase-2, and cytokines expression (TNF-α and interleukin-1β); and 4) apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and Bax and Bcl-2 expression). In a separate set of experiment, we have also clearly demonstrated that TNF-α inhibitor significantly ameliorated the recovery of limb function (evaluated by motor recovery score). Taken together, our results clearly demonstrate that treatment with etanercept reduces the development of inflammation and tissue injury events associated with spinal cord trauma.

The role of the peroxisome proliferator-activated receptor-α (PPAR-α) in the regulation of acute inflammation

The peroxisome proliferator‐activated receptor‐α (PPAR‐α) is a member of the nuclear receptor superfamily of ligand‐dependent transcription factors related to retinoid, steroid, and thyroid hormone receptors. The aim of the present study was to evaluate the role of the PPAR‐α receptor on the development of acute inflammation. To address this question, we used two animal models of acute inflammation (carrageenan‐induced paw edema and carrageenan‐induced pleurisy). We report here that when compared with PPAR‐α wild‐type mice, PPAR‐α knockout mice (PPAR‐αKO) mice experienced a higher rate of the extent and severity when subjected to carrageenan injection in the paw edema model or to carrageenan administration in the pleurisy model. In particular, the absence of a functional PPAR‐α gene in PPAR‐αKO mice resulted in a significant augmentation of various inflammatory parameters (e.g., enhancement of paw edema, pleural exudate formation, mononuclear cell infiltration, and histological injury) in vivo. Furthermore, the absence of a functional PPAR‐α gene enhanced the staining (immunohistochemistry) for FAS ligand in the paw and in the lung and the expression of tumor necrosis factor α and interleukin‐1β in the lungs of carrageenan‐treated mice. In conclusion, the increased inflammatory response observed in PPAR‐αΚΟ mice strongly suggests that a PPAR‐α pathway modulates the degree of acute inflammation in the mice.

Glycyrrhizin attenuates the development of carrageenan-induced lung injury in mice.

Glycyrrhizin is a triterpene glycoside, a major active constituent of licorice (Glycyrrhiza glabra) root and numerous pharmacological effects like anti-inflammatory, anti-viral, anti-tumour and hepatoprotective activities has been attributed to it. In this study we evaluated the anti-inflammatory activities of glycyrrhizin in mice model of acute inflammation, carrageenan-induced pleurisy. We report here that glycyrrhizin (given at 10 mg/kg i.p. 5 min prior to carrageenan) exerts potent anti-inflammatory effects in this model. Injection of carrageenan into the pleural cavity of mice elicited an acute inflammatory response characterized by fluid accumulation in the pleural cavity which contained a large number of neutrophils (PMNs) as well as an infiltration of PMNs in lung tissues and subsequent lipid peroxidation (as determinated by thiobarbituric acid-reactant substances measurement) and increased production of tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta). All these parameters were attenuated by glycyrrhizin. Furthermore, carrageenan induced an upregulation of the expression of intercellular cell adhesion molecule (ICAM-1), P-selectin, as well as an increase in the amounts of nitrotyrosine and poly(ADP-ribose) (PAR), as determined by immunohistochemical analysis of lung tissues. The degree of staining for the ICAM-1, P-selectin, nitrotyrosine and PAR was significantly reduced by glycyrrhizin. Additionally, we demonstrate that these inflammatory events were associated with the activation of nuclear factor-kappaB (NF-kappaB) and signal transducer and activator transcription-3 (STAT-3) activation in the lung. NF-kappaB and STAT-3 activation were significantly reduced by glycyrrhizin treatment. Taken together, our results indicate that prevention of the activation of NF-kappaB and STAT-3 by glycyrrhizin reduces the development of acute inflammation.

Signal transduction pathways involved in protective effects of melatonin in C6 glioma cells

Abstract:  Melatonin (N‐acetyl‐5‐methoxytryptamine), an indole hormone, is the chief secretory product of the pineal gland and is an efficient free radical scavenger and antioxidant, both in vitro and in vivo. The role of melatonin as an immunomodulator is, in some cases, contradictory. Although melatonin is reported to influence a variety of inflammatory and immune responses, evidence supporting its effects on important glioma cells‐derived mediators is incomplete. We studied, in rat glioma cell line (C6), the role of melatonin (100 μm‐1 mm) in the regulation of the expression of nitric oxide synthase (NOS) caused by incubation with lipopolysaccharide (LPS)/interferon (IFN)‐γ (1 μg/mL and 100 U/mL, respectively) and defined the mode of melatonin’s action. Treatment with LPS/IFN‐γ for 24 hr elicited the induction of inducible (iNOS) activity as determined by nitrite and nitrate (NOx) accumulation in the culture medium. Preincubation with melatonin abrogated the mixed cytokines‐mediated induction of iNOS. The effect of melatonin was concentration‐dependent. Moreover, Western blot analysis showed that melatonin inhibited LPS/IFN‐γ‐induced expression of COX‐2 protein, but not that of constitutive cyclooxygenase. Inhibition of iNOS and COX‐2 expression was associated with inhibition of activation of the transcription factor nuclear factor kappa B (NF‐κB). The ability of melatonin to inhibit NF‐κB activation was further confirmed by studies on the degradation of the inhibitor of NF‐κB, IκB‐α. Increased production of lipid peroxidation products using thiobarbituric acid assay were found in cellular contents from activated cultures. Lipid peroxidation was decreased by melatonin treatment in a concentration‐dependent manner. Moreover, several genes having roles in heat‐shock response were downregulated in melatonin‐treated cells, such as 70 proteins, reflecting the reduced oxidative stress in these cells. The mechanisms underlying in vitro the neuroprotective properties of melatonin involve modulation of transcription factors and consequent altered gene expression, resulting in downregulation of inflammation.

TNF-alpha blockage in a mouse model of SCI: evidence for improved outcome.

The aim of our study was to evaluate in vivo the therapeutic efficacy of genetic inhibition of TNF-&agr; 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-&agr;, we also investigated the effect of infliximab, a TNF-&agr;-soluble receptor construct, on spinal cord damage. Pharmacological and genetic TNF-&agr; 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-&agr;), (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-&agr; inhibition significantly ameliorated the recovery of limb function (evaluated by motor recovery score). Taken together, our results demonstrate that inhibition of TNF-&agr; reduces the development of inflammation and tissue injury associated with spinal cord trauma, suggesting a possible role of TNF-&agr; on the pathogenesis of spinal cord injury.

Effects of etanercept, a tumour necrosis factor‐α antagonist, in an experimental model of periodontitis in rats

Etanercept is a tumour necrosis factor antagonist with anti‐inflammatory effects. The aim of our study was to evaluate, for the first time, the therapeutic efficacy of in vivo inhibition of TNF‐α in an experimental model of periodontitis.

Melatonin modulates signal transduction pathways and apoptosis in experimental colitis.

Abstract:  Various evidences have documented that the pineal secretory product melatonin exerts an important anti‐inflammatory effect in different experimental models including colitis. The aim of the present study was to evaluate whether melatonin regulates the inflammatory response of experimental colitis in rats at the level of signal transduction pathway. Colitis was induced by intracolonic instillation of dinitrobenzene sulfonic acid (DNBS). Four days after DNBS administration, a substantial increase of colon TNF‐α production was associated with the colon damage. In DNBS‐treated rats, the colon injury correlated with a significant rise of apoptosis (evaluated by TUNEL coloration) which was associated with a significant increased expression of proapoptotic Bax and decreased colon content of antiapoptotic Bcl‐2. This inflammatory response was also related to activation of nuclear factor‐κB (NF‐κB) and phosphorylation of c‐Jun as well as FAS ligand expression in the colon. Treatment with melatonin (15 mg/kg daily i.p.) was associated with a remarkable amelioration of colonic disrupted architecture as well as a significant reduction of TNF‐α. Melatonin also reduced the NF‐κB activation and phosphorylation of c‐Jun as well as the Fas ligand expression in the colon. Furthermore, melatonin reduced the expression of Bax and prevented the loss of Bcl‐2 proteins as well as the presence of apoptotic cells caused by DNBS. The results of this study show that melatonin administration exerts beneficial effects in inflammatory bowel disease by modulating signal transduction pathways.

Pharmacogenetics of Antidepressants

Up to 60% of depressed patients do not respond completely to antidepressants (ADs) and up to 30% do not respond at all. Genetic factors contribute for about 50% of the AD response. During the recent years the possible influence of a set of candidate genes as genetic predictors of AD response efficacy was investigated by us and others. They include the cytochrome P450 superfamily, the P-glycoprotein (ABCB1), the tryptophan hydroxylase, the catechol-O-methyltransferase, the monoamine oxidase A, the serotonin transporter (5-HTTLPR), the norepinephrine transporter, the dopamine transporter, variants in the 5-hydroxytryptamine receptors (5-HT1A, 5-HT2A, 5-HT3A, 5-HT3B, and 5-HT6), adrenoreceptor beta-1 and alpha-2, the dopamine receptors (D2), the G protein beta 3 subunit, the corticotropin releasing hormone receptors (CRHR1 and CRHR2), the glucocorticoid receptors, the c-AMP response-element binding, and the brain-derived neurotrophic factor. Marginal associations were reported for angiotensin I converting enzyme, circadian locomotor output cycles kaput protein, glutamatergic system, nitric oxide synthase, and interleukin 1-beta gene. In conclusion, gene variants seem to influence human behavior, liability to disorders and treatment response. Nonetheless, gene × environment interactions have been hypothesized to modulate several of these effects.

Proinflammatory role of glucocorticoid-induced TNF receptor-related gene in acute lung inflammation

Glucocorticoid-induced TNFR-related gene (GITR) participates in the immune/inflammatory response. Because GITR expression has been described in cells other than T lymphocytes, we investigated whether it also modulates acute inflammatory response. Using GITR-deficient (GITR−/−) mice, we analyzed the role of GITR in the development of carrageenan-induced lung inflammation (pleurisy) by studying several proinflammatory markers 2–8 h after carrageenan injection. When compared with GITR+/+, GITR−/− mice exhibited decreased production of turbid exudate containing a lower number of leukocytes. This was correlated with the reduction of inflammatory markers (including TNF-α, IL-1β, myeloperoxidase, inducible NO synthase, and cyclooxygenase 2) in the pleural exudate and/or in the lung. Moreover, endothelial cells expressed lower levels of adhesion molecules. In lungs of GITR+/+ mice, GITR ligand expression was not modulated during pleurisy, while that of GITR increased, as a consequence of increased infiltration by GITR-expressing cells and of GITR up-regulation in macrophages and endothelial cells. Finally, cotreatment of GITR+/+ mice with carrageenan and Fc-GITR fusion protein decreased the number of inflammatory cells (pleural macrophages and lung neutrophils) as compared with carrageenan treatment alone, confirming that GITR plays a role in the modulation of pleurisy.

Glycogen Synthase Kinase-3β Inhibition Reduces Secondary Damage in Experimental Spinal Cord Trauma

Glycogen synthase kinase-3 (GSK-3) has recently been identified as an ubiquitous serine-threonine protein kinase that participates in a multitude of cellular processes and plays an important role in the pathophysiology of a number of diseases. The aim of this study was to investigate the effects of GSK-3β inhibition 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 (SCI) in mice resulted in severe trauma characterized by edema, neutrophil infiltration, production of a range of inflammatory mediators, tissue damage, and apoptosis. Treatment of the mice with 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), a potent and selective GSK-3β inhibitor, significantly reduced the degree of 1) spinal cord inflammation and tissue injury (histological score); 2) neutrophil infiltration (myeloperoxidase activity); 3) inducible nitric-oxide synthase, nitrotyrosine, and cyclooxygenase-2 expression; and 4) and apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and Bax and Bcl-2 expression). In a separate set of experiments, TDZD-8 significantly ameliorated the recovery of limb function (evaluated by motor recovery score). Taken together, our results clearly demonstrate that treatment with TDZD-8 reduces the development of inflammation and tissue injury associated with spinal cord trauma.